United States
Environmental Protection
Agency
Health Effects Researtfi
laboratory
Research Triangle Park NC 2771 1
Research and Development
&EPA
Interagency
Program in
Energy-Related
Health and
Environmental
Effects Research
Project Status Report
Interagency
Energy/Environment
R&D Program
Report
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the INTERAGENCY ENERGY-ENVIRONMENT
RESEARCH AND DEVELOPMENT series. Reports in this series result from the
effort funded under the 17-agency Federal Energy/Environment Research and
Development Program. These studies relate to EPA's mission to protect the public
health and welfare from adverse effects of pollutants associated with energy sys-
tems. The goal of the Program is to assure the rapid development of domestic
energy supplies in an environmentally-compatible manner by providing the nec-
essary environmental data and control technology. Investigations include analy-
ses of the transport of energy-related pollutants and their health and ecological
effects; assessments of, and development of, control technologies for energy
systems; and integrated assessments of a wide'range of energy-related environ-
mental issues.
This document is available to the public through the National Technical Informa-
tion Service, Springfield. Virginia 22161.
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EPA-600/7-79-009
January 1979
INTERAGENCY PROGRAM IN ENERGY-RELATED HEALTH
AND ENVIRONMENTAL EFFECTS RESEARCH
Project Status Report
Health Effects Research Laboratory
Office of Health and Ecological Effects
Office of Research and Development
U.S. Environmental Protection Agency
Research Triangle Park, N.C. 27711
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
OFFICE OF HEALTH AND ECOLOGICAL EFFECTS
HEALTH EFFECTS RESEARCH LABORATORY
RESEARCH TRIANGLE PARK, N.C. 27711
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DISCLAIMER
This report has been reviewed by the Health Effects Research
Laboratory, U.S. Environmental Protection Agency, and approved for
publication. Mention of trade names or commercial products does
not constitute endorsement or recommendation for use.
n
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FOREWORD
The many benefits of our modern, developing, industrial society
are accompanied by certain hazards. Careful assessment of the relative
risk of existing and new man-made environmental hazards is necessary
for the establishment of sound regulatory policy. These regulations
serve to enhance the quality of our environment in order to promote the
public health and welfare and the productive capacity of our Nation's
population.
The Health Effects Research Laboratory, Research Triangle Park,
conducts a coordinated environmental health research program in toxicology,
epidemiology, and clinical studies using human volunteer subjects.
These studies address problems in air pollution, non-ionizing
radiation, environmental carcinogenesis and the toxicology of pesticides
as well as other chemical pollutants. The Laboratory participates in
the development and revision of air quality criteria documents on
pollutants for which national ambient air quality standards exist or
are proposed, provides the data for registration of new pesticides or
proposed suspension of those already in use, conducts research on
hazardous and toxic materials, and is primarily responsible for providing
the health basis for non-ionizing radiation standards. Direct support
to the regulatory function of the Agency is provided in the form of
expert testimony and preparation of affidavits as well as expert advice
to the Administrator to assure the adequacy of health care and surveillance
of persons having suffered imminent and substantial endangerment of
their health.
When energy and material resources are extracted, processed, converted
and used, waste products are emitted which may have a significant impact
upon public health and the environment. A highly integrated, multidiscipli-
nary research and development effort is required to assess this impact. The
research projects reported on in this document constitute this Laboratory's
input to this Interagency Program.
F. G. Hueter, Ph. D.
Acting Director,
Health Effects Research Laboratory
m
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ABSTRACT
This report summarizes research supported by the EPA Health Effects
Research Laboratory at Research Triangle Park, NC, under the Federal Inter-
agency Energy/Environment R & D Program. The EPA has had the lead responi-
bility for the planning, coordination and implementation of this program
since fiscal year 1975.
Projects reported in this document are grouped under one of four major
research areas. The first area is identification of hazardous agents
associated with non-nuclear energy technologies. These projects involved
the development of qualitative methods for the identification of hazardous
materials. The second area is development of more rapid and sensitive
methods to evaluate dose to man. These projects focused on the development
of quantitative methods for measuring degree of toxicity of various pol-
lutants. The third area is determination of the metabolism and fate of
hazardous agents associated with energy technologies. These projects in-
volved determination of the physiological activities of several known carcin-
ogens. The fourth research area is evaluation of hazards to man. In addi-
tion to studies of the effects of certain pollutants on humans, several of
the projects concerned preparation of standard pollutant samples for use in
future studies to increase the comparability of results.
A list of additional studies funded under this program is included.
IV
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CONTENTS
Foreword iii
Abstract iv
1. Identify Hazardous Agents Associated With Non-Nuclear Energy
Technologies 1
Development of Methods for Determination of Carcino-
genes is by Bacterial Mutagenesis Employing Crude
Material from Alternate Energy Sources 1
Determination of the Influence of Materials Concerned
with the Extraction of Ores 9
Study of Metabolic and Physiologic Measurements in
Populations with Long-Term Exposure to Pollution
from Coal Conversion 12
Implementation of Screening Tests for Potentially
Hazardous Airborne Particulate Material 45
2. Develop More Rapid and Sensitive Methods to Evaluate Dose
to Man 48
Development of Test System to Assess Potential
Toxicity and Neoplastic Transformation 48
Enzymatic Characterization of Metabolic Activation and
DNA Binding of Presumptive Carcinogens in Short-Term
Assay Systems for Environmental Carcinogens 52
Development of Bioindicators to N02 and S0£ Exposure . . 54
In Vitro Screening^f Selected Air Pollutants for
Potential Carcinogenicity 57
In Vitro Screening of Selected Air Pollutants for
Potential Carcinogenicity Using Microbial Systems ... 61
Develop Cellular Model System to Determine Cytotoxicity
from Alternate Energy Sources 66
Development of Automated Behavioral Testing Method Test-
ing Methodologies for Study of Coal Conversion and
Utilization Products 67
Application of Automated Behavioral Testing System to
Monkeys Exposed to Coal Conversion Pollutants 68
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Detection of Genotoxic Effects of Environmental
Chemicals in Cultured Liver Cells
Biological Assessment of Exposure to Sulfur Dioxide
and Acid Sulfate .................... 79
3. Determine the Metabolism and Fate of Hazardous Agents
Associated with Energy Technologies ............. 87
The Effects of Whole Animal Exposure to Acid Mists
and Particulate on the Pulmonary Metabolsim of
Benzo(a)Pyrene in Isolated Perfused Lung Model ..... 87
Evaluate Influence of Inhalation of Acid Areosols
(^SO^, S03, HN03) and Particulate Production of
Chrome Lung Disease in Rats, Guinea Pigs, and gQ
Primates ........................
Determination of the Effects of Material from Alternate
Energy Sources on Upper Respiratory Tract Clearance
Mechanisms ....................... 91
Comparison of Pulmonary Carcinogenicity of Known
Carcinogens With and Without Added I^SO^ Mists, Air-
borne Respirable Particles, and Gases . ........ 94
Compare the Effects of Respirable Particles, Gases,
and Mists Using Small Airway Resistance in Donkeys as
the Model fqr Pulmonary Irritation ...... ..... 114
Studies on the Relationship Between Carcinogen Metabolism
in the Alveolar Macrophage and the Induction of Lung
Cancer ......................... 116
4. Evaluation of Hazards to Man ................. 120
Effects of Coal Gasification Prpcjuc£s on the Pulmonary
Defense System Against Infectious Disease ....... 120
To Determine Effects of Pollutants from Alternate Energy
Sources on Pulmonary Antiviral Mechanisms ....... 122
Quality Control for Assessment of Human Exposure .... 124
Evaluate Hazards of Exposure to Biological Active Agents
Associated with Energy Technology ........... 127
Studies of Health Effects Resulting from Increased In-
door Air Pollution from Energy Conservation ...... 134
Chemical Repository for Alternate Energy Source Materials 136
Preparation and Characterization of Fine Particulate En-
vironmental Contaminants for Biological Experimentation 142
Effects of Material from Alternate Energy Sources on
Whole Animal Defense Mechanisms
Environmental Mutagens Studies Utilizing a Drosophila . 145
VI
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To Evaluate Existing and Improved Methods for Sampling,
Transport, Storage and Analysis of Biological Specimens
Which Might Serve as Indicators of Contamination by
Effluents from Energy Technologies 148
Effect of Pollutants from Coal Burning and Coal Gasi-
fication on the Immune System 152
5. Other Category Projects 155
vn
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SECTION I
IDENTIFY HAZARDOUS AGENTS ASSOCIATED WITH NON-NUCLEAR ENERGY TECHNOLOGIES
A. TASK TITLE:
Development of Methods for Determination of Carcinogenesis by
Bacterial Mutagenesis Employing Crude Material from Alternate
Energy Sources.
HERL/RTP TASK NO: 8151
CONTRACTOR: Washington Universtiy
CONTRACT NO: 68-02-2287
Summary
Ames originally developed an in vitro mutagenic screening method using
special strains of Salmonella typhimurium. These strains have a histidine-
negative genome and include other genetic factors that render them specific-
ally sensitive to chemical mutation. Mutation causes them to revert to a
histidine-positive genome, so that the presence of a mutagenic agent can be
determined by counting the number of colonies that appear when bacteria are
inoculated on a nutrient medium that lacks histidine.
The original system has been employed primarily to assay pure compounds
or fairly simple mixtures. The technique has been modified under this pro-
ject in order to effectively use it to analyze crude mixtures or organic com-
pounds which may occur in coal and oil shale conversion processes; and as a
biological screening technique. Significant modifications include:
1. development of a liquid culture method as opposed to the conventional
plate method;
2. development of improved methodology for the analysis of mutagenic
urinary metabolites of carcinogens based on the liquid/culture
method;
3. development of techniques for producing dose response curves of
mutagenic activity, and;
4. development of a procedure for comparing the "relative mutagenic
activity: of different samples.
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The modified techniques have been tested and employed on analyses of
samples of coal liquification products, atmospheric particulate samples from
Los Angeles and Pittsburgh, and human urine samples.
Scope and Objective
Determine the carcinogenicity of crude alternate energy source effluents
and products by means of in vitro systems previously developed in this project;
includes whole animal and other means of detecting carcinogenic metabolites in
the urine for eventual application of clinical and population studies of groups
exposed to various carcinogenic hazards.
Background and Approach
Most carcinogenic compounds are not mutagenic toward Salmonella unless
they are converted to metabolic intermediates by the action of suitable tissue
microsomal preparations. The Salmonella method can generally distinguish
between presumptive carcinogens (compounds which have been shown to cause can-
cer in laboratory animals, but which may or may not cause cancer in people)
and noncarcinogens. However, the Salmonella mutagenesis systems now are em-
ployed primarily to assay pure compounds or relatively simple mixtures. Cer-
tain modifications of the technique are necessary in order to effectively use
it to analyse crude mixtures of organic compounds, particularly as they occur
in the various stages of coal conversion processes and shale oil processing.
The initial approach as outlined below has been modified during the course of
the project, as indicated under Research Accomplished.
In the conventional Salmonella test procedure, the sample is mixed with
the liver homogenate and the bacterial inoculum and spread over the entire
surface area of the test plate. In one modified procedure, aliquots of ex-
tracts from the crude sample are evaporated from a suitable solvent onto small
pieces (1 cm diameter) of sterile filter paper. The microsomal preparations
and the bacterial inoculum are mixed and spread over the agar plate and the
filter discs carefully placed on the top of the agar surface. The appearance
of colonies of revertant mutants in the medium, in statistically significant
numbers (compared to controls discs which lack the test compound) surrounding
the disc, indicate the presence of presumptive carcinogens. Because this pro-
cedure concentrates the sample in the filter paper, it can enhance the overall
sensitivity of the method, and also give some indication of the presence of
active substances which differ in their rates of diffusion.
In the preliminary screening, all samples are tested against a maximum
of five different strains of Salmonella typhimurium. including TA 1535, Ta 100,
TA 1537, TA 1538, and TA 98, and any additional strains of superior character-
istics that may become available during the course of the work. A minimum of
three different rat liver microsomal preparations (induced by sodium pheno-
barbital, PCB, and where possible, the crude sample itself) which differ in
their inducing agent are used for each test. The microsomal preparations are
isolated by standard biochemical methods.
The standard bacteriological plates contain a suitable agar nutrient
medium that lacks histidine. These plates are prepared in duplicate, together
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with duplicate control plates (identical with the above but lacking the crude
test substance on the filter paper disc). An additional control consists of a
plate inoculated with a mixture of bacteria and the substance being tested—
i.e., with no added microsomal preparations. After 48 hours incubation at
37°C, counts of revertant colonies are made in a circular zone surrounding the
disc and compared with those of the control discs to determine mutagenic acti-
vity.
If the sample shows mutagenic activity in this test, it is further tested
to determine dose-response relationships by the standard Salmonella test. The
strain-microsomal combinations which yielded the highest mutagenic activity in
the paper disk method are selected and retested by the standard Salmonella
test at a series of different concentrations of the crude mixture to establish
dose-response relationships.
In addition to the direct Salmonella test of the whole crude sample des-
cribed above, each of the samples is fractionated by thin layer chromatography.
Then, strips of the chromatographic fractions are placed on plates seeded with
the bacterial strains and the microsomal preparations. The test plates are
incubated for 48 hours at 37°C and zones of revertant colonies surrounding
the paper strips are located (by comparison with appropriate control paper
strips with no test compound). Plots of the number of colonies as a function
of distance along the strip are prepared when appropriate.
Each of the crude test samples is subjected to thin layer chromatography
using at least two different solvent systems, and is tested against the same
five different strains of Salmonella and three different microsomal prepara-
tions. Each of the tests is made in duplicate together with duplicate control
plates (which lack the crude test substance on the TLC)—and also an additional
control plate which lacks the microsomal preparation.
If the TLC paper strip shows zones of mutagenic activity, the correspond-
ing zone from the main chromatogram is cut out and extracted using an appro-
priate solvent such as benzene, hexane, or chloroform. The extract is taken
to dryness and analyses undertaken to identify the chemical component respon-
sible for the mutagenic activity. Studies to identify mutagenically active
compounds include one or more of the following physico-chemical methods:
U.V.; NMR; mass spectrometry; ESR hyperfine labelling and comparison of chro-
ma tographic properties with known standards.
The results of tests on mixed crude samples are compared with the results
of bacterial mutagenesis tests on the separate fractions of the thin layer
chromatogram to determine the effect of synergistic interactions and inter-
ference among the substances present in the mixed samples.
The Salmonella test relies on the now well established idea that while
some carcinogenic substances appear to be the proximately active agents which
directly induce the process of carcinogenesis, many such substances must be
converted metabolically before they are active, proximate carcinogens. Sub-
stances of the latter type are mutagenic in the Salmonella test system only
after being converted to active metabolites by a microsomal preparation which
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is included in the i£ vitro test system for that purpose. In the standard
Salmonella test this activation system consists of the 9,000-g supernate of a
rat liver homogenate, to which has been added NADPH and glucose-6-phosphate,
as a means of supplying an electron source for the oxidative process.
It is clear, however, that the activity of the microsomal preparation is
much more complicated than a simple conversion of a carcinogenic substance
into a given active metabolite. In previous tests it was found that some pre-
sumptive carcinogens are "false negatives." That is, they apparently are not
converted into active metabolites by the microsomal preparation as it is now
used. These observations indicate that our knowledge of the activation pro-
cess, as it occurs in the crude microsomal system, is rather superficial, and
needs to be refined in order to maximize the sensitivity and reliability of
the Salmonella test system.
One way to improve the activation process is to administer the test com-
pound to whole animals and to determine the presence of carcinogenic metabo-
lites in the body fluids of the animal, such as urine, blood, or bile. Stu-
dies have shown that mutagenic metabolites appear in the urine of laboratory
animals that have been fed two of the carcinogens that were "false negatives"
in the standard in vitro Salmonella test (N,N-dimethylaminoazobenzene and
1,2,5,6-dibenzathracene). The approach then will be to administer the crude
test sample to laboratory animals (either by injection or feeding) and to test
their urine by means of the Salmonella method. If any mutagenically active
metabolites are formed, they will be fractionated by the thin layer chromato-
graphic procedure. Attempts are made to identify the active compound(s).
Since such metabolites may not always be excreted in urine, it may be neces-
sary to also analyse blood or bile samples for mutagenic activity.
Research Accomplished
Using the Ames test as a basis, a liquid test system was developed in
which not only mutation, but the actual replication of mutated bacterial cells
takes place in a liquid culture medium. After a period of incubation the num-
bers of non-revertant and mutant (revertant) cells are then determined by
spreading aliquots of the incubated culture on agar plates containing an ex-
cess of histidine and no histidine, respectively. The effectiveness of the
new liquid method was compared with a procedure in which the urine is hydro-
lyzed with B-glucuronidase to free metabolites from their conjugates; it is
then extracted with ether or a mixture of benzene and isopropanol and the ex-
tract, after being reduced to dryness, is taken up in DMSO and aliquots are
tested for mutagenicity by the standard plate method. The extraction step is
essential in order to remove histidine (often present in urine) which would
otherwise support the growth of non-revertant bacteria and thus obscure the
counts of revertant bacteria on the test plate. The liquid method obviates
the need for removing histidine, so that the entire process can be compressed
into a single step, in which urine together with B-glucuronidase is added to
an incubation medium containing an appropriate Salmonella strain and micro-
somes (if desired).
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In order to validate this procedure, the capability of the liquid and the
conventional plate method was compared in detecting mutagenic material in
benzene-isopropanol extracts of urine. The results were found to be generally
similar with both methods. In extracts of urine from AAF-fed rats, some acti-
vity is observed by the plate method in the absence of microsomes (indicative
of inherently active mutagens); in this circumstance the liquid method appears
to yield no activity. Much more activity is observed by both methods when
microsomes are present (indicative of activatable mutagens). If the sample is
first treated with B-glucuronidase the activity is considerably enhanced.
The ultimate aim of the urine test is to provide a rapid and inexpensive
method of analyzing human urine in order to determine whether an individual is
being exposed to (and metabolizing) an environmental carcinogen. The original
urine technique (i.e., based on conventional plate tests of urine extracts)
was applied to a group of human urine samples, and showed that most samples
exhibit no activity, with a small percentage of samples exhibiting a positive
effect. In an initial test of the new liquid method on human urine, essen-
tially all samples were negative with respect to mutagenic activity. The
variation among the samples was about the same in the two methods.
The following three procedures developed under this project were tested
on human samples from farmers with one group using pesticides and the other
not.
1. Whole urine/liquid method: In this method a sample of urine (steri-
lized by passage through a millipore filter) is added to a mixture
consisting of nutrient broth, an inoculum of a suitable Salmonella
strain, a microsome preparation, and a solution of B-glucuronidase.
(The B-glucuronidase hydrolyzes conjugates of mutagenic components,
releasing them in an active form. The microsome preparation oxidizes
those mutagens which require activation to form active metabolites.)
The entire mixture is incubated at 370C for 16 hours. At that time
aliquots are removed and inoculated on standard histidine-free
culture plates. Revertant colonies (i.e., those derived from cells
which have mutated to a histidine-positive genome) that are present
in the incubated culture will grow in these plates, and their number
is determined by counting duplicate plates.
2. Urine extract/liquid method: In this procedure B-glucuronidase is
added to the urine sample adjusted to pH 7.0 which is then incubated
at 37°C for 18 hours to hydro!yze conjugates. The sample (150 ml
urine) is then extracted twice with 100 ml aliquots of benzene:iso-
propanol (80:20). The aqueous residue is then adjusted to pH 2.5
and refluzed for 15 minutes to hydrolyze esters of potentially muta-
genic constituents, followed (after cooling) by extraction in benzene:
isopropanol, as above. The remaining aqueous fraction is then ad-
justed to pH 11.0 and again extracted with benzene: isopropanol.
Each of the benzene:isopropanol extracts (obtained at pH 7.0, 2.5
and 11.0) was then dried in a rotary vacuum dryer and the residues
taken up in DMSO. The residues were then added to liquid cultures
and treated as described under (1) above. Aliquots representing 1, 5
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and 10 ml or urine were tested in this way, both in the presence and
absence of microsomes.
3. Urine extract/plate method: In this procedure urine extracts were
prepared at pH 7.0, 2.5 and 11.0 as described under (2) above. Each
extract, taken up in DMSO, and aliquots representing 5 ml of urine
were then added to standard test plates in the usual way and the num-
ber of revertant colonies determined. These tests were carried out
both in the presence and absence of microsomes.
With the exception of a few samples, all results were negative, based
upon the "mutagenic activity ratio" described below. There was considerable
variation in the negative values for duplicate samples, probably due to random
error in the techniques. Recently Ames, .et _aj., reported a method for concen-
trating mutagens from human urine about 200-fold for subsequent assay in the
Salmonella mutagenesis test. With this procedure he was able to demonstrate
the presence of mutagens in the urine of cigarette smokers. In this method
the urine is put through a 1.5 cur bed volume XAD-2 column and the absorbed
material is then eluted with a few milliliters of acetone. The acetone is
taken to dryness and the residue is dissolved in DMSO. Preliminary tests to
evaluate this method were conducted under this project.
Urine samples from five males (two nonsmokers and three smokers) were
analyzed. These tests were carried out against TA 1538 with and without the
presence of microsomes. In each test a 25 ml equivalent of the urine sample
was tested per plate. In one set of experiments the urine samples were passed
through the XAD-2 column without prior treatment with B-glucuronidase and in
the second set of experiments the urine was pretreated with B-glucuronidase
(the urine sample adjusted to pH 7.0 was incubated with B-glucuronidase 600
units/ml urine for 18 hours to hydrolyze the sugar conjugates) prior to pass-
ing through the XAD-2 column.
The results showed differences in the mutagenic activity of the urine
samples from smokers and nonsmokers. The two nonsmokers did not yield values
significantly higher than their respective controls. In the absence of B-
glucuronidase treatment and microsomal activation, the three smokers also did
not show activity much higher than the respective controls. However, after
B-glucuronidase treatment one smoker showed somewhat higher mutagenic activity
in the absence of the microsome preparation. In the presence of the microsome
preparation the urine samples from all three smokers yielded higher activity
with or without B-glucuronidase treatment. In two instances B-glucuronidase
treatment had little or no influence on mutagenic activity.
The results may be summarized as follows:
1. In the absence of the microsome preparation none of the samples
yielded values significantly higher than the controls under the dif-
ferent test conditions.
2. In the presence of the microsome preparation the nonsmokers did not
show positive mutagenic activity under the different test conditions.
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3. Among smokers, in general, the unhydrolyzed urine showed higher muta-
genic activity followed by B-glucuronidase hydrolyzed urine. Acid
hydrolyzed and B-glucuronidase plus sulfatase hydrolyzed urine sam-
ples showed consistently lower activity. In the case of smokers,
acid hydrolysis, as well as sulfatase treatment, resulted in toxic
effects (i.e., decreased mutagenic activity). Based on the foregoing,
it would appear that it is advisable to use unhydrolyzed urine in the
XAD-2 method.
On the basis of these preliminary results, it would appear that the XAD-2
absorption would be the method of choice for analyzing the human urine samples.
The XAD-2 absorption method (modified by substituting a 3 cm3 bed volume of
XAD-2 for the 1.5 cm3 bed volume originally used) was tested on urine samples
from Chicago steel workers (smokers and nonsmokers) and from organic farmers
(nonsmokers). The results from the organic farmers were all negative. All
positive results were found with the steel workers who smoked, although not
all smokers yielded positive results. These preliminary results indicate
that the XAD-2 absorption method is capable of detecting exposure to mutagenic
material in human urine associated with cigarette smoking. However, this is
based on a small sample and does not reflect the extent of individual varia-
tion in response.
Test data have shown a non-linear response of the Salmonella system to
most mutagens. This makes it difficult to determine from dose-response curves
how the mutagenic activity of different samples compare. Therefore, a proce-
dure was employed to provide a value for the "relative mutagenic activity"
(RMA) of a sample, based on the comparison of 50 known organic noncarcinogens
and 50 organic compounds that had been previously shown to be carcinogenic
toward laboratory animals. In this comparison a "mutagenic activity ratio"
was computed from the quotient (E - C)/CAv> where E is the number of mutant
colonies obtained from the experimental sample; C is the control value (i.e.,
the number of mutant colonies observed when the experimental material is not
included) obtained on the day of analysis; and C/\v is the "historical" control
value, or the average control value for all runs carried out during the course
of the study. Studies have shown that in the presence of liver microsomes
93% of the non-carcinogens yield mutagenic activity ratios of 2.0 or less and
83% of the carcinogens yield ratios greater than 2.0. About 98% of the noncar-
cinogens yield ratios of 2.0 or less if microsomes are absent. In reporting
analyses, a sample is regarding as possessing statistically significant mu-
tagenic activity if at some concentration it yields a mutagenic activity ratio
greater than 2.0. This type of analysis was conducted on air samples from
Los Angeles and Pittsburgh.
Several of the air samples were devoid of mutagenic activity toward bac-
teria strains TA 1538 and strain TA 100. These samples were primarily ali-
phatic fractions, MAP (Middle A.P. [heavy]), CD (composite dust sample, Alleg-
heny County) and the top stage (20-3.5/*) of the atmospheric sample (AP). There
was considerable variation in the relative mutagenic activities of different
sample fractions. The highest level of mutagenic activity occurred in aroma-
tic fraction II. Oxy-neutral fractions II and III and aromatic fraction I were
somewhat less active, while oxy-neutral fraction I had a low activity and the
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aliphatic fraction had zero activity. The Pittsburgh acid fraction (PHS-2)
and organic extracts were very active. The relative mutagenic activity of the
air particulates increased significantly with decreasing particle size, as
shown both in Pittsburgh and Los Angeles.
Results were obtained with two different Salmonella strains: TA 1538,
which is particularly responsive to frame-shift mutations, and TA 100, which
is particularly responsive to base-pair mutations and to certain types of
frame-shift mutations. With two exceptions, the relative mutagenic activities
of different samples are roughly parallel in the two strains. The aromatic
fraction II samples from Pittsburgh showed a disproportionally high effect
with TA 100 (in the presence of the microsome preparations), suggesting that
it contains a relatively high concentration of base-pair mutations. The Pitts-
burgh acid fraction (PHS-2) was only slightly active toward TA 100, suggesting
that base-pair mutagens are absent, and that the activity was due to frame-
shift mutagens which affect TA 1538, but not TA 1,00.
In some samples, inherently active mutagens (i.e., those which do not
require microsomal activation) were apparently present. Where such mutagens
occur, the interpretation of the mutagenic activity of the sample when the
microsomal preparation is present must remain qualitative. This is based on
the fact that some inherently active mutagens are inactivated by the microso-
mal preparation, while others are not affected. Thus, the mutagenesis test
responds to three general classes of mutagens: (a) inherently active mutagens
which are not inactivated by the microsome preparations; (b) inherently active
mutagens which are inactivated by the microsome preparation; (c) mutagens
which are activated by the microsome preparation. Tests conducted in the ab-
sence of the microsome preparation measure the activity due to mutagens of
classes (a) and (b). Tests conducted in the presence of the microsome pre-
paration are indicative of activity due to mutagens of class (c) plus the
activity observed in the absence of the microsome preparation, less the acti-
vity of mutagens of class (b). Thus, the value observed in the presence of
the microsome preparation is a measure of the mutagens of class (c), only if
there is no appreciable activity when the microsome preparation is absent.
These constraints are particularly applicable to the values obtained from
Pittsburgh organic extract samples, and the atmospheric particulate samples.
The relative mutagenic activities computed from the Los Angeles air parti-
culate samples generally showed an inverse relationship between particle size
and mutagenic activity. The relationship between the mutagenic activities of
samples collected upwind and downwind from the source are less consistent,
although more often than not the activity of the downwind sample is somewhat
higher than the comparable upwind one.
Mutagenesis tests on six synthetic fuel oil samples were conducted. Du-
plicate plates were prepared with successive amounts of the sample dissolved
in DMSO. The samples were tested with five different strains of Salmonella,
both in the presence and absence of microsomes. None of the samples showed
activity toward strains TA 1353, TA 1537, or TA 100. None of the samples
showed significant mutagenic activity in the absence of microsomes. A light,
hydro-treated oil sample failed to show a positive mutagenic response against
8
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any strain. In the concentration range of 100 to 1000 M9> samples of heavy
oil, heavy, hydrotreated oil, and filtered, raw pyrolysis oil showed positive
mutagenic responses toward strains TA 1538 and TA 98 in the presence of micro-
somes. The "syncrude" samples showed a weak positive response against the
same strains at 1000 jig. The results of the mutagenesis tests of a sample
of MADE #4 (fuel oil) showed that in the presence of microsomes the sample was
mutagenic toward TA 1538 and TA 1537. Activity toward TA 98 was borderline.
In the absence of microsomes the sample showed positive response toward TA
1538 at 1 /xg and toward TA 1537 at 50,000 Mg. The sample failed to show any
mutagenic activity toward strains TA 1538 or TA 100. Based on thin layer
chromatography (TLC) analysis this sample contained at least two mutagenically
active components, one active toward TA 1538 and the other toward TA 1537.
Bibliography
Commoner, B., J. I. Henry, J. C. Gold, M. J. Reiding and A. J. Vithayathil.
1976. Reliability of Bacterial Mutagenesis Techniques to Distinguish
Carcinogenic and Noncarcinogenic Chemicals. EPA-600/1-76-022.
Commoner, B., P. Madyzstha, A. Bronsdon, and A. J. Vithayzthil. 1978. Envir-
onmental Mutagens in Urban Air Particulates. Journal of Toxicology and
Environmental Health. 4:59-77.
Related Research
There is a wide range of related research on in vitro screening techni-
ques both within the Interagency Program and other research agencies and in-
stitutions.
B. TASK TITLE:
Determination of the Influence of Materials Concerned with the
Extraction of Ores
HERL/RTP TASK NO: 8152
CONTRACTOR: Northrop Services, Inc.
CONTRACT NO: 68-02-2566
Summary
Commercial asbestos of various types are known to cause human disease,
notably pulmonary fibrosis, pleural plaque and chronic fibrosis, lung cancer
and malignant tumors of the pleura and peritoneum, and possibly cancers in the
gastrointestinal tract. These occur in asbestos miners, manufacturers of as-
bestos products, and workman utilizing such products. Additionally, a number
of persons in peripheral contact with mining or mine workers have developed
malignancies.
Asbestos is a fibrous silicate which occurs in a number of varieties.
Other forms of silicates which occur abundantly in the earth's crust have
-------
varying resemblance (in the formation of either fiber or long narrow crystals)
to commercial asbestos. Such material is being constantly turned up in vari-
ous forms by mining, quarrying, road building, etc., it is of great import-
ance to know whether persons exposed to such material are at risk in a simi-
lar manner to those exposed to asbestos.
The importance of this sort of information to EPA is attested to by the
large number of inquiries, litigations, and the like with which the Agency
has to deal when such materials are reported. For the above reasons a re-
search project on the potential toxicity of the non-asbestos fibrous minerals
was in October, 1975, when building of a facility was begun. Concomitant
with this, a provisional protocol was circulated to informed individuals for
comment. These comments were reviewed by an ad hoc study section and a for-
mal protocol was approved in June, 1976. The preliminary biological experi-
mentation began in July, 1976 and intrapleural and intratracheal studies were
begun.
Scope and Objectives
This study comprises the following tasks:
1. Intratracheal incoulation of 2000 rats.
2. Intrapleural incoulation of 450 rats.
3. Toxicity in tracheal transplants.
4. l£ vitro studies
a. RBC lysis tests
b. Macrophage lysis tests
c. Toxicity to human fibroblasts W138
d. Toxicity to Chinese hamster ovary cells (CHO)
5. Retention of fibrous minerals in lung.
6. Relation of fibrous minerals in lung.
7. Influence of mineral fibers on biology of pulmonary macrophages.
8. Interaction of mineral fibers and organic carcinogens.
The objectives are (1) the determination of toxicity of mineral fibers
in the elicitation of chronic disease, cancer of the lung, mesatheliomas, and
related diseases as related to their mineralogical characteristics and com-
pared to the variations with the asbestos similar parameters, and (2) estim-
ation of health risk of exposure to such fibers during mining, quarrying, and
related operations.
Background and Approach
There is a growing body of evidence suggesting the health effects
10
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associated with exposure to asbestos may not be unique to the commercial
forms, but may also be associated with asbestos-like fibers found in other
rocks, and even man-made fibers. It is now known that the external gross
appearance of the mineral may not be the determinant but rather its micro-
fibrillar nature which is expressed after crushing or grinding. The toxicity
potential of the silicate mineral may, therefore, be related to its procli-
vity to form.micro-crystals of a certain configuration during crushing and
grinding. Thus, it is likely that a mode of toxicity, hitherto thought to be
associated only with commercial asbestos, may be present at a greater or
lesser degree in other fibrous amphiboles.
The approach consists of a closely integrated mineralogical-biological
project which compares minerals from various sites containing amphiboles of
differing positions on the cummingtonite-grunerite scale, and other minerals.
These are selected on the basis of thorough geological study and a certain
number are designated for possible biological experimentation. The samples
are then prepared uniformly in a manner to assure release of a maximal num-
ber of fine crystals, or fibers. Biological experimentation with such mate-
rial would make possible (1) selection of the worst possible case on the
basis of geological prediction, (2) determination of the biological activity
on the basis of the specific geological type, and (3) assurance of a concen-
tration of the specific geological characteristics in any given biological
experiment. The latter approach maximizes the possibility of determining
significant biological activity and relating that activity by dose-response
to the type of amphibole rock. This information would be of value not only
in a specific locale, but would have wide application in rock operations
generally.
The biological study relates the presence or absence of lung cancer,
fibrosis in the lung, and mesotheliomas to mineralogical types, using commer-
cial asbestos as a positive control and the injection vehicle (gel saline)
as a negative control. The approach in the in vitro systems compares lysis
and other parameters on the basis of presence or absence of fibers, internal
molecular structures, etc.
Research Accompli shed
Two thousand animals have been injected with fibrous minerals by intra-
tracheal instillation and intrapleural injection. The highest internal dose
was determined, and pulmonary pathological alteration was determined by short-
term studies. Comparative retention of amphibole fibers and amosite asbestos
fibers determined.
In vitro studies have demonstrated differences in lysis activity be-
tween various closely related minerals, including fibrous and non-fibrous
varieties. Mineral properties such as surface area, surface charge, and ele-
mental analysis were correlated with the lytic action on mammalian erythro-
cytes. The same particles were tested for toxicity in rabbit alveolar macro-
phage systems and Chinese hamster ovary cell systems. Toxicity in these sys-
tems seemed to correlate with lysis in mammalian erythrocyte systems.
11
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A report of this work was published in procedings of the Asbestos Work-
shop sponsored by the National Bureau of Standards. Additional scientific
papers are in preparation. An annual progress report was submitted to EPA
by the contractor.
Bibliography
Coffin, D. L./and L. D. Palekar. 1977. EPA Study of the Biological Effects
of Asbestos-Like Mineral Fibers. Presented at the U. S. Bureau of
Standards Meeting on Asbestos, Gaithersburg, Maryland. July 18-20.
Langer, Arthur M., 1977. Study Group Report, EPA Advisory Committee for the
In Vitro Study of Fibrous Amphiboles. March 15.
Related Research
Preliminary studies are being performed on rock dusk from oil shale min-
ing and retort particles in interaction with asbestos-like minerals.
C. TASK TITLE:
Study of Metabolic and Physiologic Measurements in Populations
with Long-Term Exposure to Pollution from Coal Combustion
HERL/RTP TASK NO: 8166
GRANTEE: University of Akron
GRANT NO: R804256
Summary
This study involves an investigation of the physiologic changes assoc-
iated with long-term exposure to combustion products of coal with high sulfur
content, being carried out in Cleveland, Ohio and Elyria, Ohio. The partici-
pants in this phase of the study are from 10 through 60 years old. The
health indices compared are:
-accumulation of trace metals in human systems
-pulmonary functions
-cardiovascular function, and
-incidence of hypertension.
Also, current and historic air pollution levels are measured and/or
estimated for each of the two communities.
This study also compares frequency and severity of acute respiratory
illness in 5th and 6th grade children relative to levels of air pollutants.
This component is being carried out in Akron, Ohio. The individual school
room teachers are keeping daily diaries of each child's symptoms. Base-line
pulmonary function was established for each child at the beginning of the
study and the test repeated at six to eight week intervals throughout the
12
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school year. After each incidence of acute respiratory illness (ARI), pul-
monary function measurements were made daily for two weeks to establish a
normal.recovery rate. Procedures were developed to retest the pulmonary
function of each child subsequent to an air pollution episode. These arrange-
ments will continue in effect during the 1978-1979 school year. No air
pollution episodes occurred during the 1977-1978 school year.
Scope and Objectives
The scope of this work falls in two categories:
1. What are the long term physiological effects of exposure to com-
bustion products of coal with high sulfur content? This study
involves studying a cross section of population including ages
from 10 - 60 years.
2. What are the effects of air pollutants on the incidence and severity
of acute respiratory illness (ARI) in grade school children?
Objectives of the long-term exposure (Cleveland vs. Elyria) study are:
1. To determine the levels of accumulation of trace metals in human
systems.
2. To determine the reactions of hemoglobin (COHb, MetHb).
3. To assess pulmonary function.
4. To assess cardiovascular function.
5. To determine air quality.
6. ,To determine if there is a difference in the incidence of hyper-
tension between the two study areas.
Objectives of the acute respiratory illness (Akron) study are:
1. To determine the frequency of ARI in 5th and 6th grade children
relative to levels of air pollutants.
2. To determine the severity of ARI in 5th and 6th grade children
relative to levels of air pollutants.
3. To examine specific symptoms and possible correlations with air
pollutants.
4. To assess the recovery from an ARI relative to the return to normal
of certain respiratory volumes and capacities.
5. To determine air quality.
13
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Background and Approach
erm Exposure Study— ,
Air pollution is a problem that is confronting urban populations of the
world with increasing regularity. One of the problems that is particularly
disconcerting concerning air pollution is its possible effect on the respir-
atory system. Initial work in this area was conducted by Rosenbaum (1961)
who showed that army recruits from urban areas suffered more respiratory in-
fection while in the army than those coming from rural areas. Since the
work by Rosenbaum, a number of other authors have supported similar obser-
vations in the United States (Lebowitz 1974, Ferris 1970, Shy et al 1970,
Shy et al 1973) England (Lawther et al 1974, Holland et al 1969), Japan
(Toyahama, Nagahama et al 1969, Watanabe et al 1969), and Canada (Lefcoe
1974).
In 1970, a large population of junior high school students was studied
in Barberton and Revere, Ohio. Barberton is an industrial community, while
Revere is rural. The results of this study indicated that lung functions
such as vital capacity (VC) and forced expiratory volume (FEV^) were all
significantly lower (p<.01) in the Barberton group than the Revere group
faostardi and Martell 1975)
To further investigate this population it was decided to revaluate a
a smaller sample of the original population for VC, FEV^, maximal midexpira-
tory flow (MMF), and maximal oxygen consumption (V0«) by the indirect method.
This last parameter is a measure of an individual's aerobic capacity which
involves a number of physiological factors among which are: oxygen diffusion
capacity, cardiac efficiency, and the efficiency of cellular 02 uptake and
metabolism. Because diffusion capacity of oxygen is a function of alveolar
vasculature it was decided that this parameter could provide additional in-
formation concerning the effects of air pollution on lung parenchyma! tissue.
The results of this second study were quite similar to the first. The
Barberton group had a mean VC which was lower than the mean of the Revere
group (p<.01). Neither FEVt, nor MMF were significantly different, but
V02max was (p<.01). Values of air pollutants in Barberton, although some-
what lower than three years ago, are still considerably higher than in Revere.
It was concluded that such pollutants should be considered important contribu-
tory factors on the impairment of cardiopulmonary parameters (Mdstardi and
Loenard 1974).
The following study is designed to expand our earlier work by increasing
the age groups, geographic locations and cardiopulmonary parameters studied,
and to employ them in a cross-sectional/longitudinal investigation. The com-
parative effects of air pollutants between a number of age groups, under
different pollution conditions, over an extended period of time, has never
been attempted, and these kinds of data should provide detailed, qualitative
cause and effect relationships between air pollutants and lung function.
Initially three different age groups 10-12, 25-25, and 45-65 are being
compared between two different geographic locations of varying levels of air
pollution for the cross-sectional aspect, and each of the age groups within
14
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a location will be retested for five years, providing longitudinal data.
It is believed that within this experimental design the progressive effects
of pollutants can be assessed. In this respect these areas have had near
constant levels of pollutants for 20 or 30 years. Providing that the older
subjects have fulfilled a prerequisite of longevity within a given area,
regression equations can be applied to the different age groups within a
given location and an extrapolation can be made from age group to age group.
These regressed data can then be compared between locations, providing in-
dications of long term trends. As a result, if certain levels of pollutants
are determinants of reduced lung function and pulmonary disorders, then by
using correlative statistics, an appropriate cause and effect relationship
can be made.
Location Selection—
The state of Ohio is a very suitable area for a study designed to assess
the effects of air pollutants. In the northern and eastern parts of the
state are a number of cities in which the air quality is consistently above
the ambient air quality standards, and about 50% of the time is over the once
per year levels. There are other cities where the air quality is consistent-
ly between the primary standard and the once per year level, and there are a
few areas where the levels are consistently below the primary standard. As
a result, with a minimum of travel time large pools of subjects are available
who have been exposed to these ambient conditions for nearly a lifetime.
The eastern portion of Cleveland, Ohio has been chosen as the area of
high pollution. There is a large industrial complex west of our study zone
which includes chemical plants, steel mills, and coke ovens. This area con-
stitutes one of the heaviest polluted areas in the U.S. The clean urban
area has not yet been selected, but we currently are monitoring air quality
in Elyria for possible use as a site.
Subject Selection—
It is anticipated that approximately 125 ^ 150 individuals in each age
group will be interviewed for their availability and willingness to partici-
pate in this study. Age groups are as follows:
1. 10 - 12
2. 25 - 45
3. 45 - 65
The variables to be measured include the following:
1. Vital data - derived from adult questionnaires (see Appendix 1)
2. Pulmonary functions testing (PFT) including vital capacity (VC),
forced expiratory volume (FEVt), and maximal mid-expiratory
flow (MMF).
3. Maximal expiratory flow volume (MEFV) curves while breathing room
air and also while breathing helium-oxygen (He-02) 80%-20%.
15
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There is a considerable amount of information in the literature which suggests
that MEFV curves using both air and He-02 can provide more information con-
cerning the nature of the small airways than FEV* of MMF (Antic and Mack!em
1976, Chan-Yeung at al 1976, and Dosman et al 19/5). For this reason we have
established MEFV curves as an important aspect of this study and because it
is somewhat new to this research design, we feel that it deserves further
explanation.
A schematic drawing of the setup is shown in Figure 1. Data collection
involves the following steps. The subject is seated comfortably so that
his/her mouth is directly in from of a Collins Triple J valve with attached
mouthpiece. The subject is instructed as to how a forced expiratroy maneuver
is performed, accompanied by a demonstration by the experimenter. Then,
attached to the mouthpiece and with a nose clip in place, the subject attempts
several trial maneuvers. When the experimenter is satisfied that the man-
euver is being performed adequately, the expired air is directed into the
dry rolling seal spirometer by opening stopcock 2, and three forced expiration
efforts are recorded on the x-y recorder. When three similar curves have
been recorded using room air, the same procedure is followed using the He-02
mixture.
Prior to breathing the He-02 mixture, the subject is taken off the
mouthpiece and receives a brief description of the gas mixture, notably that
the mixture is somewhat cooler than room air and that the nitrogen normally
found in the air has been replaced by another gas. Following the explanation,
the subject is reconnected to the mouthpiece with the nose clip attached and
then after turning stopcock 1, the subject breathes the He-02 mixture out of
the 200 liter Douglas bag. To displace the N2 in the lungs, the subject per-
forms three VC maneuvers with several tidal breaths in between. Following
equilibration with the He-02 mixture the subject performs forced expiratory
maneuvers until three satisfactory maneuvers have been recorded.
The forced expiratory recording are analyzed according to the pre-
viously described method of Antic and Macklem (1976). The analysis involves
superimposing the curves for air and He-02 at total Iun9 volume (TLC). The
best FVC for air is used to define the volumes at which Vmax is measured.
Thus, the 50% and 75% expired FVC volumes from the best air FVC is used to
determine air Vmax5o^ and ^max7S% volumes from all curves. The values
Vmax50% anci Vmax75£ are calculated as the difference between the air and
He-02 curves and expressed as a percentage of the total volume on air at the
point. The Visoy point is the point at which there is clear crossing or
merging of the air and He-02 curves as opposed to an apparent converging.
4. Low level exercise tests - Electrocardiographic recordings are
carried out at rest, during exercise on a bicycle ergqmeter,
immediately at recovery, and 3 minutes post exercise. The
strips are coded using the Minnesota Method.
5. Blood withdrawal Blood is withdrawn from an antecubital vein and
aliquots are used for the following tests:
16
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|He-0,
Stopcock (1) /Triple'J\ Stopcock (2)
valve
Douglas
bag (200 L)
Spirometer
C IT
CD cn
O r
Subject
x-y recorder]
Figure 1. Schematic for recording MEFV curves
17
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a. Seven ml of blood is withdrawn in a red top Vacutainer
(no preservative). The sample is centrifuged at 2500 rpm
and the serum removed for alpha-1-anitrypsin, total protein,
and immunoelectrophoresis determinations.
b. Four ml of blood is withdrawn in a green top Vacutainer
(286 USP units of sodium heparin) and used for the methe-
moglobin determinations.
c. Four ml of blood is withdrawn into each of two lavender top
vacutainers (6 mg of EDTA and 0.008 mg potassium sorbate) to
be used for carboxyhemoglobin determinations and mercury and
cadi urn analyses.
d. Four ml of blood is withdrawn into a green top Vacutainer for
catecholamine and glucocorticoid levels.
6. Urine sample - Since we are at a given site for a month or more, it
is relatively simple to get a 24 hour urine sample. For this pur-
pose we provide 4 liter wide mouth plastic jugs containing 5 ml
0.1 NHN03 as a preservative, which are given to the subject in the
morning and returned the following morning. After measurment of
total urine volume, an aliquot ofthe urine is placed into a clean
10 ml plastic vial. This sample is sent to the Trace Metals Labor-
atory at Metropolitan General Hospital where it is analyzed for
mercury and arsenic.
All of the data are entered into an appropriate computer file for stat-
istical analysis.
Acute Respiratory Illness Study—
The effects of Acute Respiratory Illness (ARI) on pulmonary functions
in school children have not been studied extensively, nor has the degree to
which lung function returns to normal been assessed.
There is also a paucity of data relating air pollution to the incidence
and severity of ARI's in children. The study conducted by Collier et al.
(1978), in which pulmonary functions were evaluated during and one month
after upper respiratory infection, did not include correlation with air
pollution. In the study by Levy et al.(1977) the number of hospital admis-
sions for acute respiratory disease in children was positively correlated
(p<0.01) with air pollution indices.
The purposes of this particular aspect of the study are (1) to determine
if a decrease in pulmonary function is related to incidence and severity of
ARI's in school children, and (2) to determine if air pollution indices are
correlated with incidence, severity, and convelescent time.
Hypertension Study-
Evidence is accumulating which suggests that stress and social change
can influence blood pressure (Bronson and Eleftheriou 1965, Ely et al.1974,
18
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Henry et al. 1975, Henry et al. 1967, Henry et al. 1971, Nestel 1969, Nestel
and Doyle 1968, Nuckolls et al. 1972). Also there is evidence that coping
ability can influence specific neuroendocrine response patterns (Conner et al.
1971, Jones et al. 1970, Kvetnansky and Mikulaj 1970, Kvetnansky et al. 1970,
Mason 1968, and Mason 1968b). Finally there is evidence that specific be-
havior patterns can influence blood pressure and neuroendocrine response
patterns (Bronson and Eleftheriou 1965, Christian et al. 1965, Henry et al.
1971, Nuckolls et al. 1972). However, the cause and effect relationships
among these variables are virtually unknown.
In the present study the relationships between three behavioral para-
meters, specific neuroendocrine correlates, certain trace metals, and blood
pressure will be examined.
The technique for measuring plasma catecholamines is by Passon and
Peuler (1973) as modified by Upjohn Co., (Cat-A-kit). The method for cort-
ison determination is the fluorimetric technique of Click et al. (1964).
Air Monitoring Component--
In support of the study of air pollution on physiological parameters,
seven air monitoring stations have been established in Cleveland, Akron, and
Elyria. The stations are located in the section of each city where the
subjects for the study live, and will provide an indication of the quality
of air in the region, as well as variations in air quality over relatively
short distances. The three areas were selected, based on available data
which indicated that Cleveland was a region of very high pollution, Akron
moderate, and Elyria relatively clean air.
At each of the sites 24 hour samples of total suspended particulates
(TSP), sulfur dioxide ($02), and nitrogen dioxide (NC^) are being obtained.
The sites are a part of the "CHAMP" system and all analyses are being made
by the "CHAMP" contractors. In addition to the TSP, S02, and NC^ measure-
ments, the filters with the total suspended particulates are being analyzed
for sulfates, nitrates, polynuclear aromatics, benzene solubles, lead,
berryllium, arsenic, cadmium, selenium and mercury.
In addition to the data acquired from the above stations, the TSP, S02,
and N02 measurements from stations in the area operated by the local Air
Pollution Control Agency are being obtained and will be used to estimate the
air pollution gradient for the region.
The major pollutants in southeast Cleveland come primarily from the in-
dustrial area, which includes all levels of steel manufacturing, coke manu-
facturing, heavy inorganic chemicals, electrical generating stations and
transportation sources. The area selected for the study is located just east
of the steel and coke manufacturing facilities and based upon the Cleveland
Air Pollution data has the highest levels of Air Pollution TSP, S02, and N02
in the Cuyahoga County and in the state of Ohio.
The major pollutants in Akron come primarily from the production of
industrial steam for the rubber industry, electrical power generation, and
19
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transportation. The industrial steam is generated using either coal, gas
or oil with most coming from oil and gas. The rubber plants have other odor
causing organic emissions which are not being analyzed during this project.
Elyria is a rather small community with light industry and there are no
large heavy industrial sources of pollutants in the community. The Cleveland
Electric Illuminating Company has a large electric generating station at
Avon Lake which is about 20 miles northeast of the chosen sites and U. S.
Steel has a major installation in Lorain which is about 10 miles north of the
area being used in the study. The U. S. Steel Plant has made extensive use
of wet scrubbers and is one of the better controlled steel plants in the
country. With the prevailing winds coming from the southwest and west, these
two plants should have a minimal effect on the air quality in the region.
The areas to the south and west of Elyria are agricultural with the closest
major industrial complex being at least 60 miles south.
As part of the study, the differences which are found in the levels of
the above pollutants for the three areas will be used along with the phy-
siological data to determine if there are correlations between specific
pollutants and physiological effects. In addition, the pollution gradients
in the areas will be studied to see how the pollution varies from one lo-
cation in the area to another.
The data will be analyzed to determine if there are significant differ-
ences in not only the levels of pollutions, but the ratio of the various
pollutants. This information along with weather information obtained from
The U. S. Weather Bureau will be used to determine if correlations of air
quality, the type of pollutants, and ratios of various pollutants can be re-
lated to the wind direction and speed. Studies from New York City (Goldstein
and Landovitz 1977a, Goldstein and Landovitz 1977b) indicated that this is
not feasible, but because of the location, type of terrain and differences in
types of buildings, the New York conclusions are probably not applicable to
this study.
Studies in St. Louis (Pooler 1966) indicate that the modeling techniques
using a modified Gaussian procedure seem to be applicable for predicting the
pollutant levels if the proper choice of dispersion parameters is made.
Studies by Yen (1971) indicate that these procedures are better for valida-
ting long term averages than daily or weekly changes. Therefore an attempt
will be made to relate the pollution levels to the climatic conditions, in-
cluding wind direction, velocity, precipitation, insulation, season and per-
iod of the week or month.
Research Accomplished
Long-Term Exposure Study--
At this date, March 28, 1978, we have tested 250 adults in east Cleve-
land, Ohio. It takes about 30 minutes to complete all of the tests, and we
normally complete 10-12 per day. Most of our subjects have been in the 45-
65 age group and we are pursuing locations where we can test younger people.
Unfortunately, at this time we do not have any subjects in the 10-12 age
20
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group. This is due to the fact that the City of Cleveland School System has
refused to cooperate with us and will not allow us to seek volunteers in the
grade schools of the area. We did receive some help from the Catholic
Dioceses of Cleveland but the enrollment of the one school in the immediate
area is small and when we sent our information package home, we received
a poor return.
The data that we have collected has been processed and most of it is on
disk storage at the main university computer.
Animal Model Studies--
In December of 1977, five pregnant Balb/c females from the Kirchbalm
Memorial Mouse Colony were placed in one of our research sites in the East
Cleveland area. Within one week all five of the females bore litters, with
the total number of mice being 26. At the same time, a similar sample size
was established in Akron, Ohio and served as the control group.
After three months all of the experimental mice and 12 of the control
mice were sacrificed. Blood was collected for mercury and cadi urn determin-
ations and hair was shaved from the back for lead, mercury, beryllium, ar-
senic, selenium and cadmium determinations. The thorax was opened and the
lungs and liver were removed and weighed. Tissue samples from each lung
lobe and selected liver samples were removed and fixed at 10% buffered
formalin for histologic examination. All of these tissue samples are cur-
rently being processed.
Acute Respiratory Illness Study-
Two Akron Public City Schools (situated 2 miles apart) were chosen for
this study on the basis of their proximity to the rubber factories. Question-
naires (Appendix 2) were sent home to all 5th and 6th grade students. Those
returning completed questionnaires which included hair samples for heavy
metal analyses comprised the group for our study. All subjects were thor-
oughly coached in pulmonary function maneuvers. Spirometry was performed
with a Warren E. Collins 9 liter water-filled spirometer, measuring Vital
Capacity (VC), Forced Expiratory Volume (FEV), and Mid-Maximal Flow (MMF).
These measurements were all done in reproducible triplicate (greater than 5%
difference rated unacceptable). Prior to each spirometry maneuver, the
height and weight of the subject were also recorded.
Pulmonary Function baselines were performed on all subjects after first
determining that they were asymptomatic. Thrice weekly, the research team
checked for ARI incidents among the subjects. This was done with the aid of
both personal interviews and symptom charts which each volunteer was asked to
complete daily (See Appendix 3). Pulmonary function measurements were then
taken on subjects who indicated the presence of an ARI. During this sympto-
matic phase of the ARI, spirometry was performed twice on the subject, two
days apart. At this time each of the subject's symptoms was graded for
relative severity, and labeled light, moderate, or severe.
Careful observation was maintained on the subjects with ARI's and when
they were asymptomatic, spirometry was performed three times. These
21
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asymptomatic tests were carried out over a nine day period and the purpose
was to assess the nature and degree of convalescence.
Hypertension Study--
Presently, (March 28, 1978) we have accumulated hypertension data on
150 subjects in the Cleveland study area. These data are currently being
programmed into our computer center for detailed analysis. Statistical
tests are being performed comparing blood pressure to heavy metal levels
in blood, urine, and hair samples, life style index, plasma catecholamines,
and gluccocorticoids.
Air Monitoring Study--
The Akron stations have been run since October, the Cleveland stations
since November and the Elyria stations since December and as yet no data
have been received from the "CHAMP" contractors. It is imperative that this
data be received as soon as possible to enable the data analysis and compar-
ison work for the project to begin.
Bibliography
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Site of Airway Obstruction in Asthma. Am. Rev. of Resp. Disease
114:851-859.
Bronson, F. H. and B. E. Eleftheriou. 1965. Behavioral, Pituitary, and
Adrenal Correlates of Controlled Fighting (Defeat) in Mice. Phys.
Zool. 38:406-411.
Chan-Yeung, M., R. Abboud, M. S. Tsao, and L. Maclean. 1976. Effects of
Helium on Maximal Expiration Flow in Patients with Asthma Before and
During Induced Bronchoconstriction. Am. Rev. of Resp. Disease 113:433-
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Christian, J. J., J. A. Lloyd, and D. E. Davis. 1965. The Role of Endo-
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Collier, A., R. L. Pimmel, V. Hasselblad, W. Clyde, J. Knelson, and J.
Brooks. 1978. Spirometer Changes in Normal Children with Upper Res-
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Ely, D. L., J. P. Henry, and R. D. Ciaranello. 1974. Long-term Behavioral
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Glick, D,, C. VonRedlich, and S. Levine. 1964. Fluorometric Determination
of Corticosterone and Cortisol in 0.02 and 0.05 Millimeters of Plasma
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Goldstein, I. F. and L. Landovitz. 1977a. Analysis of Air Pollution in New
York City I. Can One Station Represent the Large Metropolitan Areas?
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York City II. Can One Assometric Station Represent the Area Surrounding
it? Atmospheric Environment. 11:53-57.
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Their Parameters and Measurement. L. Levi ed. Raven Press, New
York. pp. 469-497.
Henry, J. P., J. P. Meehan, and P. M. Stephens. 1967. The Use of Psycho-
social Stimuli to Induce Prolonged Systolic Hypertension in Mice.
Psychosom Med. 29:408.
Henry, J. P., P. M. Stephens, J. Axelrod, and R. A. Mueller. 1971. Effect
of Psychosocial Stimulation on the Enzymes Involved in the Biosynthesis
and Metabolism of Noradrenaline and Adrenaline. Psychosom Med.
33:227.
Holland, W. W., T. Halil, A. E. Bennett, and A. Elliott. 1969. Factory
Influencing the Onset of Chronic Respiratory Disease. British Medical
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and Endocrinological Response to Emotional Stress. Progress in Brain
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and J. A. W. M. Wiejnen, eds. Elsevier, Amsterdam, pp. 325-335.
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in Rats During Adaptation to Repeated Immobilization Stress. Endocrin.
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Kvetnansky, R., Weise, and I. J. Kopin. 1970. Elevation of Adrenal Tyrosine
Hydroxylase and Phenylethanolamine-N-methyl Transferase by Repeated
Immobilization of Rats. Endocrin. 87:744-749.
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Lawther, P. J., A. B. R. Brooks, P. W. Lord, and R. E. Waller. 1974. Day to
Day Changes in Ventilatory Function in Relation to the Environment.
Environmental Research. 7:27-30.
Lebowitz, M. D., P. Bendheim, G. Cristae, D. Markovitz, J. Misiaszek, M.
Staniec,and D. Van Wyck. 1974. The Effects of Air Pollution and
Weather on Lung Function in Exercising Children and Adolescents. Am.
Rev. of Resp. Disease. 109:252-273.
Lefcoe, N. M. and T. H. Wonnacott. 1974. Chronic Respiratory Disease in
Four Occupational Groups. Arch. Environ. Health. 29:143-146.
Levy, D., M. Gent, and T. Newhouse. 1977. Relationship Between Acute Respir-
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of Resp. Disease. 116:167-173.
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Mason, J. W. 1968. A Review of Psychoendocrine Research on the Sympathetic-
adrenal Medullary System. Psychosom. Med. 30:631-653.
Mostardi, R. A. and D. Leonard. 1974. Air Pollution and Cardiopulmonary
Functions. Arch. Environ. Health. 29:325-328.
Mostardi, R. A. and R. Martell. 1975. The Effects of Air Pollution of Pul-
monary Functions in Adolescents. The Ohio Journal of Science. 72:65-69.
Nagahama, F., K. Sugita, D. Obata, and T. Ogasaivara. 1969. The Effects of
Air Pollution on the Pulmonary Ventilation. Report No. 8, School of
Medicine, Hokkaido University. Hokkaido, Japan. May 19.
Nestel, P. J. 1969. Blood-pressure and Catecholamine Excretion After Mental
Stress in Labile Hypertension. Lancet. 1:692-694. April 5.
Nestel, P. J. and A. E. Doyle. 1968. The Excretion of Free Noradrenaline
and Adrenaline by Healthy Young Subjects and by Patients with Essential
Hypertension. Aust. Ann. Med. 17:295-298.
Nuckolls. K. B., J. Cassel, and B. H. Kaplan. 1972. Psychosocial Assets,
Life Crisis and the Prognosis of Pregnancy. Am. J. Epidemiol. 95:431-
441.
Passon, P. B. and J. D. Peuler. 1973. Anal Biochem. 51:618.
Pooler, F. 1966. A Tracer Study of Dispersion Over a City. Jpurnal of
Air Pollution Control Assoc. 11:677-681.
Rosenbaum. S. 1961. Home Localities of National Servicemen With Respiratory
Disease. J. of Prevent, and Social Med. 15:61-70.
24
-------
Shy, C. M., P. Creajon, M. E. Pearlman, K. E. McClain, and F. B. Benson.
1970. The Chattanooga School Children Study: Effects of Community
Exposure to Nitrogen Dioxide. J. of Air. Poll. Cont. Assoc. 20:539-545.
Shy, C. M., V. Hasselblad, R. M. Burton, C. J. Nelson, and A. A. Cohen. 1973.
Air Pollution Effects on Ventilatory Functions of U. S. School Children.
Arch. Environ. Health. 27:124-128.
Toyahama, T. Air Pollution and its Health Effects in Japan. Arch. Environ.
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Watanabe, H., et al. 1964. Effects of Air Pollution on Health. Report No.
1: Peak Flow Rate and Vital Capacity of Primary School Children. Rep.
Osaka City Instit. Hyg. 26:32-37.
Yen, K. T. 1971. Meteorological Air Pollution Modeling. 64th Annual Air
Pollution Control Assn. Mtg. June 27-July 1. Paper 71-72.
25
-------
Appendix 1. Adult Questionnaire
University of Akron Research Study
On Respiratory Disease
ADULT QUESTIONNAIRE
Please answer the questions in this questionnaire as completely as possible.
The questions can be answered by circling the number of the best answer or
by filling in a blank with a number or word.
Example: What is your sex: 1. male
(2) female
If you have any problems in answering any of the questions, a member of the
staff will be happy to help you.
ALL INFORMATION WILL BE KEPT CONFIDENTIAL
Name QID_
Address
Phone
Social Security No
Date of Birth
26
-------
Appendix 1. (Continued)
1. What is your full name?
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
Date questionnaire completed?
(month)
What sex are you?
What is your ethnic group or ancestry?
What is your marital status?
How many years of formal education or school
ing have you had? (For example, completion
of high school =12)
What is your birthdate?
How long have you lived in your present
city or town?
How long have you lived at your present
address?
Do you anticipate moving from your
present address in the next year?
Do you usually cough first thing in the
morning in bad weather? (If you usually
cough in the morning regardless of the
weather, circle Yes.
Do you usually cough other than in the
morning in bad weather? (If you usually
1.
2.
1.
2.
3.
4.
5.
6.
1.
2.
3.
4.
5.
_
mo
1.
2.
3.
1.
2.
3.
1.
2.
1.
2.
1.
2.
(day)
Male
Female
White
19
(year)
Mexican-American
Black
Indian
Oriental
Other
Married
Separated
Never Married
Wi dowed
Divorced
/
day
1-5 years
5-10 years
10 or more
1-5 years
5-10 years
10 or more
Yes
No
Yes
No
Yes
No
(years)
/
year
years
years
cough regardless of the weather,circle
Yes.)
If you answer is Yes to either questions 11 or 12, proceed to a:
1*
a. Do you cough on most days for as much 1. Yes
as three months of the year? 2. No
27
-------
Appendix 1. (Continued)
a. For how many years have you had this
cough?
b. Do you cough more or less now than you
did two years ago?
d. During the past two years, have you
seen a doctor about your cough?
13. Do you usually bring up phlegm, sputum, or
mucous from your chest first thing in the
morning in the bad weather? (If you usually
bring up phlegm, sputum, or mucous from you
chest in the morning regardless of the weather,
circle Yes)
14. Do you usually bring up phlegm, sputum, or •
mucous from your chest at other times during
the day or night in bad weather? (If you
usually bring up phlegm, sputum, or mucous
from you chest, regardless of the weather,
circle Yes)
If your answer is Yes to either question 13 or 14 proceed to a:
1.
2.
3.
1.
2.
3.
1.
2.
1.
2.
Less than 2 years
2-5 years
More than 5 years
More
Less
No Change
Yes
No
Yes
No
1. Yes
2. No
a. Do you bring up phlegm, sputum, or mucous
from your chest on most days for as much
as three months of the year?
b. For how many years have you raised phlegm,
sputum, or mucous from your chest?
c. Do you raise more or less phlegm, sputum,
or mucous from your chest now than you did
two years ago?
d. During the past two years, have you seen
a doctor about this condition?
15. Does your chest sound wheezy or whistling?
If your answer is Yes, proceed to 15a:
a. Do you get this with colds?
1. Yes
2. No
1. Less than 2 years
2. 2-5 years
3. More than 5 years
1.
2.
3.
More
Less
No change
Yes
No
1. Yes
2. No
1. Yes
2. No
28
-------
Appendix 1. (Continued)
b. Do you get this even when you don't
have a cold?
c. Do you get this on most days?
d. During the past year, has your wheezing
or whistling improved, worsened, or
stayed the same?
e. How old were you when you first started
wheezing?
1. Yes
2. No
1. Yes
2. No
1. Improved
2. Worsened
3. Stayed the same
.(age)
1.
2.
1.
2.
1.
2.
3.
4.
5.
Yes
No
Yes
No
None
A few (1-3)
Several (4-10)
Many (13 or more)
Almost every day
16. Have you ever had attacks of shortness of breath 1. Yes
with wheezing? 2. No
if your answer is Yes, proceed to 16a
a. Do you get this with colds?
b. Do you get this even when you don't
have a cold?
c. During the past year, how many attacks of
shortness of breath with wheezing did you
have?
d. How old were you when you had your first
such attack? (age)
e. How old were you when you had your last
such attack? (If you still have attacks, (age)
indicate your present age)
17. Are you more short of breath than most people 1. Yes
your age? 2. No
18. Are you troubled by shortness of breath when 1. Yes
hurrying on level ground or walking up a 2. No
slight hill?
19. Do you get short of breath walking with other 1. Yes
people of your own age on level ground? 2. No
20. Do you have to stop for breath while walking 1. Yes
at your own pace on level ground? 2. No
21. Did you have any respiratory trouble before 1. Yes
age 16? 2. No
29
-------
Appendix 1. (Continued)
22,
23.
24,
During the past three years, how much trouble
have you had with illnesses such as chest
colds, bronchitis, or pnuemonia? (Does not
refer to head colds)
Some
During the past three years, how often
were you unable to do your usual activ-
ites because of illnesses such as chest
colds, bronchitis, or pneumonia? (Does
not refer to head colds)
Do you think you have ever had any of these
chest disorders: asthma, any kind of bron-
chial trouble, emphysema?
25. Have you ever seen a doctor for asthma?
a great
deal
(Circle appropriate number)
1. Never
2. During one such
illness
3. During 2-5 such
illnesses
4. During 6 illnesses
or more
' 1. Yes
2. No
1. Yes
2. No
If you answer is Yes, proceed to 25a:
a. During the past year, have your symptoms
improved, worsened, or stayed the same?
During the past year, have you had a
period as long as two weeks in which you
had no breathing problems at all even
with exercise?
How quickly are your attacks usually
relieved by treatment?
26. Have you ever seen a doctor for chronic
bronchitis?
If your answer is Yes, proceed to 26a:
a. During the past year, have your symptoms
improved, worsened, or stayed the same?
b. Have you had medication or treatment
for it?
1. Improved
2. Worsened
3. Stayed the same
1. Yes
2. No
1. Within minutes
2. Within hours
3. Within days
4. Not relieved
5. No treatment used
1. Yes
2. No
1. Improved
2. Worsened
3. Stayed the same
1. Yes
2. No
30
-------
Appendix!. (Continued)
27
28.
29.
Have you ever had any of the following
(If uncertain circle No):
a. Emphysema
b. Bronchi ectasis
c. sinus trouble
d. pneumonia or bronchopneumonia
e. tuberculosis
f. valley fever ( cocci diodomycosis)
g. Histo (histoplasmosis)
h. any other respiratory disease
If yes, please specify
How long have you been employed
at your current occupation?
Have you in the past worked in a
dusty job or where there has been
irritating gas, chemical fumes, or
smoke?
1.
2.
3.
1.
2.
3.
1.
2.
3.
1.
2.
3.
1.
2.
3.
1.
2.
3.
1.
2.
3.
1.
2.
1.
2.
3.
1.
2.
Yes
Yes
No
Yes
Yes
No
Yes
Yes
No
Yes
Yes
No
Yes
Yes
No
'Yes
Yes
No
Yes
Yes
No
Yes
No
1-5
5-1
10
Yes
No
, I still have it
, but I no longer have
, I still have it
, but I no longer have
, I still have it
, but I no longer have
, I still have it
, but I no longer have
, I still have it
, but I no longer have
, I still have it
, but I no longer have
, I still have it
, but I no longer have
years
0 years
or more years
it
it
it
it
it
it
it
If your answer is Yes, proceed to 29a:
How many years were your employed
there?
1. 1-5 years
2. 5-10 years
3. 10 or more years
31
-------
Appendix 1. (Continued)
b. Was the exposure to the irritating gas,
chemical fumes, smoke, or dust mild,
moderate or severe?
c. How many different companies have you
worked for in the last 10 years?
30. Do you now smoke cigarettes?
If your answer is Yes, proceed to 30a:
If your answer is No, proceed to question 31
a. Do you inhale?
b. Do you smoke cigarettes with filters?
c. How many cigaretts do you usually smoke
each day at the present time? (Please
give best estimate: one pack contains
20 cigarettes).
d. How old were you when you began to smoke
cigarettes?
e. What is the usual number of cigarettes
you have smoked per day since you began to
smoke? (Please give best estimate: one
pack contains 20 cigarettes)
f. Are you smoking less now than two years ago?
g. If so, how many are you smoking now?
If you have completed this section, skip question
31 and go to question 32
31. If you do not smoke cigarettes now, did you ever
smoke them regularly or occasionally?
1.
2.
3.
1.
2,
Mild
Moderate
Severe
1. Yes
2. No
(Number)
1. Yes
2. No
1. With filters
2. Without filters
3. Both with and
without filters
number per day
(age)
number per day
Yes
No
number per day
If your answer is regularly or occasionally, proceed
a. What was the usual number of cigarettes
you smoked per day? (Please give the
best estimate: one pack cigarettes —
contains 20 cigarettes)
1. Never
2. Regularly
3. Occasionally
to question 31a:
number per day
b. Did you inhale?
1. Yes
2. No
32
-------
Appendix 1. (Continued)
c. How old were you when you began to
smoke cigarettes? (age)
d. How old were you when you stopped
smoking cigarettes regularly? (age)
e. Were you influenced to stop because 1. Yes
you had a cough, wheezing, or shortness 2. No
of breath?
32. Do you now smoke pipes or cigars? 1. Yes
2. No
If your answer is Yes, proceed to 32a:
If your answer is No, proceed to question 33.
a. How many pipefuls or cigars do you usually number per day
smoke each day?
b. How old were you when you first smoked? (age)
c. Do you usually inhale when you smoke 1. Yes
either pipes or cigars? 2. No
(If you have completed this section, skip question 33 and go to
question 34)
33. If you do not smoke cigars or pipes now, did 1. Never
you ever smoke them regularly or occasionally? 2. Regularly
3. Occasionally
If your answer is regularly or occasionally, proceed to 33a:
a. How many pipefuls or cigars did you usually number per day
smoke each day?
b. How old were you when you first smoked (age
pipes or cigars?
c. How old were you when you stopped
smoking pipes or cigars? (age)
d. Did you usually inhale when you smoked 1. Yes
either pipes or cigars? 2. No
34. Do you ever have paid or discomfort in your 1. Yes
chest brought on by exertion or excitment and 2. No
is relieved by rest?
33
-------
Appendix 1. (Continued)
35. Have you ever been told by your doctor that you 1. Yes
had angina or angina pectoris? 2. No
36. Do you have to stop to catch your breath while 1. Yes
walking up one (1) flight of stairs? 2. No
37. Have you ever been hospitalized for a heart 1. Yes
attack? 2. No
38. Are you currently taking any medication? 1. Yes
2. No
39. If Yes to question 38, what is the name of
the medication?
34
-------
Appendix 2. Pediatric Questionnaire
University of Akron Research Study
on Respiratory Disease
PEDIATRIC QUESTIONNAIRE I
We would like the questionnaire to be completed by the parent of each child
participating in the study. The questions can be answered by circling the
number of the best answer or by filling in a blank with a number or word.
All questions in this questionnaire concern only your child. Please try
to answer all questions as completely as possible.
ALL INFORMATION WILL BE KEPT CONFIDENTIAL
Name
Address
ID
City, State, Zip Code
Child's date of birth
Phone
Sex
35
-------
Appendix 2. (Continued)
1. Date questionnaire completed?
10.
11
12,
(month)
2. Does this child suffer from shortness of
breath while walking?
(day) (year)
1. Yes
2. No
3. Does this child suffer from shortness of
breath while playing?
4. Does this child ever stop to catch his
breath while walking?
5. Does this child ever stop to catch his
breath while playing?
6. Does this child get short of breath walking
with other shildren the same age on level
ground?
7. Does this child have to stop for breath while
walking at his or her own pace on level ground?
8. Does he or she become short of breath, during
normal play, more often now than two years ago?
9. During the past two years, has this child seen
a doctor because of shortness of breath?
During the past two years, how often was this
child unable to do his/or her usual activities
because of illnesses such as chest cold, bronchi-
tis, or pneumonia? (Does not refer to head colds,
During the past year, how many days has this
child been unable to do his or her usual
activities because of such illnesses?
During the past year, has this child seen a
doctor for: (Consult your physician if you are
unfamiliar with these terms.)
a. Emphysema?
b. Chronic bronchitis?
1. Yes
2. No
1. Yes
2. No
1. Yes
2. No
1. Yes
2. No
1. Yes
2. No
1. Yes
2. No
1. Yes
2. No
1. Never
2. During 1 such
illness
)3. During 2-5 such
illnesses
4. During 6 or more
illnesses
(days)
1.
2.
1.
2.
Yes
No
Yes
No
36
-------
Appendix 2. (Continued)
13.
c. Bronchiectasis?
d. Asthma?
e. Sinus trouble?
f. Pneumonia?
g. Acute bronchitis?
h. Asthmatic bronchitis?
i. Bronchi olitis?
j . Croup
k. Whooping cough?
1 . Eczema?
m. Any other respiratory disease?
If yes, specify
Has this child ever had hay fever or any other
condition that makes the nose runny or stuffy
apart from colds?
1.
2.
1.
2.
1.
2.
1.
2.
1.
2.
1.
2.
1.
2.
1.
2.
1.
2.
1.
2.
1.
2.
1.
2.
Yes
No
Yes
No
Yes
No
Yes
No
Yes
No
Yes
No
Yes
No
Yes
No
Yes
No
Yes
No
Yes
No
Yes
No
If yes to 13, proceed to 13a:
a. How old was this child when this condition was
first noticed?
b. How old was this child when he or she last
had this condition? (If he or she still has
it, indicate present age.)
(age)
(age)
37
-------
Appendix 2. (Continued)
c. Has a doctor ever said this condition was
due to an allergy?
14. During the past year, has he or she had an
allergic reaction to any food or medicine?
15. During the past year, has this child received
allergy shots? (Does not refer to allergy
skin tests.)
16. Is this child ever troubled by redness, itch-
ing, or burning of the eyes?
If yes to 16, proceed to 16a:
a. Circle the months in which these eye symptoms
are most severe.
1.
2.
1.
2.
1.
2.
1.
2.
Yes
No
Yes
No
Yes
No
Yes
No
OR:
Check here if no relation to time
of year.
123456789 10 11
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov
12
Dec
b. Does he or she have this on most days for at 1. Yes
least one month of the year? 2. No
c. Do you think this eye problem is due to 1. Yes
an allergy? 2. No
d. Has a doctor ever said it was due to an 1. Yes
allergy? 2. No
17. During the past year, has this child had a 1. Yes
chest X-ray? " 2. No
18. During the past year, has this child been 1. Yes
hospitalized for any heart or lung problem? 2. No
19. Has this child ever had any heart or lung 1. Yes
surgery? 2. No
20. Where would you like this child's test results 1. Doctor
from this study sent? 2. Myself
3. Nowhere
21. What is the doctor's name and location: Name
Address City, State, ZIP
38
-------
Appendix 2. Pediatric Questionnaire II (Continued)
University of Akron Research Study
on Respiratory Disease
PEDIATRIC QUESTIONNIARE II
ALL INFORMATION WILL BE KEPT CONFIDENTIAL
Name ID
Address
City, State, Zip Code Phone
Child's date of birth 'Sex
39
-------
Appendix 2. (Continued)
1. Date questionnaire completed?
(Month) (Day) (Year)
During the past two years, has this
child had: (If uncertain, circle No)
a. any kind of heart trouble?
b. high blood pressure?
3. Does this child have a cough when he or
she doesn't have a cold? (Either first
thing in the morning or at any other
time of the day.)
If Yes, to 3, proceed to 3a:
a. At what time of day does this cough
usually occur? (Circle correct
answer(s).)
For how many ears has he or she had a
cough?
e. Does the weather affect the cough?
1.
2.
1.
2.
1.
2.
2.
3.
4.
5.
6.
1.
2,
Are there any months in which this
child coughs on most days?
If Yes, please specify the number
of months per year.
Circle the months in which the cough
is usually most severe; or
check here if no relation to time
of year.
123456789
Jan Feb Mar Apr May Jun Jul Aug Sep
Yes
No
Yes
No
Yes
No
In the morning (shortly
after rising)
Later in the morning
Afternoon
Evening
During the night
No relation to time of
day
Yes
No
(months)
10
Oct
11
Nov
12
Dec
1.
2.
3.
1,
2.
Less than 2 years
2-5 years
More than 5'years
Yes
No
40
-------
Appendix 2. (Continued)
f. Does this child cough more or less
now than two years ago?
If Yes to 4, proceed to 4a:
a. At what time of day does this
usually occur? (Circle correct
answer(s))
b. Are there any months in which
he or she brings up phlegm,
sputum, or mucous from the
chest on most days?
If Yes, please indicate the number
of months per year
c. Circle the months in which this is
usually most severe
1.
2.
3.
g. During the past two years, has he 1.
or she seen a doctor about the cough? 2.
4. Does this child ever bring up phlegm (flem),
sputum, mucous, or any type of fluid from
the chest when he or she doesn't have 1.
a cold? (Either first thing in the 2.
morning or at any other time of the
day.)
1.
2.
3.
4.
5.
6.
More
Less
No change
Yes
No
Yes
No
In the morning (shortly
after rising)
Later in the morning
Afternoon
Evening
During the night
No relation to time of
day
1. Yes
2. No
(months)
OR
Check here if no relation to time
of year
d.
123456789
Jan Feb Mar Apr May Jun Jul Aug Sep
For how many years has he or she raised 1.
phlegm, sputu, or mucous from the 2.
chest? 3.
10 11 12
Oct Nov Dec
Less than 2 years
2-5 years
More than 5 years
e. Does the weather affect this
condition?
1. Yes
2. No
41
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Appendix 2. (Continued)
f. Does this child raise more or less
phlegm, sputum, mucous, or any type
of fluid from the chest now than
two years ago?
g. During the past two years, has he or
she seen a doctor about this condition?
5. Does this child's chest ever sound wheezy
or whistling?
If Yes, to 5, proceed to 5a:
a. Does he or she get this with colds?
b. Does he or she get this even with-
out having a cold?
c. Does he or she have a wheezy sound
on most days?
6. Has this child ever had attacks of short-
ness of breath with wheezing?
If Yes to 6, proceed to 6a:
a. Does he or she get this with colds?
Does he or she get this even with-
out having a cold?
During the past year, how many
attacks or shortness of breath
with wheezing did this child have?
d. How old was this child when he or
she had the first such attack?
e. How old was this child when he or
she had the last such attack? (If he
or she still has attacks, indicate
present age.)
Where would you like this child's test
results from this study sent?
1.
2.
3.
1.
2.
1.
2.
More
Less
No change
Yes
No
Yes
No
1. Yes
2. No
1. Yes
2. No
1. Yes
2. No
1. Yes
2. No
1. Yes
2. No
1. Yes
2. No
1. None
2. A few (1-3)
3. Several (4-10)
4. May (11 or more)
(age)
(age)
1.
2.
3.
Doctor
Myself '
Nowhere
42
-------
Appendix 2. (Continued)
8. What is the doctor's name and location?
Name
Address
City, State, Zip
43
-------
Name
Appendix 3. Pediatric Symptom Chart
Home Room
Week
Saturday Sunday
Monday
Tuesday Wednesday
Thursday
Friday
Yes
No Yes
No
Yes
No Yes
No Yes No
Yes
No
Yes No
Cough
Stuffy or
Runny Nose
Sore Throat
Chest
Congestion
(Wheezy chest)
Eye
Irritation
Cold
-------
D. TASK TITLE:
Implementation of Screening Test for Potentially Hazardous
Airborne Particulate Materials Using the Alveolar Macrophage
Test System
HERL/RTP TASK NO: 8150
CONTRACTOR: Northrop Services, Inc.
CONTRACT NO: 68-02-2566
Summary
The contractor has implemented the Rabbit Alveolar Macrophage (RAM) Test
System with model particulates as well as particulates and other samples from
conventional combustion sources and alternative energy sources. Emphasis has
been placed on particulate samples due to the macrophages capability to en-
gulf enhaled particulate. A series of toxicity end points have been compared
and optimal end points and test conditions selected for routine screening.
Using these techniques a total of 40 environmental samples have been
evaluated. Since many of these samples were found to have a low toxicity
in the RAM, current research is focusing on comparing potentially more sensi-
tive clonal cytotoxicity assays with the RAM assay using selected particulates.
Scope and Objectives
The objective of this task is to implement the RAM bioassay with model
particulate compounds in such a manner than the sensitivity is optimized and
test conditions standardized. Subsequently actual environmental samples
would be evaluated using this toxicity bioassay system. This approach will
allow evaluation not only of a series of environmental samples including
conventional and alternative combustion source particulate, but also will per-
mit evaluation of this bioassay system.
Background and Approach
Studies in this and other laboratories have defined a central role for
the alveolar macrophage in the defense of the lung against inhaled particu-
late materials. We have identified a number of metallic substances of the
type known to be present in air effluents which are relatively toxic for the
alveolar macrophage (Cd, V, Ni, Hg, etc.). Many substances such as some
metals and certain organics are emitted in the vapor phase and subsequently
preferentially condense on the surface of the smaller respirable particulate
materials. We now have a number of chemical analytical techniques and bio-
logical end points available to study the nature of the surfaces of particles
and the resulting effects of the in vitro exposure of these particles to al-
veolar macrophage. The biological end points form the basis of a valuable
screening system for potentially hazardous airborne particulate materials.
45
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In collaboration with analytical chemists who are able to define the
surface chemical properties of particulate samples, a systematic screening
effort is being carried out to define the biological response of the alveolar
macrophage to phagocytized particles in vitro. Effort has focused on the
propensity of crude particles to promote cell lysis, to affect phagocytic
activity (as determined by depression in adenosine triphosphate levels), to
alter the integrity of the lysosomes, and to induce enzyme systems capable
of activating potentially carcinogenic compounds such as the polynuclear
aromatics which may be adsorbed to particulate surfaces. The influence of
the nature of the surface of standard particles on the extent of adsorption
of materials has been examined.
Research Accomplished
Metal-coated fly ash particles were utilized as model substances in
evaluation and implementation of in vitro particulate testing with rabbit
alveolar macrophages (RAM). Several toxicity end points were measured in
the RAM cells following exposure of the cells to the metal-coated partic-
ulates in dose response and time sequence studies. Cdo-coated particles
have a significant soluble component, whereas the MnO and NiO-coated par-
ticles are insoluble. The earliest end point effected in these studies
was cellular adenosine triphosphate (ATP) levels. Cellular viability and
protein concentrations were also significantly depressed at higher concen-
trations. These particulate samples were utilized to select optimal test
conditions and end points for evaluating the relative toxicity of particu-
late samples. A standardized protocol was developed.
Forty environmental samples have been evaluated in this bioassay includ-
ing respirable particulate from:
1. conventional coal combustion
2. fluidized bed combustion
3. coal gasification
None of these particulates were found to be highly toxic. In fact it was
generally impossible to determine ECso values due to the low toxicity to the
RAM.
Clonal assays with continuous cell lines which are generally more sen-
sitive toxicity test systems have been considered inappropriate for testing
particulates. As a part of this research, clonal assays are being examined
with particulate samples. Preliminary data show that these cells are cap-
able of engulfing particulate and do provide a more sensitive bioassay tool.
Bibliography
Campbell, J. A., H. F. Stack, M. R. Williams, D. Tillery, N. Custer, B F
Russell, S. W. King, E. B. Siegel, N. E. Garrett. Cellular Toxicity of
Four Liquid Effluent Samples from Textile Mills: Studies on the Rabbit
Alveolar Macrophage, WI-38 Human Fibroblast, and Chinese Hamster Ovary
In Vitro. Northrop Services, Inc. Report.
46
-------
Campbell, J. A., H. F. Stack, M. R. Williams, D. Tillery, N. Custer, B. F.
Russell, S. W. King, and N. E. Garrett. Cellular Toxicity of Environ-
mental Samples from Coal Gasification Processes: Studies on the Rabbit
Alveolar Macrophage In Vitro. Northrop Services, Inc. Report.
Campbell, J. A., H. F. Stack, M. R. Williams, D. Tillery, N. Custer, B. F.
Russell, S. W. King, and N. E. Garrett. Cellular Toxicity of Twelve
Fluidized Bed Combustion Samples: Studies of the Rabbit Alveolar Macro-
phage In Vitro. Northrop Services, Inc. Report
Waters, M. D., J. D. Huisingh, and N. E. Garrett. 1978. Cellular Toxicity
of Environmental Chemicals. Symposium on Application of Short-term
Bioassays in the Fractionation and Analysis of Complex Environmental
Mixtures. Williamsburg, Virginia, February 21-23.
Related Research
Research is ongoing to compare the response of the RAM in vitro to that
of the whole animal as the result of inhalation of a variety of particulates
from stationary sources.
47
-------
SECTION 2
DEVELOP MORE RAPID AND SENSITIVE METHODS TO EVALUATE DOSE TO MAN
A. TASK TITLE:
Development of Test Systems to Assess Potential Toxicity and
Neoplastic Transformation - Improvement of Scoring of Chem-
ical Transformation of C3H/10T1/2 Cells (Substituted for)
Development of Test Systems to Assess Toxicity and Neoplastic
Transformation using Type 1 and Type 2 Alveolar Epithelial
Cells In Vitro.
HERL/RTP TASK NO: 8153
GRANTEE: University of Southern California
GRANT NO: R-805208-01
Summary
The grantee plans to improve the quantitative oncogenic transformation
by chemical carcinogens and score for transformation at earlier times so
that the C3H10T1/2 mouse fibroblast system can be useful as a rapid pre-
screen for environmental pollutants. Individual compounds, as well as mix-
tures, will be studied. The use of single cells in individual dishes as the
basic system will be improved, and the inhibitory influence of cell density
on transformation frequency will be accurately quantitated. Transformed
C3H/10T1/2 cells have a different morphology in the scanning electron micro-
scope (SEM). This property will be used to develop an alternative assay
for transformation and to determine at what time after carcinogen treatment
cells become transformed. Other parameters of oncogenic transformation will
be correlated with morphological changes observed by SEM and light micro-
scopy. A test for environmental samples which may serve as promoters of
carcinogenesis will be established.
Scope and Objectives
At the present time several systems have been developed for obtaining
quantitative data on the oncogenic transformation of cultured cells by chem-
ical carcinogens. It seems clear that such systems, although more compli-
cated and tedious than a bacterial mutagenesis assay, may be more relevant
as prescreens for carcinogenic activities. They also provide valuable test
materials for studying the cellular and molecular mechanisms of chemical
48
-------
carcinogenesis; such information will be of practical value in providing
means to prevent human cancer.
The grantee has developed a system for studying chemical oncogenesis in^
vitro with the C3H/10T1/2 mouse embryo fibroblasts. However, before this
system can be validated as a potential prescreen by testing it fora larger
number of carcinogenic and noncarcinogenic chemicals, additional research
must be done to perfect its quantitative application.
The overall purpose of the present study is to perfect the quantitation
of chemical transformation of C3H/10T1/2 cells in culture. The timetable
and objectives under this grant will be: Year 1. Determine the optimal
conditions for transformation of C3H/10T1/2 cells using the several criteria
mentioned below. Year 2. Compare the quantitation of transformation by the
optimal conventional methods and the single cell methods, and begin to
screen known and unknown samples of environmental carcinogens with and with-
out liver hemogenate-mediated activation, and environmental promoters with
the purpose of validating the systems. Year 3. Continue validating the
systems and screen unknown environmental samples provided by the EPA and
other interested sources.
The perfection and validation of a system that determines and quanitates
oncogem'c transformation will produce the following benefits:
1) A reliable pre-screen for environmental carcinogens (or mixtures
thereof) is one of the highest priorities in environmental sur-
veillance.
2) Such a pre-screen can be widely applied to test large numbers of
samples (pure compounds and mixtures) of environmental pollutants
for their carcinogenic activity.
3) The determination if the oncogem'c transformation of cultured animal
cells is more relevant to human carcinogenesis than measuring muta-
genesis in bacteria or other biological or biochemical parameters.
4) The cost, space, and manpower requirements to test individual
samples would be expected to be less than 1% of those required to
carry out adequate tests of carcinogenesis in living rodents.
Background and Approach
For many years the grantee has been interested to ascertain the cellular
and molecular mechanisms for the initiation of cancer, particularly with
polycyclic aromatic hydrocarbons (PAH) on the skins of mice. However, he
recognized the severe limitations of working in vivo, and turned his atten-
tion to developing systems for the study of chemical carcinogenesis in vitro.
During initial exploratory studies, the pioneer papers of Berwald and Sachs
appeared, which clearly demonstrated that quantitative studies of carcino-
genesis could, indeed, be carried out in cell cultures. Since then, the
field has developed to the point where several systems have been developed,
49
-------
are in use, and are realizing their potential by providing useful tools both
for the study of fundamental mechanisms and for potential primary screening
systems for environmental carcinogens. This grant speaks primarily to the
latter objective.
In o.rder for the C3H/10T/2 mouse cell system to be perfected as a pre-
screen for environmental carcinogens, it is necessary that the transformation
frequency (TF) be accurately quantitated. At the present time, this parameter
depends entirely upon the method used for carrying out the transformation
assay, the time interval before fixation and staining (which has been ar-
bitrarily set at 6 weeks), and the method of scoring and counting of trans-
formed colonies. The grantee has already obtained some data to indicate
that the transformation frequency (TF) varies inversely with the number of
cells plated. This needs to be repeated in a much more thorough way with
particular attention to numbers of cells plated between 1 and 10. Another
problem in quantitating the TF is that there is a migration of cells from
one focus to form another. If such migration occurs, the TF obtained by
counting foci at 6 weeks will be too high. This migration needs to be
studied more thoroughly. The grantee has, as mentioned previously, arbi-
trarily fixed, stained, and scored for transformation at 6 weeks. He needs
to determine how the number of transformed foci would vary with time between
treatment and fixation, assuming that the problem of spreading of foci is
solved. The major purpose of this grant application is to study these
parameters so that the optimal conditions for elucidation of the TF can be
determined. This is of tremendous importance in quantitative screening of
environmental pollutants that may contain both strong and weak carcinogens.
Another problem with the cell culture system is that it is not known at
what time malignant transformation takes place after a single treatment with
a carcinogen. There are striking changes in the surface morphology of these
cells on transformation, as seen in the scanning electron microscope (SEM).
If such changes occur within a few days, it should be possible to greatly
shorten the time of the transformation assay by use of the SEM as the pri-
mary method of scoring, instead of waiting for 6 weeks to score for trans-
formed foci. This will be investigated both qualitatively and quantitatively.
Finally, systematic investigations will be carried out on a series of ad-
ditional parameters of transformation which may add to the precision of and
decrease the subjectivity of scoring for oncogenic transformation. These
parameters include: a) quantisation of the ability of a large number of
nontransformed and transformed clones to grow in soft agarose as a means to
determine loss of anchorage dependence; b) agglutination by plant lectins
such as concanavalin A; c) acquisition of fibrinolytic activity; d) per-
meability to 2-deoxyglucose; and e) appearance of tumor-specific trans-
plantation and embryonic antigens by lymphocyte-mediated cytotoxicity tests.
In all cases, these parameters will be compared in individual transformed
clones with their piled-up phenotype in foci, and with their ability to
produce fibrosarcomas on inoculation into syngeneic mice.
The grantee has succeeded in applying the same principles of liver
homogenated-mediated activation of polycyclic hydrocarbons and aflatoxins
to produce mutations at the GHPRT locus in Chinese hamster V79 cells (D. F
Krahn and C. Heidelberger, Mutation Res. 4£, 27, 1977). In research
50
-------
supported elsewhere, the grantee is applying this method of activation to
oncogenic transformation of C3H/10T1/2 cells. The grantee will apply the
same activation system in future years of this grant to the testing of en-
vironmental samples.
Research Accomplished
The grant has been in effect since October 1977. The grantee has in-
vestigated the time required to score the maximum frequency of transformation
in the C3H10T1/2 mouse fibroblast system. Following a single treatment with
3-methylcholanthrene, cultures were scored for transformed foci from the 4th
to the 10th week. Foci began to appear starting with the 4th week and re-
mained approximately constant from the 5th to the 10th week. Scoring will
be performed at 6 weeks as in previous studies. The grantee has sought a
better way of scoring transformation frequency in the 10T1/2 system. Initial
experiments with multiwell culture plates were unsuccessful due to detach-
ment of confluent cell monolayers before individual colonies would be scored.
The grantee will experiment with growing single cells on glass cover slips
to permit direct observation with precise scoring of transformation frequency.
Preliminary investigation of surface morphology of normal and malignant cells
has begun.
Bibliography
Mondal, S., D. W. Brankow, and C. Heigelberger. 1976. Two-Stage Chemical
Oncogenesis in Cultures of C3H/10T1/2 Cells. Cancer Research.
36:2254-2260.
Heigelberger, C.
44:79-121.
Heigelberger, C.
18:317-366.
1975. Chemical Carcinogenesis. Ann Rev. Biochemistry.
1973. Chemical Oncogenesis in Culture. Adv. Cancer Res.
Reznikoff, C. A., J. S. Bertram, D. W. Drankow and C. Heigelberger. 1973.
Quantitative and Qualitative Studies of Chemical Transformation of
Cloned C3H Mouse Embryo Cells Sensititve to Postconfluence Inhibition
of Division. Cancer Res. 33:3231-3238.
Reznikoff, C. A., J. S. Bertram, D. W. Brankow, and C. Heigelberger. 1973.
Quantitative and Qualitative Studies of Chemical Transformation of
Cloned C3H Mouse Embryo Cells Sensitive to Postconfluence Inhibition of
Cell Division. Cancer Res. 33:3239-3249.
Related Research
In Vitro and In Vivo-In Vitro Systems for Determining Potential Carcin-
ogenicity of Environmental Agents. In-House Task No. 8318 under Program
Element 601F, Carcinogenesis.
51
-------
B. TASK TITLE:
Enzymatic Characterization and DNA Binding of Carcinogens in
Short-Term Bioassays
HERL/RPT TASK NO: 8156
GRANTEE: Columbia University
GRANT NO: R805482-01
Summary
A series of short-term bioassays including the Salmonella typhimurium
(toes) microbial mutagenesis bioassay and the several neoplastic transform-
ation bioassays are being compared to in vivo tissue and human cells with
respect to their ability to metabolize pro-carcinogens. This research will
determine whether the carcinogen bound DNA adduct found in these test systems
is similar to that found in intact human tissue. Enzymatic characterization
of the carcinogen metabolizing capability present in these short-term bio-
assays is being conducted concurrently.
Scope and Objectives
The objective of this research is to determine if the activation of a
known carcinogen and its covalent binding to DNA within the cell is quali-
tatively and quantitatively similar in several in vitro test systems to that
which occurs in human cells and in the intact animal. The technical approach
includes a comparison of:
A. isolated and characterized benzo(a)pyrene nuclesoside adducts from
DNA
B. microsomal enzyme activities
C. metabolite profiles
Emphasis in this project is on the carcinogen DNA adducts since this provides
more definitive information on the ultimate carcinogen generated in each
system and bound to DNA.
This work is being conducted in three phases. First, Salmonella
typhimurium will be examined as it is utilized in the standard Ames Assay
utilizing Arochlor 1254-induced rat liver microsomal S-9 activation. During
the initial phase the following assays for neoplastic transformation will
also be examined:
A. Syrian hamster embryo system
B. C3H10T1/2 cell system
52
-------
The second phase of this work will include studies with intact cells as
metabolizers of carcinogens and the use of co-cultivation assays.
The third phase which has not been funded would utilize epithelial and
human cell systems which are currently in the developmental of validation
state.
Background and Approach
It is now recognized that animal bioassays are inadequate for monitoring
the thousands of environmental agents which require screening as possible
carcinogens. Several short-term in_ vitro tests for mutagenicity and carcin-
ogenicity are currently being evaluated (by governmental agencies, private
industry and various research groups). Since many environmental mutagens
and carcinogens require prior metabolism or whole cells to exert their
effects, some of these short-term assays use a rat liver microsomal fraction
for activation of the chemical being tested. There is a paucity of infor-
mation, however, on whether or not the activitation of a known carcinogen
and its covalent binding to cellular DNA in these in vitro systems is
quantitatively and qualitatively similar to that which occurs in intact
mammalian cells, or in the whole animal. The objective of this proposal is
to examine the modified DNA from cells used in these assays after exposure
to the ubiquitous carcinogen benzo(a)-pyrene (B[a]P). The approach will be
to incubate tritium labeled B[a]P with microsomes and Salmonella typhimurium
tester strains, or with various mammalian cell lines. Cellular DNA will then
be extracted, analyzed for radioactivity and fluorescense, and then hydro-
lyzed to nucleosides which will be analyzed by high pressure liquid chroma-
tography, utilizing appropriate B[a]P-nucleoside derivatives prepared chem-
ically as markers to determine the nature of the B[a]P-nucleoside adducts
present. Parallel assays will be done for mutagenicity and transformation
and of aryl hydrocarbon hydroxylase and epoxide hydratase activities. The
data will be correlated with our results obtained in intact human tissues
where it has been possible to determine the structure of the major adduct
formed.
Research Accomplished
The DNA bound benzo(a)pyrene (B[alP) adducts from the 10T1/2 oncogenic
(neoplastic) transformation bioassay have been isolated and characterized.
The major bound adduct is the 7, 8-dihydro diol 9, 10-epoxide of B[a]P.
Further characterization of these adducts is in progress. This is also the
major adduct in Syrian hamster embryo cells in vitro, bovine and human bron-
chial mucosa in vitro, and skin in vivo. This finding provides evidence
that the in vitro 10T1/2 mouse cell oncogenic transformation bioassay meta-
bolized B[a]P to the same major DNA bound adduct as do both an in vivo mouse
system and an in vitro human organ culture system. The DNA bound B[alP
adducts are novTbeing isolated from the Salmonella typhimuriurn/microsome
(Ames) bioassay.
Bibliography
Papers produced as a direct result of these funds are in progress.
53
-------
Related Research
EPA is currently funding the evaluation of several in vitro test systems
to screen environmental chemicals for potential carcinogenic and mutagenic
activity. In order to detect activity in chemicals which require activation,
either a liver microsomal fraction is added to the test or cell systems are
utilized which retain ability to metabolize carcinogens. It is important
to know whether the activation in each of these systems metabolizes carcino-
gens by the same route and to the same ultimate carcinogen that occurs in
whole animals and humans. This is crticial to the utilization of in vitro
systems as valid predictors of responses in animals or human populations.
This project proposes to answer these questions in the short-term tests
currently being either employed or evaluated by EPA.
C. TASK TITLE:
Development of Bioindicators to N02 and S02 Exposure
HERL/RTP TASK NO: 9172
CONTRACTOR: Southwest Foundation for Research and Education
CONTRACT NO: 68-02-2279
Summary
During the initial year of this investigation, efforts were concentrated
towards screening for potential bio-indicators centering around exposures to
S02. Areas of research pursued included effects of this pollutant on lymph-
ocyte responsiveness, circulating plasma neutral lipids, biogenic amines and
hormones, in addition to protein alterations induced by inhalation.
Based on findings in these preliminary studies, it was decided that the
approach showing most promise for continuation in years II and III would be
a thorough evaluation of altered immunological function as well as circulat-
ing biogenic amines and hormones with focus on dose and temporal effects.
The assessment of lumphocyte function through determinations of mitogen
responsiveness was to be pursued. It was shown that j_n vitro treatment of
globulin with bisulfite produces structural alterations' of protein which
could hopefully be used as Immunogen for attempting to produce and isolate
specific antibodies.
Short-term continuous exposure studies designed to examine acute and
latent effects in adult male Wistar-Lewis rats as a result of varying atmo-
spheric concentrations (2.5, 5 and 10 ppm) of S02 and N0? are continuing.
Assays were performed on blood samples obtained from animals in experimental
and control groups at each of several time intervals during both exposure
and recovery. Separate experiments for the determination of effects on
normal diurnal variation of corticosterone were conducted. Initial experi-
ments have now been performed for each of the exposure levels and will be
duplicated for evaluation of pooled data.
54
-------
For comparison with accumulating evidence that short-term exposures
have different effects than the same dose over longer periods, chronic
intermittent exposures will be conducted during the final year of this
contract. In an efforts to stimulate the normal work week of the human,
exposures will run twelve hours per day, five days per week for five weeks.
Scope and Objective
The intention of this research effort is to examine the biochemical
action of N02 and S02 on lung metabolism and to develop alterations that
occur into a dose-response bioindicator. Phase I: Biochemical investigations
of lipid and protein metabolism of both the lung and distal organs after
nitrogen dioxide and sulfur dioxide exposure. Phase II: To measure release
of bioactive compounds and/or their metabolites after N0? and S09 exposures.
To develop any detected alterations into bioindicators.
Background and Approach
The current standard for N02/S02 rests mainly on epidemiological
studies and their toxicity is less well defined than for the other major air
pollutants. Concern exists that exposure to these pollutants resulting from
sources such as automobile exhausts, power plants, orburningof coal may
create a major health hazard. Studies have previously shown that edematous
and hemorrhagic lungs are produced by nitrogen oxide irritants involving
primary reaction with lipid and proteinaceous material and causing damage
to the lung cells and surrounding capillaries. Acute toxicity of occupational
related nature has occurred in welders, firemen and nitric acid plant workers.
Additionally in vitro experiments have demonstrated alterations in gluco-
proteins of macrophage and lymphocyte cell membrane. Such changes may affect
the immune response and/or produce disturbances in hormonal action at the
cellular level.
Inhalation of S02 has resulted in absorption and penetration into the
systemic circulation. This has been demonstrated utilizing 35$o2, the -"$
of which diffuses through the lung into the circulatory system and is dis-
tributed throughout the body. Interaction of these gases with glycoproteins
at the cellular level could potentially alter neurohormonal mechanisms con-
trolling endocrine systems, particularly the pituitary-adrenal axis. Dis-
ruption of either the CNS or target organ cellular membrane could alter the
cyclic hormone cascade, which might be demonstrated through changes in ACTH
and/or glucocorticoid pattern. It was then suggested that plasma ACTH and
corticosterone be examined in light of their role as important chemical
mediators.
It was proposed, therefore, to evaluate the effect of exposure to high
level sub-lethal doses of N02/S02 through measurement of chemical parameters
of blood. Initial screening should include biogenic amines and their meta-
bolites, corticosterone and ACTH, CAMP, prostaglandins, histamine, and
neutral lipids. Effects of dose and duration should then be determined for
those indices showing and effect.
55
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The second category under investigation is that of immune response. In
vitro studies of blast transformation by circulating blood lymphocytes are
being conducted for detection of alteration of responsiveness possibly con-
tributing to compromised immunocompetence. Assessment of lymphocyte blasto-
genesis through mitogen stimulation is being examined using PHA, ConA and
IPS. Alteration of ability to respond to mitogen stimulation is an indication
of detrimental influence on defense mechanisms.
Research Accomplished
As of this date the first two years of investigation under this contract
have been completed. Major milestones planned and achieved include the
screening of potential bioindicators in rats subjected to sulfur dioxide ex-
posure at high sub-lethal levels and selection of appropriate assays. The
effects of five-day exposures of rats to 5 ppm of both S02 and N02 have been
evaluated in two separate groups. Initial exposure to 2.5 and 10 ppm for each
of these pollutants have been conducted. They will be repeated subsequently.
Available data indicate a possible acute effect of the two gases on the
serotonergic system at certain time points during exposure. In addition, a
residual effect appears to be exerted by 2.5 and 5 ppm SOg as evidenced by
elevated mean levels of serotonin during the recovery period. These obser-
vations were not augmented by increasing the S02 level further. Exposure to
5 ppm N02 revealed changes interpreted to indicate primary exposure response
of serotonin and epinephrin during the experimental testing followed by a
secondary recovery upon termination of exposure. More labile effects were
exhibited in the sympathetic system. Plasma corticosterone was significantly
depressed at 14 days post exposure to 10 ppm S02 while ACTH was elevated.
Negligible effects of both gases were seen in other parameters of cAMP, hist-
amine, lipids and prostaglandin.
The most striking result of immunological indicators to date was that of
a depressed mitogenic response to Con A immediately after exposure to 5 ppm
and remaining through 28 days after the highest dose of S02. At 2.5 ppm no
lasting alterations were found. Decreased response was observed for all
mitogens tested after 24 hours exposure to 10 ppm and 120 hours to 5 ppm N02-
There is some indication that the observed changes might be more persistent
at the higher dose level.
Bibliography
Allen-Rowland, C. J., Catherine Yndo-Vriend, J. Padilla, J. P. Allen and
M. F- San Miguel. 1978. The effect of a Single Continuous Exposure to
Various Atmospheric Concentrations of Sulfur Dioxide on Circulating
Biogenic Amines and Hormones. To be presented at the Endocrine Society
Meeting, June 16 - 18. Miami, Florida.
Yndo-Vriend, Catherine, Catherine Allen Rowlands, and Jorge Padilla. Effects
of Short Term Exposure to NO? Gas on Circulating Biogenic Amines in the
Rat. Presented at American Association of Anatomy Meeting, Van Couver
British Columbia, April 2 6.
56
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Related Research
NASA contract recently awarded to Dr. Irving Geller of Southwest Founda-
tion and project director of Contract #68-02-2279 for a study of S0?/N0?
Behavioral Effects. (Personal Communication)
D. TASK TITLE:
In Vitro Screening of Selected Air Pollutants for Potential
Carcinogenicity.
HERL/RPT TASK NO: 8190
CONTRACTOR: Microbiological Associates
CONTRACT NO: 68-02-2271
Summary
A tiered approach to the screening of environmental agents has been de-
veloped and implemented. This approach includes the use of bacterial muta-
genesis, mammalian cell mutagenesis and neoplastic transformation bioassays.
Agents which have been tested in this tiered system include pesticides, as-
bestos-like fibers, polycylic aromatic hydrocarbons and insoluble metal par-
ti culates.
This contractor has studied the feasability of coupling metabolic acti-
vation systems (e.g. rat liver homogenate or primary embryo cells) with
mammalian cell mutagenesis and oncogenic transformation bioassays and have
developed these combined systems which give increased sensitivity to the
detection of genotoxic agents. The most significant accomplishment has been
the development of a bioassay which simultaneously measures mutagenesis and
oncogenic transformation in mammalian cells with the addition of exogenous
metabolic activation. This system is now in the validation stage and will be
an important tool in the screening of large numbers of environmental agents.
Scope and Objectives
The scope of this contract is encompassed in three subobjectives:
A. To screen for the potential mutagenic and carcinogenic activities
of selected air pollutants and environmental chemicals provided by
the Environmental Protection Agency.
B. To determine the feasibility of combining iji vitro mammalian cell
bioassay systems with various mammalian activation systems (both
intact cellular and sub-cellular) to enhance the sensitivity to low
concentrations of biologically potent chemicals or chemicals of poor
biological activity.
C. To develop an assay which measures simultaneously both mutation and
oncogenic transformation.
57
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The tiered approach being employed to evaluate the biological activity
of the compounds supplied by the EPA is being developed along the following
lines. The mutagenic potential of each chemical agent is first determined in
the Salmonella typhimurium mutagenesis assay developed by Ames. These com-
pounds which prove to be negative in this test are then retested in the
presence of a source of mammalian metabolism in the form of the 9000 x g
supernatant (S-9) derived from rat hepatic tissue. Agents which are found to
be positive mutagens in the bacterial prescreen are then assessed for their
cytotoxic and mutagenic potential to establish in vitro mammalian cell lines.
Those cell systems presently being evaluated include the V-79 Clone 8 (V-79)
Chinese hamster lung line the BALB/c 3T3 Clone A-31-1 (3T3-1) mouse line and
the mouse C3H10T 1/2 Clone 8 (10T 1/2) line.
The incorporation of mammalian metabolic activity in order to increase
the sensititivity of these assay systems both in terms of detection of weak
carcinogens or low doses of strong carcinogens, as well as detection of di-
verse classes of carcinogens, are being developed along three lines:
A. Cocultivation of target cells with rodent S-9 fractions
B. Cocultivation of target cells with freshly derived rodent hepato-
cytes.
C. Cocultivation of target cells with X-irradiated mitotically treated
metabolically active hamster embryo cells.
Preliminary assessments are being made of the inherent levels of polycyclic
aromatic hydrocarbon metabolizing activity (in terms of aryl hydrocarbon
hydroxylase (AHH) and epoxide hydrase (EH)) in each of these cellular and
sub-cellular sources of enzyme activity. The inherent cytotoxicity of these
preparations for the in vitro model cell systems are also being evaluated.
Incorporation of these preparations into mammalian mutation and transformation
assay systems will be done with the appropriate S-9's as determined by the
above preliminary tests.
Evaluations are being made as to the feasibility of utilizing one or
more mammalian cell systems, in the presence or absence of an exogenous
source of metabolic enzymatic activity, to assay for the chemical induction
of ouabain-resistance mutation and transformation.
Background and Approach
From the time Sir Percivall Pott recorded in 1775 his observations of
"soot-wart", the scrotal and testicular cancer of chimney sweeps of Old
England, enviromental chemicals have been recognized as potential etiological
carcinogenic agents to man. The earliest reported experimental induction of
animal tumors, however, did not appear until the twentieth century when
Yamagiwa and Ichikawa produced carcinomas on the skin of rabbits painted with
chimney soot or coal tar. Finally, approximately 15 years later, Kennaway
and Hieger and Cook obtained extracts of benzo(a)pyrene and 1,2,5,6-diben-
zanthracene from such sources as coal tar, and found these polycyclic hydro-
carbons to ellicit neoplasms in vivo. Several other classes of chemical
58
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agento were also soon recognized as potential environmental carcinogens, in-
cluding aminoazodyes, aromatic amines, nitrosamines, carcinogenic metals, etc.
Polycyclic aromatic hydrocarbons, benzo(a)pyrene, in particular (a known po-
tent carcinogen), has been shown to be expelled into the atmosphere in the
range of 1320 tons per year in the United States alone. Furthermore, the
smoke of 40 cigarettes has been reported to deliver 1 g benzo(a)pyrene. In •
view of these-data and the fact that as much as 90c; of all human cancers have
been attributed to chemical agents, an appropriate method by which to screen
such environmental chemicals for their carcinogenic potential is of utmost
importance. Considering the vast number of compounds that would need to be
tested, the sole use of in vivo animal systems becomes an impossibility in
terms of finances and trained personnel. In addition, tumorigenicity in vivo
is affected by the species of animal and its genetic background, presence of
pathogens in the strain, promoting agents in the diet, drug metabolizing
enzymes, dosage and duration of treatment, route of inoculation, age and life
span of the animals, and hormonal variations. Reliable and reproducible TJT^
vitro test systems offer the following advantages as a preliminary screen:
1. Relatively Tow cost per compound; 2. a test which usually can be
initiated and completed in less than three months; and 3. microgram quant-
ities of rare or expensive compounds could be tested, whereas much larger
quantities are needed for bioassay in animals.
Compounds which transform the cells in vitro can then be further evalu-
ated for in vivo carcinogenicity using the long-term animal systems.
Major tissue culture transformation systems currently available include
the hamster embryo system, the C3H 10T1/2 clone 8 system, and the BALB/3T3
systems. Since many of the enzyme systems which can change the different
chemicals to their proximate or ultimate carcinogenic forms are unknown or
may be absent in the cell culture, more complex cell- or enzyme-mediated
assays need to be developed.
Research Accomplished
A. Bacterial Mutagenesis—
1. Screening— A number of various different types of samples were tes-
ted according to the procedure of Ames including the pesticides Captan, Fol-
pet, and asbestos-like fibers Amosite and Chrysotile. The former agents were
positive while the latter negative in this test system. An insoluble carcin-
ogenic metal, nickel subsulfide, was also tested and was negative.
2. Developmental— Studies have been initiated to examine the (AHH)
aryl hydrocarbon hydroxylase (a carcinogen metabolizing enzyme) activity of
different S-9 preparations and to relate the AHH activities to the induced mu-
tation frequency of S^. typhimurium tester strain TA 100. The S-9's were
generated from Fischer 344 male rat hepatic tissue, either from control rats
or from rats induced by intraperitoneal injection with 500 mg Aroclor-1254 per
kg body weight once 48 hours prior to sacrifice, or with 50 mg phenobarbital
(PB) per kg body weight once each day for five days prior to sacrifice. AHH
activity was monitored by the conversion of benzo(a)pyrene (B[a]P) to the
59
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phenol, 3-OH B[a]P. Mutation of TA 100 was monitored by reversion from histi-
dfne auxotrophy to prototrophy, using 6-aminochrysene as the mutagen. Cyto-
toxicity determinations were also run in parallel in order to develop a mu-
tation frequency. In terms of AHH activity, Aroclorinduced S-9 gave the
highest metabolic conversion of B[a]P to 3-OH B[a]P. PB-induced S-9 had about
1/3 the activity of Aroclor-S9. Control (noninduced) S-9 showed about 1/5
the AHH activity of Aroclor-induced S-9. However, the mutation frequency in-
duced in the presence of each of these S-9 preparations did not parallel AHH
activity; PB-induced S-9 gave the highest mutation frequency per AHH.
B. Mammalian Cell Mutagenesis and Oncogenic Transformation--
The feasibility of coupling mammalian metabolic activation systems with
in vitro mammalian cell bioassay systems was explored. The following two
mammalian bioassay systems were cultivated with induced rat liver homogenate
and benzo(a) pyrene:' Chinese hamster cells V-79 (mutagenesis) and BALB 3T3
mouse embryo fibroblasts (cytotoxicity, mutagenicity, and oncogenic trans-
formation). In all cases an increase was observed in the biological activity
of each of the systems incubated with the homogenate and B[a]P when compared
to B[a]P alone indicating that the liver homogenate activated the carcinogen
to forms which interacted with the cells.
Other metabolic activation systems were also examined. These were whole
cell preparations which included primary Syrian hamster embryo cells, primary
rat hepatocytes, and various strains of irradiated embryonic cells.
C. Amplification assay for enhanced detection of chemically-induced morpho-
logical transformation--
The standard 3T3 and 10T 1/2 focus transformation assays have proven to
be routinely reliable in ascertaining the neoplastic potential of model chem-
ical carcinogens. There are certain limitations in both systems; one of
these is the fact that similar treatment of target cells with potential car-
cinogens does not always result in comparable transformation responses. In
addition, not every replicate dish treated with a given carcinogen gives rise
to Type III transformed foci. This failure in uniformity of response suggests
the possibility that certain potential carcinogens may be incorrectly scored
as negative if they fail to induce focus formation in the standard assay.
In pilot studies with 3T3 cells it has been observed that formation of
morphologically transformed foci in dishes generated from a single subculti-
vation of cells (treated with transforming doses of carcinogen) derived from
dishes which showed no foci after the standard four week assay. Based upon
these results a procedure for amplifying the presence of phenotypically trans-
formed 3T3 cells potentially capable of forming Type III transformed foci, has
been devised and validated with standard carcinogens.
D. Development of a Simultaneous Mutagenesis and Transformation Bioassay
with Exogenous Metabolic Activation--
Another approach which was employed with the 3T3 cell system was to
simultaneously monitor both chemically-induced mutagenesis (at the ouar locus)
60
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and transformation (Type III focus formation). These studies were carried
out using 3-methylcholanthrene (3-MC) as the mutagen/carcinogen in the ab-
sence of any exogenously supplied metabolic enzyme (S-9) preparation.
To date, the model chemicals examined in the standard plate assay have
included 3-methycholanthrene (3-MC), N-methyl-N'-nitro-N-nitrosoquandiine
(MNNG) and 6-aminochrysene (6-AC). Each time morphological transformation
(Type III foci) was observed, mutagenesis at the ouabain-resistance (ouar)
locus was observed, and vice versa, when 3-MC or MNNG were tested. Using
the 3T3 system, an induced mutagenic event coincided with a transformation
event with each of 3 classes of chemicals surveyed. Also a number of pesti-
cides were tested in these test systems for transforming ability and muta-
genesis. These agents included Folpet and Captan, two fungicides.
Bibliography
Kouri, Richard E. and Leonard M. Schectman. 1977. State of the Art In Vitro
Metabolic Activation Systems. Presented to the Environmental Mutagen
Society Annual Meeting. July.
Schectman, Leonard M., and Richard E. Kouri. 1977. Control of Benzo(a)pyrene
Induced Mammalian Cell Cytotoxicity, Mutagenesis and Transformation by
Exogenous Enzyme Fractions. Progress in Genetic Toxicology. Eds.
D. Scott, B. A. Bridges and F. H. Sobels. 307-317.
Related Research
In Vitro and In Vivo-In Vitro Systems for Determining Potential Carcin-
ogenicity of Environmental Agents. In-House Task No. 8318 under Program
Element 601F, Carcinogenesis.
Development of Test Systems to Assess Potential Toxicity and Neoplastic
Transformation - Improvement of Scoring of Chemical Transformation of
C3H/10T1/2 Cells (Substituted for) Development of Test Systems to Assess Tox-
icity and Neoplastic Transformation using Type 1 and Type 2 Alveolar Epithe-
lial Cells In Vitro. Grant No. R-805208-01, University of Southern Califor-
nia, Task No. 8153, Program Element No. 625F, Energy.
E. TASK TITLE:
In Vitro Screening of Selected Air Pollutants for Potential
CaTrcinogenicity Using Microbial Systems.
HERL/RTP TASK NO: 8191
CONTRACTOR: Research Triangle Institute
CONTRACT NO: 68-02-2724
Summary
Solvent fractionation is used to separate components of particulate
61
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matter in air collected by means of a Battelle Massive Volume Air Sampler.
The crude material and resulting fractions are subjected to bioassay (muta-
genesis) via a Salmonella typhimurium reverse mutation detection system.
Positive results form the basis for chemical composition determinations,
further fractionation and additional bioassay. High pressure liquid chrom-
atography (gel permeation, adsorption and partition modes) is used for
additional separation with chemical structural analysis accomplished yja^
chromatographic retention time determinations, spectral (UV adsorption,
fluorescence excitation and emission) comparison with standards, and dir-
ect probe mass spectrometry coupled with a computerized mass spectral search
system. Volatile vapors and aerosols are collected from the atmosphere us-
ing sampling cartridges containing a sorbent material. After collection the
materials are thermally desorbed for chemical identification and bioassay
purposes.
Scope and Objectives
Industrial emissions and wastes include agents which are known to be
mutagenic/carcinogenic and thus constitute a health hazard to man in the
form of cancer, birth defects and heritable disease. In addition to the
known agents there are probably present many undetermined mutagenic/carcino-
genic substances as well.
Clearly, the most reasonable approach to the evaluation of complex
environmental samples is a judicious combination of chemical identification,
fractionation and biotesting. It is presently not possible to specify a
detailed protocol which incorporates all these aspects and which is maximally
efficient and cost effective. Thus, the purpose of the proposed work is to
develop such a protocol and in so doing, define a minimal biological and
chemical methodology which will function as an effective screen for potential
mutagenicity/carcinogenicity (and other related hazards) of complex mixtures
occurring as air pollutants.
Background and Approach
Recent work suggests that a limited set of mutagenesis tests may be the
best of presently available methods to use as indicators for the most serious
human effects. These have the ability to indicate, with mutation as the test
end point, a variety of possible effects that involve the disruption of bio-
chemical genetic mechanisms which are shared generally by all forms of life.
Recently there have been demonstrations of correlation between carcin-
ogenesis and mutagenesis (Miller and Miller, 1971; Ames, 1972) and it has
been implied that most cancers may be due to somatic mutations (Ames, 1972;
Ames, et al_., 1973). The numbers of compounds that have been demonstrated to
be mutagenic and also carcinogenic and/or teratogenic (Miller and Miller,
1971; Kalter, 1971) recommend mutagenesis tests as indicators.
Given that mutagenesis tests deserve consideration for evaluating the
biological impact of contaminating substances in the environment, there re-
mains the selection of particular mutagenesis tests and adapting them to
the study of mixtures of airborne materials. One of the most promising
62
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current methods is the Salmonella typhimurium histidine reverse mutation sys-
tem described by Ames, McCann and Yamasaki TT975). Already more than three
hundred chemicals have been evaluated for mutagenic activity with this system.
There are two simplistic approaches which could be used for the evalu-
ation of mutagenic potential of crude air samples. One is to perform very
thorough and exacting chemical analyses. Then on the basis of mutagenesis
tests of the pure compounds identified by the analysis, decide whether or not
a hazard exists. This approach is not very satisfactory because it is the
mixture to which humans are exposed and not the individual components, and
there may be interactions which make the mixture more or less active than
the individual agents evaluated separately. There is, too, the possibility
of sample matrix effects which may limit the availability of particular
agents to the biological test material. Another simplistic way of approach
would be to directly subject, the crude materials to biotesting without re-
gard for chemical analysis. Also very unsatisfactory, this would allow
hazardous substances, temporarily masked by inhibition, or interaction, etc.,
to pass unnoticed. Additionally, if there are effects other than mutageni-
city for some agents in the mixture, it is quite possible for the effect of
one compound to prevent the detection of another. Thus, a toxic effect of
one agent could prevent (because effective concentrations could not be
reached) the detection of mutagenicity of another. This task represents a
compromise between these two approaches such that a limited but effective
fractionation scheme will be performed in conjunction with a simplified but
complete bioassay protocol.
The bioassays will depend most heavily on three strains of Salmonella
typhimurium (TA-1535, TA-1537, and TA-1538) which are histidine deficient and
with which reversion to prototrophy is indicative of mutation. Testing will
be done both with and without the inclusion of a metabolic activation system
for the detection of directly and indirectly acting mutagens.
The methods selected for the collection of pollutants from ambient air
will provide a representative continuum from the more volatile organic vapors
and aerosols to the semi-volatile and non-volatile compounds associated with
particulate. Sample collection strategy will be designed to allow the co-
llection of sufficient quantities of materials by each collection method.
The two collection methods proposed are the sampling cartridge containing
a sorbent material (Tenax GC) for the concentration of the volatile vapors
and aerosols from the atmosphere and the Battelle Maxi Air Sampler for the
collection of particulate matter.
The volatile vapors trapped on Tenax GC sampling cartridges will be
removed by thermal desorption to recover the trapped organic vapors for
subsequent bioassay or for chemical characterization by high resolution gas
chromatography/mass spectrometry/computer analysis. Thermal desorption sys-
tems of varying design have been described for recovering trapped vapors with
their subsequent analysis by glc and glc/ms. The Contractor proposes to
recover the adsorbed trace organic vapors utilizing an inlet-manifold de-
veloped at RTI (EPA Contract No. 68-02-1228) Pellizzari, 1974).
63
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The fractionation sequence for particulate is divided into three levels
of separation. Initial separation of the particulate sample into groups con-
taining the inorganic matrix, polar organics, and non-polar organics occurs
in the first level. Beyond this point, primary emphasis is placed on the
biological evaluation of the non-volatile organic constituents of the partic-
ulate sample. Further evaluation of the inorganic fraction with respect to
effects on the bio-availability of organic components is considered to be an
ancillary objective in this proposal.
The second level of fractionation divides the drude organic mixtures
into general chemical classes containing polar and non-polar acidic, basic
and neutral components, respectively, with the non-polar neutrals being
further sub-classes into parafins and aromatics. Al_l fractions produced in
levels 1 and 2 will be subjected to bioassay in order to evaluate the exist-
ence of synergistic relationships between constituent classes, including the
inorganic matrix.
Level 3 involves the separation of the class mixtures from level 2 into
their respective components as required for the qualitative and quantitative
analysis of mutagenic agents. High pressure liquid chromotography (HPLC)
will be utilized extensively at this level.
Although the initial class separation and subsequent resolution of
individual components is a requirement for the evaluation of synergisms and
identification of active components, considerable shortening of the fraction-
ation scheme may be possible for the routine quantisation of specific com-
pounds in the particulate sample. This aspect will be examined as part of
the quantitative evaluation.
Research Accomplished
A "well method" for mutagenesis screening in a modified Ames test has
been validated using known mutagens (2-anthramine, 2_acethlaminofluorene,
benzo(a)pyrene, B-naphthylamine, sodium azide, wuinacridine hydrochloride,
9-aminoacridine and 2-nitrofluorene) with five strains of S.. typhimurium.
The "well test" proved as good or better than spot and disc tests and com-
pared favorably with the pour plate test. The principal advantage is a
greatly reduced sample size. The protocol for S-9 microsome preparation has
been finalized. One time induction using 500 mg Arochlor/kg body weight
proved superior to multiple injections at lower doses.
The chemical fractionation scheme for ambient particulate samples has
been modified to reduce the total number of fractions to six as compared with
the 13 originally produced with larger amounts of material in each fraction.
The efficacy of the scheme was assessed by subjecting a mixture containing
•known amounts of compounds to the partition procedure. Recoveries were
determined gravimetrically. TLC scans indicated no "spill over" of com-
pounds into other fractions.
Using known mutagens, tests of the effects of varying microsome con-
centration, bacterial concentration, and histidine concentration were con-
ducted in both pour and well procedures. Histidine concentrations of 20-40
micrograms/plate appeared to be the optimum. Variation of bacterial
64
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concentration in the range of 1.00 1.30 (OD at 420 nm) has little or no
effect. Microsome concentration (.08 2.0 mg/plate) markedly affected re-
sponse. Work is continuing to devise a mechanism to confine vapors and
gases in such a way that they can be released quantitatively into the muta-
genesis assay.
One proposed approach for the Ames testing of air vapors involves the
transfer of vapors from Tenax cartridges (as collected in the field) to acti-
vated charcoal . The charcoal with the adsorbed vapors would then be placed
in an agar well and mixed with a solvent that would desorb and disperse the
vapors into the culture medium. Critical to this approach is the choice of
desorbing solvent. Nine potential solvents were tested for compatibility
with the Ames procedure. Of these only 2 were acceptable in terms of tox-
icity in relation to volume, DMSO and ethylene glycol.
It appears that DMSO is the only solvent that is both compatible with
the Ames assay and capable of releasing significant quantities of carbon-
adsorbed material such as EDB. Even in DMSO, a volume of 0.5 ml probably
represents an upper limit in regard to what can be tolerated in the bioassay
procedure. Because of these difficulties, the analytical/bioassay approach
for vapors based on the use of carbon adsorption bears re-examination.
Other experimental approaches are under consideration. The most promis-
ing to date involves transfer of the vapors from Tenax cartridges into hollow-
fiber filters (2000-50,000 molecular weight retention) which can be sealed
at the ends and incorporated into the agar plates.
Bibliography
Ames, B. N. 1972. A bacterial System for Detecting Hutagens and Carcinogens.
Mutagenic Effects of Environmental Contaminants. Ed. H. E. Sutton and
J. I. Harris. Academic Press, New York.
Ames, B. N., W. E. Durston, E. Yamasaki and F. D. Lee. 1973. Carcinogens
are Mutagens: A Simple Test System Combining Liver Homogenates for
Actiation and Bacteria for Detection. Proc. National Acad. Sci.
2281.
Ames, B. N., J. McCann and E. Yamasaki. 1975. Methods for Detecting Car-
cinogens and Mutagens With the Salmonella/Mammalian-Microsome Mutageni-
city Test. Mutation Research. 31:347-364.
Kalter, H. 1971. Correlation Between Teratogenic and Mutagenic Effects of
Chemicals in Mammals. Chemical Mutagens, Principles and Methods for
Their Detection. I Ed. A. Hollaender. Plenum, New York.
Miller, E. C. and J. A. Miller. 1971. The Mutagenicity of Chemical Carcin-
ogens. Chemical Mutagens, Principles and Methods for Their Detection.
I. Ed. A. Hollaender, Plenum, New York.
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Pellizzari, E. D. 1975. Development of Analytical Techniques for Measuring
Ambient Atmospheric Cercingoenic Vapors. EPA-600/2-75-076. Contract
No. 68-02-1228.
Pellizzari, E. D. 1974. Development of Method for Carcinogenic Vapor
Analysis in Ambient Atmosphere. EPA-520/2-74-121. Contract No.
68-02-1228.
Stedman, R. L. et. al. 1968. Chem. and Ind. 394 pp.
Related Research
NIEHS/EPA Collaborative study on mutagenicity of ambient air using
Tradescantia plant system in a mobile van.
F. TASK TITLE:
Develop Cellular Model System to Determine Cytotoxicity from
Alternate Energy Sources
HERL/RTP TASK NO: 8193
CONTRACTOR: Dr. William E. Bowers, Rockefeller University
CONTRACT NO: 68-02-2426
Summary
A quantitative in vitro bioassay system to evaluate the effects of
pollutants on lymphocyte cytotoxic activity is being developed.
Scope and Objective
The objective of this research is to develop a quantitative model sys-
tem to determine the effects of in vitro pollutant exposure on lymphocyte
cytotoxic activity. This will involve identification of the bioenergetic
and biosynthetic pathways believed to be of importance to cytotoxicity and
determination of the nature of any substance elaborated by lymphocytes in-
volved in foreign cell destruction. After development of the model system,
its suitability for use in environmental toxicity studies will be evaluated.
Background and Approach
The immune system has a variety of defensive functions against both in-
fectious and neoplastic disease. The development of new energy processes
raises the possibility that some fossil fuel emissions may have the po-
tential of being immunosuppressive agents. However the large number of
different substances which may be emitted calls for a screening research
approach that will rank toxicity for later jn vivo testing. Due to the
complexity of the immune system, one of the better approaches to an in
vitro study is to investigate effects on lymphocytes which are sensitive to
the action of a variety of treatments.
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The general features of lymphocyte physiology fundamental to the develop-
ment of useful bioassasys are the following: small lymphocytes transform into
large-sized lymphocytes as a result of their encounter with tumors and with
foreign cells or as a response to stimulation by antigens and mitogens. These
transformed lymphocytes then divide several times to give rise to progeny
which carry out specific processes. For example, the"progeny which result
from the division of lymphocytes transformed by tumor or allogeneic cells are
capable of killing specifically the tumor or allogeneic cells. Thus, trans-
formation lies on a direct pathway to the production of effector cells having
specific functions, and the entire sequence of events occurring in vivo--
recognition of foreign surfaces, transformation, division, and production of
specifically cytotoxic progeny—can be reproduced in vitro. Such an in vitro
system makes an ideal object for the study of cytological and biochemical
changes accompanying the production of cytotoxic lymphocytes and for the bio-
chemical processes essential for the killing of tumor or allogeneic cells.
Two of the important aspects of this system, namely transformation and cyto-
toxicity, will be made the subject of bioassays.
Research Accomplished
To date, a lymphocyte transformation and cytotoxicity assay has been
established. The methodology has been improved by interfacing cell-sizing
instrumentation to a microprocessor unit which will permit large numbers of
samples to be evaluated sensitively and accurately. An automated method for
determining 3H-uridine incorporation into transforming lymphocytes has been
developed. This will allow assessment of effects on earlier biochemical
events in lymphocyte transformation, and it will complement the already de-
veloped method for measuring ^H-thymidine incorporation. Work is progressing
on identification of biochemical events associated with lymphocyte transfor-
mation and cytotoxicity. When the bioassay developmental phase is completed,
pollutants will be added to the system.
Bibliography
None
Related Research
To my knowledge this investigator is not being funded by any other
source to conduct the same research. The particular field of immunology
involved in this project is subject to intensive investigation by the
scientific community. Therefore, while I am not familiar with any dupli-
cation of effort, the possibility does exist.
G. TASK TITLE:
Development of Automated Behavioral Testing Methodologies for
the Study of Coal Conversion and Utilization Products.
HERL/RPT TASK NO: 8155
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TASK TITLE:
Application of the Automated Behavioral Testing System to
Monkeys Exposed to Coal Conversion Pollutants - RFP-B116
HERL/RTP TASK NO: 8165
CONTRACTOR: Iowa State University
CONTRACT NO: 68-02-2288
Summary
The nervous system is a target organ for a number of substances which
are emitted from energy related sources. Until recently little research has
been specifically aimed at identifying neurotoxicity, especially as it re-
lates to subtle behavioral effects brought about by chronic low-level ex-
posures. The computer-automated pattern recognition and operant testing
paradigms developed under the energy research program have proven to be be-
havioral testing procedures highly sensitive to the effects of low level
d-amphetamine exposure in Macaca fascicularis. It is expected that these
procedures will be invaluable in determining the neurotoxic effects of a
number of energy related pollutants on complex bahavioral processes. Further,
the primate appears to be an excellent animal model for examining the neuro-
behavioral effects of psychoactive drugs, and should represent the animal of
choice when examining the effects of neurotoxicants on complex behavioral
processes.
Background
The nervous system is a target organ for a number of substances which
are emitted from energy related sources. For example, it is estimated that
approximately 600 million tons of coal are consumed yearly in the United
States. Of this total, in, excess of 360 million tons are burned in central
power plants. Several investigators have documented the presence of many
neurotoxic trace elements (nickel, lead, cadmium, manganese, mercury, thallium
and tin) in coal. Although many of these toxic trace elements are present in
small quantities in coal, the extremely large volume of coal burned yearly
can result in the mobilization of significant quantities of these elements
into the environment.
Although toxicologists have for some time recognized the commonality of
the nervous system as a target organ for a variety of toxic agents, until
recently very little research has been specifically aimed at identifying
neurotoxicity, especially as it relates to subtle behavioral effects brought
about by chronic low level exposure. In the field of behavioral toxicology
a particular need has existed for the development and validation of testing
methods which are sensitive indicators of low level exposure, specifically
long-term low level exposure. This need for the development and validation
of testing methodologies formed the basis for the initiation of the present
tasks.
68
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Scope and Objectives
The main thrust of the research effort described in these tasks centers
around the following objectives: (1) the use of the non-human primate as the
experimental animal, and (2) the development and validation of computer based
behavioral testing procedures.
Much of the current effort in methods development has focused on the use
of the rodent as the test subject. The point to be made here is that a need
exists for primate research in the area of behavioral toxicology. For ex-
ample, the highly developed central nervous system and complex behavioral
patterns make the primate highly suitable for some types of testing. Only
recently have researchers begun the task of examining the effects of CNS
toxicants on complex behavioral processes in animals. It is imperative that
the effects of neurotoxicants on such complex behaviors as: perception,
learning, storage and recall of information, the use of language, social
interaction, development, motivation, and emotion be understood. It is only
in the non-human primate that some of these processes are observed. One of
the behavioral paradigms developed under Phase 1 of this effort examines a
spectrum of complex behavioral activity patterns in the primate. This tech-
nique has been successfully used in behavioral pharmacology (and recently
in behavioral toxicology) but these studies have employed the rat as the
test subject. A second paradigm developed under Phase 1 research examines
the ability of the primate to store and recall stimulus information over a
long time interval—a task too difficult for a rat to perform.
The testing procedures referenced above have been employed in a non-
automated environment. However, this application of these procedures im-
poses severe limitations on what are otherwise very informative behavioral
testing methods. The computer automation of these tests would make pract-
ical the use of these and related procedures in screening a number of energy
related toxicants for neuro-behavioral effect.
The objectives of these research tasks were therefore: (1) to develop
computer-automated behavioral testing methods using the primate, (2) to
validate these methods using pharmacological agents with known psychoactive
properties, and (3) to apply these methods to toxicity testing of candidate
pollutants.
Research Accomplished
Currently work on Task 8155 is scheduled for completion on 31 July, 1978,
with work on Task 8165 to begin 1 August 1978 with completion scheduled for
31 July 1980.
To date, the computer automated pattern recognition system for the
assessment of neuro-behavioral changes in the spontaneous behavioral act-
ivity of primates is complete. Testing of this system with primates ex-
posed to a stimulant drug (dextro-amphetamine) are completed, and tests with
a depressant drug (chlorpromazine) are currently being conducted.
69
-------
The objective of developing a computer-automated pattern recognition
system has been achieved. A computer system equipped with three closed cir-
cuit video cameras has been programmed to identify 40 separate primate be-
havioral acts. In addition to identifying each act, the system is capable
of determining the frequency, duration and sequence of occurrence of each
behavioral act. To a point, a "seeing computer" has been substituted for
the human in the task of observing and classifying complex primate behavioral
activities. This computer system has proven to be a highly reliable observer
of primate behavior when compared to similar observations performed by
trained human observers. Recent comparisons between the computer system and
trained observers, show that the computer and the human observers agree in
their observations approximately 85% of the time. However, when disagree-
ments do occur, the human observer invariably agrees that the computer's
initial observation was correct. In short, the computer is not only a more
reliable observer from one occasion to the next than the human observer, it
is generally a better observer.
Tests of the computer pattern recognition system using primates exposed
to the stimulant drug, d-amphetamine, have produced some surprising and very
encouraging results. Initial data analysis indicates that the computer
pattern recognition system is highly sensitive to amphetamine produced
changes in primate behavior. For example, if the computer system is pro-
grammed to identify the location of the primate within it's environment,
rather dramatic differences are observed in the behavior of amphetamine ex-
posed monkeys compared to controls. Figures 1 - 4 illustrate where, in a
test cage, monkeys are located at each 1/2 second interval during the 13-
minute observation period. It can be clearly seen that amphetamine, as
dosage increases, causes a rather severe limitation in which parts of it's
environment an amphetamine exposed monkey will occupy. This effect is clear-
ly evident at the very low exposure of 0.11 mg/kg. In addition to constrain-
ing where the monkey goes in its environment, amphetamine also causes a dose
related decrease in the total number of behavioral acts initiated by exposed
monkeys during the observation period (hypoactivity). Early analysis also
indicates that amphetamine causes the behavior of monkeys to be less struct-
ured than that of control animals. Sequences of ordered pairs, triplicates
and quadruplicates of behavioral acts occur less frequently, overall, in
amphetamine exposed primates. The duration of behavioral acts is also
altered, however, this data is currently being analyzed and the specific
nature of the alterations have yet to be determined.
Tests of the amphetamine exposed primate's ability to respond to stim-
uli over delay intervals varying from 0 to 24 seconds also shows dramatic
changes. Data presented in Table 1 clearly show that monkeys exposed to
0.33 or 1.0 mg/kg d-amphetamine stop responding on a delayed response test.
This task is easily mastered under control conditions.
The data collected thus far demonstrates that the computer-automated
behavioral paradigms are clearly sensitive to amphetamine produced changes
in primate behavior, and that rather dramatic alterations occur at very
low levels of exposure.
It is expected that these computer-automated procedures will be
70
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Figure 4. Location of primates in the observation cage as seen from above.
74
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01
TABLE 1 DELAYED RESPONSE PARADIGM
DEXTRO-AMPHETAMINE - NUMBER OF CORRECT RESPONSES
Replication 1
Replication 2
Monkey
QJ
IO
0)
m
E
Ol
3
4
5
11
12
30
18
25
26
X"
Control
Low
Middle
High
Control Low
38
40
41
33
42
38
40
9
23
33
= (sal
= 0.11
11
39
38
0
38
31
40
30
36
.8 29.2
ine i.m
mg/kg d-amphetamine
= 0.33 mg/kg d-amphetamine
= 1.00
mg/kg d-amphetamine
Middle
18
0
0
0
23
21
0
0
25
9.7
i .m.
i .m.
i .m.
High
0
0
0
0
0
0
0
0
0
0
Control
36
36
34
25
41
38
37
29
36
35.0
Low
42
41
38
31
39
35
21
33
35
35.0
Middle
42
0
0
0
0
33
10
0
13
10.8
High
0
0
0
0
0
0
0
0
0
0
-------
sensitive detectors of behavioral changes which might be produced by neuro-
toxicants mobilized into the environment through energy related sources. To
test this hypothesis, tests on primates exposed to thallium and methylmercury
(two trace elements found in coal) will begin in the near future. It is
anticipated that relatively low exposure to these neurotoxic agents will
result in significant alterations in the behavior of the exposed primates.
A final point, the functional capability of the central nervous system
is an important index of toxic effects, and functional alterations measured
as behavioral changes can and do often occur in the absence of a measureable
clinical effect. The procedures developed and tested under these tasks
should provide needed information concerning the effects of a number of en-
vironmental neurotoxicants on the functional capability of the central ner-
vous system.
Bibliography
Since the work for these tasks is currently being conducted, manu-
scripts are presently being prepared for publication.
Related Research
Lead-Induced Behavioral Changes in the Neonatal Primate - Contract
68-03-2524. Negotiated Contract Branch, Environmental Protection Agency,
Cincinnati, Ohio. Four year project, funded for $433,661.00.
The scope of this research is to obtain a controlled, systematic pic-
ture of the behavioral and physiopathologic effects of prenatal and early
postnatal lead exposure in the non-human primate. The specific objectives
of this task are to: (1) assess the effects of in utero and early postnatal
lead exposure on the central nervous system of the non-human primate as
measured through tests of sensorimotor coordination, spontaneous activity
and operant learning behavior, (2) evaluate the feasibility of catecholamine
turnover, and the potential paradoxical reactions of amphetamine and pheno-
barbitol in lead exposed primates, and (3) to determine the suitability of
the cynomolgus monkey as a primate model for studying the probable neurologic
and physiologic effects of environmental lead exposure to humans.
H. TASK TITLE:
Detection of Genotoxic Effects of Environmental Chemicals
in Cultured Liver Cells
HERL/RTP TASK NO: 8197
CONTRACTOR: American Health Foundation
CONTRACT NO: 68-02-2483
Summary
Several iji vitro liver culture systems have been developed and are
being validated and implemented to bioassay environmental chemicals for
76
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genotoxic effects. This contractor has developed the following two sys-
tems.
A. Induction of autoradiographic unscheduled DNA synthesis (DNA
repair) in primary nonreplieating liver cell cultures. This
system has been validated with 27 chemicals from 5 chemical
classes requiring metabolic activation.
B. Mammalian liver cell mutation assay using continuous epithelial
cultures derived from rat liver.
Scope and Objectives
The main objective of this proposal is to develop and validate i_n_
vitro liver cell culture systems for assessing the genotoxic effects of
chemicals and subsequently to apply those systems to identification of en-
vironmental effluents possessing such hazardous properties.
Specific objectives of this research are:
A. To ascertain and maximize the sensitivity of primary nonprolifer-
ating rat liver cell cultures as a bioassay system responding to
genotoxic agents as measured by DNA repair.
B. To ascertain the potential of long-term proliferating rat liver
cell cultures to respond to genotoxic agents as measured by DNA
breakage, chromosomal damage, mutagenesis, and neoplastic trans-
formation.
C. In validated systems, to test environmental agents.
Background and Approach
The consequences of the interaction of chemicals with genetic material,
i.e., DNA, include toxic, lethal and heritabel effects which have been re-
ferred to by the general expression "genotoxic". Genotoxic effects may,
thus, result in chronic disease through damage to somatic cells, in mutations
and birth defects through heritable genetic effects on germinal cells, and
in cancer, perhaps as a result of interaction with genetic material in som-
atic cells. Therefore, the genotoxic effects of environmental chemicals
require careful surveillance.
The agents which are expected to pose the greatest hazard to humans
are those which are stable, i.e. non-reactive in the environment, but are
metabolically activated by an exposed organism. Thus, systems for screening
for genotoxic agents must possess broad capability for metabolizing chemicals,
Recent review of the metabolism of chemical carcinogens has revealed that
liver possesses all the enzyme systems involved in metabolism of carcinogens
known to require enzymic activation. Also, liver has been recognized as the
best general enzyme source for activating chemicals to their mutagenic meta-
bolites. These facts indicate that liver cells are the cells of choice for
obtaining in vitro systems with broad metabolic capabilities.
77
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In a metabolically active cell culture system, the genotoxic effects
of chemicals can be detected by, at least, five end points as follows: the
induction of DNA repair in primary cultures as evidence of DNA damage; the
production of DNA or chromosomal damage in long-term cultures; the induction
of mutations in long-term cultures; and the induction of transformation in
long-term cultures. Thus, the present proposal concerns the development of
rat liver cell culture systems for quantitative assessment of genotoxic ef-
fects of chemicals.
The objectives of this contract will be pursued by studies involving
the following two rat liver cell culture systems: (A) short-term primary
nonreplieating cultures and (B) long-term continuous epithelial cultures.
The ability of carcinogens to elicit DNA repair will be measured by the
induction of autoradiographic unscheduled DNA synthesis in primary cultures.
This work will focus on validation of the system and means of enhancing
sensitivity such as modification of treatment conditions, preinduction of
drug-metabolizing enzymes, and utilization of additional species. The
ability of the cells to metabolically activate procarcinogens will also be
evaluated by study of parameters of drug metabolizing enzyme systems such as
cytochrome P450 levels and benzo(a)pyrene metabolism. In addition, scintil-
lation counting of DNA repair incorporation will be examined as a possible
simplification of the assay. The mutagenicity of carcinogens will be
studied in the continuous cultures using induction of 8-azaguanine and 6-
thioguanine resistance. Efforts will be made to develop additional markers
such as thymidine kinase deficiency. Upon satisfactory development of the
assays, validation will be performed and testing of environmental samples
undertaken.
Research Accomplished
The following two assays have been implemented:
A. Unscheduled DNA repair in primary liver cells (hepatocytes).
B. Liver cell mutagenesis assay in continuous rat liver cell lines.
The DNA repair assay in primary cultures of rat liver cells has been
implemented and validated for five classes of chemicals by testing a total
of 27 chemicals from the following classes: aromatic amines, aminoazo dyes,
mycotoxins, polycyclic aromatic hydrocarbons and nitrosamines. In each chem-
ical class, chemicals were selected based on the available whole animal car-
cinogenicity data so that positive, weakly positive and negative compounds,
where possible, were tested in each chemical class. Most of the chemicals
tested required metabolic activation to produce the reactive (mutagenic or
carcinogenic) species. The metabolic activation was accomplished without an
added activation system, indicating that these primary liver cells are cap-
able of activating a variety of pro-carcinogens or pro-mutagens of different
chemical class. The 15 positive carcinogens were all positive in the DNA
repair assay. Of the three weakly positive carcinogens tested, two were
positive in the DNA repair and one, benzanthracene, was negative. All nine
structurally related noncarcinogens were inactive, except 4-acetylamino-
fluorene, which is mutagenic in Ames Salmonella typhimurium metagenesis assay.
78
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In the mutagenesis assay the mutants have been characterized and the optimal
expression time determined.
Bibliography
long, C. and G. M. Williams. Submitted for Publication. Induction of purine
analog-resistant mutants in adult rat liver epithelial lines by metabolic
activation-dependent and-independent carcinogens. Mutation Research.
Williams, G. M., C. long and 0. J. Berman. In Press. Characterization of
analog resistance and purine metabolism of adult rat liver epithelial
cell 8-azaguanine-resistant mutants. Mutation Research.
Williams, G. M. In Press. Further Improvements in the Hapatocyle Primary
Culture DNA repair Test for Carcinogens: Detection of carcinogenic bi-
phenyl derivatives. Cancer Letters.
Related Research
We are evaluating a variety of i_n vitro test systems to screen enviro-
nmental chemicals for potential carcinogenic and mutagenic activity. Most
of these test systems require the addition of an exogenous metabolic acti-
vation system. The liver cell systems offer the potential advantage of an
internal activation system.
I. TASK TITLE:
Biological Assessment of Exposure to Sulfur Dioxide and Acid
Sulfate
HERL/RPT TASK NO: 8187
CONTRACTOR: Duke University
CONTRACT NO: R0805622-01
Summary
Because the aim of this project is to explore the possibility of devis-
ing a biological test of human S02 exposure, assay procedures for detection
of S-sulfonates are being developed. These procedures will then be applied
to experimental animals for quantitating S02 exposure.
Scope and Objectives
Investigations of the killer smogs of London, Donora and the Meuse Valley
together with the mortality studies of Schimmel and colleagues in the 1960's
and the C.H.E.S.S. studies of the late 1960's and early 1970's have all
implicated atmospheric S02 as a danger to public health. Controls of SO?
emissions have been put in force as a result of this data. These controls
have essentially banned the use of cheaper fossil fuels in metropolitan areas.
A severe problem with all these morbidity and mortality correlation studies
79
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is the tenuous connection between the ambient air concentrations of S02 as
measured at fixed air quality monitoring stations and the SC>2 levels to which
the affected individuals are actually exposed. Not only do ambient levels
vary considerably by location within the same city (Goldstein), but those who
seem most affected, individuals with pre-existing cardiorespiratory impair-
ments, are predominantly located indoors.
The use of personal air samplers in the large studies needed to examine
the low levels now being discussed in relation to air pollutants have obvious,
severe drawbacks. Earlier work (Gunnison; Irreverre et^al_.) has shown that
absorbed and endogenous S02 results in the formation of free and protein-
bound sulfonated (-S-SOs) cysteine both in the blood and tissues. If a
sufficiently sensitive assay for these sulfonates could be developed, it may
be possible to correlated cumulative exposure to S02 to the S-sulfonate levels
in single blood samples. It is to that end that we are working.
Such an assay could also be used in monitoring occupational exposures.
Workers in pulp mills, metal ore smelters, acid plants and lime plants are
exposed to higher and even more variable levels of sulfites than general ur-
ban populations. A simple blood test for cumulative exposure might be useful
in these settings for both research and routine monitoring.
Background and Approach
It has previously been shown that sulfite injected in vivo (Gunnison,
1971) and S02 inhaled by humans (Gunnison, 1974) results in a proportionate
rise in plasma S-sulfonates. An assay for these sulfonates has been develop-
ed (Gunnison, 1973), but in human exposure experiments it was found that the
precision of the method in the 1-10 ppm range of plasma S-sulfonates was no
better than - 25%. The human data (Gunnison, 1974) showed that approximately
a change of 1.1 nanomoles per ml (roughly 0.1 ppm) of plasma S-sulfonate
resulted from each 1.0 ppm increment in S02 exposure. Obviously this assay
is not adequate for environmental monitoring where the present air quality
standard requires levels to be held to the ppb range.
In order that our assay be as cheap, reliable and simple to perform as
possible, we have chosen to release the sulfite from all the plasma S-sul-
fonate at one time so that one assay for sulfite would be all that is neces-
sary. We accomplish this by adding cyanide in large excess and at high pH.
The cyanide displaces the sulfite from all the sulfonated -SH groups all at
one time (Gunnison, 1973; Nor and Tabatabai).
Sulfite Assays
le began the project by conducting an extensive search of the environ-
, occupational health, analytical chemistry and toxicology literature
il'fii-Q accat/c nf c\ iff i fi ont cone T +• i \/n tw Couaval nv»rimi o-i rtn m^+Un^J..
We
mental
for sulfite assays of sufficient sensitivity. Several promising methods
were found and are listed below with brief descriptions.
1. West and Gaeke Method as Modified by Gunnison
This is a colorimetric technique using acid bleached pararoseniline'
Its lack of sensitivity and precision at levels of interest is
80
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mentioned above.
2. Ellman Reagent (Johnston, Murray and Cain)
This assay was developed to follow the production of sulfite in
bacterial colonies. It, like the pararosaniline method, is inter-
fered with by thiols and probably cyanide also, but the authors
describe techniques by which they claim the interference can be
eliminated. Sensitivity is claimed to be in the 0.1 micromole per
ml level.
3. Polarography
Aulenbach and Balmat have used a simple D.C. polarographic technique
for the detection of sulfite in sewage sludge down to the 1.1 ppm
level. Brinkman Instruments has technical papers available describ-
ing a simple and apparently commonly used method in the paper and
pulp industry for determinations below 1.0 ppm.
4. Radioactive Labeled Adducts
S. Harvey Mudd developed a very sensitive method for urine and
tissue extracts using ^C-N-ethylmal^imide in conjunction with his
studies of a child with a congenital deficiency of sulfite oxidase.
Nakamura and Tamuta have used both N-ethylmaleimide (NEM) and p-
aminobenzoic acid (PABA) to adduct with 35$03= to assay sulfite
released by a bacterium. Labeled PABA is not as readily available
from commercial suppliers is NEM, however.
5. Sulfite Oxidase
This is an enzyme that our laboratory has much experience with. It
catalyzes the oxidation of sulfite to sulfate and can use oxidized
cytochrome £ as an electron acceptor. By following the production
of reduced cytochrome c_ spectrophotometrically the activity of the
enzyme or the concentration of sulfite can be quantitated.
Research Accomplished
Most previous workers have used the Segel and Johnson or the Clark meth-
od to produce S-sulfocysteine. Both of these methods produce a sulfonate
heavily contaminated with cupric ions. While the cupric ion is used to
catalyze the formation of the sulfonate it also catalyzes its degradation
(Sorbo). Since plasma has little unbound copper present, we need a product
free of catalyst to be sure that our cyanolysis of the synthetic sulfonate
is applicable to the conditions found in plasma. Inglis and Liu developed
a method of synthesis without using any cupric catalyst. We have success-
fully repeated their procedure and have produced basically Cu^-free S-
sulfocysteine.
It has been our ultimate goal to develop several assays from the above
possibilities and then compare them "head-to-head" on identical samples to
81
-------
determine which offers the most in sensitivity and precision for the least
cost in time and money. We have been able to perform the pararosaniline
assay (Gunnison, 1973) with sensitivity comparable to that reported.
An assay for sulfite in plasma using sulfite oxidase has been developed.
It is sensitive down to a level of 1.0 nanomole per ml. A severe problem
arose when attempting to use the enzyme to assay for S-sulfonate however.
Cyanide reacts readily with cytochrome £ producing a prohibitve amount of
interference even in a dual-beam spectrophotometer. At the moment the enzyme
offers a sensitive method for assaying sulfite in plasma, but as yet is not
applicable for measuring levels of sulfonate.
Much to our surprise the differential pulse polarogram of out-dated
human, blood bank plasma turned out to be flat in the region of the anodic
wave of sulfite. After acidification, added sulfite produces a measurable
wave even down to nanomoles/ml levels. The technique is so sensitive, in
fact, that every unit of out-dated blood we have looked at shows a sulfite
wave after incubation with alkaline cyanide. Presently our. efforts are
directed at producing sulfonate-free plasma so that we may run a standard
curve in human plasma. A standard curve run in normal saline showed sensiti-
vity and reproducibility down to micromolar sulfite concentrations. While
the need for absolute deoxygenation makes the polarographic technique slow,
there is a machine available from Princeton Applied Research which could
automatically perform the assay. Thus this technique could still be quite
useful for large scale screening programs.
Bibliography
None.
Related Research
In epidemiological studies on S02 toxicity it is essential to recognize
the fact that sulfite oxidase plays a role in the detoxification of SO? by
oxidizing it to sulfate. Since the activity of the enzyme is totally depend-
ent on the presence of molybdenum, tissue levels of the enzyme would be
governed by the molybdenum nutritional status of human populations. Popu-
lation studies on sulfite oxidase levels would, therefore, be of great use in
this connection. We have found serum and various blood constituents do not
contain sulfite oxidase activity in detectable levels, making this readily
available source useless for such studies. We have, however, found that
human fat tissue, also used as a source by epidemiologists, does contain sul-
fite oxidase. We are also testing the possibility that sulfite oxidase act-
ivity could be induced in activated human lymphocytes.
J. TASK TITLE:
]_n Vivo Methods for Assessing Neurotoxicity
HERL/RTP TASK NO: 8185
82
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GRANTEE: U. of Cincinnati
GRANT NO: R80569310
Background and Approach
Because of increasing concern over the growing number of potentially
neurotoxic agents which are entering our environment, there has been an in-
creased emphasis on investigations pertaining to the neurochenical, neurophy-
siological and behavioral effects which arise as a result of exposure to such
neurotoxins. In many cases this research has been characterized by a high
degree of empirical data collection with little or no attempt to formulate
unifying principles which allow us to extrapolate beyond the original data.
One of the primary reasons for this is the failure to employ research strat-
egies which employ hypothesis testing and the development and modification of
conceptual models of the systems under consideration.
An additional problem has been our inability to extrapolate between vari-
ous levels of analysis. In order to facilitate the translation of observa-
tions at one level of analysis, e.d. behavior, to another level of analysis,
e.g. biochemical mechanisms responsible for the behavioral changes, one needs
a test system which incorporates the following characteristics:
1. In order to minimize the cost, both in money and teime, necessary to
carry out an investigation, the procedures should be simple. This
rules out behaviors which require weeks of elaborate training, or
relatively sophisticated surgical manipulations such as precise
lesioning or the chronic implantation of cannula or electrodes.
2. The behavior should be a discrete "unit of behavior" mediated at the
central level (since this is the target organ of concern) but ana-
logous to the discrete sensory-motor reflect arc seen at the spinal
level; i.e., stereotyped and easily and reproducibly elicited.
3. A "unit of behavior" for which the neuronal circuitry is fairly
well known. This of necessity tends to restrict the selection
of behaviors to those tied fairly closely to the motor end of
the sensory-motor chain. At higher levels of sensory-motor
integration the complexity is such that our understanding of
neuronal circuitry is extremely sketchy.
4. A method must be available for the delivery of the toxic agent
to the intended test system without disrupting the function of
other systems which might interact indirectly with the system
under study.
5. In order to facilitate hypothesis testing, it is necessary that
the behavior to subject to environmental and pharmacological
manipulations which would lead to predictions of the nature of
the biochemical lesion prior to biochemical determinations.
83
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Unless the system can be manipulated in such a manner, we will
be left with nothing but the concurrent observation of one
behavioral change and one biochemical change and no means of
demonstrating a cause-effect relationship. While the collection
of empirical observations at all levels of analysis is necessary
to provide an adequate data bank, we must move beyond this approach
and begin hypothesis testing.
There are several possible behaviors mediated by the extrapyramidal
system, e.g. tremor or rotational behaviors, which meet these criteria and
might be developed into useful test systems for the study of neurotoxins.
Of these two, rotational behavior appears to be the most promising. This
behavior provides a way to study synaptic function as it is reflected in a
simple, quantified behavior that is empirically linked to dopamine trans-
mission.
Studies to be proposed here will be restricted to an investigation of the
neurotoxic effects of inorganic metals including lead, cadmium, mercury, ma-
nganese, copper and zinc. The metal to be most extensively examined will be
lead. The reasons for this selection are as follows: (1) Heavy reliance
on fossil fuels will result in the introduction of numerous metals into our
immediate environment. These include: lead, manganese, arsenic, fluoride,
nickle, cadmium, zinc, and copper. (2) Past experience tells us that lead
ranks very high on any list of those agents which constitute a significant
health hazard to a significant segment of our population. (3) The behavior-
al and biochemical effects of low level chronic exposure to lead have been
investigated extensively by this applicant for the last two and one-half
years, and (4) data from several laboratories suggest that chronic exposure
to inorganic lead produces alterations in both the catecholamine and acetyl-
choline systems. Since these systems are also intimately involved in the
mediation of rotational behavior, this test system should be sensitive to
the neurotoxic effects of lead. Cadmium and mercury will also be examined
in later studies in order to determine if a common mechanism of action can
be established to account for the neurotoxicity of this group of heavy met-
als. The essential trace metals, manganese, copper and zinc may be evalu-
ated in interaction studies with the heavy metals to determine if it is
possible to alter the neurotoxic actions of the heavy metals.
Scope and Objectives
The primary objective of this proposed research effort is to develop a
methodology for the in vivo testing of known and/or potential neurotoxic
agents. This approach will rely heavily upon a procedure, developed by
Anden et^ al_. in which the functioning of the extrapyramidal system is re-
flected in an animal's asymmetric posture and locomotion. This "rotational
behavior" will be used (1) to evaluate the effects of acute intracerebral
administration of neurotoxins and (2) to unmask covert changes in neural
function which may arise as a result of long-term low level systemic expos-
ure to neurotoxins. The aim is to develop an approach which lies between
strict behavioral analyses of neurotoxicity which do not lend themselves to
studies of mechanism of action, and the more classical iji vitro biochemical
and neurophysiological assays which do not readily permit extrapolation to
84
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the behavioral level. Initial studies will focus on the usefulness of this
methodology in the assessment of neurotoxicity arising from exposure to
heavy metals, particularly inorganic lead, cadmium and mercury.
Objectives —
The specific objectives are designed to provide information relevant to
the following general hypothesis: rotational behavior provides a sensitive
test system for the evaluation of the extent and mode of action of neuro-
toxins. Furthermore, the test system is well suited to the formulation and
testing of hypotheses pertaining to the neurochemical site of action of neuro-
toxins. To explore this hypothesis, the following specific aims are pro-
posed:
1. Develop a homogenous test population of rotating C57B1/6J mice by
the technique of intrastriatal injection of 6-OHDA.
2. Define the pharmacological effects and biochemical extent of such
lesions.
3. Evaluate the sensitivity of this behavioral test system to the
influence of intrastriatallyinjected lead or chronically ingested
lead.
4. Evaluate the usefulness of this test system in predicting the
presence of biochemical changes following exposure to lead.
5. Compare the sensitivity of this test system with more commonly used
behavior test systems such as locomotor activity or learning stud-
ies.
6. Examine cadmium and mercury toxicity with the rotational test sys-
tem. Compare findings with those obtained with lead in order to
detect any common mode of action.
Expected Benefits
Over the years there has been a steady increase in the number of po-
tentially neurotoxic agents being introduced into the environment, e.g.
heavy metals, pesticides and solvents. Before appropriate and reasonable
controls can be instituted, it is necessary to identify which agents repre-
sent a threat. This is no small task when one considers the number of
agents to be tested, the chemical diversity of the agents to be tested and
the complexity of the target system. There is need for a test system for
screening purposes comparable to those being developed in the field of car-
cinogenesis and mutagenesis. The usefulness of such a screening test could
be greatly enhanced if the test system also permitted the study of mechanism
of action.
The rotational model described in this proposal could fulfill the need
for a relatively rapid in vivo screening test. In addition, if developed to
its full potential, the test system should permit hypothesis testing, which
85
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is necessary for identifying the site and mechanism underlying the observed
behavioral toxicity. It may eventually permit the development of unifying
concepts based on common mechanisms of action. For example, the following
questions can be asked: Do all heavy metals (or solvents or pesticides) pro-
duce the same pattern of effects on rotational behavior? Do all demyelinating
agents produce the same pattern? Do anions produce uniquely different effects
than cations? These questions have been asked before, but not IP an in vivo
behavioral test system which permits this degree of experimental control.
In addition, the test system may permit the evaluation of potential
therapeutic approaches, e.g. displacement of heavy metals by essential trace
metals or the development and testing of new chelating agents with special
reference to the CNS.
Obviously not all neurotoxins have the striatum as their principal site
of action. However, this region is neurochemically representative of the
neural elements and supportive tissue which are usually regarded as neuro-
toxic target tissue. Evaluation of the neurotoxicity observed in this region
will suggest what to look for in other brain regions when the agent is admin-
istered systemically.
The test system is being used as a model system for testing drugs used
to treat Huntington's disease and Parkinson's disease. Hopefully it will be
at least as useful in identifying and studying environmental agents which
have neurotoxic potential.
86
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SECTION 3
DETERMINE THE METABOLISM AND FATE OF HAZARDOUS AGENTS
ASSOCIATED WITH ENERGY TECHNOLOGIES
A. TASK TITLE:
The Effect of Whole Animal Exposure to Acid Mists and Parti -
culates on the Pulmonary Metabolism of Benzo(a)pyrene in the
Isolated Perfused Lung Model
HERL/RTP TASK NO: 8147
CONTRACTOR: University of Cincinnati
CONTRACT NO: 68-02-1678
Summary
The isolated lung perfusion (IPL) technique was used to study in an "i_n_
vivo" situation, the effects of S02, crude air particulate (CAP), and
benzo(a)pyrene (B[a]P) treatment on benzo(a)pyrene metabolism. The influ-
ences of these agents on the metabolism of benzo(a)pyrene, an environmental
carcinogen, may explain differences in the carcinogenicity of this agent.
S)«, CAP and B[alP pretreatment increased B [a]P metabolism in the IPL and
increased the formation of specific activated metabolites indicating that
these agents may have cocarcinogenic activities.
Scope and Objectives
The long term goal of this research is to assess the effects of environ-
mental contaminants on the pulmonary metabolism and distribution of the
carcinogen benzo(a)pyrene. This research attempts to clarify whether a
change in metabolic rate, metabolic pathway or distribution in the tissues
could account for differences in the carcinogenic response.
An isolated perfused lung preparation, developed previously by the invest-
igators of this contract, is being used for this study. Various combinations
of crude air particulates, microsomal enzyme inducers, and acid mists are
used as a pretreatment regimen before the intratracheal administration of
C-benzo(a)pyrene (alone or in combination with crude air particulate).
Blood taken at various times throughout the perfusion and tissues are
extracted with organic solvents and concentrated. The metabolites are
chromatographed and quantitated using liquid scintillation counting.
87
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The metabolites of B[a]P, their rates of formation, and their distribution
in various tissues are determined.
Background and Approach
Inhalation has been the main mode of exposure of humans to agents known
to be casually associated with an increased incidence of respiratory cancer.
Epidemiological and experimental evidence indicates that the interplay
of multiple environmental factors is responsible for the induction of lung
cancer. Man is exposed to a complex mixture of potentially hazardous mate-
rials including specific carcinogens and a variety of agents which may modify
the manner in which the lung disposes of inhaled materials. It is well
established that the lungs are capable of binding and metabolizing such agents.
One such carcinogen is benzo(a)pyrene, a ubiquitous environmental pollutant
formed during the destructive distillation of coal and in other processes
involving incomplete combustion of organic materials. BtalP occurs as both
a common contaminant of the urban environment and as a constituent of tobacco
smoke. Its metabolites exhibit varying degrees of mutagenicity, carcino-
genicity and toxicity.
A major requirement for understanding the mechanism of B[a]P carcino-
genesis is a detailed knowledge of the rate and pattern of formation of
metabolites, and the factors controlling their formation. Such factors
include particulate matter which carries a multitude of chemicals, including
B[a]P, which may be deposited in various regions of the respiratory tract.
The ambient air of both occupational and urban settings contain many such
small particles. It has been established experimentally that B[alP in
combination with ferric oxide produces tumors of bronchogenic origin with
an incidence of up to 100%. Carbon particles with B[a]P also produces
a high incidence of lung tumors. The particulate effect has been suggested
as a means of providing longer residence times at the target tissue, but
the biochemical effect has not been fully investigated.
There is, however, no way to study the pulmonary metabolism of B[a]P
in vivo because of the metabolic influence of other organs. In vitro
tissue preparations, such as slices and homogenates, are not satisfactory
for studies involving concurrent administration of multiple agents in
different physical forms, distribution determinations or binding of compounds
throughout the pulmonary system. Therefore, the isolated perfused lung
(IPL) appears to be the best ijn vivo preparation for investigating
pulmonary metabolism of foreign compounds especially compounds adsorbed onto
particulate. An important aspect of current work is the assessment of the
rate of formation and types of metabolites formed when B[a]P is administered
with ferric oxide or crude air particulate (CAP) on the IPL.
Research Accomplished
SOo Inhalation - So far, rather startling and surprising results have
been ootained using low concentrations of S02 (1-2 ppm) in two animals.
-------
When compared to control (no pretreatment), the inhalation of 1-2 ppm
S02 in vitro results in (1) a large increase in the total rate of formation
of the metabolites of BtalP and (2) a change in the distribution of the
metabolites. There also appears to be a lot more nonextractable material in
the S02 experiments than the control. This is all the more interesting
because there is no pretreatment of the S0? group.
This very large rate of formation of metabolites in the S02 experiments
is similar to BtalP pretreatment. However, the distribution is quite
different. There is more 7,8-dihydrodiol and less 9,10-dihydrodiol, diones
and monohydroxylated metabolites in the S0? experiments than the BtalP
pretreatment experiments. This indicates that S0?, in conjunction with BtalP,
affects the metabolic pathway in ways which are different from the BtalP
pretreatment or control (no pretreatment).
CAP Treatment—
(1) Crude air particulate (CAP), when administered with 14C-B[a]P to
the IPL, decreased the total metabolic rate in comparison to the appropriate
control.
(2) CAP administered intratracheally (IT) increases the total metabolic
rate of Bfa]P in the IPL in comparison to the appropriate control.
(3) CAP, when administered IT either as a pretreatment regimen or
in vitro to the IPL, causes an increase of the 7,8 and 9,10-dihydrodiols
and a slight decrease of the monohydroxylated and dione metabolites when
compared to the appropriate control.
(4) CAP when administered IT either as a pretreatment regimen or
in vitro to the IPL causes a decrease in nonextractable material except
when Bla]P is given as a pretreatment.
All of these conclusions indicate that the CAP affects the metabolite
pathway of BtalP such that more 7,8 and 9,10-diol are present. CAP also
affects the rates of formation of the metabolites depending on the treatments.
These are at present only preliminary conclusions. Statistical analyses
are being done on the data using various model systems.
BtalP Treatment—
Both BtalP administered intraperitoneally (IP) and IT pretreatment
increase the metabolic rate of BlalP administered IT to the IPL preparation.
A change of the relative percentages of the metabolites, especially the
9,10-dihydrodiol, is evident in both pretreatment groups compared to the
control. The IP BtalP pretreatment increased rate versus IT BtalP can be
accounted for by the corn oil administration. Both IP and It BtalP
pretreatment and 3-methylcholznthrene stimulate 9,10-dihydrodiol, whereas
the corn oil increases the 7,8-dihydrodiol and the nonextractable metabolites.
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Bibliography
Binaham, E., D. Warshawsky, and R. W. Niemeier. 1977. The metabolism of
B[a]P in the Isolated Perfused Rabbit Lung Following n-Dodecane Inhala-
tion Exposure. Presented at Symposium on Mechanisms of Tumor Promotion
and Cocarcinogenesis. Gatlinburg, Tennessee, March 28-31.
Niemeier, R. W. 1977. Isolated Perfused Rabbit Lung: A Critical Appraisal.
Environmental Health Perspectives. 16:67-71
Niemeier, R., D. Warshawsky, and E. Bingham. 1977. Influence of Pretreatment
on B[a]P Metabolism in the Isolated Perfused Lung. Presented at the 16th
Annual Society of Toxicology Meeting. Toronto, Canada, March 29.
Warshawsky, D., R. W. Niemeier, and E. Bingham. 1977. Influence of Particu-
late and S02 on a B[a]P Metabolism. Presented at EPA Catalyst Research
Program's Sulfuric Acid Research Review Conference. Hendersonville,
North Carolina, January 31-February 3.
Warshawsky, D., R. W. Niemeier, and E. Bingham. 1977. Influence of Particu-
lates on Metabolism of Benzo(a)pyrene in the Isolated Perfused Lung.
Presented at Second International Symposium on Polynuclear Aromatic
Hydrocarbons. Batelle Labs, Columbus, Ohio, September 28-30.
Warshawsky, D., R. W. Niemeier, and E. Bingham. 1978 (in press). Influence
of Particulates on Metabolism of Benzo(a)pyrene in the Isolated Perfused
Lung. Polynuclear Aromatic Hydrocarbons. Freudenthal and Jones, Ed.
Raven Press.
Related Research
None.
B. TASK TITLE:
Evaluate Influence of Inhalation of Acid Aerosols, H2S04, S03,
HN03 and Particulates on Production of Chronic Lung Disease in
Rats, Guinea Pigs and Primates.
HERL/RPT TASK NO.: 8148
CONTRACTOR: University of California at Davis
CONTRACT NO.: 68-01-1721
Scope and Objective
The objective of this project is to evaluate the nature and extent of
functional and morphological dose-related responses of the respiratory sys-
tem resulting from inhalation of small droplet H2S04 aerosols as a function
of mass concentration, droplet size, duration of exposure, and duration of
post-exposure period. Initially, rats and subsequently guinea pigs, and non-
human primates will be studied. Physiology (pulmonary function), morphology
(transmission electron microscopy, scanning electron microscopy, and histo-
chemistry), biochemistry (glycoprotein synthesis), and pulmonary clearance
will be evaulated in control and exposed groups.
90
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Research Accomplished
The exposure facility has been developed and four groups of 100 rats
each are being exposed 24 hrs/day for 180 days to the following atmospheres:
(1) submicron H2S04 mist (0.05 /iM, CMD) at 1 mg/m3; (2) ozone, 0.5 ppm;
(3) HoSOd plus ozone; and (4) control air. These animals will be studied at
90 and 180 days for morphological changes (using SEM, TEM, and histochemistry
for mycopolysaccarides), biochemical changes (rate of glycoprotein synthesis
by in vitro systems) and physiological changes (pulmonary function tests of
lung volumes and mechanics). New methods of increased sensitivity have been
developed to determine mucopolysaccaride histochemically.
Related Research
Thi,s project is supplementary to on-going in-house research into the
health effects of these environmental pollutants. The EPA in-house research
primarily deals with ultrafine ^$04 singly and as produced by a complex
reaction with S02, 03 and a hydrocarbon.
C. TASK TITLE:
Determination of the Effects of Material from Alternate Energy
Sources on Upper Respiratory Tract Clearance Mechanisms.
HERL/RTP TASK NO: 8149
CONTRACTOR: Dr. Dorothy Adalis
CONTRACT NO: 68-02-2295
Summary
The investigator will test the cytotoxicity of a number of environmental
chemical substances employing an in vitro model that is designed to measure
subtle changes in upper respiratory tract clearance mechanisms. The follow-
ing bio-indicators of effect will be measured; ciliary frequency activity,
morphological and histological alterations, and biochemical changes. Dose-
response studies will be conducted using various concentrations and length
of exposure.
Scope and Objective
In general, the objective of this research project is to screen a variety
of chemical substances in order to determine their potential toxic effect on
mucociliary activity. An in vitro model using isolated tracheal rings of the
hamster will be employed to determine significant adverse effects on ciliary
frequency activity, morphological and structural changes and some biochemical
assay of the functional state of these isolated cells.
Samples to be tested may include materials derived from the shale oil,
coal gasification and liquefaction plants and particulate effluents from both
mobile and stationary sources. Additional substances, both soluble and in-
soluble may also require testing. These substances shall include, but not
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be limited to, metallic substances found in the atmosphere.
The contractor shall test the cytotoxicity of a variety of substances
on ciliary activity. Several concentrations will be tested in order to deter-
mine the dose-response relationship.
Background and Approach
It is expected that a number of new substances will be emitted into the
environment when new alternate sources of energy are developed and utilized.
Many of these substances which might be released during the utilization
process have not been toxicologically evaluated for their potential health
effects. Thus, it becomes necessary to screen a number of these substances
for their detrimental effect on the upper respiratory clearance. The material
expected to be tested may include, but not be limited to, substances derived
from shale oil, coal gasification and liquefaction plants and from particu-
late effluents from power stations, stationary engines and motors from various
mobile sources. These substances could be extremely chemically complex,
since they are capable of participating in a wide number of atmospheric
reactions, both with particles and with gaseous pollutants. Once these
substances are inhaled and deposited within the pulmonary racemus, they may
exert a toxic effect on the host via one or more of these mechanisms:
(1) the substances may be intrinsically toxic due to their inherent chemical
or physical properties; (2) the substances may interfere with one or more
of the clearance mechanisms in the respiratory tract; and (3) the substances
may act as carriers of an adsorbed toxic substance.
Once deposited upon the tissue, these substances are then brought into
intimate contact with very specialized host cells, i.e., cilia and alveolar
macrophage, whose function is to rid or clear the body of inhaled substances.
If these defense systems of the host are reduced in activity then an accumu-
lation of both viable and nonviable inhaled substances would occur, which in
turn would jeopardize the health of the host.
Generally, this study employs an in vitro model which will imitate the
above occurrence in an organ culture system and allow the investigator to
measure the toxicity of a large number of substances on the respiratory
cilia of the hamster's trachea. Based on this in vitro data, whole animal
exposure could then be conducted, which would vaTidate the iji vitro findings.
The objective shall be to screen a variety of substances for their toxic
effect on mucociliary activity using an in vitro Hamster Model system to
determine significant adverse effects on normal ciliary activity and normal
ciliated epithelium. A variety of parameters shall be measured including:
.ciliary frequency activity, morphological and structural changes and some
biochemical assay of the functional state of these isolated cells.
Research Accomplished
In general, the research conducted has followed the original scope of
work in that it has evaluated the effects of a number of substances on the
92
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upper respiratory clearance mechanism. During this period of performance, the
investigator has developed and is using the proposed in vitro animal (hamster)
model system for evaluating the cytotoxicity of a variety of substances.
The studies being conducted and planned may be classed as two types:
(1) physiological, and (2) histological and cytological. The physiological
studies include techniques of ciliary beating frequency determinations, oxygen
consumption and ATP content. The histological studies are of two types:
microscopic pathology and ultra-structure pathology.
Preliminary studies were conducted to find dose-level responses of a
number of pure chemicals that might be found in the effluents of alternate
energy sources.
The metals that apparently were non-toxic at 100 ng/m~\ were molybdenum,
barium, lead, chromium (in +3 valance state) sulfite, and magnesium. The
metals showing toxicity at 100 g/ml were mercury, zinc, nickel, manganese
and cadmium. The most toxic materials in order of increasing toxicity were
cobalt, copper and chromium (as chromate). Cobalt required 2 days to show
toxicity at 10 ng/m] ; copper was lethal in 24 hours at 10 Mg/ml and chromium
lethal in 24 hours at a concentration of 1
Samples of material from the electrostatic preci pita tor of a coal fired
power plant, ammonium chloride, and ammonium sulfate were tested for toxicity
and, for these samples, concentrations greater than 1 mg/ml were required to
cause significant toxicity.
ATP content of rings treated with zinc sulfate, zinc ammonium sulfate
or ammonium sulfate were measured. The ATP content of rings treated with
ZnS04 shows a linear dose-response relationship. An even greater decrease was
found for ZnS04-(NH4)2 at the same concentration of zinc ion.
Bibliography
The following publications and presentations resulted from support of
the program.
(1) Toxic effects of cadmium on ciliary activity using a tracheal ring
model system. Environ. Res. 13:111-120, 1977
(2) Cytotoxic effects of Ni on hamster tracheal ring organ culture.
Proceed, of International Congress on Toxicology, Toronto, Canada.
(3) Effect of nickel on upper respiratory tract clearance. Submitted to
Am. Rev. of Respiratory Disease, 1978.
Related Research
To my knowledge, this investigator is not being funded by any other source
to conduct any similar type of research.
93
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D. TASK TITLE:
Comparison of Pulmonary Carcinogenicity of Known Carcinogens
With and Without Added H2S04 Mists, Airborne Respirable Parti-
cles and Gases
HERL/RTP TASK NO. 8158
CONTRACTOR: New York University Medical Center
CONTRACT NO: 68-02-1750
Summary
This is a four year project which began in 1974. The project began with
the development of a primary model for evaluation of pulmonary carcinogen!city
of known carcinogens and their relationship to potential co-factors of air
pollution. The co-factors being studied are: sulfuric acid mist; other sul-
fur oxides; airborne particulates; and gases. Exposure is accomplished by
first administering carcinogens to Syrian Golden hamsters via intubation
followed by inhalation exposure to various co-factors. Initial studies con-
centrated on benzo(a)pyrene as the primary carcinogen and sulfuric acid mist
as the co-factor. Phase I, in 1974, involved range-finding studies of H2S04
mist, which led to the selection of 100 mg/m3 as the level to be used in com-
bined inhalation-intubation studies.
Phase II, initiated in 1975, includes combined intubation-inhalation
studies with benzo(a)pyrene and sulfuric acid mist. Due to the complexity of
this phase, the experiment has been segmented into two parts relating to two
distinct problems in carcinogenesis: (!) initiation-promotion in the case of
single intubation; and (2) co-carcinogenesis in the case of multiple intuba-
tions. Each segment has involved 600 hamsters.
Phase III involves the addition of other co-factors alone or in conjunc-
tion with sulfuric acid mist.
Phase IV involves the inhalation exposure of animals to the by-products
of sulfur dioxide passed through a vanadium pentoxide catalytic converter.
Autopsy and histopathology will be performed on all animals. Phase IV required
the design and construction of a catalytic reactor for the conversion of sul-
fur dioxide to sulfur oxides. The reactor consists of a temperature-regulated
oven capable of maintaining constant compartmental temperatures, a tubular
catalyst bed, and a 13.5 liter reservoir from which samples are periodically
drawn for analysis. The conversion efficiency of this catalyst was evaluated.
The vanadium pentoxide catalyst was found to be most effective in converting
S02 to other sulfuric oxides. Animal studies will be designed and performed
to characterize the toxicity of the product mixtures produced by the cataly-
tic reactor and aerosol combinations.
Histological examinations have been completed on a limited number of
exposed animals. The major lung histology includes early findings of hemor-
rhage, congestion, and edema, both in single and multiple intubation studies.
94
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A high percentage of tumors in the respiratory tract were found in animals
receiving the multiple dose intubations of B[a]P with or without sulfuric acid.
Scope and Objective
The primary objective of this project is to study the response of animals
(male golden hamsters) exposed to combinations of environmental contaminants
and known carcinogens. Two segments of the research are: (1) initiation-
promotion of carcinogenesis as simulated by a single intubation of a carcino-
gen (benzo (a)pyrene [B[a]P]) followed by lifetime exposure to sulfuric acid
mist; and (2) co-carcinogenesis as simulated by multiple intubations of a
carcinogen (benzo(a)pyrene) followed by lifetime exposure to sulfuric acid
mist. The study will also include response after exposure to B[a]P and metal
oxides (vanadium or iron).
Background and Approach
Among the critical problems in air pollution toxicology, the most impor-
tant appear to be related to the interaction of irritant gases and particu-
late materials. The frequent occurrence of high humidity and specific parti-
culate materials are conditions which lead to the formation of sulfur oxide
intermediates and sulfuric acid aerosols. Among the common atmospheric com-
ponents which catalyze these transformations are carbon, vanadium oxides,
soluble metal salts, and nitrogen oxides.
Examination of the literature indicates the extreme difficulty in assess-
ing the effects of ambient levels of sulfur oxides on human health. The avail-
able toxicological data on sulfur oxidation products indicate that sulfuric
acid and sulfates are more potent as irritants than sulfur dioxide. This has
been demonstrated in studies using mortality and lung pathology as criteria,
as well as in studies using alteration in pulmonary functions in animal and
human subjects.
In considering problems relating to the development of more serious
chronic diseases, cancer induction appears to be the most important indicated.
Carcinogenic materials have been identified in the atmosphere of all large
cities of the world. The incomplete combustion of organic materials is the
major source of a variety of carcinogenic polynuclear aromatic hydrocarbons,
of which benzo(a)pyrene is a prime example. The experimental induction of
carcinomas in rats has been demonstrated by combined inhalation of benzo(a)-
pyrene and sulfur dioxide. These facts, along with the knowledge that oxides
of sulfur and their corresponding acids and salts are major common pollutants,
have formed the basis for studies with these compounds in combination with
known carcinogens.
A model has been developed in which hamsters were exposed to a carcino-
gen by intratracheal-intubation and to sulfur dioxide by inhalation. The
finding of cancer in these studies reinforces the direct inhalation studies
reported from rats by other investigators.
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This study will utilize the intubation-inhalation technique for the
exposure of male Syrian Golden hamsters to a carcinogen followed by inhalation
exposures to the various co-factors. These co-factors include sulfuric acid
mist and other sulfur oxides, alone and in combination with aerosols. Because
of the complexity of the developmental work required for these studies, the
program is divided into several phases.
Phase I involves range-finding studies to determine tolerable exposure
levels of sulfuric acid mist. Phase II includes combined intubation-inhala-
tion studies with benzo(a)pyrene and sulfuric acid mist. Phase III involves
the addition of other co-factors alone or in conjunction with sulfuric acid
mist. Phase IV involves the inhalation exposure of animals to the by-products
of sulfur dioxide passed through a vanadium pentoxide catalytic converter.
In the development of the inhalation studies, the primary considerations
were the generation of atmospheres which could be controlled with respect to
mass, concentration, and particle size distribution. In the method of genera-
tion used, concentrated sulfuric acid was spray-atomized against the walls of
a 500 ml glass baffle chamber, using a stainless steel nebulizer developed in
this laboratory. The aerosol was then introduced into the air intake of the
1.3 cubic meter animal exposure chamber. Particle size and concentration
could be controlled by variations in baffle characteristics, spray pressure
and acid concentration.
Chamber concentrations were routinely sampled, using a midget impinger,
at half-hour intervals during exposure. The analytical technique involved
titrating the sample with a standardized sodium hydroxide solution, using
methyl red as the indicator. The analytical precision of this method, as
determined by 9 replicate samples, is better than ± 0.001 mg/ml which corres-
ponds to a precision of ± 0.5 mg/m3 in the chamber atmosphere. The sampler
efficiency, as determined by 10 repetitive tandem samples, is 94.5% with a
standard deviation of 0.3%. Repeated tests showed that interference from
carbon dioxide is negligible.
Alternate analytical techniques were used occasionally as independent
verification of chamber concentration. These techniques are ASTM D:516, non-
referee methods A and B, for the determination of sulfate.
Extensive studies were performed to characterize the particle size dis-
tribution of the sulfuric acid mist. The importance of such a determination
is well known, since this defines the site of deposition of the aerosol and
its physiochemical behavior. Several measurements of the particle size dis-
tribution of the sulfuric acid mist were made using the modified Casella
Cascade impactor and the Andersen Model 10-000 impactor. A significant disa-
greement was observed when the two distributions were compared. It was,
therefore, necessary to verify the calibration of these two instruments,
using the ultramicroscope and the Royco Model 202 particle counter.
The hydroscopic nature of sulfuric acid mist and its corrosive properties
indicated a need for a more stable aerosol for comparative calibration. An
alternate aerosol of 10* glycerine in water was selected, incorporating
flourescein as an analytical tag. The particle size distributions obtained
96
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for this aerosol by the Casella and the Andersen impactors were similar to
those obtained for the sulfuric acid mist. Also, the same variations between
the two methods were observed.
Optical determinations using the ultramicroscope were made on 497 parti-
cles. The parameters of the distribution obtained with the glycerine-water
aerosol were a mean aerodynamic diameter of 1.6 Mm with a geometric standard
deviation of 1.5. The distribution parameters obtained by"the Casella impac-
tor were a mean diameter of 1.8 urn with a geometric standard deviation of 1.8
and those for the Andersen impactor were a mean diameter of 0.9 with a geo-
metric standard deviation of 1.8. It appears that a correlation exists be-
tween the mean diameters obtained by the ultra -microscope and Casella type
impactor.
Particle size distributions were obtained simultaneously with the Royco
particle counter and samples were collected by the Casella and the Andersen
impactors. The results obtained suggested that the count median diameter
obtained by the Royco corresponds very closely with the median obtained by the
Andersen impactor.
The range finding study consisted of a single 6-hour exposure of a group
of 10 Syrian Golden hamsters to concentrations of 300 and 500 mg/m3 of sul-
furic acid mist. Based on the observations of the single exposure studies,
a 30-day inhalation study was conducted at a concentration of 100 mg/m3.
Forty male hamsters were exposed for 6 hours per day, 5 days per week. Twenty
colony controls were selected to parallel this study.
Segment 1 of the Phase II combined intubation-inhalation studies invol-
ved a single intubation of relatively high levels of carcinogen (10 mg or 40
mg Bla]P) followed by a life-time exposure to sulfuric acid mist. The exper-
imental outline for Segment 1 is given in Table 1. The suspension of B[a]P
alone and in combination with other materials were prepared by ball mill
grinding. Particle size determinations of the suspensions were performed by
optical microscopy. The geometric count median diameter was found to be 2.1
with a standard deviation of 2.3.
Segment 2 (Phase II) involved the multiple intubation of 1 and 4 mg B[a]P
on a schedule of 1 intubation per week for 15 consecutive weeks. Following
the first intubation, the animals were exposed for life to 100 mg/m3 sulfuric
acid. Control groups were included. The experimental outline for Segment 2
is given in Table 2.
In order to generate a multi compartment atmosphere, a catalytic reactor
was designed and developed to convert S02 to other sulfur oxides. The reactor
consists of a temperature-regulated oven capable of maintaining constant com-
partmental temperature, a tubular catalyst bed, and a 13.5 liter reservoir
from which samples are periodically drawn for analysis. Sulfur dioxide con-
centrations entering the reactor are regulated by metering known volumes of
compressed air and SO?. In order to determine relative percent conversion of
SO?, bubbler samples for S02 and total SOX were collected from the reservoir.
To optimize the S02 conversion, the effects of the catalyst composition, gas
97
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temperatures, and sulfur dioxide concentrations were examined. After optimi-
zing the'S02 conversion conditions, characterization of the oxide product will
be accomplished using spectrophotometry, gas chromatography, and mass spec-
trometry. Tests will be conducted at increased humidities to determine the
extent and particle size of sulfuric acid mist resulting from S02 conversion.
Subsequent to these analyses, an aerosolized material will be generated into
the reservoir along with the sulfur oxides and the determination of resultant
products will be made. The aerosolized material will consist of metals, metal
oxides, carbon and combinations of these materials as available. Where feasi-
ble, animal studies will be designed and performed to characterize the toxi-
city of suspect product mixtures.
Research Accomplished
Range finding experiments were completed, and on the basis of the results
obtained, a concentration of 100 mg/m3 of sulfuric acid mist was selected for
the combined intubation-inhalation studies. The initial responses of the
animals to the single exposure experiment were nasal and eye irritation,
slight dyspnea, and a body weight loss of 3 to 4 grams for approximately one
week. Subsequent weight gain began to parallel that of the controls, but the
initial loss was not recovered. No deaths occurred in animals exposed to
either concentration. Two animals per exposure group were sacrificed at the
end of the 15th experimental day. One of the sacrificed animals from each
of the exposure groups was found to have partial atelectasis and focal emphy-
sema. The remaining animals from both groups were sacrificed on the 30th
day post-exposure. All showed slight thickening of the alveolar septa, irre-
spective of exposure concentration. However, there was no abnormality ob-
served in the bronchial epithelium. Based on the observations of the single
exposure studies, a concentration of 100 mg/m3 of sulfuric acid mist was
selected for the 30-day inhalation study. In this study, 40 male hamsters
were exposed for 6 hours per day, 5 days per week. Twenty colony controls
were selected to parallel this study. During the initial 1 to 2 weeks of
exposures, the animals exhibited slight respiratory irritation. Thereafter,
the animals appeared to have adapted to the experimental atmosphere. The
weight gain of the experimental animals was depressed following the first week
of exposure. Thereafter, the weight gain of the exposed animals was compara-
ble to that of controls. No deaths occurred during the 30-day sulfuric acid
mist exposures. These animals were kept for further observation. Interim
sacrifice of 5 hamsters was performed on the 57th experimental day. Histo-
pathological examination of the lungs revealed no exposure related abnor-
malities. Major lung histology and mucosal changes appeared to be congestion,
hemorrhage, and edema. No significant differences in findings in the bron-
chial or broncho-alveolar region were observed. Of interest, however, were
the findings of 2 largyngeal hyperplasias and 2 laryngeal squamous metaplasias
in animals exposed to sulfuric acid mist.
The chronic studies (Phase II) involving the combined intubation-inhala-
tion exposures with benzo(a)pyrene (B[a]P) and sulfuric acid mist are nearing
completion. The experimental outlines for Phase II are shown in Tables 1 and
2. The first segment of this phase involved single intubations of B[a]P fol-
lowed by lifetime exposures to sulfuric acid mist. The-cumulative data to
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date indicates that all of the animals had died by 116 weeks in Segment 1.
Weight change in animals receiving intubations alone was markedly lower than
that of the animals receiving sulfuric acid mist exposure. Weight change of
the air controls was similar to sulfuric acid mist exposure groups, while the
weight change of the untreated controls was similar to that of the groups
receiving intubations alone.
Major lung histology for this segment is shown in Table 3 and includes
early findings of hemorrhage and edema. The major mucosal changes are given
in Tables 4 and 5.
Three benign and two malignant tumors were found in the respiratory
tract of animals receiving 40 mg B[a]P alone, compared to one benign tumor
in the group receiving both 40 mg B[a]P and sulfuric acid. In the group
receiving 10 mg B[a]P once with sulfuric acid, two out of 45 animals deve-
loped malignant tumors and one developed a benign tumor in the lung, whereas
the animals receiving 10 mg B[a]P alone developed only one benign tumor in
the lung. The highest tumor incidence was seen in the positive group receiv-
ing 4 mg B[a]P with 4 mg ferric oxide where 11 of 30 animals (37%) developed
tumors in the respiratory tract. The tumor incidences for all groups are
given in Table 6. The detailed morphological types are described in Table 7.
Non-respiratory tract tumors have also been observed and these findings are
given in Table 8.
All animals receiving multiple intubations (Segment 2) with or without
sulfuric acid mist exposures were dead by the 100th week. In the remaining
groups, all animals were dead by the 120th week with the exception of one
animal from the group receiving sulfuric acid alone. This animal was still
alive and being exposed at the 128th week, although loss of weight was evi-
dent from the 120th week onward. The histological findings for this segment
are shown in Table 9. Major mucosal findings are summarized in Tables 10
and 11. A high percentage of tumors in the respiratory tract were found in
animals receiving the multiple dose intubations of B[a]P (4mg B[alP x 15)
with or without sulfuric acid mist. A slight increase in the tumor incidences
was found in animals receiving B[a]P alone. Eighty percent of the animals in
the group receiving B[a]P alone showed tumors in the respiratory tract com-
pared to 72% in the animals receiving both B[a]P and sulfuric acid mist. In
contrast, in the group receiving the lower dose (1 mg B[a]P x 15), a much
higher incidence of respiratory tract tumors was seen when sulfuric acid mist
was given (24%) than when the B[a]P was given alone (12%).
The segmental distribution in the respiratory tract and morphological
classifications of the individual tumors are given in Tables 12 and 13. The
non-respiratory tract tumors found in these groups are listed in Table 14.
The conversion efficiency of a vanadia molybdate catalyst at higher
concentrations of SOz was evaluated. At ambient temperature or at 400°C, the
molybdate catalyst does not appear to increase the conversion substantially.
Upon an average input of 64.2 ppm S02, at ambient temperature, it varies from
29 to 11 4% At 400°C, it varies from 3.0 to 8.8% When moisture was intro-
duced into the system, it was 7.9% at ambient and 10.0% at 400°C. Neither
99
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absorption nor desorption were observed.
In summary, the vanadia molybdate catalyst'is not as efficient as the
vanadium pentoxide catalyst in converting S02 to higher state sulfur oxides.
Elevated temperature, moisture and high concentration of SOg do not appear to
increase the conversion efficiency.
Since the vanadium pentoxide catalyst appears to be most effective in
converting SOg to other sulfur oxides among three catalysts evaluated, this
catalyst was used to oxidize S02 to other sulfur oxides, at 400°C. with mois-
ture. The products of this process were then analyzed by using several meth-
ods, such as thermometric methods, gas chromatography, mass spectroscopy,
spectrophotometry and conventional wet chemistry. Iron oxide particulates
also introduced into the system during this period, since iron oxide particu-
lates are abundant in the atmosphere and are capable of oxidizing S02.
The iron oxide aerosol was generated using a DeVilbiss nebulizer and
introduced into the 13.5 liter reservoir, mixed with the gases which had
been passed over the catalyst. The particulates were then collected on two
successive millipore membrane filters and subsequently analyzed for the sulfur
species on the filter by thermometric methods. The gaseous products were
analyzed by the iodine titration and thermometric methods and the characteri-
zation will be accomplished using gas chromatography, mass spectroscopy and
spectrophotometry.
The iron oxide particulates produced by the DeVilbiss nebulizer were
collected on the microscopic grid by a point to plane electrostatic precipi-
tator developed by this laboratory. The particles were counted and sized
by scanning electron microscopy. The iron oxide appears to be mostly spheres.
It has a mass median diameter of 0.34 MHI, a surface median diameter of 0.2 //mo,
and a count median diameter of 0.02 jini with og of 1.29.
Later in this study, the particulates will be mixed with the gases coming
out from the catalyst, and the efficiency and products will be analyzed.
Bibliography
Drew, R. T., S. Laskin. 1973. Environmental Inhalation Chambers. Methods Of
Animal Experimentation, W. I. Gay, Ed., IV, Env. and Special Senses,
Academic Press, N. Y.
Laskin, S.,M. Kuschner, A. Sellakumar, and G. Katz. 1976. Combined Carcino-
gen-Irritant Animal Inhalation Studies. Air Pollution and the Lung. Eds.
E. B. Aharonson, A. Ben-David, M. A. Klingberg, Halsted Press, John Wiley
& Sons, N. Y.
Laskin, S., M. Kuschner, A. Sallakumar, G. Katz. 1975. Combined Carcinogen-
Irritant Animal Inhalation Studies. OHOLO Biological Conference, Ness
Ziona, Israel. March.
Quarterly and Annual Reports.
100
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Related Research
A number of toxocological studies using various sulfur compounds of
complexes have been conducted which relate to the problem of acute and chron-
ic toxicity.
TABLE 1 EXPERIMENTAL OUTLINE FOR COMBINED INTUBATION-INHALATION STUDIES*
PHASE 2 - SEGMENT 1
Number of
Animals Intubation Inhalation
60 40 mg/0.2 cc BP x 1 +100 mg/m3 H2S04 x life
60 40 mg/0.2 cc BP x 1
60 10 mg/0.2 cc BP x 1 + 100 mg/m3 H2S04 x life
60 10 mg/0.2 cc BP x 1
60 0.1 mg/0.2 cc Gel x 1 +100 mg/m3 H2S04 x life
60 0.1 mg/0.2 cc Gel x 1
60 — 100 mg/m3 H2S04 x life
60 Air Control
60 Colony Control
30 4 mg/0.2 cc BP + Fe203 x 15
30 1 mg/0.2 cc BP + Fe203 x 15
* Repeat Series
101
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TABLE 2 EXPERIMENTAL OUTLINE FOR COMBINED INTUBATION-INHALATION STUDIES
PHASE 2 - SEGMENT 2
Number of
Animals Intubation Inhalation
60 4 mg/0.2 cc BP x 15 + 100 mg/m3 H2S04 x life
60 4 mg/0.2 cc BP x 15
60 1 mg/0.2 cc BP x 15 + 100 mg/m3 H2S04 x life
60 1 mg/0.2 cc BP x 15
60 0.1 mg/0.2 cc Gel x 15 + 100 mg/m3 H2S04 x life
60 0.1 mg/0.2 cc Gel x 15
60 — 100 mg/m3 H2S04 x life
60 Air Control
60 Colony Control —
30 4 mg/0.2 cc BP + Fe203 x 15
30 1 mg/0.2 cc BP + Fe«0, x 15
102
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TABLE 3 COMBINED INTUBATION-INHALATION STUDIES
PHASE 2 - SEGMENT 1 - SINGLE INTUBATION SERIES
MAJOR LUNG HISTOLOGY*
Inhalation9 Intubation
H2S04 40 mg BP x 1
40 mg BP x 1
H2S04 10 mg BP x 1
10 mg BP x 1
H2S04 0.1 mg Gel x 1
0.1 mg Gel x 1
4 mg BP + 4 mg
Cp r\ **
he2u3
1 mg BP + 1 mg
Cp n *
he2U3
H2S04
Air Control
Colony Control
Hemorrhage
Congestion
Edema
50/56
54/60
41/45
58/59
58/59
58/60
25/30
29/29
58/60
58/59
50/57
Pneumonia
23/56
31/60
20/45
26/59
26/59
43/60
16/30
13/29
27/60
38/59
26/57
Infiltration
0/56
5/60
0/45
1/59
1/59
0/60
2/30
0/29
0/60
1/69
4/57
*Denominator shows ovservations to date
*H2S04 100 mg/m3 x life
*15 intubations
103
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TABLE 4 COMBINED INTUBATION-INHALATION STUDIES
PHASE 2 SEGMENT 1 - SINGLE INTUBATION SERIES
MAJOR UPPER RESPIRATORY TRACT MUCOSAL CHANGES**
Type of Exposure
Laryngeal
Trachael
Inhalation Intubation Hyperplasic
H2S04 40 mg BP x 1
40 mg BP x 1
H2S04 10 mg BP x 1
10 mg BP x 1
H2S04 0.1 mg Gel x 1
0.1 mg Gel x 1
4 mg BP + 4 mg
Fe2°3
1 mg BP + I'mg
Fe2°3
H2S04
— Air Control
— Colony Control
9/56
9/58
7/42
7/57
34/57
4/58
4/29
2/28
16/58
12/59
11/57
Squamous
Metaplasia
1/56
0/58
0/42
1/57
2/57
0/58
0/29
0/28
0/58
0/59
0/57
Hyperplasis
13/56
6/58
1/45
3/59
17/59
4/60
4/30
0/29
14/60
7/59
10/57
Squamous
Metaplasia
0/56
0/58
0/45
0/59
0/59
0/60
0/30
0/29
0/60
0/59
0/57
*Denominator shows observations to date
aH2S04 TOO mg/m3 x life
**15 Intubations
104
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TABLE 5 COMBINED INTUBATION-INHALATION STUDIES
PHASE 2 - SEGMENT 1 - SINGLE INTUBATION SERIES
MAJOR LOWER RESPIRATORY TRACT MUCOSAL CHANGES*
Type of
Inhalation3
H2S04
—
H2S04
—
H2S04
—
—
—
H2S04
—
—
Exposure
Intubation
40 mg BP x 1
40 mg BP x 1
10 mg BP x 1
10 mg BP x 1
0.1 mg Gel x 1
0.1 mg Gel x 1
4 mg BP + 4 mg
Fe203**
1 mg BP + 1 mg
Fo 0
—
Air Control
Colony Control
Bronchial
Hyperplasia
41/56
44/60
33/45
54/59
50/59
42/60
22/30
17/29
41/60
47/59
33/57
Squamous
Metaplasia
0/56
0/60
0/45
0/59
0/59
0/60
3/30
1/29
1/60
0/59
0/57
Broncho-Alveolar
Hyperplasia
0/56
7/60
1/45
5/59
2/59
1/60
9/30
5/29
4/60
1/59
2/57
Squamous
Metaplasia
0/56
0/60
0/45
0/59
0/59
0/60
2/30
0/29
0/60
0/59
0/57
*Denominator shows observations to date
aH2S04 100 mg/m3 x life
**15 intubations
105
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TABLE 6 INTUBATION-INHALATION EXPOSURES WITH HAMSTERS
PHASE 2 - SEGMENT 1
Respiratory Tract Tumors
Type of Exposure TBA/Number Observed Larynx
40 mg BP x 1 + H2SO. x Life
40 mg BP x 1
10 mg BP x 1 + H2S04 x Life
10 mg BP x 1
0.1 mg Gel x 1 + H2S04 x Life
0.1 mg Gel x 1
4 mg BP + 4 mg Fe203 x 15
1 mg BP + 1 mg Fe203 x 15
100 mg/m3 H2S04 x Life
Air Control x Life
Untreated Control
1/56 (2%)
5/60 (8%)
2/45 (4%)
1/59 (2%)
0/42 (0%)
0/60 (0%)
11/30 (37%)
2/30 (7%)
0/60 (0%)
0/59 (0%)
0/57 (0%)
0
1
0
0
0
0
0
0
0
0
0
of:
Trachea Lungs
1
2
0
0
0
0
2
0
0
0
0
0
2
3
1
0
0
10
2
0
0
0
Other
2
7
3
10
3
2
10
5
6
1
7
106
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TABLE 7 COMBINED INTUBATION-INHALATION STUDIES
PHASE 2 - SEGMENT 1 - SINGLE INTUBATION SERIES
Type of Exposure
No. Tumor-Bearing Animals
No. Animals Observed
Type of Tumor
Day First
Tumor Observed
o
-o
40 mg BP + H2S04 x Life
40 mg BP
10 mg BP + H2S04 x Life
10 mg/ BP x 1
4 mg BP + 4 mg
x 15
1 mg BP + mg Fe203 x 15
1/56
5/60
2/45
1/59
11/30
2/30
1 Polyp-Trachea
2 Papilloma-Trachea
1 Papilloma-Larynx
1 Adenocarcinoma-Lung
1 Undiff.-Car.-Lung
1 Adenoma-Lung
1 Sq. Cell Car.-Lung
1 Adenocarcinoma-Lung
1 Adenoma-Lung
1 Papilloma-Trachea
1 Polyp-Trachea
3 Sq. Cell Car.-Lung
3 Adenocarcinomas-Lung
3 Mixed Carcinomas-Lung
1 Adenoma-Lung
1 Sq. Cell Car.-Lung
1 Adenocarcinoma-Lung
353
477
577
705
722
286
286
506
538
366
504
338
389
367
481
423
324
-------
TABLE 8 COMBINED INTUBATION-INHALATION STUDIES
PHASE 2 - SEGMENT 1 - SINGLE INTUBATION SERIES
Type of Exposure
Non-Respiratory Tract Tumors
Day First
Type of Tumor Tumor Observed
40 mg BP x 1 + H2S04 x Life
40 mg BP x 1
10 mg BP x 1 + H2S04 x Life
10 mg BP x 1
4 mg BP + 4 mg Fe20_ x 15
1 mg BP + 1 mg
x 15
0.1 mg Gel x 1 + H2SO. x Life
0.1 mg Gel x 1
100 mg/m3 H2S04 x Life
Air Control
Untreated Control
1 Papilloma-Fore-Stomach
1 Malignant Lymphoma
1 Malignant Lymphoma
6 Papillomas-Fore-Stomach
3 Papillomas-Fore-Stomach
1 Malignant Lymphoma
9 Papillomas-Fore Stomach
8 Papillomas-Fore-Stomach
1 Malignant Lymphoma
1 Adrenal-Cortical Adenoma
1 Malignant Lymphoma
2 Papillomas-Fore-Stomach
1 Adrenal-Cortical Adenoma
1 Fibromatosis-Sub-cutaneous
2 Papillomas-Fore-Stomach
1 Adrenal-Cortical Adenoma
2 Papi11oma-Fore-Stomach
1 Malignant Lymphoma
2 Papilloma-Fore-Stomach
1 Adrenal-Cortical Adenoma
1 Fibroma
1 Papilloma-Fore-Stomach
5 Papillomas-Fore-Stomach
1 Adrenal Cortical Adenoma
1 Malignant Lymphoma
419
527
395
480
384
329
382
225
481
389
435
629
514
629
634
598
634
114
503
568
657
668
690
675
702
108
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TABLE 9 COMBINED INTUBATION-INHALATION STUDIES
PHASE 2 SEGMENT 2 - MULTIPLE INTUBATION SERIES
MAJOR LUNG HISTOLOGY*
Inhalation3 Intubation
H2S04 4 mg BP
4 mg BP
H2S04 1 mg BP
1 mg BP
H2S04 0.1 mg Gel
0.1 mg Gel
4 mg BP + 4 mg
Fe2°3
H2S04
— Air Control
— Colony Control
Hemorrhage
Congestion
Edema
51/60
41/50
51/58
56/59
56/59
49/55
26/30
52/60
58/60
51/58
Pneumonitis
34/60
31/60
27/58
40/59
32/59
33/55
17/30
26/60
41/60
24/58
Lymphocytic
Infiltration
0/60
0/60
1/58
3/59
2/59
4/55
2/30
0/60
0/60
6/58
*Denominator shows observations to date
aH2S04 100 mg/m3 x life
b!5 intubations
109
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TABLE 10 COMBINED INTUBATION-INHALATION STUDIES
PHASE 2 - SEGMENT 2 - MULTIPLE INTUBATION SERIES
MAJOR UPPER RESPIRATORY TRACT MUCOSAL CHANGES*
Type of Exposure
Inhalation3 Intubation
H2S04 4 mg BP
4 mg BP
H2S04 1 mg BP
1 mg BP
H2S04 0.1 mg Gel
0.1 mg Gel
4 nig BP + 4
Laryngeal
Hyperplasia
7/59
15/60
7/55
9/55
12/59
5/54
mg 8/29
Squamous
Metaplasia
5/59
0/60
1/55
0/55
1/59
0/54
1/29
Trachea 1
Hyperplasia
13/50
15/60
7/58
7/58
7/59
4/54
5/29
Squamous
Metaplasia
0/60
1/60
0/58
0/58
0/59
0/54
0/29
H2S04
Fe2°3
1 mg BP + 1 mg 7/29
•2U3
Air Control
Colony Control
11/57
12/58
9/58
0/29
0/57
1/58
0/58
5/29
7/60
8/59
7/58
0/29
0/60
0/59
0/58
* Denominator shows observations to date.
a H2S04 100 mg/m3 x life
15 intubations
110
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TABLE 11 COMBINED INTUBATION-INHALATION STUDIES
PHASE 2 SEGMENT 2 - MULTIPLE INTUBATION SERIES
MAJOR LOWER RESPIRATORY TRACT MUCOSAL CHANGES*
Type of
a
Inhalation
H2S04
—
H2S04
—
H2S04
__ _
Exposure
k
Intubation
4 mg BP
4 mg BP
1 mg BP
1 mg BP
0.1 mg Gel
0.1 mg Gel
4 mg BP + 4 mg
Hyperplasia
38/60
37/60
46/58
51/59
38/59
38/55
24/30
Bronchial
Squamous
Metaplasia
6/60
10/60
0/58
0/59
1/59
0/55
1/30
Metaplasia
21/60
28/60
30/58
27/59
2/59
1/55
12/30
Fe203
1 mg BP + 1 mg
Fe203
26/29
0/29
4/29
H2S04
Air Control
Colony Control
44/60
39/60
37/58
0/60
0/60
0/58
1/60
2/60
2/58
Denominator shows observations to date.
H2S04 100 mg/m3 x life
15 intubations
111
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TABLE 12 INTUBATION-INHALATION EXPOSURES WITH HAMSTERS
PHASE 2 - SEGMENT 2
Respiratory Tract Tumors of:
Type of Exposure
4 mg BP x 15 + H2S04 x
4 mg BP x 15
1 mg BP x 15 + H2S04 x
1 mg BP x 15
0.1 mg Gel x 15 + H2S04
0.1 mg Gel x 15
4 mg BP + 4 mg Fe203 x
1 mg BP + 1 mg Fe203 x
100 mg/m3 H2S04 x Life
Air Control
Untreated Control
TBA/Number Observed
Life 43/60 (72%)
48/60 (80%)
Life 14/58 (24%)
7/59 (12%)
x Life 0/59 (0%)
1/55 (2%)
15 8/30 (27%)
15 2/29 (7%)
0/60 (0%)
0/60 (0%)
0/58 (0%)
Larynx
0
0
0
0
0
0
1
0
0
0
0
Trachea
5
7
3
1
0
1
0
1
0
0
0
Lungs
47
58
13
6
0
0
7
1
0
0
0
112
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TABLE 13 COMBINED INTUBATION-INHALATION STUDIES
PHASE 2 - SEGMENT 2 - MULTIPLE INTUBATION SERIES
Respiratory Tracr Tumors
Number of/ Day
Tumor-/ Number of First
Bearing/Animals Tumor
Animals/ Observed Type of Tumor Observed
4 mg BP x 15 + H2$04
43/60 28 Adenocarcinomas-Lung
9 Sq. Cell Car. -Lung
6 Mixed Carcinomas
3 Adenomas-Lung
3 Papillomas-Trachea
2 Polyp-Trachea
1 Frirosarcoma-Lung
270
292
358
394
371
387
389
4 mg BP x 15 48/60 3 Adenomas-Lung 341
29 Adenocarcinomas-Lung 295
14 Sq. Cell Car.-Lung 244
11 Mixed Carcinomas-Lung 325
1 Sq. Cell Car.-Trachea 407
3 Papillomas-Trachea 387
1 Undiff. Car.-Trachea 347
1 Papilloma-Bronchus 474
1 Anaplastic Car.-Trachea 534
1 Polyp-Trachea 600
1 mg BP x 15 + H9SO. x Life 14/58 6 Adenocarcinomas-Lung 310
* ^ 6 Adenomas-Lung 345
1 Adenocarcinoma-Trachea 481
2 Papilloma-Trachea 544
1 Anaplastic Car.-Lung 697
1 mg BP x 15 7/59 4 Adenocarcinoma-Lung 399
1 Mixed Carcinoma-Lung 336
1 Adenoma-Lung 448
1 Polyp-Trachea 602
4 mg BP + 4 mg Fe000 x 15 8/30 3 Adenocarcinoma-Lung 500
23 5 Sq. Cell Car.-Lung 280
1 Papilloma-Larynx 392
1 Mixed Car.-Lung 621
1 mg BP + 1 mg Fe-O- x 15 2/29 1 Adenoma-Lung 318
3 2 3 1 Papilloma-Trachea 653
0.1 mg Gel x 15 1/55 1 Papilloma-Trachea 674
113
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TABLE 14 COMBINED INTUBATION-INHALATION STUDIES
PHASE 2 - SEGMENT 2 - MULTIPLE INTUBATION SERIES
Type of Exposure
Non-Respiratory Tract Tumors
Day First
Type of Tumor Tumor Observed
4 mg BP x 15 + H2S04 x Life
4 mg BP x 15
1 mg BP x 15 + H2S04 x Life
1 mg BP x 15
4 mg BP + 4 mg Fe^O- x 15
1 mg BP + 1 mg Fe203 x 15
0.1 mg Gel x 15 + H2S04 x Life
0.1 mg Gel x 15
100 mg/m3 H2S04 x Life
Air Control
Colony Control
19 Pipillomas-Fore-Stomach
4 Malignant Lymphomas
1 Fibrosarcoma-S.C.
1 Adenocarcinoma-Kidney
1 Adenocarcinoma-Adrenal
1 Hemangioma-Spleen
22 Papillomas-Fore-Stomach
1 Fibrosarcoma
1 Sq. Cell Car.-Fore-Stomach
2 Hemangioma-Spleen
1 Fibroma-S.C.
1 Malignant Lymphoma
7 Papillomas-Fore-Stomach
1 Malignant Lymphoma
9 Papillomas-Fore-Stomach
2 Malignant Lymphoma
13 Papillomas-Fore-Stomach
2 Malignant Lymphomas
5 Papillomas-Fore-Stomach
1 Neuroblastoma-Adrenal
1 Papilloma-Fore-Stomach
1 Malignant Lymphoma
2 Adenomas-Adrenal
2 Papillomas-Fore-Stomach
2 Papillomas-Fore-Stomach
1 Hamangioma-Spleen
3 Malignant Lymphomas
2 Papilloma-Fore-Stomach
1 Adenoma-Adrenal
1 Malignant Lymphoma
1 Hamangioma-Spleen
1 Papilloma-Fore-Stomach
270
270
275
515
387
421
273
266
348
335
428
457
424
440
283
263
310
387
600
531
610
572
537
481
473
556
440
658
461
348
420
626
114
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E. TASK TITLE:
Compare Effects of Respirable Particles, Gases and Mists Using
Small Airway Resistance in Donkeys as the Model for Pulmonary
Irritation
HERL/RTP TASK NO: 8199
CONTRACTOR: New York University
CONTRACT NO.: 68-02-1732
Summary
The investigations of the effects of (NH4)2S04 and H2S04 aerosols on
pulmonary function, regional deposition, and bronchial clearance will be pur-
sued in order to clearly establish the nature of the effects produced and
their dose-response relationships. This will provide a sound basis for sub-
sequent tests with the same aerosols on human volunteers.
Airway resistance, dynamic compliance, and regional particle deposition
were selected as indicators of physiological response. They will indicate
transient effects attributable to the inhalation of airborne irritants at
levels which produce no pathological effects. Effects on these physiological
functions in donkeys should be essentially similar to those produced by the
same irritants in humans. A major purpose is, therefore, to clarify the dose-
response relationship for pulmonary irritation resulting from transient ele-
vations in the ambient pollution aerosol concentration.
The intrabronchial deposition patterns of (NH4)2S04 and ^$04 aerosols
will also be measured in hollow bronchial casts of donkeys and human airways.
The effect of water vapor concentration on the rate of aerosol growth and sub-
sequent deposition in hollow casts will be tested in order to evaluate mathe-
matical predictions of droplet growth from physiochemical factors.
Scope and Objectives
Changes in pulmonary function, mucociliary clearance, and the regional
deposition of an inert test aerosol will be studied to characterize the dose-
effects relationships produced in donkeys by inhaled sulfuric acid and ammon-
ium sulfate aerosols. The donkey will be exposed without sedation or rigid
restraint and represents an analogue for man. The human dose-response to a
one hour exposure at low concentrations (<1 mg/m3) of sulfuric acid mists
0.5 /urn in diameter will be evaluated. Measurements will be made of pulmonary
function, mucociliary transport and aerosol deposition.
Research Accomplished
,Animal Studies—Two animals have undergone six month exposure to TOO
pg/m3 H2S04 acid mist (0.3 /urn) for one hour a day for five days a week. Two
additional donkeys have been evaluated for normal (base line) measurements
115
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and are presently undergoing an identical six month exposure regimen. Bron-
chial clearance rates were slower initially and were faster thereafter. How-
ever, there is considerable variation and no definite pattern has emerged.
Human Studies—Human testing has commenced with each subject being ex-
posed to 0, 100, 300, or 1000 ng/m^ of H2S04 in a random fashion in a blind
test. Studies of the second human volunteer are presently underway.
Related Research
This project is complimentary to the Research activities in the Clinical
Studies Division in Chapel Hill, North Carolina.
F. TASK TITLE:
Studies on the Relationship Between Carcinogen Metabolism in
the Alveolar Macrophage and the Induction of Lung Cancer
HERL/RTP TASK NO: 8157
CONTRACTOR: Northrop Services, Inc.
CONTRACT NO.: 68-02-2566
Summary
Rabbit alveolar macrophages were examined for their ability to metabolize
or activate procarcinogens and promutagens to their active forms. It has been
demonstrated that rabbit alveolar macrophage can activate the mutagen 2-anthra-
mine using the Salmonella typhimurium bacterial mutagenesis assay of Ames.
This activity is within an order of magnitude of that found in rat liver, a
startling finding. This metabolic activity is mediated be NADPH and oxygen
and is partially located in the endoplasmic reticulum (microsomal fraction) of
the macrophage.
Primary rat hepatocytes have been used to establish a cell mediated
bacterial mutagenesis bioassay using strains of Salmonella as the indicator
organism. This bioassay is sensitive to a number of hepatocarcinogens,indi-
cating that it may possess organotropic specificity. Benzo(a)pyrene has been
found to be negative in this system and the explanation for this phenomena
is being sought through metabolism studies using high pressure liquid chroma-
tography. Once the techniques and protocols for this new assay have been
established they will be applied to cocultivation of rabbit alveolar macro-
phage and Salmonella.
Scope and Objective
The objective of this task is to study the relationship between carcino-
gen metabolism in the alveolar macrophage and the induction of lung cancer.
Using in vitro methods the course of metabolic activation/detoxification of
procarcinogens will be investigated in alveolar macrophages obtained from rats
116
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and hamsters. Biochemical methods will be used in monitoring induction of
metabolic activation/detoxification systems. Microbial mutanenesis will be
used to monitor the extent of metabolic activation/detoxification by macro-
phages. Once the conditions for maximal activation/detoxification of carcin-
ogens have been established using in vitro methods, whole animal studies will
be initiated employing intratracheal instillation techniques to administer
particulates and appropriate procarcinogens. The metabolic activation of
procarcinogens will be monitored as described above using macrophages obtained
by saline lavage of carcinogen-exposed animals. When optimal conditions have
been established, experiments will be performed to demonstrate the relation-
ship between elicitation of macrophage influx, metabolic activation/detoxifi-
cation of carcinogens and the induction of lung tumors in animals. This is
a long-range project that will be approached sequentially with continuation
from year to year being dependent upon demonstrated success of initial studies.
Background and Approach
Pioneering work by Safiotti and others has demonstrated the importance
of the participate component in experimental pulmonary carcinogenesis in
rodent models. Ambient air particulate is known to contain procarcinogenic
compounds such as benzo(a)pyrene. The alveolar macrophage constitutes the
first line of defense against inhaled particulate, having been shown many
years ago to avidly phagocytize foreign materials including air particulates
which find their way to the deep lung. The half-life of particles which
penetrate to this level of the respiratory tract is months to years. There-
fore, the alveolar macrophage is in a unique position to sequester and poten-
tially to metabolically activate procarcinogenic compounds associated with
airborne particulate material. Using in vitro methods as previously described,
it is possible to determine whether the macrophage is competent enzymatically
to carry out metabolic activation and to determine whether activated compounds
are released from the intact macrophage to cause damage to the DNA of adjacent
cells. Thence, it should be possible to determine, under optimal conditions,
where cancer incidence is greater in experimental animals.
Research Accomplished
Initial studies with procarcinogens utilizing in vitro methodology are
investigating the metabolic activation and detoxification capabilities of the
alveolar macrophage. Experiments performed thus far demonstrate that a micro-
somal fraction from alveolar macrophages is capable of metabolizing the pro-
carcinogen 2-aminoanthracene (2-anthramine) to a mutagenically active species.
Macrophages stimulated with Bacillus-Calmette-Guerin were found to have less
metabolic activation capability when tested with anthramine in the Salmonella
typhimurium bacterial mutagenesis bioassay. Biochemical assays for aryl
hydrocarbon hydroxylase, cytochrome P-450 and other microsomal enzymes show
the activity to be several orders of magnitude below that found in liver.
However, the mutagenic activity of 2-aminoanthracene using macrophage S-9
preparation was within an order of magnitude of that obtained from liver.
The question was raised whether the activation of anthramine by rabbit
alveolar macrophage (RAM S-9.) is mediated by NADPH and 02 requiring mixed-
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function oxidases? This question has not been completely resolved. In the
absence of NADPH the reversion rate returned to the spontaneous (background)
levels. A 100,000 xg supernatant prepared from the S-9 had about the same
activity as the microsomes isolated from the same spin on a per mg protein
basis. The total activity of the 100,000 xg supernatant and the microsomes
was about equal to the S-9 from which it was prepared. Hence, the activation
of anthramine requires NADPH, but the enzymes effecting this process are not
solely located in the microsomes. It was determined that RAM S-9 also acti-
vates 2-aminofluorene but attempts to activate benzo-a-pyrene were unsuccess-
ful.
Since macrophages had the ability to activate promutagens to their active
mutagenic forms,the development of a cell-mediated bioassay system using
macrophage as the metabolizer cell and Salmonella typhimurium as the indicator
cell line was undertaken. Due to the difficulty in obtaining macrophages at
that time, primary rat hepatocytes were substituted in their place as the
technical problems associated with cell mediated mutagenesis bioassays would
be somewhat the same whether macrophage or hepatocytes were used. Primary
rat hepatocytes were then used to establish a system for utilizing microbial
mutagenesis in screening for carcinogenic metabolites of whole cells. S_.
typhimurium strain TA1538 is placed in a dialysis bag and incubated with the
hepatocytes and mutagen. The bacteria are then removed, plated on minimal
media, and scored 2 days later for revertant colonies. Incubation of increas-
ing numbers of hepatocytes with anthramine and Tal538 resulted in increasing
numbers of revertant colonies. Incubation times were varied with 1 hour
determined to be best. Dose curves were constructed using fixed cell concen-
trations and varying anthramine concentrations. Number of revertants was a
function of anthramine dose. Studies are underway to determine the optimum
number and volume of cells needed for the cocultivation test. The relation-
ship of protein concentration to 2-anthramine activation by rabbit alveolar
macrophage S-9 and rat liver S-9 was analysed. Protein dose curves were
constructed using a set concentration of 2-anthramine. Rat liver S-9 showed
optimal numbers of revertant colonies per plate at O.Bmg S-9 protein/plate.
RAM S-9 showed increasing numbers of revertants/plate with increasing protein
concentrations up to 2mg/plate but no optimum number.
The ability of primary rat hepatocytes to metabolize benzo(a)pyrene
B[a]P into nine individual metabolites was determined using an HPLC procedure
developed in this laboratory in order to assess the utilization of these
cells for metabolic activation purposes. Hepatocytes in culture 1.5 and 24
hours were incubated with B[a]P for 4, 8, 12, and 24 hours. In general the
longer the incubation time the less metabolites were formed per cell (probably
due to the toxic nature of some of the B[a]P metabolites). Conversion to
B[a]P water soluble metabolites also increased markedly with the longer incu-
bation times. 1.5 hour old hepatocytes were more effective (38 pmoles meta-
bolites formed/hr/million cells) in oxidizing B[a]P to organic soluble meta-
bolites than were 24 hour old hepatocytes based on an 8 hour incubation time
(15 pmoles metabolites formed/hr/million cells). Six mutagens were tested
in the hepatocyte mediated S_. typhimurium suspension test at two concentra-
tions of cells and 4 doses of chemical. 2-aminoanthracene, 2-aminofluorene,
and aflatoxin-B gave positive results and dose-response; acetylaminofluorene
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was weakly .positive, while B [a ]P was negative with Ta1538. Dimethyl ami noazo-
benzene gave no effect with TAQ3.
Bibliography
None
Related Research
The effect of whole animal exposure to acid mists and participates on
the pulmonary metabolism of benzo(a)pyrene in the isolated perfused lung model
is being studied at the University of Cincinnati. The isolated perfused lung
(IPL) appears to be the best in vivo preparation for investigating pulmonary
metabolism of foreign compounds especially compounds adsorbed onto particulate.
An important aspect of current work is the assessment of the rate of formation
and types of metabolites formed when B[a]P is administered with ferric oxide
or crude air particulate (CAP) on the IPL.
The isolated lung perfusion technique was used to study in an "in vivo"
situation, the effects of S02, crude air particulate (CAP), and benzoTa)pyrene
treatment on benzo(a)pyrene metabolism. The influences of these agents on the
metabolism of benzo(a)pyrene, an environmental carcinogen, may explain differ-
ences in the carcinogenicity of this agent. S02, CAP and B[a]P pretreatment
increased B[a]P metabolism in the IPL and increased the formation of specific
activated metabolites, indicating that these agents may have cocarcinogenic
activities.
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SECTION IV
EVALUATION OF HAZARDS TO MAN
A. TASK TITLE:
Effect of Coal Gassification Products on the Pulmonary Defense
System Against Infectious Disease
HERL/RTP TASK NO.: 8162
GRANTEE: Catherine Aranyi
GRANT NO.: 805317
Summary
This is one of several tasks designed to investigate the effects of par-
ti cul ate effluents from energy processes on host defense mechanisms against
infectious disease. In this particular task, mice will be exposed via inhala-
tion to particulates from conventional and advanced coal power sources and
challenged with viable microbes. Subsequent evaluation of the animals will
measure resistance to the induced pneumonia. The total, as well as specific
defense systems (i.e. pulmonary bactericidal activity and alveolar macrophages)
will be examined. The experiments will be designed to elucidate dose-response
relationships. Since the work began recently, results are too preliminary to
report.
Scope and Objective
The objective is to determine the effects of particulate effluents from
coal utilization processes on host defenses against pulmonary infectious
disease. Several biological endpoints will be used to define the dose-
response relationships for the aerosols to be tested, to establish the effects
of duration of exposure (acute and intermittent), and to determine the time
required for recovery from exposure. Depending upon the availability of par-
ticulate effluents, several samples will be tested, with the emphasis being
placed on advanced coal conversion processes.
Background and Approach
The role of air pollutants as a predisposing factor to respiratory infec-
tion has been well recognized. Extensive work with 03 and N02, and auto
exhaust and limited work with selected metals and sulfates has indicated that
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mpacf oTJhe nuh??rSh P?llUta,n,!:V0 Pulmonary infectious disease may have
b «Srla?Sd Sfth f' Whlle many of these Po^tants are known to
be associated with energy-related effluents, no research has addressed the
?°1ttSj fr°m these Presses. Because of the wfde range
*5' Sft?5 f unknown che™stry, emitted from energy-
thepr *Wa? deClded that a reasonable aPP^ch would be to study
of S™« c£c*J? -fUal Pa^lculate effluent. By using biological endpoints
of proven sensitivity in environmental inhalation toxicology to study these
effluents, it is thought that the data derived can be used to assist in the
assessment of the health effects of these compounds.
The principal experimental approach will use the infectivity model.
With this system, mice exposed to air or to various concentrations of parti-
culates for various lengths of time are challenged with aerosols of viable
microbes (bacteria or virus). The animals are then held in clean air for 15
days during which time mortality is recorded. Prior work with this model has
indicated that its sensitivity is most probably due to its ability to reflect
the net effect of subtle alterations in a number of host defense parameters.
These parameters, which will also be investigated separately, include:
pulmonary bactericidal activity, and changes in the number, distribution and
viability of free pulmonary cells which can be obtained by lavage. Based upon
the results of these studies, a decision will be made as to whether additional
related endpoints should be added, such as functional and biochemical integ-
rity of the alveolar macrophage, a cell primarily responsible for sterility
of the deep lung.
Research Accomplished
To date, the major emphasis of this recently awarded contract has been
to set up facilities and ensure that the model systems are working properly.
Aerosol generation and monitoring techniques have been finalized, key person-
nel have been trained, and preliminary experiments have been conducted with
most of the model systems to be used. Facilities for aerosolization of
radiolabeled bacteria (necessary for the bactericidal studies) are being
built, with no cost for capital equipment to EPA. When this facility is com-
pleted, the bactericidal technique will be modified and perfected for this
particulate study.
Preliminary work has been conducted with acute exposure of mice to aero-
sols of the first sample, a size fractionated (^3 urn) particulate from an
electrostatic precipitator from a conventional coal power source. This same
sample has been sent to other researchers (Task Nos. 8163, 8149, 8173, 8198).
The preliminary nature of the work precludes a report at this time.
Related Research
Task Nos. 8162, 8149, 8163, 8173, and 8198 are all related in that each
is designed to study various aspects of the effects of actual particulates
from energy sources on host defense mechanisms. It is intended that all the
work will be conducted on the same particulate sample so that results of all
the studies can be compared and used to develop a better assessment of effects.
In isolated instances, sample availability problems may cause some deviations
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from this approach.
In addition, the participate sample used in this study will also be used
for Task No. 8317, entitled "Effect of Industrial Particulate Emissions on
Alveolar Macrophage," which is designed to correlate the in vitro alveolar
macrophage system with the infectivity model. Under this Air Health Task, the
sample will be used in the in vitro portion of the study only, but results
will be compared to the in vivo model of Task 8162. The sample will also be
assessed under Task No. THuisingh) for mutagenic and carcinogenic potential
using in vitro screening systems.
Please see the individual task reports for details of these studies.
B. TASK TITLE:
To Determine Effects of Pollutants From Alternate Energy Sources
on Pulmonary Antiviral Mechanisms
HERL/RTP TASK NO.: 8163
GRANTEE: Leonard Schiff
GRANT NO.: R-805049
Summary
This task is one of several which will compare the effects of particu-
lates from conventional and advanced energy processes on host defense mechan-
isms. In this project, the structure and function of mucociliary transport
processes which are involved in pulmonary clearance are the focus of investi-
gation. The ability of the particulate-exposed trachea! tissue to respond
to viral infection will also be examined. Initially since particulate samples
were unavailable, a study was conducted with H2S04 and carbon, alone and in
combination. Currently, particulate exposures are being made.
Scope and Objective
The purpose of this task is to compare the effects of conventional and
advanced coal utilization processes on trachea! mucociliary transport para-
meters. Both in vitro and in vivo inhalation exposures will be conducted. In
this way the validity of the in vitro test can be assessed for suitability as
a rapid screening model. Dose-response studies of acute and intermittent
exposures will be conducted and recovery from any adverse effects of this
treatment will be examined.
Background and Approach
Due to the proliferation of both conventional and advanced energy pro-
cesses, it becomes Increasingly important to examine any health effects that
may result from fugitive emissions from these processes. Actual particulate
effluent samples will be tested to gather information in a faster and more
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relevant manner than testing each chemical species of the complex participate
separately. Dose-response and recovery studies will be conducted.
Depending on particle size, many of the particulates will deposit on the
ciliated epithelium of the lung. This surface is covered with mucus and is
responsible for clearing the lung of inhaled matter (both viable and non-
viable), thus'protecting it from a variety of insults. A proven sensitive
model for determination of effects on this pulmonary surface will be used and
involves examination of ciliary beating frequency and the morphology of ex-
posed isolated trachea! rings or explants. Cialiated epithelium also defends
the host against viral infections so several related endpoints of this inter-
action will be examined also (viral replication, interferon production, and
morphology).
Research Accomplished
Until actual effluent particulates were available, work was conducted
using H£S04 (1.1 mg/nr) and carbon (1.5 mg/m3) alone and in combination, a
model type pollutant exposure relevant to energy processes. Immediately
after a 3 hr. exposure, there was a significant depression in ciliary beating
in both the acid and acid-carbon exposure groups. Beating was depressed for
up to 24 hrs. after exposure to the acid-carbon treatment and up to 72 hrs.
after exposure to acid. In vitro exposure to these pollutants also caused
decreased ciliary activity.
Immediately after exposure to the acid-carbon mixture, the tracheal
epithelium showed greater morphological alteration than when exposed to air,
carbon or acid mist alone. The examination involved light and scanning elec-
tron microscopic techniques.
In addition to refining methods for viral exposure and measurement of
viral effects, recent work has focused on the effects of in vitro exposure
(1 hr/day, 5 days/wk for 10 days) of hamster trachea to particulates (< 3 Mm)
from an electrostatic precipitator of a conventional coal-burning power source.
At >100 g/ml there was a depression in ciliary beating frequency after the
first exposure. At >50/*g/ml, morphological alterations were observed.
Tracheal explants exposed to 10 ng/ml fly ash for 3 hr/day experienced a de-
crease in ciliary beating frequency by day 8 and cytotoxic and histological
effects at day 7. Work in progress includes combination exposures to virus
and particulate. In vivo particulate exposures are scheduled to begin short-
ly.
Bibliography
Schiff, L. J., et al. 1978. Cytotoxic Effects of Sulfuric Acid and Carbon
Particle Mixtures on Hamster Tracheal Epithelium. 29th Annual Mtg.
Tissue Culture Assoc.
Schiff, L. J., et al. 1978. Scanning Electron Microscopic Study of Develop-
ing Hamster Tracheal Epithelium in Organ Culture. 29th Annual Mtg.
Tissue Culture Assoc.
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Related Research
Task Nos. 8162, 8149, 8163, 8173, and 8198 are all related in that each
is designed to study various aspects of the effects of actual particulates
from energy sources on host defense mechanisms. It is intended that all the
work will be conducted on the same particulate sample so that results of all
the studies can be compared and used to develop better assessment of effects.
In isolated instances, sample availability problems may cause some deviations
from this approach.
In addition, the particulate sample used in this study will also be used
for Task No. 8317, entitled "Effect of Industrial Particulate Emissions on
Alveolar Macrophage," which is designed to correlate the in vitro alveolar
macrophage system with the infectivity model. Under this Air Health Task, the
sample will be used in the in vitro portion of the study only, but results
will be compared to the in vivo model of Task 8162. The sample will also be
assessed under Task No. THuisingh) for mutagenic and carcinogenic potential
using in vitro screening systems.
Please see the individual task reports for details of these studies.
C. TASK TITLE:
Quality Control for Assessment of Human Exposure
HERL/RTP TASK NO.: 8164
CONTRACTOR: Northrop Services, Inc.
CONTRACT NO.: 68-02-2788
Summary
This task was established to insure that the Clinical Environmental Labo-
ratory and the Mobile Clinical Laboratories located in Chapel Hill, N. C. are
operated according to predetermined standards of performance with respect to
safety and data validation. The purpose of these laboratories is to assess
the effects of exposure to common air pollutants on human test subjects.
There are humanitarian and legal ramifications associated with the use of
human beings as test objects. The safety and well being of both the people
being tested, and the people conducting the test, is of primary importance.
It goes without saying that every precaution must be taken to verify personnel
safety from overexposure to pollutants or electrical accidents.
The data collected at this facility will be used to establish clean air
standards, and will almost certainly be challenged. In view of the possibi-
lity of accident or challenge to the data, every step practicable must be
taken to insure that the laboratory operates according to predetermined
standards of performance with respect to safety and data validity.
The range of activities designed to accomplish this has been termed
Quality Assurance (QA). A contract was awarded to Northrop Services, Inc.
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on September 30, 1977 to design, develop and implement performance audits on
the experimental systems used in these laboratories. The audits will include:
1. examination of operations and documentation
2. comparison of actual operations and established requirements
3. recommendations for remedial and preventive action
4. independent measurement of performance
The Clinical Environmental Laboratory is identified by the acronym CLEANS
for Clinical Laboratory for Evaluation and Assessment of Noxious Substances.
CLEANS is a system of two environmentally controlled clinical laboratories
where human subjects can be housed for periods of time ranging from hours to
weeks. Precisely controlled concentrations of gaseous and water soluble
particulate pollutants, found in the environment, can be introduced, either
singly or in combinations, into the clinical exposure laboratories. In addi-
tion, to the pollutants, the temperature, humidity, lighting, and air flow
simulate natural ambient conditions. While in the controlled environment,
the test subjects will undergo a regularly scheduled program of noninvasive
pulmonary function tests, exercise electrocardiography, and systolic time
interval measurements, along with physical examination and biological specimen
collection. All aspects of these tests as well as the operation of the Clini-
cal Laboratory are controlled and recorded by dedicated computers and peri-
pheral equipment.
The Mobile Clinical Laboratories are identified by the acronym CLEVER
for Clinical Laboratory for ^valuation and Validation of Epidemiologic
Research. CLEVER consits of two forward control bus-type motor vehicles that
house mobile clinical laboratories that contain the same or similar medical
examination equipment, dedicated computers, and peripheral equipment as the
CLEANS clinical laboratories. Where the CLEANS system was designed to meas-
ure and process noninvasive human physiological parameters in a controlled
environment, the CLEVER systems were designed to make the same physiological
measurements in the uncontrolled outside ambient environment. The normal
home base for the CLEVER vehicles is at the Chapel Hill Clinical Studies
Division. If the vehicles are in use at another location when scheduled for
a QA audit, the Contractor is expected to transport the necessary equipment
and personnel to that location to perform the required audit.
Scope and Objectives
The objective of this task is to obtain expertise from a Contractor to
conduct independent Quality Assurance audits of all components of the CLEANS/
CLEVER Systems which may affect the assessment of human exposures. In addi-
tion to his expertise, the contractor will use the "Quality Assurance Hand-
book for Air Pollution Measurements Systems" and the "Health Effects Research
Laboratory Quality Assurance Manuals" as guides in developing a comprehensive
Quality Assurance (QA) program that will cover all of the appropriate QA
elements and responsibilities outlined in these documents. The main thrust of
the QA program is:
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1. To conduct a System Survey Audit to gather information on the current
operations of the laboratories, to evaluate the Quality Control pro-
cedures now being used and to identify major problem areas.
2. To design verifiable audit test procedures and develop an auditors'
handbook incorporating the QA program and test procedures.
3. To conduct periodic performance audits of the chambers and laboratory
systems.
Background and Approach
A Request for Proposal (RFP) to develop and implement a comphrehensive
Quality Assurance Program for complex and diversified experimental systems
used to measure the effects of air pollutants on humans was published on May
27, 1977. The RFP No. DU-77-B154 was sent to thirty-one potential contract-
ors, but only one responded with a proposal. The cost proposal was several
times the amount budgeted for this project. Consequently, a revised proposal
was requested from the contractor during negotiations. The cost of the re-
vised proposal was within the budgeted funds for the project and was techni-
cally acceptable. The contractor had simply overestimated the project Scope
of Work in his original proposal.
A contract was awarded to Northrop Services, Inc. on September 30, 1977.
A comprehensive quality assurance program is equally important to the
quality control procedures that are an integral part of any responsible
research protocol. The QA program corroborates the experimental results pro-
duced by the laboratory by providing a means for independently assessing and
documenting that the experimental data has precision, accuracy, validity, is
representative of the condition being measured, and is complete, A QA pro-
gram also provides a means to evaluate the relevance of a task to laboratory
and agency objectives.
The data obtained from exposing human subjects to various air pollutants
in the CLEANS chambers will be used to support Federal Standards for limits
of exposure. A well designed and implemented QA program is intrinsic to an
experimental system that must produce data that is supportable and allows com-
parability among groups.
Accomplishments
Since the award of the QA contract to Northrop Services, Inc. the follow-
ing tasks have been completed:
1. Original Work Plan describing the various tasks, the approach to be
used to meet the objectives, and the projected schedule of the acti-
vities, was submitted to the Project Officer on October 28, 1977.
2. A System Survey of the CLEANS System Operation and Maintenance was
completed February 24, 1978.
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3. Monthly Progress Reports have been issued since the inception of the
QA contract starting with the period October 1, 1977 through October
o 15 i y / /.
Bibliography
Quality Assurance Handbook for Air Pollution Measurement Systems 1976
Principles I EPA-600/9-76-005.
Management Policy for the Assurance of Research Quality Health Effects Re-
search Laboratory. 1977. RTP, N.C. EPA-600/1-77-036.
Development of Quality Assurance Plans for Research Tasks, Health Effects
Laboratory. 1978. RTP, N.C. EPA-600/1-78-012.
Original Work Plan - Technical Services for Development and Implementation of
a Comprehensive Quality Assurance Program for Assessment of Human Ex-
posure. 1978. Northrop Services, Inc. RTP. N.C. SP-410-1878.
System Survey - Technical Services for Development and Implementation of a
Comprehensive Quality Assurance Program for Assessment of Human Exposures.
1978. Northrop Services, Inc. RTP, N.C. RT9470-1.
Monthly Progress Reports - Technical Services for Development and Implement-
ation of a Comprehensive Quality Assurance Program for Assessment of
Human Exposures. 1978.
Related Research
This task directly supports the research tasks of the CLEANS/CLEVER ex-
perimental systems by providing the total Quality Assurance program required
by the Health Effects Research Laboratory/RTP, N.C., management policy.
Personnel of the Clinical Studies Division, HERL, are responsible for the
development and implementation of exposure research protocols using the
CLEANS/CLEVER system supported by the Operation and Maintenance Contractor,
Rockwell International Corp. The objectives of the CLEANS/CLEVER research
protocols are to determine and measure the health effects of humans from the
exposure of gaseous and inhaleable particulate matter found in the emissions
from alternate energy sources such as low grades of coal. The purpose of this
research is to provide information necessary to formulate environmental regu-
latory policies to protect or improve public health and welfare while at the
same time enhancing the nation's continuing productivity. This project re-
quires periodic independent QA performance audits.
D. TASK TITLE:
Evaluation of the Hazards of Exposure to Biological Active
Agents Associated with Energy Technology
HERL/RTP TASK NO: 8168
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CONTRACTOR: University of Illinois
CONTRACT NO: 68-02-2492
Summary
A "Model for Measuring the Health Impact From Changing Levels of Air
Pollution" is a federally funded EPA contract with the University of Illinois,
School of Public Health. The award was originated February 15, 1977 with a
proposed deadline of May 15, 1978. A time extension was sought for a new
deadline of October 15, 1978. The study involves creation of a model to
quantitate the relationship between ambient air concentrations of various
individual air contaminants and health in a large metropolitan area in the
Midwest.
The study encompasses both a mortality and morbidity component. The
mortality component involves examination of residents of the city of Chicago
whose deaths occurred between January 1, 1971 and December 31, 1975, and were
registered in a respiratory or cardiovascular category (8th Revised I.C.D.).
By comparing an individual's place of residence within the boundries of the
city with normally occurring and periodic levels of air pollution, the con-
tribution of certain pollutant parameters to these deaths may be ascertained
and quantified. Climatological indices as well as socio-economic variables
are accounted for in a statistical equation. In the morbidity component ill-
nesses from respiratory or cardiovascular causes is used as an index of health.
For one year, April 1977 until April 1978, visits by residents of Chicago
to two city hospital emergency rooms were monitored. Each resident's address
was cross-referenced to one of seventy-six community areas designated by the
U. S. Census Bureau. Contaminant levels in a specific location and various
health effects are quantified.
Scope and Overall Objectives
The overall objective of this continuing research is to examine the
strength of the relationship between concentrations of various ambient air
contaminants and health indices. Mortality rates and emergency room visit-
ation rates for cardiac, vascular and respiratory diseases in an urban, in-
dustrial, energy intensive area are being examined.
The following questions are addressed and answered in the stated research:
1. How much does each air pollutant contribute to cardiac and respira-
tory disease mortality?
2. How much does each air pollutant contribute to the incidence of car-
diac and respiratory disease?
3. Are the national primary ambient air quality standards appropriate
for the protection of public health?
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Multiple regression analysis plays a vital role in finding each pollu-
tant s contribution to mortality and morbidity in tandem with description
statistics and the analysis of variance. Regression analysis describes the
collective and separate contribution of two or more independent variables
(pollutants, socio-economic variables, or climatological factors in the
study) to the variation of a dependent variable (mortality index or morbidity
index).
To statistically test the hypothesis that air pollution affects health,
it is important to rule out the possibility of spurious correlations between
some other variables and both air pollution and higher mortality rates. For
example, there are several reasons why the mortality rate is higher in an
urbanized area than in a less urbanized area (e.g. tension, stress, unhealthy
personal habits). Thus, an urbanized and industrial area contributes simul-
taneously to air pollution and health status, and there is the possibility
that any correlation between air pollution and health status would be spur-
ious for this reason.
We would propose four factors affecting health status in a geographical
area:
1. demographic characteristics - age, sex, race distributions
2. socio-economic characteristics - income distribution, housing dens-
ity, occupation, and education levels,
3. environmental factors air pollution levels, climatological
characteristics, and energy utilization factors (heating fuels,
etc.)
4. personal factors - smoking habits, medical care (quality and
quantity) exercise habits, nutrition and genetic characteristics.
These factors are not all included in the model. However, those demo-
graphic variables provided in the 1970 census reports will be utilized, as
will all climatological variables and air pollution parameters, in the
development of a mathematical equation.
Background and Approach
The literature has clearly identified a positive correlation between
levels of air pollution and rates of illness and death. Examination of am-
bient air concentrations of various pollutants can determine which factors
influence mortality and morbidity rates. Once the various influencing vari-
ables have been determined, it is possible, through statistical technique,
to determine the degree of influence of each variable.
As the study is divided into two components, each shall be dealt with
separately. The first section is a three-fold mortality analysis, being
conducted to test the relationship between death and air pollution:
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1. Descriptive trend analysis is being conducted to ascertain the
mortality trend in Chicago and downstate Illinois. The following
tasks have been completed:
a. comparison between Chicago area death rates by cause, by age,
and by race with those rates in downstate Illinois for the years
1967 to 1975;
b. annual death rates for 1967 to 1975, from all causes save
accidents, homicides, and suicides, for all ages and races,
were compared among seven chosen communities of varying size
and location throughout Illinois;
c. using the same seven cities, annual air contaminant measurements
(TSP and SO?* where available) were examined and compared, thus
delineating citywide annual trends for 1967 to 1975;
d. standardized age-adjusted death rates in Chicago for all causes
were computed for 1970 to 1975.
e. annual TSP and SOg values have been charted from 1970 to 1975,
at each monitoring station in the city of Chicago. Thus, this
data may be directly compared with the trend of mortality data
for the city.
2. The next technique is long term cross-sectional analysis. Based on
ten leading causes of death (that is, excluding accidents, homicides,
and suicides), age-adjusted death rates for 1970 to 1975 are com-
puted for each of the seventy-six community areas. At the same time
annual air pollutant measurements (TSP and SO^) are computed for the
same five year period at each of the thirty-five monitoring sites.
Each site operated at varying time intervals. Air contaminant
values are interpolated to each community area, based on location of
the site. Each area is then assigned to one of three air pollution
categories (high, medium, low) based on its air contaminant value.
To determine each air pollutant's contribution to cardiac and res-
piratory disease mortality, multiple regression is applied. The
dependent variables in the equation are the death rates, by cause,
during the five year period from 1971 to 1975. Independent vari-
ables included the average concentration levels of air pollutants
(TSP, S02, N0£, NO, CO, and ozone), weather factors, and socio-
economic indices, percent of families with income below poverty
level, number of white collar workers, level of education, and
breakdown of ethnic groups. (This information is ascertained from
the 1970 Census Report). The proportion of each pollutant's con-
tribution to the total variance of the death rate is calculated
after controlling socio-economic indices.
3. A time/series analysis is used to examine more closely the acute
health effects of variations in the concentrations of ambient air
pollutants. Weekly, monthly, and seasonal analyses are conducted,
based on the availability of air pollution data. The dependent
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- variable is death rate from cardiac and respiratory diseases (for
the appropriate time period). All disease categorizations are in
accordance with the 8th Revised International Classification of
Disease specifications. Independent variables are the average
exposure levels of the deceased to the air pollutants and averages
of climate-logical indices, such as temperature, windspeed, humidity,
and sunshine. The average exposure level corresponding to each
death is determined by correlating the person's place of residence
with the estimated pollution level in that area. The exposure
levels of all deaths are combined, and then an average exposure
level is obtained by dividing the sum of the individual exposure
levels by the number of deaths on that day (week, etc.).
The morbidity component of this study attempts to establish a cause-
effect relationship between air pollution levels in Chicago and an increase
in the number of cardiac and respiratory illnesses. The sources of morbidity
patient data include two hospitals, Cook County (CC) and University of
Illinois (UI). Cook County was chosen because it was found in an earlier
study that CC registered at least as many patients per week as 13 other hos-
pitals in the Chicago area. Also, virtually all of the patients were resi-
dents of the Chicago area and most were residents from the Chicago inner city
and used CC as a primary care facility.
The UI Hospital was selected because in 1977, while data collection was
in its initial stages, a doctor's strike at CC prompted most of CC patients
to be routed to UI. To be assured of the continuity and availability of all
necessary data, it was considered safer to continue monitoring both hospitals.
Within these hospitals, data are collected from three areas; the
Emergency Room, Admissions and Pediatrics. Information is sought from these
areas because the largest majority of these study subjects are non-appoint-
ment patients who need medical attention for acute care. Another very
significant reason is that data obtained from these patients can be consid-
ered highly reliable because a doctor's diagnosis accompanies each patient
registered and treated.
Data are collected by trained personnel Tuesday, Wednesday, and Thursday
of every week. By omitting collection on weekends, days surrounding weekends
(Fridays and Mondays), and holidays, a fairly regular pattern of admissions
is established.
A standard form, entitled Hospital Record of Patients with Respiratory
and Cardiac Conditions, is filled out to record information on all persons
whose illness is applicable to the study. The following information is
gathered on each form:
1. Sequence/number
2. Date
3. Time of Visit
4. Patient's Address
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5. Patient's Age
?
6. Patient's Race
7. Patient's Sex
8. Patient's Complaint
9. Physician's Diagnosis
In addition to the patient data, other information is also collected.
Total number of persons seeking medical help is recorded daily for each
hospital area. A tally is keptof all patients who are pre-admissions, trans-
fers, referrals, rechecks, refills, or dead-on-arrival (DOA'S). This number
is subtracted from the total and the resulting number of study subjects is
designated as the disease population number.
The forms are then number coded for community area, census tract,
patient's complaint and physician's diagnosis. After the sequence number is
stamped on each form, the forms are checked and sent out to be keypunched.
Keypunched cards are listed and checked against the data forms for possible
errors, then data forms, cards and lists are filed for later analysis.
Research Accomplished
Mortality—
To date, progress has been limited due to unforeseen major problems in
collecting and processing air pollution data, as cited below. Preliminary
trend analysis has been completed, verifying a downward trend in mortality,
as well as in air pollution measurements in Chicago.
Cross-sectional analysis and time/series analysis have been delayed un-
til recently, due to unforeseen problems in cleaning, processing and organi-
aing a master file for daily pollutant averages.
The air monitoring network based in Chicago is a complex one. A wide
variety of air contaminants are monitored including total suspended partic-
ulate, sulfur dioxide, nitrogen dioxide, carbon monoxide, nitric oxide,
oxidants, methane, hydrocarbons and various metals. The network itself is
comprised of 37 sites under the sanction of the city of Chicago Department
of Environmental Control, the U.S. EPA, the State of Illinois EPA, the
Department of Air Pollution Control, the Department of Commerce or the U.S.
Public Health Service. Each pollutant is monitored by one of four different
instruments. The time interval between collection periods at any one monitor-
ing site may be 1, 2, 4, 8, or 24 hours, or 3 or 6 days.
Because the variety of contaminants, systems and collection periods is so
wide, a petition was made to the U. S. EPA for a complete data set, on tape,
which would contain daily continuous levels of particulate, S02» N02> NO,
CO, 0 and COM for all monitoring stations, regardless of controlling agency,
located within the city's limits.
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The tape received contained three files: the first contained continuous
TSP, SO? and N02 data from 1970 to 1976; the second, hourly SO?, NO, N02, 07
and CO data for 1970 to 1973; with the third covering the years 1974-1976 for
the same contaminants. (At the same time, a petition was made to both the
city agency (DEC) and the U. S. EPA for printed copies of data in their files.
After projecting much data manipulation (time being spent to clean and organ-
ize this master daily file), it was felt that any error found might be due to
excessive handling by the project data processors. Both agencies complied
and provided such a listing of data.
First, this tape was dumped, to ascertain exactly what material was
located therein. Separate files were made for each pollutant in the primary
task of calculating daily averages for each site over the entire 5 year time
frame (January 1, 1971 to December 31, 1975). In cleaning these files, care-
ful precautions had to be taken to account for all monitoring specifications,
day, site location and code, monitoring agency, duration, type of instrument-
ation, and units of computation, in order to provide an accurate comprehensive
listing of all air pollutant data.
As the tape figures were checked against those published by the city,
many inaccuracies were found. Unknown instrumentation codes were listed, as
were two values for one site (with all coding intact) at one time, missing
values (for as long as eight months), stations miscoded to mimic the same
values of another site, extra values not found on any other record and two
copies of all the same parameters reporting different values.
Approximately eight months were spent adjusting and correcting this data
by the supplementation of additional values, deletions to the file, or re-
placement with a new file entirely.
Cross-sectional analysis is about to begin. After computing annual means
for each site with no missing values for each parameter, an air pollution
field has been determined through interpolation to each community area.
Descriptive analysis should be completed within the next quarter.
Morbidity-
Si nee the start of hospital data collection on 12 April 1977, numerous
questions and problems have surfaced. Problems of interpreting hospital
records and selecting applicable information decreased considerably after the
collectors became more familiar with the total process.
In December 1977, the data from CC pediatrics was cancelled. The log
records available to us could not guarantee even a small degree of reliability
because the doctors' diagnoses were not available.
The last day of data collection will be 14 April 1978. At this point,
we project that by 16 June 1978 the collection data will have been taken
through the final states of completion (i.e. processed, cleaned and on tape)
and will be ready for final analysis. This mass of data will be collapsed
into large categories of total numbers for sex and age, race, complaints,
diagnoses, etc. The complaints and diagnoses will be further subdivided into
major respiratory and cardiac illnesses and cross-referenced, as previously
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described, to air pollution values. As in the mortality section, morbidity
data will be analyzed by the use of multiple regression analysis and analysis
of co-varfance.
E. TASK TITLE:
Studies of Health Effects Resulting From Increased Indoor Air
Pollution From Energy Conservation
HERL/RTP TASK NO: 8169
CONTRACTOR: Geomet, Inc.
CONTRACT NO.: 68-02-2294
Summary
By May 31, 1978 the contractor will have completed a 27-month effort to
develop a tool for measuring indoor air pollution exposure. The major activ-
ity of the project consisted of extensive indoor/outdoor air pollution moni-
toring. These monitoring measurements comprised the basis for developing
mathematical models for predicting indoor air quality from pollutant levels
recorded at community monitoring stations where outdoor ambient air quality
levels were measured.
Scope and Objectives
The objectives of the contract were to:
1. Review and assess indoor air pollution literature
2. Conduct indoor/outdoor air pollution monitoring
3. Develop mathematical models to predict indoor air quality
4. Develop a mobility/health document
Background and Approach
Very little information exists which relates indoor air pollution levels
to the many factors that affect such levels. Elevated levels of specific
pollutants have been measured in indoor environments, but little effort has
been expended to determine the relationship between indoor levels/outdoor
levels, indoor levels/indoor source strength, indoor levels/energy conser-
vation measures, and indoor levels/house activities. It is necessary to know
these relationships in order to be able to make decisions ranging from health
effects of indoor pollution to the consequences of making a building energy
efficient.
The goal of this project was to be able to estimate the indoor pollution
based on known factors such as outdoor pollution levels, indoor sources and
source strengths, house activity, and energy conservation measures. Taking
all of these inputs, the contractor had to develop a model or series of models
that could be used to predict indoor pollution levels-.
In order to be able to determine what parameters were most important to
134
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indoor levels and how they interacted, a monitoring program was established.
Houses of varying designs, locations, and pollution exposure were each mon-
itored continuously for a two week period. This information was used first
to develop the required relationships and then to verify the accuracy of the
model. To verify that the model was equally applicable during all seasons,
return visits were made to several houses during different heating/cooling
periods.
Research Accomplished
Preliminary results indicate the model is most accurate for the less
reactive pollutants. It also seems to work equally well in both hot and
cold seasons.
During the monitoring period no violation of the ambient air standards
was observed inside a dwelling. However, under certain combinations of events,
the model would indicate there could be very high levels of indoor pollution.
Inside concentrations levels of two pollutants were particularly noteworthy.
Carbon dioxide was observed at over three times the ambient level, and very
high levels of aldehydes were observed. The elevated CC>2 occurred when
several people were present in a room at the same time. Aldehydes were ob-
served in most buildings, but were particularly high in the mobile homes.
Bibliography
1. The status of Indoor Air Pollution Research - 1976 (published)
2. Survey of Indoor Air Quality Health Criteria and Standards (publish-
ed).
3. The Geomet Indoor-Outdoor Air Pollution Model: A Scientific Report
(in clearance process)
4. Final Contract Report Phase 2 (submitted for review and approval)
"Indoor Air Pollution in the Residential Environment". Volume 1:
Data Collection, Analysis and Interpretation; Volume 2: Prototype
Epidemiological Study; Volume 3: Supportive Documents.
Related Research
EPA's Health Effects Research Laboratory at the Research Triangle Park
has under contract a projected title: "The Acute Respiratory Disease Study".
It is a 12-month study to measure the incidence and severity of acute re-
spiratory disease symptoms related to total subject exposure: exposure within
the home and home neighborhood, exposure while traveling to and from work,
and exposure in the work environment.
The U. S. Department of Energy is presently investigating the relation-
ship of energy conservation measures in MED (minimum energy dwelling) houses
to pollution levels found inside the buildings. The project title is "Air
Quality Measurement in Energy-Efficient Buildings". This is a two-year pro-
ject and is concerned with schools, hospitals, and other buildings of this
type.
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The American Society of Heating, Refrigeration and Air Conditioning
Engineers (ASHRAE) has several committees devoting time to the indoor/outdoor
pollution problem as affected by various heating and air conditioning meas-
ures. This organization is supporting a project of the Department of Mechani-
cal Engineering at Iowa State University entitled "Experimental Energy-Con-
serving Building". This continuing project includes constructing a house that
will be fully instrumented for measuring pollution levels and energy consump-
tion parameters.
F. TASK TITLE:
Chemical Repository for Alternate Energy Source Materials
HERL/RTP TASK NO.: 8170
CONTRACTOR: Oak Ridge National Laboratory
CONTRACT NO.: ERDA(DOE) No. 40-60176 EPA No. IAG-D7-0129
Summary
This project provides physical and chemical support to health-effects
research addressing alternate fossil energy technologies. Samples provided
by the EPA and those acquired by the repository staff are made available to
qualified researchers. Samples of particular interest to the EPA are charac-
terized in terms of physical properties, elemental composition, and organic
chemical composition. Biological evaluations of highest priority samples are
supported through the preparation, including physical .or chemical separation,
of materials for biotesting and the identification of constituents responsible
for observed bioactivities. Coal liquefaction, gasification, and combustion
and oil shale processing are currently emphasized. The facilities may be
used to archive samples of particular interest to the EPA or to acquire analy-
tical characterizations of related materials undergiong study in environmental
and/or health assessments of alternative fossil fuels technologies. Results
of chemical and biological studies are forwarded to those contributing sam-
ples to the respository.
Scope and Objectives
The objective of this project is to improve the cost-effectiveness of
research on the health effects of synthetic fuels by increasing the availabi-
lity of research materials, improving the transfer of data between technology
developers and health effects researchers, and improving interlaboratory com-
parability through chemical characterization and bioassay materials prepara-;
tion services. Biological studies at the Oak Ridge National Laboratory sup-
ported by Pass-Thru funds and studies elsewhere chosen by the EPA project of-
ficer currently receive priority attention.
Background and Approach
The development of bioassay .methods and their application to health
assessments of new fossil fuels technologies .requires the availability of
136
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3. A particle-sized fraction of ash from coal combustion has been pre-
pared and distributed to EPA-specified bioassay contractors. The
elemental composition of the ash has been determined; organic anal-
yses are underway.
4. A comparison was made of available subcontractors for the size frac-
tionation of bulk quantities of particulates for bioassay research.
Small scale particulates size fractionation facilities were estab-
lished in-house and materials were supplied to EPA researchers.
5. Methodologies made available to support bioassay research included
(a) the analysis of particle size distribution (b) the chemical
separation of syncrudes for biotesting (c) the glass capillary col-
umn gas chromatographic analysis of organic constituents (d) the
separation of multialkylated derivatives of polycyclic aromatic
hydrocarbons from parent and simple alkylated derivatives, and (3)
the isolation of highly mutagenic azaarenes from syncrudes.
6. On-site observation and assistance in experimental design was pro-
vided for the sampling of an above ground oil shale retorting exper-
iment for materials potentially available for inhalation exposure.
7. Assistance was provided for a joint Laramie Energy Research Center,
Pittsburgh Energy Research Center, Oak Ridge National Laboratory
study of the combustion charactersitics of shale-derived oils. In-
sufficient quantities of combustion particulates were available for
collection for biological study. Emissions measurement results are
being compiled.
8. Studies addressing the comparability of bacterial mutagenesis
response with in vivo response following intratracheal instillation
and the utility of bacterial mutagenesis assay for discriminating
between shale-derived crude oils are underway. Repository staff
are responsible for the preparation of materials under study.
Bibliography
This provides general assistance to EPA programs. Results are reported
in informal documents to the EPA project officer. Six such "topical reports"
addressing the current inventory of materials, analyses of specified samples
and the results of requested studies have been issued in the past 12 months.
Metholologies specifically developed in support of biological experi-
ments and the results of applying these methods are to be submitted for pub-
lications. Reports resulting from the use of materials now in the repository
(not funded by the repository agreement) include:
Clark, B. R., N. A. Goeckner, and I. B. Rubin. 1978. Organic Chemical Char-
acterization of a Crude Shale Oil. Submitted for presentation at Confab
78: Government, Industry and Academic Technical Conference on Fossil
Fuel Chemistry and Energy. Saratoga, Wyoming, July 25-28.
137
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Clark, B. R. and M. R. Guerin. 1976. Chemical Characterization and Monitor-
ing Studies of Effluents from Emerging Fossil Fuel Processes. Presented
at Air Pollution Control Association's Conference on Toxic Substances.
Cambridge, Massachusetts, November 8.
Clark, B. R. and M. R. Guerin. 1977. Chemical Characterization of Organic
Constituents in Oil Shale Materials. Second ERDA Meeting and Workshop
on Oil Shale Environmental Research. Richland, Washington, November 1-2.
Clark, B. R., C. -h. Ho, and A. R. Jones. 1977. Chemical Class Fractionation
of Fossil-Derived Materials for Biological Testing. Symposium on Analy-
tical Chemistry of Tar Sands and Oil Shale. New Orleans, Louisana,
March 20-25.
Clark, B. R., C. -h. Ho, and A. R. Jones. 1977. Approaches to Chemical Class
Analyses of Fossil Derived Materials. Symposium on Analytical Chemistry
of Tar Sands and Oil Shale. American Chemical Society, Division of Pet-
roleum, Inc. Preprints, 22:(2): 811-812.
Clark, B. R., I. B. Rubin, C -h. Ho, and M. R. Guerin. 1976. Chemical-Bio-
logical Chacterization of Coal Conversion Liquids. 81st National
Meeting of the American Institute of Chemical Engineers. Kansas City,
Missouri. April 11-14.
Clark, B. R. 1976. Chemical-Biological-Environmental Characterization of
Fossil Fuel Materials. Workshop on Standard Reference Materials for
Coal Gasification and Liquefaction, National Bureau of Standards.
Gaithersburg, Maryland, January 20-21.
Clark, B. R., I. B. Rubin, C -h. Ho, M. R. Guerin, J. L. Epler, and A. A.
Hardigree. 1977. Testing for Health Hazards in Coal Liquids. Coal
Processing Technology 3:37.
Epler, J. L. and M. R. Guerin. 1978. Mutagenic Components of Alternate
Energy Sources. Symposium on Health Effects of Alternate Energy Sources,
Air Pollution Control Association National Meeting. Houston, Texas.
June 25.
Epler, J. L., B. R. Clark, C -h. Ho. M. R. Guerin, and T. K. Rao. 1978.
Short-Term Bioassay of Complex Organic Mixtures. Part II. Mutagenicity
Testing, Symposium on Application of Short-Term Bioassays in the Frac-
tionation and Analysis of Complex Environmental Mixtures. Williamsburg,
Virginia. February 21-23.
Epler, J. L., T. K. Rao, and M. R. Guerin. Evaluation of Feasibility of
Mutagenic Testing of Shale Oil Products and Effluents. 1977. U. S.
Soviet Workshop on Health Effects of Shale Oil Development. Denver,
Colorado, May 18-20.
Epler, J. L., F. W. Larimer, C. E. Nix, T. Ho, and T. K. Rao. 1977. Compar-
ative Mutagenesis of Test Materials from the Synthetic Fuel Technologies.
138
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Second International Conference on Environmental Mutagens. Edinburgh,
July 11-15.
Epler, J. L., J. A. Young, A. A. Hardigree, T. K. Rao, M. R. Guerin, I. G.
Rubin, C -h. Ho. and B. R. Clark. Analytical and Biological Analyses
of Test Materials from the Synthetic Fuel Technologies. I. Mutagenicity
of Crude-Oils Determined by the Salmonella typhimuriurn/Microsomal Activa-
tion System.
Epler, J. L., F. W. Larimer, T. K. Rao, C. E. Nix, and T. Ho. 1978. Energy
Related Pollutants in the Environment: The Use of Short-Term Tests for
Mutagenicity in the Isolation and Identification of Biohazards. Presen-
ted at Higher Plant Systems as Monitors of Environmental Mutagens;
Workshops cosponsored by the National Institute of Environmental Health
Sciences, Department of Energy. Marineland, Florida, January 16-18.
Goeckner, N. A., and W. H. Griest. 1977. Determination of Methyl Chrysenes
in a Coal Liquefaction Product. The Science of the Total Environment.
8:187-193.
Griest, W. H., H. Kubota, and M. R. Guerin. 1976. PAH Profiling Analysis
by GLC. First ORNL Workshop on Polycyclic Aromatic Hydrocarbons.
Characterization and Measurement with a View Towards Personnel Protec-
tion. Oak Ridge National Laboratory, Oak Ridge, Tennessee. February 26.
Griest, W. H., G. Olerich, J. L. Epler, and T. K. Rao. Characterization of
Multialkylated Polycyclic Aromatic Hydrocarbons in Energy Related Mate-
rials. 1978. To be presented at Third International Symposium on Poly-
nuclear Aromatic Hydrocarbons, Battelle Columbus Laboratories, Columbus,
Ohio. October 25-28.
Guerin, M. R., C. -h. Ho, B. R. Clark, J. L. Epler, and T. K. Rao. 1978.
Separation of Mutagenic Components in Synthetic Crudes. 175th National
ACS Meeting. Anaheim, California, March 12-17.
Guerin, M. R. and J. L. Epler. 1976. Determining Emissions Measurements
Needs for an Emerging Industry-Advanced Fossil Fuels Utilization. Sympo-
sium on Fugitive Emissions Measurement and Control. May, EPA-600/2-76-
246.
Guerin, M. R., W. H. Griest, C. -h. Ho, and W. D. Shults. 1975. Chemical
Characterization of Coal Conversion Pilot Plant Materials. Third ERDA
Environmental Protection Conference. September 25.
Guerin, M. R., J. L. Epler, W. H. Griest, B. R. Clark, and T. K. Rao. 1978.
Polycyclic Aromatic Hydrocarbons from Fossil Fuel Conversion Processes.
Carcinogenesis, Volume 3: Polynuclear Aromatic Hydrocarbons. Editors
P. W. Jones and R. J. Fruedenthal. Raven Press, New York. pp. 21-33.
Guerin, M. R. 1978 (in press). Energy Sources of Polycyclic Aromatic Hydro-
carbons. Polycyclic Hydrocarbons and Cancer. Academic Press.
139
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Guerin, M, R., B. R. Clark, C. -h. Ho, J. L. Epler, and T. K. Rao. 1978.
Short-Term Bioassay of Complex Organic Mixtures. Part I. Chemistry.
Symposium on Application of Short-Term Bioassays in the Fractionation
and Analysis of Complex Environmental Mixtures. Williamsburg, Virginia.
February 21-23.
Ho, C. -h., B. R. Clark, and M. R. Guerin. 1976. Direct Analysis of Organic
Compounds in Aqueous By-Products from Fossil Fuel Conversion Processes:
Oil Shale Retorting, Synthane Coal Gasification and COED Coal Liquefac-
tion. J. Environ. Sci. Health. 7:481-489.
Jones, A. R., M. R. Guerin, and B. R. Clark. 1977. Preparative-Scale Liquid
Chromatographic Fractionation of Crude Oils Derived from Coal and Shale.
Anal. Chemistry. 49:1766.
Jubota, H., W. H. Griest, and M. R. Guerin. 1975. Determination of Carcin-
ogens in Tobacco Smoke and Coal-Derived Samples Trace Polynuclear
Aromatic Hydrocarbons. Reprinted from Trace Substances in Environmental
Health-IXi A Symposium. D. D. Hemphill Ed., University of Missouri,
Columbia, pp. 281-289.
Parkhurst, B. R., C. W. Gehrs, and I. B. Rubin. 1977. Chemical Fractionation
with Acute Toxicity Testing for Identifying the Toxic Components of Com-
plex Aqueous Effluents. ASTM Second Annual Symposium on Aquatic Toxi-
city. Cleveland, Ohio.
Rubin, I. B., M. R. Guerin, A. A. Hardigree, and J. L. Epler. 1976. Frac-
tionation of Synthetic Crude Oils from Coal for Biological Testing.
Environmental Research. 12:358-365.
Related Research
1. Analytical Chemical Support of Synthetic Fuels-Related Bioassay
Research (Department of Energy). Methods are developed and applied
to the fractionation of synfuels-related materials in a manner suit-
able for biological testing. Collaborating biologists identify
biologically active subfractions which are subsequently further frac-
tionated in search of individual constituents responsible for
biological response. Polycyclic aromatic hydrocarbon isolates from
syncrudes have been isolated and characterized for mouse dermal
bioassay.
2. Chemical Characterization of Synthetic Fuels (Department of Energy).
Analytical methods are developed and applied to the characterization
of organic constituents of synfuels and synfuel production waste
streams. Phenolics, paraffins, bases, and polycyclic aromatic
hydrocarbons have received primary attention.
3, Polycyclic Aromatic Hydrocarbons in the..Aqua-tic Environment (Depart-
ment of Energy).In a collaborative project supported through the
Environmental Sciences Division, multicomponent methods are being
developed to isolate and quantify polycyclic aromatic
140
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nS ov TK 6d materlals for S^dy. Perfectly representative samples do
not exist because none of the technologies under study have achieved the
state of development where commercial scale plants are operating. A mechan-
ism was required which would provide research materials for bioassay methods
development while maximizing feedback to the technology developers and mini-
mizing premature extrapolations to overall technology assessments.
Environmental and health considerations are to be taken into account in
identifying the most suitable synthetic fuels technologies. Chemical and
physical analyses or treatments of materials required to support biological
research are generally well beyond the scope of analyses required for pro-
cess development. A mechanism was required whereby materials subjected to
the most intensive biological study could be physio-chemically characterized
to the degree required by the health effects research.
Some materials prove to be more useful than others in elucidating the
biological Characteristics of synthetic fuels technologies and in developing
suitable bioassay systems. A mechanism was required to ensure the maximum
utilization of these materials. Maximum utilization can only be achieved if
the chemical and biological histories of the material are defined. Defined
storage and chemical history also allows an assessment of the comparability
of bioassay results between laboratories and of the degree to which bioassay
results may be:-extrapolated to the technology as a whole.
These objectives are being met by combining the efforts underway within
the Bio/Organic Anal sis Section of the Analytical Chemistry Division, sponsor-
ed by the EPA, DOE, and EPRI, to the degree that joint interests allow. Re-
search materials acquired in the execution of these programs are maintained
within the repository and made available to all programs, subject to the
approval of the technology developer supplying the sample and of the agency
project officers involved. Measurement and sample preparation methodologies
developed as results of these studies are transferred to the repository per-
sonnel for supporting EPA use of repository materials. Direct support of
EPA health effects research is provided through input into design, prepara-
tion of materials for bioassay, characterization of materials undergoing bio-
assay, and assistance in acquiring sample materials of particular interest.
A mechanism is being established to ensure that sample contributors are in-
formed of physical, chemical, and biological data resulting from the use of
samples provided.
Research and Services Accomplished
1. The repository currently contains 108 sample materials derived from
five coal liquefaction or purification processes, two coal gasifi-
cation processes, three shale oil recovery processes, and four coal
combustion sources. Recent acquisitions include suites of samples
from shale oil recovery operations in the USA and USSR. Material
storage and inventory facilities are being upgraded.
2. A total of 110 samples have been distributed to 22 investigators
in 32 requests.
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hydrgparbons in stream water and sediment from the vicinity of a
coal coking operation.
4. Polycyclic Organic Matter of Fly Ash (Electric Power Research In-
stitute).Polycyclic organic matter adsorbed on fly ash is being
compared with that found on electrostatic precipitator ash to de-
termine whether bulk ashes can be utilized as surrogates for chem-
ical study of fly ash and to determine the nature of organics
available for ash pond leaching.
5. Inhalation Bioassay Monitoring (National Cancer Institute). Tobac-
co smoke inhalation bioassays carried out at other laboratories for
the Smoking and Health Program are supported through the collection
and analysis of samples taken at the bioassay laboratory, the devel-
opment of monitoring and dosimetric methodologies and instrumenta-
tion, and general troubleshooting. Aerosols provided to the animals
for inhalation by the various systems in use are chemically and
physically characterized to define exposures.
6. Tobacco Smoke Analysis (National Cancer Institute). The smokes and
smoke condensates produced by experimental cigarettes undergoing
biololical study are characterized through the quantitative deter-
mination of particulate and vapor phase constituents. Special
studies and analytical services are provided as required by the
project officer.
6. TASK TITLE:
Preparation and Characterization of Fine Particulate Envir-
onmental Contaminants for Biological Experimentation.
HERL/RTP TASK NO.: 8171
CONTRACTOR: IIT Research Institute
CONTRACT NO.: 68-02-2451
Summary
Contract 68-02-2451 supports health effects studies conducted by EPA
and by EPA contractors by providing preparation and characterization of parti-
culate matter in the respirable size range. Several types of particles pro-
duced by energy-related processes have been provided. This contract termina-
ted on March 31, 1978. Future plans for this type of contract depend com-
pletely on the plans for future health effects studies.
Scope and Objective
This contract was initiated for the purpose of providing well-charac-
terized particulate matter resulting from various energy processes for use in
in vivo and in vitro toxicity studies. 'Therefore, this contract functions in
a supporting role to other contracts. The particulate matter supplied has
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included mineral fibers-amosite, fibrous amphiboles from the Peter Mitchell
Pit (PMP) of Reserve Mining Company, and Cummingtonite-benzo(a)pyrene/lead
oxide suspensions in gelatine-saline, and benzo(a)pyrene/ferric oxide combin-
ations in a powdered state. The prepared materials are delivered to the EPA
or to designated EPA contractors.
The aforementioned particulate matter is reduced in size if necessary
into the respirable size range and then is characterized as to particle size
distribution and chemical properties.
Background and Approach
This contract is essentially a continuation of similar work done by the
same contractor on Contract No. 68-02-1687 which was a 3 year effort. The
contractor served as a centralized source for preparation and characterization
of selected particulate materials. As such the contractor developed much ex-
pertise in the area of mixing benzo(a)pyrene and metal oxides to produce ei-
ther suspensions or dry powders for in vitro testing. Both ball milling and
co-precipitation were examined and evaluated as methods for making these mix-
tures.
Work on mineral fibers was begun in the last year of Contract No. 68-
02-1687. Fibers were extracted laboriously from taconite rock found in the
Peter Mitchell Pit. These fibers were reduced in size to be comparable to
samples found polluting"the environment.
Research Accomplished
Fibrous amphiboles-both PMP fibers and UICC amosite-were supplied accor-
ding to schedule for use in in vitro studies conducted by Northrop Services
Inc. (NSI) Research Triangle Park, North Carolina. This preparation did in-
volve a research effort as it had not been attempted previously. Ambient air
samples were collected in areas of contamination. Fibers of similar chemical
composition to those in the air samples were separated from the rocks contain-
ing them and then milled to produce a particle size distribution similar to
that found in the air samples. These samples were supplied to NSI in a mul-
titude of small sealed vials containing an inert atmosphere. Physical and
chemical properties were determined. Standard UICC amosite was also supplied
in the same manner, and its properties were also determined.
Suspensions of benzo(a)pyrene/metal oxide were prepared by ball-mi 11 ing
and supplied for carcinogenesis studies. For each batch that was supplied, a
particle size distribution and the percentage of benzo(a)pyrene and metal
oxide were determined.
A powder of benzo(a)pyrene in combination with ferric oxide was sup-
plied to EPA for use in in vitro studies. Ferric oxide was sintered at dif-
ferent temperatures to obtain groups of particles having different surface
areas; then the fraction of each batch between 1 and 5 fim aerodynamic diameter
was separated. These three batches of ferric oxide having the same particle
size range but different surface areas were then combined with benzo(a)pyrene
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by precipatton of the organic compound from solution onto the ferric oxide.
The particle size distribution of each batch and percentage ferric oxide and
benzo(a)pyrene were determined.
Finally, work was conducted to set up a system for aerosol generation
and monitoring of fly ash from a conventional power plant. This work was per-
formed for an EPA Contractor.
The significance to the energy program of the above-described work is
directly related to that of the health effects research projects utilizing the
prepared particles and the generation systems.
Bibliography
As of yet there have been no publications resulting from this work
other than the required quarterly reports.
H. TASK TITLE:
Effect of Material from Alternate Energy Sources on Whole
Animal Defense Mechanisms
HERL/RTP TASK NO.: 8173
CONTRACTOR: Southwest Research Institute
CONTRACT NO.: 68-02-2286
Summary
This task is one of several which will investigate the effects of par-
ti cul ate effluents from conventional and advanced energy processes on host
defense mechanisms. This project will utilize both in vitro and in vivo
inhalation exposure models to assess effects on alveolar macrophages, the
primary defense cell of the deep lung. By comparing the results of the jn.
vitro exposure of guinea pig and baboon alveolar macrophage to the in vivo
exposure of guinea pigs, it may be possible to predict the likelihood of using
the in vitro system as a screening tool. The inhalation exposure will be used
to determine dose-response effects and the presence of any delayed effects
and recovery. Because of delays in acquiring particulate samples for testing,
only preliminary tests have been conducted to date.
Scope and Objective
The purpose of this project is to compare the effects of conventional
and advanced coal utilization processes on pulmonary disease, primarily in^
fectious disease. Animals will be exposed acutely and intermittently to par-
ticulate effluents and examined immediately and at various times after expo-
sure to allow observation of possible delayed effects or recovery. Various
biological endpoints, including the alveolar macrophage, will be studied. Iji
vitro exposures will also be conducted on alveolar macrophages from 2 animal
species to permit correlations to validate possible in vitro models. Dose-
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response studies will be conducted.
Background and Approach
With increasing use of new, as well as conventional, energy technolo-
gies, it is important to define any possible association between particulate
by-products of these processes and risk to the public health. Since these
pollutants would most likely be delivered to man via inhalation, the lung is
a possible target site. The alveolar macrophage which is primarily respon-
sible for clearing debris and maintaining sterility of the gaseous exchange
regions will be the focus of the experiments. This essential host-defense
cell is susceptible to damage by 03, N02, and metallic particles and will most
likely phagocytize energy-related particulates, thereby increasing the likeli-
hood of adverse effects. Histamine content of the lung will also be measured.
If effects are found, this endpoint, which would be related to asthma, will
be investigated further.
Guinea pigs will be exposed via inhaltion to various doses of the ef-
fluent sample. Alveolar macrophages from guinea pigs and baboons will be ex-
posed in vitro. From the in vivo and in vitro exposures of guinea pig alveo-
lar macrophages, it can be determined if the in vitro system is relevant. If
it is, then by comparing the results of the guinea pig and baboon macrophages,
improved predictions of possible human effects may be possible. In addition,
if the in vitro testing is validated, future tasks could be designed for
screening. Due to the large variety of particulate effluents and the diffi-
culities in obtaining large quantities of sample, in vitro screening would be
helpful in ranking samples for later in vivo testing.
Several functions of the alveolar macrophage will be investigated to
permit better determination of the biological significance of any observed
effects. These functions include: phagocytosis, response to lymphokines
(macrophage migration inhibition factor which is involved in keeping the cell
at the site of inflammation so that is may cause destruction of the cause of
inflammation), tumoricidal capability, and bactericidal activity. Enzyme
activities of the alveolar macrophage which relate to bactericidal activity
and the cell's role in fibrosis will also be studied.
Research Accomplished
Due to the delay of EPA in obtaining particulates for testing, work was
conducted at a very slow pace until recently and involved development of meth-
odologies. Animals are now being exposed to particulate (Onm) from an
electrostatic precipitator of a conventional power plant. Data are being col-
lected on the parameters listed above, but, as yet, the number of animals
tested is too small for accurate statistical analyses.
Related Research
Task Nos 8162, 8149, 8163, 8173, and 8198 are all related in that each
is designed to study various aspects of the effects of actual particulates
from energy sources on host defense mechanisms. It is intended that all the
work will be conducted on the same particulate sample so that results of all
145
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the studies can be compared and used to develop better assessment of effects.
In isolated instances, sample availability problems may cause some deviations
from this approach.
In addition, the particulate sample used in this study will also be
used for Task No. 8317, entitled "Effect of Industrial Particulate Emissions
on Alveolar Macrophage," which is designed to correlate the in vitro alveolar
macrophage system with the infectivity model. Under this Air Health Task, the
sample will be used in the in vitro portion of the study only, but results
will be compared to the in vivo model of Task 8162. The sample will also be
assessed under Task No. Wuisingh) for mutagenic and carcinogenic potential
using in vitro screening systems.
Please see the individual task reports for details of these studies.
I. TASK TITLE:
Environmental Mutagens Studies Utilizing a Drosophila Test
System
HERL/RTP TASK NO.: 8189
CONTRACTOR: Dr. John Baum
CONTRACT NO.: IAG D701207
Summary
The investigation will test the feasibility of adding the Drosophila
system to the battery of tests presently utilized in the assessment of envir-
onmental mutagens. Current efforts are directed toward development of dose-
response information and comparison of sensitivity of various wild type Dro-
sophila strains.
Scope and Objectives
The initial objective of this research is to evaluate alternative ap-
proaches for adding Drosophila mutagenesis experiments to complement the bat-
tery of tests currently utilized to evaluate mutugenic effects of ambient air
pollutants. This evaluation will proceed in the following steps:
1. Several wild type Drosophila strains will be tested for sensitivity
(sex-linked recessive lethal test) to ethylene dibromide and other
appropriate index mutagens. Experimentation will be conducted
under a variety of dosage schedules to determine the feasibility
of performing field experiments simultaneously and in the same
mobile exposure facilities with Tradescantia.
2. If the above experimentation demonstrates that any strains studied
are sufficiently sensitive to be appropriate for field tests,
deploy the tests to approximately one site per month to test for
mutagenic effects from direct exposure to ambient air pollutants.
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3. If no suitable wild type strain is identified, an effort will be
undertaken to identify other more sensitive, e.g. repair deficient
strains. Upon demonstration that any such strain is suitable for
deployment in the field, tests will be conducted as in (2) above.
4. If no strains are appropriate for field experiments, feeding exper-
iments will be undertaken as follows: The material collected from
high volume air samplers will be removed and mixed with Drosophila
food. The Drosophila will be raised from egg stages to adult
stages on this mixture. The effect of this treatment will be eval-
uated by sex-linked recessive lethal test.
5. An attempt will be made to develop and utilize a protocol to inject
in Drosophila a portion of the material collected from the filters.
The insects treated in this manner will be subjected to sex-linked
recessive lethal test.
Background and Approach
For several years Brookhaven National Laboratory, through an agreement
with National Institute for Environmental Health Sciences, has been working on
the development of a Tradescantia test system which would be sensitive to air
pollutants. Following successful experiments in controlled environments, a
mobile facility was constructed which would allow testing under actual, as
opposed to simulated environments. Because of the need for simultaneous air
measurements during field tests, HERL was asked to collaborate.
Because of our continuing efforts to determine appropriate locations
for epidemiologic studies of environmental carcinogenesis, it was decided that
HERL should support the effort so that experiments could be conducted in areas
under consideration for epidemiologic studies either because of high cancer
rates or because of known or suspect carcinogens in environment.
In addition to the provision of a mobile van with a standard complement
of aerometric equipment, HERL has developed methods for collection and trans-
port of substantial quantities of both particulate and vapor phase pollutants
for chemical analyses and for additional mutagenicity tests.
The battery of tests which are currently available for further assess-
ment of these samples includes bacteria systems, tissue cultures and neoplas-
tic transformation. Addition of the Drosophila system is desirable for sever-
al reasons: heritable rather than somatic mutations can be assessed, Droso-
phila can be placed in the exposure chambers and tested simultaneously with
with the plants, Drosophila can carry out metabolic activation as it occurs in
mammalian liver, and detailed analysis of the various types of genetic changes
can be undertaken to further understand the mechanisms and possible relation-
ships between mutagens, teratogens and carcinogens.
A close working relationship has been established between the two
groups of geneticists who will be working on this project through a similar
ongoing research effort supported by ERDA to study the mutagemc effects of
magnetic fields in both Tradescantia and Drosophila. Provision of facilities
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acquired in the course of this ERDA supported research constitutes a sub-
stantial contribution to this agreement.
Research Accomplished
Dose-response studies which have been performed to date seem to indicate
that the sensitivity of this system is inappropriate for deployment into the
field. This finding would not preclude laboratory tests of concentrated
pollutant samples. Tests of relative sensitivity of ten wild type strains
do not provide a basis for selection.
Bibliography
No publications or presentations have been prepared to date as a result
of this research.
Related Research
This research project is closely coordinated with the ongoing Trade-
escantia studies involving Brookhaven National Laboratory, National Institute
for Environmental Health Sciences and EPA. Samples of pollutants collected
at the various study locations are provided to Dr. Michael Waters, ETD, HERL
for further bioassay.
J. TASK TITLE:
To Evaluate Existing and Improved Methods for Sampling, Trans-
port, Storage and Analysis of Biological Specimens Which Might
Serve as Indicators of Contamination by Effluents from Energy
Technologies
HERL/RTP/TASK NO.: 8194
CONTRACTOR: National Bureau of Standards
CONTRACT NO.: IAG-D5-0568
Summary
The Specimen Bank is a national program using a systematic approach and
standardized protocols to collect, analyze and store environmental and bio-
logical samples and data to be used to assess the accumulation and movement
of harmful chemicals in the biosphere.
The concepts of the National Environmental Specimen Bank (NESB), real
time monitoring and retrospective analytical capabilities are derived from its
dual function. First, representative portions of samples included in the
bank would be analyzed at the time of their introduction to provide real time
monitoring and evaluation of pollutant trends.. Evaluation of these trends
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would servers early warning sentinels, triggering proper control measures
to halt rising human body burdens before irreversible damage could occur.
Second, a specimen bank would enable analytical scientists to use torn-
morrow s more sensitive and specific methods of chemical analysis on today's
samples. The improved measurement methodology would enable health scientists
to determine accurate levels for substances that would be either undetectable
or poorly analyzed by today's less sensitive methodology. The existence of
a specimen bank would provide the opportunity to determine what the body
burden of newly recognized toxic substances was in the past and to determine
if their levels had changed with time.
The methodology developed under this program is now at the stage where
it should be field tested. A Specimen Bank Pilot Program is scheduled to
start in FY'79.
Scope and Objectives
The intention of this research, in collaboration with the National
Bureau of Standards (NBS), is to evaluate and develop, where necessary,
methods for sampling, transport, storage and analysis of biological specimens
which might serve as indicators of contaminants by effluents from energy
technologies, industry, and agriculture.
Background and Approach
Industrialized nations are being constantly reminded about the potential
dangers to human health and the environment by the ever increasing inflex of
new man-made substances into our ecosystem, Kepon and PBB being recent ex-
amples. The U. S. EPA is especially aware of this situation and is presently
studying the feasibility of establishing a program, the NESB, that would pro-
vide a formalized, systematic approach to assess the environmental impact of
these substances at a national level.
The adverse effects of some environmental agents on human health are
already well established, although for many diseases, an environmental etio-
logy is only suspected, and, in the remaining cases, the cause is unknown.
In order to assess the hazards of pollution for populations at risk, it is
necessary to have a knowledge of the pollutant exposure. This exposure of
human populations to environmental agents can be assessed by an integrated
approach to monitoring. In this manner, the total exposure of a population
at risk can be evaluated in terms of the relative contributions from each
route of exposure. Once the relative contribution of each environmental
pathway has been determined, routine monitoring, for assessment purposes,
can be limited to the critical routes, making monitoring more cost-effective.
Environmental monitoring of pollutant levels (air, water, food etc.)
provides one approach to the assessment of human exposure. Another approach
is provided by biological monitoring; the determination of pollutant levels
in human samples. The immediate advantage of biological monitoring compared
to environmental monitoring is that, in principle, it makes it possible to
obtain a direct measure of dose of exposure. It is through environmental
149
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and biological monitoring that information of the magnitude of pollutant ex-
posure in man is obtained. That information must then be related to the
health effects produced by the pollutant.
The NESB concept of a national program functioning under a formalized
systematic approach is in sharp contrast to the many individual biological
and environmental monitoring programs being conducted in this country. The
salient feature of the NESB program is the generation of validated data and
the ability to compare data from one region of the country with that of
another. At present, with a myriad of sampling, analysis and storage proto-
cols being used, validation and comparison of data is often impossible,
making most data speculative at best.
EPA's NESB is a well defined system of collection, analysis and long-
term storage of selected environmental samples, providing two important out-
puts. First, the analytical portion of the NESB will provide real time mon-
itoring data for pollutant trend analysis. This data will provide infor-
mation on the adequacy of our present control technology and criteria
standards. In addition, the real time monitoring data locates potential
pollutant "hot-spots", thus serving as an environmental alarm system. Second,
the NESB will provide properly stored samples for retrospective analysis en-
abling health scientists to determine accurate levels of substances that
would be either undetectable or poorly analyzed by today's less sensitive
techniques.
Research Accomplished
The FY 78 funds have been used to partially fund an interagency agree-
ment with the NBS for the development of state-of-the-art methodologies for
sampling, collection, preparation, analysis, and storage of biological
indicator specimens which reflect contamination by effluents from energy
technologies and other sources. The ultimate goal of the project is the
development of the NESB.
Methodology development has been undergoing extensive laboratory test-
ing over the past three years. Accomplishments to date include: identifica-
tion of sample container material, sample handling techniques, sample prep-
aration for trace element analysis and standardization of analytical tech-
niques for twelve trace elements. In addition, investigations are underway
to study the effects and conditions of long-term storage on sample integrity.
The scientific effort is at the point now where it is necessary to scale up
the developed protocols from the "lab-bench" operation to a modified banking
program—the Environmental Pilot Bank. The pilot bank effort would give the
scientists actual working experience-through all stages of the banking effort,
including: (1) specimen collecting; (2) preparation; (3) analysis and; (4)
storage. The pilot program would concentrate on a limited number of samples,
collected, analyzed and stored in a central facility. The approach would
allow for strict control and constant evaluation over all operational pro-
cedures. Problems encountered in any aspect of the program would undergo
extensive review, detailing the extent of the deficiencies so that corrective
measures may be initiated and validated.
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,-„ The initiation °f the Pilot Bank Program is scheduled for FY 79. During
FY 78, the following tasks are scheduled:
1. Samples of human liver and marine mussel tissue will be collected
and_analyzed for trace elements, and stored in a low temperature
facility. Evaluation of the homogenization and sample splitting
techniques will be made. Sample subsets will be freeze-dried and
checked for elemental losses and contamination.
2. Continuation of protocol development for sampling and storage
techniques in human tissue, food grain, aquatic intergrator and
air intergrator matrices.
3. Initiate studies for organic contaminants in the above mentioned
matrices, including sampling, storage, and analysis protocol
development.
4. Modification of NBS clean-room facility for Pilot Bank sample
preparation and storage. Install ultra-low temperature storage
equipment.
Bibliography
Becker, D. A. 1976. Environmental Sample Banking-Research and Methodology.
Trace Substances in Environmental Health-X. A symposium. D. D.
Hemphill, Ed.
Becker, D. A. and E. J. Maienthal. 1977. Evaluation of the National Envir-
onmental Specimen Bank Survey. EPA-600/1-77-015. February 15.
Gills, T. E., H. L. Rook, and P. D. LaFleur. 1978. Evaluation and Research
of Methodology for the National Environmental Specimen Bank. EPA-000/1-
78-015. February.
Goldstein, G. M. 1977. The National Environmental Specimen Bank, Its Con-
cepts, History and Objectives. International Workshop on The Use of
Biological Specimens for the Assessment of Human Exposure to Environ-
mental Pollutants. Luxembourg, April 18 - 22.
Goldstein, G. M. 1978. Plan for a National Environmental Specimen Bank.
EPA-600/1-78-022. March.
Mavrodineau. R. 1977. Procedures Used at the National Bureau of Standards
to Determine Selected Trace Elements in Biological and Botanical Mater-
ials. NBS special publication 492. November.
Rook, H L., and G. M. Goldstein. 1978. The National Environmental Specimen
Bank. NBS special publication 501. February.
Rook H L and G M. Goldstein. 1977. Recommendations of the EPA/NBS
'workshop on the National Environmental Specimen Bank. EPA-600/1-77-020.
Apri1.
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Van Hook, R. I., and E. E. Huber. 1976. National Environmental Specimen
Bank Survey. EPA-600/1-76-006. January.
Related Research
This project is a cost sharing exterprise in which the NBS participates
in the funding. In addition, the EPA has a bilateral agreement with the
Environmental Agency of the Federal Republic of Germany to jointly share in
the scientific investigation, leading to the development of the NESB.
K. TASK TITLE:
Effect of Pollutants from Coal Burning and Coal Gasification
on the Immune System
HERL/RTP TASK NO: 8198
CONTRACTOR: Pennsylvania State University
College Park, Pennsylvania 16802
CONTRACT NO: 68-02-2472
Summary
This task is one of several designed to investigate the effects of
particulates from conventional and alternate energy sources on host defense
systems. This particular task is focused on effects of chronic particulate
exposure on the systemic and pulmonary immune system. To date, preliminary
studies have been conducted on the effects of a continuous 56 day exposure
to carbon, fly ash (bag-house, conventional power) and particulate from an
electrostatic precipitator (conventional power). It would appear from pre-
liminary tests that the humoral immune system is more affected than the cell
mediated immune system. The differences between the effects of the 3 types
of particulates could be due to differences in concentrations of exposure.
Scope and Objective
The objective of this task is to determine the effects of particulate
effluents from conventional and alternate energy sources on the immune sys-
tem. Mice will be chronically exposed to either particulate effluent, car-
bon or air prior to assay of the humoral and cell mediated immune system.
Both the pulmonary and systemic immune system will be examined. The phago-
cytic activity of alveolar macrophages from the exposed animals will also be
tested.
Background and Approach
Previously, it has been shown that chronic inhalation exposure to carbon
can cause alterations of the immune system which is responsible for defending
the host against infectious and neoplastic disease. If carbon can induce
these changes, it would be prudent to determine if particulates from con-
ventional or alternate energy processes exert a similar effect. It would
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also be helpful to regulatory decision making to compare the effects of car-
bon to energy-related particulates. Therefore, such studies are being con-
ducted using the same model system used for the original work with carbon.
Mice will be exposed chronically and examined periodically during exposure.
Parameters for cell mediated immunity include: mitogen-induced trans-
formation of T cells from the spleen and mediastinal lymph nodes which drain
the lungs, functioning of recognitive T cells using mixed lymphocyte culture
techniques, and ability of T cells to kill tumor cells. For humoral immunity
the following paramters will be examined: mitogen-induced transformation of
P cells from the spleen and mediastinal lymph nodes, antibody production by
lymphocytes from the spleen and mediastinal lymph nodes following aerosol
immunization with bacteria, and circulating antibody titers after aerosol
immunization. Alveolar macrophage phagocytic activity will also be investi-
gated since it plays a role in pulmonary immunity.
Research Accomplished
Due to unavailability of an EPA-supplied particulate effluent, work was
begun with carbon and fly ash from a bag house of a conventional power source,
Recently, an EPA supplied particulate from an electrostatic precipitator of a
conventional power plant has been used. Since exposures are for 56 days,
only 2 replicates have been conducted and statistical analysis is not yet
complete. Therefore, all results should be considered to be preliminary.
Since results are preliminary, for brevity, only approximate exposure
concentration ranges will be given here. Some endpoints were examined on
different tests, hence, the range of concentrations. The concentrations
were as follows: Penn State-supplied fly ash: .66-93 mg/m3 (< 2.1 urn); a
total of 1.9-2.3 mg/m3 (< 5 jzm). EPA-supplied particulate, .37-.69 mg/m3
(<2.1 Mm); a total of 1.1-3.1 mg/m3 (<5M"0 carbon .84-1.5 mg/m3 (<2.1 urn);
a total of 5.4-2.5 mg/m3 (< 5 urn). Generally, exposures caused few effects
on cell-mediated immunity. However, for the number of antibody producing
spleen cells after aerosol bacterial immunization, carbon caused a depression
which was observed at days 7-56. The Penn State-supplied fly ash caused a
reduction at day 21 only and the EPA-supplied particulate caused no effects
after 35 or 56 days of exposure. Mediastinal lumph nodes did not appear to
be affected by any of the treatments. Serum antibody titers were reduced
after 7, 21, and 56 days of carbon exposure. The Penn State supplied fly
ash was effective at 21 and 56 days, but the EPA supplied fly ash only caused
depression after 56 days of exposure. Mitogen-induced transformation of B
and T cells did not appear to be afftected by 35 days of exposure to carbon
or Penn State-supplied fly ash. EPA-supplied particulate caused no effects
after 1 week of exposure. Results of the studies on alveolar macrophages
and lymphocyte tumoricidal capability are too preliminary to report at this
time.
Related Research
Task Nos. 8162, 8149, 8163, 8173, and 8198 are all related in that each
is designed to study various aspects of the effects of actual particulates
from energy sources on host defense mechanisms. It is intended that all the
153
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work will be conducted on the same particulte sample so that results of all
the studies can be compared and used to develop better assessment of effects.
In isolated instances, sample availablility problems may cause some deviations
from this approach.
In addition, the particulate sample used in this study will also be used
for Task No. 8317, entitled "Effect of Industrial Particulate Emissions on
Alveolar Macrophage," which is designed to correlate the in vitro alveolar
macrophage system with the infecticity model. Under this Air Health Task,
the sample will be used in the in vitro portion of the study only, but results
will be compared to the in vivo model of Task 8162. The sample will also be
assessed under Task No (Huisingh) for mutagenic and carcinogenic potential
using j_n_ vitro screening systems.
Please see the individual task reports for details of these studies.
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SECTION 5
OTHER CATEGORY PROJECTS
Title Status*
The Pharmacodynamics of Certain Endogenous Mammalian A
Antioxidants During N02 Exposure
Contractor: Stanford Research Institute
Contract No. 68-02-1713
HERL/RTP Task No.
Human Biochemical and Physiological Response to Acute A
Photochemical Air Pollution Exposure
Contractor: Copley International
Contract No. 68-02-1724
HERL/RTP Task No. 8175
Chromosomal Aberrations fn Peripheral Lymphocytes of A
Students Exposed to Air Pollutants
Contractor: University of Utah
Contract No. 68-02-1730
HERL/RTP Task No.
Nitrogen Dioxide Trends in Selected Chattanooga Communities A
Contractor: Research Triangle Institute
Contract No. 68-02-1737
HERL/RTP Task No.
Chromosomal Abnormalities Associated with Known Low Level
Occupational Ozone Exposure in Welders
Contractor: Columbia University
Contract No. 68-02-1738
HERL/RTP Task No.
The Operation and Maintenance of Controlled Environmental C
Labs (CEL) and Mobile Physiologic Medical Vehicle (MPMV)
CLEANS
Contractor: Rockwell International
Contract No. 68-02-2485
HERL/RTP Task No.
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Determine Relative Irritant Potency of (a) Participates A
Resulting from Oxidation to Sulfur Oxide, (b) Inert
Particles Interacting with Sulfur Oxide.
Grantee: Harvard School of Public Health
Grant No. RO-802030
HERL/RTP Task. No. 6307
Evaluate Effects of Chronic or Intermittent Exposure
to Respirable Particles and Mists Using Mouse Pulmonary
Infectivity Model
Contractor: IIT Research Institute
Contract No. 68-02-1717
HERL/RTP Task No. 6711
Cytotoxicity Evaluation of Selected Sulfates and of A
Source and Ambient Air Samples
Contractor: Northrop Services
Contract No. 68-02-1567
HERL/RTP Task No. 6713 and 5101
Implementation of Screening Tests for Potentially A
Hazardous Airborne Particulate Material
Contractor: Northrop Services
Contract No. 68-02-1566
HERL/RTP Task No. 8150
Workshop on Screening Systems for Alternate Energy Sources A
Contractor: Kappa Systems
Contract No. 68-02-2435
HERL/RTP Task No. 8154
Operation and Maintenance of Community Health Air Monitoring C
Program to Quantitate Air Pollution Exposure in Selected
Health Study Areas
Contractor: Xonics, Inc.
Contract No. 68-02-2493
HERL/RTP Task No. 7167
•
Interactions of Various Pollutants on Causation of Pulmonary A
Disease
Contractor: IIT Research Institute
Contract No. 68-02-2274
HERL/RTP Task No. 7174^
Human Biochemical and Physiological Response to Acute
Photochemical Air Pollution Exposure
Contractor: Copley International
Contract No. 68-02-1724
HERL/RTP Task No. 8175
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Field Testing—Application of Improved Combustion
Technology to Power Generation Combustion Systems
Contractor: Exxon
Contract No. 68-02-1415
HERL/RTP Task No. 7184
Calibrate Cyclone Used to Obtain Large Quantities of
Size Separated Particulate
Contractor: IERL (Piggyback)
Contract No. 68-02-2131
HERL/RTP Task No. 7186
Chemical Characterization and Specimen Preparation of
Fibrous Amphiboles and Refined Particles
Contractor: GCA
Contract No. 68-02-2771
HERL/RTP Task No. 8188
Furnish Rabbits
Contractor: Pel-Freeze BioAnimals
Contract No. 68-02-1638
HERL/RTP Task No.
Assessment of the Postnatal Development and Function of
the Central Nervous System of Monkeys Exposed to Tritiated
Water from Conception to Birth or Weaning
Contractor: SRI
Contract No. 68-02-2280
HERL/RTP Task No.
Investigate Neoplastic and Life Span Effects on Potentially
Sensitive Populations of Rats Chronically Exposed to Tritiated
Water
Contractor: Dawson Research Corporation
Contract No. 68-02-2289
HERL/RTP Task No.
Addition of Mobile Air Monitoring Field Stations and Portable
Air Pollution Monitors to CHAMP System
Contractor: Rockwell International
Contract No. 68-02-0759
HERL/RTP Task No.
Operation, Calibration and Maintenance of Total CHAMP
System
Contractor: Rockwell International
Contract No. 68-02-1745
HERL/RTP Task No.
157
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Effects of N02 on Lung Function in Human Subjects with A
Asthma and Chronic Bronchitis
Contractor: University of Maryland
Contract No. 68-02-1745
HERL/RTP Task No.
Stationary and Mobile Facilities for Study of Health B
Effects of Environmental Contaminants (CLEANS/
CLEVER)
Contractor: Computer Sciences
Contract No. 68-02-0768
HERL/RTP Task No.
Scanning Electron Microscopic Examination of the Effects A
of Air Pollutants on Pulmonary Systems
Contractor: IIT Research Institute
Contract No. 68-02-0761
HERL/RTP Task No.
Preparation and Characterization of Fine Particular A
Environmental Contaminants for Biological Experiments
Contractor: IIT Research Institute
Contract No. 68-02-1687
HERL/RTP Task No.
Addition of Equipment for Generation and Monitoring Aerosols B
in the CLEANS Clinical Exposure Chambers
Contractor: Environmental Research Tech
Contract No. 68-02-2300
HERL/RTP Task No.
The Effects of Low Level N02, 03, and Ambient Air, A
Separately and in Combination, on Cardiac, Pulmonary
and Peripheral Circulatory Functions in Adult Males,
in Response to Heat Stress at Rest and During
Moderate Exercise
Contractor: University of California
Contract No. 68-02-1723
HERL/RTP Task No.
Collection and Characterization of Naturally Occurring A
Airborne Particulates
Contractor: NBS
EPA IA6 D4-F531
HERL/RTP Task No.
Operation and Maintenance of CLEANS C
Contractor: Rockwell International
Contract No. 68-02-2485
HERL/RTP Task No.
158
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Human Biochemical and Physiologic Response to Acute
Photochemical Air Pollution Exposure
Contractor: Copley International Corporation
Contract No. 68-02-1724
HERL/RTP Task No. 8175
159
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TECHNICAL REPORT DATA
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i
1. REPORT NO.
EPA-600/7-79-009 J
:3. REC.f-'.l-.NT'S A-'' LSSiON NO
4. TITLE AND SUBTITLE ;3 r EFORT U
INTERAGENCY PROGRAM IN ENERGY-RELATED HEALTH AND j January 1979
ENVIRONMENTAL EFFECTS RESEARCH - Project Status Report t--p6rtFOHMIKG -"^AN.,:AT.oNcoob
7. AUTHOR(S)
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Health Effects Research Laboratory
Office of Health and Ecological Effects
U.S. Environmental Protection Agency
Research Triangle Park, N.C. 27711
8. PE^I-Ofil^ING ORGAN! ZATi-_.
I
MO FFOCM \M ELEMENT NO.
_
'. i CONTRACT/GRANT HO.
12. SPONSORING AGENCY MAME AND ADDRESS
Office of Health and Ecological Effects
Office of Research and Development
U.S. Environmental Protection Agency
Washington. DC 20460
I .3 TYFL Or REPORT AND PER.OC COVE
l« SPONSORING AGENCY CODE
EPA 600/1 ?
15. SUPPLEMENTARY NOTES
16. ABSTRACT
ABSTRACT
This report summarizes research supported by the EPA Health Effects
Research Laboratory at Research Triangle Park, NC, under the Federal Inter-
agency Energy/Environment R & D Program. The EPA has had the lead responi-
bility for the planning, coordination and implementation of this program
since fiscal year 1975.
Projects reported in this document are grouped under one of four major
research areas. The first area is identification of hazardous agents
associated with non-nuclear energy technologies. These projects involved
the development of qualitative methods for the identification of hazardous
materials. The second area is development of more rapid and sensitive
methods to evaluate dose to man. These projects focused on the development
of quantitative methods for measuring degree of toxicity of various pol-
lutants. The third area is determination of the metabolism and fate of
hazardous agents associated with energy technologies. These projects in-
volved determination of the physiological activities of several known carcin-
ogens. The fourth research area is evaluation of hazards to man. In addi-
tion to studies of the effects of certain pollutants on humans, several of
the projects concerned preparation of standard pollutant samples for use in
future studies to increase the comparability of results.
A list of additional studies funded under this program is included.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
bioassay
hazardous agents
energy
environments
metabolism
carcinogens
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI i'iuld/GrOUp
06 F, T
18. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19 SECURITY CLASS (Tim Report)
UNCLASSIFIED
21. NO. OF PAGES
167
20 SECURITY CLASS ,Thif i^age)
UNCLASSIFIED
22 PRICE
EPA Form 2220-1 (Rev. 4-77)
Ol TlO>. i .*, C Hr*' -FT T h
160
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