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Identification and
Evaluation of
Potential
Physiological
Toxicity Assays
Final
Report
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IDENTIFICATION AND EVALUATION
OF POTENTIAL PHYSIOLOGICAL
TOXICITY ASSAYS
George H. Kidd, John M. Rice, Melanie E. Davis,
Mark A. Hurst, Mickey F. Arthur, Steven E. Pomeroy
and Martin L. Price
BATTELLE
Columbus Laboratories
505 King Avenue
Columbus, Ohio 43201
Final Report
Contract No. 68-01-5043
Prepared for
U.S. Environmental Protection Agency
Office of Pesticides and Toxic Substances
Washington, D.C.
January 1980
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DISCLAIMER
This report has been reviewed by the Office of Pesticides and Toxic
Substances, U.S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily reflect
the views and policies of the U.S. Environmental Protection Agency, nor does
mention of trade names or commercial products constitute endorsement or
recommendation for use.
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SUMMARY
Battelle's Columbus Laboratories has contracted with the Office of
Pesticides and Toxic Substances, U.S. Environmental Protection Agency, to
develop a list of physiological assays as potential toxicity screening tests
and to assess the strengths and weaknesses of these assays (Contract No.
68-01-5043). After an extensive literature search, Battelle has compiled a
list of 24 assays, covering all of the categories cited by OPTS/EPA in its
Technical Directive. Brief descriptions of assay methods and tables
containing critiques .of each assay are presented, along with literature
references for all of the assays.
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TABLE OF CONTENTS
Page
Disclaimer i
Summary ii
INTRODUCTION 1
Initial Identification and Screening of Potential
Assays 2
Organization of Information About Assays 7
Explanation of Data Tables Accompanying Each Assay 7
Assays From Woodard (1976) 11
DISCUSSION AND RECOMMENDATIONS 15
POTENTIAL TOXICITY ASSAYS—DESCRIPTIONS, METHODS, AND
ASSESSMENTS 19
Nitrogen Fixation 19
Photosynthesis 20
Respiration 28
High-Energy Phosphate Production 30
Growth and Cell Division 34
Catalysis (Enzymatic Activities) 47
Other Cellular Processes 55
Other Potential Physiological Toxicity Assays 67
TABULAR COMPARISON OF CRITERIA 71
REFERENCES 78
APPENDIX: LITERATURE SEARCH METHODS A-l
LIST OF TABLES
Table 1. Assays Considered but Not Evaluated 3
Table 2. Potential Toxicity Assays Addressed in This
Report 5
Table 3. Commercial Sources of Test Organisms 6
Table 4. Acetylene Reduction 21
Table 5. Hill Reaction 23
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LIST OF TABLES (CONT'D.)
Page
Table 6. Greening 25
Table 7. RuDP Carboxylase Activity 27
Table 8. Photosynthetic Oxygen Evolution 29
Table 9. Respiration in HeLa Cells 31
Table 10. Adenylate Energy Charge 33
Table 11. Cloning L929 Mouse Cells 36
Table 12. Protozoan Clonal Viability 38
Table 13. Human KB Cell Growth Rate 40
Table 14. Human Embryonic Lung Fibroblast (WI-38)
Cytotoxicity 42
Table 15. Mitogen Stimulation of Lymphocytes 44
Table 16. Chick Embryo Development 45
Table 17. Trypan Blue Dye Exclusion by Human KB Cells 48
Table 18. RNA Polymerase Activity 50
Table 19. Adenyl Cyclase Activity 52
Table 20. Lysosomal Enzyme Release 54
Table 21. Macromolecular Synthesis in Human KB Cells 56
Table 22. Cyclosis 58
Table 23. Hemolysis 59
Table 24. Protozoan Vacuole Contraction . 62
Table 25. Protozoan Motility 64
Table 26. Phagocytosis by Alveolar Macrophages 66
Table 27. Ami no Acid Transport 68
Table 28. Source of Test Organism 72
i v
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LIST OF TABLES (CONT'D.)
Page
Table 29. Test Organism and Organismal Level or
Parameter Evaluated 73
Table 30. Special Equipment 74
Table 31. Times,' Cost, and Technician Skill 75
Table 32. Data Base and Comments 76
LIST OF FIGURES
Figure 1. Schematic representation of viability test
adapted to toxicity testing 37
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INTRODUCTION
In this report, Battelle's Columbus Laboratories develops a list of
potential cellular, organellar, and enzymatic toxicity tests (here collec-
tively referred to as physiological toxicity tests or assays) for rapid
screening of potential toxicants by the Office of Pesticides and Toxic
Substances-Environmental Protection Agency (OPTS/EPA, Contract No.
68-01-5043). This study also documents, on the basis of a review of the
published scientific literature and of ongoing research efforts, the strengths
and weaknesses of selected physiological toxicity test methods.
At present, the testing strategies proposed for premanufacture
evaluation of chemical hazards to ecological systems utilize 96- or 48-hour
acute toxicity tests on fish or invertebrates, respectively, as the primary
(or only) screening methods to identify the need for further testing. There
are several problems with these assays. First, such acute tests are usually
poor predictors of the results of chronic studies, phytotoxicity tests, behav-
ioral tests, and multispecies ecological studies. Second, the standard short-
term acute tests depend significantly, not only on the toxic biological activ-
ity of a chemical substance, but also on its solubility, dispersability,
and/or ability to penetrate target cells or organs in the test organisms.
Such penetration is highly variable among species. Third, the time and cost
of the standard acute tests mentioned above are great.
To circumvent the problems arising from the fish and invertebrate
toxicity tests, a battery of rapid, in vitro, physiological tests could be
developed as a first screening tier of a step-sequenced testing strategy for
assessing ecological effects of chemicals. These physiological assays would
measure effects on major metabolic and cellular functions, and from these
functions, would attempt to predict effects on various test species and on
ecosystem function. It is further assumed by this approach that chronic
effects are the result of chemical toxicity at the cellular or subcellular
level, and that these rapid, physiological tests can predict chronic effects.
In sum, physiological assays might best serve as preliminary tests in ecologi-
cal effects test schemes.
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Described below are the theory and experimental procedures of these
potential physiological toxicity assays. Each assay method is evaluated by
several criteria, including the advantages and limitations, response to chemi-
cals, and special equipment required. Pertinent references are included along
with the criteria for each assay, as well as in bibliographic form at the end
of the report.
Initial Identification and Screening of Potential Assays
In the initial stage of Battelle's literature search (see Appendix),
some 45 potential toxicity tests were identified for consideration by
OPTS/EPA. Potential assays were either of particular interest to OPTS/EPA,
referenced in Woodard (1975), or independently identified by Battelle.
Of the 45 physiological assays considered, 21 have little promise for
development as rapid toxicity screening tests (Table 1). The assays marked
with an asterik (*) in Table 1 were not considered strong candidates for rapid
toxicity screens because they required use of isolated organs and tissues.
Isolated organs and tissues have been very useful in studying the mechanisms
of toxic action and in providing an understanding of mechanisms by which chem-
icals exert toxic effects. In this role, such systems should become increas-
ingly important. However, the use of isolated organs has serious limitations
for studying physiological or toxicological effects, partly because of the
modulating systems existing in the whole organism which can either increase or
decrease a chemical effect. The usefulness of organ systems for rapid toxi-
cological screening remains limited, primarily because such systems are most
useful for screening large numbers of chemicals for a site-specific effect as
opposed to screening a chemical for multiple biological effects. Also, in
most cases, isolated organ assay systems use almost as many animals as an in
vivo test would require. Consequently, there are little savings in total
animal usage. Given these considerations, no rapid, physiological toxicity
assays using tissue or organs are critiqued in this report.
Other potential assays (oxidative activity in rabbit endothelium,
chromosome breakage in human leukocytes, oxygen consumption by human
leukocytes) were rejected because of GLP problems. Endothelia and leukocytes
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TABLE 1. ASSAYS CONSIDERED BUT NOT EVALUATED IN DETAIL IN THIS REPORT
Plant callus growth
Inhibition of cell division of plant suspension cultures
Glycogen conversion in perfused rat liver*
Malting
Inhibition of axon myelation*
Invertase activity in duodenum culture*
Epithelial growth in mouse kidney tissue*
Cholinesterase inhibition in cerebral cortex*
Collagen synthesis in human pleura cultures*
Vitamin 8^2 uptake in monkey illeum*
Trachea! muco-ciliary transport rate*
Aldosterone synthesis in the adrenal gland*
Fatty acid synthesis in adipose tissue*
Mutagenesis in Saccharomyces**
Mitotic frequency in duck embryonic lung cells (L132) t
Oxidative activity in rabbit endothelium t
Chromosome breakage in human leukocytes t
Enzyme leakage in perfused liver*
Oxygen consumption by human leukocytes t
Osmotic and ionic changes in leaf guard cells
Inhibition of regeneration in Hydra
* Assays described in Woodard (1976)—Organs and Tissues.
** Assays described in Woodard (1976)--Bacteria, Fungi,
Protozoa, and Plant Cells.
t Assays described in Woodard (1976)—Mammalian and Avian
Cell Culture Systems.
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are genetically and developmentally heterogeneous, and thus create problems
with quality control and uniformity of response. Malting, osmotic and ionic
changes in leaf guard cells and inhibition of regeneration in Hydra were not
considered promising assays because a very small, if any, data base on toxic
chemical effects existed. Those potential toxicity screens involving plant
tissue cultures (plant callus growth, inhibition of cell division of plant
suspension cultures) were rejected because a very long time (as much as 2
months) is required to complete a toxicity assay involving callus or suspen-
sion cultures. Also, plant cell cultures are easily contaminated, especially
since aseptic conditions must be maintained without antibiotics for the
duration of the assay. The final tv/o rejected assays [mutagenesis in
Saccharomyces and mitotic frequency in duck embryonic lung cells (L132)] may
be effective for screening a wide variety of mutagens. But, while all
mutagens are toxicants, not all toxicants are mutagens. These assays are
probably responsive only to chemicals that interact with or influence DNA.
The remaining assays, which are evaluated in this report, appeared
reproducible, well -documented, and straightforward (Table 2).
The selection of these final 24 assays was based on several criteria.
First, the test organism in each of these assays is either commercially
available or easily grown or cultured from commercially available materials
(e.g., seeds) (Table 3). Second, the methods for each assay are well docu-
mented and have been performed in many laboratories worldwide. For example,
estimates of RuDP carboxylase activity have been made in many laboratories
throughout the U.S., Japan, Europe, and many other locations. Third, many of
the physiological processes measured by these assays have been tested with
some chemicals for possible toxic effects. So, at least some data base on
toxic chemical effects is available for each of the 24 assays. Fourth, the
selected assays are generally more rapid (assays per unit time) than the
rejected candidate assays.
Each of these protocols is currently the most streamlined process
available. With more research and development, the potential for further
streamlining exists.
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TABLE 2. POTENTIAL TOXICITY ASSAYS ADDRESSED IN THIS REPORT
Gel 1 ill ar process
Specific assay
Nitrogen fixation*
Photosynthesis*
Respiration*
High-energy phosphate production
Growth and cell division*
Catalysis (enzymatic activities)
Other cellular processes
Acetylene reduction
Hill reaction
Greening
RuDP carboxylase activity
Photosynthetic oxygen evolution
Respiration in HeLa cells**
Adenylate energy charge
Cloning L929 mouse cells**
Protozoan clonal viability
Human KB cell growth rate
Human embryonic lung fibroblast
(WI-38) cytotoxicity
Mitogen stimulation of lymphocytes
Chick embryo development**
Trypan blue dye exclusion by
human KB cells
RNA polymerase activity
Adenyl cyclase activity
Lysosomal enzyme release
Macromolecular synthesis in KB cells
Cyclosis*
Hemolysis*
Protozoan vacuole contraction*
Protozoan motility*
Phagocytosis by alveolar macrophages
Ami no acid transport
*Subjects mentioned in EPA directive to Battelle.
**Subjects mentioned in Woodard (1976).
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TABLE 3. COMMERCIAL SOURCES OF TEST ORGANISMS
Organism
Source
Algae:
Scenedesmus
Euglena
Chlorella
Chlamydomonas
Chara
Elodea
NlteHa
Bacteria:
Azotobacter vinelandii
Escherlchia coll
Clostrldlum pasteurianum
Protozoan:
Tetrahymena pyrlformls
Human Cell Lines
HeLa
KB
WI-38
Human erythrocytes
Other mammalian Cell Lines:
Mouse L929
Mouse lymphocytes
Rabbit alveolar macrophages
Rat erythrocytes
Higher Plant (seeds):
Spinach oleracea
Hordeum vulgare
Phaseolus vulgaris
Canavalia ensiformis
Higher Animal:
Mice
Rabbits
Rats
Chickens
ATCC*
ATCC
ATCC
ATCC
Starr**
Starr
Starr
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
ATCC
Calbiochem
ATCC
Mouse colony
Rabbit colony
Rat colony
DeKalb Agresearch t
DeKalb Agresearch
DeKalb Agresearch
DeKalb Agresearch
Jackson Labs, Bar Harbor, ME
Jackson Labs, Bar Harbor, ME
Jackson Labs, Bar Harbor, ME
Reliable local hatchery
*ATCC—American Type Culture Collection.
**Dr. Richard Starr, algal culture collection, University of Texas at
Austin.
t Or other company with genetically homogeneous seeds [good sources
can be confirmed by the American Seed Trade Association (ASTA)].
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Organization of Information About Assays
In the following pages, 24 physiological assays which could be devel-
oped for rapid toxicity testing are described and assessed. As shown in Table
2, assays are organized under seven different cellular processes which were
suggested by OPTS/EPA.
The information on each assay is divided into two parts. First,
there is a brief description of each assay along with the biological meaning
of the test results. Second, data tables accompany each assay description for
easy access of relevant information. The protocols and data tables are com-
plementary, so both should be considered for objective evaluation of the
individual assays. The information organization described above allows easy
evaluation and comparison of individual assays.
A tabulation summarizing pertinent data on the assays is presented in
the Discussion and Recommendations section at the beginning of the report.
Explanation of Data Tables Accompanying Each Assay
Each potential toxicity assay has been assessed according to seven
criteria. The scope and meaning of each criterion are given below.
Test Organism—
A representative test organism (or organisms) is suggested for each
assay. Each organism mentioned in this report has been the object of most of
the particular studies in toxicity evaluations. Observations have been made
on these organisms, and on others mentioned in certain assays, in terms of
growth and survival, hallmark metabolic process, or changes in gross morphol-
ogy or ultramorphology. These test organisms were also selected because
relatively large quantities of these cells or tissues can be quickly grown or
inexpensively purchased (Table 3). Enzymes used in certain toxicity assays
(e.g., RNA polymerase activity) were selected because of their abundance in
particular tissues, commercial availability, stability of activity, or rele-
vance of the metabolic process in which the enzyme participates.
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Advantages and Limitations—
Some advantages and limitations of each method,are listed in the
following tables and these are straightforward in interpretation.
One factor considered was the level of technical skill needed to per-
form each assay. We identified three different levels of technical competen-
cy. Highly skilled technicians are defined as individuals with master's
degrees and research experience. Skilled technicians are individuals who have
had college or university research experience and who hold a technical bache-
lor's degree. Unskilled technicians are defined as individuals with a minimal
scientific background (e.g., associate or junior college degree). If un-
skilled technicians could be used to perform an assay, this fact was listed as
an advantage. On the other hand, if a skilled or highly skilled technician
was required, this fact was considered a limitation. As each particular assay
method is standardized and becomes routine for a given laboratory, a lesser
degree of technical skill than mentioned in the table for that assay may be
utilized.
Response to Chemicals—
Each potential toxicity assay was assessed with regard to its re-
sponse to certain chemicals. Most of these assays have not been developed as
toxicity tests as such, but were used to study certain physiological pro-
cesses. Any toxicity testing has been incidental (i.e., to determine physio-
logical effect of a chemical, not to determine a chemical's toxicity).
A sampling of chemicals and chemical classes has been included in
each table to give an idea about the range of chemicals that affect the assay.
Other chemicals may (or may not) affect the physiological process in each
assay, but these chemicals have either not been studied or were not revealed
during our literature search.
In every assay listed (except hemolysis and lysosomal enzyme re-
lease), the term "response to chemicals" refers to chemical inhibition of the
observed physiological process. For instance, in respiration in HeLa cells,
malonate lowers (or in sufficiently high concentrations, stops) mitochondrial
oxygen uptake. The chemicals listed in hemolysis, however, promote the lysis
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of red blood cells rather than inhibit it. In each assay the chemicals listed
affect the physiological process in a concentration-dependent manner.
When available, the concentrations of some chemicals affecting the
assays were included. Affecting chemical concentrations v/ere abbreviated EC,
I, LC, or MEC. ECX (effective concentration) is the concentration that
induces detrimental effects in x percent of the test organisms. For example,
in the hemolysis assay, £659 would be a chemical concentration that causes
50 percent of the red blood cells to lyse. Ix is defined as the chemical
concentration causing x percent inhibition of a physiological process (e.g.,
greening or enzymatic catalysis activity). LCX (lethal concentration) is
the chemical concentration causing the death of x percent of the test cells or
organisms. For instance, LCX is used for chemicals causing chick embryo
death. MEC (minimal effective concentration) is defined as the lowest chemi-
cal concentration at which toxic effects are first observed. MEC is used in
the mammalian cell culture assays.
In the assays examined in this report (Table 2), the test chemicals
have elicited a unidirectional physiological response (e.g., inhibition of
respiration, inhibition of KB cell growth rate). It is possible that future
studies may reveal chemicals having an opposite effect (e.g., stimulation of
respiration, stimulation of KB cell growth rate). Would a chemical that
stimulated, instead of inhibited, a cellular process be considered toxic?
Assay Time—
The times required for each assay (including preparation time, tech-
nician time, etc.) were assessed and are included in each data table. There
are four numbers listed opposite Assay Time in each table. The first number
is the time (in hours) to perform an assay set (i.e., one chemical, three
replicates of each of five concentrations). If several replicates of diffe-
rent chemicals could be assayed simultaneously, this was considered an advan-
tage. It was considered a limitation when an assay set required long periods
of time or when only one individual assay could be completed at a time. The
second number listed in the Assay Time category is the total time for an assay
set to be completed. This includes cell growth period, solution preparation,
data recording, and laboratory cleanup and is an estimate of time from
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start-up to expression of results. The third number is total technician time.
Since technicians can do several operations simultaneously (e.g., prepare
solutions during cell growth period) and some processes may continue unsuper-
vised overnight, this number may be significantly less than the total assay
time. The last number is the administrative time required. This includes
Ph.D. supervision time, managerial time, data analysis, and reporting of re-
sults. It was necessary to separate this from technician time since a more
highly trained person is usually required for administration. These times are
estimated for assays that are in the late developmental stage. As the assay
method is standardized and becomes routine, times would probably become
shorter.
Specialized Equipment--
Specialized equipment needed to accomplish each assay is listed in
the table accompanying each assay description. Not all equipment required for
each test is presented. All of these potential toxicity assays can be per-
formed routinely only in a laboratory equipped with basic analytical instru-
ments (centrifuges, balances, etc.), minimal cell culture equipment (incuba-
tors, culture dishes or flasks, etc.), and biochemicals (buffers, metabolites,
etc.). If the other necessary special equipment was rare or costly, this was
considered a limitation. For example, mammalian cell culture facilities,
needed in many of the mammalian cell cytotoxicity assays, require a sterile
working area such as laminar flow hood or transfer room. Such apparatus is
probably not standard in most laboratories and may cost as much as $7000.
Other special equipment, such as a spectrophotometer or colorimeter, is rela-
tively inexpensive and is found in many laboratories. Such special equipment
is listed in the data tables but is not considered a limitation.
The assays listed in Table 2 already involve certain levels of auto-
mation. For example, protozoan motility utilizes a microphotography unit and
RNA polymerase activity uses a multipurpose filtration manifold. Many other
assays (e.g., mitogen stimulation of lymphocytes, adenyl cyclase activity,
amino acid transport) are partially automated by using scintillation counters
with statistical data analyzers. The need for automation of any particular
assay is dependent on the volume of chemicals to be tested--if many chemicals
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are to be tested, automation or development of automation v/ould ensue. In
several assay descriptions we suggest potential points for automation (e.g.,
use of computer and TV in protozoan motility assay).
Cost—
The estimated cost for an assay set is included in each data table
as well as in a comprehensive comparison table at the end of the report
(Table 29).
To calculate the estimated costs, the technician's hourly v/age was
multiplied by the total number of technician hours, and this was multiplied by
a factor of 2.64. This is the estimated labor factor for Battelle's Bioenvi-
ronmental Sciences Section, and is used for determining the approximate total
cost for performing a task, including labor, supplies, use of equipment, and
use of other facilities (e.g., electricity, water, maintenance).
The technicians' hourly wages are based upon average pay for similar
technicians at Battelle, including 2 weeks annual vacation and other fringe
benefits. Annual salaries of these technicians are: highly skilled techni-
cian, $18,000; skilled technician $12,000; and unskilled technician, $9,500.
The cost of supervision by a Ph.D. level research scientist and managerial
costs are also included. These annual salaries are estimated to be $25,000
and $35,000 for a Ph.D. scientist and manager, respectively.
The approximate costs listed in this report are only for purposes of
comparison of assays. Actual costs may vary 20 to 25 percent from these
figures at different laboratories. Developmental work on the assays would be
considerably higher than these estimates for semi routine testing. As the test
comes into routine use, however, costs could decline sharply because of sim-
plified and standardized methods (disregarding inflation).
Data generated in any one of the assays described in this report
would be analyzed by routine statistical methods (e.g., variance analysis).
Assays from Woodard (1976)
In the report by Woodard (1976) to OPTS/EPA, potential physiological
toxicity assays for studies on chemicals were reviewed under four categories:
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(1) use of fertilized eggs, (2) use of isolated organs and tissues, (3) use of
mammalian and avian cell cultures, and (4) use of bacteria, fungi, protozoa,
and plant cells. In the following paragraphs, we consider in vitro tests
derived from each of Woodard's test groupings.
The literature on the use of fertilized eggs in studies on chemicals
focuses almost exclusively on the development and use of fertilized chicken
eggs as a toxicity bioassay. The production of abnormalities in the devel-
oping embryo as a result of the administration of thallium was first demon-
strated by Karnofsky in 1950 using the fertile chicken egg. However, the
chick embryo development assay requires a long period of time to complete
(1 month), calls for expensive specialized equipment, and does not have a
universally standardized end point such as embryo death or abnormal limb
development (Table 16). In general, the use of fertilized chicken eggs has
enjoyed some attention in studies on the teratogenic potential of chemicals,
but this method is still regarded by toxicologists as only marginally useful
in screening for other types of toxic effects.
Toxicity tests have also been conducted using two types of inverte-
brate eggs as test subjects. However, few data exist concerning chemical
effects on hatching of brine shrimp or on the early development of sea urchin
embryos, and a tremendous amount of developmental research would be needed to
adapt these assays for routine toxicity testing. As for present data on these
two test systems, the inhibitory or stimulatory effects on hatching or devel-
opment apparently do not correlate with the carcinogenic effects of known
chemical compounds tested (Woodard, 1976). So these assays are apparently not
immediately useful as potential toxicity screens.
Woodard's category on the use of isolated organs and tissues in
studies on chemicals was reviewed. The advantages and limitations of these
bioassay systems are discussed on page 2 in relationship to Table 1.
Several rapid, potential toxicity assays using mammalian cell cul-
ture, bacterial, protozoan, and plant test systems are critiqued later in this
report.
Cytotoxicity assays employing mammalian cells in culture measure
quantitatively cellular and metabolic impairment or death resulting from in
vitro exposure to soluble and particulate toxicants. Mammalian cells derived
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13
from various tissues and organs can be maintained as short-term primary
cultures or, in some cases, as continuous cell strains or lines. Primary
cultures exhibit many of the metabolic and functional attributes of the
original tissue. Some of these attributes may be lost after a prolonged time
in culture.
There are certain requirements basic to any assay that requires the
use of mammalian cells in vitro. Paramount among these are aseptic facilities
for the propagation and handling of cultured cells and qualified personnel
trained in safe and proper cell culture technique.
There are many advantages in using mammalian cell culture systems in
toxicity assays. First, they are generally more rapid and less costly than
whole animal tests. Second, a lesser quantity of potential toxicant is
required for these in vitro tests. Third, specific physiological or biochem-
ical alterations are more easily evaluated in cell culture systems, and
fourth, the systems provide useful information about the relative cellular
toxicity of unknown samples (Woodard, 1976).
Cell culture toxicity screens also have several drawbacks. Since the
assays employ isolated cells and not intact animals, they can provide only
preliminary information about the ultimate health hazards of toxic chemicals.
In many instances, some metabolic action in an animal renders a chemical toxic
or nontoxic. So, a chemical which appears toxic at the cellular level may
actually be innocuous at the tissue or higher level because of metabolic
deactivation. Likewise, a toxic chemical could appear nontoxic at the cellu-
lar level since metabolic activation of a chemical to a toxic form could occur
in vivo but might not occur in cell culture.
Another disadvantage is that cell culture test systems may become
contaminated with latent viruses or Mycoplasma sp., which can alter cellular
metabolism. Also, media constituents (such as calf serum) must be carefully
monitored and controlled since they may affect cellular metabolism or form
complexes with the test chemical (Woodard, 1976).
Both neoplastic (tumor-derived) and nonneoplastic (primary) cell
lines are utilized in assays described in this report. Although neoplastic
cells are abnormal and have probably lost some metabolic capabilities as
compared with primary cultures, they respond equally well in many cytotoxicity
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14
assays. Neoplastic cells are generally used because they grow rapidly and are
more readily propagated than primary cultures. The only neoplastic cell lines
used include human HeLa and KB. Nonneoplastic cell lines utilized include
human WI-38, rabbit alveolar macrophages, mouse L929, and mouse lymphocytes.
Other mammalian cell types can be used as alternatives to these cell lines.
Several of the cell culture assays described here could be combined
to form one assay which could assess several parameters. This would provide a
more cost-effective means for using cell culture systems for screening toxic
chemicals.
As described by Woodard (1976), the use of nonmammalian cell systems
in toxicity testing is now well established. The potential for bacteria, pro-
tozoan, and plant systems in physiological assays is also great. Most assays
involving these systems are more rapid and less expensive than mammalian
systems. Also, many potential toxicants can generally be screened simulta-
neously, and often only unskilled technicians are required to perform the
test.
A drawback to the use of plant, bacteria, and protozoan systems in
toxicity screens is the questionable extrapolation of data obtained from these
systems to mammals. The converse is also true in that mammalian systems as
toxicity screens cannot always be extrapolated to plant or microbial systems.
Although all cells have certain structural and metabolic properties in common,
certain processes which only occur in whole animals or plants (e.g., uptake
and transportation of potential toxicants) still are not fully understood.
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15
DISCUSSION AND RECOMMENDATIONS
The objective of this report was to identify potential physiological
toxicity tests in the literature and to assess each on the basis of several
criteria. Even though each assay has advantages and disadvantages, it is
difficult to rank them on the basis of a literature review alone. Before any
final decision on the utility of any assay is made, laboratory evaluation is
necessary. However, on the basis of the literature review, it is possible to
approximate the degree of laboratory development needed to adapt and validate
these protocols as routine toxicity screens.
We have identified three levels of assay development: those requiring
minimal development, those requiring some development, and those requiring
significant development. Assays are placed in one of these categories on the
basis of the criteria listed in Tables 28 through 32.
Assays that would require little development for use as toxicity
assays (i.e., immediate validation) include greening, hemolysis, human KB cell
growth rate, phagocytosis by alveolar macrophages, macroinolecular synthesis in
human KB cells, RNA polymerase activity, and human embryonic lung fibroblast
(WI-38) cytotoxicity. Assays that would require some development for use as
toxicity assays include acetylene reduction, the Hill reaction, RuDP carboxyl-
ase activity, adenylate energy charge, chick embryo development, protozoan
clonal viability, cloning L929 mouse cells, trypan blue dye exclusion by human
KB cells, protozoan motility, and amino acid transport. Assays that would
require extensive development include cyclosis, protozoan vacuole contraction,
photosynthetic oxygen evolution, respiration in HeLa cells, mitogen stimula-
tion of lymphocytes, lysosomal enzyme release, and adenyl cyclase activity.
Some assays naturally drop from consideration. These tests meet few
(or none) of the criteria used for assay evaluation (simplicity, rapidity,
cost effectiveness, documentation, reproducibility, etc.). For example,
cyclosis is one of the most expensive and time-consuming assays. It has a
poor data base, and results are probably not ecologically significant.
Protozoan vacuole contraction, photosynthetic oxygen evolution, and respira-
tion in HeLa cells require extensive development and have been rejected for
immediate use because several of the criteria are not optimum. Adenyl cyclase
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16
activity, mitogen stimulation of lymphocytes, and lysosomal enzyme release
have poor data bases with regard to toxic chemical effects. In addition, it
appears difficult to relate results from adenyl cyclase activity to cellular
or tissue toxicity. Mitogen stimulation of lymphocytes requires maintenance
of an expensive mouse colony.
Those assays in the second category (some development) are more dif-
ficult to evaluate since their advantages and limitations are more equally
balanced. In some assays (Hill reaction, chick embryo development) very good
data bases on toxic chemical effects exist. However, there are GLP problems
with the Hill reaction since chloroplast activity may vary. Chick embryo
development requires a long time to complete and lacks a standardized end
point. RuDP carboxylase activity has a poor data base and GLP problems, even
though it is rapid and inexpensive. Protozoan motility is time consuming and
expensive, but automation could make test results easier to obtain. The
clonal assays (cloning L929 mouse cells and protozoan clonal viability) both
have good data bases and are simple. However, each requires a long time to
complete.
The membrane assays (amino acid transport, trypan blue dye exclusion)
were not considered easily developed assays because they have poor data bases.
Development of these assays should be considered because tests results can be
extrapolated to all membranes. Even though acetylene reduction monitors a
vital physiological and ecological process, the current assay method needs
streamlining (e.g., use of a multisample gas chromatograph).
The remaining assays are rapid, simple, reproducible, cost-effective,
and well documented. In many cases, it is advisable to combine several tests
(or give a single test multiple end points) and to correlate the results to
give the responses to chemicals broader ecological or biological meaning.
Greening, Hill reaction, chlorophyll fluorescence, and a growth test (e.g.,
seedling growth) could possibly be combined to give a good indication of
phytotoxicity if the same organism were used in all assays (Kratky and Warren,
1971).
It also would be possible to combine RNA polymerase activity and
macromolecular synthesis in human KB cells to detect chemical inhibition of
RNA synthesis. Phagocytosis by alveolar macrophages and amino acid transport
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17
(and/or trypan blue dye exclusion by human KB cells) could be combined to
detect inhibition of mammalian membrane function.
Hemolysis appears to be one of the best potential toxicity assays
because it is cost-effective and can be performed by unskilled technicians.
Also, the lysis of erythrocytes is a generally accepted standard of toxicity
because mammalian tissues depend on hemoglobin for transport of gases and
nutrients. Human KB cell growth rate and human embryonic lung fibroblast
(WI-38) cytotoxicity also should require little development since they are
already used as toxicity assays by the National Cancer Institute. Since these
assays are well documented, simple, and inexpensive, they could probably be
quickly validated as toxicity screens, with implementation following.
Comments
In evaluating and ranking these 24 potential physiological toxicity
test methods, there are several points that merit consideration.
Physiological test methodologies have proved very useful for studying
mechanisms of toxic action and for evaluating large numbers of toxic chemi-
cals. Physiological tests (predominately in vitro) have several advantages
over in vivo methods (e.g., time, cost, and quantisation of results), but
results from physiological tests can at best give preliminary information on a
chemical's toxicity. As described on page 13, an in vivo system may mediate a
chemical's toxic activity by metabolic activation or deactivation. This can-
not occur in in vitro systems. Hence, physiological methodologies could give
false positive or negative results.
Results obtained from cellular, organellar, or enzymatic test systems
cannot usually be extrapolated to ecosystem effects for several reasons.
First, only one physiological parameter of a single test organism is monitored
in each assay. These assays are by no means an intensified ecological study,
and no direct extrapolations from these tests could effectively be made to
ecosystem effects. Second, test organisms such as mammalian cells or algae
are genetically homogeneous because these test cells are clonally derived.
Since cells and tissue systems differ greatly in whole animals, these cellular
systems are usually not good indicators of in vivo responses. Third, a
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18
selected test organism may be unusually sensitive or insensitive to certain
chemicals and give inconclusive (or false) results. Physiological test
systems, both mammalian and nonmammalian, could be effectively used as toxi-
city screens to identify the need (or lack of need) for further testing.
Information obtained from one cellular physiological test can often
be extrapolated to a more complex multicellular system because of certain
structural and functional similarities. All cells are enclosed by virtually
identical semipermeable membranes, contain DNA, and respire. It is not usual-
ly safe to extrapolate beyond this, however.
In summary, since most previous toxicity test methods have been in
vivo, the effectiveness of cellular or subcellular test methods has yet to be
demonstrated. The in vitro test methods have several advantages over in vivo
ones (e.g., time and cost), but they still have certain practical and scien-
tific limitations involving correlation of toxic effects on cellular metabol-
ism to toxic effects in ecological systems.
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19
POTENTIAL TOXICITY ASSAYS—DESCRIPTIONS, METHODS, AND ASSESSMENTS
NITROGEN FIXATION
Acetylene Reduction
The conversion of atmospheric nitrogen into organic compounds by
living organisms is called nitrogen fixation. This process is carried out by
microorganisms, including the free-living bacteria, blue-green algae, and
bacteria associated in a symbiotic condition with plant roots. The enzyme
nitrogenase catalyzes the transfer of electrons from an electron source to ni-
trogen, resulting finally in the production of ammonium ions. The acetylene-
ethyl ene assay for nitrogen fixation is based on the nitrogenase-catalyzed
reduction of acetylene to ethylene. Ethylene concentration is determined by
using a gas chromatograph equipped with a hydrogen-flame analyzer.
This assay involves incubation of bacteria with an energy source and
reductant in a flask sealed with a serum cap. After repeated flushing with a
source of acetylene, the bacteria are added aseptically through the cap. The
reaction mixture is incubated on a rotary shaker at 30 C for 30 minutes, and
the incubation is stopped by addition of 0.5 ml 6N sulfuric acid. Samples of
the gas phase are then measured with a hydrogen2-flame ionization detector
after gas chromatographic separation.
As described by Hardy et al (1968), the complete assay system con-
tains 4 ml liquid volume and 36 ml gas volume. The liquid reaction mixture
includes 50 mM Tris-HCl, 56 mM creatine phosphate, 5 mM ATP, 5 mM magnesium
chloride, 20 mM disodium thiosulfate, 0.2 mg of creatine kinase, and 4 mg of
heated extract of ammonia-grown _A. vinelandii. The gas phase of the reaction
mixture contains 0.1 atmosphere of acetylene and 0.9 atmosphere of helium.
Chemicals to be tested are added to the reaction vessels at various concen-
trations. Inhibition of acetylene reduction, expressed as a percentage of
control values, can be calculated for the various levels of a test chemical.
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20
Monitoring the effects of toxic chemicals on nitrogen fixation is
important because captured atmospheric nitrogen is converted into amino acids,
the building blocks of all proteins.
Details on acetylene reduction are summarized in Table 4.
PHOTOSYNTHESIS
Hill Reaction
Robin Hill discovered that light-induced oxygen evolution can be
observed in cell-free granular preparations (chloroplasts) extracted from
green leaves. Illumination of such chloroplast preparations in the presence
of artificial electron acceptors, such as ferricyanide or reducible dyes,
causes evolution of oxygen and simultaneous reduction of the electron
acceptor, according to the general equation
H20 + Ah+ AH2 + 1/2 02
where A is the electron acceptor and AH2 is its reduced form. In a photo-
synthesizing plant, A is nicotinamide adenine dinucleotide phosphate (NADP).
NADP accepts the electrons, and the reduced form of NADP is used to reduce
carbon dioxide into sugars. Hov/ever, in the in vitro Hill assay described
below, dyes are used to accept the electrons liberated from water. As the
dyes are reduced, they change color and this color change is quantitated.
For the Hill assay, chloroplasts are isolated from plants grov/n under
controlled conditions or from batch cultures of Euglena or Chlorella. Accord-
ing to the chloroplast isolation method of Wald et al (1966), algae or leaves
are homogenized with 0.5 M sucrose solution at 0 C for 30 seconds in a Waring
Blendor. The suspension is then filtered through tv/o layers of cheese cloth.
The filtrate is centrifuged at 50 g for 10 minutes. The supernatant is then
decanted and centrifuged for 10 minutes at 600 g. The supernatant is decanted
and discarded. The pellet at the bottom, containing the chloroplasts, is
suspended in 0.5 M sucrose. It is important to keep the chloroplasts at 0 C
because they deteriorate rapidly at higher temperatures.
It is advisable to examine the chloroplast preparation under a micro-
scope to ensure that the chloroplasts are of uniform size, intact, and free of
other cellular debris.
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21
TABLE 4. ACETYLENE REDUCTION
Criteria
Critique/Comments
Test Organisms
Advantages
Limitations
Response to Chemicals
Assay Time, hours
Special Equipment
Cost §
References
Either Azotobacter vinelandii or
Clostridium pasteurianum may be used.
The analytical method can detect as little
as 1 picomole of ethylene.
The tests organisms are simple to culture.
The assay may be utilized in either the
field or laboratory.
The phase of the potential toxicant may
be solid, liquid, or gas.
A short time is required to obtain
the results of this assay.
Acetylene is a very explosive gas and
requires care in handling.
Nonnitrogenase catalysis of the reduction
may occur.
Specialized equipment is needed.
The assay and gas chromatography must be
performed by skilled technicians.
Chlorinated aliphatics [trichloroacetic
acid, ethylene glycol bis(trichloroacetate)]
Arsenicals (cacodylic acid, disodium
methanearsonate)
Metabolic inhibitors (2,4-dinitrophenol)
Gases (carbon monoxide-I^gg is 0.18 atm)
2.5*, 54**, 12t, 8t
Gas chromatograph with a hydrogen-flame
analyzer
$620
Hardy et al, 1968
Rubinstein, et al, 1975
*Time for one assay set—three replicates of each of five concentrations
of ore chemical.
**Total assay set time, including cell growth, solution preparation, and
data recording.
t Total technician time, including GLP, performing assay, and solution
preparation.
t Administrative time (Management, Ph.D. supervision, data analysis, and
reporting).
§ Estimated cost for comparative purposes. Actual costs may differ 20 to
25 percent (see p. 11).
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22
The Hill reaction assay mixture contains 2 ml of 0.1 M phosphate
buffer (pH 6.5), 2 ml of 2.5 x 10-4 M dichlorophenolindophenol, 0.1 of
chloroplast suspension, 1 ml of toxicant or chemical to be tested, and 5 ml of
distilled water. Chemicals to be tested are incubated with the chloroplast
suspension prior to addition of the electron acceptor. Each reaction mixture
is then exposed to the same bright light for 10 minutes. Over the course of
the 10 minutes, the absorbance of the dye is monitored at 620 nm (Wald et al,
1966). The effect of a potential toxicant on the rate of this photosynthetic
reaction is reflected by the rate at which the dye is reduced and turns from
blue to clear.
Details on the Hill reaction are summarized in Table 5.
Greening
Potential toxicants alter plants' chlorophyll content by a number of
mechanisms. Chlorophyll biosynthesis is affected by specific chemical stimu-
lation or inhibition of DNA, RNA, or protein synthesis. Some chemicals affect
chloroplast development or structure, resulting in an altered chlorophyll
content. Other chemicals degrade or induce the degradation of the chlorophyll
molecule (Wolf, 1977).
To measure the effect of a chemical on chlorophyll accumulation,
dark-grown plants are subjected to a series of chemical concentrations prior
to greening. In detail, etiolated barley plants 7 to 9 days old are sprayed
(misted) with solutions of a chemical. The spray is directed at the coleop-
tiles (or hypocotyl hooks) from above. For every 200 seedlings, about 50 ml
of solution is used. Alternatively, seedlings could be grown in soil amended
with toxicant. The seedlings are transferred to an irradiation chamber 1 hour
after being treated. The plants are irradiated for 24 hours by white fluores-
cent lamps at an intensity of 1000 ftc. Except during white-light irradia-
tion, plant material is handled in dim green light (Margulies, 1962).
Chlorophyll is extracted from 2-g leaf samples by heating in boiling
water for 30 seconds, and then by grinding in a Virtis-type homogenizer with
80 percent acetone. The macerate is centrifuged, and the chlorophyll content
of the resulting supernatant is measured spectrophotometrically at 663, 645,
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23
TABLE 5. HILL REACTION
Criteria
Critique/Comments
Test Organisms
Advantages
Limitations
Response to Chemicals
Assay Time,* hours
Special Equipment
Cost*
References
oleracea), Chlorella,
Spinach (Spinacia
Euglena
The test organisms are simple to grow or are
readily available.
A short time is required to obtain the
results of this assay.
The assay is capable of detecting very
minute quantities of potential toxicants.
The assay can be performed by unskilled
technicians.
Chloroplast activity varies among
preparations and declines with age.
Reducing or oxidizing agents may interfere
with and produce variation in the assay.
Antibiotics (chloramphenicol-Igg is
4 mg/ml)
Ureas (3-cyclooctyl-l,1-dimethylurea,
l-(2-methylcyclohexyl)-3-phenylurea)
Herbicides (2-chloro-4,6-bis(isopropylamino)-
s-triasine, 2-methoxy-4,6-bis(ethylamino)-
s-triazine)
Inorganic salts (ammonium chloride)
Inorganic ions-heavy metals (cadmium, zinc)
3, 57, 18, 11
Spectrophotometer (Beckman Spec 20)
or colorimeter
$820
Hill, 1937
Margulies, 1962
Morel and and Hill, 1962
Anderson and Boardman, 1964
Wald et al, 1966
Brown and Haselkorn, 1972
Hamp et al, 1975
Rubinstein et al, 1975
*See time and cost explanation, pp. 9-11 in text.
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24
and 626 nm by the method of Anderson and Boardman (1964). This method takes
into account the absorbance of protochlorophyll , the precursor of chlorophyll,
as well as chlorophylls a and b. The following equations can then be solved
to give the individual pigment concentrations in yg/ml:
C = 12.67E663 - 2.65E6« - 0.29E626
a
Cb = -4.23E663 + 23.60Es« - O.SSEsas
P = -3.99Es63 - 6.76E6« + 29.60Es26 -
This bioassay is especially sensitive to photosynthetic and respira-
tory inhibitors, but results do not usually correlate with results from growth
Assays (Kratky and Warren, 1971).
Details on the greening assay are summarized in Table 6.
RuDP Carboxylase Activity
Ribulose-l,5-diphosphate carboxylase (RuDPCase) is a soluble enzyme
localized in the chloroplast stroma of vascular plants. This enzyme catalyzes
the primary fixation of carbon dioxide during photosynthesis in some monocots
and in most dicots.
This assay employs cell-free extracts of leaves or algae. Leaves are
obtained from spinach plants grown under controlled conditions and the algae
Euglena and Chlamydomonas are easily grown in batch cultures. As described in
the greening assay, test organisms are exposed to a chemical prior to isola-
tion and determination of RuDPCase. Alternatively, chemicals to be tested can
be incubated with the enzyme preparation prior to additon of the other reac-
tion reagents (see below).
To prepare an extract, as described by Goldthwaite and Bogorad
(1971), 1 g of leaves or algae is ground in a small Waring Blendor for 2 min-
utes in 2 ml of an ice-cold buffer containing 0.2 M sodium bicarbonate (pH
8.0), 1 percent polyvinyl pyrrol idone, and 1 mM dithiothreitol. The homoge-
nate is filtered through cheesecloth and Miracloth and then is centrifuged at
35,000 g for 15 minutes at 4 C. The resulting supernatant is assayed for
enzymatic activity.
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25
TABLE 6. GREENING
Criteria
Critique/Comments
Test Organisms
Advantages
Limitations
Response to Chemicals
Assay Time,* hours
Special Equipment
Cost*
References
Barley seedlings (Hordeum vulgare) are
the major test organism.
Pinto beans (Phaseolus yulgaris) and jack beans
(Canavalia ensiformis) may also be used.
The test is capable of identifying many
different chemicals as potential toxicants.
A relatively short time is required to obtain the
results (chlorophyll determinations) of this assay.
The test organisms are simple to grow.
There is a direct relationship between the
concentration of the chemicals investigated
and percentage of chlorophyll inhibition.
The assay can be performed by unskilled technicians.
Specialized equipment is needed.
The total time, including greening and chlorophyll
determinations, is lengthly.
Antibiotics (streptomycin, chloramphenicol)
Nucleic acid analogues (5-fluorouracil,
Z-thiouracil-Igy is 5 mM)
Amino acid analogues (ethionine, p-fluorophenylalanine)
Plant hormones (2,4-dichlorophenoxy acetic
acid, naphthalene acetic acid, abscisic acid)
Herbicides (amino triazole, paraquat, atrazine)
Growth retardants (coumarin, N,N-dimethylamino
succinamic acid)
Ureas [diphenylurea, 3-(4-chlorophenyl)-
l-(l-dimethylurea)]
Fungal metabolites (alternaric acid, tentoxin)
Alcohols (ethanql-Iigo is 100%)
nickel, lead, and aluminum ions)
10 mM, fructose, glucose)
carbon monoxide, methane,
Inorganic ions (cobalt.
Sugars (sucrose-Igg is
Gases (carbon dioxide,
ethylene)
29, 150, 9, 8
Spectrophotometer or colorimeter growth chamber
$560
Arnon, 1949
Margulies, 1962
Anderson and Boardman, 1969
Keller and Huffaker, 1967
Kratky and Warren, 1971
Rubinstein et al, 1975
Borque et al, 1976
Wolf, 1977
*See time and cost explanation, pp. 9-11 in text.
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26
RuDPCase activity is measured by incorporation of radioactive sodium
bicarbonate (NaH^cc^) into acid-stable products in the presence of
ribulose 1,5-diphosphate (RuDP). A 25-yl aliquot of enzyme is mixed on the
surface of a planchet with 100 yl of reaction mixture containing the following
(all are final concentrations): 100 mM Tris-HCl (pH 8.0); 50 mM NaHl4co3
(sp. act. 0.20 yCi/ymole); 0.3 mM RuDP; 10 mM magnesium chloride; 6 mM reduced
gluthathione; 0.1 mM ethylenediaminetetraacetic acid. After 10 minutes at
room temperature, the reaction is stopped by addition of 6 N acetic acid. The
planchets are dried and counted in a gas-flow counter. The reaction is linear
with enzyme concentration until 30 to 50 percent of the RuDP is consumed.
Incorporation in the absence of RuDP is Tess than 2 to 3 percent of that when
PuDP is added.
Details on this assay are summarized in Table 7.
Photosynthetic Oxygen Evolution
In the presence of sunlight, algae and .terrestrial green plants
photosynthesize and thus convert carbon dioxide and water into carbohydrates
and oxygen (02). Even though 02 evolution is used as a measure of photo-
synthesis, the 02 evolved from a plant cell is equal to the 02 released by
photosynthesis minus the 02 consumed by respiration. The assay described
here is based on comparing the rates of 02 evolution from algal cells pre-
incubated with a test chemical to the rate of 02 evolution from algal cells
not treated with the test chemical.
The green, unicellular alga Scenedesmus is used in this assay.
Scenedesmus obliquus, strain 03, is grown in a glucose-yeast extract medium
until a packed cell volume of about 10 yl/ml is obtained. About 40 ml of
cells are collected and washed in 0.05 M potassium phosphate buffer (pH 6.5).
The washed cells are incubated in a buffer containing concentrations of the
test chemical. Following exposure to the test chemical, cells are washed free
of the chemical by suspension and centrifugation. Then, 2 ml of the algal
suspension is added to each of two Warburg flasks and 0.5 ml of p-benzoquinone
is added to each side arm to inhibit respiration. Also, diuron is added to
the side arm of the first flask (control) to inhibit photosynthesis, and water
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27
TABLE 7. RUDP CARBOXYLASE ACTIVITY
Criteria
Critique/Comments
Test Organisms
Advantages
Limitations
Response to Chemicals
Assay time,* hours
Special Equipment
Cost*
References
Spinach (Spinacia oleracea),
Euglena, Chlamydomonas
The test organisms are easy to grow or
obtain.
A short time is required to obtain
the results of this assay.
There is usually a direct relationship
between the concentration of the
chemicals investigated and percent of
enzyme inhibition.
The enzymatic activity varies among
preparations and declines with storage.
The growth conditions of the plants
dramatically affect enzymatic activity.
Some specialized equipment is needed.
The assay is performed by highly skilled
technicians.
Antibiotics (cycloheximide-Iioo 1S
0.01 mg/ml; puromycin, streptomycin)
Herbicides (paraquat)
Growth regulators (N-(dimethylamino)
succinamic acid)
Arsenicals (cacodylic acid)
2, 54, 12, 8
Gas flow counter or scintillation counter
$750
Keller and Huffaker, 1967
Goldthwaite and Bogorad, 1971
Rubinstein et al, 1975
*See time and cost explanation, pp. 9-11 in text.
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28
i'S added to the side arm of the second flask. The flasks are equilibrated in
the water bath of a Warburg apparatus at 25 C for 20 minutes. The contents of
the side arms of the two flasks are then tipped into the bottom of the flasks,
and immediately the measurement of 03 evolution with high light intensity is
started.
In the Warburg apparatus, volume changes are measured in an enclosed
atmosphere in direct contact with the liquid under conditions in which oxygen
is the only substance undergoing a net transfer between the liquid and gas
phases.
Results from this manometric measurement are expressed as the percent
of inhibition of 02 evolution as a function of test chemical concentration.
An oxygen electrode can be used instead of the Warburg apparatus.
Details on this assay are summarized in Table 8.
RESPIRATION
Respiration in HeLa Cells
Mitochondria are present in virtually all living cells. Both the
Krebs (tricarboxylic acid) cycle and electron transport systems, the final two
stages of cellular respiration, occur in the mitochondria. In these final
stages, oxygen is consumed, and carbon dioxide and water are evolved. The net
equation for cellular respiration is:
CCH,,,0C + 60,, -> 6CO, + 6H00 + energy.
O I i. 0 C. <- c.
To measure cellular respiration, it is possible to monitor either
consumption of oxygen or evolution of carbon dioxide. In this assay, HeLa
(human) cells are grown in Eagle's medium to a density of 4 x 10^ cells/ml.
Other mammalian cell types can be used, but it is not feasible to use algal
cells which both photosynthesize and respire. The cells and a potential
toxicant are placed in the test chamber of a precalibrated oxygen electrode,
which can be purchased from a commercial source or made as described in
Bruening et al (1970). The dissolved oxygen is monitored at 10 minute
intervals for 1 hour. A graph of dissolved oxygen versus time is plotted, and
the slope of the plot represents the rate of oxygen consumption.
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29
TABLE 8. PHOTOSYNTHETIC OXYGEN EVOLUTION
Criteria
Critique/Comments
Test Organism
Advantages
Limitations
Response to Chemicals
Assay Time,* hours
Special Equipment
Cost*
References
Scenedesmus obliquus, strain D^
The test organism is simple to culture.
Oxygen electrode can be used in place of
the Warburg apparatus.
Specialized equipment is needed.
A great deal of time is required to
equilibrate flasks and accurately
determine gas exchange rates.
This assay must be performed by skilled
technicians.
Variation in respiratory and photosynthetic
Og evolution make interpretation of
results difficult.
Herbicides (l,l-dimethyl-3-pheny1urea,
3-(p-chlorophenyl)-l,l-dimethylurea)
50, 100, 84, 11
Warburg manometric apparatus
$2330
Pratt and Bishop, 1968
Rubinstein et al, 1975
time and cost explanation, pp. 9-11 in text.
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30
To note the effect of a potential toxicant, a different concentration
of the test chemical is added to a chamber of fresh HeLa cells, and again
dissolved oxygen is monitored at specific time intervals. This is carried out
at several different chemical concentrations, and a graph is plotted for each
concentration. Inhibition or stimulation of respiration is determined by
comparing the rates of oxygen consumption for the test chemicals with those of
the standard.
Toxic chemicals that inhibit respiration would most certainly influ-
ence the metabolism and viability of the organism since respiration is the
process by which aerobic cells obtain energy from the oxidation of fuel mole-
cules by molecular oxygen.
Details on this assay are summarized in Table 9.
HIGH-ENERGY PHOSPHATE PRODUCTION
Adenylate Energy Charge
Even though the production of adenosine-51 triphosphate (ATP) is a
common goal of both anaerobic and aerobic metabolic activities, the measure-
ment of ATP alone may not be an accurate index of both biomass and metabolic
activity. The total energy level of the cell is dependent upon the balance
between the adenosine phosphates. ATP contains two high-energy anhydride
bonds, ADP contains one, and AMP none. Atkinson (1969) and Atkinson and
Walton (1967) proposed an adenylate energy charge (AEC) as a fundamental
metabolic control parameter:
acr _ ATP + 1/2 ADP
tL AMP + ADP + ATP
The expression is a measure of the anhydride-bound phosphate groups per
adenine moiety and is written so that the parameter will range in value from 0
to 1. In general, when the AEC > 0.5, ATP-utilizing systems increase their
activities; AEC < 0.5, ATP-regenerating sequence dominates (Atkinson, 1969;
Ching et al, 1974). In this assay, cells are incubated with various concen-
trations of a toxicant, and the adenosine phosphates are then isolated from
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31
TABLE 9. RESPIRATION IN HELA CELLS
Criteria
Critique/Comments
Test Organisms
Advantages
Limitations
Response to Chemicals
Assay Time*, hours
Special Equipment
Cost*
References
HeLa (human) cells
The test results are straightforward
and easily obtainable.
The test organism is easy to grow.
The assay shows the effect of
chemicals on a major metabolic
pathway, respiration.
This assay must be performed by
skilled technicians.
Only one chemical at one concentration
can be tested at a time.
Some special equipment is required.
Reducing or oxidizing agents may inter-
fere with and produce variation in
this assay.
Metabolic inhibitors (malonate)
Detergents (Triton X-100, sodium
deoxycholate)
30, 80, 51, 13
Oxygen electrode, mammalian cell
culture facilities
$2350
Bruening et al, 1970
*See time and cost explanation, pp. 9-11 in text.
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32
the cells and quantitated. This assay can be applied to bacterial, fungal,
algal, mammalian, and plant cells or to mixed cultures and microbial assem-
blages. However, for development as an in vitro toxicity assay, the rapidly
growing bacterium Escherichia coli, the green alga Euglena, or the human cell
line HeLa are excellent cellular candidates.
In this assay, a known number of cells are incubated at 37 C for 1 to
2 hours with a toxicant. Adenosine phosphates are then extracted from cells
with chloroform (Bostick and Ausmus, 1978; Nannipieri et al, 1978). Quantita-
tion of ATP is based on reactions with hexokinase and glucose-6-phosphate
dehydrogenase. In these enzymatic reactions, ATP causes the production of
reduced nicotinamide adenine dinucleotide (NADH). NADH can then be quanti-
tated fluorometrically down to 10~12 M. For determining AMP and ADP, ade-
nylate kinase and pyruvate kinase are added to the mixture to convert these
two adenosine phosphates to ATP. This ATP is then measured using the hexoki-
nase method described above (Bostick and Ausmus, 1978). A ready-made ATP
determination kit is commercially available from Calbiochem. Comparison of
calculated AEC per cell values allows conclusions to be drawn on the physio-
logical status of cell populations exposed to toxicant.
Details on this assay are summarized in Table 10.
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33
TABLE 10. ADENYLATE ENERGY CHARGE
Criteria
Critique/Comments
Test Organisms
Advantages
Limitations
Response to Chemicals
Assay Time*, hours
Special Equipment
Cost
References
Escherichia coli is the major test
organism.
Euglena or the human cell line HeLa
may also be used.
This is a rapid indication of metabolic
state.
Test organism^, coli is commercially
available and relatively inexpensive.
This assay is applicable to a wide
range of organisms and environmental
and chemical conditions.
The assay can be converted to microbially
immobilized macronutrients in the
microbial energy charge assay.
Three parameters, AMP, ADP, and ATP,
must be measured.
The test requires a skilled technician.
Analysis must be promptly performed.
Unknown
8, 62, 23, 9
Spectrophotometer
$920
Atkinson and Walton, 1967
Atkinson, 1969
Ching and Ching, 1972
Bostick and Ausmus, 1978
*See time and cost explanation, pp. 9-11 in text.
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34
GROWTH AND CELL DIVISION
Cloning 1929 Mouse Cells
The L929 cloning assay is an in vitro bioassay to examine the cyto-
toxic effect of a variety of toxicants. The toxicants may include particulate
or soluble (aqueous or limited organic) toxicants.
The L929 cell line is carried in Eagle's Minimal Essential Medium
containing 10 percent fetal calf serum, 10,000 units of penicillin per 100 ml
medium, 10,000 yg of streptomycin per 100 ml medium, and 10,000 ug of my-
costatin per 500 ml medium. Cells are cultured in 75 cm2 tissue culture
flasks. When cells are 75 to 90 percent confluent, 0.25 percent trypsin is
used to remove cells from the flask. A 1:10 dilution of cells is made u'sing
complete media, and cells are seeded into new flasks. Cells should be split
every 3 to 4 days.
A flask of L929 cells is trypsinized, and the cells are counted and
diluted to 1 x 103 cells/ml, 8 x 102 cells/ml, 6 x 102 cells/ml, 4 x
102 cells/ml, and 2 x 102 cells/ml. The dilutions of the cells are plated
onto to 60-mm dish containing 4 ml of complete medium. Twenty plates are
needed at each cell concentration for one complete test. The cells are per-
mitted to attach to the tissue culture dishes for 24 hours. The plates are
then treated with various concentrations of the test chemical. Five concen-
trations of test chemical should be assayed in each cell dilution. Therefore,
six sets of five plates should be made: one for each of the five concentra-
tions of the test chemical plus one set to be used as an untreated control.
The cells are exposed to the test chemical for 24 hours. Following the treat-
ment period, the cells are washed twice with phosphate-buffered saline and fed
normal growth medium. Microscopic examination of the plates should discern
discrete colonies in approximately 10 to 12 days. At this time the plates are
washed with phosphate-buffered saline, fixed with methanol, and stained by
Giemsa. The colonies on the plates are counted, and a plating efficiency is
determined. The plating efficiency is calculated as the number of surviving
cells expressed as a percentage of the cells planted:
£ of colonies per plate
X 100
# of cells seeded
-------
35
An evaluation of the cytotoxic effect of the test chemical may be made by
comparing the plating efficiency of the test plates with that of control
plates.
Details are summarized in Table 11.
Protozoan Clonal Viability
This assay is based on the observation that when cells are subjected
to toxicants or stresses, only a fraction of the population survives and re-
produces. Heaf and Lee (1971) first developed this method to measure the
viability of Tetrahymena after exposure to low temperatures. This viability
assay is currently being adapted for toxicity testing (Persoone and Dive,
1978).
In the assay J. pyriformis is grown axenically in the dark. The cul-
tures are then diluted to about 5 cells/ml. One ml of the dilution is placed
in each well (cup) of a 100-hole, plastic, hemagglutination tray. Also, a
toxicant is added in increasing concentrations to the wells containing
Tetrahymena. After 6 days at 28 C, cells surviving certain toxicant concen-
trations will proliferate, while those affected by other toxicant concentra-
tions will not divide. The number of wells containing growing populations, as
well as the number of organisms in each well, can be counted with the naked
eye and recorded. A schematic of the clonal viability test method is shown in
Figure 1. The details on this assay are summarized in Table 12.
Human KB Cell Growth Rate
Nephelometric measurements, such as changes in the optical density or
macromolecular complement of cell cultures, provide a basis for monitoring the
growth of cell populations. In this assay, the inhibition (or possible stimu-
lation) of mammalian cell growth is determined by measuring colorimetrically
the total protein present in dividing cells both before and after incubation
with a test chemical (Oyama and Eagle, 1956). Even though any of several cell
lines could be employed, the rapidly growing human tumor line KB or the mouse
tumor lines P388 or L1210 are excellent candidates for this assay.
-------
36
TABLE 11. CLONING L929 MOUSE CELLS
Criteria
Critique/Comments
Test Organisms
Advantages
Limitations
Response to Chemicals
Assay Time*, hours
Special Equipment
Cost*
References
L929 mouse cell line.
The test organism is simple to culture.
Several concentrations of potential
toxicants may be assayed simultaneously.
This assay must be performed by skilled
technicians.
Specialized equipment is needed.
A very long time is required to obtain
results of this assay.
Aromatic hydrocarbons (benzene, toluene-
MEC is 5 to 50 ppm)
Detergents (sodium dodecyl sulfate-MEC
is .005% w/v)
Inorganic ions-heavy metals (cobalt,
nickel-MEC is 0.11 to 1
244, 326, 15, 9
Mammalian cell culture facilities
$750
Duke et al , 1977
Richardson, et al , 1977
*See time and cost explanation, pp. 9-11 in text.
-------
37
^
r0|
r^ ^
o o
/T-- O,
1
•»•
GOG
GCG
ceo
+
^* -
COO
OGG
IGSQ
1 TOXICANT
1 1
1
1
I
, 1
LlNCL'3ATiCN
I
^^>r
GOG
COG
'-^ T\ /T\
[ Vj Op
1
1
1
|
kC
1
1
Figure 1. Schematic Representation of Viability Test
Adapted to Toxicity Testing. From Persoone
and Dive (1978).
-------
38
TABLE 12. PROTOZOAN CLONAL VIABILITY
Criteria
Critique/Comments
Test Organism
Advantages
Limitations
Response to Chemicals
Assay Time*, hours
Special Equipment
Cost*
References
Tetrahymena pyriformis
Rapidly dividing cultures of test
organisms are easily grown.
Many replicates and chemical concentrations
can be done simultaneously.
The assay is not disrupted by particulate
matter or color of the potential
toxicant.
The TLso can ^e easily determined.
The assay can be performed by unskilled
technicians.
A long time is required to obtain
the results of this assay.
Gases (ethylene oxide)
148, 210, 14, 9
None
$660
West et al, 1962
Heaf and Lee, 1971
Gardinono et al, 1973
Mouton and Hendrickx, 1974
*See time and cost explanation, pp. 9-11 in text.
-------
39
The test is conducted as follows: KB cells, seeded at 1020 - g/ml
protein (2 to 3 x 10^ cells/ml), and the appropriate concentrations of test
chemical are mixed and incubated for 72 hours at 37 C. After incubation,
total protein is determined in the test and untreated control cultures as de-
scribed by Lowry et al (1951). For significance, untreated control cultures
must go through at least six cell divisions. The number of cultures in the
control group varies according to the formula 2Vn~T where n is the test cul-
tures or number of chemicals being tested. A positive control, cells treated
with 6-mercaptopurine, exhibits an ED 50 between .05 and 0.5 pg/ml.
Criteria for cytotoxicity of test chemicals would be any inhibition
of growth caused by the test chemical. The influence of a toxicant on cell
growth rate could be possibly extrapolated to the development and prolifera-
tion of tissues and organs (e.g., KB cell growth rate to nasopharynx lining
proliferation).
Details of this assay are summarized in Table 13.
Human Embryonic Lung Fibroblast (HI-38) Cytotoxicity
This assay is used to measure growth-inhibition effects of various
toxicants on mammalian cells.
Human embryonic lung fribroblasts (WI-38) are cultured in 75-cm2
Falcon flasks with Eagle's Minimum Essential Medium plus mycostatin (10,000
units/500 ml medium), penicillin (10,000 units/500 ml medium), streptomycin
(10,000 ug/100 ml medium), and heat-inactivated fetal calf serum (10 percent).
Cells are incubated in 10 percent carbon dioxide-humidified atmosphere at 37
C. Only cells between the 15 and 35 subculture should be used.
After the cells reach about 90 percent confluence, one flask of cells
is used for conducting this assay. There are eighteen 60-mm plates per assay.
After assembling medium, cells, and plates, 4 ml of medium is pipetted into
each plate. The stock cells in a 75-cm2 Falcon flask are then trypsinized.
After cell counts and dilutions are made, a 2 x 10^ cells/ml suspension is
seeded into each 60-mm plate.
After cells have grown to 100 percent confluence (4 to 5 days), test
toxicant is added. Cells and test chemicals are then incubated for 20 hours.
-------
40
TABLE 13. HUMAN (KB) CELL GROWTH RATE
Criteria
Critique/Comments
Test Organisms
Advantages
Limitations
Response to Chemicals
Assay Time*, hours
Special Equipment
Cost*
References
Human KB eel Is
The test organism is easy to culture.
Many potential toxicants at several
concentrations may be tested
simultaneously.
Inhibition of cell growth is usually an
accepted standard of toxicity.
National Cancer Institute routinely
uses this assay for cytotoxicity
screening.
Specialized equipment is needed.
A skilled technician is required to
perform this assay.
Inorganic ions-heavy metals (cadmium,
nickel-MEC is 0.1 to 10 ug/ml)
Nucleotide analogues (deoxy-adenosine
S'-triphosphate-MEC is > 5 x lO'5 M)
Aromatic hydrocarbons (toluene, benzene-MEC
is 1 to 5 ppm)
Detergents (sodium dodecyl sulfate, Triton
X-100-MEC is .001 to .005%)
Carcinogenic nitrosamines (dimethyl-
nitrosamine-MEC is 5 to 20 ug/ml)
75, 125, 12, 8
Mammalian cell culture facilities,
spectrophotometer
$620
Oyama and Eagle, 1956
*See time and cost explanation, pp. 9-11 in text.
-------
41
Following incubation, the three plates of cells per test chemical concentra-
tion (five different concentrations) are washed in phosphate-buffered saline
twice and then trypsinized. The cells are collected by centrifugation at 500
g for 5 minutes. The supernatant is poured off, and 1 ml of medium is added
to the centrifuge tube. The cells are suspended and counted with a hemocytom-
eter. The average number of cells recovered from each test chemical concen-
tration and from control plates is determined. Cell counts from the plates
are averaged for all the concentrations of the test chemicals and the con-
trols. The average counts from the test chemical plates are then expressed as
a function of the number of cells obtained from the control plates. A cyto-
toxicity curve for the test chemical is constructed. The curve is an expres-
sion of the cellular survival as a function of concentration of the toxicant.
Effects of toxic chemicals on fibroblasts could be extrapolated to
effects on human connective or pulmonary tissue.
Details on this assay are summarized in Table 14.
Mitogen Stimulation of Lymphocytes
Blastogenic transformation of lymphocytes is considered to be a mani-
festation of lymphocytes in cellular immunity. Measurement of the effect of
test chemicals on this mitogen-induced blast transformation is a measurement
of the effects on immune function. Thymus, spleen, or lymph-note cell suspen-
sions are cultured in the presence of mitogens such as Concanavalin A (Con A),
phytohemagglutinin-P (PHA), and Pokewood mitogen (PWM). Certain cells within
these populations respond to the presence of mitogens by undergoing blastogen-
esis. The response is quantitated by monitoring ^H-thymidine incorporation
in mitogen stimulated and nonstimulated cultures.
Varying concentrations of the test substance are added to microlym-
phocyte cultures in Falcon microtest II multi-well plates. Each well is a
microculture of 5 x 10^ lymphocytes growing in the presence or absence
(control) of a mitogen (PHA, 50 yg/ml final; Con A, 100 pg/ml final). Each
dosage of the test chemical is tested in quadruplicate with the lymphocyte
cultures. Incubation is pulsed with 1 yCi of 3H-thymidine. Twenty-four
hours after pulsing, the cultures are harvested on glass-fiber filters using a
-------
42
TABLE 14. HUMAN EMBRYONIC LUNG FIBROBLAST
(WI-38) CYTOTOXICITY
Criteria
Critique/Comments
Test Organisms
Advantages
Limitations
Response to Chemicals
Assay Time*, hours
Special Equipment
Cost*
References
Human embryonic lung fibroblasts
(WI-38)
An automatic cell counter can be used
to simplify this assay.
Many replicates and chemical concentrations
can be tested simultaneously.
The assay result's are easily obtained
by direct counting.
A skilled technician with experience in
microscopy is required.
Some specialized equipment is needed.
The possibility of human mistakes due
to fatigure and boredom exists
because of the tedious nature of
the data collection.
Inorganic ions-heavy metals (nickel,
cadmium-MEC is 0.1 to 1 yg/ml)
Gases (carbon monoxide)
Aromatic hydrocarbons (benzene, toluene,
ethyl benzene-MEC is 0.5 to 50 yM)
27, 176, 14, 9
Mammalian cell culture facilities,
binocular microscope (optional),
hemocytometer
$730
Baiile and Hardegree, 1970
*See time and cost explanation, pp. 9-11 in text.
-------
43
multisample harvesting unit. ^-thyroidine incorporation is determined by
counting the filters, using liquid scintillation spectrophotometry.
To analyze the data, one must (1) compute mean radioactive counts per
minute (cpm) and standard error of mean for all control values, i.e., PHA, Con
A, and medium; (2) compute mean cpm and standard error of mean for each quad-
ruplicate cell control, i.e., cells and medium; (3) compute mean cpm and stan-
dard error of mean for each quadruplicate PHA- and Con A-stimulated cultures;
(4) for stimulation index, divide each of the quadruplicate cpm values for
PHA-stimulated cells by the mean cpm value of the same cells nonstimulated,
and average the four indices determined in this manner for a mean stimulation
index (repeat for Con A and PHA); and (5) compare index of test-substance
treated and untreated cultures.
Impairment of lymphocyte function by toxic chemicals may foreshadows
the impairment of antibody formation and immune response in mammals.
Details of this assay are summarized in Table 15.
Chick Embryo Development
The assay is an attempt to predict toxicologic or teratologic (tera-
togenic) effects on higher vertebrates based on responses of chick embryos to
potentially harmful compounds. Fertilized white leghorn eggs are candled to
locate the air cell. A hole drilled through the shell over the air cell is
the site of aseptic injection of 0.1 ml of a test chemical into the yolk of
the developing embryo. The hole is covered with tape and the eggs are incu-
bated at 38 C and periodically candled. Dead embryos are pathologically
examined and surviving chicks are examined over a 2 to 6-week period for
weight change, gross abnormalities, and mortality. At least 20 eggs are used
for each chemical concentration tested to add statistical significance to the
results. Eggs hatch after 21 days so the entire procedure may be performed in
approximately 1 month.
Details are summarized in Table 16.
-------
44
TABLE 15. MITOGEN STIMULATION OF LYMPHOCYTES
Criteria
Critique/Comments
Test Organisms
Advantages
Limitations
Response to Chemicals
Assay Time*, hours
Special Equipment
Cost*
References
Mouse lymphocytes
The effect of a potential toxicant is
defined for a developmental parameter
as well as a growth parameter.
It is possible to determine a
developmental change before a loss
in viability or growth potential of
test eel Is.
Some variation of responsivenss of
lymphocyte preparations may interfere
with this assay.
Great expense is incurred maintaining
a mouse colony as a source of
lymphocytes.
Specialized equipment is needed.
The assay must be performed by highly
skilled technicians.
Fungal toxins (aflatoxins-MEC is
5 to 20 ug/ml)
Inorganic ions-heavy metals (nickel,
cadmium-MEC is 0.1 to 1 ug/ml)
76, 76, 11, 8
Scintillation counter, spectrophotometer,
animal rearing facilities, mammalian
cell culture facilities.
$820
Save! et al, 1970
*See time and cost explanation, pp. 9-11 in text.
-------
45
TABLE 16. CHICK EMBRYO DEVELOPMENT
Criteria
Critique/Comments
Test Organisms
Advantages
Limitations
White Leghorn chick embryos
Large numbers involved make the results
of this assay statistically meaningful.
Responses of test organism correlate well
with other animal responses to
traditionally toxic chemicals (lead
acetate, mercury II chloride).
Since eggs are incubated under controlled
conditions, maternal influence is not a
variable, as it is in placental animals.
The rapidly dividing cells may reduce the
time necessary to elicit a response
to possible toxicants.
This assay can be performed by unskilled
technicians.
There is a lack of standardized methods
in this assay.
An extremely long time is required to
obtain the results of this assay.
Since responses are dependent on critical
periods of development, responses may
vary with each test chemical.
One species of test organism selected,
White Leghorn, may be unrealistically
sensitive or insensitive to some
chemicals.
Response depends on several different
variables: specific gravity, solubility,
pH, ionic concentration, and coagulating
effect.
Negative results may not be significant.
The lack of a placental barrier gives a
questionable correlation between
responses of chick embryos and responses
exhibited by mammals.
Specialized equipment is needed.
-------
46
TABLE 16. (Continued)
Criteria
Critique/Comments
Response to Chemicals
Assay Time*, hours
Special Equipment
Cost*
References
Inorganic ions-heavy metals (lead,
mercury, cobalt-LC/i is 0.1 mg)
Food additives (monosodium glutamate,
sodium benzoate)
Nucleic acid analogues (5-fluorouraci1)
Antibiotics (tetracycline, methacycline,
doxycycline)
Dithiocarbamates [bis(dimethyl
thiocarbamoyl)-disulfide]
Organic solvents (carbon tetrachloride,
n-butanol)
Hallucinogens [lysergic acid diethylamide,
(LSD)]
Lathyrogenic agents (B-aminopropionitrile-
LC62 is 0-63 mg)
Metabolic inhibitors (2,4-dinitrophenol)
720 (1 month), 720, 108, 11
Incubators, hatching facilities, rearing
facilities, sterile injecting facilities.
$2510
Feldman et al, 1958
Mclaughlin et al, 1963
Gebhardt and Van Logten, 1968
Kury and Crosby, 1968
Hall, 1972
Pagnini et al, 1972
Flick et al, 1973
Messier, 1973
Palmer et al, 1973
Hulbert and Klawitter, 1974
Hall, 1976
Swartz, 1977
Zagris, 1977
Lee, 1978
Loomis, 1978
*See time and cost explanation, pp. 9-11 in text.
-------
47
Trypan Blue Dye Exclusion by Human KB Cells
The ability of cultured human cells to exclude the dye trypan blue is
a measure of a functioning cell membrane.
Monolayer cultures of KB cells are incubated with a test chemical for
24 hours. Following the incubation period, the cells are removed from the
plates with a rubber policeman. The cell suspension is placed in 15 ml coni-
cal centrifuge tubes and centrifuged at 600 g for 5 minutes. The supernatant
is discarded, and the cells are resuspended in 2 ml of phosphate buffered
saline. An 0.66-ml aliquot of the cell suspension is mixed with 0.66 ml of a
0.4 percent trypan blue solution. A cell count and a viability determination
are carried out for each concentration level, using a hemocytometer or
cytograf. Viable cells are those cells that do not take up the trypan blue
dye. Viability is calculated by:
No. of viable cells
: X 100 = percent viability
total cell #
A Viability Index is also calculated as follows:
u- U-T-J. T j mean total cell count of test . , .,.
Viability Index = mean tota1 Ce11 count of Contro1 X mean percent^viability
Information from this assay may be extrapolated to other cellular or
subcellular membranes because other cells are enclosed by semi permeable
membranes which are structurally and functionally similar to the lipid bilayer
surrounding KB cells.
Details of this assay are summarized in Table 17.
CATALYSIS (ENZYMATIC ACTIVITIES)
RNA Polymerase Activity
RNA polymerase is a multimeric enzyme that catalyzes the synthesis of
RNA chains from the nucleoside triphosphates ATP, CTP, GTP, and UTP. The
synthetic reaction has an absolute requirement for a divalent metal ion and
-------
48
TABLE 17. TRYPAN BLUE DYE EXCLUSION BY HUMAN KB CELLS
Criteria
Critique/Comments
Test Organisms
Advantages
Limitations
Response to Chemicals
Assay Time*, hours
Special Equipment
Cost*
References
Human KB cells
This assay can be partially automated
by the use of a hemocytometer.
The test results are straightforward
and easily obtainable.
A relatively short time is required
to obtain the results of this assay.
Specialized equipment is needed.
A skilled technician with experience
in microscopy is required.
Detergents (Triton X-100), sodium
dodecyl sulfate-MEC is .001 to
.005%)
Polycyclic aromatic hydrocarbons
(naphthalene, anthracene-MEC is
0.5 to 75 uM)
Inorganic ions-heavy metals (cadmium,
lead-MEC is 0.1 to 1 ug/ml)
30, 77, 11, 8
Binocular microscope, mammalian cell
culture facilities, hemocytometer
$600
Corning and Firth, 1969
*See time and cost explanation, pp. 9-11 in text.
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49
normally requires DNA as a template. RNA polymerase recognizes and binds to
certain base sequences in DNA, initiates RNA synthesis, elongates the RNA
chain, and finally terminates RNA synthesis with a release of a new RNA
molecule.
The standard wheat germ RNA polymerase II assay mixture, in a final
volume of 0.25 ml, contains 2.5 umol of Tris-HCl (pH 7.9); 0.25 umol of manga-
nese chloride; 12.5 ymol of ammonium sulfate; 100 nmol each of GTP, CTP, and
ATP; 1 yCi of (5-3H)UTP diluted to a specific radioactivity of 1 uCi/0.1
nmol; 50 ug of heat-denatured calf thymus DNA; and 125 ug of bovine serum
albumin.
The assays mixture is 'incubated for 15 minutes at 25 C, and the RNA
is precipitated by adding 2 ml of 5 percent (w/v) ice-cold trichloroacetic
acid containing 25 mM sodium pyrophosphate. After 5 minutes at 0 C, the pre-
cipitates are collected on Whatman GF/C filters and are washed under suction
with five 4-ml rinses of ice-cold 2 percent trichloroacetic acid containing 10
mM sodium pyrophosphate followed by 2 ml of 95 percent ethanol. After the
filters are dried under a heat lamp, they are assayed for radioactivity by
liquid scintillation counting.
By changing only the template and the radioactive nucleotide, one can
assay for several other polymerase activities involved in gene replication and
expression. These enzymatic activities include DNA polymerase, poly(A)-
polymerase, and polynucleotide phosphorylase. In all of these polymerase
assays, test chemicals can be added to the reaction mixture prior to the ad-
dition of the enzyme.
By effecting RNA polymerase activity, a toxic chemical would be
modifying the mechanism by which all new cellular proteins and enzymes are
produced. Since RNA polymerase is present in all living organisms, results
could be extrapolated to all life forms.
Details of this assay are summarized in Table 18.
Adenyl Cyclase Activity
Adenyl cyclase is a hormonally activated surface membrane enzyme
which catalyzes the conversion of adenosine triphosphate (ATP) to cyclic
-------
50
TABLE 18. RNA POLYMERASE ACTIVITY
Criteria
Critique/Comments
Test Organisms
Advantages
Limitations
Response to Chemicals
Assay Time*, hours
Special Equipment
Cost*
Reference
Either RNA polymerase purified from wheat
germ or Escherichia coli may be used.
RNA polymerase is present in every living
organism.
All assay components as well as E. coli
and wheat germ enzymes are commercially
available and are relatively inexpensive.
Hundreds of assays can be completed daily. .
Many replicates and chemical concentrations
can be done simultaneously.
Noting the effects of chemicals on RNA
polymerase will contribute to pinpointing
molecular mechanisms of chemical action.
A very short time is required to obtain the
results of this assay.
Colored chemicals and precipitates inter-
fere with this assay.
A high degree of technical skill and
training is necessary to assay for RNA
polymerase.
Specialized equipment is needed.
Metal carcinogens (cobalt-Iso is 0.5
mM, lead)
Antibiotics (actinomycin D, proflavine,
mithramycin, rifamycin)
Carcinogenic nitrosamines (dimethyl-
nitrosamine, azobenzene derivatives)
Polyanions (heparin, polyethylenesulfonate)
Metal mutagens (lithium-Igg is 0.2 M)
Fungal toxins (alpha-amanitin, aflatoxin)
Nucleotide analogues (2'-0-methyl-adenosine
S'-triphosphate)
1.5, 5, 5, 8
Millipore filtration manifold, liquid
scintillation counter
$530
Polya, 1973
Jendrisak and Burgess, 1975
Hoffman and Niyogi, 1977
Glazer, 1978
*See time and cost explanation, pp. 9-11 in text.
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51
adenosine monophosphate (cAMP). The latter (cAMP) is an important regulatory
molecule in both prokaryotic and eukaryotic cells.
WI-38 human diploid cells are seeded at 2 x 106 cells/60-mm dish.
Various doses of the test chemical are added at the time of culture seeding.
Cultures are incubated for 24 hours. Treated and control cells are then
processed to determine adenyl cyclase activity. After trypsinization, cell
pellets are obtained by centrifugation and washed three times in a 10 mM Tris
buffer (pH 7.4) containing 0.1 mM dithiothreitol at 0 to 4 C. After the final
wash, buffer is added followed by ice-cold magnesium chloride and sucrose to
final concentrations of 3 mM and 250 mM, respectively. Better than 99 percent
breakage of WI-38 cells is observed. Broken cells are subsequently centri-
fuged at 2000 g for 15 minutes, and the pellet is suspended in buffer contain-
ing 250 mM sucrose, 3 mM magnesium chloride, 0.1 mM dithiothreitol, and 10 mM
Tris buffer (pH 7.4)'.
The broken cell pellet is used for the adenyl cyclase assay.
Reaction mixtures contain 2 mM ATP, 6.6 mM magnesium chloride, 1.0 mM dithi-
othreitol, 40 mM Tris buffer (pH 7.4), and 0.05 M sucrose in a final volume of
0.5 ml. Reactions are initiated by adding 40 to 80 g of protein to the cell
preparation. After incubation at 37 C for 15 minutes, the reaction is stopped
by adding trichloroacetic acid to a final concentration of 5 percent. After
centrifugation, the supernatant is treated three times with ethyl ether to
remove the trichloroacetic acid. The solution is analyzed for cAMP using a
radiotracer competitive protein binding method (commercially available). An
ATP regenerating system composed of 10 mM creatine phosphate and five units of
creatine phosphokinase/0.5 ml of incubation mixture is used in all experi-
ments. Enzymatic activity is expressed as the amount of cAMP produced.
Despite the fact that cAMP plays a key role in controlling biological
processes, the correlation between a chemical effect on adenyl cyclase and
cellular or tissue toxicity would require extensive research and development.
Details of this assay are summarized in Table 19.
Lysosomal Enzyme Release
The destabilization of internal cellular membranes produced by toxic
chemicals can be assessed by measuring the release of certain enzymes from the
-------
52
TABLE 19. ADENYL CYCLASE ACTIVITY
Criteria
Critique/Comments
Test Organisms
Advantages
Limitations
Response to Chemicals
Assay Time*, hours
Special Equipment
Cost*
References
WI-38 human diploid cells
The assay measures a specific enzymatic
activity at a surface membrane. This
enzyme plays a key regulatory role in
cellular metabolism.
This assay assesses a specific physio-
logical process as well as an
organellar function.
A relatively short time is necessary to
obtain the results of this assay.
Screening large numbers of chemicals
is time consuming due to the
requirement for processing cell membranes
before the enzyme assay, followed by the
cAMP assay.
Some specialized equipment, is needed.
This assay must be performed by highly
skilled technicians.
Inorganic ions-heavy metals (lead,
nickel-MEC is .05-10 ug/rnl)
Detergents (Triton X-100-MEC is .001-.05%)
29, 79, 14, 9
Mammalian cell culture facilities,
liquid & scintillation counter
$880
Klein et al, 1978
*See time and cost explanation, pp. 9-11 in text.
-------
53
lysosomes into the cytoplasm. In this assay, the release of the lysosomal
enzyme acid phosphatase is measured histochemically.
KB cells, seeded at 10$ cells per 60-mrn dish, are pipetted onto
coverslips and then various doses of a test chemical are administered. After
treatment for 24 hours, coverslips are washed in isotonic saline and then are
incubated for 15 minutes in a medium of 10 mM g-glycerophosphate and 50 mM
acetate buffer (pH 5.0) containing 4 mM lead nitrate and 50 mM sodium chlor-
ide. Following incubation, the coverslips are fixed for 10 seconds in one
percent acetic acid, transferred to hydrogen sulfide-saturated water for
5 minutes, washed in distilled water, and mounted. Staining of acid phos-
phatase is indicative of damaged lysosomal membranes because intact lysosomal
membranes are impermeable to the g-glycerophosphate substrate.
Other mammalian cell lines (HeLa, mouse L) can be used in this assay.
It should be noted that this assay measures the effect of a toxicant
on an organellar membrane inside the cell. This assay does not measure cell
lysis.
This assay measures the perturbation of an important cellular com-
ponent. So, the enzymes, released from lysosomes ruptured by a toxicant, can
kill cells and cause tissue necrosis. This assay can be used as quick, quali-
tative screen. Microspectrophotometric techniques could be used to quantitate
this assay.
Details are summarized in Table 20.
Macromolecular Synthesis in KB Cells
This assay is used to assess the effects of test chemicals on the
syntheses of macromolecules. The rate of incorporation of radiolabelled pre-
cursors into an acid precipitable form (macromolecular form) is used as a
measure of the synthetic rate, ^^-uridine incorporation is used to measure
RNA synthesis, ^H-thymidine incorporation to measure DNA synthesis, and
3H-leucine incorporation to measure protein synthesis. The protocols for
measuring any of these three parameters are the same since the radiotracer is
the only variable.
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54
TABLE 20. LYSOSOMAL ENZYME RELEASE
Criteria
Critique/Comments
Test Organisms
Advantages
Limitations
Response to Chemicals
Assay Time*, hours
Special Equipment
Cost*
References
Human KB cells
This assay measures a specific membrane
alteration before a loss in viability
occurs.
The test results are straightforward and
easily obtainable.
A relatively short time is required to
obtain the results of this assay.
This assay requires carefully controlled
conditions of incubation to prevent
nonspecific lysosomal damage or staining.
A skilled technician with experience in
microscopy is required.
Specialized equipment is needed.
Abrasives (silica)
Detergents (Triton X-100-MEC is .05-1%)
29, 76, 9, 8
Microscope, mammalian cell culture
facilities
$560
Grasso et al, 1973
*See time and cost explanation, pp. 9-11 in text.
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55
Approximately 2.0 x 10^ KB cells are seeded into 60-mm petri
dishes. Various dosages of the test chemical are added at the time of seeding
of the cultures. Untreated controls are included. After 24 hours of incuba-
tion, the cultures are pulsed with 1 uCi/ml of the appropriate radiotracer
(^H-thymidine, ^H-uridine, or 3n_-|euc-jne). After a 2-hour pulse, the
monolayer is rinsed with cold physiological saline, trypsinized, and resus-
pended in saline. Aliquots of the suspensions are taken for cell count. The
remaining cells are lysed by the addition of sodium deoxycholate to a final
concentration of 0.5 percent. An equal volume of cold 10 percent trichloro-
acetic acid is added to the suspension, and the resulting precipitate is
collected on glass-fiber filters. The filters are then dried, and the
radioactivity is determined by liquid scintillation spectrophotometry. The
results are calculated as counts per minute of isotope incorporated per cell.
This assay could be combined and correlated with other biochemical or
enzymatic assays. For example, the chemical inhibition of cellular RNA
synthesis could be correlated with the effect of that chemical on RNA poly-
merase activity. Also, this assay protocol can be expanded to monitor other
cellular syntheses by simply utilizing other labelled precursors (e.g., -fy-
acetate for fatty acid synthesis).
Details of this assay are summarized in Table 21.
OTHER CELLULAR PROCESSES
Cyclosis
Cyclosis or protoplasmic streaming is the regular, cyclic movement of
particles within a cell. Lucas (1977) has devised an assay to measure the
inhibition of cyclosis caused by various levels of ammonium sulfate. This as-
say could be adapted for toxicity testing.
In this procedure, internodal cells of the al ga Chara are cut from an
algal mat 1 day prior to the experiment. After cutting, the cells are soaked
in 1.0 mM sodium bicarbonate buffer (pH 9.0) and are subjected to a regime of
13 hours of light and then 11 hours of dark. All cells are illuminated under
fluorescent lights (10 W/sqm) for 2 hours before the start of an experiment.
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56
TABLE 21. MACROMOLECULAR SYNTHESIS IN KB CELLS
Criteria
Critique/Comments
Test Organisms
Advantages
Limitations
Response to Chemicals
Assay Time*, hours
Special Equipment
Cost*
References
KB Human cell cultures
This assay measures specific functional
properties of cells and can be
indicative of functional alteration
or loss before general toxic effects
(such as death) are observed.
A relatively short time is required to
obtain the results of this assay.
This assay must be performed by skilled
technicians.
Specialized equipment is needed.
Inorganic ions-heavy metals (nickel,
lead-MEC is 0.1 to 10 yg/ml)
Polycyclic hydrocarbons (naphthalene-
MEC is 1 to 50 uM)
Detergents (hexachlorophene-MEC is
5 to 100 ug/ml)
34, 78, 11, 8
Liquid scintillation counter, mammalian
cell culture facilities
$720
Carr and Ligaton, 1973
*See time and cost explanation, pp. 9-11 in text.
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57
At the beginning of the test, cells are soaked for 1 hour in a solution of the
chemical to be tested. After exposure to the chemical, cells are examined,
and cyclosis is measured with a binocular microscope having an ocular microm-
eter. The time required for a standard-size cytoplasmic particle to traverse
1000 urn is measured with a stopwatch. Rates of cyclosis are measured in 10
cells and are expressed as the mean _+ standard error.
It would be difficult to correlate a specific chemical effect on
algal cyclosis with a chemical effect on metabolic processes in mammalian
cells and tissues.
Details on this assay are summarized in Table 22.
Hemolysis
This bioassay is capable of identifying the hemolytic effect of
various potential toxicants. To measure this hemolytic effect, solutions of
varying concentrations of the suspected toxicant, and a buffered saline
solution containing 0.1 mM ethylenediaminetetraacetate (EDTA), are prepared
and washed rat erythrocytes are added. The concentration of these cells
should be 0.5 percent (volumetrically). The solutions are incubated for 1
hour and then centrifuged for 10 minutes at 1000 g to remove intact red cells.
The supernatant fraction is then spun for 15 minutes at 20,000 g to remove any
remaining particulate matter. The optical densities of the final supernatant
fractions are measured at 542 nm to estimate hemoglobin. Addition of a like
amount (0.5 percent) of red blood cells to water gives the value for 100 per-
cent hemolysis.
Information from this assay may be extrapolated to other cellular and
subcellular membranes because other cells are enclosed by semipermeable mem-
branes which are structurally and functionally similar to the lipid bilayer
surrounding erythrocytes. Also, since erythrocytes contain hemoglobin which
transports oxygen to tissues, any erythrocyte aberation would cause detrimen-
tal effects elsewhere in the body.
Details on this assay are summarized in Table 23.
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58
TABLE 22. CYCLOSIS
Criteria
Critique/Comments
Test Organisms
Advantages
Limitations
Response to Chemicals
Assay Time*, hours
Special Equipment
Cost*
References
Chara coral!ina is the major organism.
Nitella translucens and Elodea may also
be used.
None
Inhibition of cyclosis goes from 0 to
100 percent with a small change in the
concentration of certain interfering
chemicals.
The test organism is very difficult to
culture.
A skilled technician experienced in
microscopy is necessary.
A relatively long time is required to
obtain the results of this assay.
Some chemicals may cause increased,
instead of decreased streaming.
Only one assay at one test chemical
concentration can be performed in
one hour.
There is a variable rate of streaming
which is dependent on cell volume.
Specialized equipment is needed.
Metabolic inhibitors (2,4-dinitrophenol).
Chlorinated aliphatics (chloroform).
Inorganic salts (ammonium sulfate-
125 is 0.5 mM).
Gases (oxygen)
Sugars (mannitol, sucrose)
30, 141, 58, 11
Binocular microscope with ocular
micrometer
$1810
Pfeffer, 1938
Thaine, 1964
Geis and Morrison, 1971
Lucas, 1977
*See time and cost explanation, pp. 9-11 in text.
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59
TABLE 23. HEMOLYSIS
Criteria
Critique/Comments
Test Organisms
Advantages
Limitations
Response to Chemicals
Assay Time*, hours
Special Equipment
Cost
Rat or human erythrocytes
\
This assay can be performed by an
unskilled technician.
A very short time is required to obtain
the results of this assay.
The test results are straightforward
and easily obtainable.
Erythrocytes from several sources are
commercially available.
Many chemicals and varying chemical
concentrations may be tested
simultaneously.
Rat and human erythrocytes vary from
batch to batch because of nutritional
and genetic differences in donors.
With some chemicals it is difficult to
establish a dose-response relationship.
Sulfhydryl inhibitors (p-chloromer-
curibenzoic acid-EC4Q is 37.4 mg)
Inorganic ions-heavy metals (lead, mercury)
Hormones (epinephrine, prostaglandin £2)
Peroxides (peroxidized microsomal lipids,
hydrogen peroxide)
Abrasives (silica)
Arylhydrazines (phenyldrazine-ECyy is
100% solution, m-toylhydrazine)
Detergents (Triton X-100)
Inorganic ions-halides (iodide)
Ionic surfactants (alkyltrimethylammonium
halides)
Chlorinated antibacterials (hexachloro-
phene)
Industrial particulates (asbestos)
Buffers (Tris-HCl-ECioo is 100% solution)
2, 4, 4, 8
Spectrophotometer or colorimeter
$440
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60
TABLE 23. (Continued)
Criteria Critique/Comments
References Sheets et al, 1956
Allen and Rasmussen, 1971
Lessler and Walter, 1973
Itano et al, 1974
Klebanoff and Clark, 1975
Luthra et al, 1975
Majer, 1975
Light and Wei, 1977
Summerton et al, 1977
Pesh-Iman et al, 1978
Zaslavsky et al, 1978
*See time and cost explanation, pp. 9-11 in text.
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61
Protozoan Vacuole Contraction
This assay is based on the observations that contractile vacuoles
function in regulating osmotic pressure (Rifkin, 1973) and expelling waste
substances from the cell. Nilsson (1974) developed an assay to measure the
effect of a foreign substance on vacuolar contraction in Tetrahymena. Under
favorable conditions, the vacuole contracts at regular intervals. However,
when the protozoan is stressed, the timing of the intervals may be altered.
In the assay, J_. pyriformis is grown axenically in an enriched
medium. Aliquots of the cells are then incubated at 28 C in the test chemical
solution. Observations are made using a light microscope, a Reichert anoptral
optical system or a similar viewing system, during the 1-hour period, with
expulsion intervals of vacuoles from several cells being recorded. The time
required to reach normal size and the expulsion intervals for control cells
are also recorded. The time intervals for individual cells are recorded
separately.
It would be difficult to correlate results from this assay with data
obtained from mammalian cells and tissues. However, if vacuolar contraction
were developed as a toxicity assay, those results could complement data ob-
tained from the protozoan clonal viability assay and the protozoan motilty
assay.
The use of protozoans as test organisms in toxicity studies would
appear to bridge the gap between undifferentiated prokaryotic organisms, such
as bacteria, and the more complex metazoa (Woodard, 1976).
Details on this assay are summarized in Table 24.
Protozoan Motility
Bergquist and Bovee (1974) conceived an original method for measuring
the motility of ciliates by microphotography.
Tetrahymena pyriformis is centrifugally pelleted and separated from
axenic growth medium, washed, and again pelleted centrifugally. Then it is
introduced by pipette into a holding chamber. For the assay, greater than 98
percent of the organisms should be motile. The test chamber is covered at its
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62
TABLE 24. PROTOZOAN VACUOLE CONTRACTION
Criteria
Critique/Comments
Test Organisms
Advantages
Limitations
Response to Chemicals
Assay Time*, hours
Special Equipment
Cost*
References
Tetrahymena pyriformis
The test organism is easy to obtain
commercially and culture.
The effects of potential toxicants can
be easily observed.
Vacuolar contraction can be standardized
by controlling temperature, the age of
cells, and the nutritional state of
eel Is.
Response times may vary even in a single
cell.
A long time is required to obtain the
results of this assay.
The assay must be performed by skilled
technicians with experience in
microscopy.
Vacuolar contraction varies with vacuole
size and a cell may contain more than
one vacuole.
Numerous observations must be made.
Dipolar solvents (dimethyl sulfoxide)
17, 143, 30, 10
Light microscope, temperature control unit
$1130
Rifkin, 1973
Nilsson, 1974
Patterson and Sleigh, 1976
*See time and cost explanation, pp. 9-11 in text.
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63
bottom by #5 Whatman filter paper and suspended in 200 ml of test solution.
The pH is monitored and readjusted to 7.0 as needed. After 1 hour, samples
are pipetted onto clean slides for microscopic examination. Multiple-exposure
photographs are taken stroboscopically using a perforated aluminum disc
attached to a stirring motor, which is equipped with a variable-speed reduc-
tion gear, permitting optimal image-spacing. The revolving perforated disc is
interposed between a Zeiss RA microscope and the removable light, permitting
full use of the microscope's optics and lighting. Negative-image films are
then projected onto a frosted glass screen and maximal-speed paths and linear
spacings are measured. As an end point, the distances between the ciliates in
the multiexposed photomicrographs are measured very easily by projection of
the negatives onto a large screen. This method permits large data samples to
be obtained quickly and easily. The results are then tested statistically for
comparative and descriptive purposes. This assay might be automated by em-
ploying a computer and a TV.
A chemical inhibition of protozoan flagellar or ciliary function
might be similar to effects on mammalian tracheal tissues. The advantage of
using protozoans in toxicity studies is mentioned in the description of the
vacuole contraction assay.
Details on the assay are summarized in Table 25.
Phagocytosis by Alveolar Macrophages
This assay employs a primary cell line, alveolar macrophages, to
define the acute cellular toxicity of particulates and other chemicals. Toxic
effects are assessed by measuring a macrophage function, phagocytosis.
Rabbit alveolar macrophages are harvested. The cellular composition
should contain a minimum of 95 percent alveolar macrophages. The cell suspen-
sion is then adjusted to a concentration of 1 x 105 cells/ml.
One-mi aliquots of the cell suspension are placed in 60-mm tissue
culture plates and the macrophages allowed to adhere. After 2 hours, the
medium is decanted and 5 ml of fresh medium is added to each plate.
The test chemical is diluted to the desired concentration with cell
culture medium and 1-ml aliquots are added to each plate. Initially, three
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64
TABLE 25. PROTOZOAN MOTILITY
Criteria
Cri t i que/Comments
Test Organisms
Advantages
Limitations
Response to Chemicals
Assay Time*, hours
Special Equipment
Cost*
References
Tetrahymena pyriformis is the major
test organism.
Paramecium caudatum may also be used.
The test organisms are easy to culture.
The decrease in motility is usually
related to the concentration of the
potential toxicant.
Large data samples may be easily obtained.
Precipitates or particulate matter may
interfere with this assay.
Specialized equipment is needed.
There is a lengthy film development
period in microphotography.
This assay requires a skilled technician
with experience in microscopy.
Heavy metals (nickel, cadmium)
Ionic detergents (sodium dodecylsulfate,
sodium stearate)
23, 136, 27, 9
Microphotography unit
$1000
Andrivon, 1968, 1972
Dryl and Bujwid-Cwik, 1972
Berquist and Bovee, 1973, 1974
Perkins and Cieresko, 1973
*See time and cost explanation, pp. 9-11 in text.
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65
concentrations of test chemical are used with three replicates per dose. The
plates are incubated in a humidified 95 percent air, 5 percent carbon dioxide
atmosphere at 37 C for 20 hours. The plates may be rotated for the first hour
to ensure uniform exposure of the test material. At the end of the incubation
period, the medium is decanted and fresh medium added.
Phagocytic activity is measured by addition of 1.1 urn polystyrene
latex particles to alveolar macrophages cultured in Lab-Tek four-chamber
microslides (approximately 25 particles/cell in 1 ml of medium). One hour
after the addition of latex particles, the slides are then exposed for an
additional 5 to 6 minutes with 1:1 aqueous dilution of Wright's stain. After
air drying, the slides are placed in xylene for one hour to dissolve extra-
cellular particles. Following an additional drying step, the slides are
mounted with permount. Phagocytic activity is determined under oil immersion
by scoring a minimum of 200 cells. Each cell that contains at least one
particle is considered phagocytically active. Typically, 80 to 90 percent of
the cells in control cultures ingest one or more particles.
This assay is already in limited use as a toxicity assay.
Details on this assay are summarized in Table 26.
Ami no Acid Transport
A measurement of plasma membrane function is active transport and
exchange of molecules into and out of the cell. This assay tests membrane
function by measuring the active transport of the amino acid histidine into KB
cells.
For this assay, 10^ KB cells in 3 ml of suspension culture medium
are exposed to various doses of the test chemical for 24 hours. At the end of
the exposure period, the cells are washed and then suspended in incubation
medium containing 131 mM sodium chloride, 5.2 mM potassium chloride, 1.3 mM
magnesium sulfate, and 1.0 mM calcium chloride in 10 mM sodium phosphate
buffer (pH 7.4). One uCi of ^H-histidine is added, and the mixture is in-
cubated with agitation at 37 C. Samples are taken every minute for 5 minutes
for radioactivity and cell number determinations. For measurements of histi-
dine uptake, the cells in each sample are washed in cold saline to remove
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66
TABLE 26. PHAGOCYTOSIS BY ALVEOLAR MACROPHAGES
Criteria
Crit1que/Comments
Test Organisms
Advantages
Limitations
Response to Chemicals
Assay Time*, hours
Special Equipment
Cost*
References
Rabbit alveolar macrophages
The assay measures a functional process
of macrophages, phagocytosis, and
indicates specific functional alterations
produced by test substances.
A relatively short time is required to
obtain the results of this assay.
Alterations in phagocytosis occur before
any general loss in cellular viability.
A great expense is incurred maintaining a
rabbit colony as a source of macrophages.
The assay can be performed by skilled
technicians.
Other specialized equipment is needed.
The assay is labor intensive in the
preparation of macrophages and in
monitoring results.
Macrophage preparations may vary in
responsiveness from day to day.
Gases (cigarette smoke, fly ash-MEC is
10 to 100 ug/ml)
Inorganic ions-heavy metals (cadmium,
zinc-MEC is 25 to 100 mM)
27, 27, 17, 9
Microscope, animal rearing facilities,
mammalian cell culture facilities
$890
Green and Carol in,
Duke et al, 1977
1967
*See time and cost explanation, pp. 9-11 in text.
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67
extracellular histidine. The total radioactivity in the cells is determined
by liquid scintillation counting. Rates of histidine transport are then
determined and compared by plotting the concentration of intracellular,
labelled histidine against the extracellular concentration by the classic
method of Lineweaver and Burke. Toxicity is indicated by decreases in the
rate of histidine transport as compared to untreated controls.
Information from this assay may be extrapolated to other cellular or
subcellular membranes because other cells are enclosed by semi permeable mem-
branes which are structurally and functionally similar to the lipid bilayer
surrounding KB cells.
Details on this assay are summarized in Table 27.
OTHER POTENTIAL PHYSIOLOGICAL TOXICITY ASSAYS
Several other assays show promise as toxicity screens and are cri-
tiqued below. In each case, few data on the assay or on effects of known
toxicants exist. For the chlorophyll fluorescence assay, much of the
information is in press and will appear in the near future. However, it is
impossible for us to develop a complete protocol for these assays with the
information presently available.
The Microtox bacterial luminescence assay designed by Beckman
Instruments, Carlsbad, California, shows promise as a potential toxicity
screen.
All living organisms have certain structural and metabolic similari-
ties. It is often possible to extrapolate studies performed on one organism
to other living systems because of these similarities. Cells of luminescent
bacteria are structurally and functionally similar to other living cells. The
semipermeable membranes surrounding all cells are quite similar. In addition,
certain metabolic processes (e.g., respiration) are common to all cells.
According to Beckrnan, the Microtox system is simple, rapid, inexpen-
sive, and accurate. From the data available, this appears to be true.
Preliminary test results also seem to correlate with results of the 96-hour
fish acute toxicity test. Although this assay appears to be an effective
toxicity screen, the methods and results still need to be validated by
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68
TABLE 27. AMI NO ACID TRANSPORT
Criteria
Critique/Comments
Test Organisms
Advantages
Limitations
Response to Chemicals
Assay Time*, hours
Special Equipment
Cost*
References
Human KB cells
Simple kinetics (one or tv/o points)
could allow time for several chemical
concentrations to be tested
simultaneously.
The test organism is easy to culture.
The assay can detect alternations in a
specific membrane process before
generalized toxicity.
A relatively short time is required to
obtain the results of an assay.
If detailed kinetic measurements are made,
only one concentration of one specific
chemical can be tested at a time.
A highly skilled technician is required
to perform this assay.
Many measurements are required to obtain
the results of this assay.
Some specialized equipment is required.
Detergents (Triton X-100, sodium
deoxychloate-MEC is .001 to .05%)
Inorganic ions-heavy metals (nickel,
cadnrium-MEC is 10 to 50 mM)
Inorganic salts (magnesium chloride,
calcium carbonate)
29, 53, 11, 8
Liquid scintillation counter, mammalian
cell culture facilities
$810
Matthews et al, 1970
*See time and cost explanation, pp. 9-11 in text.
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69
independent researchers. After significant testing with known toxicants, an
objective decision can be made about the utility of the Microtox system.
Another promising assay is the chlorophyll fluorescence assay de-
veloped by Arntzen, Steinback, and others at the USDA laboratory at the
University of Illinois, Urbana.
Fluorescence of living leaves has been known for over 100 years,
having been recognized by Muller in 1874 as a pathway of energy dissipation
which competes with energy utilization in photosynthesis and with heating of
the leaf. Since Muller's time, studies of in vivo chlorophyll fluorescence in
algae, in leaves, and in chloroplasts of higher plants have improved our pres-
ent understanding of the light reactions of photosynthesis.
Quite recently, in vivo chlorophyll fluorescence measurement has also
begun to be recognized as a means of detecting damage in intact plants sub-
jected to environmental stresses and deleterious agents. The technique is an
attractive one because data collection is easy, fast, and nondestructive, and
can be done in the field. The potential applications of fluorescence measure-
ment at the whole plant level probably will not be limited to detection of
stress-induced damage but may be extended to use as a versatile diagnostic
tool in plant pathology and as a screening tool in plant genetics. This
fluorescence assay could be used to complement results from the Hill reaction
and/or greening assays and to provide a broad data base about toxic effects on
plants.
McFarlane, Rogers, and Bradley at U.S. EPA, Environmental Monitoring
and Support Laboratory, Las Vegas, are developing a rapid toxicity assay
involving tritium oxidation by soil microorganisms. In this assay, v/ater is
added to air-dried soil in a reaction vessel, and the slurry is incubated
overnight. Tritium is injected into the reaction vessel and, after a fixed
time, the amount of tritium oxidized to water is determined using a liquid
scintillation spectrophotometer. Toxicity is determined by adding a fixed
amount of potential toxicant to the reaction vessel prior to the addition of
the tritium. The oxidation rate of this test sample is compared with that of
the standard (untreated) sample. Even though several chemicals (e.g., silver
nitrate, monuron, cadmium chloride) have been tested using this assay, the
test organisms are not standardized, and different soil samples may contain
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70
different populations of microbes. This toxicity assay could probably be used
only as a pretest to indicate the need for further testing.
From the limited available information, these assays appear to be
excellent candidates for use as toxicity assays. Since we do not have suffi-
cient information to evaluate them by the same criteria used to evaluate other
assays in this report, any decision about their utility as toxicity screens
would be premature. However, it appears that little laboratory development of
these assays would be necessary. As more information on these assays becomes
available, objective ratings will be possible.
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71
TABULAR COMPARISON OF CRITERIA
On the following pages are five tables (Tables 28 through 32) which
show the relative strengths and weaknesses of these 24 physiological assays.
These tables provide, at a glance, comparative data on which specific recom-
mendations are based (see Discussion and Recommendations). With the exception
of the "data base" category (Table 32), the criteria used in these five tables
are defined and discussed in the introduction. The "data base" category in
Table 32 refers to the relative amount of data available about chemical
effects on a physiological process. The data base for each assay was rated as
good (+++), fair (++), or poor (+). This is merely a subjective rating based
upon sources revealed during our literature search (see Appendix).
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72
TABLE 28. SOURCE OF TEST ORGANISM
Culture Animal Commercially
Assay or grow facility prepared
Acetylene Reduction X
Hill Reaction X
Greening X
RuDP Carboxylase Activity X
Photosynthetic Oxygen X
Evolution
Respiration in HeLa Cells X
Adenylate Energy Charge XX X
Cloning L929 Mouse Cells X
Protozoan Clonal Viability X
Human (KB) Cell Growth Rate X
Human Embryonic Lung Fibroblast X
(WI-38) Cytotoxicity
Mitogen Stimulation of X
Lymphocytes
Chick Embryo Development X
Trypan Blue Dye Exclusion by X
Human KB Cells
RNA Polymerase Activity X X
Adenyl Cyclase Activity X
Lysosomal Enzyme Release X
Macromolecular Synthesis in X
KB Cells
Cyclosis X
Hemolysis X X
Protozoan Vacuole Contraction X
Protozoan Hotility X
Phagocytosis by Alveolar Macrophages X
Ami no Acid Transport X
-------
TABLE 29. TEST ORGANISMS AND ORGANISMAL LEVEL OR PARAMETER EVALUATED
Test organism
Level or parameter
Assays
Other Higher
Human vertebrate Protozoan plant Alga Bacteria Organising.! Cellular Qrcjanellar Enzymatic
Acetylene Reduction
Hill Reaction
Greening
RuDP Carboxylase
Activity
Photosynthetic Oxygen
Evolut ion
Respiration in HeLa
Cells
Adenylate Energy Charge
Cloning L929 Mouse Cells
Protozoan Clonal Viability
Human KB Cell Growth Rate
Human Embryonic Lung Fibro-
blast (WI-38) Cytotoxicity
Mitogen Stimulation of
Lymphocytes
Chick Embryo Development
Trypan Blue Dye Exclusion
by Human KB Cells
RNA Polymerase Activity
Adenyl Cyclase Activity
Lysosomal Enzyme Release
Macromolocular Synthesis
in KB Cells
Cyclosis
llemolysis
Protozoan Vacuole
Contraction
Protozoan Moti1ity
Phagocytosis by Alveolar
Macrophages
Ami no Acid Transport
-------
TABLE 30. SPECIAL EQUIPMENT
Assays
Scintillation
Centrifuge Microscope Spectrophotonietcr Counter
Mammalian
cell culture
facilities
Other
equipment
Acetylene Reduction
Hill Reaction
Greeninu
HuDP Carboxylase Activity
Photosynthetic Oxygen
Evolution
Respiration in HeLa Cells
Adenylato Energy Charge
Cloning L9i!9 Mouse Cells
Protozoan Clonal Viability
Human (KB) Cell Growth Rate
Human Embryonic Lung Fibro-
blast (WI-38) Cytotoxicity
Hitogen Stimulation of Lympho-
cytes
Chick Embryo Development
Tryjian Blue Oye Exclusion by
Human Kli Cells
RNA Polymeraso Activity
Adenyl Cyclase Activity
Lysosomal Enzyme Release
Macromoleciilar Synthesis
in KI3 Cells
Cyclosis
llemolysis
Protozoan Vacuole Contraction
Protozoan Hotility
Phagocytosis liy Alveolar
Macrojihacjc's
Ami no Acid Transport
Gas chromatograph
Warburg apparatus
Oxygen electrode
llemocytometer
Aniu:al rearing
facilities
Incubators, hatching and
rearing facilities,
sterile injecting
facilities
llemocytometer
Mil 1 ipori> filtration
manifold
Microphotography unit
Animal rearing facilities
-------
Ji . I ihi
LUil'ilLiAii SKILL
Assay
Acetylene Reduction
Hill Reaction
Oreening
RuOP Carboxylase Activity
Photosynthetic Oxygen
Evolution
Respiration in HeLa Cells
Adcnylatc Energy Charge
Cloning L929 Mouse Cells
Protozoan Clonal Viability
Human (KB) Cell Growth Rate
Hunan Embryonic Lung Fibro-
blast (WI-38) Cytotoxicity
Hitogen Stimulation of
lymphocytes
Chick Embryo Development
Trypan Blue Dye Exclusion
by Human (KB) Cells
UNA Polymerase Activity
Adenyl Cyclase Activity
Lysosouidl Enzyme Release
Macromolecular Synthesis in
KB Cells
Cyclosis
llemolysis
Protozoan Vacuole
Contraction
Protozoan Motility
Phagocytosis by Alveolar
Macrophages
Amino Acid Transport
Assay Set
time*, hours
2.5
3
29
2
50
30
8
244
148
75
27
76
720 (1 month)
30
1.5
29
29
34
30
2
17
23
27
29
Total assay set
fime**, hours
54
57
150
54
100
80
62
326
210
125
176
76
720
77
5
79
76
78
141
4
143
136
27
53
Total
technician Technician
timet, hours Skill t
12 ++
18 *
9 t
12 t++
84 ++
51 ++
23 +t
15 ++
14 t
12 ++
14 ++
11 +++
108 +
11 ++
5 t++
14 +++
9 ++
11 ++
58 + +
4 +
30 tt
27 ++
17 ++
11 +++
Other
(administrative)
time5, hours
8
11
8
8
11
13
9
9
9
8
9
8
11
8
8
9
8
8
11
8
10
9
9
8
Other Approximate
costs and total cost*.
consideration
$35 (greenhouse
fee)
$100 (animal
facility fee and
rearing
$150 (animal
facil ity fee and
rearing)
$30 (special algal
culture facilities)
$100 (animal
facil ity fee and
rearing)
.
$
620
820
560
750
2330
2350
920
750
660
620
730
820
2510
600
530
880
560
720
1810
440
1J30
1000
890
810
--J
en
•Three replicates of five chemical concentrations.
**Including cell growth, solution preparation, organelle or enzyme preparation, and data recording.
'''Including GLP, running assay, and solution preparation.
fHated from + (unskilled) to >n (highly skilled).
'Including Ph.D. supervision, managerial time, data analysis, and reporting.
"costs are estimated for the purpose of comparison only. Actual costs may vary to 20 to 25 percent
from these figures at different laboratories.
-------
76
TABLE- 32. DATA BASE AND COMMENTS
Assay
Data base*
Comments
Acetylene Reduction ++
Hill Reaction +++
Greening +++
RuDP Carboxylase Activity +
Photosynthetic Oxygen
Evolution
Respiration in HeLa Cells
Adenylate Energy Charge ++
Cloning L929 Mouse Cells ++
Protozoan Clonal Viability ++
Human (KB) Cell Growth Rate ++
Human Embryonic Lung Fibro-
blast (WI-38) Cytotoxicity
Mitogen Stimulation of Lympho
cytes
Chick Embryo Development
Trypan Blue Dye Exclusion by +
Human KB Cells
RNA Polymerase Activity ++
Adenyl Cyclase Activity
very explosive.
activities
Acetylene gas is
Chloroplasts and
vary
This assay has already been
used to test many chemicals.
This assay is rapid and rela-
tively inexpensive, but
activity varies.
Respiratory and photosynthe-
tic rates may vary.
An extensive time and great
cost are required to com-
plete an assay set.
No chemical effects on this
assay are known, but it
measures a universal meta-
bolic process.
A long time is required to
complete an assay set.
A long time is required to
complete an assay set, but
many assays can be per-
formed simultaneously.
This assay has already had
limited use as a toxicity
screen.
Toxic chemical effects could
be extrapolated to human
pulmonary tissues.
A great expense is incurred in
maintaining a mouse colony.
Extensive time and great cost
are required to complete an
assay set. There is no
standardized endpoint.
Results from this assay can
be extrapolated to all
membranes.
Hundreds of assays can be
completed daily and correl-
ated to all life forms.
It is difficult to correlate
assay results with cellular
or tissue toxicity.
-------
77
TABLE 32. (Continued)
Assay
Data base*
Comments
Lysosomal Enzyme Release
Macromolecular Synthesis in ++
KB Cells
Cyclosis
Memolysis +++
Protozoan Vacuole Con- +
traction
Protozoan Motility ++
Phagocytosis by Alveolar +++
Macrophages
Ami no Acid Transport +
Results from this assay can
be extrapolated to cellular
and tissue levels.
This assay could be combined
with RNA polymerase activity
to detect chemical inhibition
of RNA synthesis.
This assay has little relevance
to mammalian cell and
tissues.
The assay is rapid, inexpen-
sive, and results can be
extrapolated to many systems.
Results may be correlated to
other assays involving
protozoans.
A long time and great expense
is required to complete an
assay set.
This assay is already in limited
use as a toxicity screen.
The results of this assay can
be extrapolated to other
membranes.
*Rated +++ (good), ++ (fair), and + (poor).
-------
78
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-------
79
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-------
80
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Zagris, N., and J. G. Georgatsos. 1977. Applications of chemicals in early
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Carr, J., and M. Ligaton. 1973. Hexachlorophene-induced alterations in the
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82
Glazer, R. I. 1978. Comparisons of the fidelity of transcription of RNA
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-------
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General Reference
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Toxic Substances, Washington, D.C., 110 pp.
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APPENDIX
LITERATURE SEARCH METHODS
-------
A-l
APPENDIX
LITERATURE SEARCH METHODS
LITERATURE SEARCH
To obtain information on physiological assay methods and on the
effects of potential toxicants on these assays, manual literature searches of
published articles, reviews, abstracts, and bibliographies as well as com-
puterized searches of selected data bases were conducted. This literature
search was conducted over a period of 6 weeks from May 7, 1979, to June 15,
1979, by the methods outlined in Van Voris, et al (1979).
Hand Searches
In the hand literature searches, 50 of the most widely used and sub-
scribed journals in biochemistry, molecular biology, enzyrnology, cell biology,
pharmacology, botany, zoology, toxicology, ecology, and microbiology were
scanned over the past year (May, 1978-April, 1979) for references pertaining
to rapid physiological toxicity assays (Table A-l). Additional references
were located by reviewing the abstract and bibliographic publications Biblio-
graphy of Agriculture, Environmental Abstracts, Biological Abstracts, and
Chemical Abstracts. These bibliographic publications were searched over a
5-year period (usually 1974-1979).
The applicability of the indexed articles was based upon the titles
only, or the abstracts when available. In many instances it was necessary to
make subjective decisions as to whether an article pertained to in vitro
physiological assays, particularly when abstracts were not available. Addi-
tional references were reviewed on the basis of bibliographic citations in
individual papers. Several articles familiar to the authors of this report
were also reviewed.
-------
A-2
Computer Searches
Computer searches were conducted on data bases simultaneously with
the hand searches. The rationale of the data bases selected was to give
coverage to all types of literature reporting ecological effects of toxic
chemicals. The bases searched were:
• Bioscience Information System (BIOSIS) which focuses on life
sciences worldwide since 1964. The data base is journal pub-
lications containing the entire life sciences and including
microbiology, plant and animal sciences experimental medicine,
agriculture, pharmacology, ecology, bioengineering, biochemistry,
and biophysics. The producer is BioSciences Information Service
of Biological Abstracts.
• Toxicology Information On-line (TOXLINE) which gives worldwide
coverage to toxicology studies on animals and humans since 1971.
The data base includes toxicology studies on environmental
pollutants and chemicals, adverse drug reactions, and other toxic
materials. It is produced by the National Library of Medicine.
BIOSIS, searched from 1969 to the present, printed only titles. We
found this data base to be particularly useful because it has controlled
vocabularies with articles referenced by both key words and topics (e.g., Hill
reaction and photosynthesis) so that all references pertaining to a desired
subject under a topic are printed. TOXLINE was the more comprehensive data
base (searched since 1971), overlapping somewhat with BIOSIS.
The information retrieved from computer searches is dependent on
words entered into the computer by the user as well as the key word descrip-
tors used by authors or reviewers of articles used in the different data
bases. In many cases, the key words used by a reviewer omit important facets
of the article or refer to lightly covered topics because of misleading
titles. The key words to be entered in the computer by the searchers v/ere
determined by assay title, test organism, and cellular processes involved.
Key words (assays, organism, and processes) were taken from Table 2 of this
report. For example, for protozoan vacuole contraction, the key words
protozoan and contractile vacuole were among those entered in the computer.
Articles that appeared relevant were then reviewed and specific information
was extracted from them.
-------
A-3
TABLE A-l. JOURNALS SCANNED IN LITERATURE SEARCH
Agricultural and Biological Chemistry
Analytical Biochemistry
Annual Review of Microbiology
Applied and Environmental
Microbiology
Biochemical and Biophysical Research
Communications
The Biochemical Journal
Biochemistry
Biochimica et Biophysica Acta (Nucleic
Acids, Enzymology, Reviews on Cancer,
Lipids, Bioenergetics)
Botanical Gazette
Botanical Review
Canadian Journal of Biochemistry
Carbohydrate Research
Cell
Developmental Biology
Ecology
Ecotoxicology and Environmental Safety
Environmental Science and Technology
European Journal of Biochemistry
Experimental Cell Biology
Experimental Cell Science
Experientia
FEES Journal
Food and Cosmetics
Toxicology
Histochemistry and Cyto-
chemistry
Journal of Bacteriology
Journal of Biological
Chemistry
Journal of Cell Biology
Journal of Cell Science
Journal of Cellular
Physiology
Journal of Experimental
Botany
Journal of General Micro-
biology
Journal of Molecular
Biology
Journal of Protozoology
Methods in Enzymology
Molecular and General
Genetics
Molecular Pharmacology
Nature
Nucleic Acids Research
Parasitology
Pesticide Biochemistry
and Physiology
-------
A-4
TABLE A-l. (Continued)
Physiologia Plantarum
Plant Physiology
Plant Science Letters
Planta
Proceedings of the National Academy
of Science
Proceedings of the Society for
Experimental Biology and
Medicine
Sabouraudia
Science
Toxicology and Applied
Pharmacology
Virology
Weeds
-------
A-5
REFERENCE
Van Voris, P., S. Pomeroy, H. Grotta, and A. Rudolph. March, 1979.
Literature Evaluation of Field-Observed Effects of Toxic Chemicals. OPTS/EPA
Contract No. 68-01-5043. 27 pp.
-------
TECHNICAL REPORT DATA
(Please read Instructions on ihe reverse before completing)
1. REPORT NO.
EPA-560/11-80-001
2.
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
Identification and Evaluation of Potential
Physiological Toxicity Assays
5. REPORT DATE
February 1980
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
G. H. Kidd, J. M. Rice, M. E. Davis, M. A. Hurst,
M. F. Arthur, S. E. Pomeroy, and M. L. Price
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Battelle-Columbus Laboratories
505 King Avenue
Columbus, OH 43201
10. PROGRAM ELEMENT NO.
11. CONTRACT/GRANT NO.
68-01-5043
12. SPONSORING AGENCY NAME AND ADDRESS
EPA, Office of Pesticides and Toxic Substances
401 M Street, S. W.
Washington, D.C. 20460
13. TYPE OF REPORT AND PERIOD COVERED
Final Report: 4/79 - 2/80
14. SPONSORING AGENCY CODE
EPA-560/H
15. SUPPLEMENTARY NOTES
EPA
i_ civic IN i Mn T r*u \ co
project officer for this report is Ronald A. Stanley
16. ABSTRACT
Battelle's Columbus Laboratories has contracted with the Office of Pesticides and
Toxic Substances, U.S. Environmental Protection Agency, to develop a list of
physiological assays as potential toxicity screening tests and to assess the
strengths and weaknesses of these assays. After an extensive literature search,
Battelle has compiled a list of 24 assays, covering seven physiological cate-
gories cited by OPTS/EPA. Those categories included nitrogen fixation, photo-
synthesis, respiration, high-energy phosphate production, growth and cell div-
ision, catalysis, and other cellular processes. Brief descriptions of assay
methods and tables containing critiques of each assay are presented along with
literature references for all of the assays. Assays that are simple, rapid,
cost-effective, reproducible, and well-documented are highlighted.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
t. COSATI Field/Group
Analyzing/Assessing
Bioassay
Physiological/Cellular Toxicity
Physiological Toxicity
Tests
Biological Toxicity
Cellular and Subcell-
ular Mechanisms of
Toxicity
18. DISTRIBUTION STATEMENT
Unlimited
19. SECURITY CLASS (This Kcportj
Unclassified
21. NO. OF PAGES
93
20. SECURITY CLASS (Timpage)
Unclassified
22. PRICE
EPA Form 2220-1 (R»». 4-77) PREVIOUS EDITION is OBSOLETE
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