Below are the first 10 and last 10 pages of uncorrected machine-read text (when available) of this chapter, followed by the top 30 algorithmically extracted key phrases from the chapter as a whole.
Intended to provide our own search engines and external engines with highly rich, chapter-representative searchable text on the opening pages of each chapter.
Because it is UNCORRECTED material, please consider the following text as a useful but insufficient proxy for the authoritative book pages.
Do not use for reproduction, copying, pasting, or reading; exclusively for search engines.
OCR for page 141
10
Conclusions and Recommendations
There is a paucity of information on
the prevalence of naturally occurring
reproductive abnormalities and the preva-
lence of reproductive abnormalities in-
duced by physical agents or toxic chemicals
in the human male. Two major reproductive
health outcomes are of concern when human
males are exposed to toxic agents: patho-
physiologic changes (e.g., reduction in sper-
matozoal concentration, decrease in mo-
tility, increase in proportion of sperm
with abnormal forms), which might or might
not be associated with fertility status,
and heritable genetic damage. Chromosomal
and genie abnormalities in parental germ
cells may lead to reduced fertility, early
or late pregnancy loss, congenital malfor-
mations, or other defects and diseases
in the offspring, some of which may not have
a health effect until later in adult life.
The biologic markers used to assess patho-
physiologic changes in human males in
response to exposure to toxic chemicals
are potency, fertility, serum concentra-
able genetic damage in the human male ge-
nome in response to exposures can include
pregnancy outcome, presence of sentinel
phenotypes (e.g., that of achondropla-
sia), chromosomal damage, and physico-
chemical changes in macromolecules of
offspring (e.g., occurrence of electro-
phoretic variants of red blood cell en-
zymes). These genetic markers have thus
far been used to measure the spontaneous
rate of human germinal mutations but have
Proved ineffective for assessing exposure
to or effects of suspected mutagenic chemi-
cals in humans.
One of the major problems with the use
of animal models is the difficulty in ex-
trapolation of their results to human
beings. Some of the reasons follow, and
research to address these questions is
encouraged:
· Few toxicologic studies in laboratory
animals have been directed at male repro-
ductive health, relative to the large num-
t~ons of gonadal steroids and pituitary ber of potentially toxic chemicals to
hormones, and semen characteristics. which people are exposed.
Blood serum concentration of gonadal ster- · Studies vary in their experimental
old and pituitary hormones and several
of the semen markers, especially sperm
count, motility, and morphology, have been
used to identify human male reproductive
hazards.
Currently available markers of herit-
141
. .
design, species used, dose and route of
administration of toxic chemical, and
choice of markers. That makes it difficult
to compare results of different studies
and results from different laboratories.
· Few animal studies have yielded infor-
OCR for page 142
142
mation on the correlation of markers
with fertility or reproductive outcomes.
· Techniques for extrapolating data
from laboratory animals to human males
are rudimentary and not validated.
New biologic markers of reproductive
and genetic toxicity in the laboratory
human male are needed. The male reproduc-
tive system consists of several organs
that interact in a complex manner with each
other and with the neural and endocrine
systems. A medical history and a physical
examination, although important, are
unlikely to detect exposure to a variety
of toxic chemicals or effects on male re-
production. However, case reports that
tentatively link infertility or abnormal
reproductive outcome with particular
occupations, chemicals, exposures, or
drugs might be valuable indicators that
lead to identification of new human male
reproductive toxicants. Limiting the
analysis to individual markers might be
an oversimplified approach. Instead, a
battery of markers that reflects a wide
array of reproductive functions and herit-
able genetic damage is required, in com-
bination with a thorough medical history
and physical examination. Finally, it
must be remembered that some changes in
markers could be so subtle that important
alterations in response to toxic insult
will be seen only in a large population.
The subcommittee makes the following
general recommendations:
· Extensive basic research in labora-
tory animals must continue to identify
additional markers of physiologic func-
tion and heritable genetic damage.
· Markers of exposure to toxic chemicals
must be correlated with markers of effect
and with changes in reproductive health
in the human male.
· Mechanisms of toxicity must be inves-
tigated in laboratory animals and in vitro
culture systems and related to the human
response.
· Controlled reproductive toxicologic
studies must be completed on several spe-
cies to validate markers.
· Risk assessment procedures must be
developed, to allow data from laboratory
AfALE REPRODUCTIVE TOXICOLOGY
animals exposed to toxic chemicals to be
extrapolated to human males.
· Human markers that measure the effec-
tive magnitude of toxic exposure or
effect on germ cell, epididymis, and other
reproductive organs must be identified.
· New markers must be evaluated with
cross-sectional and longitudinal data
for individuals and for groups (the latter
stratified by regions, occupations,
races, etc.), to establish baselines.
· Develop assays in semen, saliva, and
urine or use noninvasive externally de-
rived signals, such as from ultrasound,
that can be applied to the screening of
large populations.
· Continue to develop more invasive
techniques limited to use in subgroups,
to gain insight into mechanisms of
toxicity.
The subcommittee focused its specific
recommendations, which follow, on the
identification of markers that could be
used to detect exposures or their effects
on male reproductive function and herit-
able genetic damage, criteria for the de-
velopment of markers of male reproduction,
and the development of strategies for
testing the effects of toxic chemicals
on markers of male reproduction.
IDENTIFICATION OF MARKERS OF
ABNORMAL PHYSIOLOGIC
FUNCTION
Most of the potentially useful biologic
markers of male reproductive function
are in the developmental stage in the la-
boratory. Research needs to be continued
in various subjects, as follows, in an
effort to identify additional markers of
specific physiologic functions.
Testes
· Evaluate the use of noninvasive physi-
cal measurements, such as magnetic res-
onance imaging and ultrasound, for provid-
ing signals that can be used to assess such
criteria as size, consistency, blood flow,
and function.
· Investigate the capacity of the
testis to metabolize xenobiotics.
OCR for page 143
CONCLUSIONS AND RECOMMENDATIONS
· Investigate the pharmacokinetics
of toxic chemicals and their metabolites
with respect to transport into the inter-
stitium and seminiferous tubules.
· Carry out basic research on spermato-
genesis, with special consideration of
stem cell numbers, division, and clonal
nature.
· Test the effect of toxic agents on
markers of Leydig cells, Sertoli cells,
and specific germ cells; special attention
must be given to the time course of toxic
effects and the potential recovery period.
· Develop assays for inhibin, androgen-
binding protein, Mullerian inhibiting
substance, etc., as biologic markers of
Sertoli cell function.
· Identify and characterize additional
molecules that are produced by Leydig,
Sertoli, and germ cells and secreted into
the blood and semen.
· Carry out basic research and develop
probes for testicular RNA, DNA, and macro-
molecules; these can be used to develop
assays for quantifying specific steps in
the development of germ cells.
· Continue basic research on mechanisms
of intratesticular communication among
Leydig, Sertoli, and germ cells with the
goal of identifying additional markers
of testicular function.
· Compare the toxic responses of human
and laboratory animal germ cells, to iden-
tify useful laboratory animal models of
human effects.
Epididymis
· Carry out basic research to determine
which facets of spermatozoa change during
epididymal transit (e.g., specific sur-
face antigens).
· Evaluate the effect of toxic chemicals
on spermatozoa! epididymal transit time,
site of acquisition of motility, and fer-
tilizing potential.
· Evaluate the effect of toxic chemicals
on epididymal structure, integrity of the
blood-epididymis barrier, and function
of specific cells.
· Evaluate the capacity of the epididy-
mis to metabolize xenobiotics.
· Continue basic research on the func-
tional interaction between the immune
system and the epididymis.
143
· Develop molecular probes of epididy-
mal function applicable for blood or semen
evaluation.
Accessory Sex Organs
· Identify specific molecular markers
and their cDNA probes for individual acces-
sory sex organs (e.g., prostatein).
· Continue basic research on the regula-
tion of the biosynthesis and secretion
of those molecular markers, especially
their composition in seminal plasma.
· Develop ultrasound and other physical
measurements to provide externally de-
rived signals that can be used to assess
site and function of specific accessory
sex organs.
· Investigate the pharmacokinetics
of toxic chemicals in individual accessory
sex organs.
SEMEN MARKERS OF ABNORMAL
PHYSIOLOGIC FUNCTION
In principle, semen can be used to evalu-
ate the cellular product of spermatogene-
sis, the function of the somatic supporting
testicular cells, the function and integ-
rity of the efferent duct system (including
the epididymis), the function of the acces-
sory glands, hormonal state, and perhaps
the xenobiotic exposure of the male organ-
ism. Several available markers have been
evaluated (Table 7-2) and some have shown
detrimental effects in people exposed to
radiation or chemicals. For some markers,
baselines have been established, but they
have not been evaluated in exposed people.
Most markers remain inadequate for quan-
titatively assessing male reproductive
health at this time. More research is
required for elucidating underlying
molecular mechanisms, for evaluating the
predictive value of individual markers
in relation to fertility, and for identify-
ing the utility of markers as indicators
of exposure to xenobiotic agents.
Chapter 7 highlighted several promising
concepts (Table 7-2), including automa-
tion of sperm measurements; markers of
sperm function; immunologic reagents
for molecular studies of spermatogenesis;
recombinant probes of spermatogenic
genes; and semen markers of Sertoli-
OCR for page 144
144
cell, epididymal, prostatic, and seminal
vesicle function. The list is not intended
to be complete; rather, it gives examples
of research subjects with early promising
results. In the near future, we hope to
have markers of cell differentiation and
function that are specific for events at
the molecular level.
The following are specific recommenda-
tions for semen studies:
· Develop in vitro sperm assays that
measure the capacity of animal and human
spermatozoa to fertilize oocytes.
· Develop seminal plasma molecular
markers of individual accessory sex
organ function.
· Develop monoclonal-antibody assays
of specific functional domains on sper-
matozoal plasma membranes.
· Develop computer-based and automated
techniques for making quantitative sperm
measurements and for sorting sperm, e.g.,
on the basis of numbers, motility, morphol-
ogy, domains, and enzyme function.
· Study the pharmacokinetics of toxic
chemicals and their metabolites in seminal
plasma and the capacity of semen and sperm
to carry toxic agents to the female and the
site of fertilization.
· Develop semen-based assays for Ser-
toli cell and Leydig cell function.
NEED FOR IMPROVED MEASURES
OF FERTILITY STATUS AND
EXPOSURE
In addition to the need for improved
semen markers, improved approaches for
assessing their validity are needed. The
evaluation of a semen marker's utility
for fertility assessment requires sensi-
tive quantitative measures of fertility
for comparison. Further work is required
to quantify fertility, including further
evaluation of standardized fertility
ratios and time required to conception.
Another measure of a male's fertility might
be the immunologic detection of,8-chori-
onic gonadotropin in his mate's urine,
which indicates pregnancy about 10 days
after conception (Table 9-3~. This measure
of fertility has disadvantages-it is in-
sensitive during the first 10 days of preg-
nancy, and it obviously requires that cou-
MALE REPRODUCTIVE TOXICOLOGY
pies plan to have children. At present,
it appears to be the most sensitive in-
dicator of early pregnancy, and, on further
evaluation, could become the fertility
reference of choice for determining the
predictive value of selected semen
markers.
The assessment of a semen markers utili-
ty as an indicator of exposure to xenobiot-
ic agents requires sensitive quantitative
exposure measures for each agent to be
evaluated. Although a crucial aspect of
the validation process, the ability to
quantify human exposure varies dramati-
cally with agent and circumstances. Sev-
eral approaches are possible, including
biologic, physical, and chemical dosime-
try and exposure history. However, with
the exception of very few agents for which
exposure can be determined with reasonable
certainty (e.g., therapeutic ionizing
radiation and chemotherapeutic drugs),
human exposure assessments are usually
little more than guesses. It might not be
possible generally to determine the quan-
titative dose-response relationship be-
tween a semen marker and human exposure,
as required for validation. However, sev-
eral solutions are possible: select a
single agent to model the human dose-
response relationship and extrapolate
to other human exposures, develop improved
biologic dosimetry (with sensitive meth-
ods for detecting adduction of DNA and
proteins), and perform detailed dose-
response evaluations in animal models and
extrapolate the results to humans.
IDENTIFICATION OF MARKERS OF
GERMINAL GENETIC TOXICITY AND
HERITABLE MUTATIONS
Recommendations for research needs
in the development of markers of germinal
genetic toxicity and heritable mutations
include studies with human and animal tis-
sues:
· Study whether integration and exci-
sion of transposable elements constitute
one mechanism for inducing germinal muta-
tions.
· Determine the extent to which trans-
posable elements exist in humans.
OCR for page 145
CONCLUSIONS AND RECOMMENDATIONS
· Carry out basic research on selective
pressures for and against chromosomal
defects during spermatogenesis.
· Compare the consequences of protein
and DNA adducts in various types of germ
and somatic cells in mice.
· Study the extent and conditions of
induction of aneuploidy in male germ
cells.
· Investigate and compare the molecular
nature of stem cell and post-stem-cell
mutations, both spontaneous and induced,
· —
In mice.
· Develop sperm-based markers ofgenet-
ic damage in mice, such as alkaline elu-
tion, cytogenetic abnormalities, aneu-
ploidy, and gene mutations analogous to
those in humans.
· Evaluate the effect of toxic chemicals
on the capacity of epididymal spermatozoa,
as opposed to testicular germ cells, to
produce genetically normal offspring.
· Develop additional animal models
for studying the role of toxic chemical
metabolism, the mechanism of toxic-chemi-
cal damage, and repair mechanisms in the
induction of germinal and heritable muta-
tions.
· Develop and validate human semen mark-
ers of genetic toxicity and induced muta-
tions, including methods for detecting
gene mutations, aneuploidy, chromosomal
aberrations, and DNA adducts in mature
sperm and in immature germ cells.
· Develop and validate DNA markers of
human heritable mutations including Ler-
ner gels, restriction-fragment-length
polymorphisms (RFLPs), subtractive hy-
bridization, and RNAse digestion.
CRITERIA FOR DEVELOPMENT AND
VALIDATION OF MARKERS OF
MALE REPRODUCTION
Identifying biologic markers that rep-
resent exposure to toxic chemicals or the
effects of exposure on specific male repro-
ductive tract functions and heritable
genetic damage is only the first step. The
subcommittee has compiled a series of steps
that should be followed when a potentially
useful marker is to be validated.
· Establish normal baseline values
and distribution of each marker in labora-
tory animals and humans.
145
· Evaluate the sensitivity and speci-
ficity of each marker to predict a health
outcome (e.g., fertility) and heritable
genetic damage.
· Understand in detail the dose-re-
sponse relations and time course of
response of each marker to a given toxic
chemical, with special attention to the
recovery process.
· Evaluate new markers in studies, in-
cluding existing proven markers of patho-
physiologic functions and heritable ge-
netic damage, and understand the relative
value of each marker to others in the bat-
tery.
· Develop a strategy and consensus
for the use of multiple species in toxico-
logic studies.
~ Test the effect of toxic chemicals
in several species, including mice and
rats; mice are particularly useful for
genetic studies, because they have been
widely studied for mutagenesis, but murine
metabolism of some chemicals might differ
markedly from human metabolisms, in which
case species more closely resembling hu-
mans must be studied.
· Encourage the development of animal
markers with human correlates so that risk
extrapolation models can be developed and
evaluated.
STRATEGY FOR TESTING EFFECT
OF TOXIC CHEMICALS ON
MARKERS OF MALE
REPRODUCTION
It is suggested that a task force be es-
tablished to develop and carry out a stra-
tegy for evaluating biologic markers in
laboratory animals. The task force might
have the following functions:
· Select a battery of markers of male
reproductive functions and heritable
genetic damage.
· Select a number of toxic chemicals
and nontoxic analogs.
· Select a single source for production
of highly purified chemicals.
· Design important aspects of a proto-
col, such as species to be used and dosage,
duration, and route of chemical adminis-
tration.
OCR for page 146
146
· Select specific laboratories that
have the expertise to carry out the meas-
urements required for testing a specific
marker. No single laboratory would carry
out all the measurements at this stage,
because unique methods might be required.
· Serve as a clearinghouse for data anal-
M'4LE REPRODUCTIVE TOXICOLOGY
ysis and evaluation of a marker of specifi-
city, sensitivity, precision, and ac-
curacy in reflecting exposure to toxic
chemicals and the effects of exposure on
reproductive function or heritable genet-
ic damage in laboratory animals.
Representative terms from entire chapter:
genetic damage
