In utero exposure to the environmental androgen trenbolone masculinizes female Sprague-Dawley rats.

Recently, the occurrence of environmental contaminants with androgenic activity has been described from pulp and paper mill effluents and beef feedlot discharges. A synthetic androgen associated with beef production is trenbolone acetate, which is used to promote growth in cattle. A primary metabolite, 17beta Trenbolone (TB), has been characterized as a potent androgen in both in vitro and in vivo studies with rats. The current study was designed to characterize the permanent morphological and functional consequences of prenatal TB exposure on female rats compared with those produced in an earlier study with testosterone propionate (TP). Female rat offspring were exposed to 0mg/day, 0.1mg/day, 0.5mg/day, 1.0mg/day, or 2.0mg/day TB on gestational days 14-19. The 0.5mg/day, 1.0mg/day, or 2.0mg/day TB groups displayed increases in neonatal anogenital distance (AGD) which persisted in the high dose group. Puberty was delayed in the high dose group and there were increased incidences of external genital malformations and the presence of male prostatic tissue in the 0.5mg/day, 1.0mg/day, or 2.0mg/day groups. These changes were associated with amniotic fluid concentrations of TB that compare favorably with concentrations known to be active in both in vitro systems and in fish.

Xenoendocrine disrupters-tiered screening and testing: filling key data gaps.

The US Environmental Protection Agency (EPA) is developing a screening and testing program for endocrine disrupting chemicals (EDCs) to detect alterations of hypothalamic-pituitary-gonadal (HPG) function, estrogen (ER), androgen (AR) and thyroid hormone synthesis and AR and ER receptor-mediated effects in mammals and other animals. High priority chemicals would be evaluated in the Tier 1 Screening (T1S) battery and chemicals positive in T1S would then be tested (Tier 2). T1S includes in vitro ER and AR receptor binding and/or gene expression, an assessment of steroidogenesis and mammalian (rat) and nonmammalian in vivo assays (Table 1). In vivo, the uterotropic assay detects estrogens and antiestrogens, while steroidogenesis, antithyroid activity, (anti)estrogenicity and HPG function are assessed in a 'Pubertal Female Assay'. (Anti-) androgens are detected in the Hershberger Assay (weight of AR-dependent tissues in castrate-immature-male rats). Fish and amphibian assays also are being developed. The fathead minnow assay can identify EDCs displaying several mechanisms of concern, including AR and ER receptor agonists and antagonists and inhibitors of steroid hormone synthesis. An amphibian metamorphosis assay is being developed to detect thyroid-active substances. Several alternative mammalian in vivo assays have been proposed. Of these, a short-term pubertal male rat assay appears most promising. An in utero-lactational screening protocol also is being evaluated. For Tier 2, the numbers of endocrine sensitive endpoints and offspring (F1) examined in multigenerational tests need to be expanded for EDCs. Consideration should be given to tailoring T2, based on the results of T1S. Tier 1 and 2 also should examine relevant mixtures of EDCs. Toxicants that induce malformations in AR-dependent tissues produce cumulative effects even when two chemicals act via different mechanisms of action.

Description and evaluation of a short-term reproduction test with the fathead minnow (Pimephales promelas).

Due to the time and expense associated with full life-cycle testing, most current toxicity tests with fish do not explicitly consider reproductive output as an endpoint but, rather, focus on early life-stage survival and development. However, some classes of chemicals could adversely impact reproduction at concentrations below those that affect development. Further, estimates of the effects of toxic compounds on reproductive output can be critical to the ecological risk assessment process. In this manuscript, we describe a short-term reproduction test with the fathead minnow (Pimephales promelas) and evaluate the test using two model reproductive toxicants, methoxychlor (an estrogenic compound) and methyltestosterone (an androgenic chemical). The test is initiated with reproductively mature animals and is comprised of a pre-exposure phase of 14 to 21 d, followed by a chemical exposure of up to 21 d. During and at completion of the test, several endpoints related to reproductive fitness and endocrine function are assessed. Both chemicals evaluated in our study caused a significant decrease in fecundity of the fish at nominal concentrations of 5.0 micrograms/L (methoxychlor) and 0.2 mg/L (methyltestosterone). Methoxychlor decreased plasma concentrations of one or more steroids (testosterone, 11-ketotestosterone, beta-estradiol) in both sexes and caused a significant induction of plasma vitellogenin in males, a response consistent with activation of the estrogen receptor by the pesticide (or its metabolites). Methyltestosterone decreased plasma concentrations of sex steroids and adversely affected gonadal status (as evaluated by relative weight and histopathology) in both sexes. The androgenic nature of methyltestosterone was clearly expressed as masculinization of exposed females via formation of nuptial tubercles, structures normally present only in reproductively active males. The chemical also caused a significant induction of plasma vitellogenin in both males and females; this unexpected estrogenic response was most likely due to aromatization of the androgen to a form capable of binding to the estrogen receptor. These studies demonstrate the utility of this short-term assay for identifying chemicals that exert reproductive toxicity through alterations in endocrine systems controlled by estrogens and androgens.

Amendment of sediments with a carbonaceous resin reduces bioavailability of polycyclic aromatic hydrocarbons.

We evaluated the effectiveness of Ambersorb, a carbonaceous resin, in reducing bioavailability of polycyclic aromatic hydrocarbons (PAHs) in contaminated sediments collected from the field. In laboratory studies, sediment pore-water concentrations of eight unsubstituted PAHs were significantly decreased after resin addition. Reduced PAH concentrations in oligochaete tissues from a laboratory bioaccumulation test, along with increased survival/reproduction and reduced photo-enhanced toxicity and sediment avoidance, also resulted from sediment treatment with Ambersorb. Resin amendment also decreased pore-water PAH concentrations in field deployed sediments but did not improve benthic invertebrate colonization. Prediction of partitioning of PAHs between solid and aqueous phases in the test sediments was complicated by the presence of coal and soot. However, accurate predictions of bioavailability were achieved based on pore-water chemistry. Overall, these studies show that the addition of high affinity sorbents effectively reduces pore-water PAH concentrations and bioavailability and suggests that sorbent addition may serve as an option for in situ remediation of some contaminated sediments.

Stage- and species-specific developmental toxicity of all-trans retinoic acid in four native North American ranids and Xenopus laevis.

Within the last decade, there have been increasing reports of malformed amphibians across North America. Recently, it has been suggested that hind-limb malformations are a consequence of xenobiotic disruption of developmental pathways regulated by retinoids. To assess the validity of this hypothesis, the developmental toxicity of all-trans retinoic acid (RA) was examined in Xenopus laevis and four North American anurans, at several life stages. To determine the effects of RA on embryogenesis, mid-blastula stage embryos were exposed to 0, 6.25, 12.5, 25, or 50 ng RA/ml for 24 h. To evaluate the effects of RA on hind-limb development, early- and mid-limb bud stage tadpoles were exposed to RA concentrations of 0, 250, 500, 750, 1000, or 1250 ng RA/ml for 24 h. Mid-blastula RA exposure resulted in a concentration-dependent increase in dysmorphogenesis and mortality in the three species examined (R. clamitans, R. septentrionalis and X. laevis). RA exposure at stage 51 in X. laevis and stage 28 in R. sylvatica resulted in concentration-dependent increases in reductions and deletions of the hind limb. However, RA was ineffective at inducing hind-limb abnormalities in stages 26 and 28 of R. pipiens, stage 28 in R. clamitans, or stage 48 in X. laevis tadpoles. These results indicate that mid-blastula stage embryos are more sensitive to RA-induced dysmorphogenesis and mortality than limb-bud stage tadpoles. The significance of these findings is discussed in the context of the possible occurrence of retinoid mimics in the environment.

Additive toxicity of binary mixtures of phototoxic polycyclic aromatic hydrocarbons to the oligochaete Lumbriculus variegatus.

Toxicity of some polycyclic aromatic hydrocarbons (PAHs) can increase by an order of magnitude, or more, in the presence of solar ultraviolet (UV) radiation. In the environment, PAHs exist as complex mixtures, which generally would include multiple PAHs that could cause photoinduced toxicity. Hence, to accurately predict the potential ecological risk of phototoxic PAHs, it is critical to understand their joint toxicity. In this study, we exposed the oligochaete Lumbriculus variegatus to the phototoxic PAHs anthracene, fluoranthene, and pyrene, both singly and as binary mixtures for 96 h. Following this, the animals were exposed to UV light for an additional 96 h, during which periodic observations of mortality were made. Time-dependent phototoxicity of the binary PAH mixtures, expressed as a function of the product of UV light intensity and PAH dose (in the tissue of the animals), was adequately described using a concentration addition model. Given the probability that the PAHs examined acted via a common mechanism of action, this result was consistent with expectations. These data highlight the need to consider the combined photoactivation potential of PAH mixtures and provide the technical basis for a modeling approach to predict their ecological risk.

Toxicity of 4-nonylphenol in a life-cycle test with the midge Chironomus tentans.

A life-cycle test with the macroinvertebrate Chironomus tentans was conducted with 4-nonylphenol. The chironomids were exposed to a series of concentrations of 4-nonylphenol via the water, in an intermittent (2 times/day) water renewal system. The test included evaluation of a number of developmental (e.g., growth) and reproductive (e.g., emergence, fecundity, viability) endpoints through parental and into F1 generations. Reductions in survival were observed in 20-day-old larvae at the highest test concentration, which corresponded to no-observable- and lowest-observable-effect concentrations of 42 and 91 microg/liter, respectively. No significant effects on larval growth (20 days), organism survival past 20 days, emergence success or pattern, sex ratio, fecundity, or egg viability were observed at any treatment level. Qualitative observations indicated an increase in deformed egg masses at the highest test concentrations; however, the biological significance of this is uncertain.