Inhibition of rat and human steroidogenesis by triazole antifungals.

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.

Effects of storage, RNA extraction, genechip type, and donor sex on gene expression profiling of human whole blood.

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.

Success and failure in human spermatogenesis as revealed by teratozoospermic RNAs.

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.

Disruption of testosterone homeostasis as a mode of action for the reproductive toxicity of triazole fungicides in the male rat.

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.

Biomarkers of ovulation, endometrial receptivity, fertilisation, implantation and early pregnancy progression.

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.

Effect of conazole fungicides on reproductive development in the female rat.

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.

Gene expression in head hair follicles plucked from men and women.

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.

Gene expression profiling in the liver of CD-1 mice to characterize the hepatotoxicity of triazole fungicides.

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.

Gene expression profiling in liver and testis of rats to characterize the toxicity of triazole fungicides.

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.

Reproductive and genomic effects in testes from mice exposed to the water disinfectant byproduct bromochloroacetic acid.

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.

Biomarkers of reproductive toxicity.

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.

Gene expression patterns associated with infertility in humans and rodent models.

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.

Overview of an interlaboratory collaboration on evaluating the effects of model hepatotoxicants on hepatic gene expression.

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.

Clofibrate-induced gene expression changes in rat liver: a cross-laboratory analysis using membrane cDNA arrays.

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.

Confirming microarray data--is it really necessary?

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.

Surrogate tissue analysis: monitoring toxicant exposure and health status of inaccessible tissues through the analysis of accessible tissues and cells.

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.

Prospective pregnancy study designs for assessing reproductive and developmental toxicants.

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.

The value of home-based collection of biospecimens in reproductive epidemiology.

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.

Biomarkers for assessing reproductive development and health: Part 1--Pubertal development.

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.

Exploiting genome data to understand the function, regulation, and evolutionary origins of toxicologically relevant genes.

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.