vvEPA
United States
Environmental Protection
Agency
Office of Health and
Environmental Assessment
Washington DC 20460
Research and Development
Workshop
Proceedings:
Approaches for
Improving the
Assessment of
Human Genetic
Risk—Human
Biomonitoring
EPA-600/9-84-016
August 1984
PROTECTION
AGENCY
DALLAS, TEXAS
HURT
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EPA-600/9-8^-016
August
WORKSHOP PROCEEDINGS: APPROACHES FOR IMPROVING THE ASSESSMENT OF
HUMAN GENETIC RISK—HUMAN BIOMONITORING
December 13-15, 1982
Washington, DC
John R. Fowle III*
Reproductive Effects Assessment Group
Office of Health and Environmental Assessment
U.S. Environmental Protection Agency
Washington, DC 20460
Work sponsored by the U.S. Environmental Protection Agency in
cooperation with the Department of Energy/Brookhaven National
Laboratory and the Council for Research Planning in Biological
Sciences under Interagency Agreement No. AD89F2A165.
*Workshop participants are listed on page viii. They all contributed to the
preparation of this report.
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DISCLAIMER
This document has been reviewed in accordance with U.S. Environmental
Protection Agency policy and approved for publication. Mention of trade names
or commercial products does not constitute endorsement or recommendation for use.
ii
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CONTENTS
Page
Preface v
Abstract vii
Participants and Reviewers viii
I. Introduction 1
II. Methods for Biomonitoring Exposed Populations 3
A. Direct Method 3
B. Indirect Method(s) 6
1. Genetic Endpoints 7
a) Germ Cells 7
b) Somatic Cells 8
(1) Gene Mutations 8
(2) Chromosomal Aberrations 12
a) Chromosome Breakage and Rearrangement 12
b) Micronucleus Test 13
2. Measurements of DNA Damage 14
a) Sister Chromatid Exchanges 14
b) DNA Repair 15
c) Measurement of Chemical Binding to DNA 18
(1) Radiolabeled DNA Probes or Mutagens 18
(2) Immunoassays 18
(3) Fluorescence 19
(4) Measuring Damage in DNA by Gas Chromatography-
Mass Spectrometry 19
III. Biomonitoring Assays Available for Mammalian Experimentation .... 20
A. Direct Method 21
1. Gene Mutations 21
a) Specific Locus Test 21
b) Tests for Dominant Mutations 23
2. Chromosomal Aberrations 23
a) Heritable Translocation Test 23
b) Sex Chromosomal Abnormalities 24
c) Dominant Lethal Test 25
B. Indirect Method (Mouse Spot Test) 26
C. Limitations of Animal Test Data for Estimating Human
Genetic Effects 27
IV. Identification of Human Populations Exposed to Chemical Mutagens. . . 27
m
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CONTENTS (cont.)
Page
V. Discussion of Approaches for the Improvement of Mutagenicity
Risk Assessment 28
A. Bridging Human Biomonitoring Endpoints with Animal Experimentation
(Defining the Relationships) 30
B. Other Types of Testing Needed 33
1. Cell-Specific Effects 36
2. Homeostatic Mechanisms 37
C. Need for Coordination of Efforts 37
VI. References 40
iv
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PREFACE
This report explores ways to improve the ability to predict genetic risk to
humans. Certain Federal laws administered by EPA require that a pollutant's
negative impacts on society be weighed against its benefits in arriving at
regulatory decisions. To accomplish this one must somehow quantify the MSKS
and benefits for comparative purposes. When cause and effect relationships can
be demonstrated in humans after exposure to a toxicant, quantifying risks and
benefits is a straight-forward process. But for genetic diseases, chronic
effects observed in the offspring of exposed individuals, it is very difficult
and requires many assumptions. There are no data linking pollutant exposures
to the induction of heritable mutations in humans, although this has been
demonstrated in experimental studies using mice. Furthermore, even though
data can be obtained from exposed humans showing that adverse genetic effects
are occurring, these can only be measured in somatic tissue, not germinal
tissue. Thus, in order to arrive at estimates of the risk of heritable genetic
diseases in humans after exposures to mutagens, data on heritable effects from
experimental rodent systems and/or data from humans on somatic cell effects
must be somehow extrapolated to heritable effects likely to occur in humans.
This requires assumptions about species-to-species relationships and tissue-to-
tissue relationships. Furthermore, high- to low-dose extrapolations are usually
invoked when using the experimental data because such studies are usually
conducted at much higher doses than humans are exposed to in the environment.
Because of these uncertainties, a workshop was held to examine the assumptions
implicit in making quantitative estimations of heritable human mutation
frequencies using human and animal data. Emphasis was placed on ways to assess
the correctness of some of the assumptions and ways to verify the risk assessment
approaches.
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In so doing it was pointed out that the experimental animal data must be
anchored to comparable human data. Recommendations were made to study somatic
and heritable damage in individuals from high risk human populations (e.g.,
cancer chemotherapy patients) and to study the corresponding effects in parallel
experiments using mice receiving the same treatment.
It is believed that the information in this report will be particularly
useful to risk assessors, genetic toxicologists, epidemiologists, and research
planners. It is hoped that the report will stimulate collaborative efforts
among government agencies, and among government agencies and other organizations
as well. Continued examination and refinement of risk assessment approaches
will provide a mechanism for more accurate prediction of human genetic risk.
It is hoped that the efforts described in this report will serve as a springboard
for generating a firm data base to support future risk assessment efforts.
Because of the dynamic nature of this subject, this report cannot be
considered comprehensive or complete. New questions continue to arise about
the assessment of mutagenic risk as chemical interactions with cellular
macromolecules (such as DNA) become better known and as interspecies and
intertissue relationships begin to unravel. This report provides a reasonable
outline of the state-of-the-subject in 1983. It should be considered a guide
for planning work, hopefully, to develop a scientifically credible approach
for using human biomonitoring data for genetic risk assessment.
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ABSTRACT
Federal laws require a consideration of adverse health effects, including
mutagenicity, in arriving at regulatory decisions on chemical substances.
Certain laws require balancing the consequences of these risks with the benefits
provided by the use of chemical substances. This requires that risk be
quantitatively assessed. Estimates of human genetic risk can be made indirectly
based on data from animal experimentation and human somatic cells, but it is
not practical to estimate genetic risk directly based on data from human germ
cells. The indirect estimates are highly debated because of uncertainties
about interspecies and interorgan extrapolations. Uncertainties in extrapolating
from effects observed in animals at high experimental doses to effects likely
to occur in humans at much lower environmental levels further complicate genetic
risk assessment. Comparative studies are needed to define the relationships
between somatic cell and germ cell events and between experimental animals and
humans. This may involve selecting at least one high risk human population
for study. These efforts will require a long-term coordination of efforts
among the Federal agencies and among government agencies, industrial concerns,
and the academic community.
vn
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PARTICIPANTS AND REVIEWERS
Organizing Committee
John R. Fowle III, Co-Project Officer
U.S. Environmental Protection Agency
Raymond R. Tice, Co-Project Officer
Brookhaven National Laboratory
Vicki L. Vaughan-Dellarco
U.S. Environmental Protection Agency
Ernest R. Jackson
U.S. Environmental Protection Agency
Other Participants
K.S. Lavappa
U.S. Environmental Protection Agency
Sheila L. Rosenthal
U.S. Environmental Protection Agency
Michael D. Waters
U.S. Environmental Protection Agency
Speakers/Participants
Richard J. Albertini
University of Vermont
J. Grant Brewen
Allied Chemical Co.
Gerald L. Chan
Dana-Faber Cancer Institute
James E. Cleaver
University of California
San Francisco
Roger W. Giese
Northeastern University
George R. Hoffman
Holy Cross College
James V. Neel
University of Michigan
James A. Swenberg
Chemical Industry Institute of
Toxicology
Lawrence R. Valcovic
U.S. Food and Drug Administration
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I. INTRODUCTION
A considerable genetic disease burden has been recognized in the human
population. It is estimated that perhaps 10% of all human disease has a
significant genetic component, resulting from changes in the composition,
arrangement, or number of genes and chromosomes (BEIR 1980, Flamm 1977, UNSCEAR
1977). Humans are exposed to a large and increasing number of chemical substances,
some of which have mutagenic effects in other organisms and may pose a genetic
risk to people. Because induced genetic diseases can only be expressed in
future generations, much effort has gone into designing methods for detecting
mutagenic agents. Recently, combinations of tests that are quite effective at
identifying mutagenic chemicals have been developed. However, these tests are
not useful in monitoring humans for heritable mutations, and thus, the magnitude
of the contribution that chemical mutagens may make to human genetic disease is
highly debated. Despite the lack of definitive evidence, there is no reason
to doubt that chemical mutagens can induce heritable, germ-line mutations in
human beings.
Concerns about the ability of man-made chemical substances to alter the
environment led to the passage of Federal laws to protect against such effects.
All of these laws require a consideration of adverse health effects in arriving
at regulatory decisions. Some, such as the Toxic Substances Control Act,
require that specific effects of chemical substances, including mutagenicity,
be considered in light of the benefits provided by those chemicals in order to
ensure that human exposure does not result in an unreasonable risk. This
means that the extent of the potential risk must be quantified before decisions
are made.
The task of quantifying potential mutagenic risks associated with exposure
to chemical mutagens is very complex, and current capabilities require that
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many assumptions be made. Extrapolations must be made between species if
animal data are used to estimate human genetic risk or between tissues in
order to use data from somatic cell biomonitoring to estimate heritable genetic
risk. In addition, uncertainties about the exposures that humans receive and
extrapolations from effects at high experimental levels to the types of effects
expected from much lower environmental levels further compound the problems.
Thus, quantitative mutagenicity risk assessments are not scientifically rigorous,
because the data base needed to support the extrapolations is not yet complete.
A workshop entitled Approaches for Improving the Assessment of Human
Genetic Risk: Human Biomonitoring was held in December 1982, to identify the
types of experimental approaches that are required to eliminate some of the
assumptions and uncertainties of mutagenicity risk assessment. The approaches
identified for using biomonitoring data as a basis for building bridges between
experimental mammals and humans are discussed in this paper with the goal of
providing direction for the future research that will be required to improve
the scientific basis for mutagenicity risk assessment. Emphasis was placed on
practical ways to obtain data that are useful in estimating genetic risks.
The workshop analyzed available techniques, their usefulness, their limitations,
and possible methods for improvement. The impact that increases in the mutation
frequency may have on the incidence of human genetic disease was not considered
to be in the scope of this workshop. The discussions presented here focus on
the workshop proceedings. However, it should be noted that several related
publications dealing with the monitoring of humans for detection of genetic
damage and/or assessment of genetic risk have appeared since the workshop was
held (e.g., ICPEMC 1983 a-f, Streisinger 1983, Ramel 1983, Hook 1983, Miller
1983, Matsunaga 1983, Ashby 1983, Ehrenberg et al. 1983, and Lyon 1983).
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II. METHODS FOR BIOMONITORING EXPOSED POPULATIONS
Methods of monitoring human populations for purposes of genetic risk
assessment may be classified as direct or indirect, depending on whether
extrapolations are necessary for estimating an effect in humans.
A. DIRECT METHOD
The direct method involves the search for mutational effects in human
populations exposed to a potential mutagen. It can be used when there is a
large population of children of exposed persons available for study. This
method was first applied to the study of children whose parents had been exposed
to the atomic bombs (Neel and Schull 1956). These children are monitored with
respect to a battery of phenotypes, and, when variants are encountered, studies
are undertaken to determine whether they are mutations. The observations that
might be made on children are of three types: morphological, cytological, and
biochemical (Bloom 1980; NAS 1982; Neel 1971, 1981, 1983; Neel and Rothman 1981).
The "morphological" observations include the frequency of dominant mutations,
congenital defects and stillbirths, altered physical growth and development,
and reduced survival. The cytological data include scoring for an array of
chromosomal abnormalities. The biochemical approach involves a search for
mutant proteins not present in either parent. Because most genetic diseases
involve protein alterations, the biochemical approach yields less ambiguous
results than the morphological and cytological approaches. However, this method
requires a higher level of technology.
Until recently, the biochemical approach in humans and experimental
animals has employed one-dimensional electrophoresis and quantitative enzyme
level determinations (Neel 1979; Neel et al. 1979; 1980a, 1980b; Satoh et al.
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1983). A development of potentially great importance for biochemical
monitoring is the advent of two-dimensional polyacrylamide gel electrophoresis
(2-D PAGE). This method permits the separation of proteins on the basis of
both charge and size on a slab gel; as many as 1000 of the different polypeptides
contained in a single cell-type can be detected. Although not all of these can be
scored unequivocally for genetic variation (electrophoretic or quantitative),
it appears that at least 200 polypeptides potentially suitable for monitoring
purposes can be identified from the components of a venous blood sample (Neel
et al., in press). Computer algorithms for both the enhancement of these
images and their scoring are under development in several laboratories (e.g.,
Skolnick et al. 1982, Miller et al. 1982, Brown and Ezer 1982).
These developments, if they realize their early promise, could dramatically
improve the monitoring of human populations for genetic damage. There are
limitations, however. Given the low spontaneous frequency of the mutational events
detectable in such gels (3-5 x 10~6/locus/generation), the population size
required for an adequate test of an altered mutation rate is massive in cases
of low-level exposures. Based on an ability to screen for 200 proteins, the
number of observations necessary to test for an increase in the mutation rate
of the order of 100% would be at least 10,000 to 12,000 offspring. Such large
populations are not likely to exist for the majority of exposure situations
that one would like to assess. Because of the requirement for large populations
and the fact that most exposures to mutagens will involve low dosages, this
method is expensive and will be of limited value scientifically in routine
biomonitoring of small groups. However, with appropriate exposures, it may be
useful in a coordinated effort pooling offspring from several high risk groups.
Despite the technical difficulty, studies of human germinal mutation rates
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are essential in order to understand the increases (if any) in transmitted
genetic damage following exposure to mutagens. Such studies are also necessary
as part of the basis for extrapolating from animal studies to humans. Thus,
it is essential that at least one comprehensive study of a sufficiently large
or appropriately pooled "worst case" population be conducted in conjunction with
observation on a variety of "presumptive" indicators of mutation. This proposition
will be discussed more fully later.
Three pilot studies involving the application of the 2-D PAGE technology
either to the evaluation of the effects of a potentially mutagenic exposure or
to developing baseline data are being initiated. One involves the children
of the atomic bomb survivors of Hiroshima and Nagasaki, who already have been
subjected to extensive studies including one-dimensional electrophoresis of
blood proteins (Neel et al. 1980b). This pilot study should therefore yield
data on the relative efficiency of the two-dimensional method in comparison to
the one-dimensional approach. The second pilot study involves children born
to Japanese parents who, prior to and during World War II, were engaged in the
manufacture of sulphur mustard and other military gases (Neel et al., in press).
The third, in Ann Arbor, Michigan, utilizes samples from the placentas of
newborn infants and blood samples from their parents.
Because the task of conducting a study using the 2-D PAGE approach to
determine human mutation frequencies will be formidable, one may question the
wisdom of initiating such studies. To place this in perspective, however, it
is necessary to examine an alternative direct method for estimating genetic
risk—phenotypic monitoring. The scoring of sentinel phenotypes has been
discussed for many years, but the limitations of this approach (i.e., the rarity
of individual genetic diseases necessitating the use of large populations)
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are recognized to be severe, particularly because the diagnosis for some of
the relevant genetic disorders is not always unequivocal. In order to define
the mutation frequency in humans, relatively sensitive, direct, and unambiguous
approaches must be used. At this time, the 2-D PAGE method seems to be the
most feasible approach.
The recent advances in molecular genetics begin to raise the possibility
of an approach for estimating mutation rates at the DNA level. A wide battery
of restriction-site endonucleases is now available, with each enzyme able to
recognize a somewhat different site at which it will cleave DNA. The action
of these endonucleases is to cut DNA into fragments of varying lengths. A DNA
mutation appearing for the first time in a child would manifest itself as a
DNA fragment in an endonuclease digest of that person's DNA which was different
in size from any of the fragments obtained from the parents. Although the
theory is simple, much experimentation will be necessary to determine which
enzymes in combination with which DNA probes yield the most suitable material.
A potential problem is translating the implications of any finding with reference
to its phenotypic impact on the child. For this reason, it would seem wise to
work with defined probes, e.g., probes such as those that exist for the a - and
3 -chains of hemoglobin or for a number of different enzymes.
B. INDIRECT METHOD(S)
In an indirect approach, tissue samples from exposed persons are analyzed
for genotoxic damage, or body fluids are tested for the presence of mutagens.
Extrapolations from tissue to tissue are then made in order to predict the
risk of genetic disease in future generations. Available methods involve the
study of genetic endpoints, including mutations and chromosomal aberrations,
or determinations of chemical interactions with DNA.
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1. Genetic Endpoints
a) Germ Cells—
For obvious reasons measurements of mutations or other genotoxic effects
in germ cells are more appropriate for assessing heritable genetic risk than
are similar measurements in somatic tissues. There are at least six indirect
tests that can be performed on germinal tissue (i.e., semen samples) from
human populations. Four of these tests measure effects on sperm cells, including
azoospermy or alterations in sperm motility, morphology, or capacitation (Wyrobek
et al. 1983a, b). The remaining approaches utilize other cytological techniques
(i.e., analysis of the number of fluorescent Y body chromosomes per sperm
[YFF] and hamster egg fertilization with human sperm). Most of the tests for
endpoints that may have a genetic basis, such as the putative Y-chromosomal
aneuploidy of YFF sperm (Beatty 1977, Kapp et al. 1979) and alterations in
sperm morphology (Wyrobek et al. 1983a), are not yet well characterized genetically.
Besides this limitation, all of these tests are not applicable to females
because of the inability to study ova.
Based on mouse radiation data, the frequency of mutations recovered from
females following long-term exposures is considerably lower than that recovered
from males exposed in a comparable fashion (Russell 1977). Preliminary data
indicate that this is also true for exposures to the chemical mutagen
ethylnitrosourea (Russell 1982). It has been proposed that this decreased
mutational expression is due to more extensive repair of premutational DNA
lesions in the oocyte than in male germ cells. In the absence of conflicting
data, the same situation is assumed to exist in humans. Thus, studies restricted
to male germ cells may overestimate the true risk for the entire population.
Based on these considerations, available human germ cell systems are not yet
adequate for predicting genetic risks.
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b) Somatic Cells—
There are several genotoxic endpoints that can be measured in somatic tissue
(Table I), and the information generated from such measurements can be used to
indirectly monitor human populations for heritable genetic damage. Some of
the tests measure direct genetic alterations, gene mutations or chromosomal
aberrations, while the remainder measure other endpoints indicative of genotoxic
damage. Most of these tests can be conducted in both humans and experimental
animals, providing a means for correlating epidemiological and clinical data
with respect to adverse health outcomes. Because these tests monitor for j_n_
vivo events, they offer several advantages: (i) they detect genotoxicity from
agents whose in vivo effects are dependent upon metabolic or pharmacokinetic
factors, (ii) they potentially are able to determine the effects of complex
mixtures, and (iii) for humans and animals, they may detect heterogeneity for
individual susceptibilities to genotoxicants.
(1) Gene mutations—Several somatic cell mutagenicity tests for in vivo
studies have already been developed, or are in the developmental stage. These
include: (i) the detection of 6-thioguanine resistant (TGr) T-lymphocytes
arising in vivo in humans (Strauss and Albertini 1979, Albertini 1982) or in
animals (Recio et al. 1983, Gocke et al. 1983), (ii) the enumeration of mutant
hemoglobin-containing mature red blood cells (RBCs) arising in vivo in humans
or animals (Stamatoyannopoulos and Nute 1983, Bigbee et al. 1983), and (iii)
the recognition of surface antigen changes arising in vivo on human RBCs
(Bigbee et al. 1983). Furthermore, both spontaneous and induced HLA antigen
loss mutants can be quantified iji vitro in humane -lymphoblasts (Pious and
Soderland 1977, Kavathas et al. 1980), thus suggesting a method whereby analogous
mutants arising in^ vivo in human T-lymphocytes might also be determined.
8
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Two methods currently are available for detecting TGr T-lymphocytes arising
in vivo in humans--i.e., autoradiography (Strauss and Albertini 1979, Albertini
1982) and cloning (Albertini et al. 1982, Albertini 1982, Strauss 1982, Morley et
al. 1983). Although each has its advantages, the cloning technique allows recovery
and study of the TGr cells and has allowed a biochemical demonstration of
the mutant character of these cells (Albertini et al. 1982, Morley et al. 1983).
Lymphocytes usually are obtained for testing by obtaining peripheral blood.
However, other sources, such as skin, liver, various tumors, and/or lymph node
tissues, can be used. The organ specificity of toxic and/or mutagenic agents
potentially can be assessed by quantitating TGr T-lymphocytes using these
techniques because of restricted lymphocyte migration in vivo (Gallatin et
al. 1983). Some population studies have already been conducted, and "mutagenic"
effects have been demonstrated in vivo in humans (Strauss and Albertini 1979,
Albertini 1982) and in mice (Gocke et al. 1983). Human-animal comparisons are
feasible and gene cloning techniques allow for the possibility of comparisons
being made at the DNA level.
Many mutant hemoglobins (HbG) have been identified and structurally
characterized in humans. Several of these could be useful marker mutants (i.e.,
hemoglobins S, C, E, Wayne and Cranston). The various mutant hemoglobins result
from a variety of structural changes in either the alpha or beta chain of
hemoglobin. Identification of these mutant hemoglobins, therefore, is based on
the knowledge of their specific changes. Such identification avoids the possible
confounding effects of "phenocopies" inherent in assays that detect protein
deficiencies. With respect to experimental animals, the ability to test for
mutant HbGs is not as developed as it is for humans, although a mutant hemoglobin
in the mouse has recently been described (Popp et al. 1983) and schemes for
10
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using interspecies structural differences among HbGs have been suggested
(Stamatoyannopoulos and Nute 1983). Different mouse strains have different
HbGs, including several with known amino acid differences. Potentially,
systems could be constructed with affinity antibodies developed in order to
test for mutant HbGs in mice.
For purposes of qauntifying heritable genetic risks, the various tests
using somatic tissues are limited. The most obvious restriction is that they
are performed with somatic tissue. Thus, tissue to tissue extrapolations
must be made in order to make predictions of transmissible genetic risk.
Additionally, for risk assessment purposes, mutational events rather than mutant
cells are of interest. However, in somatic cell tests, it is mutant frequencies
rather than mutational events that are quantified. It will be difficult to
quantify the latter because little or nothing is known about in vivo cell
generations or cell kinetics (e.g. clonal expansion, in vivo selection, sizes
of the populations at risk, cell killing, etc.).
There is also the general difficulty with somatic cell tests in defining
the genetic basis of the phenotypic changes at the somatic cell level. Although
this difficulty has been overcome for TGr T-lymphocyte and mutant HbG tests,
several potentially useful somatic tests have been abandoned because of the
presence of "phenocopies." An early candidate test determined the frequency of
antigen-loss variants (ABO antigens) of mature human RBCs (Atwood 1958; Atwood
and Scheinberg 1958, 1959). Another test proposed that mutation of the X-
chromosomal gene for the enzyme glucose-6-phosphate dehydrogenase (G6PD) could
be detected in polymorphonuclear white blood cells (WBCs) by cytochemical
methods (Sutton 1972, Sutton 1974). Also, rare variant RBCs containing fetal
hemoglobin (HbF) in amounts sufficient to be detected were found in human
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peripheral blood (F-cells) (Sutton 1972, Sutton 1974, Stamatoyannopoulos et
al. 1975, Wood et al. 1975). It was postulated that these F-cells arose because
of a mutation in the structural gene for the beta chain of normal hemoglobin,
an event which occurred in rare erythroid precursor cells. Therefore, F-cells
were proposed as indicators of somatic cell mutation occurring in vivo. All
three endpoints have been rejected as indicators of somatic cell mutations
because non-genetic factors such as physiological or pathological conditions,
or changes in assay methods could result in cells with the indicated phenotypes
(Atwood and Fetter 1961, Stamatoyannopoulos 1979). In none of these systems
could "phenocopies" be differentiated from true mutant cells.
(2) Chromosomal aberrations—It is possible to study cells in various
tissues for chromosomal aberrations and micronuclei.
(a) Chromosome breakage and rearrangements—Several approaches are available
for studying the frequency of chromosomal aberrations in peripheral blood
lymphocytes, bone marrow cells, and germ cells (Preston et al. 1981). Such
cytogenetic studies allow comparisons between effects in somatic cells and
effects in germ cells, as well as comparisons between species. Chromosomal
aberrations provide unequivocal evidence of genetic damage and thus constitute
a relevant endpoint for reproductive hazards. Furthermore, many carcinogens
have been shown to be clastogens. Generally accepted principles for the conduct
of tests and the scoring of results have been developed (Preston et al. 1981).
Considerable research has been conducted to assess spontaneous frequencies,
the clastogenic effect of physical and chemical agents, and to define the
technical variables in the techniques. The limitations of cytogenetic analysis
are that it is labor intensive and requires a high level of experience for
accurate scoring. In addition, the data base on interindividual variation,
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persistence of lesions, and the sensitivity of peripheral blood lymphocytes to
various classes of chemicals is relatively small.
(b) Micronucleus test—Micronuclei are small membrane-bound nuclear fragments
that arise from acentric chromosomal fragments or from entire chromosomes which
are excluded from within one of the daughter nuclei during cell division (Heddle
et al. 1983). Lagging fragments result from clastogenic damage while lagging
chromosomes result from a disturbance of the mitotic apparatus. Micronuclei
can be scored in any proliferating cell population, but the most common procedure
involves the scoring of micronuclei in the polychromatic erythrocytes (PCEs)
in mammalian bone marrow (Schmid 1976, Heddle et al. 1983). However, because
of the short lifespan of PCEs (approximately 24 hr) and the requirement for
bone marrow samples, this approach is largely restricted to tests that use
acute exposure regimes in experimental mammals. Although rbcs with micronuclei
can be detected in the peripheral blood of mice, the ability of the spleen to
remove such rbcs in most other mammals, including humans, precludes the use of
this approach for studies of environmental effects. Attempts to study micronuclei
in other cell populations, such as buccal smears (Stich et al. 1982) or biopsy
material from endoscopic examinations (Heddle et al. 1983), have been conducted
with only partial success. The usefulness of micronucleus tests for detecting
somatic genotoxic damage, therefore, remains in question.
Although it is possible to perform somatic cell biomonitoring tests on
several different cell-types, the difficulty of obtaining most tissues
necessitates that peripheral blood will be the main source of tissue for human
biomonitoring studies. Blood tissue is readily available, and thus somatic
cell systems offer the key advantage of permitting the collection of data from
small populations of individuals. However, measurements of mutational events
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in somatic cells also have deficiencies. Among these are the limited data base
for chemicals, the insensitivity of some of the endpoints as indicators of
genotoxic damage, the lack of appropriate bridging models to predict heritable
effects, and the lack of evidence for a correlation between elevated levels of
mutations in somatic cells and an increased risk for adverse health effects.
2. Measurements of DNA Damage
Various endpoints that potentially indicate mutagenesis can be detected in
both somatic and germinal tissue. These include sister chromatid exchanges (SCEs),
chemical interactions with DNA, and DNA repair. Of these three approaches,
the detection of SCEs is at the most advanced stage of development. Difficulties
are encountered in all of these methods, however, because of varying replication
rates and repair capabilities in different cell-types and because of the restriction
of germ cell measurements to males. Furthermore, positive findings with these
tests cannot be equated with an increase in the frequency of mutations. Nonetheless,
the measurement of SCEs in peripheral lymphocytes is a relatively easy and
sensitive test, and several sensitive techniques are being developed for measuring
DNA damage. These approaches may be used to provide information on internal
dosages resulting from human exposures to chemical substances, and as such may
be employed to provide a common denominator for tying human biomonitoring and
animal testing methods together.
a) Sister Chromatid Exchanges--
SCEs are sensitive indicators of certain types of DNA damage (Latt et al.
1981). SCE tests are easy to conduct in vitro, and most mutagens induce SCEs.
In vivo tests are also possible, allowing interorgan and interspecies comparisons
to be made. The most useful approach for interspecies comparisons involves
combined in vivo - in vitro tests, where exposure to the mutagen occurs jijl
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vivo, but bromodeoxyuridine (BrdUrd) treatment and cell replication occur in
vitro. Certain classes of agents are more effective inducers of SCEs than
others. Agents that cause DNA base damage (e.g., ultraviolet light and alkylating
agents) are good inducers of SCEs, while agents that primarily break the DNA
backbone (e.g., X-rays and bleomycin) are poor inducers of SCEs. Variability
in test parameters and a lack of understanding about the mechanism of SCE
formation limit the use of the technique in risk assessment. Variability in the
measurement of SCEs must also be considered. Biological variability may be
associated with sex, age, genotype, or diet, and technical variability may be
associated with various culture conditions (e.g., the amount of BrdUrd used,
the source and type of serum, temperature).
b) DNA Repair—
The repair of DNA damage involves a sequence of biochemical steps that can be
studied by techniques that detect the formation and removal of damaged bases,
the insertion of new bases during repair synthesis, or the formation and sealing
of DNA breaks during excision. Few of these techniques are amenable to rapid,
simple, and inexpensive use in screening large numbers of individuals. Peripheral
lymphocytes, which are the most available cell type in population studies, are
not ideal for the measurement of repair; their repair capacity is depressed in
comparison to many other somatic cell types, probably because they have low
levels of DNA polymerase alpha (Scudiero et al. 1976).
Available techniques for measuring DNA damage/repair include: (i) alkaline
sucrose gradients for single strand breaks; (ii) tritiated thymidine incorporation,
isopycnic gradients, and BrdUrd photolysis for repair replication; (iii) direct
measurement of loss of adducts with labeled mutagens; (iv) and analysis of DNA
monomers by high pressure liquid chromatography (HPLC). With the exception of
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Initiated thymidine incorporation to measure repair replication, these methods
are time-consuming, need specialized skills, and can be expensive (Cleaver
1974). Some of the methods, such as direct measurement of the loss of radiolabeled
adducts, are not suitable for use in humans. Alkaline elution has potential
(Petzold and Swenberg 1978) but is subject to complex artifacts, and interpretations
of the resulting data are rarely simple.
The most readily applicable method for assessing DNA repair in screening
studies is the measurement of the patching step of repair. Measurements can
only be made by autoradiography or by scintillation counts of the incorporation
of 3H-thymidine (3H-TdR) into the DNA of cells in which semi conservative DNA
replication is negligible (Cleaver 1974). A drawback to this technique is
that 3H-TdR uptake may differ between individuals, not only because of DNA
repair differences but also because of differences in thymidine kinase,
phosphorylases, or pyrimidine nucleotide pools (Cleaver 1967). Autoradiography
is more time-consuming but does allow cell by cell comparisons of unscheduled
DNA synthesis (UDS). The most rapid method for measuring UDS is scintillation
counting of cells exposed to 3H-TdR in the presence of inhibitors of DNA
replication (e.g., hydroxyurea). However, scintillation counting only registers
a net change in counts without indicating the amount in each cell. Residual
replicative DNA synthesis can therefore confuse the results obtained by scintillation
counts, but not those obtained by autoradiography. Furthermore, high concentrations
of hydroxyurea can also interfere with repair synthesis, further confusing
interpretation of the results. Additionally, levels of UDS can vary greatly
between cell types and differences in UDS between individuals may not reflect
only differences in DNA, but also differences in levels of enzymes responsible
for the incorporation of exogenous TdR. Finally, although many mutagens elicit
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an increase in UDS, some known promutagenic lesions, such as 06-methylguanine
do not. The failure to detect the repair of such lesions further complicates
extrapolation of UDS data to human health risks.
The use of DNA repair to detect genotoxic damage should be viewed with
reservations. DNA repair primarily occurs during the initial 12-24 hours after
exposure making a delayed assessment of repairable damage extremely difficult.
Screening for DNA repair also presents a conceptual problem, because the techniques
do not measure damage resulting in mutagenesis per se. Rather, they measure
the cell's attempts to correct such damage. The correspondence between repair
and genetic damage is complex and depends on parameters such as the cell cycle
and type and extent of damage induced. In replicating cells there is, in a
sense, competition between replication and repair. Mutations may result even
if error-free repair occurs, if the damaged DNA replicates first. In addition,
if the type or extent of DNA damage is such that it is not repaired and persists,
it can cause disturbances in DNA replication and produce sister chromatid
exchanges or chromosomal aberrations, mutations, carcinogenic transformation,
etc. On the other hand, certain kinds of DNA alterations that lead to mutation
and/or cancer either do not stimulate repair processes at all, or do so to
such a small extent that detection is not practicable. Thus, difficulties
exist in using methods for detecting DNA repair to predict genetic risk.
Mutagenic chemicals, such as the intercalating agents ethidium bromide,
acriflavine, actinomycin D, and adriamycin, do not invoke DNA repair synthesis
even at high doses (Cleaver 1968, Painter 1978, Painter and Howard 1978).
Similarly, X rays are mutagenic and carcinogenic but induce very little repair
synthesis (Painter and Young 1972, Regan and Setlow 1974). Also, some metals
that are suspected carcinogens do not elicit detectable repair (Painter 1979).
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Thus, while detection of repair synthesis is useful after exposure to some
mutagens or carcinogens, resolution of important differences within a population
that are relevant to mutagen/carcinogen damage will not always be possible by
this method.
c) Measurement of Chemical Binding to DNA--
The major, current methods for measuring DNA lesions are radioactivity,
immunoassays, and fluorescence. Each of these techniques has certain strengths,
but also some major weaknesses that limit their usefulness.
(1) Radiolabeled DNA probes or mutagens—DNA can be uniformly radiolabeled
by feeding cells radioactive precursors to yield DNA probes which can then be
reacted with modifying agents. These techniques are not applicable for the
detection of DNA lesions in human specimens from environmental exposure. It
is also unfortunate from a practical standpoint that radiolabeled substances,
particularly those of high specific activity needed for ultratrace work, present
hazard, inconvenience, cost, and disposal problems. Post-labeling techniques
with radiolabeled probes can be used to detect DNA lesions in human specimens
(Franklin and Haseltine 1983, Randerath et al. 1981) with high sensitivity
and fingerprinting capability (Gupta et al. 1982), but the same practical
limitations remain because they require the use of radioisotopes.
(2) Immunoassays—Radioimmunoassay and related procedures are important
techniques for ultratrace analysis of DNA lesions. For example, Hsu et al. (1981)
detected 3 fmol (3 x 10~15 moie) of a benzo(a)pyrene-DNA adduct with an immunoassay,
Poirier et al. (1979) similarly detected various acetylaminofluorene-DNA
adducts with detection limits, depending on the immuno-substrate, from 0.5 to
160 pmol, and achieved a detection of 1-2 fmol of platinum DNA adducts per 50
ug of DNA (Poirier et al. 1982, Poirier 1983). Because immunoassays are indirect
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methods, there is a potential for inaccuracy (Houck et al. 1980, Julliard et
al. 1980). Another limitation is that a different imnumoassay is needed for
each DNA modification.
(3) Fluorescence—Detection of complexes between mutagens and DNA
by fluorescence has recently been reviewed (Vigny and Dusquesne 1979). The
major limitations of this approach are that different structures can have
similar flourescent spectra. In addition, some structures are not very fluorescent,
and fluorescent contamination can confuse the results.
(4) Measuring damage in DNA by gas chromatography-mass spectrometry—
McCloskey and co-workers used gas chromatography (GC) with fused silica columns for
the analysis of nucleic acid bases (Gelijkens et al. 1981). The bases were
converted into N^-peralkyl (methyl or ethyl )N-trifluoroacetyl derivatives,
and, after GC separation, were detected by either flame ionization detection,
nitrogen-phosphorous detection, or electron impact mass spectrometry. These
solutes showed good GC behavior, including a good sensitivity (low pg level,
10'10g). There is a potential for higher sensitivity by gas chromatography
with electron capture detection (GC-ECD) and gas chromatography with negative
ion chemical ionization detection (GC-NICI-MS). Detection limits at the femtogram
level for the analysis of standards of cytosine and 5-methylcytosine have
recently been established (Nazareth et al., in preparation; Gemal et al., in
preparation).
The methodology being developed to measure damage to DNA will consist of
the following steps: (1) purification of the DNA; (2) enzymatic or acid hydrolysis
of the DNA, releasing monomers; (3) separation of damaged monomers from normal
monomers by HPLC; and (4) chemical labeling of the damaged monomers with "direct
electrophones," followed by characterization and quantification of these
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electrophone-labeled monomers by GC-ECD or GC-NICI-MS.
Preliminary results with GC-ECD and GC-NICI-MS methodology suggest, at
least for certain standards, that damaged DNA bases can potentially be quantified
at the 10~16 mole level. This translates into an ability to measure nearly
100 damaged monomers per human cell given a sample of 2 x 106 cells or 10 ug
of DNA (a typical number of cells or amount of DNA from a typical human microbiopsy)
This amount of DNA corresponds to that obtainable from about two Petri dishes of
cultured cells. However, this technology has not yet been applied to the
analyses of actual samples.
Much remains to be learned about specific DNA lesions, their repair, and
their implications for mutagenesis and carcinogenesis. Despite the complexities,
simple models can prove valuable in making the necessary first steps to improve
genetic risk assessment. For initial studies with experimental animals, total
DNA alkylation may serve as useful indicators of dose. Eventually, the specific
type of alkylation product, rate of specific adduct repair, amount of cell
replication, and the probability of mispairing of specific adducts needs to be
considered. Although we are presently incapable of accomplishing this goal,
the methodology for conducting such studies is developing rapidly.
III. BIOMONITORING ASSAYS AVAILABLE FOR MAMMALIAN EXPERIMENTATION
Both the direct and indirect approaches can be used to estimate the frequency
of heritable mutations associated with mutagen exposures in experimental mammals.
This is required for the bridge building approaches described later. In animal
studies, exposures can be carefully identified and controlled, and cause and
effect relationships can be demonstrated. Application of the indirect methods for
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both human and animal studies has been described previously. Direct and indirect
methods applicable only for animal experimentation will be described in this
section. These methods are useful for defining intertissue relationships and
for making comparisons with human data in order to strengthen the basis for
extrapolating between species.
Whole mouse tests for putative heritable gene mutations are generally
considered the most valid experimental approaches for making quantitative
mutagenicity risk assessments (NAS 1982, Lyon 1983). Among these are the
morphological and biochemical specific locus tests and tests for dominant
mutations causing skeletal defects or cataracts. Other available tests score
for chromosomal aberrations; these tests include the heritable translocation
test, dominant lethal test, and X chromosome loss test. All of these tests,
except perhaps the dominant lethal test, which cannot be shown to respond only
to mutagenic events, may be used for quantifying genetic risk. An indirect
estimation of heritable genetic effects in mice can be performed using the
mouse spot test.
A. DIRECT METHOD
1. Gene Mutations
a) Specific Locus Test —
The morphological specific locus test has several advantages that arise
from its long historical use in genetic toxicology and genetic risk assessment
(Russell et al. 1981a). For this test, there are good historical control data
with reliable spontaneous mutation frequencies. However, much of the
available information comes from radiation studies (Russell 1951). The data
base on chemicals evaluated in the specific locus test in intact mammals is
small. Only 25-30 chemicals have been evaluated, and of these, there are
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adequate data for only about 15; of the 15, roughly half are inconclusive. There
are dose-response data in specific locus tests on only four chemicals: procarbazine,
mitomycin C, triethylenemelamine, and ethylnitrosourea. It is difficult to
decide on the meaning of negative results in such tests, because large numbers
of animals are required to preclude modest (two- to fourfold) increases in
mutation frequencies. Nevertheless, the specific locus test has the advantage
that the genetic events are reasonably well defined, and it does seem to detect
both gene mutations and deletions.
In the morphological specific locus test, mutations are detected at only
seven loci, and the number of loci is not readily expandable. The ability to detect
genetic events at more loci is one of the advantages of the electrophoretic
specific locus test (Valcovic and Mailing 1973; Johnson et al. 1981). In this
test, there are currently 10 loci at which the two tester strains of mice
differ, permitting the detection of both null mutants and electromorphs. There
are at least 10 additional loci that can be screened for electromorphs only
with the possibility of further expansion. Another advantage of the electrophoretic
specific locus test is that it is directly comparable to the electrophoretic
monitoring system for human populations that has been developed by Neel et al.
(1979) and described above. A real limitation at this stage in the development
of the electrophoretic specific locus test is that the data base on chemically-
induced mutations is small. No spontaneous mutants have yet been recovered in
about 300,000 locus tests, which would suggest a mutation frequency less than
or equal to that of the morphological specific locus test. The system described
here uses the C57BL/6 and DBA/2 inbred mouse strains; other inbred strains
could be used but the number of polymorphic loci would be reduced.
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b) Tests for Dominant Mutations--
Tests for dominant mutations that cause skeletal defects (Ehling 1966,
Selby and Lee 1981) or cataracts (Kratochvilova 1981; Kratochvilova and Ehling 1979)
have been proposed for use in genetic risk assessment. It has been argued
that these tests better approximate the dominant human disease syndromes that
might be expected in the first few generations after significant mutagenic
exposure. The argument has also been made, however, that this advantage may be
overstated for the dominant skeletal test (Lovell et al., submitted) and may
even be trivial. There may be a serious disadvantage in detecting dominant
mutations in particular strains of inbred experimental animals and extrapolating
the result to outbred populations; polygenic traits can appear as simple dominants
in a particular cross that produces a genotype that is near the threshold of
expression. Another factor to consider is that the data base on chemicals is
extremely limited for these tests. So far, there are data only for ethylnitrosourea.
Relative to testing for skeletal mutations, the test for cataracts offers the
advantage that the detection is noninvasive, thereby permitting follow-up
breeding experiments to verify the genetic nature of the alteration.
2. Chromosomal Aberrations
a) Heritable Translocation—
The heritable translocation test detects symmetrical reciprocal exchanges
of chromosomal material between nonhomologous chromosomes which are transmitted
from parents to offspring (Generoso et al. 1980). Translocation heterozygotes
are viable and are detected by sterility or semi-sterility. Confirmation is
made by chromosome analysis. Because heritable translocations are by definition
scored in live progeny, they provide a very definitive and unequivocal
measure of clastogenic effects in the germ cells. Consequently, they are
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generally considered to be the most important clastogenic endpoint with respect
to genetic risk assessment.
The spontaneous occurrence of chromosome breakage-related anomalies in
humans is estimated to be 2,400 per million live births (35), many of which
result from translocations. For example, approximately 20% of the trisomy 13
disorders (Patau's syndrome) are caused by translocations (Magenis et al.
1968, Taylor et al. 1970), which usually involve the transfer of material from
chromosome 13 to chromosome 14. The phenotypic manifestations of translocations
in human carriers are varied, ranging from no perceptible effect to mentally-related
handicaps (UNSCEAR 1977). The exchange usually involves the transfer of material
from chromosome 13 to chromosome 14. Although the total contribution that
heritable translocations make toward all chromosomal disorders has not been
determined adequately, it may be assumed for purposes of estimating human
risk that all newly-occurring translocations are deleterious, if not directly
to the carriers, then at least to some of their conceptuses.
b) Sex Chromosomal Abnormalities--
Approximately O.fi% of human newborns have congenital anomalies which result
from chromosomal aberrations. These may be unbalanced segregation products of
translocations or numerical aberrations (BEIR 1980). Numerical anomalies are
known for both autosomes and sex chromosomes.
A numerical sex chromosome-loss test using mice has been described by
Russell (1976, 1979a, b). Potentially mutagenic agents can be tested in either
sex, and numerical chromosomal disorders can be scored in the Fj generation.
As is the case with most other whole mammal tests, large numbers of animals
are necessary to obtain dose-response relationships at low exposure levels,
necessitating the use of relatively high experimental doses. If experimental
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data on an environmental agent are known, then an estimate of human risk can
be attempted by extrapolation. For example, if mouse experiments show that a
certain exposure to a chemical agent doubles the incidence of sex chromosome
loss (XO), then the incidence of Turner's syndrome in humans may be multiplied
by the same factor following "equivalent" exposure.
One has to assume that an agent causing a positive response in a numerical
sex chromosome study would also cause numerical anomalies (monosomy or trisomy)
among autosomes. Therefore, humans exposed to the chemical agent would be at
risk to other diseases caused by autosomal numerical aberrations. Only four
chemicals (triethylenemelamine, methyl methanesulfonate, isopropyl methanesulfonate,
and hycanthone) and ionizing radiation have been tested in the mouse numerical-
sex chromosome anomaly system (Russell 1976). All of these agents are also
known to cause dominant lethals. The sex chromosome loss test is the only
available test that assesses the capacity of an agent to cause germ cell aneuploidy
in vivo which results in recognizable offspring.
c) Dominant-Lethal Test—
Dominant-lethal mutations are genetic changes in parental germ cells that
cause death of first-generation embryos. It is generally believed that induced
dominant-lethal mutations are due to chromosome breakage events (Bateman and
Epstein 1971; Generoso, in press). Because most point mutations will not be
detected, the dominant-lethal test can only be used to assess an agent's
mutagenic potential in qualitative terms. In general, there appears to be a
close correlation between the induction of dominant-lethal mutations and the
induction of heritable translocations. There are examples, however, of
chemicals that effectively induce dominant lethal mutations with very few or
no heritable translocations (Generoso et al. 1979). A definitive positive
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response in a dominant lethal test serves as a strong indication that a chemical
causes genetic damage in male germ cells. However, the dominant lethal test
is not a sensitive assay and negative responses are not conclusive.
B. INDIRECT METHOD (MOUSE SPOT TEST)
In the mouse spot test, somatic cell mutations occurring in melanocytes
during embryonic development are detected after birth as patches or spots of
altered fur color. It is thought that this technique is capable of detecting
gene mutations, large and small chromosomal deficiencies, nondisjunction or
other chromosome loss, and somatic recombination (Russell 1978, 1979a; Russell
and Major 1957). Pregnant mice are treated about 10 days after conception at a
time when there are an estimated 150-200 melanocyte precursor cells/embryo at
risk. Depending on the crosses used, the fetuses are heterozygous at three or four
coat color loci. At birth, offspring are checked for externally visible morphological
features and examined for spots 12-14 days later. The animals are then re-checked
at least once at 4-5 weeks of age before they are discarded. Data are recorded
as the percentage of animals with marker spots. Positive responses in this
test demonstrate the test agent is a mammalian mutagen, but because somatic
cell events are scored, the data cannot be used to estimate heritable genetic
risk directly. Thirty substances have been tested in this manner. Three were
solvents used in testing the other chemicals. Of the remaining 27 chemicals,
16 were positive, 6 were negative, and 5 were inconclusive (Russell et al.
1981b). Historical control values vary significantly depending on which solvent
was used to administer the test agent. Thus, it is important to compare
experimental results with appropriate controls. It is recommended that a
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concurrent solvent control of at least 150 animals be used (Russell et al.
1981b). Appropriate animal facilities and properly trained personnel are
needed to conduct this test. The test is subject to false positive results.
For instance, certain coat color spots resulting from chemical treatment do
not have a genetic basis but appear to be due to cell-killing. Errors of
differentiation that are not due to genetic damage are also detected in the
test.
C. LIMITATIONS OF ANIMAL TEST DATA FOR ESTIMATING HUMAN GENETIC EFFECTS
The close biological and evolutionary relationship between humans and
other mammals is the basis for estimating heritable human genetic risk from
mouse and rat data. However, there are several limitations associated with
using studies in animals for predicting human responses. One is the difficulty
of accounting for differences in metabolism, repair, and cell cycle kinetics.
Another is the need to extrapolate from high acute dosages, often involving
long sterile periods, to dosage levels that would be more typical of human
exposures. Another limitation in essentially all assays for mutagenesis in
germ cells is the shortage of information on females; the great majority of
the available information comes from males. Consequently, many assumptions
must be made in attempting to project human genetic risk. This leads to the
inescapable conclusion that there is no substitute for genetic data from humans
to calibrate the experimental systems for risk assessment purposes.
IV. IDENTIFICATION OF HUMAN POPULATIONS EXPOSED TO CHEMICAL MUTAGENS
Considerable effort will be required to collect human data for assessing
genetic risk for just one chemical substance will be great, and all sources of
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information ought to be drawn upon to select the appropriate human population
for study. A number of populations at greatest risk should be identified for
potential studies. One is the children of cancer chemotherapy patients. For
1983 it was estimated that there would be in excess of 160,000 cancers that
may have long remissions/cures following effective chemotherapy (Table II)
(Silverberg and Lubera 1983). Around 29,000 of these cancer patients will be
treated at large cancer centers, most with defined protocols of drug administration.
A large number of treated individuals will be of child-bearing age. It is
reasonable to expect similarly large populations in the years to come. Even
if a small fraction of such individuals were employed in an epidemiological
study, it is likely that a meaningful cohort could be assembled in a short
time. This population is clearly not typical of a normal healthy population,
but it provides a model situation where persistent damage to DNA in humans
could be investigated and related to the incidence of mutations in their offspring.
There are other analogous populations as well, and careful thought should be
given in the design of a program to identify and select the most appropriate
ones. Information from these studies would be useful for defining the extent
of genetic hazard and in validating the animal models as predictors of human
risk.
V. DISCUSSION OF APPROACHES FOR THE IMPROVEMENT OF MUTAGENICITY RISK ASSESSMENT
There are many available tests for identifying chemical mutagens. Data
from combinations of tests provide a basis for making qualitative assessments
of the ability of chemical substances to cause gene mutations, chromosomal
aberrations, and other effects that are indicative of interaction with DNA.
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TABLE II. ESTIMATES OF INCIDENCES OF CANCERS WITH LONG REMISSIONS/CURES
Tumor Incidence
Breast Cancers 114,000
Ovarian Cancers 18,200
Hodgkin's Lymphomas 7,100
Non-Hodgkin's Lymphomas 23,600
162,900
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However, only a few tests (i.e., heritable gene mutation and heritable translocation
tests in mice) can be used by themselves for quantitatively assessing genetic risk.
They are not routine tests, however, and they cannot be used to estimate human
genetic risk directly. By default, assessment of genetic risk must be done
qualitatively for most chemical substances.
For decision-making by the Federal government, it is no longer adequate to
merely qualitatively assess genetic risk; quantitative assessments are needed
to balance the risk associated with exposure to a chemical against the benefit
of its use. Because our understanding of interorgan relationships is inadequate,
human monitoring data cannot be used for such purposes. Many assumptions have
to be made before genetic risk can be estimated quantitatively, and the assessments
are not scientifically rigorous. Because of practical and legal considerations,
it is important to make optimal use of all sources of information in future
genetic risk assessment efforts and to develop the science to a point where
rigorous assessments can be made.
A. BRIDGING HUMAN BIOMONITORING ENDPOINTS WITH ANIMAL EXPERIMENTATION
(Defining the Relationships)
Each test system has advantages and limitations for assessing genetic
risk to humans. It is only by determining how the endpoints measured in these
tests relate to events that occur in humans that full advantage can be taken
of each. Comparative experimentation involving different endpoints, test
systems, and chemicals is required to build "intellectual bridges" between the
systems.
Studies in mice can be used by themselves to predict mutagenic effects
in humans because the same range of steps between external exposure and production
of mutant offspring occurs in all mammals (Jackson, in preparation) (Figure 1).
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Figure 1. An approach for examining the process leading from external exposure
to expression of genetic disease and somatic cell effects is outlined in this
Figure. It is derived into five major steps (i.e., external exposure, internal
exposure, dose, potential risk, and expressed risk) each represented by a
separate column. The top row represents events occurring in the germinal
tissue of males, while the bottom row represents the tissue of those occurring
in females. The middle row corresponds to somatic cell events. The squares
represent potentially measureable events. The circles represent processes
that cannot be measured but that can be estimated from measurements of the
events represented by squares.
31
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32
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Measurements of somatic cell and germ cell events should be performed in
mice in such a way that the relationships between biomonitoring markers and
health outcomes of concern (i.e., genetic disease, cancer, and birth defects)
can be determined (Figure 2). Such an approach will provide a basis for using
biomonitoring endpoints to predict adverse health effects. It wil1 also enable
comparisons to be made between different health outcomes in order to determine
their sensitivity of expression and predictability.
It is necessary to conduct at least one study in such a manner so that the
data generated in an experimental animal study can be compared to the results
obtained from similarly exposed humans. Until this is done, the applicability
of animal data for human risk assessment will be unknown. A large number of
tissues are available for routine study in animal tests, but only a limited
variety and amount of tissue will be available for study in "routine" human
biomonitoring studies (i.e., blood and sperm). However, for "bridge-building"
purposes a larger variety and amount of tissue is potentially available (e.g.,
tissue that normally would be discarded after surgery on cancer patients).
B. OTHER TYPES OF TESTING NEEDED
Because it has not been nor will it likely be possible to study genetic
damage in more than a very few human populations, it will be necessary to rely
heavily on animal experimentation and short-term biomonitoring tests to predict
human risk. This requires bridge building between human heritable mutagenicity
data, other human biomonitoring data, and animal heritable mutagenicity and
biomonitoring data. Although the specific models that can be used to make
bridges remain controversial, there seems to be some agreement that ratios
(or parallelograms as they are sometimes called) involving dosimetry should be
explored further (Sobels 1981 a, b; Mailing 1981; Lyon 1983). As such
33
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Figure 2. Relationships between biomonitoring endpoints (markers) and disease
outcomes of concern. Understanding these relationships is required to be able
to use biomonitoring data for estimating risk. This requires comparative
experimentation measuring different markers and outcomes in the same or similarly
exposed individuals. This diagram shows a comparative approach for defining
interorgan and interspecies relationships.
34
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studies are conducted, knowledge of biological processes rather than statistical
models can start to drive the risk assessment procedure. Flexibility should
be maintained so that the risk assessment process works differently when different
amounts of data are available and so that the level of sophistication can be
increased as better data become available. In addition to providing more
precise risk assessments, maintaining the flexibility needed to better risk
assessments would foster the application and support of basic research for the
risk assessment procedure.
1. Cell-Specific Effects
The effects caused by a chemical in one cell-type might not predict
its effects in a different cell-type. Thus, it is important to consider
cellular specificities of certain chemicals. For example, based on studies
investigating carcinogen metabolism, it is known that the metabolic competence
of different cell populations within the target organ can affect the extent of
DNA adduct formation as well as toxicity-induced compensatory cell proliferation.
For example, removal of covalently bound 2-AAF has been shown to be similar in
hepatocytes and nonparenchymal cells of the liver (NPC), whereas major differences
have been demonstrated for removal of O^-alkylguanine (Swenberg et al. 1984).
In the latter case, cell specificity is also dependent on chemical reactivity
with DNA. The major premutagenic lesion induced in DNA by SNj methylating
agents is O^-methylguanine (0^ MG) (Singer 1975). In contrast, ethylating
agents produce a greater proportion of 0-alkylated premutagenic pyrimidines,
relative to 0-alkylation of guanine. These differences can perhaps be best
appreciated if one considers some recent data (Swenberg et al. 1984) which has
shown that O^-ethylthymidine progressively accumulates in hepatocytes over 4
weeks of exposure to diethylnitrosamine (DEN). In contrast, O^-ethylguanine
36
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does not accumulate. Although these relationships have been elucidated only
in the liver cells, they likely occur also in cell-types of other organs.
2. Homeostatic Mechanisms
Attention also should be paid to the homeostatic mechanisms of humans and
experimental animals that may be important with respect to disease outcomes.
The stage of the cell cycle, level of differentiation, and location in the
body all affect a tissue's response to toxic insults. Somatic cell mutations,
induced in the cells of a tissue with great regenerative potential, may ultimately
compromise the health of exposed individuals more severely than if the toxic
insult had resulted in cell death. For instance, mutagenesis of pluripotent
stem cell lines or committed cell lines of the bone marrow may result in the
appearance of late onset diseases such as myeloid disorders (e.g., anemias,
polycythemias), myeloproliferative and lymphoproliferative disorders (e.g.,
leukemias and cyclic neutropenia), and irregular immunological processes (e.g.,
cytopenia, immune complex disease, toxic epidermal necrolyis, lymphadenopathy,
and autoimmune diseases).
C. NEED FOR COORDINATION OF EFFORTS
To ensure maximum efficiency in collecting relevant data, it is desirable
to search systematically for information on existing efforts related to genetic
risk assessment rather than to attempt to set up overlapping independent studies.
Although effective collaboration among agencies is still sporadic, there are
some promising developments. For example, the coordination between the U.S.
Environmental Protection Agency and the National Toxicology Program (NTP) to
obtain dosimetric information on chemicals being tested in the mouse specific
locus tests should enable existing NTP studies to better be used for purposes
37
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of genetic risk assessment. It now seems appropriate that an oversight committee
be formed for guidance on needs in human biomonitoring and to facilitate the
coordination of efforts in genetic risk assessment.
The research effort required to answer major questions in genetic risk
assessment must be cumulative rather than episodic, and funding for these
efforts should endure over many years. With proper support, an oversight
committee could help to ensure that this is accomplished. Investigators studying
exposure or genotoxic damage in different tissues and different organisms must
somehow integrate and focus their efforts. More effective sharing of valuable
materials would certainly be useful in this respect. Within the purview of
an oversight committee, a repository for biological materials obtained from
animals and humans that have been exposed to putative mutagens should be established.
A series of'regularly scheduled workshops should be inaugurated to facilitate
collaboration. These workshops should include investigators using the biological
materials in the repository and should provide for a cumulative review, comparison
of results, and the identification of research needs.
It may be worthwhile to consider the selection of a few key compounds for
concentrated, long-term efforts in genetic risk assessment. Since a long-term
effort to collect data on chemical mutagenesis in human germ cells is a major
undertaking, with important implications for genetic risk assessment, it should
not be undertaken lightly. There are other issues that could be considered by
an oversight committee.
The scientific and administrative issues in genetic risk assessment
are too complex to be resolved here, and consequently, follow-up efforts are
warranted. This entails coordinating efforts among the Federal agencies and
among government agencies, industrial concerns, and the academic community*
38
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Some preliminary efforts are underway, and it is hoped that these efforts will be
expanded.
39
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VI. REFERENCES
Albertini RJ (1982): Studies with T-lymphocytes. An approach to human mutagen-
icity monitoring. In Bridges BA, Butterworth BE, Weinstein IB (eds):
"Indicators of Genotoxic Exposure." Banbury Report 13. Cold Spring Harbor,
New York: Cold Spring Harbor Laboratory Press, pp 393-412.
Albertini RJ, Castle KJ, Borcherd WR (1982): T cell cloning to detect the
mutant 6-thioguanine-resistant lymphocytes present in human peripheral
blood. Proc Natl Acad Sci USA 79:6617-6621.
Ashby J (1983): ICPEMC Working Paper 1/1. The unique role of rodents in the
detection of possible human carcinogens and mutagens. Mutat Res 115(2):
177-213.
Atwood KC (1958): The presence of A£ erythrocytes in AI blood. Proc Natl Acad
Sci (USA) 44:1054-1057.
Atwood KC, Petter FJ (1961): An erythrocyte automosaicism in some persons of
known genotype. Science 134:2100-2102.
Atwood KC, Scheinberg SL (1958): Somatic variation in human erythrocyte antigens.
J Cell Comp Physiol 52:97-123.
Atwood KC, Scheinberg SL (1959): Isotope dilution method for assay of
inagglutinable erythrocytes. Science 129:963-964.
Bateman AJ, Epstein SS (1971): Dominant lethal mutations in mammals. In
Hollaender A (ed): "Chemical Mutagens-Principles and Methods for Their
Detection," Vol 2. New York: Plenum Press, pp 541-568.
Beatty RA (1977): F-bodies Y chromosome markers in mature human spermheads: A
quantitative approach. Cytogenet Cell Genet 18:33-49.
BEIR (1980): "Biological Effects of Ionizing Radiation III." The effects on
populations of exposure to low levels of ionizing radiation. Washington,
DC: National Academy Press, pp 91-181.
Bigbee WL, Branscomb EW, Jensen RH (1983): Counting of RBC variants using
rapid flow techniques in environmental sciences research. In deSerres FJ,
Sheridan W (eds): "Utilization of Mammalian Specific Locus Studies in
Hazard Evaluation and Estimation of Genetic Risk". New York and London:
Plenum Press, pp 39-54.
Bloom AD (1980): The genetics task force of New York. Prototype organization
for state-wide delivery of genetic services. Am J Hum Genet 32:A99.
Brown WT, Ezer A (1982): A computer program using Gaussian fitting for evaluation
of two-dimensional gels. Clin Chem 28:1041-1044.
Cleaver JE (1967): Thymidine metabolism and cell kinetics. Amsterdam, The
Netherlands: North Holland Publishing Company, 259 pp.
40
-------
Cleaver JE (1968): Repair replication of mammalian DNA: Effects of compounds
that inhibit DNA synthesis or dark repair. Radiat Res 37:334-348.
Cleaver JE (1974): Repair processes for photochemical damage in mammalian
cells. In Lett JT, Adler H, Zelle M (eds): "Advances in Radiation
Biology," Vol. 4. New York: Academic Press, pp 1-75.
Ehling UH (1966): Dominant mutations affecting the skeleton in offsprir.g of
x-irradiated mice. Genetics 54:1381-1389.
Ehrenberg L, Moustacchi E, Osterman-Golkar S, with an Appendix II by Ekman G
(1983): Working paper 4/4. Dosimetry of genotoxic agents and dose-response
relationships of their effects (MTR07163). Mutat Res 123(2):121-182.
Flamm WG (1977): Approaches to determining the mutagenic properties of chemicals:
Risk to future generations. J Environ Pathol Toxicol 1:301-352.
Franklin WA, Haseltine WA (1983): The use of post-labeling methods to detect
and characterize infrequent base modifications in DNA. In Friedberg ED,
Hanawalt PC (eds): "DNA Repair." Vol. II. New York: Marcel Dekker,
pp 161-171.
Gallatin WM, Weissman IL, Butcher EC (1983): A cell-surface molecule involved
in organ-specific homing of lymphocytes. Nature 304:30-34.
Gelijkens CF, Smith DL, McCloskey JL (1981): Capillary gas chromatography of
pyrimidines and purines: N,0-peralkyl and trifluoroacetyl-N,0-alkyl
derivatives. J Chromatography 225:291-299.
Gemal A, Nazareth A, Joppich M, Giese RW, Vouros P: In preparation.
Generoso WM, Bishop JB, Gosslee GD, Newell GW, Sheu CJ, von Halle E (1980):
Heritable translocation in mice. Mutat Res 76:191-215.
Generoso WM, Huff SW, Cain KT (1979): Relative rates at which dominant-lethal
mutations and heritable translocations were induced by alkylating chemicals
in post-meiotic male germ cells of mice. Genetics 93:163-171.
Gocke E, Eckhardt K, King MT, Wild D (1983): Autoradiographic detection of
6-thioguanine resistant lymphocytes of mice: A novel system in somatic
mutagenesis testing. Mutat Res 113:455-456.
Gupta RC, Reddy MV, Randerath K (1982): 32P-postlabeling analysis of non-radio-
active aromatic carcinogen-DNA adducts. Carcinogenesis 3(9):1081-1092.
Heddle JA, Hite M, Kirkhardt B, Mavourin K, MacGregor JT, Newell GW, Salamone MF
(1983): The induction of micronuclei as a measure of genotoxicity. A report
of the U.S. Environmental Protection Agency Gene-Tox Program. Mutat Res
123:61-118.
41
-------
Hook EB (1983): International Commission for Protection Against Environmental
Mutagens and Carcinogens working paper 5/3. Perspectives in Mutation
Epidemiology: 3. Contribution of chromosome abnormalities to human morbidity
and mortality and some comments upon surveillance of chromosome mutation
rates. Mutat Res 114(3) :389-423.
Houck JC, Kimball C, Chang C (1980): Placenta! g -endorphin-1 ike peptides.
Science 208:78-79.
Hsu I, Poirier MC, Yuspa SH, Grunberger D, Weinstein IB, Yolken RH, Harris CC
(1981): Measurement of benzo(a)pyrene-DNA adducts by enzyme immunoassays
and radioimmunoassay. Cancer Res 41:1091-1095.
International Commission for Protection Against Environmental Mutagens and
Carcinogens (ICPEMC) (1983a): Editorial introduction to papers produced by
Committee 4 of ICPEMC on "Risk Estimates of Genotoxic Chemicals." Mutat
Res 114(1):91.
International Commission for Protection Aganist Environmental Mutagens and
Carcinogens (1983b): Committee 1. Final report: Screening strategy for
chemicals that are potential germ-cell mutagens in mammals. Mutat Res
International Commission for Protection Against Environmental Mutagens and
Carcinogens (1983c): Committee 3. Final report: Regulatory approaches to
the control of environmental mutagens and carcinogens. Mutat Res 114(2):
179-216.
International Commission for Protection Aganist Environmental Mutagens and
Carcinogens (1983d): Editorial introduction to papers produced by Committee
5 of ICPEMC on epidemiological studies on populations exposed to mutagenic
and carcinogenic agents. Mutat Res 114(3) :387-388.
International Commission for Protection Against Environmental Mutagens and
Carcinogens (1983e): Committee 5. Final report: Mutation epidemiology
review and recommendations (MTR07157). Mutat Res 123(1):1-11.
International Commission for Protection Aganist Environmental Mutagens and
Carcinogens (1983f): Editorial introduction to papers produced by Committee
4 of ICPEMC on risk estimates of genotoxic chemicals (MTR07163i). Mutat
Res 123(2):119-120.
Jackson EJ: Basic parameters to be considered in animal to human mutagenicity
extrapolations. In preparation.
Johnson FM, Roberts GI, Sharma RK, Chasalow F, Zweidinger R, Morgan A, Herdren
RW, Lewis SE (1981): The detection of mutants in mice by electrophoresis:
Results of a model induction experiment with procarbazine. Genetics
97:113-124.
Julliard JH, Shibasaki T, Ling N, Guillemin R (1980): High-molecular-weight
immunoreactive g -endorphin in extracts of human placenta is a fragment of
immunoglobulin G. Science 208:183-185.
42
-------
Kapp RW, Jr., Picciano DO, Jacobson CB (1979): Y-chromosomal nondisjunction in
dibromochloropropane-exposed workmen. Mutat Res 64:47-51.
Kavathas P, Bach FH, DeMars R (1980): Gamma ray-induced loss of expression of
HLA and glyoxalase I alleles in lymphoblastoid cells. Proc Natl Acad
Sci (USA) 77:4251-4255.
Kratochvilova J (1981): Dominant cataract mutation detected in offspring of
gamma irradiated male mice. J Hered 72:302-307.
Kratochvilova J, Ehling UH (1979): Dominant cataract mutations induced by gamma
irradiation of male mice. Mutat Res 63:221-223.
Latt SA, Allen J, Bloom SE, Carrano A, Falke E, Kram D, Schneider E, Schreck R,
Tice R, Whitfield B, Wolff S (1981): Sister-chromatid exchanges: A report
of the gene-tox program. Mutat Res 87:17-62.
Lovell DP, Leverton D, Johnson FM: Lack of evidence for skeletal abnormalities
of offspring of mice exposed to ethyl nitrosourea (ENU). Submitted.
Lyon MF (1983): Problems in extrapolation of animal data to humans. In deSerres
FJ, Sheridan W (eds): "Utilization of Mammalian Specific Locus Studies in
Hazard Evaluation and Estimation of Genetic Risk." New York: Plenum Press,
pp 289-306.
Magenis RE, Hecht F, Mulham S (1968): Trisomy 13 (D) Syndrome: Studies of
parental age, sex ratio, and survival. J Pediatr 73:222-228.
Mailing HV (1981): Perspectives in mutagenesis. Environ Mutag 3:103-108.
Matsunaga E (1983): International Commission for Protection Against Environmental
Mutagens and Carcinogens working paper 5/5. Perspectives in mutation
epidemiology: 5. Modern medical practice versus environmental mutagens:
their possible dysgenic impact. Mutat Res 114(3)-.449-457.
Miller JR (1983): International Commission for Protection Against Environmental
Mutagens and Carcinogens working paper 5/4. Perspectives in mutation
epidemiology: 4. General principles and considerations. Mutat Res 114(3):
425-447.
Miller MJ, Vo PK, Nielsen C, Geiduschek EP, Xuong NH (1982): Computer analysis
of two-dimensional gels: Semi-automatic matching. Clin Chem 28:867-875.
Morley AA, Trainor KJ, Seshadera R, Ryall RG (1983): Measurement of in vivo
mutation in human lymphocytes. Nature 302:155-156.
National Academy of Sciences (NAS) (1982): Identifying and estimating the
genetic impact of chemical environmental mutagens. Washington, DC: National
Academy Press, 316 pp.
Nazareth A, Joppich M, Abdel-Baky S, O'Connell K, Sentissi A, Giese RW:
Electrophone-labeling and alkylation of nucleic acid pyrimidine bases for
analysis by GC-ECD. In preparation.
43
-------
Neel JV (1971): The detection of increased mutation rates in human populations.
Perspect Biol Med 14:522-534.
Neel JV (1979): Some trends in the study of spontaneous and induced mutation
in man. Genetics 92:S25-S37.
Neel JV (1981): In quest of better ways to study human mutation rates. In
Hook E (ed): "Human Mutation: Biological and Population Aspects." New
York: Academic Press, pp 361-378.
Neel JV (1983): Frequency of spontaneous and induced "point" mutations in
higher eukaryotes. J Hered 74:2-15.
Neel JV, Schull WJ (1956): The effect of exposure to the atomic bombs on
pregnancy termination in Hiroshima and Nakasaki. Publication No. 461.
Natl Acad Sci National Research Council, Washington, DC, pp XVI and 241.
Neel JV, Rothman E (1981): Is there a difference among human populations in the
rate with which mutation produces electrophoretic variants. Proc Natl Acad
Sci USA 78:3108-3112.
Neel JV, Mohrenweiser HW, Satoh C, Hamilton HB (1979): A consideration
of two biochemical approaches to monitoring human populations for a change
in germ cell mutation rates. In Berg K (ed): "Genetic Damage in Man Caused
by Environmental Agents." New York: Academic Press, pp 29-47.
Neel JV, Mohrenweiser HW, Meisler MW (1980a): Rate of spontanenous mutation at
human loci encoding protein structure. Proc Natl Acad Sci USA 77:6037-6041.
Neel JV, Satoh C, Hamilton HB, Otake M, Gorik K, Kageoka T, Fujita F, Neriishi
S, Asakawa J (1980b): Search for mutations affecting protein structure in
children of atomic bomb survivors. Preliminary report. Proc Natl Acad S.ci
USA 77:4221-4225.
Neel JV, Nishimoto Y, Goriki K, Satoh C, Fujita M, Yoshimoto Y: Epidemiological
studies of germ cell mutation in human populations, with particular reference
to groups with unusual chemical exposures. In Vouk VB (ed): "Proceedings,
Workshop on Quantitative Estimation of Risk to Human Health from Chemicals."
Rome: International Program for Chemical Safety. In press.
Painter RB (1978): DNA synthesis inhibition of HeLa cells as a simple test
for agents that damage DNA. J Environ Pathol Toxicol 2:65-78.
Painter RB (1979): DNA-synthesis inhibition in HeLa cells as a simple test for
agents that damage DNA. Environ Pathol 2:65-78.
Painter RB, Young BR (1972): Repair replication in mammalian-cells after x-
irradiation. Mutat Res 14:225.
Painter RB, Howard R (1978): A comparison of the HeLa DNA synthesis inhibition
test and the Ames test for screening of mutagenic carcinogens. Mutat
Res 54:113-115.
44
-------
Petzold GL, Swenberg JA (1978): Detection of DNA damage induced in vivo follow-
ing exposure of rats to carcinogens. Cancer Res 38:1589-1594.
Pious D, Soderland C (1977): HLA variants of cultured human lymphoid cells:
Evidence of mutational origin and estimation of mutation rate. Science
197:769-771.
Poirier MC (1983): Immunoassays of cis-platinum bound to DNA in vivo a^d JJT_
vitro. Subcommittee on Environmental Mutagenesis (SEM) meeting on DNA
adducts as potential dosimeters for monitoring human populations exposed
to environmental mutagens and carcinogens. National Institutes of health,
Bethesda, MD, September 23, 1983.
Poirier MC, Dubin MA, Yuspa SH (1979): Formation and removal of specific
acetylaminofluorene-DNA adducts in mouse and human cells measured by
radioimmunoassay. Cancer Res 39:1377-1381.
Poirier MC, Lippard SJ, Zwelling LA, Ushay HM, Kerrigan D, Thill CC, Santella
RM, Gruneberger D, Yuspa SH (1982): Antibodies elicited against cis-
diamminedichloroplatinum (Il)-modified DNA are specific for cis-
diamminedichloroplatinum (II)-DNA adducts formed in vivo and in vitro.
Proc Natl Acad Sci USA 79:6443-6447.
Popp RA, Bailiff EG, Skow LC, Johnson FM, Lewis SE (1983): Analysis of a mouse-
globin gene mutation induced by ethyl nitrosourea. Environ Mutagen 5:495.
Abstract.
Preston RJ, Au W, Bender MA, Brewen JG, Carrano AV, Heddle JA, McFee AF, Wolff
S, Wasson JS (1981): Mammalian jn_ vi_vp_ and i_n_ \rvtro cytogenetic assays. A
report of the U.S. Environmental Protection Agency's Gene-Tox Program.
Mutat Res 87:143-188.
Ramel C (1983): International Commission for Protection Against Environmental
Mutagens and Carcinogens publication No. 8. Polygenic effects and genetic
changes affecting quantitative traits. Mutat Res 114(1):107-116.
Randerath K, Reddy MV, Gupta RC (1981): 32P-labeling test for DNA damage.
Proc Natl Acad Sci USA 78:6126-6129.
Recio L, Chastenay B, Albertini RJ, Hsie AW (1983): Detection of 6-thioguanine
resistance spleenocytes from Chinese hamsters. Environ Mutagen 5:401-402.
Abstract.
Regan JD, Setlow RB (1974): Two forms of repair in the DNA of human cells
damaged by chemical carcinogens and mutagens. Cancer Res 34:3318-3325.
Russell LB (1976): Numerical sex-chromosome anomalies in mammals: Their
spontaneous occurrence and use in mutagenesis studies. In Hollaender A
(ed): "Chemical Mutagens," Vol. 4. New York: Plenum Press, pp 55-91.
Russell LB (1979a): In vivo somatic mutation systems in the mouse. Genetics
92:S153-S163.
45
-------
Russell LB (1979b): Meiotic nondisjunction in the mouse. Methodology for genetic
testing and comparison with other methods. Environ Health Perspect
31:113-128.
Russell LB, Major MH (1957): Radiation-induced presumed somatic mutations in
the house mouse. Genetics 42:161-175.
Russell LB, Selby PB, von Halle E, Sheridan W, Valcovic L (1981a): The mouse
specific-locus test with agents other than radiations. Mutat Res
86:329-354.
Russell LB, Selby PB, von Halle E, Sheridan W, Valcovic L (1981b): Use of the
mouse spot test in chemical mutagenesis. Interpretation of past data and
recommendations for future work. Mutat Res 86:355-379.
Russell WL (1951): X-ray induced mutations in mice. Cold Spring Harbor Symp
Quant Biol 16:327-336.
Russell WL (1977): Mutation frequencies in female mice and the estimation of
genetic hazards of radiation in women. Proc Nat! Acad Sci USA 74(8):3523-
3527.
Russell WL (1982): Factors affecting mutagenicity of ethylnitrosourea in the
mouse specific-locus test and their bearing on risk estimation. In Sugimura
T, Kondo S, Takabe H (eds): "Environmental Mutagens and Carcinogens."
Proceedings of the 3™ International Conference on Environmental Mutagens,
Tokyo, Mishima, and Kyoto. September 21-27, 1981. New York: Alan R. Liss
Inc., pp 59-70.
Satoh C, Neel JV, Yamashita A, Goriki K, Fujita M, Hamilton HB (1983): The
frequency among Japanese of heterozygotes for deficiency variants of
11-enzymes. Am J Hum Genet 35(4):656-674.
Schmid W (1976): The micronucleus test for cytogenetic analysis. In Hollaender
A (ed): "Chemical Mutagens. Principles and Methods for Their Detection,"
Vol. 4. New York: Plenum Press, pp 31-54.
Scudiero D, Novin A, Karran P, Strauss B (1976): DNA excision-repair deficiency
of human peripheral blood lymphocytes treated with chemical carcinogens.
Cancer Res 36:1397-1403.
Selby PB, Lee SS (1981): Sensitive-indicator results show that ethylnitrosouf-ea
is also a super mutagen for dominant skeletal mutations. Environ Mutagen
3:373.
Silverberg E, Lubera JA (1983): A review of American Cancer Society estimates
of cancer cases and deaths. Ca-A Cancer Journal for Clinicians 33(l):2-25.
Singer B (1975): The chemial effects of nucleic acid alkylation and their
relation to mutagenesis and carcinogenesis. Progr Nucleic Acid Res Mol
Biol 15:219-284.
46
-------
Skolnick MM, Sternber SR, Neel JV (1982): Computer-programs for adapting 2-
dimensional gels to the study of mutations. Clin Chem 28:969-978.
Sobels FH (1981a): Establishment of requirements for estimation of risk for the
human population. In deSerres FJ, Shelby MD (eds): "Comparative Chemical
Mutagenesis." New York: Plenum Press, pp 1067-1101 (Chapter 37).
Sobels FH (1981b): The parallelogram. An indirect approach for the assessment
of genetic risks from chemical mutagens. In Bora KC, Douglas PR, Nestman
ER (eds): "Progress in Mutation Research." Amsterdam: Elsevier/North
Holland, pp 323-328.
Stamatoyannopoulos G (1979): Possibilities of demonstrating point mutations
in somatic cells as illustrated by studies of mutant hemoglobin. In Berg K
(ed): "Genetic Damage in Man Caused by Environmental Agents." New York:
Academic Press, pp 49-52.
Stamatoyannopoulos G, Nute PE'(1983): Detection of somatic mutants in hemoglobin
in environmental sciences research. In deSerres FJ, Sheridan W (eds):
"Utilization of Mammalian Specific Locus Studies in Hazard Evaluation and
Estimation of Genetic Risk," Vol 28. New York and London: Plenum Press,
pp 29-38.
Stamatoyannopoulos G, Wood WG, Papayannopoulou TH, Nute PE (1975): An atypical
form of hereditary persistance of fetal hemoglobin in blacks and its
association with cycle cell traits. Blood 46:683-692.
Stich HF, Lam P, Lo LW, Koropatnick DJ, San HC (1982): The search for relevant
short-term bioassays for chemical carcinogens. The tribulation of a modern
sisyphus. Genet Cytogenet 17:471-492.
Strauss GH (1982): Direct mutagenicity testing. The development of a clonal
assay to detect and quantitate mutant lymphocytes arising in vivo. In
Bridges BA, Butterworth BE, Weinstein IB (eds): "Indicators of Genotoxic
Exposure." Banbury Report 13. Cold Spring Harbor, New York: Cold Spring
Harbor Laboratory Press, pp 423-441.
Strauss GH, Albertini RJ (1979): Enumeration of 6-thioguanine-peripheral blood
lymphocytes in man as a potential test for somatic cell mutations arising
in vivo. Mutat Res 61:353-379.
t
Streisinger G (1983): International Commission for Protection Against Environ-
mental Mutagens and Carcinogens working paper 4/2. Extrapolations from
species and from various cell types in assessing risks from chemical
mutagens. Mutat Res 114(1):93-105.
Sutton HE (1972): In Sutton HE, Harris MI (eds): "Mutagenic Effects of Environ-
mental Contaminants." New York: Academic Press, pp 121-128.
Sutton HE (1974): Somatic cell mutations in birth defects. In Motulski AC, Lenz
W (eds): "Proceedings of the Fourth International Conference." Amsterdam:
Excerpta Medica, pp 212-214.
47
-------
Swenberg JA, Dyroff MC, Bedell MB, Popp JA, Huh N, Kirstein U, Rajewsky MF
(1984): 0^-ethyldeoxythymidine, but not 06-ethyldeoxythymidine accumulates
in DNA of heptocytes of rats exposed continuously to diethylnitrosamine
Proc Natl Acad Sci USA 81(6):1692-1695.
Taylor MB, Juberg R, Jones B, Johnson WA (1970): Chromosomal variability in
the D! trisomy syndrome. Am J Dis Child 120:374-381.
UNSCEAR (1977): United Nations Scientific Committee on the Effects of Atomic
Radiation." Sources and effects of ionizing radiation. Report to the
General Assembly, with Annexes Publ. E771X1, (United Nations, NY), 725 pp.
Valcovic LR, Mailing HV (1973): An approach to measuring germinal mutations in
the mouse. Environ Health Perspect 6:201-205.
Vigny P, Dusquesne M (1979): Fluorimetric detection of DNA-carcinogen complexes,
In Grover PL (ed): "Chemical Carcinogens and DNA." Boca Raton, Florida:
CRC Press, pp 85-110.
Wood WG, Stamatoyannopoulos G, Lim G, Nute PE (1975): F-cells in the adult.
Normal values and levels in individuals with hereditary an acquired
elevations of HbF. Blood 46:671-682.
Wyrobek AJ, Gordon LA, Burkhart JG, Francis MW, Kapp, RW Jr., Letz G, Mailing
HV, Topham JC, Whorton MD (1983a): An evaluation of the mouse sperm
morphology test and other sperm tests in non human mammals. Mutat Res
115:1-72.
Wyrobek AJ, Gordon LA, Burkhart JG, Francis MW, Kapp, RW Jr., Letz G, Mailing
HV, Topham JC, Whorton MD (1983b): An evaluation of human sperm as
indicators of chemically induced alterations of spermatogenic function.
Mutat Res 115:73-148.
48
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