**2.1 High-throughput assays**

High-throughput assays evaluate the effect of a test substance on a single biologic process using an automated manner that allows thousands or tens of thousands of compounds to be tested in a short time at a reasonable cost. Robotics and genetic engineering make it possible to produce large quantities of receptors or genetically engineered cells for use in these assays. Knowledge about mechanisms of toxicity is often central to the strategy of highthroughput assays. For example, cells are being developed that are bioengineered to express human hormone receptors for estrogens and androgens. These cells can be used for highthroughput chemical screening for steroid hormone receptor affinity or the potential to act as endocrine disruptors. Both isoforms of recombinant human estrogen receptor and human androgen receptor are commercially available for this purpose. Based on the same principles, other batteries of high-throughput assays are available to screen for activity against various receptors and cytochrome P-450 enzyme isoforms (Lawson et al., 2003). The

Environmental Toxicants Induced

Sperm fertilizing ability

agents (Lawson et al., 2003).

Sperm DNA

Male Reproductive Disorders: Identification and Mechanism of Action 495

and a possible surrogate for sperm measures (Anderson and Sharpe 2000). The recognition that sperm functional tests are also desirable has led to development of various new tests that have only recently been applied to toxicology. Biomarkers of the genetic integrity of sperm are designed to identify risks for paternally mediated developmental effects. Sperm proteins are being tested as biomarkers of fertility to detect specific deficits in sperm function (as opposed to decreased sperm output). Although details of such tests are beyond the scope of this review, **Table 1.** provides a list of new tests and references regarding methodology and examples of use. Further research is needed to make these tests more practical and more cost effective and to determine their ultimate utility for hazard

identification and elucidation of modes and mechanisms of toxicant action.

Sperm maturity Cytoplasmic droplets

Sperm count surrogate Inhibin-B

**2.5 Estimating occupational exposure** 

Target Bioassay Function assessed

Sperm function Acrosome reaction

Chromatin damage DNA damage

Endocrine feedback of spermatogenesis.

Translocation

Heat shock protein A2 Sperm morphology

SP-22 protein Sperm antigens Ubiquitin

CMA3 staining COMET DNA adducts TUNEL SCSA

Sperm chromosomes FISH Aneuploidy, Brakage

Abbreviations: CMA3 staining, Chromomycin A3 staining; COMET, Single-cell gel electrophoresis assay; FISH, Fluorescence in situ hybridization; SCSA, Sperm chromatin structure assay; TUNEL, Terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (Lawson et al., 2003) Table 1. The Biomarkers for the assessment of male reproductive toxicity by Chemical

Establishing that a significant number of workers or members of the general population are or will be exposed to a potential reproductive toxicant is central to priority setting. NIOSH's National Occupational Hazard Survey and National Occupational Exposure Survey conducted in 1972-1974 and 1981-1983, respectively, has been used extensively to identify substances of common exposure (NIOSH 1978, 1988). These surveys are the only comprehensive assessments of general industry where the number of workers potentially exposed to chemical agents has been estimated. However, these databases are outdated and of limited use because they indicate only potential exposure. NIOSH is currently planning a new hazard surveillance activity that will target industry sectors on a rolling basis, beginning with the health care sector. Public health researchers will continue to require updated exposure surveys to keep up with the changing workplace exposures and monitor

availability and application of these assays will undoubtedly expand as we understand more about the relevance of each protein in toxicologic processes.

#### **2.2 Structure-activity prediction**

Methods for predicting activity from structure continue to be developed and refined. Computer programs use available empirical information about the toxicity of existing compounds and their chemical characteristics to predict whether a new compound will have similar toxicity. These programs have not performed well in the area of reproductive and developmental toxicity, probably because reproductive processes are complex and effects may be elicited through multiple modes and mechanisms. As science progresses and we learn more about mechanisms of toxicity at the molecular level, however, structure-activity computer programs will become more exact and predictive. The best examples are the programs that are being developed and refined for estrogen receptor binding (Tong et al., 1997).

#### **2.3 Integration of human studies and tests of model species**

Though 4,000 chemicals have been tested in model species, few chemicals have been adequately evaluated for reproductive effects in humans and contain a partial list of known human developmental and adult toxicants. Because the interpretation of studies of model species is often not straightforward and because field studies are labor and resource intensive, a systematic approach is needed to select and prioritize chemicals for epidemiologic studies. Moorman et al., (2000) recently proposed a process for selecting chemicals for human field studies. In this process, information gained from model species testing conducted by the National Toxicology Program (NTP) was reviewed for significant adverse reproductive effects and potency of the toxicants. The evaluative process then combined this information with human exposure information available in public databases to arrive at a list of high-priority candidates for studies in humans.

#### **2.4 New biomarkers for humans and model species**

In 1977, men exposed to dibromochloropropane (DBCP), a pesticide that is now banned in the United States, were found to be azoospermic and oligospermic (Whorton et al., 1977). Currently, a variety of biomarkers are used to assess the potential adverse reproductive effects due to toxic chemical exposures. Bioindicators of sperm production and quality (semen volume, sperm concentration, sperm motility, sperm morphology) are routinely evaluated in ejaculated semen samples in men and in suspensions of epididymal sperm from test species (epididymal sperm reserves, sperm motility, and sperm morphology) (Moline et al., 2000; U.S. EPA 1998a). During the past decade, computer-assisted methods developed to improve and automate the evaluation of sperm motion and morphology have been added to the battery of routine sperm measures, and guidance for their use and interpretation has been made available through a number of workshops (ILSI 1999; Seed et al.,1996). Furthermore, baseline data on the relationship between various semen or epididymal sperm measures and fertility have emerged from a number of large studies designed to address this question (Zinaman et al.,2000). Thus, these measures are widely accepted biomarkers of adverse reproductive effects that are suitable for application in both human and model species studies. Serum hormone measures can also be determined in humans and test species. Inhibin B has been proposed as an indicator of testicular function

availability and application of these assays will undoubtedly expand as we understand

Methods for predicting activity from structure continue to be developed and refined. Computer programs use available empirical information about the toxicity of existing compounds and their chemical characteristics to predict whether a new compound will have similar toxicity. These programs have not performed well in the area of reproductive and developmental toxicity, probably because reproductive processes are complex and effects may be elicited through multiple modes and mechanisms. As science progresses and we learn more about mechanisms of toxicity at the molecular level, however, structure-activity computer programs will become more exact and predictive. The best examples are the programs that are being developed and refined for estrogen receptor binding (Tong et al.,

Though 4,000 chemicals have been tested in model species, few chemicals have been adequately evaluated for reproductive effects in humans and contain a partial list of known human developmental and adult toxicants. Because the interpretation of studies of model species is often not straightforward and because field studies are labor and resource intensive, a systematic approach is needed to select and prioritize chemicals for epidemiologic studies. Moorman et al., (2000) recently proposed a process for selecting chemicals for human field studies. In this process, information gained from model species testing conducted by the National Toxicology Program (NTP) was reviewed for significant adverse reproductive effects and potency of the toxicants. The evaluative process then combined this information with human exposure information available in public databases

In 1977, men exposed to dibromochloropropane (DBCP), a pesticide that is now banned in the United States, were found to be azoospermic and oligospermic (Whorton et al., 1977). Currently, a variety of biomarkers are used to assess the potential adverse reproductive effects due to toxic chemical exposures. Bioindicators of sperm production and quality (semen volume, sperm concentration, sperm motility, sperm morphology) are routinely evaluated in ejaculated semen samples in men and in suspensions of epididymal sperm from test species (epididymal sperm reserves, sperm motility, and sperm morphology) (Moline et al., 2000; U.S. EPA 1998a). During the past decade, computer-assisted methods developed to improve and automate the evaluation of sperm motion and morphology have been added to the battery of routine sperm measures, and guidance for their use and interpretation has been made available through a number of workshops (ILSI 1999; Seed et al.,1996). Furthermore, baseline data on the relationship between various semen or epididymal sperm measures and fertility have emerged from a number of large studies designed to address this question (Zinaman et al.,2000). Thus, these measures are widely accepted biomarkers of adverse reproductive effects that are suitable for application in both human and model species studies. Serum hormone measures can also be determined in humans and test species. Inhibin B has been proposed as an indicator of testicular function

more about the relevance of each protein in toxicologic processes.

**2.3 Integration of human studies and tests of model species** 

to arrive at a list of high-priority candidates for studies in humans.

**2.4 New biomarkers for humans and model species** 

**2.2 Structure-activity prediction** 

1997).

and a possible surrogate for sperm measures (Anderson and Sharpe 2000). The recognition that sperm functional tests are also desirable has led to development of various new tests that have only recently been applied to toxicology. Biomarkers of the genetic integrity of sperm are designed to identify risks for paternally mediated developmental effects. Sperm proteins are being tested as biomarkers of fertility to detect specific deficits in sperm function (as opposed to decreased sperm output). Although details of such tests are beyond the scope of this review, **Table 1.** provides a list of new tests and references regarding methodology and examples of use. Further research is needed to make these tests more practical and more cost effective and to determine their ultimate utility for hazard identification and elucidation of modes and mechanisms of toxicant action.


Abbreviations: CMA3 staining, Chromomycin A3 staining; COMET, Single-cell gel electrophoresis assay; FISH, Fluorescence in situ hybridization; SCSA, Sperm chromatin structure assay; TUNEL, Terminal deoxynucleotidyl transferase-mediated dUTP-biotin end-labeling (Lawson et al., 2003)

Table 1. The Biomarkers for the assessment of male reproductive toxicity by Chemical agents (Lawson et al., 2003).

### **2.5 Estimating occupational exposure**

Establishing that a significant number of workers or members of the general population are or will be exposed to a potential reproductive toxicant is central to priority setting. NIOSH's National Occupational Hazard Survey and National Occupational Exposure Survey conducted in 1972-1974 and 1981-1983, respectively, has been used extensively to identify substances of common exposure (NIOSH 1978, 1988). These surveys are the only comprehensive assessments of general industry where the number of workers potentially exposed to chemical agents has been estimated. However, these databases are outdated and of limited use because they indicate only potential exposure. NIOSH is currently planning a new hazard surveillance activity that will target industry sectors on a rolling basis, beginning with the health care sector. Public health researchers will continue to require updated exposure surveys to keep up with the changing workplace exposures and monitor

Environmental Toxicants Induced

development of a large database.

(Khoury & Dorman 1998).

**2.10 Communication** 

**2.7 Gene-environment interactions** 

differences in their ability to metabolize a toxicant.

understand the role of these genes for reproductive toxicity.

Male Reproductive Disorders: Identification and Mechanism of Action 497

the data to identify changes related temporally, by dose, or by metabolic pathway. The second challenge will be to separate those changes in gene expression pivotal to the toxic response from those that are more generalized responses to any stimulus. The third challenge will be to quantitatively relate changes in expression of critical genes with toxicity, which is manifested at a more complex level of biologic organization (i.e., the cell, organ, or organism). Real comfort in this genomic approach will come only with experience and the

Reproductive toxicants can affect human populations over the total life span, including the *in utero* and perinatal periods, childhood, puberty, and adulthood. Thus, extending research efforts to address stage-specific sensitivity is recommended. Another emerging approach allows the identification of populations at potentially increased risk from toxicant exposure by characterizing genetic polymorphisms of metabolizing enzymes in exposed cohorts. Such methods may identify vulnerable subpopulations on the basis of inherent (genetic)

Genetic factors that elevate risk for disease can be grouped into two categories: those for which having a particular allele conveys a high risk for the disease regardless of other (e.g., environmental) influences and those associated with only small increases in risk of the disease. The latter, termed susceptibility genes are being identified at an increasing rate. The interaction of these alleles with environmental agents or other susceptibility alleles ultimately determines whether the disease will be manifested. Much work has been done to

If epidemiologic studies could identify genetic-toxicant interactions by comparing the prevalence of a particular genetic marker (polymorphism) or a group of markers in affected and unaffected populations, this information could be used to target environmental, behavioral, or medical interventions (Khoury 1997). Ultimately, validation of genetic testing to link a particular genotype with exposure to a specific chemical to the increased prevalence of a particular reproductive disorder would require epidemiologic confirmation

An essential component of future reproductive studies will be improved communication. Because of the complex mechanisms involved in reproductive research, collaboration across scientific disciplines must be conducted. In addition, notification of research results and recommendations must be communicated to workers and the affected public in a manner that is timely, accessible, and easily understood. A primary goal of reproductive research is to reduce the high percentages of adverse outcomes such as infertility, pregnancy loss, and congenital malformations. Although certain limitations exist that are unique to reproductive research, many advances in technology and methodologies have been recently developed that will aid researchers in their efforts to *a*) understand mechanisms by which toxicants

**2.8 Identifying genes that increase sensitivity to reproductive toxicants** 

**2.9 Potential information from genetics to advance epidemiologic studies** 

new exposures that may be potential reproductive toxicants. New technologies such as geographic information systems (GIS) allow mapping of industries and specific chemical exposures. Use of GIS to identify geographic areas with high volume of use of suspect chemicals might be an effective method of identifying populations with greater potential occupational and environmental exposures. Biomonitoring is a valuable tool for estimating occupational exposure. The National Report on Human Exposure to Environmental Chemicals is a new and ongoing assessment of the U.S. population's exposure to environmental chemicals. The first edition of the report presents levels of 27 environmental chemicals, including metals (e.g., lead, mercury, and uranium), cotinine (a marker of tobacco smoke exposure), organophosphate pesticide metabolites, and phthalate metabolites. This is a significant step forward in assessing the potential human toxicity of a class of chemicals known to be reproductive and developmental toxicants in rodents. Improved methods for analysis of exposure, especially of age and time effects, are likely to impact the characterization of occupational exposure in these studies (Richardson and Wing 1998). Current research approaches usually consider the action of single, unique toxicants on outcomes of interest, creating yet another challenge to drafting a reproductive hazards agenda. The more common human exposure scenario is to mixtures of toxicants at low concentrations, episodically and over the long term. Attention to cumulative exposure over years of a working lifetime and total aggregate exposure to toxicants from multiple exposure sources, as well as classical considerations of exposure routes, must also be addressed. Methodologic approaches must enlarge and mature to consider the effects and modulation of effects mediated by both exposure to mixtures of toxicants and the complexities of exposure mode at low dose and over prolonged duration.

#### **2.6 Mechanistic research**

Understanding mechanisms of action of toxicants is important for a number of reasons, including *a*) supporting the biologic plausibility of an observed association between chemical exposure and adverse outcome; *b*) uncovering common pathways of actions of different agents; *c*) extrapolating across species for risk assessment; *d*) improving the predictability of human morbidity from responses of model species; and *e*) predicting responses to mixed exposures (Lawson et al., 2003). Mechanistic studies are not new in toxicology; however, new tools in genomics, proteomics, and bioinformatics present unprecedented opportunities to advance our understanding of toxicant action at a molecular level. Genomic information and the ability to screen most or all of the genome of an increasing number of organisms for changes in gene expression are revolutionizing the way in which biologic effects data are gathered. It is now possible to determine the effects of a toxicant exposure on gene expression of most of the genome of mice and rats. This will allow us to generate testable hypotheses about the mechanism of action of toxicants. It will also open up the possibility of identifying markers of exposure or effect specific to a particular insult that can be used in field studies. As with any new technology, a number of problems will need to be overcome for the promise of genomics to be realized. The first will be to manage the large volume of information produced by gene expression experiments. Gene chips may contain thousands or tens of thousands of sequences. Experience shows that any perturbation in a biologic system leads to numerous changes in gene expression. An entire field of bioinformatics is being developed to help collect, organize, and manage the data to identify changes related temporally, by dose, or by metabolic pathway. The second challenge will be to separate those changes in gene expression pivotal to the toxic response from those that are more generalized responses to any stimulus. The third challenge will be to quantitatively relate changes in expression of critical genes with toxicity, which is manifested at a more complex level of biologic organization (i.e., the cell, organ, or organism). Real comfort in this genomic approach will come only with experience and the development of a large database.
