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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-

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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.

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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-

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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.

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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.

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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.