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This review considers antiviral nucleoside analog drugs, including ribavirin, favipiravir, and molnupiravir, which induce genome error catastrophe in SARS-CoV or SARS-CoV-2 via lethal mutagenesis as a mode of action. In vitro data indicate that molnupiravir may be 100 times more potent as an antiviral agent than ribavirin or favipiravir. Molnupiravir has recently demonstrated efficacy in a phase 3 clinical trial. Because of its anticipated global use, its relative potency, and the reported in vitro "host" cell mutagenicity of its active principle, β-d-N4-hydroxycytidine, we have reviewed the development of molnupiravir and its genotoxicity safety evaluation, as well as the genotoxicity profiles of three congeners, that is, ribavirin, favipiravir, and 5-(2-chloroethyl)-2'-deoxyuridine. We consider the potential genetic risks of molnupiravir on the basis of all available information and focus on the need for additional human genotoxicity data and follow-up in patients treated with molnupiravir and similar drugs. Such human data are especially relevant for antiviral NAs that have the potential of permanently modifying the genomes of treated patients and/or causing human teratogenicity or embryotoxicity. We conclude that the results of preclinical genotoxicity studies and phase 1 human clinical safety, tolerability, and pharmacokinetics are critical components of drug safety assessments and sentinels of unanticipated adverse health effects. We provide our rationale for performing more thorough genotoxicity testing prior to and within phase 1 clinical trials, including human PIG-A and error corrected next generation sequencing (duplex sequencing) studies in DNA and mitochondrial DNA of patients treated with antiviral NAs that induce genome error catastrophe via lethal mutagenesis.

Background: A gastroschisis incidence decline has recently been reported in Liaoning, China, from 2006-2015; we suspect that this decline could be associated with the fall in industrial manufacturing activities. Methods: The outcome variable, gastroschisis incidence, was from a previous publication. The industrial manufacturing activities were measured by relative job participation and production in the primary and secondary economic sectors, and the corresponding data were collected in 14 cities from 2005 to 2015. The general linear model and a random-effect Poisson regression model were performed to assess the association and time-series trend of gastroschisis with the manufacturing activities. Results: The relative job participation in the first economic sector was marginally associated with gastroschisis incidence in 2006 (p=0.070), but more significantly associated (Beta=-0.040; p=0.014) in the cross-sectional and time-series analysis with one-year lag in the outcome variable. Conclusion: The gastroschisis incidence decline in Liaoning is likely due to the reduced job participation in manufacturing and heavy industry, which might be an indication of reduced risk exposure associated with the industrial manufacturing activities.

Coronavirus disease 2019 (COVID-19) is a new infectious respiratory disease that has caused the ongoing global pandemic. The primary purpose of this article is to describe evolving clinical epidemiology of COVID-19, including 1) infection and testing, 2) clinical spectrum including classification of clinical type, asymptomatic cases, severe cases and comorbidity, and clinical and immunological response, 3) regional variation in clinical presentation, 4) population distribution by age, sex, and occupation, and finally, 5) case-fatality. This content may provide important information on detailed clinical type and presentation of the disease, in which appropriate clinical outcomes can be derived for developing prevention strategies and clinical studies or trials that aim to test potential therapeutics or products for different patient populations.

We received commentaries that included comments on our paper. Here we provide replies to the comments in the order received. To avoid confusion, we made a revision in the Table 1 to assure that level of significance and power at specified effect size are used appropriately.

There have been a series of recent discussions and debates on the p-value and statistical significance. These discussions, including publications of more than 40 papers in a special issue of the American Statistician, provide an excellent opportunity to think about some technical measures for practical implementation in grant applications and publications. While several factors have been discussed, it may be the rigor of a study that determines the p-value for reporting study results and judging a consistent replication of research. Both p-values and power, which integrate Fisherian and Neyman-Pearson methods, should be used for hypothesis testing. We propose new criteria, which can be implemented without fundamental changes in existing statistics, to reduce false positives and irreplicability of studies that are either inadequately powered or overpowered.

Background: Both genetic and epidemiological studies have indicated that environmental factors play an essential role in the development of autism spectrum disorder (ASD). We conducted this study to identify maternal exposure to environmental factors, in particular during the fetal development or perinatal period, associated with ASD. Methods: Two independent samples of children with ASD and typical developed (TD) were from distinct regions in China. Multiple logistic regression analysis was performed to identify factors associated with ASD in each sample and then in the combined sample. Results: Five factors were consistently associated with ASD in both samples. In the combined sample, maternal chemical exposure (odds ratio [OR] =4.50; 95% CI: 2.38-8.52), use of medication (OR = 3.19; 95% CI: 2.19-4.65), maternal infection (OR = 2.68; 95% CI: 1.99-3.61), threatened abortion (OR = 2.37; 95% CI: 1.61-3.50), and induced abortion before having the child (OR = 2.07; 95% CI: 1.65-2.60) showed strong associations with ASD; moreover, five factors explained 10-15% of the variation in the risk of ASD. A significant interaction between maternal infection and the use of medication during pregnancy was consistently detected in both independent and combined samples together. Conclusion: Two novel risk factors of maternal chemical exposure and induced abortion may have important implications for understanding the etiology of ASD, particularly in China. Prospective studies are needed to validate these findings, and necessary interventions are recommended to reduce the risk of ASD. KEYWORDS Autism spectrum disorder, chemical exposure, induced abortion, maternal infection

Background: Gastroschisis has increased globally over recent decades and this increase is not explained by demographic changes in maternal age. Implicated risk factors for this increase include lifestyle behaviors, environmental exposures, low-er socioeconomic status, lower body mass index, poor nutrition, smoking tobacco, using illicit drugs, alcohol, or analgesics and genitourinary infections. Methods: Selective review of the literature. Results: Present hypotheses would only suggest avoidance of suspect exposures as protective interventions. To identify safe and efficacious protective therapies, new cellular/molecular modes-of-action need to be considered. Plausible develop-mental modes-of-action include a) changes in epigenetic programming of relevant stem or progenitor cells; b) mechanical forces (cellular mechanosensitivity and mechanotransduction) signaling; and c) ephrin–Eph receptor multimodal signali-ng. These developmental modes-of-action present plausible options for “druggable” molecules that could be developed into protective or mitigative therapeutic agents for gastroschisis. Conclusion: Possible interventions for modifiable factors in gastroschisis include 1) Delay childbearing. 2) Improve nutri-tion for younger gravidas. 3) Pre-conceptional counseling to reduce embryonic exposures to the range of implicated lifest-yle, environmental and medical factors. 4) Urge research colleagues to investigate the cellular and molecular mechanisms underlying gastroschisis and to translate those insights into one or more safe and efficacious preventive or mitigative thera-pies.

Transparency in reporting the results of clinical and preclinical research is critical for unbiased publications. Funding agencies, publishers, and regulators have the responsibility to advocate and implement reporting standards for rigorous design. While individual study protocols may have included these standards, the items reported in the respective publications have often been inconsistent or lack transparency. This editorial intends to provide some specific guidelines for reporting results of clinical research with standards required for rigorous study design. We recommend that reporting clinical research should include sufficient information on study design and analysis plan that contains data processing, quality assurance, and appropriate methods used for rigorous statistical analysis or modeling. Any discrepancy between publications and original study design should be disclosed and discussed. Additionally, recent advances in the analysis of outcome with repeated measurements and statistical modeling should be employed to obtain unbiased estimates. Finally, we briefly discuss some issues reporting real-world evidence in clinical research.

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder typically diagnosed in children in the first few years of life. Genetic studies have demonstrated a moderate to high heritability of ASD, but only a limited number of single nucleotide polymorphisms (SNPs) have been identified. Meanwhile, numerous single de novo rare variants and copy number variations have been detected in patients with ASD, which are likely caused by environmental factors. Here we provide an overview of genetic and environmental factors that may contribute to the risk of ASD and we recommend that further study should be focused on both genes and environmental factors, as well as their interactions with the expectation that epigenetic studies will lead to understanding the link between the environment and risk of ASD.

Comments on Maternal exposure to triclosan constitutes a yet unrecognized risk factor for autism spectrum disorders. Cell Res. 2019. https://www.nature.com/articles/s41422-019-0220-1, PMID:31462724

To investigate the effect of dietary chromium (Cr) as Cr methionine (CrMet) on growth performance, carcass traits, pork quality, meat colour and expression of meat colour-related genes in growing-finishing pigs, 189 crossbred Duroc×(Landrace×Yorkshire) growing-finishing pigs (male, castrated, average initial BW 74.58±1.52 kg) were selected and randomly allocated into four groups. Dietary treatments per kg of feed were as follows: 0 (CT), 0.3 mg/kg (T1), 0.6 mg/kg (T2) and 0.9 mg/kg (T3) Cr (in the form of CrMet; as-fed basis), and each treatment was replicated five times with 8 to 10 pigs per replicate pen. During the 28 d of the experiment, both the ADG and the ADFI increased linearly (p

The neonatal hormone environment influences the sexually differentiated patterns of development. Estrogens, derived from intracerebral aromatization, promote male pattern development of the central nervous system. The purpose of this study was to determine the effects of neonatal exposure to environmental estrogens on luteinizing hormone (LH) secretion and development of the sexually dimorphic nucleus of the medial preoptic area (SDN-POA) in castrated adult rats. Neonatal rats of both sexes received injections of either corn oil, 0.1 microgram diethylstilbestrol (DES), 3 micrograms beta-sitosterol (B1), 30 micrograms beta-sitosterol (B2), 0.1 microgram coumestrol (C1), 1 microgram coumestrol (C2), or 10 micrograms coumestrol (C3) on Day 1-10 of life and were castrated on Day 21. Right heart catheters were placed on Day 42, and GnRH (50 ng/kg) was administered. Blood was sampled for LH at 0-, 5-, 10-, 15-, and 30-min intervals. All doses of beta-sitosterol and coumestrol elicited increased basal levels of LH in females. In males, B1, B2, C2, and C3 increased basal levels of LH. The GnRH-induced LH increase was prevented in females treated with diethylstilbestrol and 10 micrograms of coumestrol. Males in all treatment groups exhibited GnRH-induced LH surges. The animals were sacrificed by decapitation on Day 49. Volumes of the SDN-POA of the groups were compared. Treatment with the agents did not result in significantly increased SDN volume in females; nor was there a difference in SDN size among the male groups. These data show that exposure to environmental estrogens early in development alters both postpubertal pituitary response to GnRH and basal LH secretion in females and alters only basal LH secretion in males. No significant enlargement (i.e., masculinization) of the SDN-POA was exhibited.

Since presentation of our view of translational developmental toxicology in 2013, numerous investigations from a wide range of approaches have added to the evidence underlying our core premise; namely that as the potential adverse developmental effects from a range of exposures are progressively defined, preventative or mitigative therapies can be conceived, assessed and ethically implemented. The spectrum of studies reported in recent years span a wide range of exposure categories and various developmental outcomes ranging from molecular and cellular to the organismal level up to and including human behavior. Since human exposures to chemicals, physical agents and social factors are inevitable, the human fetus is subject to effects that can have lifelong consequences. In order to apply the translational concept to developmental toxicology, established or potential therapeutic obstetrical, neonatal, childhood and adolescent interventions will be required. Those that undergo testing during developmentally sensitive intervals will likely derive from generally-regarded-as-safe (GRAS) or well-established/repurposed pharmaceutical options. Ultimately if we are to translate environmental health discoveries into safe and effective interventions, we must assert and characterize valid, applicable therapies such as GRAS treatments and eventually “ethical pharmaceuticals” for the protective care of these highly vulnerable young people. We can create a safe and efficacious environmental health portfolio of interventional options to improve human health that include both reduction/avoidance of exposure and specific preventative/mitigative/restorative therapeutics. In this chapter we will broadly update new insights that have been gained over the last 2 years regarding the progress of translational developmental toxicology toward becoming a therapeutic discipline.

Background: The association of DDT (dichlorodiphenyltrichloroethane) with breast cancer is controversial, but animal studies directly linking DDT to risk are lacking. Concerns with DDT reside in its environmental persistence, bioaccumulation in breast adipose tissue, and endocrine-disrupting actions. Whereas most attention has been focused on estrogenic congeners, we tested the cancer-inducing potential of the antiandrogen, p,p´-DDE [1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene], the most prevalent and persistent DDT metabolite. Objectives: We aimed to determine whether developmental exposure to p,p´-DDE stored in adipose tissue surrounding the cancer-prone mammary epithelium of MMTV-Neu mice influences tumor development. Methods: For localized delivery, Elvax 40P pellets containing p,p´-DDE were implanted into the mammary fat pads of prepubertal female mice. We compared mammary tumor development with p,p´-DDE with development in response to its estrogenic isomer, o,p´-DDE [1,1-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl) ethylene], and a mixture of both isomers. Results: p,p´-DDE implants significantly accelerated mammary tumor onset compared with vehicle Elvax implants. o,p´-DDE had similar results, but only at ≤ 10 months of age. Lipid-adjusted levels of p,p´-DDE in mammary adipose tissue and serum in young mice were within the ranges of human exposure, whereas concentrations in aged mice were low to undetectable. Exposure to a 2:1 ratio of p,p´-DDE:o,p´-DDE did not result in the younger latency observed with the individual isomers. Conclusions: p,p´-DDE exposure at concentrations relevant to human exposure accelerates mammary carcinogenesis in mice, possibly through hormonal and/or other actions. These data suggest that DDE exposure would promote, but not cause, mammary tumorigenesis. Developmental exposure in immature mammary tissue continues to affect tumor onset even after p,p´-DDE levels have declined. Future studies are needed to determine whether early exposure to p,p´-DDE correspondingly predisposes women to early-onset breast cancer.

Reproductive function and fertility are thought to be compromised by behaviors such as cigarette smoking, substance abuse, and alcohol consumption; however, the strength of these associations are uncertain. Furthermore, the reproductive system is thought to be under attack from exposure to environmental contaminants, particularly those chemicals shown to affect endocrine homeostasis. The relationship between exposure to environmental contaminants and adverse effects on human reproductive health are frequently debated in the scientific literature and these controversies have spread into the lay press drawing increased public and regulatory attention. Therefore, the objective of the present review was to critically evaluate the literature concerning the relationship between lifestyle exposures and adverse effects on fertility as well as examining the evidence for a role of environmental contaminants in the purported decline of semen quality and the pathophysiology of subfertility, polycystic ovarian syndrome, and endometriosis. The authors conclude that whereas cigarette smoking is strongly associated with adverse reproductive outcomes, high-level exposures to other lifestyle factors are only weakly linked with negative fertility impacts. Finally, there is no compelling evidence that environmental contaminants, at concentrations representative of the levels measured in contemporary biomonitoring studies, have any effect, positive or negative, on reproductive health in the general population. Further research using prospective study designs with robust sample sizes are needed to evaluate testable hypotheses that address the relationship between exposure and adverse reproductive health effects.

Ovarian hormones regulate pituitary gonadotropin secretion across the menstrual cycle via negative and positive feedback mechanisms. The contribution of individual hormones is complex and is a continuing area of research.The aim of the study was to identify relationships between LH/FSH and estradiol, progesterone, inhibin A, inhibin B, and anti-Mullerian hormone (AMH) in ovulatory menstrual cycles across reproductive age.Serum ovarian and pituitary hormones were studied in a group of young (35 yr; n = 21) and older (45 yr; n = 55) women. The slopes of the regression lines relating the ovarian and pituitary hormones were determined by multiple linear regression analysis and expressed with 95% confidence intervals for each ovarian hormone, with FSH and LH as independent variables. Both simultaneous and delayed (time lagged) relationships were examined.Clear associations were evident for the lagged prediction of FSH, with significant negative associations being evident with inhibin B and AMH in the follicular phase and with estradiol, inhibin B, progesterone, and AMH in the luteal phase. For the lagged prediction of LH, significant positive and negative associations were observed with estradiol and inhibin B, respectively, in the follicular phase and a negative association with progesterone and inhibin B in the luteal phase.It is concluded that in the follicular phase, inhibin B is a major feedback regulator of FSH and may also be a negative feedback regulator of LH. AMH may be indirectly involved in FSH regulation.

To date, the endocrine changes accompanying female reproductive aging have been quite variable and unpredictable. The first standardized guidelines were drawn up at the Stages of Reproductive Aging Workshop (STRAW) in 2001. The present investigators attempted to describe comprehensively the endocrinological features associated with STRAW stages. Healthy women 21-35 and 45-55 years of age submitted 3 blood samples each week during a single menstrual cycle. The 21 younger women served as mid-reproductive age (MRA) controls with regular menstrual cycles. The 56 older women were classified at STRAW stage 3, late reproductive age (LRA) with regular cycles (n = 16); STRAW stage 2, early menopause transition (EMT) with cycles of varying length (n = 17); and STRAW stage 1, late menopause transition (LMT) with at least 1 intermenstrual interval of 60 days or longer (n = 23). The numbers of anovulatory cycles were 9, 1, 0, and 2, respectively, in the LMT, EMT, LRA, and MRA groups. Ovulatory-cycle levels of follicle-stimulating hormone (FSH), luteinizing hormone, and estradiol all increased with the progression of STRAW stage. Mean luteal-phase levels of serum progesterone declined. Early-cycle (ovulatory and anovulatory) levels of inhibin B declined steadily across STRAW stages, and it was largely undetectable during elongated ovulatory and anovulatory cycles in the menopausal transition. Levels of anti-Mullerian hormone decreased markedly by as much as 15-fold across the STRAW stages. These findings, although not conclusive, suggest that both inhibin B and anti-Mullerian hormone may be preferable to FSH for predicting STRAW stage with respect to the onset and progression of the menopausal transition.

The National Toxicology Program (NTP) and the National Institute of Environmental Health Sciences (NIEHS) established the NTP Center for the Evaluation of Risks to Human Reproduction (CERHR) in June 1998. The purpose of CERHR is to provide timely, unbiased, scientifically sound evaluations of human and experimental evidence for adverse effects on reproduction and development caused by agents to which humans may be exposed. Hydroxyurea was selected for evaluation by a CERHR expert panel because of (1) its increasing use in the treatment of sickle cell disease in children and adults, (2) knowledge that it inhibits DNA synthesis and is cytotoxic, and (3) published evidence of its reproductive and developmental toxicity in rodents. Hydroxyurea is FDA-approved for reducing the frequency of painful crises and the need for blood transfusions in adults with sickle cell anemia who experience recurrent moderate-to-severe crises. Hydroxyurea is used in the treatment of cancer, sickle cell disease, and thalassemia. It is the only treatment for sickle cell disease aside from blood transfusion used in children. Hydroxyurea may be used in the treatment of children and adults with sickle cell disease for an extended period of time or for repeated cycles of therapy. Treatment with hydroxyurea may bemore » associated with cytotoxic and myelosuppressive effects, and hydroxyurea is mutagenic.« less

In addition to estrogen dependence, endometriosis is characterized by chronic pelvic inflammation. The impact of the chronic pelvic inflammatory state on other organ systems and women’s health is unclear. Endometriosis associated chronic inflammation and potential adverse health effects across the lifespan render it imperative for renewed research vigor into the identification of novel biomarkers of disease and therapeutic options. Herein we propose a number of opportunities for research and development of new therapeutics to address the unmet needs in the treatment of endometriosisper seand its ancillary risks for other diseases in women across the lifespan.

This study determined the impact of breastfeeding on hypoestrogenic symptoms among women in the postpartum period and correlated these findings with the Estrogen Threshold Hypothesis, which postulates that the hypoestrogenic symptoms experienced are related to circulating estrogen levels.Using a survey instrument that combined previously validated assessments of postpartum mood changes and menopausal symptoms, women were evaluated in the immediate postpartum period, prior to hospital discharge, and at 3 and 6 weeks postpartum. Each time period was analyzed independently, in a cross-sectional design, where women were categorized as "breastfeeding" or "bottle feeding."Of 236 women recruited, 171 (72.5%) intended to breastfeed, and 62 (26.3%) intended to bottle feed. At both the 3- and 6-week postpartum evaluations, a similar percentage of women in the breastfeeding and bottle-feeding groups reported hot flashes. However, breastfeeding women were more likely to report vaginal dryness than those who did not breastfeed: 20/150 (13.3%) versus 3/80 (3.8%) at 3 weeks, p0.05; 25/143 (17.5%) versus 2/87 (2.3%) at 6 weeks, p0.001.The Estrogen Threshold Hypothesis accurately predicts the findings of increased reported vaginal dryness but not hot flashes during lactation.

Ovarian endometriomas are a common manifestation of endometriosis that can represent a more severe stage of the disease. There is much debate over the treatment of these cysts in infertile women, particularly before use of assisted reproductive technologies. Evidence exists that supports surgical excision of ovarian endometriomas, as well as evidence that cautions against surgical intervention. Certain factors need to be examined closely before proceeding with surgery or continuing with expectant management. These include the patient’s symptoms, age, ovarian reserve, size and laterality of the cyst, prior surgical treatment, and level of suspicion for malignancy. The most recent evidence appears to suggest that certain patient profiles may benefit from proceeding directly to in vitro fertilization (IVF). These include symptomatic infertile patients, especially those that are older, those that have diminished ovarian reserve, those that have bilateral endometriomas, or those that have had prior surgical treatment. Although endometriomas can be detrimental to the ovarian reserve, surgical therapy may further lower a woman’s ovarian reserve. Nevertheless, the presence of an endometrioma does not appear to adversely affect IVF outcomes, and surgical excision of endometriomas does not appear to improve IVF outcomes. Regardless of treatment plan, infertile patients with endometriomas must be counseled appropriately before choosing either treatment path.

Bisphenol A (BPA) is the monomer used to manufacture polycarbonate plastic, the resin lining of cans, and other products, with global capacity in excess of 6.4 billion lb/year. Because the ester bonds in these BPA-based polymers are subject to hydrolysis, leaching of BPA has led to widespread human exposure. A recent report prepared by the Harvard Center for Risk Analysis and funded by the American Plastics Council concluded that evidence for low-dose effects of BPA is weak on the basis of a review of only 19 studies; the report was issued after a delay of 2.5 years. A current comprehensive review of the literature reveals that the opposite is true. As of December 2004, there were 115 published in vivo studies concerning low-dose effects of BPA, and 94 of these report significant effects. In 31 publications with vertebrate and invertebrate animals, significant effects occurred below the predicted "safe" or reference dose of 50 microg/kg/day BPA. An estrogenic mode of action of BPA is confirmed by in vitro experiments, which describe disruption of cell function at 10(-12) M or 0.23 ppt. Nonetheless, chemical manufacturers continue to discount these published findings because no industry-funded studies have reported significant effects of low doses of BPA, although90% of government-funded studies have reported significant effects. Some industry-funded studies have ignored the results of positive controls, and many studies reporting no significant effects used a strain of rat that is inappropriate for the study of estrogenic responses. We propose that a new risk assessment for BPA is needed based on a) the extensive new literature reporting adverse effects in animals at doses below the current reference dose; b) the high rate of leaching of BPA from food and beverage containers, leading to widespread human exposure; c) reports that the median BPA level in human blood and tissues, including in human fetal blood, is higher than the level that causes adverse effects in mice; and d) recent epidemiologic evidence that BPA is related to disease in women.

A panel convened by the Harvard Center for Risk Analysis (HCRA) evaluated the weight of evidence for potential developmental and reproductive toxicity of bisphenol A (BPA, CASRN 80-05-7) in animals at doses well below the Lowest Observed Adverse Effect Level (LOAEL) of 50 mg/kg-day previously identified by the U.S. Environmental Protection Agency (US EPA) and even US EPA's reference dose (RfD) of 0.05 mg/kg-day. The effects are hypothesized to occur through an endocrine-modulating mode of action, specifically through estrogen receptors. The panel focused on potential male reproductive effects but also examined other endpoints possibly associated with hormone-like effects. The review considered studies published through April 2002. A formal deliberation framework focused on consistency, generalizability, and biological plausibility. The panel found no consistent affirmative evidence of low-dose BPA effects for any endpoint. Inconsistent responses across rodent species and strains made generalizabi...

The human population in the industrialized world is ubiquitously exposed to complex mixtures of persistent pollutants that contaminate food, water, and air. A large number of these contaminants have been shown to cause significant toxicity to the hypothalamic-pituitary-thyroid (HPT) axis in laboratory animal studies, through a variety of mechanisms, although these effects occur at levels of exposure greatly in excess of common human exposure. While many of the mechanisms of thyroid toxicity of these substances are potentially complementary, little is known of the degree of interaction of common persistent contaminants on responses of the HPT axis. To investigate the potential effects of a complex, environmentally relevant mixture on the HPT axis, sexually mature male rats were administered a mixture of 16 common organochlorines (dichlorodiphenoxytrichloroethane [DDT], p,p'-dichlorodiphenoxydichloroethylene [p,p'-DDE], hexachlorobenzene [HCB], tetrachlorodibenzo-p-dioxin [TCDD], polychlorinated biphenyls [PCBs], methoxychlor, endosulfan, heptachlor, hexachlorocyclohexane, dieldrin, aldrin, mirex, and several chlorinated benzenes, and metal contaminants [lead, cadmium]). The doses of the mixture that were administered were related to minimum risk levels or tolerable daily intakes of these substances, as derived by risk assessment with the 1x, 10x, 100x, and 1000x groups receiving mixture components at doses equivalent to 1x, 10x, 100x, or 1000x the minimum risk level (or tolerable daily intake, reference dose), respectively. After 70 daily treatments by gavage, endpoints related to circulating thyroid hormone (serum thyroxine [T(4)], triiodothyronine [T(3)], thyroid stimulating hormone [TSH], and serum T(3) uptake [T(3)-up]), thyroid gland histomorphology (thyroid follicle cross sectional area, epithelial height, follicle roundness or aspect ratio, colloid/epithelial ratio) and hepatic metabolism of thyroid hormone (UDP-glucuronyl transferase [UGT] and outer-ring deiodinase [ORD]) were assessed. All examined endpoints were significantly altered by the mixture albeit with great variability between endpoints in the sensitivity. While most endpoints examined did not show significant changes at mixture doses below 1000x, 2 endpoints, TSH and hepatic outer ring deiodinase activity, were significantly increased and decreased, respectively, by 1x dose and showed dose-related increases in severity with increasing dose. Median thyroid follicle cross sectional area was also increased by the lowest dose of the mixture but decreased with subsequent increases in dose until, at the highest dose, this parameter was significantly reduced relative to control. The relative sensitivity of endpoints of thyroid function in detecting toxicity of the mixture was TSH = ORD = median follicle area >> T(3) > all other endpoints. These results demonstrate that low doses of ubiquitous environmental contaminants can alter HPT physiology in sexually mature males.

BACKGROUND Oral contraceptive (OC) use is associated with a reduced risk of ovarian cancer. An OC component, progestin, induces apoptosis in the primate ovarian epithelium. One regulator of apoptosis is transforming growth factor-beta (TGF-beta). We determined the effect of progestin on TGF-beta expression in the primate ovarian epithelium and examined the relationship between TGF-beta expression and apoptosis. METHODS Female cynomolgus macaques were randomly assigned to receive a diet for 35 months containing no hormones (n = 20); the OC Triphasil (n = 17); or each of its constituents, ethinyl estradiol (estrogen, n = 20) or levonorgestrel (progestin, n = 18 ), alone. Ovarian sections were immunostained with monoclonal antibodies against TGF-beta1 or TGF-beta2 plus TGF-beta3 (TGF-beta2/3) isoforms. The expression of TGF-beta isoforms in four ovarian compartments (epithelium, oocytes, granulosa cells, and hilar vascular endothelium) was compared among treatment groups. The association between TGF-beta expression and apoptosis, as determined by morphology and histochemistry, was examined in ovarian epithelium. All statistical tests were two-sided. RESULTS Compared with ovaries from the control and estrogen-only-treated monkeys, the ovaries of progestin-treated monkeys showed 1) a marked decrease in the expression of TGF-beta1 and a concomitant increase in the expression of the TGF-beta2/3 isoforms in the ovarian epithelium (P

Evidence from experimental studies suggests that fetal exposure to the endocrine-disrupting chemical bisphenol A (BPA) has adverse reproductive effects in both males and females. Studies from our laboratory suggest that exposure to the developing female fetus produces a unique, multigenerational effect. Specifically, maternal exposure affects the earliest stages of oogenesis in the developing fetal ovary, and the resulting subtle meiotic defects increase the likelihood that embryos produced by the exposed female in adulthood (i.e., the grandchildren) will be chromosomally abnormal. To understand the impact of BPA on the developing ovary, we conducted expression studies to characterize gene expression changes in the fetal ovary that result from BPA exposure. We first tested the validity of the approach, asking whether we could reliably detect temporal changes in expression levels of meiotic genes in controls. As anticipated, we were able to identify appropriate increases in expression in meiotic, but in few other, genes. Intriguingly, this analysis provided data on a small set of genes for which timing and expression changes suggest that they may have important and heretofore unrecognized meiotic roles. After verifying the utility of our approach, we focused our analysis on BPA-exposed animals. We found modest, but significant, changes in gene expression in the fetal ovaries from exposed fetuses. The first changes were evident within 24 h of exposure, and the most extensive changes correlated with the onset of meiosis. Furthermore, gene ontology analysis suggested that BPA acts to down-regulate mitotic cell-cycle genes, raising the possibility that fetal BPA exposure may act to limit expansion of the primordial germ cell population.

Objective To assess the effects of a red clover-derived isoflavone extract on the Ki-67 proliferative marker of endometrial biopsies in 45-to 50-year-old perimenopausal women. We hypothesized that we would be able to detect a decrease in the Ki-67 proliferative index during the late follicular phase after a 3-month course of approximately 50 mg red clover isoflavones. Isoflavones have been found to have some antiestrogenic effects, and an antiproliferative effect during the perimenopausal period may be especially useful owing to the excessive endometrial proliferation often characteristic of this period. Design In a double-blind, randomized, controlled study, 30 women between the ages of 45 and 50 years consented to an endometrial biopsy before and after a 3-month course of either placebo or active isoflavone extract. The biopsies were timed as close as possible to days 7-11 of the menstrual cycle, and simultaneous measurements of transvaginal endometrial thickness, uterine artery Doppler, hormone profiles, lipids, and bone markers were performed. Results Of 30 women, 2 did not return for a second biopsy, and a third had an unsuccessful second biopsy. Four subjects were excluded from the Intention to Treat analysis because they did not have a menstrual bleed within the time frame of the study (3 subjects) or were tested on day 13 instead of between days 7 and 11 of the cycle (1 subject). There was no change in the Ki-67 proliferation index after treatment in either group. Eight subjects in the placebo group and eight in the P-07 group had proliferative endometrial biopsies that were synchronized with estradiol levels at baseline and post-treatment, and analysis of these subjects revealed no detectable change in the relationship between estradiol levels and Ki-67 with treatment in either group. There was no change in fasting lipids, bone markers, uterine Doppler resistance, or pulsatility index. Conclusion In this small pilot study, we did not find, using immunohistochemical quantification of the Ki-67 antigen, that red clover isoflavones had an antiproliferative effect in the endometrium. Small sample size, examination of a relatively short interval in the menstrual cycle, and isoflavone formulation may have contributed to our lack of findings; however, we believe that the issue of isoflavones and their possible antiproliferative effect is deserving of further study. A simpler physiological model with less hormonal variability, such as healthy, recently menopausal women on predetermined doses of estrogen, may prove to be more informative.

If endocrine active chemicals (EACs) adversely affect human development, then there must be evidence of effects in animal models at properly scaled levels of exposure during pertinent sensitive periods as derived from quantified exposures of the human fetus. Our recent studies attempt to address both effects and exposures. First Study: Dams were gavaged from Gestation Day (GD) 14 through weaning on Post-Natal Day (PND) 21 with either corn oil alone (unexposed controls) or Low DES (0.5 mg/kg BW); High DES (5.0 mg/kg BW); GEN (15 mg/kg BW); GEN + DES (GEN at 15 mg/kg BW and DES at 0.5 mg/kg BW). No treatments affected duration of gestation, litter size or birth anogenital distance / birth body weights ((bAGD/bBW) or ratios of bAGD/cube root of bBW of pups of either sex. The ratio of weaning AGD to weaning body weight (wAGD/wBW) differed significantly between the control group and each of the estrogenic treatments in both sexes with larger wAGD/wBW values associated with each of the estrogenic treatments. Ma...

Oral contraceptive (OC) therapy has long been known to produce hypoandrogenemia. However, androgens are not part of any OC therapy available to women. This project was designed to evaluate the effects of low-estradiol containing OC, with or without methyltestosterone (MT), on cell proliferation and progesterone receptor (PgR) expression in mammary gland epithelia of virgin female rats. Sixty rats were divided into four groups. One group received OCs, whereas a second group received OC plus MT. A third group of rats was treated with an antiandrogen to mimic the hypoandrogenemic effects caused by OC therapy. All treated groups were compared with age-matched untreated controls. After 15 weeks of treatment, no inflammatory, precancerous, or cancerous lesions were observed in any treatment group. OC plus MT therapy caused significant suppression of epithelial proliferation, a reduction in the number of proliferating cell nuclear antigen-labeled cells, and an increase in the number of PgR-labeled cells. Our results suggest that a medication containing an estrogen-progestin-androgen combination has antiproliferative effects in mammary glands of experimental animals that could prove to have breast-protective potential in women.

OBJECTIVE The apoptosis pathway is a vital mechanism in vivo that functions to eradicate genetically damaged cells prone to malignancy. The purpose of this study was to determine whether oral contraceptives, which confer significant protection against subsequent epithelial ovarian cancer, induce apoptosis in the ovarian epithelium. METHODS Female cynomolgus macaques (N = 75) were randomized to receive a diet for 35 months containing either no hormones, the oral contraceptive Triphasil (Wyeth-Ayerst Laboratories, Philadelphia, PA), the estrogenic component of Triphasil (ethinyl estradiol) alone, or the progestin component of Triphasil (levonorgestrel) alone, each administered in a cyclic fashion. At study termination, the animals underwent ovariectomy and the ovarian epithelium was examined morphologically and immunohistochemically for apoptosis. The percentage of ovarian epithelial cells undergoing apoptosis was measured in each animal and compared between the treatment groups. RESULTS The median percentage of ovarian epithelial cells undergoing apoptosis by treatment was control (3.8%), ethinyl estradiol (1.8%), Triphasil (14.5%), and levonorgestrel (24.9%). Compared with control and ethinyl estradiol-treated monkeys, a statistically significant increase in the proportion of apoptotic cells was noted in the ovarian epithelium of monkeys treated with the oral contraceptive Triphasil (P < or = .01) or levonorgestrel (P < .001), with a maximal effect (six-fold) seen in the group treated with levonorgestrel alone. CONCLUSION Oral contraceptive progestin induces apoptosis in the ovarian epithelium. Given the importance of the apoptosis pathway for cancer prevention, an effective chemopreventive strategy may be possible using progestins or other agents that selectively induce apoptosis in the ovarian epithelium to prevent the development of ovarian cancer.

Mathematical models of the hypothalamus-pituitary-ovarian axis in women were first developed by Schlosser and Selgrade in 1999, with subsequent models of Harris-Clark et al. (Bull. Math. Biol. 65(1):157–173, 2003) and Pasteur and Selgrade (Understanding the dynamics of biological systems: lessons learned from integrative systems biology, Springer, London, pp. 38–58, 2011). These models produce periodic in-silico representation of luteinizing hormone (LH), follicle stimulating hormone (FSH), estradiol (E2), progesterone (P4), inhibin A (InhA), and inhibin B (InhB). Polycystic ovarian syndrome (PCOS), a leading cause of cycle irregularities, is seen as primarily a hyper-androgenic disorder. Therefore, including androgens into the model is necessary to produce simulations relevant to women with PCOS. Because testosterone (T) is the dominant female androgen, we focus our efforts on modeling pituitary feedback and inter-ovarian follicular growth properties as functions of circulating total T levels. Optimized parameters simultaneously simulate LH, FSH, E2, P4, InhA, and InhB levels of Welt et al. (J. Clin. Endocrinol. Metab. 84(1):105–111, 1999) and total T levels of Sinha-Hikim et al. (J. Clin. Endocrinol. Metab. 83(4):1312–1318, 1998). The resulting model is a system of 16 ordinary differential equations, with at least one stable periodic solution. Maciel et al. (J. Clin. Endocrinol. Metab. 89(11):5321–5327, 2004) hypothesized that retarded early follicle growth resulting in “stockpiling” of preantral follicles contributes to PCOS etiology. We present our investigations of this hypothesis and show that varying a follicular growth parameter produces preantral stockpiling and a period-doubling cascade resulting in apparent chaotic menstrual cycle behavior. The new model may allow investigators to study possible interventions returning acyclic patients to regular cycles and guide developments of individualized treatments for PCOS patients.

Contemporary translational biomedical research strives to move from discoveries in models (in silico, in vitro and in vivo) into human clinical trials to expeditiously develop specific therapeutics. Translational toxicology must similarly strive to identify applicable therapeutics that can safely and effectively mitigate potential harm from exposures. Since human exposures to chemicals, physical agents and social factors are inevitable, the human fetus is subject to effects that can have lifelong consequences. In order to apply the translational concept to developmental toxicology, we are guided by the modest number of established and accepted obstetrical therapeutics used primarily for fetal benefit. These established or potential therapeutic obstetrical interventions suggest that early steps into testing or implementing developmental translational toxicology therapies during the in utero and early neonatal period will likely derive from Generally-Recognized-As-Safe (GRAS) options. If we are to translate environmental health discoveries into safe and effective interventions, we must assert and characterize valid, applicable therapies such as GRAS treatments and eventually “ethical pharmaceuticals” for the protective care of these highly vulnerable young patients. We can create a safe and efficacious environmental health portfolio of interventional options to improve human health that include both reduction/avoidance of exposure and specific preventative/mitigative/restorative therapeutics.

The ability of foreign compounds to affect the functioning of various endocrine systems is currently thought responsible for a wide variety of effects. The presentations in this Symposium reviewed the evidence for and against the involvement of endocrine systems in several different aspects of reproduction. The mechanism behind the ability of a triazine herbicide to cause enhanced appearance of mammary tumors in one strain of female rats is reviewed by Stevens. The data suggest that enhanced aging, not direct mammary modulation, is responsible. Dietary phytoestrogens, the mediators of their actions, their effects in various biological systems, and the relationships between phytoestrogen producers and consumers are all provocatively and succinctly reviewed by Hughes. Kelce presents the strategy used to dissect the mode and mechanisms of action of a fungicide that opened a new awareness in reproductive toxicology: the possibility of xenobiotics being antiandrogens. Finally, to heighten our understanding of the interplay among hormonal systems in vivo, Hess reviews the data that show that androgens are not the only hormones important in the development of the male reproductive system: the pituitary is shown to play a critical role at specific stages of development. The breadth of these presentations, and the implications of their findings, should make us pause and realize how much there is still to discover about the interaction between the reproductive system and anthropogenic compounds.

Many edible plants contain natural estrogens called phytoestrogens. These compounds possess mixed estrogen agonist-antagonist properties that are organ-specific in vivo. We have focused on estrogenic soybean isoflavones because of their potential extensive dietary availability. In this article, we review the clinical and experimental evidence for the possible benefits and risks of ingestion of estrogenic isoflavones throughout the life span, and highlight areas needing further elucidation.

Exposure to naturally occurring estrogens during critical periods of development can alter morphologic and physiologic markers of sexual differentiation. The current experiment characterizes the effects of in utero treatment with genistein, an isoflavonoid phytoestrogen, on birth weight, anogenital distance (AGD) at birth. GnRH stimulated luteinizing hormone (LH) secretion, volume of the sexually dimorphic nucleus in the preoptic area of the hypothalamus (SDN-POA), puberty onset, and vaginal cyclicity. Pregnant Charles River CD rats were injected sc daily on gestation day 16-20 with either 25,000 micrograms genistein (G25), 5,000 micrograms genistein (G5), 5 micrograms diethylstillbestrol (DES), 50 micrograms estradiol benzoate (E), or corn oil alone for controls. Birth weights and anogenital distance was taken and exposed progeny were subsequently used in two experiments. In Experiment 1 intra-atrial catheters were placed in adult castrated rats, GnRH was given iv, serial blood samples were drawn and sera were assayed for LH by radioimmunoassay (RIA). Brains obtained by subsequent decapitation were saved for histology. In Experiment 2, females were monitored for timing of vaginal opening as a marker of puberty onset, and vaginal smears were taken to monitor cyclicity. G25-treated females and DES- and E-treated animals of both sexes had decreased weights at birth compared with controls. G5- and E-treated animals of both sexes and DES males had smaller AGD than controls. No significant differences in pituitary responsiveness to GnRH were found among treatment groups. There was a nonsignificant decrease in SDN-POA volume in G5-treated females while DES- and E-treated females had increased SDN-POA volume compared with controls. G5-treated females had delayed puberty onset, and DES-treated females had atypical vaginal cycles in comparison with controls. The results confirm that prenatal exposure to estrogens in the environment can influence sexual differentiation. Our previous experiments have demonstrated that castrate female rats exposed as neonates to genistein have decreased pituitary responsiveness to GnRH challenge and enlarged SDN-POA volume in comparison with controls. Prenatal genistein at these dosages did not significantly alter these markers. However, genistein did mimic other estrogens' effects on AGD and birth weight and had a unique influence on puberty onset. Not only are genistein's effects different from other estrogens, but dosage and timing of exposure during development appear to be important factors in genistein's ability to modify these end points.

The estrogen receptor α (ERα) splicing variant with an in-frame deletion of exon 3 (ERΔ3) is frequently expressed in the normal breast, but its influence on tumorigenesis has not been explored. In vitro, ERΔ3 has dominant negative activity, suggesting it may suppress estrogen stimulation in the breast. ERΔ3 may inhibit classical signaling on estrogen response element (ERE)-regulated genes as well as activate non-classical pathways at Sp1 and AP-1 sites. Transgenic mice were developed that express mouse ERΔ3 in all tissues examined, including the mammary gland. To investigate if ERΔ3 expression affects tumorigenesis, ERΔ3 mice were crossbred with MMTV-Neu mice. Mammary tumor onset was significantly delayed in ERΔ3/Neu versus MMTV-Neu females and metastatic incidence and burden was significantly reduced. Consequently, ERΔ3 expression suppressed tumor development and metastasis in this aggressive model of HER2/Neu-positive breast cancer. To determine if ER ligands with anticancer activity may augment ERΔ3 protection, the bitransgenic mice were treated with tamoxifen and soy isoflavones starting at age 2 months. Soy protein with isoflavones (181 mg/1,800 kcal) did not affect tumor development in MMTV-Neu or ERΔ3/Neu mice; however, metastatic progression was not inhibited in soy-treated ERΔ3/Neu mice, as it was in untreated ERΔ3/Neu mice. In contrast, tamoxifen (20 mg/1,800 kcal) significantly enhanced tumor prevention in ERΔ3/Neu versus MMTV-Neu mice (98 % vs. 81 % tumor free). The results in ERΔ3/Neu mice demonstrate that ERΔ3 influences estrogen-dependent mammary carcinogenesis and, thus, may be protective in women expressing ERΔ3 in the breast. However, exposure to different estrogens may augment or block its beneficial effects.

To investigate the effect of dietary allicin on health and growth performance of weanling piglets, at 21 days of age. Two hundred and twenty-five piglets were weaned and randomly allocated into five groups. Piglets in the control group were fed diets supplemented with antibiotics. Those in the treatment groups were fed diets without antibiotics, but supplemented with allicin product (25% pure allicin oil) in the proportion of 0.10 g/kg, 0.15 g/kg, 0.20 g/kg and 0.25 g/kg in the diet, respectively. During the 28 days of the experiment, average daily weight gain increased linearly (P < 0.0001) and quadratically (P = 0.0014) as the level of dietary allicin increased. The feed gain ratio decreased linearly (P < 0.0001) and quadratically (P < 0.0001). As the dietary allicin level increased, the incidence of diarrhoea in the treatment piglets, especially female piglets decreased linearly (P = 0.0003) and tended to decrease quadratically (P = 0.0716). The number of flies alighting on the surface of the faeces of the piglets at each counting time point decreased linearly (P < 0.0001), quadratically (P < 0.0001) and cubically (P < 0.0001) as the dietary allicin level increased. In conclusion, supplementation of the diet with allicin may improve growth performance, reduce the incidence of diarrhoea and possibly improve their local environmental conditions by reducing the attractiveness of faeces to flies.

Objective To test the hypothesis that high galactose consumption and low activity of galactose-1-phosphate uridyl transferase (transferase) is associated with early ovarian senescence among nongalactosemic women. Design Cross-sectional study. Data collection consisted of a self-administered questionnaire with sections on diet (food frequency data to measure galactose consumption), reproductive, and medical histories. One blood sample was collected to measure FSH and transferase activity; FSH was used as a measure of ovarian senescence. Among women who were having menstrual periods at least every 8weeks, the blood sample was drawn in the early follicular phase (days 2 to 4) of a menstrual cycle. Participants Two hundred ninety-five women volunteers ages 38 to 49years who had not had a hysterectomy or oophorectomy were recruited through posters and advertisements. Main Outcome Measure Serum FSH concentrations. Results Controlling for age, smoking, and body mass, transferase activity and FSH were unrelated. However, FSH levels were 29% higher (95% confidence intervals, 9% to 52%) among women who reported consuming ≥6g galactose/d. Conclusion These data do not support the hypothesis that low transferase activity represents a genetic predisposition for early ovarian senescence, as measured by FSH levels in women ages 38 to 49years. However, the hypothesized positive association between galactose consumption and FSH was supported.

Quantification of exposure to environmental contaminants such as endocrine active chemicals (EACs) during critical periods of development, particularly in utero, remains largely unexplored. Therefore, we tested the hypothesis that EACs can be detected and quantified in second trimester human amniotic fluid. Amniotic fluid was obtained from women (n=175) undergoing routine amniocentesis between 14 and 21 weeks gestation. Samples were assayed by gas chromatography/mass spectrometry (GC/MS) for common organochlorine contaminants and dietary phytoestrogens. The DDT metabolite p,p'-DDE was found in approximately 25% of amniotic fluid samples (mean±S.D., 0.15±0.06 ng/ml) whereas the dietary phytoestrogens, genistein and or daidzein were found in 96.2% of samples tested (0.94±0.91 and 1.08±0.91 ng/ml, respectively). Our results demonstrate that: (1) human amniotic fluid is a suitable biological medium to evaluate developmental exposure to EACs, and (2) fetuses are exposed to biologically active levels of EACs in mid pregnancy.