United States
Environmental Protection
Agency
Office of the Administrator
Science Advisor/ Board
Washington DC 20460
SA8-EC-88-040D
September 1288
Final Report
REVISED OCTOBER 24, 1988
Appendix D:
Strategies for
Health Effects Research
Report of the Subcommittee
on Health Effects
Research Strategies Committee
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NOTICE
This report has b«n written as a part of the activities
of tHeScience Advisory Board, a public advisory group providing
extramural scientific information and advice to the Administrator
and other officials of the Environmental Protection Agency.
The Board is structured to provide a balanced, expert assessment
of scientific matters related to problems facing the Agency.
This report has not been reviewed for approval by the Agency,-
hence, the contents of this report do not necessarily
represent the views and policies of the Environmental Protection
Agency or of other Federal agencies. Any mention of trade
names or commercial products do not constitute endorsement or
recommendation for use.
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U.S. Environmental Protection Agency
Science Advisory Board
Research Strategies Committee
Health Effects Group
Chair
Dr. David Rail
Director
National Institute of Environmental Health Sciences
111 Alexander Drive, Bldg. 101
Research Triangle Park, NC 27709
Member
Dr. Ejla Bingham
Department of Environmental Health
University of Cincinnati Medical College
Kettering Laboratory
3223 Eden Avenae
Cincinnati, Ohio 45267
Dr. Bernard Goldstein
Chairman, Department of Environmental and Community Medicine
UMDNJ-Robert Wood Johnson Medical
675 Hoes Lane
Piscataway, New Jersey 08854-5635
Dr. David Hoel
Director, Division of Biometry and Risk Assessment
National Institute of Environmental Health Sciences
Research Triangle Park, North Carolina 27709
Dr. Jerry Hook
Vice President, Preclinical R&D
Smith, Kline and French Laboratory
709 Swedland Road
King of Prussia, PA 19406
Dr. Philip Landrigan
Director, Division of Environmental and Occupational Medicine
Mt. Sinai School of Medicine
1 Gustave Levy Place
New York, New York 10029
Dr. Donald Mattison
Director, Division of Human Risk Assessment
National Center for Toxicological Research
Jefferson, Arkansas 72079
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Dr. Frederica Perera
School of Public Health
Division ot Environmental Sciences
Columbia University
60 Haven Avenue
New York, New York 10032
Dr. E.Len Silbergeld
Chief, Toxics Program
Environmental Defense Fund
1616 P Street, N. W.
Room 150
Washington, D. C. 20036
Dr. Arthur Upton
Director, Institute of Environmental Medicine
New York University Medical Center
550 First Avenue
New York, New York 10016
Science Advisory Board Staff
Dr. C. Richard Cothern
Executive Secretary
Environmental Protection Agency
Science Advisory Board
401 M Street, S. W.
Washington, D. C. 20460 (A101)
Ms. Renee' Butler
Staff Secretary
Environmental Protection Agency
Science Advisory Board
401 M Street, S. W.
Washington, D. C., 10460 (AlOl)
Ms. Mary Winston
Staff Secretary
Environmental Protection Agency
Science Advisory Board
401 M Street, S. W.
Washington, D. C. 20460 (AlOl)
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HEALTH EFFECTS WORK GROUP REPORT OF THE AL ALM COMMITTEE
Chapter
Abstract
Chapter 1
Chapter 2
Chapter 3
Chapter 4
Title
Environmental Factors and Human Health
Kinds of Long-Term Research
Research Advances in the Toxicology of
Lead
Newer Basic/Long-Term Research with
Application to Environmental Health
Problems
Chapter 5 Population Risk/Risk Reduction
Author
Arthur Upton
James Fouts
Kathryn Mahaffey
Marshall Anderson
Frederlca Perera
Lawrence Relter
Morrow Thompson
Michael Luster
Donald Mattlson
Alan Hllcox
David Hoel
Michael Hogan
Chapter 6 Summary
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ABSTRACT
This document attempts to delineate the long-term health effects
sarch needs (both basic and applied) considered most supportive of EPA
programs. Chapter 1 provides a historical perspective , descrih" *ha
nature and sources of environmental determinants of health and '
touches on the underlying mechanisms of toxidty with implicate
research
programs. Chapter 1 provides a historical perspective , describes the
disease,
_..__. - - -j •• - - r- - - i ons for r1sk
assessment and disease prevention, and indicates some of the areas where
research support is clearly Inadequate.
Chapter 2 draws a distinction between the basic and applied long-term
health effects research needs of EPA programs by providing specific
examples that illustrate the need for research addressing "generic" issues
as well as various research activities that have application to specific
problems and specific settings but which must be carried on over a period
of several years. An attempt has been made to explain how EPA uses/depends
on basic research of the type conducted by other Federal Agencies,
particularly as it relates to the regulatory mission of the Agency.
In Chapter 3 the toxic metal lead is used as the paradigm to Illustrate
the place of and necessity for long-sustained, basic research activity 1n
the development of a foundation for constructive action 1n Important
problems 1n environmental health. Continued long-range and basic research
Investigations on lead toxlcity are at one and the same time perhaps among
the more justifiable and yet less supportable of such activities 1n the
entire field of environmental health sciences.
A .number of leading-edge/long-term basic research activities with
potential application to environmental health problems are described 1n
Chapter 4. It attempts to highlight those activities which perhaps
have the greatest promise 1n this area. Many of these include various
aspects of the "new molecular biology" research field, such as the study of
oncogenes and proto-oncogenes, the development and use of biomarkers to
determine internal dose and exposure and for relating exposure to disease.
Other newer developments in neurotoxicology, Imrounotoxicology and
reproductive toxicology are described. An important area of basic research
includes methods development and validation. Magnetic resonance imaging is
discussed as a very promising new technique that should find many useful
applications in studies of the internal structures, states, and
compositions of various biological systems.
Finally, 1n Chapter 5 the problem of estimation of population risks is
addressed, particularly as 1t relates to the role of animal data in the
quantification of possible human health risks. Factors considered here
include choice of mathematical model or extrapolation procedures, primary
versus secondary or indirect modes of action and threshold mechanism,
problems in species extrapolation and determination of biologically
effective dose. Some specific problems in human epidenlologlc studies and
population risks analysis are also described. Factors affecting the
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balanee of basic rtsearch on cancer and non-cancer endpolnts *1th1n any
Federal organization are also discussed. Long-term, basic research Into
both cancer and non-cancer endpolnts 1s recognized as being essential 1f
the EPA 1s to formulate a broad regulatory policy 1n the most accurate
manner possible.
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Chapttr 1
ENVIRONMENTAL FACTORS AND HUMAN HEALTH
Arthur Upton
HISTORICAL PERSPECTIVE
The past century has seen the conquest of those diseases which have
caused the greatest morbidity and mortality in previous generations. In
the developed countries of the world, the average life expectancy has
doubled, now surpassing the biblical ideal of "three score and ten" years
(Figures 1 and 2). This transformation, which would have seemed miraculous
to our great grandfathers, has resulted from advances in our understanding
of the relationship between health and the environment, broadly speaking.
These advances, and the resulting improvements 1n agriculture, nutrition,
sanitation, public health, and medicine, have all but eliminated infectious
and parasitic diseases as major causes of death in the Industrialized
world. Replacing such afflictions as major causes of death 1n the
industrialized world are abnormalities 1n early growth and development,
chronic degenerative diseases, and cancer (Figure 3). These diseases,
viewed until recently largely as hereditary or Inevitable accompaniments of
aging, are now attributed Increasingly to environmental causes. Our
challenge 1s to Identify the causes and to control then (4).
NATURE AND SOURCES OF ENVIRONMENTAL DETERMINANTS
OF HEALTH AN DISEASE
The "environment", defined broa-;y, encompasses all external factors
that may act on the human mind and .;dy. Many of the factors are produced
or altered by man himself. They Include chemical and physical agents In
air, food, water, drugs, cosmetics, consumer products, the home, and the
workplace. The "environment" 1s thus complex and constantly changing.
Inevitably, moreover, 1t contains a myriad of agents 1n varying
combinations and from multiple sources. Furthermore, because the effects
of different agents Interact 1n various ways, the ultimate Impacts of any
given environmental agent may depend' on the effect of other agents and the
conditions of exposure (4),
Air
Acute episodes of atmospheric pollution, such as those listed in
Table 1, have been observed to cause transitory Increases in morbidity and
mortality. The effects of chronic exposure, however, are less well
documented and may vary, depending on the pollutants 1n question and their
concentrations in the atmosphere (4).
On chronic exposure at relatively high concentrations in the workplace,
a variety of pollutants are known to cause toxic effects. Examples Include
various gases (e.g., carbon monoxide, vinyl chloride, coke oven emissions,
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radon), metals (e.g., lead, mercury, arsenic, nickel) and dusts (e.g.,
asbestos, silica, cotton fibers, coal) (5).
Also well documented are the effects of chronic exposure to cigarette
smoke. The Incidence of 1ung cancer has risen precipitously, in parallel
with the antecedent Increase in cigarette consumption (Figure 4). In
smokers, furthermore, there 1s a systematic relationship betweecn the
amount of smoke Inhaled and mortality from lung cancer (Figure 5), other
cancers, heart disease, and respiratory diseases. Lesser effects have been
tentatively attributed to passive Inhalation of cigarette smoke 1n
chronically exposed nonsmokers.
The ultimate effects of chronic low-level exposure to other widely
prevalent combustion products and their derivatives (such as sulfur
dioxide, ozone, nitrogen dioxide, benzo(a)pyrene, and various suspended
partlculates) are less well understood.
Although the air pollution produced as a result of coal combustion is a
direct cause of respiratory fatalities, there 1s no exact measure of their
number; however, several estimates have been made of the number of
fatalities attributable to the combustion of coal 1n generating electricity
(where about 70* of coal combustion occurs). Inhaber (8), for example,
estimated that between 5 and 500 fatalities result per 1000 M*e of electric
power produced each year from pollution generattd by coal fired plants. A
more recent survey by experts 1n this artt puts the estimate bttwten zero
and 1000 fatiHtles per year per 1000 Mwe of electric power produced
(9,10). On the basis of a value of 7 x 10 Mwt of electric power produced
1n the U. S. by the consumption of coal, the estimates Imply that up to
700,000 fatalities per year may result from combustion of coal 1n the U. S.
Within the uncertainties of this estimate, 1t agrees well with a recent
Inference by Wilson that "50,000 among the 2 million persons who die each
year 1n the United States may have their lives shortened by air pollution14
(11). One may question, therefore, the extent to which current ambient air
standards provide adequate protection against the potential long-term
health effects of coal combustion products, which cannot be specified with
certainty on the basis of existing knowledge (12).
It is noteworthy 1n the above context that Indoor pollution with
combustion products may lead to health effects in the chronically exposed,
especially children. Of Increasing concern Is the extent to which
elevation of the radon concentrations within houses and buildings, by
weather-stripping and other heat-saving measures, may enhance the risk of
lung cancer 1n their occupants (13-15).
Other air-borne pollutants with potential health effects Include
allergens of various kinds. Although susceptibility to such agents differs
widely among Individuals, sizable populations are at risk (4). The full
significance of air-borne agents as causes of disease 1s far fro»
established and strongly merits continued study (4).
yater
In the third world microblal contamination of drinking water still
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constHutes a raajor cause of death. Although this type of pollution no
longer exists on a significant scale 1n developed countries, the ch«1cal
composition of drinking water has been Implicated tentatively 1n the two
leading causes of death 1n the U. S.: cancer and cardiovascular disease
(4,Li). It 1s also noteworthy that water supplies have been found to be
polluted in a growing number of areas (Figure 6), owing to contamination by
metals, toxic wastes, pesticides, agricultural chemicals, and products of
chlorination or ozonizatlon.
The health impacts of small quantities of chemicals in drinking water
cannot be assessed precisely on the basis of existing knowledge. Research
is needed to elucidate the relevant causal relationships and to clarify the
pathways through which compounds affecting human health my enter the water
supply (17).
Food
There is some truth to the adage, "you are what you eat". Overall
health is undoubtedly influenced by the total intake of calories 1n the
diet, the relative Intakes of different types of foods (protein, fat,
carbohydrates), the nutritional value of the various foods that are
ingested, the presence 1n food of certain naturally occurring constituents
or contaminants, and the presence of man-made additives or pollutants (18).
In general, more 1s known about the nutritional requirements for normal
growth, maturation, and reproduction than about the optimal diet for long
11fe and vigor.
In the case of cancer, for example, there appear to be.many ways
through which the diet may affect the probability of the disease (Table 2);
however, the relative contributions of any of these hypothetical mechanisms
to the pathogenesls of a particular form of human cancer remains to be
established (18). In this connection 1t 1s noteworthy that some dietary
factors may exert protective effects which are of equal or greater
importance than the carcinogenic effects of others. Hence, the net effects
of the diet may reflect the balance between the two types of Influences.
Because of the Importance attributed to the diet in the pathogenesls of
cancer, heart disease, and other leading causes of death 1n the modern
world, the role of dietary factors strongly merits further study.
Occupation
As noted above, occupational exposure to diverse physical and chemical
agents at relatively high dose levels has been observed to cause various
diseases. Collectively, the health Impacts of these agents and of
work-related stresses may approach those caused by occupationally-related
accidents (5).
Occupational diseases are also significant in pointing to risk, factors
that may affect other populations at lower levels of exposure. In
addition, occupational disease represents a category of health effects that
1s relatively amenable to preventive strategies. To lessen the health
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impacts of occupational risk factors, research of several types deserves
further emphasis: 1) more systematic and quantitative monitoring of
physical and chemical agents 1n the workplace; 2) more complete
surveillance and recording of work-related health effects; and 3)
development of clinical and laboratory tests for ascertaining prior
exposure to disease causing agents, for Identifying high-risk groups, and
for detecting work-related diseases at early stages, when they are most
readily arrested, or reversed (4).
Toxic Wastes
Love Canal and Times Beach, to mention only two of many recent examples
(Tables 3 and 4), testify to the need for more adequate disposal of toxic
wastes. Although 1t 1s clear that disposal practices have been deficient
In many instances, the development of- optimally safe and cost-effective
techniques will require further research, as will precise assessment of the
magnitude of the risks posed by prevailing levels of contamination around
existing dump sites (21-23).
The assessment of risks cannot depend on ep1dem1olog1cal approaches
alone. This would be tantamount to making guinea pigs of exposed
populations. Instead, comparative toxlcologlcal methods Involving
laboratory assays and animal models must be exploited Insofar as possible
1n view of the paucity of toxlcologlcal data for most chemicals 1n the
human environment (Figure 7). This will necessitate research to advance
tht state-of-the-art, 1n view of existing uncertainties iboyt species
differences and the Interactive effects of the many chemicals that are
characteristically present at dump sltts.
MECHANISMS OF TOXICITY: IMPLICATIONS FOR RISK ASSESSMENT
AND DISEASE PREVENTION
Toxlcologlcal Research
As noted above, many of the Impacts of environmental agents result from
the combined effects of multiple factors, each of which may contribute
differently to the total. Furthermore, the effects of a given agent, or
combination of agents, may vary, depending on the conditions of exposure as
well as the dose. In addition, although some chemicals exert their effects
directly, many act Indirectly, through the formation of biological active
metabolites or through effects on the metabolism of other substances (4).
Because of tht complexity of these processes, 1t 1s difficult or Impossible
to assess the effects of a given agent without some understanding of Us
metabolism and mode of action. Knowledge of the comparative toxicology and
mechanisms of action of a substance is also essential 1n assessing Its
potential risks for hunans on the basis of extrapolation from Its observed
effects 1n laboratory animals, since choice of the appropriate
extrapolation model cannot be made without assumptions about the relevant
dose-effect relationships and mechanisms of action (25-26).
With respect to the dose-effect relationship, 1t must not be forgotten
that for some types of environmental Insults no thresholds art known or
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presumed to exist. These Include the mutagenlc, carcinogenic, and some of
the teratogenlc effects of ionizing radiation (14) and certain chemicals
(4). Noteworthy tn this connection 1s the growing evidence that exposure
to lead during prenatal life and early infancy may cause permanent
impairment 1n the development of the brain, the dose-effect relationship
for which extend down to doses hitherto considered nontoxic and may
conceivably have no threshold (27).
In addition, since it is not always feasible to eliminate a toxic agent
from the environment, the most practical approach for mitigating Its
noxious effects may be to arrest or reverse them in exposed individuals.
For this purpose knowledge of the mechanisms of such effects may be
crucial, as well as the ability to identify affected Individuals at early
enough stages for effective protective intervention. Methods for
monitoring exposed populations, as well as for monitoring the environment,
are thus needed.
Social and Behavioral Factors
Any consideration of the role of environmental factors 1n health should
not neglect the influence of social and behavioral
influences (28). Among these, socio-economic status is one of the most
important since 1t may affect many, if not all, other environmental
influences, directly or Indirectly. Mortality from many of the cofwon
causes of death tends to vary Inversely with socio-economic and educational
levels (29). The poor who live 1n urban ghettos exemplify the problem 1n
their high Incidence of malnutrition, congested and stressful living
conditions, vermin infestation, chronic exposure to dusts and other air
pollutants, and relegation to hazardous working conditions,. Poverty also
breeds deviant behavior. Including alcohoHsn, drug addiction, and crime,
which have enormous Impacts on health.
The importance of wholesome dally living habits 1n those who are not
economically disadvantage^ also deserves conment. Such simple hysical
exercise, adequate hours of sleep, control of body weight, abstinence from
smoking, and avoidance of excessive intake of alcohol are correlated with
marked reductions 1n overall morbidity (30). In Mormons (31) and Seventh
Day Adventlsts (32), who generally practice these habits, mortality from
cancer and many other diseases 1s appreciably lower than 1n the population
at large.
Also noteworthy 1s the Inverse correlation between level of educational
attainment and cigarette consumption (33), which points to the Importance
of education 1n motivating people not to smoke or to stop smoking. The
large numbers of people at all educational levels who continue to smoke,
however, attest to the need for further efforts to solve the problem
completely. The cigarette problem -- which accounts for more than 300,000
deaths per year 1n the U. S. from cancer, respiratory ailments, and
cardiovascular disease (33) -- exemplifies the Importance of behavioral
factors, socio-economic Influences , and political forces 1n shaping the
environment for better or for worse.
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UNOER-RECOGNITION AND UNOER-OIAGNOSIS OF ENVIRONMENTAL DISEASE
As noted above, environmental diseases encompass an extremely broad
range of human Illnesses. They Include, for example, emphysema in persons
chronically exposed to acid air pollution, leukemia in persons exposed to
benzene, lung cancer and mesothelioma in individuals exposed to asbestos,
chronic kidney disease and neurologic impairment in persons exposed to
solvents, impairment of brain development in children exposed early in life
to lead, heart disease in individuals exposed to carbon monoxide, and
impairment of reproductive function in men and women exposed to lead and
certain pesticides. Such Illnesses afflict millions of persons in the
United States.
Because such environmental diseases arise from man-made conditions,
they can be prevented through the elimination or reduction of hazardous
exposures at the source; i.e., through primary prevention. They are also
amenable to secondary prevention — i.e., early detection in presymptomatic
stages when they can still be controlled or cured; this depends, however,
on efficiently and effectively identifying populations at high risk.
Finally, their impacts may be lessened by tertiary prevention; i.e., the
prevention of complications or disability by application of appropriate
diagnostic and treatment strategies. Prevention at all three levels
requires adequate Information about the effects of specific environmental
exposures and adequate data on the places and populations affected.
Laws enacted 1n the past two decades are Intended to prevent
environmentally-Induced disease. These Include, for exaaple, the Clean Air
Act, the Safe Drinking Hater Act, the Resource Conservation and Recovtry
Act, and the Superfund legislation. In spite of this legislation, however,
environmentally-induced disease remains widespread 1n American society.
Given that such Illnesses are Important and highly preventable, why do they
still persist? A series of factors interact to maintain this situation.
1. Despite at least two decades of regulatory and scientific awareness and
effort, relatively little 1s known about the potential health effects
of most synthetic chemicals. Most attention and research have been
focused on a small number of relatively well known hazards, such as
asbestos and lead, and their associated diseases. Virtually no
information 1s available on the toxldty of approximately 80 percent of
the 48,000 chemical substances 1n commercial use (Figure 7). Even for
groups of substances which are most closely regulated and about which
most is known -- drugs and foods -- reasonably complete Information on
possible untoward effects is available for only a minority of agents
(Figure 7). Premarket evaluation of new chemical products 1s notably
inadequate.
2. Physicians are not trained to suspect the environment as a cause of
disease. Most physicians do not routinely obtain histories of
environmental exposure for their patients, which would allow them to
identify an environmental origin of disease. Recent surveys Indicate
that environmental histories are recorded on fewer than 10 percent of
hospital charts (34). In consequence, many diseases of environmental
origin are mistakenly assigned to other causes, such as old age or
cigarette smoking, and opportunities for early prevention or treatment
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are lost. This problem of Inaccuracy 1n diagnosis 1s compounded by
the fact that disease of environmental origin are typically not
clinically or pathologically different from those caused by lifestyle
and other factors.
3. Physicians do not receive adequate training 1n environmental medicine.
Very little time is devoted in American medical schools to teaching
physicians in training to recognize the symptoms of known toxins, or to
understand the known associations between environmental exposures and
disease outcomes. The average American medical student receives only
four hours of training in environmental and occupational health during
the four years of medical school (34).
4. Persons are typically exposed to more than one toxic substance in the
environment and often do not realize that they have been exposed at
all. Further, the symptoms of many environmental conditions develop
only many years after onset of exposure during this long latency
(Incubation). Persons may change addresses, may be exposed to a
variety of environmental exposures, may suffer various environmental
exposures, and finally may forget exposures which they had many years
ago. All of these Issues compound the difficulty that physicians and
environmental scientists face 1n attempting to deduce the etiology of
environmentally Induced Illness.
5. The U. S. Environmental Protection Agency (USEPA) and State
environmental agencies are enpowered to Investigate hazardous and
environmental conditions; however, severe resource limitations have
reduced the capacity of these agencies to undertake necessary
inspection and enforcement actions.
6. Fragmented, unreliable and outdated surveillance systems for
environmentally related disease produce significant underestimates of
the actual number of cases of environmentally induced Illness 1n our
society. As a result, the picture they produce does not convey an
appropriate sense of urgency about reducing the burden of environmental
disease.
In summary, a profound lack of Information on the toxldty of the
majority of commercial chewlcals. Insufficient and Inappropriate education
of physicians, and Inadequate surveillance Impede all efforts to reduce the
Impact of environmentally Induced disease 1n the United States. A coherent
plan to Improve the surveillance, prevention, diagnosis, and treatment of
environmental disease 1s sorely needed. Models which have recently been
developed for the detection, treatment and prevention of occupational
disease 1n states such as Mew York, New Jersey, and California might serve
as useful models for undertaking such an effort (35).
INADEQUACY OF RESEARCH SUPPORT
Frora the foregoing 1t 1s evident that much of the burden of Illness 1n the
U. S. today 1s attributable wholly or 1n part to environmental risk
factors. Thus, of the more than $400 billion annual health expenditures 1n
the U. S., a major part 1s spent on Illnesses that are related directly or
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1nd1rect1y to environmental causes (36) and that ire thus potentially
preventable. Although the economic Impact of such Illnesses cannot be
reckoned precisely without more adequate Information, 1t 1s obviously
enormous.
Viewed 1n the light of the enormous costs of Illness to the U. S.
population, the sums spent on research to prevent such Illness are
relatively small. In 1985, for example, only $1,180,370 of the $5,121,557
R4D funds obligated by MIH went specifically to support research on disease
prevention (37). This sum amounted to less than 0.25% of the total cost of
health care 1n the U. S. that year (37). The sum spent for the same
purpose by all other federal agencies combined was far smaller (37).
Hence, in view of EPA's mandate to protect the U. S. population against
environmental pollutants, 1t 1s clear that the Agency's strategies and
budget for the purpose need to be greatly strengthened.
SUMMARY
The major diseases in modern life result 1n large measure from the
Influence of environmental causes. Defined broadly, these causes encompass
all external Influences that may act on the hunan mind and body. Included,
among other Influences, are physical and chemical agtnts in food, water,
air, the home, and the workplace, many of which art produced by mm and/or
subject to his modification. Although some such agents produce adverse
effects only at high dose levels, others may cause .effects at lower dose
levels, conceivably without a threshold. In practice, furthermore, the
observed Impacts on human health frequently result fro* the emulative
effects of combinations of agents, 1n which additive or multiplicative
interactions among causal agents are Involved. Hence, although
environmental factors have been Implicated as major causes of disease, the
precise role of any one causal factor 1n the occurrence of a particular
disease cannot always be specified. By the sane token, 1t 1s difficult to
predict the potential risks to health that may result from a given agent at
any particular dose level. In our present state of knowledge, assessment
of such risks 1s especially uncertain when direct human evidence 1s lacking
and estimates must be based on extrapolation from observations in
laboratory animals or other assay systems.
To advance our understanding of the role of environmental factors 1n
health and disease, priority must be given to research on the following:
1) more syst«iit1c monitoring and characterization of the human
environment; 2) more adequate recording of human morbidity and mortality
rates, with record-linkage systems to enable the frequency of specific
disease to be related to possible environmental causes; 3) further
development of methods for detecting Indices of exposure to toxicants and
for Identifying high-risk subgroups; 4) refinement of laboratory tests for
characterizing the biological activity of chemical and physical agents,
especially at low doses and in combinations; 5) improvement in techniques
for human risk assessment with particular reference to comparative
toxicological methods and extrapolation from anliul data; and 6) better
understanding of the Mechanisms of environmentally-related htalth effects,
as needed for Improvements 1n risk assessment and 1n the primary ind
secondary prevention of environmentally-related diseases. In addition, more
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vigorous efforts should be directed toward the application of existing
knowledge, through: 1) public and professional education, 2)
standards-setting, 3) implementation of new and existing legislation, 4)
law enforcement, and 5) research to evaluate the efficacy of such measures.
In pursuit of Its mission EPA in coordination with other agencies and
institutions must have a long-range research strategy addressing each of
the above needs.
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Congress^ U. S. Environmental Protection Agency,
Washington, 0. C. , 1980.
13. Committee on Indoor Pollutants. Indoor Pollutants. National Academy
of Sciences, Washington, D. C. , 198T
14. Advisory Comlttee on the Biological Effects of Ionizing Radiation.
The Effects on Population of Exposure to Low Level s _ of Ionizing
Radiation" National Academy of Sciences, ~™ — —
Washington, D. C. , 1980.
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1S. National Council on Radiation Protection and Measurements. Evaluation
of Occupational and Environmental Exposures to Radon and Radon
Daughters in the united states. RCRP ReporTTJo. 78^.MartlwiaT Council
on Radiation Protection and Measurements,
Washington, D. C., 1984.
16. Dlckson, D. Toxic Synopses: United States. Lessons on Love Canal
Prompt Clean Up.. Ambio 11:46-50. 1982.
17. Committee on Safe Drinking Water. Drinking Water and Health, Vol. 4.
National Academy of Sciences, Washington, D. C. , 1982.
18. Committee on Diet, Nutrition, and Cancer. Diet, Nutrition, and Cancer.
National Academy of Sciences, Washington, 0. C., T98Z.
19. Doll, R. The Epidemiology of Cancer. In: Accomplishments 1n Cancer
Research, 1979 Prize Year General t^^rs Cancer Reseafch FoundationT
edited by J. G. Fortner and J.E. RhoadsT J- B. Llpplncott Co.,
Philadelphia, 1979, pp. 103-121.
20. Weiss, B. and Clarkson, T. Toxic Chemical Disasters and the
Implications of Bhopal for Technology Transfer. M11 bantc Quarterly
6£:216-240, 1986. ' ~~~~
21. Office of Technology Assessment. Techno!ogles and Strategies for
Hazardous Waste Control. U. S. Congress, Wasnlngton, D. C7, 1983.
22. National Materials Advisory Board. Management of Hazardous Industrial
Wastes. National Academy of Sciences,
Washington, D. C., 1983.
23. Committee on Response Strategies to Unusyal Chemical Hazards.
Assessment of Multl chemical Contamination. National Acadeay of
Sciences, Washington, D. C., 1981.
24. National Academy of Sciences - National Research Council. Toxldty
Testing. Strategies to Determine Needs and Priorities. National
Academy Press, Washington, D. C., 1984.
25. Committee on Chemical Environmental Mutagens. Identifying and
Estimating the Genetic Impact of Chemical Mutagens. NationaT Aca deny
of Sciences, Washington, 5. C., 1982.
26. Omenn, G. S. Environment Risk Assessment: Relation to Mutagenesls,
Teratogenesls, and Reproductive Effects. J. Amer. Coll. Toxlcol.
2/113-123, 1983.
27. Bellinger, D., Levlton, A., Waternaux, C., Neddlewan, H., and
Rab1now1tz, M. Longitudinal Analyses of Prenatal and Postnatal lead
Exposure and Early Cognitive Development. N. Engl. J. Med.
316:1037-1043, 1987.
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28. Elsenbud, M. Environment, Technology, and Health. Human Ecologyin
Historical Perspective. New York Universily" Press, New York, 79715—
29. Occupational Mortality: England and Wales, 1970-1972. (Decemial
Supplement).Her Majesty's Stationery Office, London, 1978.
30. Belloc, N.B. Relationship of Health Practices and Mortality. Prev.
Med. 2_:67-81 , 1973.
31. Lyon, J.L. Gardner, J.W., and West, D.W. Cancer Incidence in Mormons
and non-Mormons in Utah During 1967-1975. J. Nat. Cancer
Inst. 6_5:1055-1062, 1980. "
32. Phillips, R.L., Garfinkel, L., Kuzma, J.W., Beeson, H.I.,
Lotz, T., and Brin, B. Mortality Among California Seventh-Day
Adventists for Selected Cancer Sites. J. Nat!. Cancer In_st._
65^:1097-1108, 1980. ~——
33. Surgeon General. Smoking and Health. Department of Health, Education,
and Welfare, Washington, D. C., 1979.
34. Levy, B.S. The Teaching of Occupational Health in United States
Medical Schools: Five-Year Follow-Up of An Initial Survey. Amer.
J. Public Health 75:79-80. 1985. ~~
35. Markowitz, S.8. and Landrfgan, P.J. Occupational Disease 1 n New York
State. Hount Sinai School of Medicine, New York, 1987.
36. Institute of Medicine. Costs of Environment-Related Health Effects: A
Plan for Continuing Study!National Academy Press, Washington,D. C.,
iwn
37. National Institutes of Health. DataJBook. U. S. Printing Office,
Washington, D. C., 1986.
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Ttble 1. Some Selected Acute A1r Pollution Episodes
Estimated Nos.
of Attributed
Place Date Excess Deaths
Meuse Valley, Belgium December 1930 63
Donora, Pennsylvania October 1948 20
London, England December 1952 4,000
New York, New York November 1953 200
London, England December 1962 700
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Table 2. Ways In Which Diet May Affect Incidence of Cancer
1. By providing source of carcinogens or precardnogens:
-- Natural components of plants
-- Products of chemical, bacterial or fungal action during
processing or storage
-- Products of cooking
-- Contaminants (products of fuel combustion, pesticide
residues)
2. By affecting formation of carcinogens:
— Provision of substances for formation of nltrosamlnes
(secondary amines, nitrates, nitrites)
-- Inhibition of formation of nltrosamlnes as 1n stomach
(Vitamin C)
— Alteration of excretion of bile salts and cholesterol Into
large bowel {fat)
-- Alteration of metabolism of carcinogens (enzyme Induction
by meat, fat, indoles 1n vegetables, antloxldants)
-- Alteration of enzyme formation (trace elements)
— Affect on formation of estrogen (fits, total calories)
3. By modifying effects of carcinogens:
-- Through transport (alcohol, fiber)
— Through effect on concentration 1n bowel (fiber)
-- Inhibition of promotion (Vitamin A, beta-carotene)
!From Reference 19)
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Table 3. Some Acute Environmental Pollution Episodes
Toxic Pollutant Location Year
Mercury Mlnimata Bay, Japan 1959
PCBa Kyushu, Japan 1968
PBBa St. Louis, Michigan 1973
Lead Kellogg, Idaho 1976
Dioxina Seveso, Italy 1976
OBCPa Lathrop, California 1977
Kepone Hopewell, Virginia 1978
Multiple Agents Love Canal, Mew York 1978
Oioxin Times Beach, -Missouri 1983
Dloxin Newark, New Jersey 1983
aPCB defined as polychlorinattd blphenyls, PBB as polybronlnated
blphenyls, dloxln as 2,3,7,8-t®trach1orod1binz0-£-d1ox1n, and OBCP as
1,2-d1bromo-3-chloropropane.
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Table 4. Examples of Outbreaks of Mass Human Poisoning From
Toxic Chemicals
Date Location
1930 U.S.A.
1934 Detroit
1952 London
1952 Japan
1952 MoHnga (Japan)
955 Mlnamata (Japan)
1956 Turkey
1958 Kerala (India)
1959 Morocco
1960 Iraq
1964 Nlggata (Japan)
1967 Qatar
1968 Japan
1971 Iraq
1976 Pakistan
1981 Spain
1984 Bhopal
Chemical
Tr1orthocresyl phosphate
Lead
A1r pollutants
Parathlon
Arsenic
Methyl mercury
Hexachlorobenzene
Parathlon
Tr1orthocresylphosphate
Ethylmercury
Methylmercury
Endrin
Polychlorlnated blphenyls
Methylmercury
Malathlon
Toxic oil
01 methyl 1socyanate
No. Affected
16
4
4
1
12
1
4
000
000
000
800,
159b
000
000
828
,000
,022
646
691
,665
50,000
7,500
12.600r
2,QOOC
10
1
1
Year of onset.
These wert the estimated number of exposed babies. It was stated
that several thousand were poisoned and 131 died.
C0eaths. The full scale of lingering and permanent morbidity remains
unknown.
(From Reference 20)
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Flgure 1
Age-Specific Death Rates 1n Various Countries and
Years (From Reference 1).
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100
o
Z 75 h
QC
50 -
uu
U
oe
UJ
25 -
10 20 30 40 SO 60 70
AGE
80 90 100
Figure 2
The Increasingly Rectangular Survival Curve 1n the
U.S. About 80 percent (stippled area) of the
difference between the 1900 curve and the Ideal
curve (stippled area plus hatched area) had been
eliminated by 1980. Trauma 1s now the dominant
cause of death 1n early life. (From Reference 2).
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Pwcont of all acottii
1900 1947
Coma of daoHt 10_ 0 10 20
Dliiani of Hwhoart
AMBIMIMMf
central ncrvaiM tytfam
AN occwantt
tnflmnw or
of •arty infancy C!
Figure 3
Leading Causes of Death In the United States, 1967,
as Compared with 1900. (From Reference 3).
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Flgure 4
Time-Trends in Lung Cancer Mortality and Cigarette
Consumption 1n England and Wales. (From Reference
6).
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AMNUAL
INCIMNCI
for *f«
Pit M00M
HfN
r
i
DOH IAT1 (
Figure 5
Incidence of Lung Cancer 1n Regular Cigarette
Smokers In Relation to Number of Cigarettes Smoked
Per Day. (From Reference 7).
-------
Shaded areas = Reported pollution areas
Open areas = Areas that may not be problem-free, but the problem 1s
not considered major.
0 Industrial chemicals other than chlorinated hydrocarbons
Heavy metals, such as mercury, zinc, copper, cadmium and lead
Chlorinated hydrocarbons from treatment processes & energy
development
* Conform and other bacteria
Saline w*t*r
General municipal and Industrial waste
Figure 6
Drinking Hater Problem Areas (As Identified by
Federal and State Regional Study Teams). Source:
U. S. Water Resource Council. (From Reference 16)
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t U I* U
CtlMit
1.IU
I.U7
II.I
U.IU
a.ru
Black bars = Complete health hazard assessment possible
Dotted bars = Partial health hazard assessment possible
Slanted line bars * Minimal toxldty Information available
Horizontal line bars * Some toxldty Information available
(but below minimal)
Open space birs » No toxldty Information available
Figure 7
Adequacy of Available Data on Chemicals of Different
Categories for Health-Hazard Assessments. (From
Reference 24).
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Chapter 2
KINDS OF LONG-TERM RESEARCH
James Fouts
LONG-TERM HEALTH EFFECTS RESEARCH SUPPORTIVE OF EPA PROGRAM MEEDS
I. Basic Research
Basic research needed 1n EPA programs may or may not be directed
specifically at support of certain applied research programs. Such basic
research may seek only to understand deeper levels of the general universe
of problems attacked in the specific, discrete long-term, applied
researches (such as described Just below). The general basic research
philosophy 1s that understanding more about the ways chemicals cause
disease can lead to earlier detection or better tests for adverse health
effects (and better designs of epidemiology studies), better analytical
methods, etc. All of this can and often does lead to better bases for
regulation and, thus, better regulation. (Ste Section III below)
Some of this basic research can be directed at using some of the
"new biology" to advance our ability to assess exposure or to better
identify and quantify specific bad effects of (or bad actors 1n) complex
mixtures of chemicals occurring "naturally". Overall though, the '
distinguishing feature of this baste research 1s that 1t addresses mort
"generic" Issues, and that 1t not necessarily be tied Into any one specific
problem nor seek "quick" answers. As such, 1t must be supported for
several years to be effective and to give the kinds of findings that will
be most useful to many "applied" research programs. It 1s, however, true
that often the most useful facts and new approaches needed 1n resolving any
environmental emergency have come from turning to laboratories doing good
basic research.
There are many examples here of the kind of research that probes
deeper (and 1s more risky) than any of the applied programs. Some of these
might be:
A. New methods to detect and quantify dloxlns
Basic research has Identified and characterized an
intracellular "receptor" for
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for assaying dloxlns 1n mixtures). But it has stretched over many years,
and although never without seme merit to the most practical/applied of
objectives, has not been of Immediate value to most of the EPA needs.
B. New methods for detecting exposure to some toxic
chemicals
The cytochrome P-450s are a component of steroid, Hpid,
and xenoblotic metabolizing enzyme systems found in a variety of living
systems (from yeast to humans). Much basic research over at least 35 years
has led to some understanding of the diversity and responsiveness of these
systems in many species. The "new biology" again has given us some new
tools for quantifying and Identifying many of these pigments. It is now
possible to "fingerprint" the kinds and amounts of many different isozymes
of P-450 in tissues of many animals (Including humans) and plants. Basic
research has described in some detail the responsiveness of these P-450s to
various environmental stresses (including chemical exposures). Taken all
together then, this long-continuing, basic research program may now be
giving us tools for looking at the exposures of plants, animals, and humans
to many environmental chemicals--e.g., the amounts and types of P-450s seem
to reflect exposures to things like pesticides, smoke, solvents, etc.
Further, basic research «ork (especially 1n pharmacoklnetlcs) may even give
us a tool for assessing both acute and c$j»ulat1ve/chron1c toxic exposurts
(of species ranging from fish to humans) using these monoclonal antibodies
for specific P-450S-.
II. Applied Research
Ther® are several types of research activity which have
application to specific problems and specific settings, but which must be
carried on over a period of several years. Thtse can be divided Into 3
major categories: 1) research programs with discrete and sequential
parts/steps--where one part must usually be done before another can be
initiated/planned, 2) research programs that often take a long time, but
parts of which can be carried on concurrently, and 3) methods development
and validation.
A. Long-term research programs best done in sequential steps
This 1s usually a series of several, discrete projects, each
of which generates data useful/needed 1n other related projects—either in
their design or execution. There are many examples here, but the key
feature 1n each 1s that this 1s a long-lasting program with several stages,
and each staff fetds Into/sets up the next action:
1. The ozone layer and ozone depletion
This is a program which has continued for many years.
The human and ecological health effects Implications of this are enormous.
Human health effects of the ozone-layer depletion Include possibly large
increases 1n UV light-Induced cancers and othtr serious skin diseases.
Ecological effects on agriculture/crops may be equally huuwn
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Hfe-threatening, though less direct. There have been many stages 1n this
overall program:
a. The first studies looked at the Issue—Is there any
evidence that we are actually losing stratospheric ozone? The answer to
this (data supporting this) 1s still being gathered and debated (at least
in some quarters), but the first indications were that evidence existed to
suggest a loss; therefore, step 2 was needed.
b. The second step seemed to be: What might be causing
this loss of ozone? Is there any human contribution (e.g., chemical) which
can destroy ozone and which is likely to get to the ozone layer? Data
about the chemistry and Interactions of light, ozone, and hydrocarbons had
to be generated here first. Some experiments are stm being carried out
at this stage.
c. The next step was to gather data about the presence
of ozone-destroying materials/chemicals (e.g., halogenated hydrocarbons) 1n
the upper layers of the atmosphere. New methods for measurement*
collection of samples etc. had to be developed, validated and used.
d. Then real-life sources of these hydrocarbons had to
be sorted out and evaluated for their possible contributions to the
problem.
e. Then decisions as to which steps would be most
effective 1n changing the amount of hydrocarbons at the ozone layer had to
be decided.
Thus, many types of research wert/are Involved
here—chemistry, biochemistry, ecology, climate, stratospheric, marketing,
sociological/psychological, and political. However, the steps to be taken
next in the overall strategy of dealing with this problem depended on the
outcome of those studies made just before and on most of those preceding.
2. The ecologlc and health effects of add rain
A number of Issues have been raised here, but they all
concern whether add rain or another source of pollution has caused the
effects, and what these effects really are. Add rain 1s believed to be
formed primarily from Industrial sources, though others are also possible
and constitute another subset of evolving Issues. One example 1n this
problem arta 1$ whether the damage to trees (and other flora, here and in
Europe) 1s due to add rain from factories and electric power generation or
1s caused by pollutants from cars/traffic, etc. A series of studies has
been made and others are continuing. It 1s becoming obvious from some of
the results that the answer 1s "yes" to both; tree damage (and crop
effects/human health effects) may result from add rain and car exhaust.
This answer comes from a series of sequential and evolving researches
carried out over several/many years. One of the most recent reports on all
this (Including some limited assessment of human health effects of add
aerosols) 1s probably the National Add Precipitation Assessment Program
report Issued 1n September 1987. Human health effects of add aerosols
were recently re-assessed at an EPA-MIEHS sponsored symposium held at NIEHS
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1n October 1987. The report of this will be published 1n Envirormental
Health Perspectives 1n 1988. This research effort 1n both ecology and
human healtn effects of add rain has gone on for years/decades, and some
answers are only now becoming barely visible.
B. Long-term studies with concurrent steps
These are studies that just take a long time—the objectives
are such that the study just can't be done in short time frames. Many
"purely" epidemiology studies fall here—where the questions concern health
effects of low level, chronic exposures or seek to determine endpolnts
resulting only years after exposure or 1n populations that must "age" to
have detectable effects. Most studies on possible causes of cancer or on
carcinogenic effects of chemicals are here. So are evaluations of the
causes of many other slowly developing effects/diseases (e.g., emphysema,
kidney failures, liver damage, and CVS or CMS effects). These evaluations
involve multiple studies done at the same time but continuing for a long
time on the same populations. Chronic toxiclty studies in animals are a
subset of this kind of approach. There are many examples here:
1. The "Six Cities Studies" of health effects of air
pollution—comparing various Indices of health 1n persons living 1n 6
cities of widely varying degrees of pollution. This Study has been going
on for years now. Some part of the Increasing clarity 1n this Study
results from more data—accumulated now over more than 10 years, but some
part 1s the adding of new tests and better data analysis to the screens for
health effects. The point 1s that this Study required/used repeated
studies of the same populations/regions over several years to establish
effects and to clearly associate these health effects with the changes 1n
air pollution (which occurred during the years of the study) 1n these 6
"regions". The principal effects now being seen are those on the lung
(lung function decrements), but other systems (e.g., kidney, CVS) may be
shown to be affected as these studies continue.
2. The effects of maternal polychlorlnated biphenyl (PCB)
exposures on childhood development. This began with several accidents both
in the U.S. and elsewhere (e.g., cooking oil contaminations 1n Taiwan and
Japan and accidents like the dumping of waste oil contaminated with PCBs
along highways, and the exposures of persons living near, or walking along
these highways). From both short- and long-term animal studies 1t was
known that many serious effects of PCBs were not seen acutely but were
instead delayed 1n onset and subtle. Therefore, several epidemiology
studies were begun to follow (for several years) health in populations of
PCB-exposed persons and especially in any children they might have. The
effects of various levels of maternal exposure to PCBs on childhood
development are now being described in some detail but only because these
accidentally-exposed populations and a large number of "less-exposed" and
"normaT'/unexposed women and children were followed for many years.
3. The effects of polybromlnated biphenyl (PBB) exposure.
Again, this began with an accident—the mixing of PBBs Into animal feed and
the spread of this chemical/mixture among many farms and Into many parts of
the food chain 1n Michigan. Heavily-exposed persons are still being
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monltored for effects, since again, animal studies show that these effects
are delayed and subtle.
C. Development and validation of test methods
In many cases the methods for detecting and quantifying new
environmental toxins/problem chemicals do not exist at the time such
"problems" are first discovered. This set of "long-term" research
activities is vital in any program seeking to understand and affect
environmental health hazards. There are many examples here, but only a few
can be given:
1. Dioxins (PCDDs) and dlbenzofurans (PCDFs)
Chemical methods for detecting, separating, and
quantifying these "families" of toxic materials did not exist when the
first "poisoning" episodes in humans occurred. The amounts of these
materials present in samples from most accidents 1s very small, and yet, in
animals, these chemicals show toxic effects at extremely low
concentrations. We are only now getting the methods needed to detect,
quantify and selectively identify and separate the wide variety of these
chemicals found 1n most real life exposures. Some of the newest 1n
analytical techniques were developed to meet this problem/series of
problems. The best of separation and analytical methods were required to
identify the dlbenzofurans as contaminants of the PCBs and dloxln mixtures
and also as contributors to some of the toxlcologlcal effects/problems
associated with these mixtures. This long-term research has stretched over
at least twenty years and 1s not ended yet. Validation of all these
methodological advances 1s still occurring.
2. Lead
W1th/1n several environmental problems we need some
measure of the toxic material 1n "deep" body tissues. Getting at these
without painful surgery/biopsy or the use of autopsy material 1s a must 1f
the amounts of information we need are to be generated—particularly for
long-term studies, or for uncovering chronic effects (although this
information may also be vital for acute emergencies). Lead, like several
other metals, tends to stay only briefly 1n readily accessible body tissues
and fluids. Stores of lead and several other chemicals occur 1n relatively
inaccessible tissues like bone, teeth (or deep fat, etc.). Methods to
measure these "deep" stores of toxic chemical are urgently needed.
Non-invasive methods are especially useful/attractive for
screening/repeated measurements. Newer methods for this In the case of
lead may be possible now with X-ray fluoroscopy. Validation of this method
1s now taking pi ace--total time from concept to use will be about
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ten years 1f all goes well — a long-term effort typical here of several
others.
111. How EPA Uses/Depends on Basic Research Conducted by Other
Federal Agencies
Health research within the EPA 1s ultimately directed toward the
regulatory mission of the Agency. While such research 1s often of an
"applied" and/or "immediate" nature which answers specific problems that
the Agency must deal with in an expeditious manner, sound basic or
fundamental research is the only method of Improving the scientific
rationale underlying regulatory decisions. It is vital that the EPA
scientific staff maintain current awareness of relevant basic research by
performing such research within the Health Effects Research Laboratory and
by closely following the latest developments 1n toxlcological research.
The Agency cannot effectively accomplish Its research mission without
scientists who have competence 1n and knowledge of the tools of basic
research. Without this competence and knowledge health scientists within
the Agency would be unable to effectively translate the findings of
fundamental research into the applied research areas most supportive of the
Agency's regulatory mission. However, since basic research performed by
EPA represents only a small fraction of that which 1s necessary to support
its regulatory mission, the Agency must rely heavily on basic research
information developed by other Federal agencies particularly by the various
Institutes of the Department of Health and Human Services. These
organizations have been reponslble for many of the scientific breakthroughs
In molecular biology, genetics, biochemistry, Immunology, and cancer
research that have enabled development of applied methods for exposure
monitoring, dosimetry, toxlcological testing, and biochemical epidemiology.
Basic research performed through programs developed at the
National Institutes of Health has substantially Impacted the Agency's
regulatory approaches and policies. Research on the molecular basis of
mutation, xenobiotic metabolism, pharmacoklnetlcs, and molecular dosimetry
performed at the National Institute of Environmental Health Sciences has
found applications at EPA 1n genetic bloassay development and Improved
metabolic activation systems for 1n vitro test systems, molecular
techniques for exposure monitoring, and advanced methods for human
biochemical epidemiology. Fundamental research by the National Cancer
Institute on mechanisms of carcinogenesls and Immune surveillance has
contributed directly to the development of toxlcologlcal test methods and
guidelines for cancer risk assessment promulgated by the EPA Office of
Health and Environmental Assessment. EPA 1s benefiting directly from
widely and federally-funded basic research 1n the area of neurotoxlcology.
The discovery of biochemical differences among various cell types within
the central nervous system (and their concomitant differential
vulnerability) 1s leading to an Improved understanding of mechanisms of
neurotoxidty and Improved methods for the assessment of adverse
neurotoxicologic responses. These methods will undoubtedly contribute to
future Agency guidelines for neurotoxlcity testing.
In addition to the use which the Agency makes of basic research
information generated by other Federal agencies through Indirect means
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(infortMtion appearing in the literature and discussed at scientific
forums), the Agency also depends upon active research collaborations which
take advantage of basic findings and/or expertise.
EPA scientists frequently engage in collaborative studies with
scientists in other governmental agencies as well as their colleagues in
academia who may be funded by these agencies. These research efforts often
take advantage of expertise in new technologies and new findings that may
have applications to the regulatory mission of the Agency. As an example,
research on mechanisms involved in the successful fertilization of the
oocyte has led to interagency collaborative research to improve methods for
the evaluation of maie fertility. Other research efforts delineating the
fundamental factors involved in dermal absorption have led to joint
interagency research projects centered on the development of improved
methodologies for the assessment of the kinetics of such exposure.
Clearly, it would be possible to extend this list of relevant
examples since much of the scientific information utilized by the Agency
for regulatory decision-making and guidelines formulation rests on a
foundation of basic research.
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Chapter 3
RESEARCH ADVANCES IN THE TOXICOLOGY OF LEAD
Kathryn Mahaffey
PREAMBLE
The place of and necessity for long-sustained basic research activity
in the development of a foundation for constructive action 1n Important
problems in environmental health could be Illustrated by reference to any
of several current problem areas. We have chosen the story about lead and
its dangers or toxicity to serve this purpose. Lead as a public health
problem has been recognized for years (1f not centuries). Yet how, what,
and when to do something about both preventing Its health effects and
treating those not prevented have been obvious only recently, and only as a
result of long-continuing basic research. For one thing, only long-range,
multidisciplinary, continuing basic research has given us the varied tools
we need to detect some of the more subtle (yet extremely important) effects
of lead. We have moved from counting dead bodies to worrying about things
like changed behavior and nerve damages 1n lead-exposed children—but only
because we now have some good tests for such effects of lead. This then 1s
the story of an environmental health research success—made possible only
because such slow-moving (and sometimes hard to explain) studies were
pursued and supported by far-sighted people who believed that long-range
research was and would continue to be extremely cost-benefit positive.
Background
Understanding the range of adverse health effects produced by lead
exposure has advanced markedly 1n this century. Research Into the toxic
effects of lead provides a paradigm that has guided the entire discipline
of clinical and laboratory toxicology for the past five decades.
Fundamental mul ^disciplinary laboratory research 1n such areas as
biochemistry and'physiology has been a major key to this progress.
Lead has long been recognized to be acutely toxic at high-dose
exposure. In addition, we now recognize, based on reearch findings 1n the
1970's and 1980's, that lead toxicity reflects two patterns of lead
exposure. Adverse neurobehavlorlal effects of lead on Infants occur at
levels within one standard deviation of the Man concentration of the
United States population. Superimposed on the general population lead
exposure 1s an additional severe problem of high-level lead exposure
concentrated aaong young children froa lower sodoeconoalc families,
particularly those froa urban areas.
In children, high-dose exposure to lead, such as results from 1ngest1on
of lead-based paint, has been shown to cause a profound neurologic syndrome
characterized by coma, convulsions, and 1n severe cases death. In adults
with high-dose exposure to lead, abdominal cramping, a syndrome termed
"wrist and ankle drop," and end-stage renal disease are the well-recognized
consequences.
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The challenge has been tc '.^rstand that the range of health problems
caused by lead was much more e -rnslve than the clinically-obvious disease.
What has made this challenge es:sc1ally difficult 1s that environmental
lead pollution has been at very high levels, producing an elevated body
burden of lead 1n a sizable portion of the population. During the 1970's
in metropolitan areas, young children frequently had blood lead
concentrations greater than 40 g/dl; a concentration now associated with
several neuropsychological impairments. The challenge 1s to perceive the
etiology and severity of health problems that are so common they are
considered "normal." In the paradigm of lead public health and preventive
medicine have progressed from enumerating mortality and morbidity (I.e.,
case reports) to understanding the disease process. This progress reflects
and has been possible only because of long-range support of environmental
research.
Among the most exciting recent findings with respect to understanding
of the toxicology of lead 1s the realization that lead 1s capable of
producing toxic effects in adults and children at relatively low levels of
exposure, i.e., levels that are insufficient to_produce grossly clinical
symptoms. Only a decade ago such levels of lead exposure were considered
"safe". Lead 1s now recognized to produce a syndrome of subcl1n1cal
toxldty.
Recent research has demonstrated that this subcl1n1cal toxldty of lead
is a many-faceted syndrome Involving multiple organ systems. The
developing red blood cells, the nervous system, ind the kidneys are the
organ systems in which these toxic effects have been more Intensively
studied.
In the early 1900's lead exposures were so high that occupational
records routinely reported lead-induced mortality statistics, for exanple,
Hoffman (1935) reported that the number of deaths attributed to lead
poisoning for the United States registration area between 1900 and 1933 was
in excess of 3400. The number of deaths among children, who are more
susceptible to the effects of lead exposure, remains largely unknown. In
the 1940's through the 1960's descriptive reports of clinical aspects of
the disease dominated the literature. Prior to the Introduction of
chelatlon therapy, severe lead poisoning with encephaloptthy had a
mortality rate of 651 (MRC, 1972).
Among survivors of lead poisoning profound neurological damage 1s the
predominant, reported effect. :*For example, Byers and Lord (1943) and other
clinicians showed long-term residual sequelae of acute pediatric lead
poisoning which included mental retardation, seizures, optic atrophy,
sensory motor deficits, and behavioral dysfunctions. Perlstein and Attala
(1966) reported such sequelae 1n 37% of children who suffered lead
poisoning without evidence of encephalopathy.
Through screening programs to Identify children with lead toxldty
before they become symptomatic, and through legal requirements to monitor
occupational exposures of workers to lead, severe clinical cases of lead
toxicity have been brought under a degree, of control; however, they have
not been eliminated. These case reports, describing clinical aspects of
intoxication, have identified which organ systems are most affected at
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high-dose exposures. The limited reversibility or 1rrevers1bil1ty his been
documented 1n many of the clinically-reported, neurologic effects. Using
these clinical studies as a guide, long-range, multldisciplinary research
has extended the understanding of lead toxldty to the current emphasis on
biomarkers of exposure, dose-response relationships for specific effects,
and identification of susceptible subgroups for these effects.
Research Findings in the 197Q's and 198Q's
The general picture of adult and pedlatrlc lead poisoning has changed
in recent decades. The overall pattern is Identification of significant
adverse health effects at progressively lower exposures. These can be
arbitrarily separated Into neurobehavloral , hetnatopoletlc, renal/endocrine,
and reproductive effects. As a part of this effort, differential
sensitivity of various subpopulatlons has been revealed. Identification of
effects occurring at environmental exposures once considered "normal" has
coincided with reducing environmental exposures to lead. Only through
reduced exposures can the results given by toxicology and epidemiology
research be evaluated in general human populations.
I. Neurobehavloral Effects
Recognition that neurobehavloral effects 1n children are produced
by lead exposures considered "normal" 1n earlier decades (e.g., blood lead
concentrations of 20-50 g/dl) has been mmnq the soft significant research
findings in the 1970's and 1980's. Longitudinal studies during the past
10-15 years built upon early case reports and cross-sectional studies. The
longitudinal prospective designs have permitted gathering Improved
information on exposure histories. Information on exposure levels and
patterns is clearly important 1n assessing effects of a cumulative toxicant
on endpoints such as neurobehavloral function that may reflect changes
induced at far earlier, but critical, developmental periods.
The most consistent finding of the prospective studies 1s that an
association exists between low-level lead exposures during developmental
periods (especially prenatally) and later deficits 1n neurobehavloral
performance. This latter 1s reflected by Indices such as the Bayley Mental
Development Index, a well-standardized test for Infant Intelligence. Blood
lead concentrations of 10-15 g/dl constitute a level of concern for these
effects (EPA, 1986). In addition, Impaired neurophyslologlcal function has
been associated with Increasing blood lead concentrations among children.
These functional deficits Include changes 1n the auditory bralnstem evoked
potentials and evidence of lead-related reduced hearing acuity (Robinson et
al. , 1985, 1987). These subcl1n1cal toxic effects of lead on the central
nervous system are generally considered to be permanent and Irreversible,
and they are associated with permanent loss of Intelligence and
irreversible alteration 1n patterns of behavior.
Bellinger et al (1987) reported significantly lower post-natal
development scores on the Mental Development Index of Infants from an
upper-middle class population when maternal blood lead levels were 1n the
rnage of 10-25 g/dl. Among adult women ages 20-40 years mean, blood lead
levels were between 10 and 12 g/dl based on the NHANES II general
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popylitlon dtU for the period 1176-1980 (Mthiffey et al, 1982). Thus, 1t
must be emphasized that these neyrobehivloral changes are associated with
blood lead levels within one standard deviation of the aean blood lead
level of the United States' population reported 1n the NHAKES II dat*.
The peripheral nervous system is also affected by lead.
Typically, adults are likely to demonstrate peripheral rather than central
nervous system effects. In the early 1960's investigators began to call
attention to "subclinical" neuropathy manifested by changes in peripheral
nerve conduction velocity in lead workers not having overt neurological
involvement (Sessa et al., 1965). In the 1970's Seppalainen et al. (1972,
1975) reported the slowing of the maximal motor conduction velocity of the
median and ulnar nerves and other electromyelographic abnormalities in
workers whose blood lead concentrations never exceeded 70 g/dl.
Investigations of the behavioral effects of lead uncovered an increased
hearing threshold, decreased eye-hand coordination, and other physiological
and psychological changes 1n workers with blood lead concentrations below
80 g/dl (Repko et al., 1975).
II. Hematopoiesis
Anemia has been a symptom of severe clinical lead poisoning 1n
both children and adults. Anemia (increased prevalence of hewotocrit
values below 35%) 1s now recognized to become evident 1n one-year-old
children at blood values of 30 g/dl. Lead Interferes with synthesis of
heme and the formation of hemoglobin at a number of metabolic steps. In
the developing red blood cells lead Inhibits the enzyme -am1nolevu!1n1c
add dehydratase to Increase levels of erythrocyte protoporphyrln 1n
children. The threshold for this effect 1n children is associated with a
blood lead concentration of 15-18 g/dl (PlotnelH et al., 1982).
Impaired heme biosynthesis produces effects 1n addition to anemia.
The accumulation of protoporphyrln IX (measured as zinc protoporphyrln or
as protoporphyrin 1n erythrocytes) is not only an Indicator of diminished
heme biosynthesis but also signals general mltochondrlal Injury. The final
step of heme biosynthesis occurs 1n the mitochondria. Such Injury to the
mitochondria can Impair a variety of subcellular processes Including energy
metabolism and homeostasls. Health Implications of such Impairment
include: reduced transport of oxygen to all tissues; Impaired cellular
energetics; disturbed Imtjunoregulatory role of calcium; disturbed calcium
metabolism; disturbed role 1n hematogenesls control; Impaired
detoxification of xenoblotlcs; and Impaired metabolism of endogenous
agonists (e.g., metabolism of tryptophan).
III. Renal Effects
Acute high-dose lead exposure 1n children produces a Fancon1-type
syndrome with glucosurla, phosphaturla and amlnoaciduria secondary to
poisoning of the proximal convoluted tubule. High-dose exposure to lead in
childhood has been associated with glomerular nephritis and renal disease
in adults. Among occupationally-exposed adults, an Increased rate for
mortality from all causes, from all neoplasms (specifically, cancers of the
stomach, liver, and lungs), from chronic nephritis, and from other
hypertensive disease (I.e., hypertension due to kidney damage and not heart
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disease) were observed 1n a longitudinal study of workers in lead battery
plants and lead smelters (Cooper, 1985).
A statistically-significant relationship has been reported between
Increases in systolic and diastolic blood presures and Increases in blood
lead among 40-to-59 year old, white wales from the NHAMES II survey
population (Pirkle et al, 1985).
Impairment of the endocrine functions of the kidney have been
reported to occur at much lower lead exposures. Recognition of these
effects required development of several areas of research:
A. Understanding the metabolic activation of Vitamin 0 to
1,25-dihydroxyvitamin D. This metabolite is critical to regulation of
calcium metabol1sm.
B. Recognition that'lead impairs various steps 1n both
biosynthesis and function of 1,25-dihydroxyv1tam1n D.
Currently, the most studied site at which these metabolic pathways
converge is the proximal convoluted tubule of the kidney. Here
25-hydroxyvitamin D, formed in liver from Vitamin 0, undergoes a second
hydroxylation which is catalyzed by the enzyme 1, ,25-hydroxyvitamin D
hydroxylase. Research using in vitro techniques (following 1n yivo
exposure of chickens to 1 ead)"~h"aT~defflonstrated that lead 1nhTb~l€s the
activity of this enzyme. Findings from a clinical Investigation among
young children indicated that plasma 1,25-dihydroxyvitamin 0 levels were
depressed 1n proportion to blood lead concentration. Chelatlon therapy to
reduce body burden of lead, resulted 1n Increasing serum concentrations of
1 ,25-dihydroxyvitamin 0 up to levels similar to those present 1n children
serving as controls (Rosen et al., 1980). Additional ep1dem1olog1cal
research has shown that 1,25-d1hydroxyv1ta»1n 0 concentrations were
decreased with increasing blood lead concentration over a broad range of
blood lead concentrations, 12 to 120 g/dl (Mahaffey et al., 1982b).
IV. Reproductive Effects
Early in the century a number of adverse effects of lead on
reproduction were reported among women with occupational lead exposures.
These included increased spontaneous abortion rate, Increased still-birth
rate, and a higher, post-natal and early childhood mortality rate among
children of such exposed women> Exposures associated with these adverse
outcomes were very high. However, longitudinal, prospective studies,
designed to evaluate neuropsychological effects of lead, have provided
important information on reproductive effects at the upper range of current
environmental levels. McMlchaels et al. (1986) found that the Incidence of
preterm deliveries (before the 37th week of pregnancy) were significantly
related to maternal blood lead at delivery. When late fetal deaths were
excluded, the strength of the asociatlon Increased. The relative risk of
preterm delivery at exposure levels reflected 1n blood lead concentrations
of 14 g/dl or higher was 8.7 times the risk at blood lead concentrations
up to 8 g/dl. Reduction in gestational age at delivery with increasing
blood lead concentrations were also reported by Dietrich et al. (1986),
Bellinger et al. (1984), Moore et al. (1982), and Bornscheln et al. (1987a,
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b). The data from Bornscheln Indicate that for each 10 g/dl Increase 1n
blood lead concentrations birth weight decrtastd between 58 and 601 grams
depending on the age of the mother.
The findings of McMlchaels et al. (1986) also Identified an excess
In miscarriages and still births fn the high-lead exposure areas. In
contrast, data from this study show that average, maternal blood lead
concentration was lower for still births than for live births. Placenta!
response to lead remains an unanswered question.
Basic research 1n the toxic effects of lead at low doses 1s of profound
importance for the fields of preventive medicine and public health. Until
recently, blood lead concentrations of 25 g/dl and below were considered
safe, and indeed, only five years ago the Centers for Disease Control (CDC)
stated that 25 g/dl should constitute a threshold level indicative of
increased lead absorption in children. Now, on the basis of recent
research it is evident that lead produces toxic effects 1n children at
levels below this guideline. Thus, recent research Into the toxldty of
lead at low doses is about to force a total re-evaluation of current
standards for assessing the exposure of American children to lead.
The importance of these basic research findings stems from the fact
that lead exposure remains extremely widespread among children 1n the
United States. Data from the Second National Health and Nutrition
Examination Survey (NHANES) Indicated that 1n 1980 9.1% of all preschool
children 1n the United States - 1.5 million children - had blood lead
concentrations of 25 g/dl or more (Mahaffey et al., 1982a). Among black
preschool children the prevalence of Increased lead absorption (high blood
lead concentrations) was 25%.
These findings on the high prevalence of Increased lead absorption
(high blood lead concentrations), when taken 1n conjunction with the data
on subcl1n1cal lead toxldty, carry a message of chilling significance.
These findings suggest that 9% of all children 1n this nation, and 25% of
minority children, may be suffering irreversible neurologic, Intellectual,
and behavioral ^-npalrment as the result of chronic, low-dose exposure to
lead. The implications of these basic research data for public health and
environmental medicine are enormous.
This then has been a very condensed story about one of the many
pervasive and Important environmental health hazards. It 1s a story that
continues beyond the present findings and their Implications. It will
reach even more successful conclusions only 1f the kind of studies which
brought us to this stage are continued. Continued long-range and basic
research Investigations on lead toxldty are at one and the same time
perhaps among the more justifiable and yet less supportable of such
activities 1n the entire field of environmental health sciences. So much
has been done before 1n lead research that 1n comparison, no other (few at
least) of all the current health hazards has received this much emphasis.
Yet it 1s obvious that this sustained effort 1n lead research has paid off
handsomely and 1s still needed. It is this "apology" for long-range, basic
research that we feel can stand for the entire field of environmental
health science, whatever may be the specific stage of development of this
research for any one hazard.
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REFERENCES
1. Bellinger D.C., Neddleman H.L, Leviton A., Waternaux C., Rab1now1tz
M.B., Nichols M.L. (1984). Early Sensory-Motor Development and
Prenatal Exposure to Lead. Neurobehav Toxlcol Teratol 6:387-402.
2. Bellinger D.C., Leviton A., Waternaux C., Neddleman H.L. , Rab1now1tz
M.B. (1987). Longitudinal Analyses of Prenatal and Postnatal Lead
Exposure and Early Cognitive Development. New England Journal of
Medicine 316:1037-1043.
3. Bornschein R.L., Succop P.A., Dietrich K.N., Krafft K., Grote J. ,
Mitchell T., Berger 0., Hammond P. B. (1987a). Prenatal-Lead Exposure
and Pregnancy Outcomes in the Cincinnati Lead Study. In: Linberg
S.E., Hutchinson T.C., eds. International Conference: Heavy Metals in
the Environment, V 1: September: New Orleans, LA: Edinburgh, United
Kingdom: CEP Consultants, Ltd., pp. 156-158.
4. Bornschein R.L., Grote J. , Mitchell T., Succop L. , Shukla R. (1987b).
Effects of Prenatal and Postnatal Lead Exposure on Fetal Maturation
and Postnatal Growth. In: Smith M., Grant L.D., Sors A., eds. Lead
Exposure and Child Development: An International Assessment.
Lancaster, United Kingdom: MTP Press.
5. Byers R.K., Lord E.E. (1943). Late Effects of Lead Poisoning on
Mental Development. Am J D1s Child 66:471-483.
6. Cooper W.C., Wong 0., Khelfets L. (1985). Mortality 1n Employees of
Lead Battery Plants and Lead Production Plants, 1947-1980. Scand J
Woj-k Environ Health 11:331-345.
7. Dietrich K.N., Krafft K.M., Bier M., Succop P.A., Berger 0.,
Bornschein R.L. (1986). Early Effects of Fetal Lead Exposure:
Neurobehavioral Findings at 6 Months. Int J. Blosoc. Res. 8:151-168.
8. U. S. Environmental Protection Agency (1986). A1r Quality Criteria
for Lead. Research Triangle Park, NC: Office of Health and
Environmental Assessment, Environmental Criteria and Assessment
Office: EPA Report No. EPA-600/8-83/028af-df. 4v. Available from:
NTIS, Springfield, VA: PB87-142378.
9. Hoffman F.L. (1935). Lead Poisoning Statistics for 1933. Am J Public
Health 25(2; Suppl):90-100.
10. Mahaffey K.R., Annest J.L. , Robert J., Murphy R.S. (1982a). National
Estimates of Blood Lead Levels. United States, 1976-1980. NHANES. New
Engl J Med 307:573-579.
11. Mahaffey K.R. , Rosen J.F. , Chesney R.W., Peeler J.T., Smith C.M.,
DeLuca H.F. (19826). Association Between Age, Blood Lead
Concentration and Serum 1,25-d1hydroxycholecolc1ferol Levels 1n
Children. Am J CUn Nutr 35:1327-1331.
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12. McMlchaels A.J., Vimpani G.V., Robertson E.F., Baghurst P.A., Clark
P.O. (1986). The Port P1r1e Cohort Study: Maternal Blood Lead and
Pregnancy Outcome, J Epidemiology Community Health 40:18-25,,
13. Moore M.R., Goldberg A., Pocock S.J., Meredith A., Stewart I.M.,
Maconesplc H., Lees R., Low A. (1982). Some Studies of Maternal and
Infant Lead Exposure 1n Glasgow. Scott Med J 27:113-122.
14. National Research Council (1972). Airborne Lead 1n Perspective.
Washington, O.C. Committee on Medical and Biological Effects of
Atmospheric Pollutants.
15. Perlsteln M.A., Attala R. (1966). Neurologic Sequelae of Plumbism in
Children, din Pedlatr 5:292-298.
16. PlomelH S., Seaman C.t Zullow 0., Curran A., Davidow B. (1982).
Threshold for Lead Damage to Heme Synthesis 1n Urban Children. Proc
Natl Acad Sci USA 79:3335-3339.
17. Pirkle J.L., Swartz J., Landis J.R., and Harlan W.R. (1985). The
Relationship Between Blood Lead Levels and Blood Pressure and Its
Cardiovascular Risk Implications. Am Jr of Ep1dem1ol 121:246-258.
18. Repko J.D., Morgan B.B., Nicholson J. (1975). Behavioral Effects of
Occupational Exposures to Lead. U. S. Department of Health, Education
and Welfare. National Institute for Occupational Safety and Health.
Washington, D. C.
19. Robinson G., Baumann S., Klelnbaum D., Barton C., Schroeder S.R.,
Musak P., Otto D.A. (1985). Effects of Low to Moderate Lead Exposure
on Bralnstem Auditory Evoked Potentials 1n Children. Copenhagen,
Denmark: WHO Regional Office for Europe: pp. 177-182. (Environmental
Health Document 3).
20. Robinson G.S., Keith R.W., Bornscheln R.L., Otto D.A. (1987). Effects
of Environmpntal Lead Exposure on the Developing Auditory System as
Indexed by Ine Bralnstem Auditory Evoked Potential and Pure Tone
Hearing Evaluations 1n Young Children. In: Llndberg S.E., Hutchlnson
T.C., eds. International Conference: Heavy Metals 1n the Environment,
VI: September, New Orleans, LA: Edinburgh, United Kingdom: CEP
Consultants, Ltd., pp. 223-225.
21. Rosen J.F., Chesney R.W., Hamstra A., DeLuca H.F., Mahaffey K.R.
(1980). Reduction in 1,25-dihdroxyvitam1n D in Children with Increased
Lead Absorption. New Engl J Med 302:1128-1131.
22. Seppalalnen A.M., Hernberg S. (1972). Sensitive Technique for
Detecting Subcllnical Lead Neuropathy. Br J Ind Med 29:443-449.
23. Seppalalnen A.M., Tola S., Hernberg S., Kock B. (1975). Subcllnical
Neuropathy at "Safe" Levels of Lead Exposure. Arch Environ Health
30:180-183.
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24. Sessa T., Ferrari E., Colucci d'A.C. (1965). Velocita de Conduzione
Nervosa Net Saturnini. Folia Med. (Napoli) 48:658-668.
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Chapter 4
NEWER BASIC/LONG-TERM RESEARCH
WITH
APPLICATION TO ENVIRONMENTAL HEALTH PROBLEMS
PREAMBLE
In this Chapter a number of authors discuss some of the newer
basic/long-term research with possible applications to current
environmental health problems (especially 1n humans). This does not
represent the whole universe of possible basic/long-range research which
will or could be of great benefit to such environmental Issues. It 1s,
,however, an attempt at careful choices of those studies which have required
such long-term support for the reaching of this stage where their
applications could have great impact on environmental health. As such
then, this is a look at the many and more probable benefits of supporting
such long-range research more adequately than has been done 1n the past.
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ACTIYATION OF PROTO-ONCOGENES BY CHEMICALS
Marshall Anderson
INTRODUCTION
Proto-oncogenes are cellular genes that are expressed during normal
growth and development processes. These genes were Initially discovered is
the transduced oncogenes of acute transforming retrovlruses (1). Recent
studies have established that proto-oncogenes can also be activated to
cancer causing oncogenes by mechanisms Independent of retrovlral
involvement (2-4). These mechanisms Include point mutations or gross DMA
rearrangements such as translocatlons or gene amplifications. The
activation of proto-oncogenes by genetic alterations results 1n altered
levels of expression of the normal protein product, or 1n normal or altered
levels of expression of an abnormal protein.
ACTIVATION OF PROTOONCOGEMES
The activation of proto-oncogenes 1n spontaneous and chemically-1ndyced
rodent tumors and 1n human tumors has been studied 1n great detail during
the past several year£ Investigations 1n rodent models for chemical
carclnogenesls Imply that certain types of oncogenes are activated by
carcinogen treatment and that this activation process 1s an early event fn
tumor Induction (5-6K Alternatively, analysis of some human and rodent
tumors suggests that oncogene activation 1s Involved 1n neoplastlc
progression (7-9). The number of proto-oncogenes that must be activated 1n
the multlstep process of neoplasla 1s unclear at present. The concerted,
low level expression of at least two oncogenes, ras and myc, are needed for
the partial transformation of primary rodent cellsIn vitro (10).
Furthermore, 1n addition to the activation of proto-oncogenes, the loss of
specific regulatory functions such as tumor suppressor genes may be a
distinct step 1n neoplastlc transformation (11). The Implication of
activated oncogenes in rodent tumor will be discussed in terms of
extrapolation of rodent carcinogenic data to human risk assessment*
The aetlvirfon of ras proto-oncogenes appears to represent one step 1n
the multlstep process "oT~carc1nogenes1s for a variety of rodent and human
tumors (5,6). The activation of ras by point mutations 1s probably an
early event 1n tumoHgenesis and may be the "Initiation" event in some
cases. Thus, a chemical that induces rodents tumors by activation of ras
proto-oncogenes can potentially Invoke one step of the neoplastlc process
in humans exposed to the chemical. Is this property alone enough to
classify the chemical as a potential human carcinogen? Dominant
transforming oncogenes other than ras have also been detected 1n
chemical-induced rodent tumors (6). The Involvement of these oncogenes in
the development of human tumors 1s unclear at present, as well as whether
the non-ras genes detected in human tumors can be activated by chemicals or
radiation (6).
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ONCOGENE ANALYSIS
Most chemicals are classified as potentially hazardous to humans on the
basis of long-term carcinogenesis studies in rodents. While these rodent
carcinogenesis studies are often designed to mimic the route of human
exposure in the environment or workplace, the dose of a given chemical is
usually higher than that which actually occurs in human exposure. Coupled
with the appearance of species- and strain-specific spontaneously occurring
tumors in vehicle-treated rodents, this complicates the extrapolation of
rodent carcinogenic data to human risk. Oncogene analysis of tumors from
spontaneous origin and from long-term carcinogenesis studies should help
determine the mechanisms of tumor formation at a molecular level. For
instance, the finding of activating mutations in different codons of the
H-ras gene in furan-induced liver tumors versus finding activating
mutations in only one codon of the H-ras gene in spontaneous liver tumors
suggest that the chemical itself actiTITed the H-ras proto-oncogene by a
genotoxic event (12). In general, comparison of patterns of oncogene
activation in spontaneous versus chemically-induced rodent tumors, together
with cytotoxic information, should be helpful in determining whether the
chemical in question is mutagenic, cytotoxic, has a receptor mediated
mechanism of promotion, or some combination of these (and other) modes of
action. This type of analysis might be of particular importance for
compounds such as furan and furfural (12,13) which are negative for
mutagenicity in short-term bioassays.
APPLICATION TO STUDY OF CARCINOGENICITY
Another approach which should be helpful in species-to-spec1es
extrapolation of risk from carcinogenic data 1s to examine oncogene
activation and expression in tumors from different species induced by the
same agent. For example, K-ras oncogenes with the activating lesion in
codon 12 were observed in both rat and mouse lung tumors induced by
tetranitromethane (14). Even though little is known about the DNA damaging
properties of this chemical, these data suggest that this compound is
acting in the same manner to induce tumors 1n both rats and mice.
The role of chemicals and radiation in the activation of proto-oncogenes by
gene amplification, chromosomal translocatlon, and other mechanisms which
can alter gene expression, is currently being investigated by several
groups. Also, as human life span increases, it becomes more important to
study chemical-Induced enhancement of the progression of benign to
malignant tumors. These and similar approaches to explore the mechanisms
by which chemicals induce tumors in animal model systems may remove some of
the uncertainty 1n risk analysis of rodent carcinogenic data.
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REFERENCES
1. Bishop JM. 1985. Viral Oncogenes. Cell; 42:23-38.
2. Varmus HE. 1984. The Molecular Genetics of Cellular Oncogenes.
Annual Rev Genet; 18:553-612.
3. Weinberg RA. 1985. The Action of Oncogenes in the Cytoplasm and
Nucleus. Science; 230:770-776.
4. Bishop JM. 1987. The Moleuclar Genetics of Cancer,
Science; 235:305-311.
5. Baroacid M. 1987. Ras Genes, Ann Rev of Biochem 56, in press.
6. Anderson M, Reynolds S. Activation of Oncogenes by Chemical
Carcinogens in: The Pathology of Neoplasia. A Sirica, ed, Plenum
Press, N.Y., N.Y. (In press 1988).
7. Brodeur GM, Seeger RC, Schwab M, Varmus HE, Bishop JM. 1984,
Amplification of N-myc in Untreated Neuroblastomas Correlated with
Advances Disease Stage; Science 224:1121-1124.
8. Seeger RC, Brodeur GM, Sather H, Dalton A, Siege! SE, Wong KY,
Hammond D. 1985, Association of Multiple Copies of the N-myc
Oncogene With Rapid Progression of Neuroblasts, The New England
Journal of Medicine; 313:1111-1116.
9. Slamon DJ, Clark GM, Wong SG, Levin WJ, Ullrich A, McGuIre WL. 1987,
Human Breast Cancer: Correlation of Relapse and Survival With
Amplification of the HER-2/neu Oncogene; Science 235:117-182.
10. Land H, Parada LF, Weinberg RA. 1983. Tumorigenic Conversion of
Primary Embryofibroblasts Requires at Least Two Cooperating
Oncogenes; Nature (London) 304:596-602.
11. Barrett JC, Oshimura M, Koi M. 1987. Role of Oncogenes and Tumor
Supressant Genes in a Multistep Model of Carcinogenesis, In:
Symposium on Fundamental Cancer Research. Volume 38 (F. Becker,
ed.,) , in press.
12. Reynolds SH, Stovers SJ, Patterson R, Maronpot RR, Aaronson SA,
Anderson MW. 1987. Activated Oncogenes in B6C371 Mouse Liver
Tumors: Implications for Risk Assessment, Science 237:1309-1316.
13. Tennant RW, Margolin BH, Shelby MD, Zeiger E, Haseman JK, Spalding J,
Caspary W, Resnick M, Stasiewica S, Anderson B, Minor R. 1987.
Prediction of Chemical Carcinogenicity in Rodents from In Vitro
Genetic Toxicity Assays, Science 236:933-941.
14. Stowers SJ, Glover PL, Boone LR, Maronpot RR, Reynolds SH,
Anderson MW. 1987. Activation of the K-ras Proto-oncogene in Rat and
Mouse Lung Tumors Induced by Chronic Exposure to Tetranitromethane,
Cancer Res 47:3212-3219.
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CARCINOGEN-OMA AND PROTEIN ADDUCTS: RESEARCH PERSPECTIVES
Frederlca P. Perera
INTRODUCTION
Advances in basic research in molecular biology and biochemistry have
permitted the development of innovative methods applicable to studies of
human populations exposed to chemical carcinogens. These highly sensitive
techniques can detect and sometimes quantify the internal dose of
carcinogens (the amount of the carcinogen or its metabolite in body tissues
and fluids) or the biologically effective dose (the amount that has
interacted with cellular macromolecules such as DNA, RNA or protein) in
target tissue or a surrogate. This latter type of dosimetry data could be
especially valuable in studies of cancer etiology by providing a
mechanistically relevant link between external exposure data on the one
hand and clinical disease on the other. Comparable molecular dosimetry
data in rodents and humans have the potential to improve Interspecies
extrapolation of risk in addition to providing early warning of a
carcinogenic hazard to humans. Successful applications of such "adducts
research" could directly address major programs/needs at EPA for better
estimates of exposure and risk to humans.
Various methods are available to monitor chemical-specific lesions
(such as immunoassays for DNA and protein adducts) as well as non-chemical
specific biologic alterations (such as cytogenetic effects or somatic cell
mutations). Table 1 gives examples of currently available methods for
measuring the biologically effective dose of carcinogens. As can readily
be seen, all pertain to endpoints associated with carcinogens that exert
genetic toxicity. Moreover, almost all available methods depend on readily
es for the actual target tissue itself. Despite these limitations,
biological markers have significant potential usefulness in cancer etiology
and risk assessment.
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Table 1. Examples of Human Biologic Monitoring Methods'2'
Sites
End Point Method F1uids(b)
Biologically effective dose
Adducts (DNA) Immunoassay, postlabeling, fluor- WBC
escence spectroraetry
Adducts (protein) Mass spectrometry, ion-exchange RBC
ami no acid analysis, HPLC, gas
chroma tography
Excised adducts HPLC, fluorescence Urine
UDS Cell culture, thymidine incorporation WBC
SCE Cytogenetic HBC
Micronuclei Cytogenetic BM.WBC
Chromosomal aber- Cytogenetic HBC
rations
Somatic cell mutation Autoradlography, light Microscopy HBC
(HGPRT)
Somatic cell mutation Itmtunoassay RBC
(glycophorln A)
Sperm quality Analyses of count, morphology, motlHty Sperm
Source: See Reference 1 (as modified)
RBC=red blood cells; BM-bone marrow; WBC»wh1te blood cells;
UDS=Unscheduled DNA Synthesis; HPLC-H1gh Performance Liquid
Chromatography; SCE'Sister Chromatld Exchange; HGPRT-Hypoxanthl ne
Guanine Phosphorlbosyl Transferase
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ADOUCTS
Carc1nogen-DNA and carcinogen-protein adducts have been the focus of
considerable research 1n the past 5 years and Illustrate a number of
strengths and limitations common to biological markers 1n general (2,3).
Biological Basis
The biologic rationale for measuring DMA adducts 1s that these lesions,
if unrepaired, can produce a gene mutation. There 1s considerable evidence
that gene mutation in somatic cells "initiates" the multistage process of
carcinogenesls (4,5); but it may also result in conversion of tumors to the
malignant state (6,7). Carcinogen-DMA adducts resulting 1n gene mutation
may also activate certain oncogenes instrumental in carcinogenesis
(8,9,10).
Protein such as hemoglobin can, in theory, act as a more readily
available surrogate for DMA. Proportionality between protein and DNA
binding has been demonstrated for a number of carcinogens (11,12,13).
Adducts are generally monitored 1n peripheral blood cells rather than
target tissue. However, for only a few carcinogens (e.g., benzo(a)pyrene
and cis platinum) is there actual experimental and/or human evidence that
comparable levels are formed at both sites (14,15).
METHODS
Techniques to measure carcinogen-DNA adducts include immunoassays using
adduct-speciflc polyclonal or monoclonal antibodies, synchronous
fluorescence spectroscopy, HPLC fluorescence spectrophotometry, and
P-postlabelling. Carcinogen-protein adducts may be determined using
antibodies and gas chromatography-mass spectrometry. The sensitivity of
the DNA to adduct methods is in the range of one adduct per 10 -10
nucleotides. Those methods aimed at carcinogen-protein adduct
quantification also appear to have adequate sensitivity for environmental
studies (16). However, unambiguous identification of particular DNA
adducts at low levels is difficult with present analytical methods.
Moreover, cross-reactivity of antibodies (such as the BPDE-I-DNA antibody
which also detects closely related polycyclic aromatic hydrocarbon (PAH-DNA
adducts) presents problems in definitive characterization of adducts (17).
ANIMAL AND HUMAN STUDIES
Experimental studies Involving acute and/or chronic exposure to diverse
carcinogens have shown that the relationship between administered dose and
macromolecular binding 1s generally linear with few exceptions (12,2,18,3).
With respect to humans, carcinogen-DNA and -protein adducts have been
investigated in human populations with exposures such as cigarette smoke,
PAHs, tobacco and betel nut, dietary aflatoxin and N-nitrosamines, cis
platinum, psoralen, 4-am1nobiphenyl , propylene oxide, vinyl chloride and
ethylene oxide (3).
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While results thus far support the feasibility and adequate sensitivity
of the methods 1n terms of human studies, they are frequently limited by
technical variability 1n the assays, small sample size, lack of appropriate
controls, and Inadequate data about exposure. However, they consistently
Illustrate that there 1s significant variability 1n the formation of
carcinogen-DNA and -protein adducts between Individuals with comparable
exposure or administered dose (15,19-25). Another consistent finding 1n
the human studies involving environmental exposure, 1s that measurable
levels of adducts are seen even 1n so-called "unexposed controls"
(19-20,26-29). Both of these observations have obvious implications for
risk assessment.
Although still largely in the validation stage, methods to monitor DMA
and protein adducts in experimental animals and humans have considerable
potential in a number of areas. These Include: hazard identification,
understanding of mechanisms involved 1n cardnogenesis, interspedes risk
extrapolation and improving the power and timeliness of epidemiology
(19,26,30-32).
Research Needs
Research is needed 1n the following areas:
A. Interlaboratory validation of methods as has been undertaken
recently for PAH-DNA Iwnunoassays (33).
B. Research on the stabililty of adducts 1n stored tissues.
C. Investigation of 1ntra-and 1nter-1ndiv1dual viHation 1n adduct
levels.
D. Research on the persistence of adducts in various cells and
tissues.
E. Comparison of adduct levels in DNA versus protein as well as in
surrogate versus target tissue for a number of different classes
of compounds.
F. Identification of critical sites or "hot spots" on DNA with
respect to the carcinogenic effectiveness of adducts.
G. Interspecies comparisons of DNA and protein adduct formation
(e.g., humans and rodents with acute and chronic exposure to the
same compound(s)).
H. Experimental and human studies on the relationship between adduct
formation, gene mutation, and oncogene activation.
I. Longitudinal studies (experimental and human) on the relationship
between adduct levels and tumor incidence/cancer risk. Examples
would be molecular epldemiological studies in model populations
(such as patients exposed to high dose chemotherapy and who
experience a high rate of secondary cancer, or heavily-exposed
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worker groups). Biologic samples could be drawn at the outset and
stored for future analysis.
J. Sample banks to serve as archives of human blood, urine, and
tissue for retrospective analysis.
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REFERENCES
1. Perera F. 1987. Molecular Cancer Epidemiology: A New Tool 1n
Cancer Prevention, J Natl Cancer Inst, 78, 887-898.
2. Wogan GN, Gorellck NJ. 1985. Chemical and Biochemical Dosimetry of
Exposure to Genotoxlc Chemicals. Environ Health Perspect, 62, 5-18.
3. Perera F. The Significance of DNA and Protein Adducts 1n Human
B1omonitor1ng Studies. Mut Res C1n press).
4. Weinsteln IB, Gatton1-Cell1 S, Klrschmeler P, Lambert M, Hsiao W,
Backer J, Jeffrey A. 1984. Multistage Carc1nogenes1s Involves
Multiple Genes and Multiple Mechanisms, Cancer cells 1. The
Transformed Phenotype, Cold Spring Harbor Laboratory. New York, pp.
229-237.
5. Harris CC. 1985. Future Directions in the Use of DNA Adducts as
Internal Dosimeters for Monitoring Human Exposure to Environmental
Mutagens and Carcinogens. Environ Health Perspec, 62, 185-191.
6. Hennings H, Shores R, Wenk ML, Spangler EF, Tarone R, Yuspa SH 1983.
Malignant Conversion of Mouse Skin Tumors 1s Increased by Tumor
Initiators and Unaffected by Tumor Promoters. Mature (London), 304,
67-69.
7. Scherer E. 1984. Neoplastlc Progression in Experimental
Hepatocarcinogenesis. Biochim Biophys Acta, 738, 219-236.
8. Beland FA, Kadlubar FF. 1985. Formation and Persistence of Arylamlne
DNA Adducts In Vivo. Environ Health Perspect, 62, 19-30.
9. Marshall CJ, Vousden KM, Phillips DH. 1984. Activation of c-Ha-ras-1
Proto Oncogene by In Vitro Modification with the Chemical Carcinogen,
Benzo(a)pyrene 01 oT^epoxTHe, Nature (London), 310, 586-589.
10. Hemminki K, Forstl R, Mustonen R, Savela K. 1986. DNA Adducts in
Experimental Cancer Research. J. Cancer Res Clin Oncol, 112,181-188.
11. Ehrenberg L, Moustacchi E, Osterman-Golkar, Ekman G. 1983. Dosimetry
of Genotoxic Agents and Dose Response Relationships of Their Effects.
Mutation Res 123, 121-182.
12. Neuman HG. 1984a. Dosimetry and Dose-response Relationships, 1n:
Berlin A, Draper M, Hemminki K, Ysainio H (Eds.), Monitoring Human
Exposure to Carcinogenic and Mutagenic Agent, IARC Sci, Pub! No 59,
Lyon, pp. 115-126.
13. Neuman HG. 1984b. Analysis of Hemoglobin as a Dose Monitor for
Alkylating and Arylatlng Agents, Arch Toxicol, 56, 1-6.
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14. Stowers SJ, Anderson MW. 1985. Fonnatlon and Persistence of
Benzol a)pyrene Metabolite-DMA Adducts. Environ Health Perspect, 62,
31-39.
15. Reed E, Yuspa SH, ZwelUng LA, Ozols RF, Po1r1er MP. 1986.
Quantitatlon of C1s-d1amm1ned1chloroplat1nutn II (els platln)
-DNA-1ntrastrand Adducts 1n Testlcular and OvarlanTancer Patients
Receiving Cisplatln Chemotherapy, J C11n Invest, 77, 545-550.
16. Tannenbaum SR, Skipper PL. 1984. Biological Aspects to the Evaluation
of Risk: Doslmetry of Carcinogens 1n Man. Fund Appl Toxlcol, 4,
S367-S370.
17. Santella RM. Application of New Techniques for Detection of
Carcinogen Adducts to Human Population Monitoring. Mutation Res
(1n press).
18. Po1r1er MC, Beland FA. 1987. Determination of Carcinogen-Induced
Macromolecular Adducts 1n Animals and Humans, Prog Exp Tumor Res, 31t
1-10.
19. Perera F, Santella R, Flschman HK» MunsM AR, Poirer M, Brenntr D,
Mehta H, Van Ryzin J. 1987a. DMA Adducts, Protein Adducts and Sister
Chromatid Exchange 1n Cigarettes Smokers and Nonsmokers. J Natl
Cancer Inst, 79:449-456.
20. Perera F, Henralnkl K, Young TL, Brenner D, Kelly G, Santell RM.
1987b. Detection of Polycycllc Aromatic Hydrocarbon-DNA Adducts 1n
White Blood Cells of Foundry Workers. (Accepted).
21. Shamsuddln AKM, Slnopoll K, Henm1nk1t Boesch RR, Harris CC. 1985.
Detection of Benzol a)pyrene-DNA Adducts 1n Human White Blood Cells.
Cancer Res, 45, 66-68.
22. Haugen A, Becher G, Benestad C, Vahakangas K, TMvers GE, Mewman MJ,
Harris. CC. 1986. Determination of Polycycllc Aromatic Hydrocarbons 1n
the Urine, BenzoCa]pyrene Diol Epoxlde-DMA Adducts 1n Lymphocyte DMA,
and Antibodies to the Adducts in Sera from Coke Oven Workers Exposed
to Measured Amounts of Polycycllc Aromatic Hydrocarbons 1n the Work
Atmosphere. Cancer Res 46, 4178-4183.
23. Bryant MS, Skipper PL, Tannebaum SR, Maclure M. 1987. Hemoglobin
Adducts of 4-aminobiphenyl in Smokers and Nonsmokers.
Cancer Res 47, 602-608.
24. Dunn BP, Stlch HF. 1986. 32p Postlabellng Analysis of Aromatic DNA
Adducts in Human Oral Mucosal Cells. Cardnogenesis 7, 111-5-1120.
25. Phillips DH, Hewer A, Grover PI. 1986. Aromatic DNA Adducts 1n
Human Bone Marrow and Peripheral Blood Leukocytes, Carcinogenesis 7,
2071-2075.
26. Bridges BA. 1980. An Approach to the Assessment of the Risk to Man
from DNA Damaging Agents. Arch Toxlcol, Suppl 3:271-281.
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27. Wright AS. 1983. Molecular Dosimetry Techniques 1n Human Risk
Assessment: An Industrial Perspective, in: Hayes AW, Schnell RC,
Miya TS (Eds.). Developments in the Science and Practice of
Toxicology. Elsevier, Amsterdam, pp. 311-318.
28. Tornqvist M, Osteraan-Golkar S, Kautiainen A, Jensen A, Fanner
PB, Ehrenberg L. 1986. Tissue Doses of Ethylene Oxide in Cigarette
Smokers Determined from Adduct Levels in Hemoglobin.
Carcinogenesis, 7, 1519-1521.
29. Everson RB, Randerath RM, Santella RM, Cefalo RC, Avitts TA,
Randerath R 1986. Detection of Smoking Related Covalent DNA Adducts
in Human Placenta, Science 231, 54-57.
30. Bridges BA, Butterworth BE, Weinstein IB. Banbury Report 1982.
Indicators of Genotoxic Exposure; Report No. 13. Cold Spring Harbor
Lab, Cold Spring Harbor, NY.
31. NAS Briefing Panel. 1983. Report on Human Effects of Hazardous
Chemical Exposures. National Acad Sci, Washington, DC.
32. Sobsel FH. 1982. The Parallelogram: An Indirect Approach for the
Assessment of Genetic Risks from Chemical Mutagens. In: Progress in
Mutation Research (Bora KC, Douglas GR, Nestmann ER. eds.). Elsevier,
Amsterdam, pp. 323-327.
33. Santella Rm, Weston A, Perera F, et al. 1987. Inter!aboratory
Comparison on Antibodies and Immunoassays for Benzo[a]pyrene Diol
Epoxide-1 Modified DNA. (Submitted).
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NEUROTOXICOLOGY
Lawrence Relter
INTRODUCTION
Epldemiological studies 1n Europe indicate that long-term exposure to
solvents can produce neurobehavioral disorders which, depending on the
length and severity of exposure, can range from loss of concentration and
memory impairment to mood and personality changes to severe and apparently
irreversable dementia. Indeed, cognitive Impairment appears to be an early
sign of solvent neurotoxldty. These studies have led the International
neurotoxicology community to call for Improved methods for identifying and
characterizing solvent neurotoxldty both in animal models and 1n human
clinical populations.
NEUROBIOLOGY OF LEARNING AND MEMORY
An area of long-term research which promises to produce powerful
applications to this problem 1s the neuroblology of learning and memory,
The goal of this field is to understand how normal memory function is
carried out by the nervous system as well as how various neuropathological
conditions, such as Alzheimer's disease and Korsakoff's syndrome, produce
cognitive dysfunction. Interest in this area of neuroscience research is
very intense. By some estimates, fully a quarter of all research in the
basic neurosclences is concerned with the neuroblology of learning and
memory. It is not surprising then that progress 1n this area is occurring
at a very rapid rate. This paper will briefly highlight some specific
recent developments in this field which should have a major future Impact
on neurotoxicologlcal assessment.
Analysis of the neuroblology of learning has been organized around
three general areas: (1) key brain regions i.e., which brain regions are
essential for different forms of memory; (2) memory "circuits" in the
brain, i.e., the delineation of neural pathways through which sensory
information results in the production of learned behavioral responses; and
(3) synaptic mechanisms, i.e., the nature of the synaptic changes that
occur during learning, and the biochemical and cellular processes which
underline them. The first of these areas has had, as one of its major
concerns, the problem of how to extrapolate from animal models of cognitive
dysfunction to human dementia. The latter two areas have been concerned
primarily with analyzing the animal model systems at more molecular levels.
In the past 5-7 years, dramatic discoveries have been made 1n all three of
these areas.
NEUROTOXICOLOGICAL ASSESSMENT
Attempts in the area of extrapolation have taken two forms. One has
been to develop behavioral tests in animals which are more analogous to
those which are used to assess cognitive function 1n humans. The other
form, and the one which we will emphasize here, has been to apply
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behavioral tests to humans which are analogous to those which are well
understood, both behavlorally and neuroblologically, 1n animals. For
example, 1t has recently been shown that delayed-non-matching-to-sample, a
task which is a sensitive indicator of memory Impairment associated with
limbic system and frontal cortical damage in rats and primates, is also a
sensitive indicator of dementia associated with similar neuropathology 1n
human clinical populations. Another example 1s the successful use of human
eyebllnk conditioning to detect learning deficits, associated with aging
and Alzheimer's disease, which were predicted by neuroblologlcal studies of
eyeblink conditioning 1n rabbits. These recent developments 1n basic
behavioral neuroscience establish a direct neurobehavioral link between the
experimental analysis of cognitive dysfunction in animals and Its
assessment to humans. Neurotoxicological research, aimed at validating the
application of these new animal models to the problem of risk assessment
will substantially advance progress 'on the question of how animal studies
can be used to characterize risk to human populations, following exposure
to solvents and other environmental pollutants.
The second important development which could greatly Increase the
sophistication of neurotoxicological assessment is the Identification of
neural circuits subserving learning. The best example of this is the
neurobiologlcal study of rabbit eyeblink conditioning. This Pavlovlan
conditioning preparation has many advantages for neurotoxicological
assessments, including: (a) the wealth of knowledge of its behavioral
properties, which makes 1t possible to study anything from simple
associative reflexes to complicated cognitive-perceptual processes 1n a
single experimental preparation; (b) the ability to directly compare
quantitative measures of both learned and unlearned behavior, on-line and
in real time; (c) the ability to directly compare the same type of
conditioning in animals and humans; (d) the ease of arranging concurrent
electrophysiological recording from discrete brain loci (or, 1n the human,
brain EEG activity recorded from scalp electrodes). However, the most
important advantage offered by this recent research development is the
wealth of knowledge that we now have about Its essential neural circuitry
in the brain stem and cerebellum. We also know a good deal about the
effect of pharmacological agents on this type of conditioning and this
greatly improves our ability to Integrate the various aspects of
neurotoxicological assessment. If an unknown compound produces a
behavioral effect, we have a good idea of where to look for Its
neurochemlcal and neuroanatomical effects, and ultimately Its mechanism(s)
of action. Conversly, if a compound produces an effect on a neurochemical
or neuroanatomical system, we know what functional consequences to look for
in terms of the types of behavioral or cognitive processes which might be
impaired. Some investigators have already begun to use Pavlovlan
techniques of this kind as animal models in the neurotoxicological
assessment process. Just this year (1987), the rabbit eyeblink preparation
has been applied to the study of dementia associated with aluminum
toxicity.
One final development which is worth mentioning is the use of the j_n
vitro brain slice technique to study neural plasticity.
Electrophysiological studies of hippocampal slices have uncovered a
phenomen, termed long term potentiation (LTP), which has become very
influential as an experimental model for studying the synaptlc mechanisms
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of learning. In LTP there 1s, 1n effect, an Increase 1n synaptlc efficacy
that occurs «1th repeated use. Investigations of the cellular and
biochemical mechanisms of LTP have revealed a special role of a particular
receptor type (the N-methyl-D-aspartate or NMDA receptor). Pharmacological
antagonists of the NMDA receptor may prevent the Induction of LTP, and may
disrupt cognitive function 1n rats. What 1s true of drugs may also be true
of other compounds with neurotoxlc potential (tg.f environmental
chemicals). It is likely that with continued research in this area,
hlppocampal slice preparations may be used as a means of screening unknown
compounds for their potential ability to produce cognitive dysfunction, and
of characterizing the neuroblologlcal mechanisms of any neurotoxlc effects
which are found.
SUMMARY
In summary, these three general areas of long-term research 1n
behavioral neuroscience create a framework for the analysis of
neurobehavloral function which 1s Integrated at both a conceptual and,
perhaps more Importantly, a practice level. With this framework, it 1s
possible to use information from diverse scientific subdiscipllnes,
including cell biology, neurochemistry, neuroanatomy, neurophyslology, and
both animal and human psychology, 1n a very direct and real way to either
(a) identify the risk that compounds with neurotoxlc potential may pose to
normal cognitive function or (b) characterize the risk of classes of
compounds, such as the solvents, which are known to produce memory loss,
dementia and other neurobehavloral disorders.
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USE OF MONOCLONAL ANTIBODIES IN NEUROTOXICOLOGY
Monoclonal antibodies provide another example of long-term research
which has promise for application to a wide variety of environmental
problem (See Chapter 2 for some others). This section will describe some
new applications in neurotoxiclty.
Background
Exposure to a foreign substance often elicits an immune response
characterized by production of antibodies. Antibodies are serum proteins
that react with antigens (antigens are foreign substances capable of
inducing antibody formation). Such antigenic substances can include
viruses, bacteria, proteins, or even complex molecules like environmental
chemicals. Antigen-antibody reactions are highly specific, indeed, among
the most specific known to biology. It is this specificity of the
antigen/antibody complex that has been exploited by the biomedical
scientist with applications ranging from curing Polio to understanding the
molecular basis of enzyme catalysis.
Antibodies are produced in the body by B lymphocytes (B-cells), each of
which produces its own unique antibody. In theory, as many as 10 million
antibodies can be produced by a mouse 1n response to a single antigen.
Each antibody reacts with a unique antigenie site (termed an epltope) and
each antigen contains several epitopes. Because one B-cell can form
antibodies against only one epltope but there are many B-cells producing
antibodies against each epitope, this 1s referred to as a polyclonal (many
cells) antibody.
The lymphocyte fusion technique of Kohler and Milsteln, for which they
received the 1984 Nobel Prize, was designed to overcome the limitations
associated with the use of polyclonal antibodies (e.g., contamination,
heterogeneity, limited supply). The antibodies produced by Kohler and
Mil stein were referred to as monoclonal because they were produced by a
single (mono) B-cell line (clone). Monoclonal antibodies have several
advantages including: 1) inherent specificiy (each clone produces only one
specific antibody); 2) unlimited supply (clones produce large amounts of
antibody and can be kept Indefinitely); and 3) purified antigens are not
required for the production of pure antibodies (monoclonals by definition
recognize only a single antigenic determinant).
Monoclonals have been used to define, localize, purify, quantify, and
modify antigens. The main distinction between the use of monoclonals, as
opposed to polyclonal antibodies, is that monoclonals confer far greater
precision and accuracy and are available as essentially immortal, off the
shelf reagents. Thus, it is now possible to define antigens with a greater
degree of certainty than ever before. This Inherent trait of monoclonals
has made it far easier to identify rare antigens both in vivo and in vitro
(e.g., nervous tissue cell types and tissue typing 1n cell cultureTOne
example of the application of monoclonals that is relevant to the EPA is
the use of specific monoclonals to identify dioxin congeners in
contaminated soils. True purfication of antigens from heterogeneous
sources (e.g., serum, tissue) also is now possible with monoclonals. Thus,
rare factors or hormones, such as interferon, can now be easily obtained in
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bulk pure form. Likewise, quantification of antigens 1n complex mixtures
1s also easier to achieve with wonoclonals than with polyclonal antibodies,
an example being human chorlonic gonadatrophln for pregnancy tests. By
targeting specific antigens with nsonoclonals, modification of toxldty or
disease states also may be realized. Examples are treatment of dlgoxln
overdose (with antibody to dlgoxln), and cancer therapy with anticancer
agents linked to monoclonals targeted to tumor cell antigens.
Applications of Monoclonals to Neurosclence/neurotoxlcology
The years of research on monoclonal antibodies that followed Kohler and
Milstein's original report 1n 1975 are now beginning to revolutionize
neurobiology by providing the tools with which to understand the complex
cellular and subcellular organization of the nervous system. Thus, the
major impact of monoclonal antibody technology on neuroscience has been the
unambiguous identification of different cell classes 1n the nervous system.
Indeed, monoclonals have now been produced which Identify previously
unknown subsets of neurons and g!1a (the major cell types of nervous
tissue) which otherwise would not appear to be different using classical
techniques of light or electron microscopy. Monoclonals have also proved
crucial for the identification and characterization of unique
macromolecules, and have been even shown to reveal important differences
within the same molecule. For example, monoclonal antibodies have now been
produced that reveal phosphate-containing versus nonphosphate-contalnlng
neurofHaments, the major structural (filament) component of all neurons.
The significance of this subtle difference, I.e., the absence or presence
of a single phosphate, is that this substitution may be related to a
variety of neurological disease states, including Alzheimer's disease, and
also may represent a general response to injury of the nervous system.
In neurotoxicology, it is known that toxicant-induced injury to the
developing or mature nervous system often 1s manifested by alterations in
the cytoarchltecture of specific neuroanatomical regions. Furthermore,
within an affected region, the response to injury may encompass several
cell types. Because antigens that distinguish the diverse cell types
comprising the mammalian nervous system have been revealed by monoclonal
antibodies, these same antibodies can be used to detect, localize and
characterize ce:-jlar responses to neurotoxlc exposures. This can be
accomplished by a technique known as 1mmunohistochem1stry, where antibodies
are used as specific probes for microscopically localizing specific
antigens within tissue obtained from toxicant-exposed animals.
Quantitative data are obtained with the same antibodies by using
monoclonal-based radioimmunoassays. Thus, through the use of monoclonal
antibodies an integrated morphological/biochemical evaluation of
neurotoxicity may eventually be achieved. The possibility also exists that
the sensitivity and specificity of monoclonal antibodies can be applied to
the detection and measurement of antigens released into the cerebrospinal
fluid and blood as a consequence of neurotoxic exposures. Theoretically,
it would then become possible to develop inexpensive monoclonal-antibody
based test kits for detecting neurotoxicity in the exposed human
population.
In summary, it is clear that current advances 1n the neurosdences will
continue to reveal the extensive cellular and subcellular heterogeneity of
the nervous system based on the use of monoclonal antibodies. The EPA, by
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actively particlpating 1n long-range research, will benefit by having the
tools with which to assess ind predict environmentally-Induced
neurotoxldty.
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MAGNETIC RESONANCE IMAGING
Morrow Thompson
INTRODUCTION
A major problem in environmental health sciences is the non-invasive
detection of small adverse effects or adverse effects at early stages.
Research applications of magnetic resonance imaging hold promise for just
such advances. In the few years since Lauterbur*s (1) paper was published,
magnetic resonance (MR) imaging has evolved rapidly into an accepted
clinical technique and, also, a research tool of enormous potential.
Systems with high field, superconducting magnets (1.5 to 4.7 Tesla) are
available commercially and are designed for human beings and laboratory
animals (separate systems). Sophisticated techniques that modulate the
effects of proton density, relaxation times, and motion permit the
acquisition of 3-d1mens1onal Images that optimize differences between
normal tissue types, define pathologic structures of areas, and allow the
measurement of blood flow or perfuslon (2-5). For reasons of abundance and
signal intensity, the hydrogen nucleus (proton) is probed for the
production of?Dract1cal1y all MR Images. The abilities.to image alternate
nuclei (e.g. Na) and chemically shifted nuclei (e.g. H in water versys
fat) have been demonstrated and show the versatility and undeveloped
potential of the technology.
Present day proton MR images of human beings and laboratory animals
contain superb anatomic detail that, 1n some applications (biologic
specimens and small animals), approaches microscopic levels. In recent
publications (6,7), images of frog eggs and plant stems have been shown
with volume elements (voxels) of 0.2 and 12.0 L, respectively. Perhaps
more impressive are experiments being conducted at Duke University in which
chemically Induced hepatic lesions as small as 100 L in volume have been
imaged 1n rats. The ability to detect such small lesions 1n live animals
requires long imaging sessions (as long as 6 hours), strong magnetic fields
and gradients, sophisticated pulse sequences, and little or no relative
motion. Because respiratory motion is transferred through the dlaphram to
the liver, the last issue (no motion) is accomplished by intubating the
animal, using a gaseous anesthetic, and synchronizing signal acquisition to
respiratory motion (8,9).
Some of the advantages of MR imaging are common to those of other
techniques, and other advantages are unique. Similar to computerized
tomography (CT) scans, MR imaging is non-invasive and may be performed
multiple times on the same animal or patient. In toxicology experiments,
for example, the incorporation of MR imaging of a group of animals could
provide important information concerning target organs, time to lesion
(e.g., tumor) development, and response to continued or modified treatment
(e.g., progression or regression of lesions). MR imaging uses fewer
animals per exepriment compared with conventional means for gathering
similar information.
While imaging techniques based on ionizing radiation are well
established, rapidly produced (a distinct advantage compared to MR imaging
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at Us present state of developnent), and excellent for demonstrating some
anatomic structures or abnormalities (e.g., bone lesions containing calcium
deposits, recent hemorrhage), MR imaging has some distinct and Important
advantages. With current and anticipated magnetic fields, gradients, and
RF signals, and with the proper precautions MR imaging 1s considered safe
for patients and technicians (10). Additionally, the MR signal, unlike the
penetrating beams of Ionizing radiation, contains information 1n addition
to that of tissue (In this case, proton) density. The signal 1s also
determined by the rates at which protons relax in relationship.to the
molecular lattice (Tl, spin-lattice, longitudinal relaxation) and to each
other (T2, spin-spin, transverse relaxation). Because these time constants
are influenced by the chemical composition of the tissue (probably by the
amount and motional freedom of water molecules), the resulting Image can
permit distinction of tissues that are similar in proton density but differ
in relaxation times.
Although not a consistent finding, malignant tumors frequently have Tl
and T2 relaxation times greater than those of benign tumors or normal
tissue. Recent disappointments concerning the apparent Inability of MR
imaging (relaxation times) to distinguish between pathologic entitles have
been expressed (11). This may be partially related to the acquisition of
the signal from tissue slices that, because of slice thickness, Include
degenerative and normal areas within and adjacent to the lesion of
interest. In animal experiments at Duke University, this possibility 1s
being explored by excising very thin (only 1.25 mm thick) tissue slices 1n
rats. While signals from such thin slices are weak and imaging sessions
are relatively long, the thin sections with high resolution greatly improve
the selectivity, and, hopefully, the discriminating ability of the method.
CURRENT AND FUTURE APPLICATIONS
In clinical medicine, MR imaging compliments and frequently exceeds the
performance of other Imaging methods. MR Imaging excells In demonstrating
neoplastic, demyelinatlng, and degenerative processes of the central
nervous system. Because of the suscetlbiHty of the thyroid and
parathyroid glands to ionizing radiation, MR imaging is a preferred method
for examination of these tissues. Respiratory and cardiac gating have been
used to produce excellent diagnostic images of the heart, thoracic blood
vessels, and lungs. MR images of liver, kidney, reproductive organs, and
pelvis routinely demonstrate a variety of neoplastic and non-neoplast1c
processes. Current and future developments will Incorporate the use of
faster scanning sequences, 3-d1mensional Imaging, measurement of perfusion
and flow, contrast agents imaging combined with in vitro spectroscopy of
different nuclei (e.g., JiP, r
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imaging. High field systems (300 MHz, 7 Tesla) are being developed and
tested that have a theoretical resolution of 10 M. Areas of active
research include the improvement of RF coil designs, and the use of
stronger field gradients, surface and implanted coils, and contrast agents.
Within a few years, increases in resolution should permit, for example, the
visualization of renal glomeruli, preneoplastic hepatocellular foci, and
nuclei in the brain. With such developments, Lauterbur's closing statement
in his 1973 paper would seem remarkably prophetic, "Zeugmatographic
techniques should find many useful applications in studies of the internal
structures, states, and compositions of microscopic objects."
-------
REFERENCES
1. Lauterbur PC. Image Formation by Induced Local Interactions:
Examples Employing Nuclear Magnetic Resonance. Nature 1973;
242:190-1.
2. Morgan CJ, Hendee WR. The Evolution of Nuclear Magnetic Resonance.
In: Introduction to Magnetic Resonance Imaging. Denver: Multi-Media
Publishing, Inc. 1984:1-12.
3. Andrew ER. A Historical Review of NMR and Its Clinical Applications.
Br Med Rev 1984;40: 115-9.
4. Damadian R. Tumor Detection by Nuclear Magnetic Resonance. Science
5. Lauterbur PC. Cancer Detection by Nuclear Magnetic Resonance
Zeugmatographic Imaging. Cancer 1986;57:1899-1904.
6. Aguayo JB, Blackband SJ , Schoeniger J, Mattingly MA, Hintermann M.
Nuclear Magnetic Resonance Imaging of a Single Cell. Nature
1986;322:190-1.
7. Johnson GA, Brown J. , .Kramer PJ. Magnetic Resonance Microscopy of
Changes in Water Content in Stems of Transpiring Plants. Proc Natl
Acad Sci USA 1987;84:2752-5.
8. Hedlund L, Dietz J, Nassar R, Herfkens R, et al . A Ventilator for
Magnetic Resonance Imaging. Invest Radiol 1986 ;21: 18-23.
9. Hedlund L, Johnson GA, Mills GI. Magnetic Resonance Microscopy of the
Rat Thorax and Abdomen. Invest Radiol 1986;21:843-6.
10. Saunders RD, Smith H. Safety Aspects of NMR Clinical Imaging. Br Med
Bull 1984;40:148-54.
11. Johnston DL, Liu P, Wismer GL, Rosen BR, et al . Magnetic Resonance
Imaging: Present and Future Applications. Can Med Assoc J
1985;132:765-77.
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IMMUNOTOXICOtOGY
Michael Luster
In a broad sense 1mmunotox1cology can be defined as the study of
adverse (inadvertent) effects of environmental chemicals, therapeutics or
blologlcals on the Inmune system. The types of effects that may occur
include Immunomodulation (I.e., suppression or enhancement),
hypersensitlvity (allergy) and, in rare Instances, auto1mmun1ty. A large
body of Information has developed over the past 10 years that exposure to
certain chemicals or therapeutics can produce Immune dysfunction and alter
host resistance 1n experimental animals following acute and subchronlc
exposure. Examples of these are listed 1n the attached table. The most
extensively studied class of environmental chemicals 1s the polyhalogenated
aromatic hydrocarbons (PHAs), including polychlorlnated blphenyls,
polybrominated blphenyls, chlorinated dlbenzofurans and the prototype of
this class, chlorinated d1benzo-p-diox1ns.
Despite the species variability associated with the toxic manifestation
of these compounds, studies 1n laboratory animals exposed during neonatal
or adult Hfe with PAHs and, in particular, dibenzo-p-d1oxins have
indicated that the immune system is one of the most sensitive targets for
toxicity. These effects are characterized by thymlc atrophy and severe and
persistent suppression of cell-mediated (T cell) Immunity and share many
features of neonatal thymectomy. Laboratory studies have further Indicated
that the target cell for 1mmunosuppress1on by PHAs 1s the thymlc epithelium
which 1s necessary for T cell maturation. Although only a limited number
of reports Indicate immune dysfunction following human exposure to PHAs,
the effects have been found to be remarkedly similar to these which occur
in animals. For example, suppression of a delayed hypersensitlvity
response and Increased susceptibility to respiratory Infections have been
found 1n patients who accidentally Ingested polychlorlnated
b1phenyl/dibenzofuran-contam1ned rice oil. Another example of this Immune
dysregulation by PHAs has been reported 1n Michigan farm residents who
inadvertently ingested polybromlnated biphenyls. These individuals also
demonstrated persistent suppression of cell-mediated Immunity with
increased numbers of null cells, possible reflecting the presence of
immature cells. Although long-term deleterious consequences of
polybrominated biphenyls remain to be determined in humans, early data
indicate a correlation between Immune alterations and Increased tumor
incidence.
Thus, 1t appears that early laboratory studies 1n rodents have provided
a very accurate account of the Immunological dysfunction that is observed
in humans following Inadvertent exposure to these compounds.
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£XAKPL£S OF 1KMONQLOGICAL ABNOBflALfTf ES ASSOCIATED
WITH
CHEMICAL EXPOSURE ZN RODENTS AMD HUMANS
Chemical
Class
Polyhalogenated
Aromatic
Hydrocarbons
Heavy Metals
Aromatic Hydro-
carbons x
(Solvents)
Polyeyelic
Aromatic
Hydrocarbons
Pesticides
Orgmnotins
Aromatic Andnes
Oxidant Gases
(Air Pollutants)
Others
Laboratory
iBcsune
Example Abnormality
TCDD 4-
PCS +
PBB «•
HCB +
Lead •*•
Cadniun +
Methyl Mercury +
Benzene +
Toluene +
DKBA 4-
SaP *
MCA +
Triaethyl Phospho- +
rothioate
Carbofuran *
Chlordane +
DOTC *
0STC *
Benzidine •»•
NO, *
soz
Asbestos *
-OWN- ;: •»•
Human Innune.
Abnormality
^
4.
4-
N.S.
_
-
-
4-
N.S
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
N.S.
4-
N.S.
4-
N.S.
4-
N.S.
N.S. - Not studied; ± - Positive and negative findings hava oe«n reported.
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HUMAN CHORIONIC GQNADOTRGPIN (HCG)
Donald Mattlson and Alan HI!cox
BACKGROUND
Public health scientists have long been concerned about the many
possible reproductive hazards of environmental pollution. One unanswered
and troubling question has been whether there are effects of toxins on the
earliest stages of pregnancy. This can Include environmentally-Induced
very early abortions/fetal wastage. If there were some way to detect the
earliest stages of pregnancy, then perhaps such effects of occupational ,
environmental, or drug exposures could be more easily defined and
addressed. It 1s known that about 15% of clinically-recognized pregnancies
end 1n recognized loss (spontaneous abortion). The risk of such loss has
been found to be higher 1n some populations with occupational,
environmental, etc., exposures. However, clinical losses don't tell the
whole story; clinically-recognized losses represent only a portion of all
pregnancy losses. There are at least twice as many earlier losses as
recognized spontaneous abortions. Thus, a technique which could detect
pregnancy very early and define its ending precisely could help pinpoint
whether chemical or other environmental exposures might have been involved
1n such ending, The application of new researches with human chorionic
gonadotrophin offers such possibilities.
METHOD
Determination of very early pregnancy loss requires sensitive and
specific methods for identifying pregnancy. The recent development of
antibodies to one component of the beta subunit of HCG has vastly Improved
the capacity of HCG assays to detect early pregnancy. HCG 1s produced by
the conceptus starting at about the seventh day after fertilization. HCG
1s quickly excreted in the mother's urine and 1s detectable by immunometric
assays. For tMs reason, HCG assays are the mainstay of studies of early
pregnancy. Thi'j 1mmunorad1ometric assay 1s reactive to the unique
carboxyterminal peptide of the HCG molecule. The assay 1s up to one
hundred times more sensitive than any previously available assay. This
added sensitivity has proved to be Important because up to three-quarters
of early pregnancy losses never reach a level of HCG secretion that could
have been detected by previous assays.
IMPLICATIONS
Early pregnancy loss may be one of the earliest signs of human exposure
to mutagens or other toxins that damage human reproduction. It should be
possible to streamline this type of study, collecting urines only on days
when early loss 1s most likely to be detected. This approach could be
extended to high-risk groups of women 1n occupational or other settings
where toxic effects on reproduction are suspected. These assays are now
able to measure HCG in urine down to the background levels that occur in
healthy non-pregnant persons. These assays are just now beginning to be
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applied in epidesniologic studies for the detection of very early pregnancy
loss. This 1s an exciting new applied research area 1n environmental
medicine which 1s the direct result of very basic research In reproductive
biology. This may be a model for future research and suggests that basic
and clinical studies are essential 1f we are to make progress 1n
understanding human reproductive vulnerability to environmental chemical
exposure.
Further basic and applied research is needed in this area — as a high
priority -- because of existing data which suggest that there are indeed
exposures which can Increase the rate of clinically-recognized, spontaneous
abortion. These may include various segments of the chemical industry and
the microelectronics Industry.
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Chapter 5
ESTIMATION OF POPULATION RISKS
David Noel/Michael Hogan
ANIMAL MODELS AND RISK ESTIMATION
Since relevant epidemiologic and clinical information are often lacking
on the potential health hazards associated with exposure to a specified
agent or chemical, laboratory animal data usually constitute the primary
basis for both qualitative and quantitative human risk estimation. The
majority of animal-based, human risk estimation is qualitative in nature.
That is, laboratory or experimental identification of a given exposure
source as a potential human health hazard is often sufficient, in and of
itself, to control or even prevent future exposure of the general public to
the agent or chemical in question, and no determination of the magnitude of
the risk involved in the anticipated exposure may be required (e.g.,
regulation of potentially carcinogenic food additives under the Delaney
Amendment). Nevertheless, it is the role of animal data in the
quantification of possible human health risks that is of greater scientific
interest and debate.
Animal-based, quantitative risk estimation almost always involves two
separate issues or problems that must be addressed: low-dose
extrapolation, necessitated by the high dose levels typically employed in
laboratory animal studies and, of course, species extrapolation, since the
ultimate concern is with the risk posed to humans. Perhaps the single most
important issue involved in low-dose extrapolation is the choice of the
specific mathematical model or extrapolation procedure to be used in
determining the low-dose risk or the acceptable exposure level for the
agent under consideration. In carcinogenesis, mathematical modeling may
have progressed as far as is possible or defensible without further
insights into the mechanisms underlying the carcinogenic process.
Certainly the need for greater emphasis on the meaningful incorporation of
molecular and biochemical data into risk models is well recognized, and it
offers an important research opportunity to those interested in the
quantification of potential human risk based on animal data. For
noncarcinogenic outcomes or endpoints there is definitely a need to
reevaluate the "safety factor" approach to risk determination, which has
been the regulatory standard since the mid-50's, and, in some instances, to
promote the development of quantitative models similar to those used in
carcinogenesis.
Regardless of the toxicologic response of interest, however, it is
clear that, increasingly, attention will be focused on making the selected
model or extrapolation procedure more closely reflect the underlying
biological mechanisms. For example, in carcinogenesis the question of
"primary" versus "secondary" or "indirect" modes of action and their
potential impact on the risk assessment process is sometimes raised with
those who assume the latter mechanism often arguing against traditional
low-dose extrapolation models (1). On the other hand, those, who out of
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convenience or convention, have relied on the safety factor approach for
determining permissible exposure levels for noncarcinogenlc toxicants, may
need to reconsider the biological Issues that underlie Its use, giving
particular attention to the question of thresholds. For example, 1f one
argues that a threshold mechanism 1s present, does the threshold represent
a true (biological),"no effect** level or merely Imply a dose or exposure
level where the observable effects are minimal? Does it apply to the
population as a whole or vary from individual to individual? (In the
latter Instance the population dose-response may be Indistinguishable from
one for which no threshold exists. That is, if threshold levels vary among
individuals, then the "population" threshold level would correspond to the
threshold for the most sensitive individual in that population, which, for
all practical purposes, might be indistinguishable from a zero exposure
level.) Another issue of concern is whether there 1s a biological (as
opposed to traditional) basis for the selection of any given safety factor
to be used with an observed/estimated threshold value in generating
estimates of acceptable human exposure levels (2).
The question of species extrapolation may well generate as much
scientific debate as the selection of the most appropriate low-dose
extrapolation procedure. Certainly, the utility of the laboratory animal
model for identifying potential human health risks is broadly recognized
within the scientific community [e.g., see the IARC Preamble (3) regarding
the interpretation of experimental results with regard to human
carcinogenic risk when ep1dem1ologic or clinical data are not available].
However, there 1s no universally accepted means of quantitatively scaling
the results observed 1n laboratory animals to hunans. What 1s usually done
is to assume that animals and humans have equivalent risks when risk 1s
expressed in terms of the appropriate dosage scale. Yet, human risk
estimates based, e.g., on mouse data can vary by as much as 40-fold (4)
depending on whether they are expressed in terms of an average lifetime
daily mg/kg dose or a total acculumated mg dose, standardized (divided) by
body weight. Furthermore, even though necessity may force one to rely on
nothing more than a comion dosage scale as the basis for extrapolating risk
estimates across species, such an approach is only an approximate
adjustment for the variety of factors that can contribute to interspecles
differences in response (e.g., differences in Hfespan, body size, kinetic
profile, genetic homogeneity, general environment, etc.). Improvements in
the quantitative extrapolation of toxicologic responses across species will
require greater emphasis on the use of molecular and biochemical data. For
example, the use of pharmacokinetics or molecular dosimetry, when
scientifically feasible, to estimate the "biologically effective dose"
could significantly reduce the uncertainty associated with Interspecles
extrapolation of observed toxicologic responses.
HUMAN STUDIES
Mathematical dose-response models for quantitative risk estimation have
been and are increasingly being applied to epidemiologlc data as well as to
laboratory animal results, particularly in the area of carcinogenesis.
Some of the better known examples include Peto's fitting of the multistage
model to Doll's smoking data (6), Day and Brown's use of the same model to
assess whether a number of human cancer risk factors such as smoking,
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asbestos and radiation affected early, late or both early and late stages
of the carcinogenic process (7), BEIR Ill's (6) use of absolute and
relative risk models to characterize the time-related distribution of
site-specific tumors among Japanese A-bomb survivors, and their use of
linear, linear-quadratic and quadratic models to predict low-dose cancer
risk associated with ionizing radiation. While the use of epldemiologic
data obviously eliminates the need for species extrapolation, such data may
not be sufficiently sensitive to allow one to chose among competing
dose-response models or, in some instances, even to determine if any health
risk appears to be associated with low or moderate levels of exposure.
A number of procedures may be employed to increase the sensitivity of
the available epidemiologic data. For Instance, initial attempts at human
risk identification and estimation could be focused on sensitive subgroups
within the general population under study, such as the very old or young,
individuals with insufficient immune response, individuals suffering from
concurrent disease or inherited deficiencies, and individuals also exposed
to other known risk factors for the toxicologic endpoint or health effect
of interest.
Recently, a new speciality has emerged in the field of epidemiology,
which is commonly known as molecular or biochemical epidemiology. One of
the primary purposes of molecular epidemiology is to adapt laboratory
procedures for the identification and characterization of biochemical
markers to epidemiologic field studies, so as to clarify the nature of
underlying dose-response relationships, i.e., relationships between
exposure and disease or toxicologic effect (8). Specifically, biochemical
markers may provide quantitative evidence of generalized exposure (e.g.,
blood lead levels), organ specific exposure (e.g., DNA adduct formation),
biologic change, and early or frank disease to replace the more subjective
and qualitative measures that have often been used in epidemiologic
investigations (e.g., determining exposure histories through questionnaire
data and then classifying study subjects as being either "exposed" or
"unexposed".)
While interest in and and even application of biochemical markers 1s
increasing rapidly, validation of their use for epidemiology is currently a
major research endeavor, and it 1s likely to continue to be so in the
future. [Among the issues that should be considered in any validation
exercise are the determination of marker sensitivity, specificity,
predlctivity, range of normal or baseline values, and whether the marker is
reflecting current or cumulative exposures, average or peak exposures, and
cumulative or noncumulative biological effects (8).]
POPULATION RISKS
The last step in the quantitative risk assessment process is the
determination of the overall risk for the population of interest or,
alternatively, the selection of an acceptable exposure level for that
population. Some of the uncertainties involved in using experimental
animal or epidemiologic data in hazard identification and, particularly, 1n
dose-response modeling and low-dose risk estimation have already been
enumerated. If a strong case can be presented for the presence of a
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threshold phenomenon and a safety factor approach fs elected, then 1t 1s
Important to remember that failure to compensate adequately for the
unknown, underlying threshold can result 1n a proportion of the exposed
population having their Individual threshold values falling below the
estimated acceptable exposure level 1n sowe Instances (2).
Another significant factor that must be addressed 1n developing
population risk estimates 1s the determination or estimation of exposure
levels within the population under evaluation. There are a number of
potential problems or uncertainties typically Involved 1n the estimation of
population exposure levels. Exposures may vary considerably among
individuals or even for a single Individual across time, so that the use of
average exposure levels may not be very representative of the exposure
histories of Individual population members. While use of worst-case
exposures may provide an upperbound on the actual levels of exposure
encountered, 1t can also lead to an overestimate of the population's health
risks and certainly engenders a great deal of uncertainty about such
estimates. The uncertainty 1s compounded when average or worst-case
exposure estimates are multiplied by the estimated average risk per unit
dose to obtain an overall estimate of population risk. For example, even
though worst-case exposure estimates may overestimate the actual exposure
experience of much or possibly all of the population of Interest, "average"
risk per unit dose estimates may significantly underestimate the risks of
the most susceptible subsets of that population.
Some argue that the uncertainties involved 1n quantitative risk
estimation and concern for the health of the exposed population have often
led to the overuse of worst-case or ypperboynd assumptions 1n quantitative
risk estimation—assumptions that result 1n what they regard as unduly
conservative estimates of the population risks. However, there are other
investigators (9) who fear that national concern about the assessment of
human health risks has tended to be focused almostly exclusively on cancer
risk, and that as a result, other (perhaps less quantifiable) forms of
human disease or dysfunction may have received Insufficient attention: (See
Appendix). If this is the case, then, 1n any specific situation the
estimated "acceptable", "virtually safe" or "minimal risk" dose for
cardnogenesls may still entail an unreasonable level of risk of other
adverse health outcomes, even when the estimation process has been based on
conservative assumptions.
The OSTP cancer document (10) and other science policy reports have
stressed the need for qualitative and quantitative characterization of the
uncertainties of specific risk estimates (e.g., consideration of the Impact
of model selection, the use of one set of laboratory data over another, the
choice of a particular species as being most representative of humans,
etc.). Also Important are considerations and specification of the
assumptions underlying a particular risk assessment (e.g., the construct of
an estimated lifetime average dally dose rate so that animals continuously
dosed at a constant rate throughout their lifetimes might be used to
estimate the risk in humans who may have received Intermittent exposures at
varying doses for only a portion of their lifespan). The continued
attention to/stress on such descriptions of specific uncertainties and
assumptions involved 1n any given risk assessment and to their potential
impact on the estimation of risks has been most helpful to those charged
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with regulatory responsibilities for more rational and reasonable decisions
about the proper fate of the agent/chemical under consideration.
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REFERENCES
3.
4.
5,
6.
7.
Hoel , D.G. , Haseman, J.K., Hogan, M.O., Huff, J., and McConnell ,
E.E. The Impact of Toxicity on Carcinogenicity Studies:
Implications for Risk Assessment. (Submitted for Publication)
Portier, C. , and Hogan, M. (1987). An Evaluation of the Safety
Factor Approach in Risk Assessment. In: McLachlan, J.A. , Pratt,
R.M. , and Markert, C.L. eds. Banbury Report 26: Developmental
Toxicology: Mechanisms and Risk. Cold Spring Harbor Laboratory,
New York.
International Agency for Research on Cancer (1985). Preamble (p.
20). In: Volume 35: Polynuclear Aromatic Compounds, Part 4,
Bitumens, Coal-tars and Derived Products, Shale-oils and Soots.
IARC, Lyon, France.
Office of Technology Assessment (1981). Assessment of Technology
for Determining Cancer Risks from the Environment. Washington,
D.C.: Government Printing Office.
Doll, R. , and Peto, R. (1978). Cigarette Smoking and Bronchial
Carcinoma: Dose and Time Relationships Among Regular Smokers and
Life-Long Non-Smokers. J. Epid. Comm. Health 32: 303-313.
Day, N.E., and Brown, C. C.
Prevention of Cancer. JNCI
(1980). Multistage Models and Primary
64: 977-989.
8.
9.
10.
National Academy of Sciences, Committee on the Biological Effects
of Ionizing Radiations (1980). The Effects of Populations of
Exposure to Low Levels of Ionizing Radiation: 1980. Washington,
D. C. : National Academy Press.
Schulte, P. A. (1987). Methodologic Issues in the Use of Biologic
Markers in Epidemiologic Research. Am. J. Epid. 126: 1006-1016.
Silbergeld, E.K.
1399.
!1987). Letters: Risk Assessment. Science 237;
U. S. Interagency Staff Group on Carcinogens
Carcinogens: A Review of the Science and Its
Principles. EHP 67: 201-282.
1986). Chemical
Associted
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APPENDIX
BALANCE OF CANCER AND NON-CANCER ENDPOINTS
Neil Chernoff and Stephen Nesnow
The balance of basic research on cancer and non-cancer endpoints within
any Federal organization 1s dependent upon a variety of factors such as
Congressional mandates, the given organization's operational policy, public
perceptions and concern, ongoing identification of potential data gaps and
to some extent, the range of disciplines represented by the organization's
scientific staff. All of these forces have an 1mp°act on scientific
managers 1n their designing and Implementing a basic research program to
meet their organization's needs now and in the future.
Generally, in enacting legislative authority Congress exhorts EPA to
evaluate a broad range of potential health effects associated with exposure
to environmental chemicals and insults. Rarely does legislation require
specific health endpoints to be addressed over other endpoints. It is,
therefore, EPA's policy which directs attention to specific endpoints of
concern for environmental exposures. Being a public institution, EPA is
influenced by the perceptions and concerns of the public and industrial
sectors regarding adverse health effects of chemicals. Consequently, EPA
molds its administrative and regulatory policy to balance these concerns.
For many years the primary environmental health concern, as perceived
by the public, was the possibility of chemically-Induced cancer. The
reasons for this concern include the prevalence and general irreversibility
of the disease, its potential for debilitation and eventual lethality, and
the knowledge that many chemicals to which there is prevalent human
exposure can cause cancer in laboratory animals. As a result of these
concerns and the body of data that has been generated over the years, the
EPA's (as well as other regulatory agencies) regulatory policy has been
largely driven by cancer as the health endpolnt of greatest severity.
Over the past several decades, however, 1t has become Increasingly
apparent that there are many other adverse health endpoints which may be
and have been induced by exposure to environmental agents. The methyl
mercury-induced epidemic of birth defects in Japan, the incidents of
delayed neuropathy in the Middle East, and the occurrence of male sterility
in workers occupationally exposed to chemicals 1n the USA all have served
to alert the public that the potential risk of exposure to environmental
agents may require consideration of many health endpoints. The outcome of
this realization has been a broadening of the areas of concern and a
simultaneous commitment of resources to these additional research areas.
Along with this commitment there has been an increasing tendency to
consider these health endpoints during the formulation of regulatory
policy.
Toxicologists in both the public and private sectors have also
identified other organ systems and susceptible populations that are at
potential risk from exposure to environmental agents. These realizations
have lead to considerable support for research in other areas such as
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inmunotoxicology, heritable disease, and prepubertal and geriatric
populations. Additionally, scientists and the public have become
increasingly concerned about the toxic potential of lifetime exposure to
relatively snail amounts of a multitude of xenobiotics. Increasing
resources have been allocated to gain a better understanding of the
potential risks from these types of exposures as well as the most accurate
ways to measure such risk.
Finally, there are two additional concerns of the general public and
the scientific community that Influence allocation of resources. The
translation of laboratory data Into human health risk assessments is an
extremely difficult process. While a safe environment is desirable, the
regulation and removal of chemicals based upon faulty assumptions may lead
to undesirable results (including their substitution with potentially more
hazardous compounds) entailing a reduction in the quality of life through
Increased expense, disease prevalence, and/or reductions of food and other
material production. Therefore, as the preceding Chapter Indicates,
considerable research resources are now and 1n the future will be devoted
to increasing the scientific basis and accuracy of risk estimates. The
development of a better understanding of the basic mechanisms responsible
for cancer and non-cancer responses is ultimately the most rational way in
which to formulate regulatory policy. This obviously leads to a continuing
requirement for long-term baste research.
The second factor which influences resources allocations concerns the
need for simpler, less expensive, and less whole-animal-oriented forms of
testing. The number of agents and complex mixtures of potential concern is
far in excess of our ability to test for toxic potential by standard
methodologies. Concerns raised by the public about the use of laboratory
animals in such studies have been a further impetus to the development of
alternative test methods.
The EPA research efforts in non-cancer endpolnts have greatly increased
over the last decade for the reasons listed above. Whether this increase
has led to a proper balance between cancer and non-cancer endpoints is
impossible to say, since there are so miny competing factors that go Into
the composition of this balance. Certainly, a resource allocation to both
cancer and non-cancer endpoints has enabled the Agency to utilize a broad
base of health endpoints In the formulation of regulatory policy.
Long-term basic research into both cancer and non-cancer endpoints is
recognized as being essential 1f the Agency is to formulate a broad
regulatory policy 1n the most accurate manner possible. Rather than
consider cancer and non-cancer effects separately, research 1n the future
will evaluate multiple toxicological responses from the same exposure.
Issues of adversity and severity of effects over time will be given greater
attention. Efforts will be made to capture and analyze toxicological data
in a more systematic fashion. These data will form the basis of improved
structure-activity and pharmacokinetlc modelling, test battery design, and
dose-response evaluation of cancer as well as non-cancer endpoints.
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Chapter 6
SUMMARY
This century has seen the emergence of abnormalities 1n early growth
and development, chronic degenerative diseases, and cancer as the major
causes of human morbidity and mortality in the industrialized nations of
the world. Initially, these diseases were often viewed as being the result
of heredity or the natural consequence of the aging process. More
recently, however, there has been a growing recognition that they
frequently have important environmental components or risk factors in their
etiology.
Many of these environmental risk factors are either produced directly
by humans or subject to their manipulation. They include chemical and
physical agents in the air, water, food supply, drugs, consumer products,
home and workplace. While detailed estimates of the Impact of these risk
factors are difficult to generate or verify, 1t has been variously
postulated that a significant number of the two million Individuals who die
each year in the United States may have had their lives shortened to some
degree by the effects of air pollution; that pollutants in our drinking
water systems may play a role 1n the onset of cancer and heart disease,
which are the two leading causes of death in this country; and that the
collective effects of work-related disease and stress may now be
approaching a level of Impact more typically associated with workplace
accidents. Therefore, federal health researchers and regulators are
increasingly being challenged to identify these environmental risk factors
and reduce or eliminate their deleterious effects.
Attention has been focused on some of the technical problems that can
be encountered when one attempts to assess the true impact of environmental
exposures on human health. Often, relevant epidemlologic and clinical
information on the potential health hazard associated with exposure to a
specific chemical or physical agent will not be available. Even when such
data is available, however, it may not be sufficiently sensitive or
specific to allow an investigator to choose among competing mathematical
models that attempt to characterize the unknown, underlying relationship
between exposure and dose. In some instances the available human data may
not even permit one to determine if any health risk appears to be
associated with low or moderate levels of exposure. As a result,
laboratory animal data will often constitute the primary basis for both
qualitative (i.e., hazard identification) and quantitative human risk
estimation.
Because of the high (often maximally tolerated) doses typically
employed in laboratory animal screening studies, quantitative risk
estimation based on laboratory data involves two separate issues that must
be addressed: low-dose extrapolation and species extrapolation. In some
instances (e.g., when the agent of concern Is a carcinogen or mutagen)
mathematical modeling will be employed to generate low-dose risk estimates,
and choice of a particular model may have a significant impact on the
magnitude of the estimated risk. In other cases a threshold phenomenon may
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be assumed and a safety factor approach used to determine acceptable
exposure levels. This approach also clearly suffers from a number of
methodological uncertainties and problems.
Ideally, the problem of species extrapolation should be addressed by
taking Into consideration all of the various species-specific factors that
could contribute to Interspedes differences 1n response to the exposure of
interest. Instead, the conventional approach to this Issue 1s to assume
that humans and the test animal 1n question will have equivalent responses
when comparisons are made on an appropriately-chosen dosage scale.
Unfortunately, choice of the most appropriate-dosage scale cannot always be
justified on biological grounds, and significant differences can result 1n
the projected human risk estimate depending on the decision reached. As
our reliance on quantitative risk estimation/assessment continues to
increase, more and more Importance 1.s being attached to the need to
characterize the uncertainties associated with these risk assessments and
to reduce these uncertainties by Improving the biological basis upon which
the risk assessment process 1s based.
In addition to technical problems related to the risk assessment
process itself, which have complicated and on occasion frustrated our
efforts to evaluate adequately the potential risks posed by various
environmental hazards, there are also a number of additional factors that
have hampered our attempts to reduce the impact of environmentally-related
disease. Among these are the lack of substantive toxlcologlc Information
on the majority of commercial chemicals that have been Introduced Into the
human environment, the Insufficient and sonetlmes Inappropriate training of
our nation's physicians with respect to environmental Issues, and the
inadequate surveillance of populations exposed or potentially exposed to
environmental hazards.
Priority must be given to research 1n a number of Important areas 1f we
are to resolve these problems and advance our understanding of the role of
environmental factors in human health and diseases. For example, more
emphasis needs to be given to the development and refinement of procedures
(particularly non-invasive procedures) for measuring low levels of human
exposure to toxic environmental agents. Similarly, we need to develop a
better understanding of the biological mechanisms that underlie
environmentally-related health effects to Improve both the quantitative
assessment of human health risks and the primary/secondary prevention of
environmentally-related diseases. A number of examples of long-term, basic
research activities in these areas that either have or may utlimately have
direct application to the types of environmental health problems that EPA
and other regulatory agencies must address on an ongoing basis are cited 1n
this document.
Comparison of patterns of proto-oncogene (I.e., cellular genes
expressed during normal growth and development processes) activation 1n
spontaneous and chemically-induced rodent tumors may provide Insight into
the mechanisms of tumor formation at the molecular level. In addition,
some of the uncertainty involved 1n species-to-species extrapolation of
carcinogenic risk estimates may eventually be removed by interspecies
comparisons of oncogene activation and expression.
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Recent advances in biochemistry and molecular biology have led to the
development of highly sensitive techniques which may allow the
quantification of the internal dose of carcinogens or in some cases the
biologically effective dose in target tissues. This ability to express
external exposure or administered dose levels on a more
biologically-relevant basis should eventually lead to a clearer
understanding of the relationships between exposure and disease or
toxicologic effect for many health hazards in the human environment.
Recognition of the potential usefulness of these biochemical markers has
led to the emergence of a new field of epidemiology, known as molecular or
biochemical epidemiology, that has as one of its major goals the adaptation
of these laboratory procedures into epidemiologic field studies.
In the fields of neurotoxicology and immunotoxicology new methodologies
promise to enable toxicologists to greatly improve our ability to assess
both central nervous and immune system deficits. The utilization of novel
techniques in molecular biology (e.g., monoclonal antibodies to specific
critical chemical components of these systems) promises to allow improved
evaluations of potential disfunctions.
In the area of human reproduction one of the most important questions
involves the potential of environmental agents to affect pre-implantation
loss. Researchers have recently identified an antibody to a subunit of the
hormone human chorionic gonadotropin. This advance enables the
identification of spontaneous abortions at an earlier stage and with
greater accuracy than was previously possible and may significantly improve
our monitoring capabilities.
In addition to identifying specific examples of long-term research
activities that either are generating or may generate results directly
applicable to the environmental health issues that EPA must address from a
regulatory viewpoint, this document also attempts to describe the
relationship between long-term and short-term (or immediate)
"problem-solving" research and to put it in perspective. For example, it
is noted that the general philosophy underlying basic health research is
that understanding more about the biologic mechanisms by which
environmental hazards such as toxic chemicals induce adverse effects will
lead, ultimately, to earlier detection of such effects, more sensitive
analytical methods for fully characterizing their potential impact on human
health, and a better understanding of how to eliminate or, at least, reduce
that impact. The distinguishing characteristic of this basic research is
that it typically addresses "generic" scientific issues and is not focused
on a specific problem or immediate concern. Furthermore, it must usually
be supported for a period of several years before it produces results that
may have a direct application to regulatory needs or problems.
The environmental health problem with the toxic metal lead is used to
illustrate the necessity of and role for long-term research activities in
the development of a sound, scientific foundation necessary for
constructive actions dealing with public health problems. Hhile lead
toxicity resulting from "high" level exposures has long been recognized as
an important public health concern, ongoing, long-term basic research has
only recently given us the technical tools to detect some of the more
subtle yet extremely important effects of low-level lead exposure.
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Even when research 1s more focused on a specific Issue or health
concern, It may need to be sustained for a considerable length of time
before any practical results or applications can be produced, research 1s
most often sequential with each new phase of the overall effort dependent
on the results from the preceding phase(s). Alternatively, even 1f the
research 1s focused and the required course of action clearly delineated
before any effort 1s expended, a considerable Investnent of time and effort
may be required before the project 1s completed. Certainly, this 1s the
case with prospective cohort studies 1n epidemiology and to a lesser extent
with laboratory-based, lifetime cardnogenidty screening experiments.
It seems clear, therefore, that while many of the health effects (or
possible health effects) Issues that confront EPA require an expeditious 1f
not immediate response, the most appropriate and in many cases the only
approach to formulating such responses will be to draw on the experience
and insights gained from long-term research. This certainly has been the
experience in dealing with most environmental crises to date. The only
approach that will enable us to engage in such long-term research 1s to
provide stable, consistent support for such a program. With continued
support and in-house expertise EPA can directly address applied research
issues with which 1t 1s particularly concerned and effectively apply both
its own long-term findings and those of other public and private
institutions to the solution of critical environmental health problems.
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