Environmental-endocrine control of reproductive maturation in gastropods: implications for the mechanism of tributyltin-induced imposex in prosobranchs.

Prosobranch snails have been afflicted globally by a condition whereby females develop male sex characteristics, most notably a penis. This condition, known as imposex, has been causally associated with the ubiquitous environmental contaminant tributyltin (TBT). Deduction of the mechanism by which TBT causes imposex has been hampered by the lack of understanding of the normal endocrine regulation of reproductive tract recrudescence in these organisms. We have reviewed the relevant literature on the environmental and endocrine factors that regulate reproductive tract recrudescence, sexual differentiation, and reproduction in gastropods. We provide a cohesive model for the environmental-endocrine regulation of reproduction in these organisms, and use this information to deduce a most likely mechanism by which TBT causes imposex. Photoperiod appears to be the predominant environmental cue that regulates reproductive tract recrudescence. Secondary cues include temperature and nutrition which control the timing of breeding and egg laying. Several hormone products of the central and peripheral nervous systems have been identified that contribute to recrudescence, reproductive behaviors, oocyte maturation and egg laying. Retinoic acid signaling via the retinoid X-receptor (RXR) has shown promise to be a major regulator of reproductive tract recrudescence. Furthermore, TBT has been shown to be a high affinity ligand for the RXR and the RXR ligand 9-cis retinoic acid causes imposex. We propose that TBT causes imposex through the inappropriate activation of this signaling pathway. However, uncertainties remain in our understanding of the environmental-endocrine regulation of reproduction in gastropods. Definitive elucidation of the mechanism of action of TBT awaits resolution of these uncertainties.

Use of the Adverse Outcome Pathway (AOP) framework to evaluate species concordance and human relevance of Dibutyl phthalate (DBP)-induced male reproductive toxicity.

Dibutyl phthalate (DBP) is a phthalate ester used as a plasticizer, and solvent. Studies using rats consistently report that DBP exposure disrupts normal development of the male reproductive system in part via inhibition of androgen synthesis. However, studies using xenograft models report that in human fetal testis DBP exposure is unlikely to impair testosterone synthesis. These results question the validity of the rat model for assessment of male reproductive effects caused by DBP. The Adverse Outcome Pathway (AOP) framework was used to evaluate the available evidence for DBP-induced toxicity to the male reproductive system. Three relevant biological elements were identified: 1) fetal rats are more sensitive than other rodents and human fetal xenografts to DBP-induced anti-androgenic effects, 2) DBP-induced androgen-independent adverse outcomes are conserved amongst different mammalian models and human fetal testis xenografts, and 3) DBP-induced anti-androgenic effects are conserved in different mammalian species when exposure occurs during postnatal life stages.

Novel text analytics approach to identify relevant literature for human health risk assessments: A pilot study with health effects of in utero exposures.

BACKGROUND: Systematic reviews involve mining literature databases to identify relevant studies. Identifying potentially relevant studies can be informed by computational tools comparing text similarity between candidate studies and selected key (i.e., seed) references. Challenge Using computational approaches to identify relevant studies for risk assessments is challenging, as these assessments examine multiple chemical effects across lifestages (e.g., human health risk assessments) or specific effects of multiple chemicals (e.g., cumulative risk). The broad scope of potentially relevant literature can make selection of seed references difficult. Approach We developed a generalized computational scoping strategy to identify human health relevant studies for multiple chemicals and multiple effects. We used semi-supervised machine learning to prioritize studies to review manually with training data derived from references cited in the hazard identification sections of several US EPA Integrated Risk Information System (IRIS) assessments. These generic training data or seed studies were clustered with the unclassified corpus to group studies based on text similarity. Clusters containing a high proportion of seed studies were prioritized for manual review. Chemical names were removed from seed studies prior to clustering resulting in a generic, chemical-independent method for identifying potentially human health relevant studies. We developed a case study that focused on identifying the array of chemicals that have been studied with respect to in utero exposure to test the recall of this novel literature searching strategy. We then evaluated the general strategy of using generic, chemical-independent training data with two previous IRIS assessments by comparing studies predicted relevant to those used in the assessments (i.e., total relevant). Outcome A keyword search designed to retrieve studies that examined the in utero effects of environmental chemicals identified over 54,000 candidate references. Clustering algorithms were applied using 1456 studies from multiple IRIS assessments with chemical names removed as training data or seeds (i.e., semi-supervised learning). Using a six-algorithm ensemble approach 2602 articles, or approximately 5% of candidate references, were "voted" relevant by four or more clustering algorithms and manual review confirmed nearly 50% of these studies were relevant. Further evaluations on two IRIS assessments, using a nine-algorithm ensemble approach and a set of generic, chemical-independent, externally-derived seed studies correctly identified 77-83% of hazard identification studies published in the assessments and eliminated the need to manually screen more than 75% of search results on average. Limitations The chemical-independent approach used to build the training literature set provides a broad and unbiased picture across a variety of endpoints and environmental exposures but does not systematically identify all available data. Variance between actual and predicted relevant studies will be greater because of the external and non-random origin of seed study selection. This approach depends on access to readily available generic training data that can be used to locate relevant references in an unclassified corpus. Impact A generic approach to identifying human health relevant studies could be an important first step in literature evaluation for risk assessments. This initial scoping approach could facilitate faster literature evaluation by focusing reviewer efforts, as well as potentially minimize reviewer bias in selection of key studies. Using externally-derived training data has applicability particularly for databases with very low search precision where identifying training data may be cost-prohibitive.

Hazards of diethyl phthalate (DEP) exposure: A systematic review of animal toxicology studies.

BACKGROUND: Diethyl phthalate (DEP) is widely used in many commercially available products including plastics and personal care products. DEP has generally not been found to share the antiandrogenic mode of action that is common among other types of phthalates, but there is emerging evidence that DEP may be associated with other types of health effects. OBJECTIVE: To inform chemical risk assessment, we performed a systematic review to identify and characterize outcomes within six broad hazard categories (male reproductive, female reproductive, developmental, liver, kidney, and cancer) following exposure of nonhuman mammalian animals to DEP or its primary metabolite, monoethyl phthalate (MEP). METHODS: A literature search was conducted in online scientific databases (PubMed, Web of Science, Toxline, Toxcenter) and Toxic Substances Control Act Submissions, augmented by review of online regulatory sources as well as forward and backward searches. Studies were selected for inclusion using PECO (Population, Exposure, Comparator, Outcome) criteria. Studies were evaluated using criteria defined a priori for reporting quality, risk of bias, and sensitivity using a domain-based approach. Evidence was synthesized by outcome and life stage of exposure, and strength of evidence was summarized into categories of robust, moderate, slight, indeterminate, or compelling evidence of no effect, using a structured framework. RESULTS: Thirty-four experimental studies in animals were included in this analysis. Although no effects on androgen-dependent male reproductive development were observed following gestational exposure to DEP, there was evidence including effects on sperm following peripubertal and adult exposures, and the overall evidence for male reproductive effects was considered moderate. There was moderate evidence that DEP exposure can lead to developmental effects, with the major effect being reduced postnatal growth following gestational or early postnatal exposure; this generally occurred at doses associated with maternal effects, consistent with the observation that DEP is not a potent developmental toxicant. The evidence for liver effects was considered moderate based on consistent changes in relative liver weight at higher dose levels; histopathological and biochemical changes indicative of hepatic effects were also observed, but primarily in studies that had significant concerns for risk of bias and sensitivity. The evidence for female reproductive effects was considered slight based on few reports of statistically significant effects on maternal body weight gain, organ weight changes, and pregnancy outcomes. Evidence for cancer and effects on kidney were judged to be indeterminate based on limited evidence (i.e., a single two-year cancer bioassay) and inconsistent findings, respectively. CONCLUSIONS: These results suggest that DEP exposure may induce androgen-independent male reproductive toxicity (i.e., sperm effects) as well as developmental toxicity and hepatic effects, with some evidence of female reproductive toxicity. More research is warranted to fully evaluate these outcomes and strengthen confidence in this database.

Quantification of the Uncertainties in Extrapolating From In Vitro Androgen Receptor Antagonism to In Vivo Hershberger Assay Endpoints and Adverse Reproductive Development in Male Rats.

Multiple molecular initiating events exist that disrupt male sexual differentiation in utero including androgen receptor (AR) antagonism and inhibition of synthesis, and metabolism of fetal testosterone. Disruption of androgen signaling by AR antagonists in utero reduces anogenital distance (AGD) and induces malformations in F1 male rat offspring. We are developing a quantitative network of adverse outcome pathways that includes multiple molecular initiating events and key events linking anti-AR activities to permanent reproductive abnormalities. Here, our objective was to determine how accurately the EC50s for AR antagonism in vitro or ED50s for reduced tissue growth in the Hershberger assay (HA) (key events in the adverse outcome pathway) predict the ED50s for reduced AGD in male rats exposed in utero to AR antagonists. This effort included in-house data and published studies from the last 60 years on AR antagonism in vitro and in vivo effects in the HA and on AGD after in utero exposure. In total, more than 250 studies were selected and included in the analysis with data from about 60 potentially antiandrogenic chemicals. The ability to predict ED50s for key events and adverse developmental effects from the in vitro EC50s displays considerable uncertainty with R2 values for HA and AGD of < 6%. In contrast, there is considerably less uncertainty in extrapolating from the ED50s in the HA to the ED50s for AGD (R2 value of about 85%). In summary, the current results suggest that the key events measured in the HA can be extrapolated with reasonable certainty to predict the ED50s for the adverse in utero effects of antiandrogenic chemicals on male rat offspring.

Cumulative effects of in utero administration of mixtures of "antiandrogens" on male rat reproductive development.

Although risk assessments are typically conducted on a chemical-by-chemical basis, the 1996 Food Quality Protection Act (FQPA) required the Environmental Protection Agency (EPA) to consider cumulative risk of chemicals that act via a common mechanism of toxicity. To this end, we are conducting studies with mixtures to provide a framework for assessing the cumulative effects of "antiandrogenic" chemicals. Rats were dosed during pregnancy with antiandrogens singly or in pairs at dosage levels equivalent to about one half of the ED50 for hypospadias or epididymal agenesis. The pairs include: AR antagonists (vinclozolin plus procymidone), phthalate esters (DBP plus BBP and DEHP plus DBP), a phthalate ester plus an AR antagonist (DBP plus procymidone), and linuron plus BBP. We predicted that each chemical by itself would induce few malformations; however, by mixing any two chemicals together, about 50% of the males would be malformed. All binary combinations produced cumulative, dose-additive effects on the androgen-dependent tissues. We also conducted a mixture study combining seven "antiandrogens" together. These chemicals elicit antiandrogenic effects at two different sites in the androgen signaling pathway (i.e., AR antagonist or inhibition of androgen synthesis). In this study, the complex mixture behaved in a dose-additive manner. Our results indicate that compounds that act by disparate mechanisms of toxicity display cumulative, dose-additive effects when present in combination.

Hazards of diisobutyl phthalate (DIBP) exposure: A systematic review of animal toxicology studies.

BACKGROUND: Biomonitoring studies indicate a trend towards increased human exposure to diisobutyl phthalate (DIBP), a replacement for dibutyl phthalate (DBP). Recent reviews have found DIBP to be a male reproductive toxicant, but have not evaluated other hazards of DIBP exposure. OBJECTIVE: To inform chemical risk assessment, we performed a systematic review to identify and characterize outcomes within six broad hazard categories (male reproductive, female reproductive, developmental, liver, kidney, and cancer) following exposure of nonhuman mammalian animals to DIBP or the primary metabolite, monoisobutyl phthalate (MIBP). METHODS: A literature search was conducted in four online scientific databases [PubMed, Web of Science, Toxline, and Toxic Substances Control Act Test Submissions 2.0 (TSCATS2)], and augmented by review of regulatory sources as well as forward and backward searches. Studies were identified for inclusion based on defined PECO (Population, Exposure, Comparator, Outcome) criteria. Studies were evaluated using criteria defined a priori for reporting quality, risk of bias, and sensitivity using a domain-based approach. Evidence was synthesized by outcome and life stage of exposure, and strength of evidence was summarized into categories of robust, moderate, slight, indeterminate, or compelling evidence of no effect, using a structured framework. RESULTS: Nineteen toxicological studies in rats or mice met the inclusion criteria. There was robust evidence that DIBP causes male reproductive toxicity. Male rats and mice exposed to DIBP during gestation had decreased testosterone and adverse effects on sperm or testicular histology, with additional phthalate syndrome effects observed in male rats. There was also evidence of androgen-dependent and -independent male reproductive effects in rats and mice following peripubertal or young adult exposure to DIBP or MIBP, but confidence was reduced because of concerns over risk of bias and sensitivity in the available studies. There was also robust evidence that DIBP causes developmental toxicity; specifically, increased post-implantation loss and decreased pre- and postnatal growth. For other hazards, evidence was limited by the small number of studies, experimental designs that were suboptimal for evaluating outcomes, and study evaluation concerns such as incomplete reporting of methods and results. There was slight evidence for female reproductive toxicity and effects on liver, and indeterminate evidence for effects on kidney and cancer. CONCLUSION: Results support DIBP as a children's health concern and indicate that male reproductive and developmental toxicities are hazards of DIBP exposure, with some evidence for female reproductive and liver toxicity. Data gaps include the need for more studies on male reproductive effects following postnatal and adult exposure, and studies to characterize potential hormonal mechanisms in females.

Proposed Key Characteristics of Male Reproductive Toxicants as an Approach for Organizing and Evaluating Mechanistic Evidence in Human Health Hazard Assessments.

BACKGROUND: Assessing chemicals for their potential to cause male reproductive toxicity involves the evaluation of evidence obtained from experimental, epidemiological, and mechanistic studies. Although mechanistic evidence plays an important role in hazard identification and evidence integration, the process of identifying, screening and analyzing mechanistic studies and outcomes is a challenging exercise due to the diversity of research models and methods and the variety of known and proposed pathways for chemical-induced toxicity. Ten key characteristics of carcinogens provide a valuable tool for organizing and assessing chemical-specific data by potential mechanisms for cancer-causing agents. However, such an approach has not yet been developed for noncancer adverse outcomes. OBJECTIVES: The objective in this study was to identify a set of key characteristics that are frequently exhibited by exogenous agents that cause male reproductive toxicity and that could be applied for identifying, organizing, and summarizing mechanistic evidence related to this outcome. DISCUSSION: The identification of eight key characteristics of male reproductive toxicants was based on a survey of known male reproductive toxicants and established mechanisms and pathways of toxicity. The eight key characteristics can provide a basis for the systematic, transparent, and objective organization of mechanistic evidence relevant to chemical-induced effects on the male reproductive system. https://doi.org/10.1289/EHP5045.

Diverse mechanisms of anti-androgen action: impact on male rat reproductive tract development.

Scientists have identified environmental chemicals that display anti-androgenic activity via multiple mechanisms of action. Early studies focused on pesticides acting as androgen receptor (AR) antagonists but it soon became apparent that was not the only endocrine mode by which compounds affected the androgen signalling pathway. Classes of chemicals currently known to interfere with the androgen signalling pathway include dicarboximide fungicides (e.g. vinclozolin), organochlorine-based insecticides (e.g. p,p'-DDT and -DDE), conazole fungicides (e.g. prochloraz), plasticizers (phthalates) and urea-based herbicides (linuron). Phthalate esters (PEs) and vinclozolin appear to act primarily via a single mechanism of action, while others such as linuron and prochloraz, appear to display dual mechanisms of action. Exposure to PEs decreases mRNA expression of key steroidogenic enzymes and also the peptide hormone insulin-like peptide 3 (insl3) from the foetal Leydig cells. Hence, both androgen- and inls3-dependent tissues are affected. Vinclozolin and procymidone act solely through binding to the AR as antagonists thus blocking the action of androgen at the cellular level but do not affect foetal testosterone synthesis or insl3 gene expression. The compounds linuron and prochloraz are AR antagonists but also inhibit foetal testosterone synthesis, although unlike the PEs, mRNA expression of steroidogenic enzymes and insl3 are not affected. All the above chemicals disrupt androgen signalling in the foetal male rat and produce some malformations in common, but the precise profiles of effects in the offspring are pathognomonic for each mode of action. For example, the 'phthalate syndrome' vs. the 'vinclozolin syndrome' each displays a profile of effects which is clearly different. In summary, as more and more molecular studies with anti-androgenic compounds are conducted, the number of mechanisms by which compounds can affect the androgen signalling pathway is likely to increase. Furthermore, the effects of mixtures of these compounds are just beginning to be explored.

Assessment of Health Effects of Exogenous Urea: Summary and Key Findings.

PURPOSE OF REVIEW: Urea has been utilized as a reductant in diesel fuels to lower emission of nitrogen oxides, igniting interest in probable human health hazards associated with exposure to exogenous urea. Here, we summarize and update key findings on potential health effects of exogenous urea, including carcinogenicity. RECENT FINDINGS: No definitive target organs for oral exposure were identified; however, results in animal studies suggest that the liver and kidney could be potential target organs of urea toxicity. The available human-subject literature suggests that the impact on lung function is minimal. Based on the literature on exogenous urea, we concluded that there was inadequate information to assess the carcinogenic potential of urea, or perform a quantitative assessment to derive reference values. Given the limited information on exogenous urea, additional research to address gaps for exogenous urea should include long-term cancer bioassays, two-generation reproductive toxicity studies, and mode-of-action investigations.

Prenatal testosterone exposure permanently masculinizes anogenital distance, nipple development, and reproductive tract morphology in female Sprague-Dawley rats.

In mammals, abnormal increases in fetal androgens disrupt normal development of the female phenotype. Due to the recent concern regarding environmental androgen-active chemicals, there is a need to identify sources of fetal androgen variation and sensitive developmental markers for androgenic activity in female rats. Anogenital distances (AGD), nipple retention, reproductive tract, and external genitalia are morphological parameters organized by prenatal androgens and are predictive of altered masculinized/defeminized phenotype in adult female mice and rats. The objectives of this study were to (1) characterize the natural prenatal androgen environment of rats including the magnitude of the intrauterine position (IUP) effect, (2) characterize the permanent effects of prenatal androgen exposure on female rats, and (3) determine the ability of AGD and areolas to predict these permanent androgenic alterations in female rats. Untreated male fetal rats had higher tissue testosterone (T) concentrations than females in the amniotic fluid, reproductive tract, gonad, and fetal body. The intrauterine position (IUP) of male and female fetuses did not affect T concentrations or AGD in male or female rats at gestational day (GD) 22. Female offspring exposed to 0, 1.5, and 2.5 mg/kg/day testosterone propionate (TP) on GDs 14-18 displayed increased AGD at postnatal day (PND) 2 and decreased nipples at PND 13 and as adults. TP-induced changes in neonatal AGD and infant areola number were reliable indicators of permanently altered adult phenotype in female rats. Further, females in the two high-dose groups displayed increased incidences of external genital malformations and the presence of prostatic tissue, not normally found in female rats.

An environmental androgen, 17beta-trenbolone, affects delayed-type hypersensitivity and reproductive tissues in male mice.

Recently, a growth promoter for farm animals, trenbolone acetate, was identified as an environmental androgen that potentially affects reproduction. Because androgens also suppress immunity, it was hypothesized that an active metabolite of trenbolone acetate, 17beta-trenbolone (TB), might impair immune responses. Castrated adult CD-1 mice were injected daily with either one of two different doses of 17beta-trenbolone (TB), testosterone propionate (TP), or corn oil (vehicle). The antigen-specific immune response was assessed by measuring delayed-type hypersensitivity (DTH) responses. Reproductive response was assessed by measuring reproductive tissue mass and determining testosterone concentrations. Mice treated with TB or TP displayed larger reproductive tissue mass than males treated with corn oil. Furthermore, males exposed to the highest dose of TB displayed a reduced DTH response compared to vehicle-treated animals. In comparison, TP, at a similar dose, only minimally reduced the DTH response. These data support the reproductive and potentially immunosuppressive effects of this environmental androgen, and raise the possibility of health concerns for individuals or populations in contact with high concentrations of TB.

Cumulative effects of antiandrogenic chemical mixtures and their relevance to human health risk assessment.

Toxicological studies of defined chemical mixtures assist human health risk assessment by establishing how chemicals interact with one another to induce an effect. This paper reviews how antiandrogenic chemical mixtures can alter reproductive tract development in rats with a focus on the reproductive toxicant phthalates. The reviewed studies compare observed mixture data to mathematical mixture model predictions based on dose addition or response addition to determine how the individual chemicals in a mixture interact (e.g., additive, greater, or less than additive). Phthalate mixtures were observed to act in a dose additive manner based on the relative potency of the individual phthalates to suppress fetal testosterone production. Similar dose additive effects have been reported for mixtures of phthalates with antiandrogenic pesticides of differing mechanisms of action. Overall, data from these phthalate experiments in rats can be used in conjunction with human biomonitoring data to determine individual hazard indices, and recent cumulative risk assessments in humans indicate an excess risk to antiandrogenic chemical mixtures that include phthalates only or phthalates in combination with other antiandrogenic chemicals.

Melatonin receptor (MT1) knockout mice display depression-like behaviors and deficits in sensorimotor gating.

Although critical for transducing seasonal information, melatonin has also been implicated in several physiological systems, as well as the regulation of behavioral and cognitive processes. Therefore, we investigated the neurobehavioral effects of mice missing the type 1 melatonin receptor (MT1). Male and female MT1 knockout (MT1-/-) and wild-type (WT) mice were tested in the acoustic startle/prepulse inhibition (PPI), open field and Porsolt forced swim tests. Male and female MT1-/- mice displayed dramatically impaired prepulse inhibition in the acoustic startle response. Female WT mice were more active in the open field than WT males. However, male and female MT1-/- mice did not differ in total locomotor activity. WT animals spent significantly more time in the center of the arena (a behavioral outcome associated with reduced anxiety-like behavior) than MT1-/- mice. Also, the sex difference between male and female WT mice in the amount of time spent in the center versus periphery was not observed among MT1-/- mice. Both male and female MT1-/- mice significantly increased the time spent immobile in the forced swim test, an indication of depressed-like behavior. The lifetime lack of MT1 signaling contributes to behavioral abnormalities including impairments in sensorimotor gating and increases in depressive-like behaviors. Taken together, MT1 receptor signaling may be important for normal brain and behavioral function.

Establishing the "Biological Relevance" of Dipentyl Phthalate Reductions in Fetal Rat Testosterone Production and Plasma and Testis Testosterone Levels.

Phthalate esters (PEs) constitute a large class of compounds that are used for many consumer product applications. Many of the C2-C7 di-ortho PEs reduce fetal testicular hormone and gene expression levels in rats resulting in adverse effects seen later in life but it appears that relatively large reductions in fetal testosterone (T) levels and testis gene expression may be required to adversely affect reproductive development (Hannas, B. R., Lambright, C. S., Furr, J., Evans, N., Foster, P. M., Gray, E. L., and Wilson, V. S. (2012). Genomic biomarkers of phthalate-induced male reproductive developmental toxicity: a targeted RT-PCR array approach for defining relative potency. Toxicol. Sci. 125, 544-557). The objectives of this study were (1) to model the relationships between changes in fetal male rat plasma testosterone (PT), T levels in the testis (TT), T production (PROD), and testis gene expression with the reproductive malformation rates, and (2) to quantify the "biologically relevant reductions" (BRRs) in fetal T necessary to induce adverse effects in the offspring. In the fetal experiment, Harlan Sprague-Dawley rats were dosed with dipentyl phthalate (DPeP) at 0, 11, 33, 100, and 300 mg/kg/day from gestational days (GD) 14-18 and fetal testicular T, PT levels, and T Prod and gene expression were assessed on GD 18. In the postnatal experiment, rats were dosed with DPeP from GD 8-18 and reproductive development was monitored through adulthood. The dose-response curves for TT levels (ED(50) = 53 mg/kg) and T PROD (ED(50) = 45 mg/kg) were similar, whereas PT was reduced at ED50 = 19 mg/kg. When the reductions in TPROD and Insl3 mRNA were compared with the postnatal effects of in utero DPeP, dose-related reproductive alterations were noted when T PROD and Insl3 mRNA were reduced by >45% and 42%, respectively. The determination of BRR levels may enable risk assessors to utilize fetal endocrine data to help establish points of departure for quantitative risk assessments.

A systematic evaluation of the potential effects of trichloroethylene exposure on cardiac development.

The 2011 EPA trichloroethylene (TCE) IRIS assessment, used developmental cardiac defects from a controversial drinking water study in rats (Johnson et al. [51]), along with several other studies/endpoints to derive reference values. An updated literature search of TCE-related developmental cardiac defects was conducted. Study quality, strengths, and limitations were assessed. A putative adverse outcome pathway (AOP) construct was developed to explore key events for the most commonly observed cardiac dysmorphologies, particularly those involved with epithelial-mesenchymal transition (EMT) of endothelial origin (EndMT); several candidate pathways were identified. A hypothesis-driven weight-of-evidence analysis of epidemiological, toxicological, in vitro, in ovo, and mechanistic/AOP data concluded that TCE has the potential to cause cardiac defects in humans when exposure occurs at sufficient doses during a sensitive window of fetal development. The study by Johnson et al. [51] was reaffirmed as suitable for hazard characterization and reference value derivation, though acknowledging study limitations and uncertainties.

Melatonin and immune function: hype or hypothesis?

Outside of the tropics, the combined challenge of food shortage and low temperature makes winter a particularly difficult time for reproduction and survival. Traditionally, vertebrate physiologists interested in seasonal phenomena have focused on reproduction and largely ignored survival. Our goal is to emphasize a new dimension to the study of seasonality by shifting the focus from reproduction to immune function and survival. Our hypothesis is that mechanisms enhancing immune function have evolved that enable individuals to anticipate predictable seasonal challenges that may adversely affect immunity. Stated differently, we visualize seasonal variations in survival as the interaction between two factors: the suppression of immune response due to changing energetic conditions and an endogenous rhythm of enhancement of immune response that is dependent on photoperiod, clocks, and melatonin. In this article, we review evidence of photoperiod influences on seasonal changes in immune function within the context of energy allocation. Further, we also examine mounting evidence that the pattern of melatonin secretion, mediated by photoperiod, directly influences immune function.

Aggressive behavior increases after termination of chronic sildenafil treatment in mice.

Recent reports to the U.S. Food and Drug Administration Adverse Event Reporting System implicate sildenafil citrate in adverse emotional and aggressive behaviors. Sildenafil citrate (Viagra) is widely prescribed for erectile dysfunction and acts by inhibiting phosphodiesterase Type-5, resulting in accumulation of cyclic-guanosine monophosphate (cGMP). Cyclic-GMP is synthesized by guanylyl cyclase that is directly activated by the messenger molecule, nitric oxide (NO), formed throughout the CNS by the enzyme nitric oxide synthase (NOS). Elevated concentrations of cGMP have been associated with increased aggressive behavior. In addition, the potential effect of cGMP accumulation on NO-mediated behavioral and neuroendocrine function through possible feedback mechanisms remains unspecified; however, neuronal NOS (nNOS) inhibition by pharmacologic agents or ablation of the gene encoding nNOS increases aggressive behavior in male mice. We tested the hypothesis that sildenafil citrate may increase aggression via its actions on cGMP and potential feedback inhibition of NO concentrations. Male C57BL/6 mice were injected with saline vehicle (0), 2, 5, 8, or 10 mg/kg of sildenafil citrate thrice weekly for 4 weeks. Latency to display aggressive behavior, frequency, and duration of aggressive behavior were recorded during neutral-arena aggression tests. No change in agonistic behavior was observed in mice during treatment with sildenafil citrate. However, sildenafil-treated mice given the highest dose were generally more aggressive 1 week post-cessation of drug treatment as compared to vehicle-treated mice. Additional investigation into potential withdrawal effects or abuse doses seems warranted.

The anogenital distance index of mice (Mus musculus domesticus): an analysis.

The anogenital distance (AGD) is sexually dimorphic in Mus musculus domesticus, with the male AGD approximately twice as long as that in female mice. Among female mice, the AGD varies as a function of prenatal androgen exposure. The anogenital distance index (AGDI) has been developed to serve as an indicator of prior androgen exposure due to intrauterine position (IUP). Concerns have been raised that the AGDI may not be an appropriate indicator of female IUP in mice. To further refine the AGDI, we have applied some commonly used and suggested transformations to the original data set of female CD-1 mice of known IUP, weaning body mass, and AGD. Our analysis suggests that the residual log transformation and untransformed body mass AGDIs are the most accurate means to predict the IUP of the pup. However, the IUP is only one mechanism by which a fetus may be exposed to hormonal variations in utero. Additional analyses revealed that the AGDI is influenced not only by the IUP of the female fetus but also by the identity of the dam (indicative of maternal influences) and the number of male fetuses found in the particular uterine horn. Therefore, the AGDI is not strictly a predictor of female IUP but of the intrauterine androgen environment in mice.

Photoperiodic regulation of circulating leukocytes in juvenile Siberian hamsters: mediation by melatonin and testosterone.

The reproductive system of Siberian hamsters (Phodopus sungorus) undergoes rapid phenotypic responses to changes in day length that occur around the time of weaning. The present experiments tested whether the immune system of Siberian hamsters is similarly photoperiodic early in life and whether photoperiodic changes in melatonin or gonadal hormone secretions mediate any such responses to day length. Circulating blood leukocyte concentrations (WBC) were measured in juvenile male Siberian hamsters that were gestated in long-days (LD), transferred to short-days (SD) on the day of birth, and subsequently either remained in SD or were transferred from SD to LD at 18 days of age (day 18). WBC values were comparable between LD and SD hamsters on day 18. Between day 18 and day 32, SD hamsters exhibited a 3-fold increase in WBC, whereas LD hamsters failed to undergo a significant increase in WBC during this interval. WBC of LD hamsters was significantly lower than that of SD hamsters on day 25 and on day 32. In LD housed males, peripheral injections of melatonin delivered so as to extend the nocturnal duration of elevated endogenous melatonin secretion (i.e., provided in late afternoon) on days 18-31 increased WBC as measured on day 32. Peripubertal (day 17) gonadectomy abolished the immunosuppressive effect of LD exposure on WBC, and treatment with silastic implants containing testosterone suppressed WBC independent of photoperiod treatment. These data indicate that juvenile Siberian hamsters are immunologically responsive to photoperiod and that the leukocyte responses to day length are the result of melatonin-mediated effects of photoperiod on testicular hormone secretion.