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Final Report: Influence of Genotype on Reproductive Effects of Air Pollution in Young Men
EPA Grant Number: R827019Title: Influence of Genotype on Reproductive Effects of Air Pollution in Young Men
Investigators: Evenson, Donald P. , Marshall, Don , Rubes, Jiri , Sram, Radim
Institution: South Dakota State University
EPA Project Officer: Deener, Kacee
Project Period: September 1, 1998 through September 30, 2000 (Extended to November 30, 2003)
Project Amount: $274,670
RFA: Interindividual Variation in Human Susceptibility to Environmentally-caused Disease (1998)
Research Category: Health Effects
Description:
Objective:The objective of this research project was to test the hypothesis that metabolic genotypes may influence the extent of DNA damage in spermcells of men who are exposed to high levels of genotoxic substances found in air pollution. If this hypothesis is true, then men with susceptible genotypes may represent a sensitive subpopulation with respect to the potential for environmental exposures to increased risk to the genetic integrity of their germ cells and thus to their children. The context of this project, as detailed below, is that serial semen samples, obtained from young men during periods of low and extremely high ambient air pollution in the Czech Republic were archived and available for further study. Preliminary evidence obtained from these samples indicated that there may be responders and nonresponders with respect to altered sperm chromatin structure. Determination of the semen donors’ genotypes for the appropriate metabolic genotypes (GSTM1, GSTT1, NAT2) would make it possible to explore a possible relationship between genotype and sperm DNA damage. In addition to completing analyses of sperm chromatin structure, several assays more specific to DNA and chromosome damage are required. Furthermore, the information obtained with respect to DNA damage was examined in the context of male reproductivehealth by integrating the findings with the existing database on semen quality and considering potential modifiers such as lifestyle and health status available from the questionnaire data.
Air pollution in the Czech Republic increased dramatically with the advent of industrialization in the 1950s, primarily the result of the increasing use of brown coal (with high sulfur content). In the Teplice District of Northern Bohemia, wintertime levels of air pollution reach extreme highs because of the use of additional coal for home heating and the episodic temperature inversions that hold the pollution in the valley (Moldan, et al., 1992). Components of this air pollution have been characterized (Stevens, et al., 1996; Watts, et al., 1994) and include high amounts of mutagenic polycyclic aromatic hydrocarbons (PAHs), some of which are known to be male reproductive toxicants (Ahlquist, et al., 1966; Ford, et al., 1963; Generoso, et al., 1982; Georgellis, et al., 1990). Furthermore, personal monitoring studies conducted on Teplice residents revealed increased levels of urinary metabolites of PAHs, as well as DNA adducts in white blood cells during periods of high pollution(Binkovà, et al., 1995; Binkova, et al., 1996).
Concern about the potential health effects of this pollution prompted the initiation of an international research program, the Teplice Program, in 1991. A number of health outcomes, including respiratory and neurological effects in children, biomonitoring of mutagens in adults, and reproductive effects, were studied (Šrám, et al., 1990). Reports that conception rates and the incidence of congenital anomalies were affected adversely by seasonal increases in air pollution (Moldan, et al., 1992) prompted studies on pregnancy outcome in women of reproductive age and semen quality in young men. Thus, these studies provided an opportunity to evaluate the potential health effects of exposure to air pollution at levels rarely encountered in the United States today.
The cohort of men consisted of all the young men who were turning 18 in the district and receiving a physical examination to determine fitness for compulsory military duty. During a 2 year study period, these men were asked to participate in this study and to provide a single semen sample. These men typically had lived in the district their entire lives but had little work experience and thus were less likely than older men to have had potentially confounding occupational exposures to reproductive toxicants. Results of 325 semen analyses showed that men living in Teplice in the winter (exposed to high air pollution) experienced a significantly increased risk of having poor sperm motility and morphology when compared with men living in Teplice in the summer (low air pollution), or living in the comparison district (low air pollution) at either time of the year (Šrám, et al., 1990; Selevan, et al., 1998).
In addition, men sampled during the period of highest pollution (a 90 day period of time when ambient SO2 levels were approximately double the U.S. annual standard) exhibited significantly higher levels of abnormal sperm DNA, as measured by the sperm chromatin structure assay (SCSA), our flow cytometric assay that detects increased denaturability of sperm DNASelevan, et al., 1998; Evenson, et al., 1994). In animal studies, abnormal SCSA has been associated with exposure to a number of testicular toxicants and germ cell mutagens, whereas in humans, chemotherapy (Fossa, et al., 1997) and high fever result in abnormal SCSA (Evenson, et al., 1994). Thus, there is a potential association between exposure to high levels of air pollution and genetic damage in sperm.
Longitudinal Study Population
These preliminary results provided the rationale for an expanded study involving men from Teplice. Semen samples were obtained from 40 men who had participated in the first study. These men agreed to donate serial samples at times when pollution is low (late September) and high (January-March). Starting in Year 1 of the project and ending in Year 3 of the project, these men provided a total of up to seven serial samples. Air pollution was monitored daily in Teplice during this entire period of time. The advantages of this study design included the fact that each man served as his own control or baseline during periods of low pollution, and those effects were evaluated with respect to the interval between exposure and sampling. The latter is critical when evaluating damage in sperm which may have originated at any previous stage of spermatogenesis.
Detailed histories were obtained at each sampling time using a questionnaire designed for use in the Czech Republic (Selevan, et al., 1998). This information allowed us to control for lifestyle and other health conditions or occupational exposures that could alter semen quality. Routine semen analyses were performed on these samples and data on sperm morphology and motility (by computer-aided semen analysis) currently are being analyzed. Urine also was analyzed for cotinine, to control for exposure to cigarette smoke. Aliquots of semen were frozen and archived. In addition, blood samples were available for genotyping.
Specific Objectives
Funding was sought to conduct assays for sperm genetic integrity using the archived semen samples. The preliminary SCSA data provided compelling evidence that the response of these men to toxicant exposure may be genotype dependent. To explore this possibility, genotype analyses was done for a subset of men. In addition to the SCSA, which we have shown to be sensitive to exposures to high levels of air pollution in the previous study, additional studies were done on SCSA sperm populations to test by morphology and Comet assay the relationship between SCSA data and pollution driven DNA damage.
Most importantly to this grant, we explored the possibility that individual variation in metabolic capability plays a role in the susceptibility of men to exposure-related, adverse reproductive effects. By determining the genotypes of these donors, we were able to evaluate whether their response to air pollution was genotype-dependent.Specifically, we looked at the genotype for GSTM1 enzyme that detoxifies the electrophilic metabolites of PAH and N-acetyl transferase, an enzyme involved in the detoxification of arylamines (also found in air pollution) (Šrám, et al., 1998). Altered metabolic genotypes, specifically GSTM1 and GSTT1 null mutations, and NAT2 polymorphisms have been associated with increased susceptibility to cancer, raising the possibility that susceptible individuals also may be at greater risk for pollution-induced DNA damage. We hypothesized that men who metabolize PAHs less efficiently (because of GSTM1 or GSTT1 null mutation, and/or NAT2 or GSTT1 polymorphisms) will be more susceptible to (i.e., have a genetic predisposition for) pollution-induced DNA damage in sperm and/or altered sperm chromosome constitution.
Summary/Accomplishments (Outputs/Outcomes):Synopsis
This research tested the hypothesis that human genotype influences the extent of damage to sperm chromatin integrity. The research was leveraged from an international environmental epidemiology study exploring the potential relationship between the exposure of young men to episodes of high air pollution (containing carcinogenic polyaromatic hydrocarbons [PAHc] in the particulate fraction) and altered semen quality. One objective of this study was to include newly developed tests for sperm chromatin integrity, DNA damage, and aneuploidy. Having these data provided a unique opportunity to explore further the possibility that polymorphisms in two genes that metabolize PAHc might confer increased susceptibility to DNA damage in sperm. A novel gene-environment interaction was found between metabolic genotype and DNA damage induced in sperm and associated with exposure to air pollution.
Background and Experimental Design
The rationale for including a measure for sperm DNA fragmentation (i.e., SCSA) was that reactive metabolites of PAHc in air pollution, if they reached sperm in the testis or epididymis, could react with sperm DNA and/or sperm protamines and induce DNA strand breaks. Alternatively or in addition, toxicants in the air pollution might interfere with spermatogenesis, particularly with meiosis, and increase the risk of sperm aneuploidy. The latter now can be evaluated using chromosome-specific cDNA probes and fluorescence in situ hybridization (FISH).
In a preliminary study, exposure to high levels of air pollution in the Teplice District of the Czech Republic was associated with increased sperm DNA fragmentation as measured by SCSA (Selevan, et al., 2000; Perreault, et al., 2000; Perreault, et al., 2001; Evenson, et al., 2001), and with increased incidence of YY sperm disomy (Rubes, et al., 2001). Based on these preliminary observations, a longitudinal study was implemented in which a group of 36 young men (19-22 years of age) living in Teplice were evaluated seven times in 2 years. This study design provided semen samples from these men collected after long intervals of low pollution and after shorter episodes of high pollution. Each man served as his own baseline, providing sufficient power to detect exposure-related changes in semen quality in a relatively small group of men. This study provided support for the conduct of SCSA and FISH in these samples. Importantly, exposure to high air pollution was shown again to be associated with increased DNA damage in sperm, as indicated by higher levels of DNA fragmentation index (%DFI), although not with increased sperm aneuploidy (Rubes, et al., 2005).
PAHc are metabolized into active metabolites that are known to damage DNA. The enzyme GSTM1 detoxifies these active metabolites and therefore is protective. On the other hand, NAT2 may or may not be protective. Polymorphisms in these two phase II enzymes are common (about 50% of Caucasians are GSTM1 null, for example) and are known to alter susceptibility to DNA damage associated with cancer risk. We therefore undertook to test the hypothesis that GSTM1 and NAT2 genotypes might modify the relationship between SCSA-%DFI and exposure to high levels of air pollution. This grant provided support such that the metabolic genotypes of the men in this study could be determined from a blood sample.
Findings
Genotype then was entered into the statistical model, along with appropriate modifiers based on the questionnaire data (e.g., smoking). For exposure and DFI (without genotype), β (95% CL) = 0.175 (0.006, 0.345); for genotype and DFI (without exposure), β = 0.251 (0.030, 0.473); and, for the interaction (exposure x genotype) and DFI, β = 0.427 (0.157, 0.697). Thus, DFI was associated with both exposure and genotype, and the interaction between genotype and exposure was even more significant. This demonstration of a gene-environment interaction supports the hypothesis that GSTM1(-) men may be at increased risk for sustaining DNA damage in their sperm and may be more susceptible than GSTM1 men to adverse reproductive effects of air pollution. In contrast, NAT2 genotype was not significant in the model, and there was insufficient power to put both genotypes into the model at once. This novel observation was reported at national (Rubes, et al., 2004) and international (Rubes, et al., 2005) meetings, and a manuscript is being prepared for publication.
Significance to the U.S. Environmental Protection Agency
These findings further strengthen the rationale for including the SCSA in environmental epidemiology studies, and for further research exploring the possibility that at least some forms of male infertility may be associated with genetic polymorphisms of this nature. The %DFI is known to correlate with male infertility and increased risk of spontaneous abortions in clinical studies. The outcome of this research suggests that men who carry the GSTM1 null mutation may exhibit increased susceptibility to pollution (toxicant, drug)-induced DNA damage in their sperm and therefore to increased risk for male infertility or male-mediated abnormal developmental outcomes.
In addition to the novel and exciting observation that genetics plays a role in the level of sperm DNA damage, as above, the other novel finding was that the SCSA data were dose responsive to higher and lower levels of air pollution in Teplice, Czech Republic (Rubes, et al., 2005 above). The classical parameters of sperm count, motility, and morphology were not dose responsive to the air pollution. The SCSA is a flow cytometric measure of altered sperm chromatin structure, mostly considered to be DNA strand breaks. Thus, of importance, the SCSA detected environmentally induced sperm DNA damage even though the classical semen parameters of count, morphology, and motility were not dose responsive. In other words, the SCSA is an independent measure of sperm DNA damage, a highly important point in the area of reproductive toxicology.
The question was asked: What is the effect of SCSA-detected DNA fragmentation on pregnancy outcome?
In a study of 294 couples attending an infertility clinic, men with high levels of DNA fragmentation (> 30% DFI) were at a greater risk for low blastocyst rates and failure to initiate an ongoing pregnancy. In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) fertilization rates were not statistically different between high- and low-DFI groups. More men with more than 15 percent High DNA Stainability (HDS) had lower IVF fertilization rates. Men with DFI levels of more than 30 percent were at greater risk for low blastocyst rates and failure to initiate an ongoing pregnancy.
These studies as well as other studies were subjected to a meta analysis (Evenson and Wixon, 2006). If the DFI is greater than 30 percent (i.e., more than 30% of sperm have measurable DNA fragmentation), there is a significant negative effect on pregnancy outcome using natural, intrauterine insemination (IUI), and routine IVF fertilization. Results from studies that have used the SCSA analysis indicate that couples were seven times more likely to become pregnant if their DFI was less than 30 percent using natural (n = 362, P = 0.0001) and IUI fertilization (n = 518, P = 0.0001). If routine IVF was used, couples were approximately two times more likely to become pregnant if their DFI less than 30 percent (n = 381, P = 0.03). For ICSI and/or routine IVF, the results showed a nonsignificant trend where infertility couples were 1.6 times (CI 0.92, 2.94) more likely to achieve a pregnancy/delivery if the DFI was less than 30 percent (n = 323, P = 0.06).
These meta-analyses show that the SCSA infertility test was significantly predictive for reduced pregnancy success using in vivo, IUI, and routine IVF, and to a lesser extent ICSI fertilization. This manuscript provided information showing that the method of fertilization had a large impact on pregnancy success—which is a new concept to many clinicians.
Studies have been made to clarify the etiology of sperm DNA fragmentation. Infection significantly contributes to higher DFI values. This was seen clearly in a study by Alverez, et al. (2002) where leukocytospermic semen samples showed increased DFI values. Treatment with antibiotics often reduced the %DFI values.
Of great interest was the characterization of the different sperm populations seen in the SCSA analysis. These populations are: (1) main population without detectable DNA fragmentation, (2) low-level DFI population, (3) high-DFI population, and (4) HDS population.
These different populations were sorted into a test tube and then split into two separate aliquots. (1) Sperm were cytocentrifuged onto a glass slide, fixed, and stained with Feulgen stain. Two hundred sperm then were evaluated by a computer-assisted image analyzer for morphology and morphometry. The data show that the moderate-DFI population was not different from the main population. The high-DFI population, however, was in a state of cell degeneration with apoptotic and necrotic events.
(2) The second aliquot was cyotocentrifuged onto a glass slide that had been prepared for Comet analysis. Immediately after centrifugation, electrophoretic gel was placed on the spot of sperm. After protease incubation, these samples were then electrophoresed and stained with a DNA dye as per the protocol for Comet analysis. The image analyzer was used to quantitate 200 sperm per sample. The results (in preparation) showed that both the moderate- and high-DFI populations had more than 50 percent comets. The important point of this observation is that even though the moderate-DFI fraction looked normal under the light microscope, this population had a high level of sperm DNA fragmentation. This fraction likely is picked up by ICSI technicians with the likely consequences of reduced pregnancy outcomes, including an increased rate of spontaneous abortions.
Quality Assurance
All of our research efforts centered on the SCSA. This assay was pioneered in our laboratory 25 years ago. Since that time we have measured more than 100,000 sperm samples from animals and humans. The reason that this assay has been cited as the only DNA fragmentation assay rigorous enough for use in the human clinic is that the acridine orange biochemistry is precise and repeatable and that the flow cytometer measures 5,000 sperm per sample, thus providing a machine-derived observation on large populations. The confidence value between repeat samples is in the 1 to 2 percent range. All experimental data are derived after the flow cytometer has been set up with a “reference” sperm sample. This reference sample consists of small aliquots (1,000 aliquots made from a single sample and frozen in liquid nitrogen) that are used to bring the green and red fluorescence values to the same channel values (+ 5 channels). Thus, there is no “control” sample, but rather “reference” samples so that all experimental samples are compared to the “reference”. Thus, when setting up the flow cytometer before each “run”, fluorescent beads first are analyzed to attain the best possible focus of the laser beam. Then the reference sample is run to achieve the correct photomultiplier settings. The reference sample is run again after every 5-7 samples to verify that the instrument is operating exactly the same for each sample. If any single sample has odd values, potentially caused by the laser beam drifting or a sample line clog, the reference sample is run again to verify whether the experimental sample data were correct or the consequence of a machine malfunction (which is rare).
References:
Selevan SG, Borkovec L, Scott VL, Zudova Z, Rubes J, Evenson DP, Perreault SD. Air pollution and semen quality in young men residing in two Czech communities, 1998.
Moldan B, Schnoor JL. Czechoslovakia: examining a critically ill environment. Environmental Science & Technology 1992;26:14-21.
Stevens RK, Pinto JP, Willis RD, Mamane Y, Novak JJ, Beneš I. Monitoring and modeling methods for developing air pollution control strategies: a case study in the Northwest Czech Republic. In: Alligrini I, DeSantis F, eds. NATO AS Series, Partnership Sub-Series. 2: Environment. Springer-Verlag, Heidelberg 1996;151-166.
Watts R, Lewtas J, Stevens R, Hartlage T, Pinto J, Williams R, Hattaway K, Míšková I, Beneš I, Kotešovec F, Šrám R. Czech- U.S. EPA health study – assessment of personal and ambient air exposures to PAH and organic mutagens in the Teplice district of Northern Bohemia. International Journal of Environmental Analytical Chemistry 1994;271-287.
Ahlquist KA. Enzyme changes in rat testis produced by the administration of busulphan and of 7,12-dimethylbenz(a)anthracene. Journal of Reproductive Fertilization 1966;12;377-379.
Ford E, Huggins C. Selective destruction in testis induced by 7,12-dimethylbenz(a)-anthracene. Journal of Experimental Medicine 1963;118:27-40.
Generoso WM, Cain KT, Cornett CV, Russell EW, Hellwig CS, Horton CY. Difference in the ration of dominant-lethal mutations to heritable translocations produced in mouse spermatids and fully mature sperm after treatment with triethylenemelamine (TEM). Genetics 1982;100:633-640.
Georgellis A, Toppari J, Veromaa T, Rydström J, Parvinen M. Inhibition of meiotic divisions of rat spermatocytes in vitro by polycyclic aromatic hydrocarbons. Mutation Research 1990;231(2):125-135.
Binková B, Lewtas J, Míšková I, Lenicek J, Šrám R. DNA adducts and personal air monitoring of carcinogenic polycyclic aromatic hydrocarbons in an environmentally exposed population. Carcinogenesis 1995;16(5):1037-1046.
Binkova B, Lewtas J, Míšková I, Rössner P, Cerná M, Mrácková G, Peterková K, Numford J, Meyer S, Šrám R. Biomarker studies in Northern Bohemia. Environmental Health Perspectives 1996;104(S3):591-597.
Šrám RJ, Rožnikoá I, Albrecht V, Beránková A, Machovská E. Monitoring congenital anomalies in populations exposed to environmental mutagens. In: Kappas A, ed. Mechanisms of Environmental Mutagenesis – Carcinogenesis. Plenum, NY: 1990;25-236.
Šrám RJ, Beneš I, Binková B, Dejmek J, Horstman D, Kotĕšovec F, Otto D, Perreault SD, Rubes J, Selevan SG, Skalik I, Stevens RK, Lewtas J. Teplice program – the impact of air pollution on human health. Environmental Health Perspectives 1996;104(S4):699-714.
Evenson DP, Jost LK. Sperm chromatin structure assay: DNA denaturability. In: Darynkiewicz Z, Robinson JP, Crissman HA, eds. Methods in Cell Biology, Vol 42, Flow Cytometry, 2nd Edition. Orlando, FL: Academic Press, Inc. 1994;159-176.
Fossa SD, De Angelis P, Kraggerud SM, Evenson D, Theodorsen L, Claussen OP. Prediction of post-treatment spermatogenesis in patients with testicular cancer by flow cytometric sperm structure assay. Communications in Clinical Cytometry 1997;30:192-196.
Šrám RJ. Effect of glutathione S-transferase M1 polymorphisms on biomarkers of exposure and effects. Environmental Health Perspectives 1998;106(S1):231-239.
Journal Articles on this Report: 11 Displayed | Download in RIS Format
| Other project views: | All 29 publications | 19 publications in selected types | All 15 journal articles |
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Alvarez JG, Sharma RK, Ollero M, Saleh RA, Lopez MC, Thomas AJ, Evenson DP, Agarwal A. Increased DNA damage in sperm from leukocylospermic semen samples as determined by the sperm chromatin structure assay. Fertility and Sterility 2002;78(2):319-329 |
R827019 (2000) R827019 (Final) |
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Evenson DP, Larson KL, Jost LK. Sperm chromatin structure assay: its clinical use for detecting sperm DNA fragmentation in male infertility and comparisons with other techniques. Journal of Andrology 2002;23(1):25-43. |
R827019 (Final) |
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Evenson DP, Wixon RL. Environmental toxicants cause sperm DNA fragmentation as detected by the sperm chromatin structure assay (SCSA®). Toxicology and Applied Pharmacology 2005;207(2, Suppl 1):532-537. |
R827019 (Final) |
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Evenson D, Wixon R. Clinical aspects of sperm DNA fragmentation detection and male infertility. Theriogenology 2006;65(5):979-991. |
R827019 (Final) |
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Evenson D, Wixon R. Meta-analysis of sperm DNA fragmentation using the sperm chromatin structure assay. Reproductive BioMedicine Online 2006;12(4):466-472. |
R827019 (Final) |
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Larson-Cook K, Brannian JD, Hansen KA, Kasperson KM, Aamold ET, Evenson DP. Relationship between the outcomes of assisted reproductive techniques and sperm DNA fragmentation as measured by the sperm chromatin structure assay. Fertility and Sterility 2003;80(4):895-902. |
R827019 (Final) |
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Ostermeier GC, Sargeant GA, Yandell BS, Evenson DP, Parrish JJ. Relationship of bull fertility to sperm nuclear shape. Journal of Andrology 2001;22(4):595-603. |
R827019 (Final) |
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Perreault SD, Rubes J, Robbins WA, Evenson DP, Selevan SG. Evaluation of aneuploidy and DNA damage in human spermatozoa: applications in field studies. Andrologia 2000;32(4-5):247-254. |
R827019 (2000) R827019 (Final) |
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Rubes J, Selevan SG, Evenson DP, Zudova D, Vozdova M, Zudova Z, Robbins WA, Perreault SD. Episodic air pollution is associated with increased DNA fragmentation in human sperm without other changes in semen quality. Human Reproduction 2005;20(10):2776-2783. |
R827019 (Final) |
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Selevan SG, Borkovec L, Slott VL, Zudova Z, Rubes J, Evenson DP, Perreault SD. Semen quality and reproductive health of young Czech men exposed to seasonal air pollution. Environmental Health Perspectives 2000;108(9):887-894. |
R827019 (2000) R827019 (Final) |
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Virro MR, Larson-Cook KL, Evenson DP. Sperm chromatin structure assay (SCSA®) parameters are related to fertilization, blastocyst development, and ongoing pregnancy in in vitro fertilization and intracytoplasmic sperm injection cycles. Fertility and Sterility 2004;81(5):1289-1295. |
R827019 (Final) |
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atmosphere, exposure, health effects, human health, sensitive populations, dose-response, cellular, population, metabolism, genetic predisposition, susceptibility, toxics, particulates, PAHs, pollution prevention, clean technologies, cleanup, public policy, decision making, public good, environmental assets, biology epidemiology, genetics, pathology, monitoring, measurement methods, Czech Republic, industry,
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ENVIRONMENTAL MANAGEMENT, Air, Scientific Discipline, Health, RFA, Susceptibility/Sensitive Population/Genetic Susceptibility, Endocrine Disruptors - Environmental Exposure & Risk, Risk Assessment, Biology, Risk Assessments, genetic susceptability, Health Risk Assessment, endocrine disruptors, air toxics, Children's Health, Biochemistry, Environmental Chemistry, Genetics, Endocrine Disruptors - Human Health, developmental processes, exposure assessment, genotype, endocrine disrupting chemicals, genetic diversity, toxicants, Czech Republic, pollution exposure, DNA damage, human health risk, race ethnicity, sperm, aromatic hydrocarbons, ethnic, reproductive effects, reproductive processes, chromatin, young men, air pollution, reproductive, reproductive health, exposure, gender, PAH, environmentally-caused disease, biological effects, genetic predisposition, health risks, human exposure, sensitive subpopulations
Progress and Final Reports:
2000 Progress Report
Original Abstract