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Environmental chemicals that alter steroid production could interfere with male reproductive development and function. Three agricultural antifungal triazoles that are known to modulate expression of cytochrome P450 (CYP) genes and enzymatic activities were tested for effects on steroidogenesis using rat in vivo (triadimefon), rat in vitro (myclobutanil and triadimefon), and human in vitro (myclobutanil, propiconazole, and triadimefon) model systems. Hormone production was measured in testis organ cultures from untreated adult and neonatal rats, following in vitro exposure to 1, 10, or 100 muM of myclobutanil or triadimefon. Myclobutanil and triadimefon reduced media levels of testosterone by 40-68% in the adult and neonatal testis culture, and altered steroid production in a manner that indicated CYP17-hydroxylase/17,20 lyase (CYP17A1) inhibition at the highest concentration tested. Rat to human comparison was explored using the H295R (human adrenal adenocarcinoma) cell line. Following 48 h exposure to myclobutanil, propiconazole, or triadimefon at 1, 3, 10, 30, or 100 muM, there was an overall decrease in estradiol, progesterone, and testosterone by all three triazoles. These data indicate that myclobutanil, propiconazole, and triadimefon are weak inhibitors of testosterone production in vitro. However, in vivo exposure of rats to triazoles resulted in increased serum and intra-testicular testosterone levels. This discordance could be due to higher concentrations of triazoles tested in vitro, and differences within an in vitro model system lacking hepatic metabolism and neuroendocrine control.

Background: Gene expression profiling of whole blood may be useful for monitoring toxicological exposure and for diagnosis and monitoring of various diseases. Several methods are available that can be used to transport, store, and extract RNA from whole blood, but it is not clear which procedures alter results. In addition, characterization of interindividual and sex-based variation in gene expression is needed to understand sources and extent of variability., Methods: Whole blood was obtained from adult male and female volunteers (n = 42) and stored at various temperatures for various lengths of time. RNA was isolated and RNA quality analyzed. Affymetrix GeneChips (n = 23) were used to characterize gene expression profiles (GEPs) and to determine the effects on GEP of storage conditions, extraction techniques, types of GeneChip, or donor sex. Hierarchical clustering and principal component analysis were used to assess interindividual differences. Regression analysis was used to assess the relative impact of the studied variables., Results: Storage of blood samples for >1 week at 4 degrees C diminished subsequent RNA quality. Interindividual GEP differences were seen, but larger effects were observed related to RNA extraction technique, GeneChip, and donor sex. The relative importance of the variables was as follows: storage < genechip < extraction technique < donor sex., Conclusion: Sample storage and extraction methods and interindividual differences, particularly donor sex, affect GEP of human whole blood.

We are coming to appreciate that at fertilization human spermatozoa deliver the paternal genome alongside a suite of structures, proteins and RNAs. Although the role of some of the structures and proteins as requisite elements for early human development has been established, the function of the sperm-delivered RNAs remains a point for discussion. The presence of RNAs in transcriptionally quiescent spermatozoa can only be derived from transcription that precedes late spermiogenesis. A cross-platform microarray strategy was used to assess the profile of human spermatozoal transcripts from fertile males who had fathered at least one child compared to teratozoospermic individuals. Unsupervised clustering of the data followed by pathway and ontological analysis revealed the transcriptional perturbation common to the affected individuals. Transcripts encoding components of various cellular remodeling pathways, such as the ubiquitin-proteosome pathway, were severely disrupted. The origin of the perturbation could be traced as far back as the pachytene stage of spermatogenesis. It is anticipated that this diagnostic strategy will prove valuable for understanding male factor infertility.

Triazole fungicides associated with a range of reported male reproductive effects in experimental animals were selected to assess potential toxic modes of action. Wistar Han rats were fed myclobutanil (M: 100, 500, or 2000 ppm), propiconazole (P: 100, 500, or 2500 ppm), or triadimefon (T: 100, 500, or 1800 ppm) from gestation day 6 to postnatal day (PND) 120. One male per litter was necropsied on PND1, 22, 50, or 92. Measurements included anogenital distance (AGD) at PND0, body and organ weights, serum hormone levels, age at preputial separation (PPS), sperm morphology and motility, and fertility and fecundity. AGD was increased by the high dose of all three triazoles, indicating hypervirilization. Triadimefon delayed PPS, consistent with delayed puberty, at 1800 ppm. Relative liver weights were increased at PND1, 50, and 92 by all three triazoles. Hepatocellular hypertrophy was present at PND50 from propiconazole and triadimefon and at PND92 from all three high-dose triazole treatments. Relative pituitary weights were decreased at PND92 by middle- and high-dose myclobutanil treatment. Absolute testis weights were increased at PND1 by myclobutanil, at PND22 by myclobutanil and triadimefon, and at PND50 by propiconazole and triadimefon treatment. Relative ventral prostate weights were increased at PND92 by myclobutanil and triadimefon treatment. Serum testosterone was increased at PND50 by triadimefon and at PND92/99 by all three triazole treatments. Insemination and fertility were impaired by myclobutanil and triadimefon treatment. In addition to the reproductive system effects, total serum thyroxine levels were decreased at PND92 by high-dose triadimefon. These reproductive effects are consistent with the disruption of testosterone homeostasis as a key event in the mode of action for triazole-induced reproductive toxicity.

Increasing interest in early preconception and periconception exposures and human developmental outcomes has led to studies that monitor subjects from before conception to gestation, birth and childhood. Monitoring ovulation, endometrial receptivity, fertilisation, implantation and gestation requires the non-invasive collection of biological information and samples, and the measurement of biochemical and biological markers (biomarkers) that are associated with the aforementioned physiological events. This paper describes some of the key features of biomarkers needed for epidemiological studies, identifies some existing and potential biomarkers and available measurement devices, and suggests some directions for identification and development of new biomarkers that might be employed in longitudinal studies involving the analysis of female reproductive function and of embryonic development.

Three triazole fungicides were evaluated for effects on female rat reproductive development. Rats were exposed via feed to propiconazole (P) (100, 500, or 2500 ppm), myclobutanil (M) (100, 500, or 2000 ppm), or triadimefon (T) (100, 500, or 1800 ppm) from gestation day 6 to postnatal day (PND) 98. Body weight (BW) and anogenital distance (AGD) at PND 0, age and BW at vaginal opening (VO), estrous cyclicity, and body and organ weight at necropsy were measured. BW at PND 0 was unaffected by treatment. AGD was increased by M2000. VO was delayed by M2000 and T1800. Estrous cyclicity was initially disrupted by P500, P2500 and T1800, but later normalized. At PND 99 there was a decrease in BW by T1800, an increase in liver weight by P2500 and T1800, and an increase in ovarian weight by M2000 and T1800. It is concluded that exposure to P, M and T adversely impacted female rodent reproductive development.

Four triazole fungicides used in agricultural or pharmaceutical applications were examined for hepatotoxic effects in mouse liver. Besides organ weight, histopathology, and cytochrome P450 (CYP) enzyme induction, DNA microarrays were used to generate gene expression profiles and hypotheses on potential mechanisms of action for this class of chemicals. Adult male CD-1 mice were exposed daily for 14 days to fluconazole, myclobutanil, propiconazole, or triadimefon at three dose levels by oral gavage. Doses were based on previous studies that resulted in liver hypertrophy or hepatotoxicity. All four triazoles caused hepatocyte hypertrophy, and all except triadimefon increased relative liver/body weight ratios at the middle and high dose levels. CYP enzyme activities were also induced by all four triazoles at the middle and high doses as measured by the dealkylations of four alkoxyresorufins, although some differences in substrate specificity were observed. Consistent with this common histopathology and biochemistry, several CYP and xenobiotic metabolizing enzyme (XME) genes were differentially expressed in response to all four (Cyp2d26 and Cyp3a11), or three of the four (Cyp2c40, Cyp2c55, Ces2, Slco1a4) triazoles. Differential expression of numerous other CYP and XME genes discriminated between the various triazoles, consistent with differences in CYP enzyme activities, and indicative of possible differences in mechanisms of hepatotoxicity or dose response. Multiple isoforms of Cyp1a, 2b, 2c, 3a, and other CYP and XME genes regulated by the nuclear receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR) were differentially expressed following triazole exposure. Based on these results, we expanded on our original hypothesis that triazole hepatotoxicity was mediated by CYP induction, to include additional XME genes, many of which are modulated by CAR and PXR.

Four triazole fungicides were studied using toxicogenomic techniques to identify potential mechanisms of action. Adult male Sprague-Dawley rats were dosed for 14 days by gavage with fluconazole, myclobutanil, propiconazole, or triadimefon. Following exposure, serum was collected for hormone measurements, and liver and testes were collected for histology, enzyme biochemistry, or gene expression profiling. Body and testis weights were unaffected, but liver weights were significantly increased by all four triazoles, and hepatocytes exhibited centrilobular hypertrophy. Myclobutanil exposure increased serum testosterone and decreased sperm motility, but no treatment-related testis histopathology was observed. We hypothesized that gene expression profiles would identify potential mechanisms of toxicity and used DNA microarrays and quantitative real-time PCR (qPCR) to generate profiles. Triazole fungicides are designed to inhibit fungal cytochrome P450 (CYP) 51 enzyme but can also modulate the expression and function of mammalian CYP genes and enzymes. Triazoles affected the expression of numerous CYP genes in rat liver and testis, including multiple Cyp2c and Cyp3a isoforms as well as other xenobiotic metabolizing enzyme (XME) and transporter genes. For some genes, such as Ces2 and Udpgtr2, all four triazoles had similar effects on expression, suggesting possible common mechanisms of action. Many of these CYP, XME and transporter genes are regulated by xeno-sensing nuclear receptors, and hierarchical clustering of CAR/PXR-regulated genes demonstrated the similarities of toxicogenomic responses in liver between all four triazoles and in testis between myclobutanil and triadimefon. Triazoles also affected expression of multiple genes involved in steroid hormone metabolism in the two tissues. Thus, gene expression profiles helped identify possible toxicological mechanisms of the triazole fungicides.

Metabolism of two triazole-containing antifungal azoles was studied using expressed human and rat cytochrome P450s (CYP) and liver microsomes. Substrate depletion methods were used due to the complex array of metabolites produced from myclobutanil and triadimefon. Myclobutanil was metabolized more rapidly than triadimefon, which is consistent with metabolism of the n-butyl side-chain in the former and the t-butyl group in the latter compound. Human and rat CYP2C and CYP3A enzymes were the most active. Metabolism was similar in microsomes prepared from livers of control and low-dose rats. High-dose (115 mg kg-1 day-1 of triadimefon or 150 mg kg-1 day-1 of myclobutanil) rats showed increased liver weight, induction of total CYP, and increased metabolism of the two triazoles, though the apparent Km appeared unchanged relative to the control. These data identify CYP enzymes important for the metabolization of these two triazoles. Estimated hepatic clearances suggest that CYP induction may have limited impact in vivo.

Characterizing gene expression in hair follicles can help to elucidate the hair growth cycle by delineating the genes and pathways involved in follicular growth and degeneration. The objectives of this study were to determine whether intact RNA could be extracted from a small number of plucked, unstaged hair follicles in sufficient quantity to conduct gene expression profiling, and to conduct global gene expression profiling. To this end, RNA was extracted from 1 to 3 unstaged follicles plucked from the scalp of 36 volunteers. The average quantifiable yield of RNA/follicle was 112.5 ng. Ribosomal ratios were lower than normally expected, but investigation indicated the RNA was intact. Ten of the samples were amplified and hybridized to Affymetrix genechips. On average, 2,567 of the total probe sets (8,500) were expressed in each sample; 1,422 were expressed in all 10 samples; 97 were significantly changed in one gender compared to the other, and 41 had high levels of interindividual variability. This study demonstrates that RNA of sufficient quantity and quality to use in microarray hybridizations can be obtained from as little as a single plucked human hair follicle. Genes expressed in all individuals are probably related to follicular growth and could form a starting set for developing signatures of toxicant exposure. The differentially expressed genes could be involved in producing gender and interindividual differences in hair growth.

A biomarker can be broadly defined as any biological index capable of being measured, which is associated with or indicative of a defined biological endpoint such as a developmental or disease stage. Identification and verification of anatomical, endocrine, cellular and molecular biomarkers is crucial for successful clinical diagnosis and treatment of toxicity and disease, as well as basic toxicological, epidemiological and other research. Various biomarkers of reproductive development and health have been identified, including those associated with pubertal development, adult reproductive health and pregnancy outcome. Herein we discuss those in situ biomarkers which have been more closely associated with toxicant action on the reproductive system. Biomarkers of toxicant exposure and susceptibility are addressed, but the majority of the review focuses on those biomarkers which may prove useful for determining current pathophysiological status or predicting future adverse outcomes. In males these are primarily associated with altered spermatogenesis and sperm parameters, and in females with altered endocrine function. We conclude that although few robust in situ biomarkers are currently available which are specific for toxicant exposure, susceptibility or effect in reproductive systems, there is expectation that post-genomic technologies offer a new paradigm for identifying and verifying such biomarkers as may exist.

A byproduct of drinking water disinfection, bromochloroacetic acid (BCA), acts as a reproductive toxicant in rats. To determine if BCA produces similar reproductive toxicity in mice, juvenile and adult C57BL/6 males were exposed to 0, 8, 24, 72 or 216 mg/kg of BCA once daily for 14 days. Five of 12 animals from each dose-group were sacrificed at the end of dosing, and testes, epididymes, and seminal vesicles harvested and weighed. Seven mice from each dose-group (including juvenile-exposed mice, following a 14-week maturation period) were used in a 40-day sequential breeding assay to determine if BCA targets a particular phase of spermatogenesis. No significant effects were observed in mice exposed to BCA as juveniles, and there were no effects on fertility by 14 weeks after dosing. However, effects were observed in adult-exposed mice over the first 10 days after BCA exposure: mean number of litters/male, percentage of litters/female bred, and total number of fetuses/male were all reduced by 72 and 216 mg/kg BCA. These results in adult mice indicate BCA disrupted differentiation of spermatids during dosing and the first 10 days of mating, and are consistent with the spermatid retention and atypical residual bodies observed in animals exposed to 72 and 216 mg/kg BCA. To investigate mechanisms involved, we utilized cDNA microarrays containing 950 testis-expressed genes to profile gene expression from Control and BCA-treated mice. Statistical analyses of microarray results identified 40 well-characterized genes differentially expressed in a dose responsive manner as a result of BCA exposure. Microarray results were supplemented with quantitative real-time PCR and Westerns for several genes and proteins. The 40 genes whose expression was altered by BCA are involved in numerous biological processes including: cell communication and adhesion, cell cycle and cell proliferation, metabolism, signal transduction, stress response, and spermatogenesis and male fertility. Modulated expression of these genes, particularly the 15 expressed in Sertoli cells and spermatids, offers new insights into potential mechanisms of BCA toxicity in the mouse testis.

Modern genomic technologies such as DNA arrays provide the means to investigate molecular interactions at an unprecedented level, and arrays have been used to carry out gene expression profiling as a means of identifying candidate genes involved in molecular mechanisms underlying a variety of phenotypes. By comparing gene expression profiles from normal and abnormal human testes with those from comparable infertile mouse models, we endeavored to identify genes and gene networks critical for male fertility. We used commercially available filter-based DNA arrays to analyze testicular gene expression from eight human testis biopsies and three different infertile mouse models (atrichosis mutation, ataxia telangiectasia knockout and CREMtau knockout). Forty-seven mouse genes exhibited differential testicular gene expression (P <0.01) associated with male infertility. These included genes involved in DNA repair (Vim, Rad23A, Rad23B), glutathione metabolism (Gsr, Gstp 1, Mgst1), proteolysis (Ace, Casp1, Ctsd), spermatogenesis (Prlr, Tmsb4 and Zfp-37) and stress response (Hsp 1, Osp94). The expression of 19 human genes was different (P<0.05) between normal and abnormal samples, including those associated with apoptosis (GADD45), gonad development (SOX9), proteolysis (PSMC3, SPINK2, TIMP3, UBE213) and signal transduction (DLK1, NAP4, S100A10). Direct comparison of differentially expressed human and mouse genes identified glucose phosphate isomerase, and the highly similar human tissue inhibitor of metalloproteinase 3 (TIMP3) and mouse Timp2. Using DNA microarrays to profile gene expression in testes from infertile animal models and humans will be useful for understanding congenital infertility, and also infertility caused by environmental exposures where the same genes and molecular mechanisms are involved.

The generation of corroborative data has become a commonly used approach for ensuring the veracity of microarray data. Indeed, the need to conduct corroborative studies has now become official editorial policy for at least 2 journals, and several more are considering introducing such a policy. The issue of corroborating microarray data is a challenging one-there are good arguments for and against conducting such experiments. However, we believe that the introduction of a fixed requirement to corroborate microarray data, especially if adopted by more journals, is overly burdensome and may, in at least several applications of microarray technology, be inappropriate. We also believe that, in cases in which corroborative studies are deemed essential, a lack of clear guidance leaves researchers unclear as to what constitutes an acceptable corroborative study. Guidelines have already been outlined regarding the details of conducting microarray experiments. We propose that all stakeholders, including journal editorial boards, reviewers, and researchers, should undertake concerted and inclusive efforts to address properly and clarify the specific issue of corroborative data. In this article we highlight some of the thorny and vague areas for discussion surrounding this issue. We also report the results of a poll in which 76 life science journals were asked about their current or intended policies on the inclusion of corroborative studies in papers containing microarray data.

DNA microarrays and related tools offer promise for identification of pathways involved in toxic responses to xenobiotics. To be useful for risk assessment, experimental data must be challenged for reliability and interlaboratory reproducibility. Toward this goal, the Hepatotoxicity Working Group of the International Life Sciences Institute (ILSI) Health and Environmental Sciences Institute (HESI) Technical Committee on Application of Genomics to Mechanism-Based Risk Assessment evaluated and compared biological and gene expression responses in rats exposed to two model hepatotoxins--clofibrate and methapyrilene. This collaborative effort provided an unprecedented opportunity for the working group to evaluate and compare multiple biological, genomic, and toxicological parameters across different laboratories and microarray platforms. Many of the results from this collaboration are presented in accompanying articles in this mini-monograph, whereas others have been published previously. (Italic)In vivo(/Italic) studies for both compounds were conducted in two laboratories using a standard experimental protocol, and RNA samples were distributed to 16 laboratories for analysis on six microarray platforms. Histopathology, clinical chemistry, and organ weight changes were consistent with reported effects. Gene expression results demonstrated reasonable agreement between laboratories and across platforms. Discrepancies in expression profiles of some individual genes were largely due to platform differences and approaches to data analysis rather than to biological or interlaboratory variability. Despite these discrepancies there was overall agreement in the biological pathways affected by these compounds, demonstrating that transcriptional profiling is reproducible between laboratories and can reliably identify affected pathways necessary to provide mechanistic insight. This effort represents an important first step toward the use of transcriptional profiling in risk assessment.

Microarrays have the potential to significantly impact our ability to identify toxic hazards by the identification of mechanistically relevant markers of toxicity. To be useful for risk assessment, however, microarray data must be challenged to determine reliability and interlaboratory reproducibility. As part of a series of studies conducted by the International Life Sciences Institute Health and Environmental Science Institute Technical Committee on the Application of Genomics to Mechanism-Based Risk Assessment, the biological response in rats to the hepatotoxin clofibrate was investigated. Animals were treated with high (250 mg/kg/day) or low (25 mg/kg/day) doses for 1, 3, or 7 days in two laboratories. Clinical chemistry parameters were measured, livers removed for histopathological assessment, and gene expression analysis was conducted using cDNA arrays. Expression changes in genes involved in fatty acid metabolism (e.g., acyl-CoA oxidase), cell proliferation (e.g., topoisomerase II-Alpha), and fatty acid oxidation (e.g., cytochrome P450 4A1), consistent with the mechanism of clofibrate hepatotoxicity, were detected. Observed differences in gene expression levels correlated with the level of biological response induced in the two in vivo studies. Generally, there was a high level of concordance between the gene expression profiles generated from pooled and individual RNA samples. Quantitative real-time polymerase chain reaction was used to confirm modulations for a number of peroxisome proliferator marker genes. Though the results indicate some variability in the quantitative nature of the microarray data, this appears due largely to differences in experimental and data analysis procedures used within each laboratory. In summary, this study demonstrates the potential for gene expression profiling to identify toxic hazards by the identification of mechanistically relevant markers of toxicity.

Genomics and proteomics have made it possible to define molecular physiology in exquisite detail, when tissues are accessible for sampling. However, many tissues are not accessible for human diagnostic evaluations or experimental studies, creating the need for surrogates that afford insight into exposures and effects in such tissues. Surrogate tissue analysis (STA) incorporating contemporary genomic and proteomic technologies may be useful in determining toxicant exposure and effect, or disease state, in target tissues at the pre- or early clinical stage. We present here a discussion of STA based on presentations given at the Society of Toxicology's 2003 annual meeting's "Innovations in Applied Toxicology" symposium. Speakers at the symposium (Box 1) discussed various potential applications of STA, including the use of peripheral blood lymphocytes (PBLs) as a source of genetic biomarkers to monitor radiation exposure; the use of gene expression analysis of PBLs and hair follicles as a means to monitor the impact of toxicants on inaccessible organs; the characterization of disease-associated gene signatures in peripheral blood mononuclear cells (PBMCs) of renal cell carcinoma (RCC) patients; the use of sperm RNA to determine genetic and environmental effects on sperm development in the testis; and the use of serum protein profiles to monitor the development and progression of various cancers. Also discussed are some of the challenges that must be overcome if the utility of STA is to be proven, and thus permit researchers to move this concept from the laboratory to the clinical environment.

The proposed National Children's Study has helped raise awareness of the issues related to children's health and the importance of monitoring the growth and development of children from preconception through adulthood. Many genetic predispositions can adversely impact the normal development process, and various environmental exposures have been linked to adverse reproductive health in rodent models and a small number of accidental human exposures. To monitor reproductive health and identify adverse effects at the earliest possible juncture, investigators must develop a network of biomarkers covering all stages and aspects of reproductive development and function. Biomarkers are biological indicators that can be measured repeatedly and are informative on one or more aspects of biological development or function. They can range from the anatomical level down to the molecular level and may provide information on the nature of an exposure, the effect of an exposure, or the susceptibility of individuals or populations to the toxic effects of an exposure. In theory, biomarkers can be used to monitor a wide variety of conditions and responses ranging from abnormal development to early indicators of late-onset disease. The main stumbling block with this theory has been finding appropriate biomarkers for particular conditions and exposures. Such biomarkers must be easily accessible, robust, and sensitive. Ideally, they will be expressed across a large section of the population, and can be monitored quickly, easily, conveniently, and with minimal cost. In this review, we discuss some of the current and emerging biomarkers of human pubertal development.

The determinants of successful human reproduction and development may act as early as periconceptionally, underscoring the need to capture exposures during these critical windows when assessing potential toxicants. To identify such toxicants, couples must be studied longitudinally prior to conception without regard to a couple's ability to ascertain a clinically recognized pregnancy. We examined the utility and feasibility of prospective pregnancy study designs by conducting a systematic review of the literature to summarize relevant information regarding the planning, implementation, and success of previously published prospective pregnancy studies. Information concerning design elements and participation was abstracted from 15 eligible studies (from a total of 20 identified studies) using a standardized form. The primary author of each study was contacted to review our summary of their work and obtain missing information. Our findings confirm the ability to recruit women/couples from diverse populations using a variety of recruitment strategies. Among the studies we reviewed, 4-97% of eligible individuals were successfully contacted, with enrollment rates ranging from 42 to 100%. Length of follow-up varied from 3 to 12 months. A high percentage of women provided urine (57-98%) and blood (86-91%) specimens and most male partners (94-100%) provided semen samples. These data support the feasibility of this design.

Detection, quantification, and prognosis of environmental exposures in humans has been vastly enhanced by the ability of epidemiologists to collect biospecimens for toxicologic or other laboratory evaluation. Ease of collection and level of invasiveness are commonly cited reasons why study participants fail to provide biospecimens for research purposes. The use of methodologies for the collection of biospecimens in the home offers promise for improving the validity of health effects linked to environmental exposures while maximizing the number and type of specimens capable of being collected in a timely and cost-effective manner. In this review we examine biospecimens (urine and blood) that have been successfully collected from the home environment. Related issues such as storage and transportation will also be examined as well as promising new approaches for collecting less frequently studied biospecimens (including hair follicles, breast milk, semen, and others). Such biospecimens are useful in the monitoring of reproductive development and function.

A letter discussing the issues surrounding post-hybridization confirmatory studies of microarray data.

The wealth of new information coming from the many genome sequencing projects is providing unprecedented opportunities for major advances in all areas of biology, including the environmental health sciences. To facilitate this discovery process, experts in the fields of functional genomics and informatics and the emerging field of toxicogenomics recently gathered at the Mount Desert Island Biological Laboratory in Salisbury Cove, Maine, site of a National Institute of Environmental Health Sciences Marine and Freshwater Biomedical Science Center, to share their ideas and latest research findings. The goal of the symposium was to highlight approaches that may be used to identify and characterize toxicologically relevant genes being discovered in the genome sequencing projects. Many of the approaches rely heavily on comparative models as a way of identifying gene homology, ontology, and physiologic function, and on the availability of databases that facilitate storage, analysis, interpretation, and widespread dissemination of relevant data.

We propose that gene expression changes in accessible tissues such as blood often reflect those in inaccessible tissues, thus offering a convenient biomonitoring method to provide insight into the effects of environmental toxicants on such tissues. In this pilot study, gene expression changes in peripheral blood leukocytes (PBL) were compared to those in the uteri of adult rats to identify genes that were altered in both tissues following estradiol treatment. Ovariectomized rats were treated with either 17beta-estradiol or vehicle control (corn oil) for 3 days. PBL and uterine RNAs were hybridized to arrays containing 1185 genes. One hundred and ninety three genes were expressed in common between the PBL and uterus. Eighteen were changed significantly in both tissues, 9 of which were treatment- but not tissue-specific (e.g., jun-D, phospholipase A2, thymidine kinase). These results demonstrate that many genes are coexpressed between PBL and uterus, and that some are coregulated by estradiol. Given the limited number of genes examined in this study and the estimated size of other mammalian genomes, we conclude that many more genes will also be coregulated and suggest that accessible tissues such as PBL can serve as surrogate tissues for observing gene expression changes in inaccessible target tissues.

Testicular heat shock was used to characterize cellular and molecular mechanisms involved in male fertility. This model is relevant because heat shock proteins (HSPs) are required for spermatogenesis and also protect cells from environmental hazards such as heat, radiation, and chemicals. Cellular and molecular methods were used to characterize effects of testicular heat shock (43 degrees C for 20 min) at different times posttreatment. Mating studies confirmed conclusions, based on histopathology, that spermatocytes are the most susceptible cell type. Apoptosis in spermatocytes was confirmed by TUNEL, and was temporally correlated with the expression of stress-inducible Hsp70-1 and Hsp70-3 proteins in spermatocytes. To further characterize gene expression networks associated with heat shock-induced effects, we used DNA microarrays to interrogate the expression of 2208 genes and thousands more expression sequence tags expressed in mouse testis. Of these genes, 27 were up-regulated and 151 were down-regulated after heat shock. Array data were concordant with the disruption of meiotic spermatogenesis, the heat-induced expression of HSPs, and an increase in apoptotic spermatocytes. Furthermore, array data indicated increased expression of four additional non-HSP stress response genes, and eight cell-adhesion, signaling, and signal-transduction genes. Decreased expression was recorded for 10 DNA repair and recombination genes; 9 protein synthesis, folding, and targeting genes; 9 cell cycle genes; 5 apoptosis genes; and 4 glutathione metabolism genes. Thus, the array data identify numerous candidate genes for further analysis in the heat-shocked testis model, and suggest multiple possible mechanisms for heat shock-induced infertility.

Background: Over the past five years, interest in and use of DNA array technology has increased dramatically, and there has been a surge in demand for different types of arrays. Although manufacturers offer a number of pre-made arrays, these are generally of utilitarian design and often cannot accommodate the specific requirements of focused research, such as a particular set of genes from a particular tissue. We found that suppliers did not provide an array to suit our particular interest in testicular toxicology, and therefore elected to design and produce our own., Results: We describe the procedures used by members of the US Environmental Protection Agency MicroArray Consortium (EPAMAC) to produce a mouse testis expression array on both filter and glass-slide formats. The approaches used in the selection and assembly of a pertinent, nonredundant list of testis-expressed genes are detailed. Hybridization of the filter arrays with normal and bromochloroacetic acid-treated mouse testicular RNAs demonstrated that all the selected genes on the array were expressed in mouse testes., Conclusion: We have assembled two lists of mouse (950) and human (960) genes expressed in the mouse and/or human adult testis, essentially all of which are available as sequence-verified clones from public sources. Of these, 764 are homologous and will therefore enable close comparison of gene expression between murine models and human clinical testicular samples.

Understanding the genetic profile of a cell at all stages of normal and carcinogenic development should provide an essential aid to developing new strategies for the prevention, early detection, diagnosis and treatment of cancers. We have attempted to identify some of the genes that may be involved in peroxisome-proliferator (PP)-induced non-genotoxic hepatocarcinogenesis using suppression PCR subtractive hybridisation (SSH). Wistar rats (male) were chosen as a representative susceptible species and Duncan-Hartley guinea pigs (male) as a resistant species to the hepatocarcinogenic effects of the PP, [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio] acetic acid (Wy-14,643). In each case, groups of four test animals were administered a single dose of Wy-14,643 (250 mg/kg per day in corn oil) by gastric intubation for 3 consecutive days. The control animals received corn oil only. On the fourth day the animals were killed and liver mRNA extracted. SSH was carried out using mRNA extracted from the rat and guinea pig livers, and used to isolate genes that were up and downregulated following Wy-14,643 treatment. These genes included some predictable (and hence positive control) species such as CYP4A1 and CYP2C11 (upregulated and downregulated in rat liver, respectively). Several genes that may be implicated in hepatocarcinogenesis have also been identified, as have some unidentified species. This work thus provides a starting point for developing a molecular profile of the early effects of a non-genotoxic carcinogen in sensitive and resistant species that could ultimately lead to a short-term assay for this type of toxicity.

The human genome contains an estimated 3 billion bases of DNA making up some 100000 genes, and the variation within this genome accounts for human diversity and, in many cases, disease. Defining and understanding the expression profile of given genotypes is essential to understanding adverse effects from acute or chronic exposure to environmental toxicants or other stimuli. DNA array technology could help researchers understand how organisms function in response to exposure by elucidating the molecular mechanisms that underlie them. DNA arrays have been developed and refined over the past 5 years and matured into a relatively accessible and affordable technology. They vary in design from membrane-based filters with a few hundred cDNAs, to glass-based 'chips' with tens of thousands of genetic elements. Mammalian DNA arrays will soon allow expression analysis on a genome-wide scale, similar to that already accomplished in some lower organisms (e.g. S. cerevisiae, E. coli). These whole-genome arrays will be powerful tools for identifying and characterizing toxicants in environmental and pharmaceutical science. This review discusses the technology behind the production of DNA arrays, the options available to those interested in applying them to their own research, and the possible toxicological applications of this exciting new technology.

DNA array technology makes it possible to rapidly genotype individuals or quantify the expression of thousands of genes on a single filter or glass slide, and holds enormous potential in toxicologic applications. This potential led to a U.S. Environmental Protection Agency-sponsored workshop titled "Application of Microarrays to Toxicology" on 7-8 January 1999 in Research Triangle Park, North Carolina. In addition to providing state-of-the-art information on the application of DNA or gene microarrays, the workshop catalyzed the formation of several collaborations, committees, and user's groups throughout the Research Triangle Park area and beyond. Potential application of microarrays to toxicologic research and risk assessment include genome-wide expression analyses to identify gene-expression networks and toxicant-specific signatures that can be used to define mode of action, for exposure assessment, and for environmental monitoring. Arrays may also prove useful for monitoring genetic variability and its relationship to toxicant susceptibility in human populations.

1. An important feature of the work of many molecular biologists is identifying which genes are switched on and off in a cell under different environmental conditions or subsequent to xenobiotic challenge. Such information has many uses, including the deciphering of molecular pathways and facilitating the development of new experimental and diagnostic procedures. However, the student of gene hunting should be forgiven for perhaps becoming confused by the mountain of information available as there appears to be almost as many methods of discovering differentially expressed genes as there are research groups using the technique. 2. The aim of this review was to clarify the main methods of differential gene expression analysis and the mechanistic principles underlying them. Also included is a discussion on some of the practical aspects of using this technique. Emphasis is placed on the so-called 'open' systems, which require no prior knowledge of the genes contained within the study model. Whilst these will eventually be replaced by 'closed' systems in the study of human, mouse and other commonly studied laboratory animals, they will remain a powerful tool for those examining less fashionable models. 3. The use of suppression-PCR subtractive hybridization is exemplified in the identification of up- and down-regulated genes in rat liver following exposure to phenobarbital, a well-known inducer of the drug metabolizing enzymes. 4. Differential gene display provides a coherent platform for building libraries and microchip arrays of 'gene fingerprints' characteristic of known enzyme inducers and xenobiotic toxicants, which may be interrogated subsequently for the identification and characterization of xenobiotics of unknown biological properties.

The technique of differential display reverse transcription-polymerase chain reaction (ddRT-PCR) has been used to produce unique profiles of up-regulated and down-regulated gene expression in the liver of male Wistar rats following short term exposure to the non-genotoxic hepatocarcinogens, phenobarbital and WY-14,643. Animals were treated for 3 days, whereupon their livers were extracted and snap frozen. mRNA was prepared from the livers and used for ddRT-PCR. Individual bands from the differential displays were extracted and cloned. False positives were eliminated by dotblot screening and true positives then sequenced and identified.

Infiltration by T lymphocytes into oesophageal carcinomas was assessed immunohistochemically, total T lymphocyte numbers by staining for CD3 and activated T lymphocytes by staining for CD25. Five squamous carcinomas and seven adenocarcinomas, resected without neoadjuvant treatment, were studied. Computer aided quantitation showed that total numbers of tumour infiltrating CD3 positive cells were highly variable (range 48-1673 cells/mm2). They were located largely in the stromal (87.9-99.2%) rather than intratumoral regions. Up to 84% of tumour infiltrating T lymphocytes were CD25 positive, although the median figure was 33%. There was no correlation between T lymphocyte infiltration or activation and expression of class I and II histocompatibility antigens, intercellular adhesion molecule-1, tumour stage or grade. These results imply that the local inflammatory response in oesophageal carcinomas is deregulated, which may be a factor contributing to the aggressive nature of the tumours.

Aim: To examine the expression of HLA-ABC and HLA-DR major histocompatibility (MHC) antigens and intercellular adhesion molecule (ICAM)-1 in normal, inflamed, metaplastic, and neoplastic oesophageal tissue and in freshly disaggregated tumours., Methods: Sequential sections of frozen tissue and cytospins of freshly disaggregated tumour were stained using the ABC peroxidase system and monoclonal antibodies specific for HLA-ABC, HLA-DR and ICAM-1., Results: Normal oesophageal tissue showed positive staining for HLA-ABC in the basal layers of the oesophageal squamous epithelium and on the epithelial cells of the submucosal oesophageal glands. HLA-DR and ICAM-1 were not detected in either of these cell types. In 20 of 37 (54%) carcinomas HLA-ABC was expressed weakly, with heterogeneous expression in nine (24%). Two tumours showed strong expression of HLA-ABC, but 15 of 37 (41%) were negative. HLA-DR and ICAM-1 were expressed weakly in six of 37 (16%) carcinomas without correlation with each other or with the expression of HLA-ABC., Conclusions: HLA-ABC is absent from a high proportion of oesophageal carcinomas (41%) and is otherwise variably and weakly expressed with strong expression in only a small fraction (3%). In other carcinomas there is a higher level of HLA-ABC expression. This discrepancy may partly explain the aggressive nature of oesophageal carcinomas. HLA-DR and ICAM-1 are not normally expressed on those cells from which oesophageal carcinomas are thought to arise. The limited expression found here could suggest a partial or inhibited immune response against oesophageal carcinoma. In vivo repressive factors may be involved.