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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                                    -2-

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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                                    -3-

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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                                    -4-

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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                                    -5-

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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                                    -6-

 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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                                   -7-

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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                                   -8-

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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                                   -9-

    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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                                    -10-

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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                                    -11-

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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                                    -12-

REFERENCES

1.  Jones, H.B.  A Special  Consideration of  the  Aging  Process, Disease, and
    11 fe Expectancy.   University  of  California ,  Radiation Laboratory,
    Berkeley, California,  1955.

2.  Fries, J.F.   Aging, Hatural Death, and  the  Compression of Mortality.
    New England J.  Hed. 303:130-135. 1980.

3.  Donabedian,  A., Axelrod,  S.J. ,  Swearingen,  C. ,  and Jameson, J. Medical
    Care Chart Book.  5th Edition.   Bureau of Public Health Economics^
    University of Michigan,  Ann Arbor, Michigan,  1972.

4.  Second Task Force for Research  Planning In  Environmental Health
    Science.   Human Heailth and the  Environment  -  Some Research  Needs.
    U.  S. Department of Health,  Education,  and  lei fare,  DHE¥ PubT. Ho.  NIH
    77-127, Washington, D. C. , 1977.

5.  Levy, B.S. and Wegman, O.H.  (Editors) Occupational  Health:  Recognizing
    and Preventing Work-Related  Disease.   Little,  Brown  and  Co.,  Boston,
6.  Cairns, J.  The Cancer Problem.   Sc1.  Anter.  233:64-78,  1975.

7.  Doll, R.  An Epldemlological Perspective of the Biology of Cancer.
    Cancer Res.  38:3573-3583, 1978.

8.  Inhaber, H.   Risk of Energy Production, ACEG-1119/REY-1 , Atonic Energy
    Control Board , 5tf awi , Canada , May 1578.

9.  Morgan, M.G. , Morris, S.C., HenHon, M. , toiaral , D.A.L. , and R1sh,  W.R.
    Technical Uncertainty 1n Quantitative Policy Analysis - A Sylfur A1r
    Pollution Example.  Risk Analysis 4:201-216. 1984=

10. Morgan, M.G. , Henri on, M. , Morris, S.C., and Arearal , D.A.L. Uncertainty
    in Risk Assessment.  Environ. Sc1. Techno!. 19:662-667, 1985.

11. Letter to the Editor.  Chernobyl  Public Health Effects.  Science
    238_:10-11, 1987.

12. Task Force on Environmental Cancer and  Heart and  Lung  Disease.
    Environmental Cancer and Hfeart and Lung Disease,  3rd Annual  Report  to
    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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                                    -13-

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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                                    -14-

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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                                    -15-
            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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                                    -16-
        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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                                    -17-



        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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                                   -18-
     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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                                   -19-
Flgure 1
Age-Specific Death Rates 1n Various Countries and
          Years (From Reference 1).

-------
                                 -20-
          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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                                     -21-
                                             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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                                   -22-
Flgure 4
Time-Trends in Lung Cancer Mortality and Cigarette
Consumption 1n England and Wales. (From Reference
6).

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                                   -23-
        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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                                    -25-
                                            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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                                    -26-

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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                                    -27-

 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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                                   -28-

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

-------
                                    -29-

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

-------
                                    -30-

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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                                    -31-
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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                                    -32-

(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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                                    -33-

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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                                    -34-

    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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                                    -35-

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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                                    -36-

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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                                    -37-

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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                                    -38-

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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                                    -39-

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2.   Bellinger D.C., Leviton A., Waternaux C.,  Neddleman H.L. ,  Rab1now1tz
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     Concentration  and Serum 1,25-d1hydroxycholecolc1ferol  Levels 1n
     Children.  Am J CUn Nutr 35:1327-1331.

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                                    -40-

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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                                    -41-

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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                                    -42-

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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                                    -43-

                 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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                                    -44-

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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                                    -45-

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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                                    -46-

         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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                                    -47-
         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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                                    -48-

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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                                    -49-

    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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                                -50-

     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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                                    -51-

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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                                    -52-

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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                                    -53-

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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                                    -54-

                              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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                                    -55-

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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                                    -56-

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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                                    -57-

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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                                    -58-

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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                                    -59-

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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                                    -60-

                         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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                                    -Sl-

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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                                    -62-

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."

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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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                                    -64-

                              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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                                 -65-
            £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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                                    -66-

                     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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                                    -67-

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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                                    -68-

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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                                    -69-

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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                                    -70-

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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                                    -71-

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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                                    -72-

with regulatory responsibilities for more rational and reasonable decisions
about the proper fate of the agent/chemical under consideration.

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                                    -73-
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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                                    -74-

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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                                    -75-

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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                                    -76-

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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                                    -77-

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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                                    -78-

    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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                                    -79-

    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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