Sex Hormone Balance Research Articles

An in vivo assay performed using multiple biomarkers related to testosterone synthesis and conversion for assessing the androgenic potency of refuse leachate

Refuse leachate is likely an important source of androgens. However, common in vitro bioassays underestimate the potential androgenic activity of leachate, owing to non-receptor-mediated mechanisms that modify the balance of sex hormones and promote the accumulation of endogenous androgens. This study aimed to develop an in vivo assay by using multiple biomarkers related to testosterone synthesis and conversion for assessing the potential androgenic activity of refuse leachate sampled from a municipal solid waste treatment plant in Qingdao, China. The results indicated that exposure to leachate increased the levels of testosterone and luteinizing hormone, but decreased those of 17β-estradiol in both male and female goldfish (Carassius auratus), suggesting a potential androgenic activity. Further, Leydig cell hyperplasia and decreased gonadal P450 aromatase mRNA levels were observed; these alterations might promote the biosynthesis of testosterone and hinder the conversion of testosterone to 17β-estradiol, which in turn enhance testosterone accumulation. Exposure to leachate also resulted in reproductive impairments, including decreased gonadosomatic index and plasma vitellogenin levels of female goldfish, as well as decreased testicular enzyme activities in male goldfish. The integrated use of biochemical, molecular, and histological markers not only improved our understanding of the androgenic effects of leachate but also verified the reliability and validity of the results. Therefore, the in vivo bioassay described in this study might allow the investigation of the androgenic effects of other complex contaminant mixtures in the future.

Effects of tris(2-butoxyethyl) phosphate exposure on endocrine systems and reproduction of zebrafish (Danio rerio)

Tris(2-butoxyethyl) phosphate (TBEOP), a widely used organophosphate flame retardant, has frequently been detected both in the environment and the biota. However, limited information is available on the effects of TBEOP on the endocrine system and its underlying mechanisms. We exposed adult zebrafish pairs to TBEOP at concentrations of 0, 2.1, 11, and 118 μg/L for 21 d, and investigated the effects on gene transcription and hormone production related to the hypothalamic-pituitary-gonadal (HPG) axis, and on reproduction. The adverse effects on the F1 generation were further examined. In male fish, plasma concentrations of 17β-estradiol were significantly increased along with up-regulation of cyp19a. Exposure to TBEOP at 118 μg/L led to a significant decrease in average egg production. Exposure of the F0 generation to TBEOP delayed hatching and lowered hatching rates in the F1 generation. The results demonstrate that exposure to TBEOP at environmentally relevant concentration levels could affect the sex hormone balance by altering regulatory circuits of the HPG axis, eventually leading to disruption of reproductive performance and the development of offspring.

Thymoquinone ameliorated elevated inflammatory cytokines in testicular tissue and sex hormones imbalance induced by oral chronic toxicity with sodium nitrite

Scientific evidence illustrated the health hazards of exposure to nitrites for prolonged time. Nitrites affected several body organs due to oxidative, inflammatory and apoptosis properties. Furthermore, thymoquinone (TQ) had curative effects against many diseases. We tried to discover the impact of both sodium nitrite and TQ on inflammatory cytokines contents in testicular tissues and hormonal balance both in vivo and in vitro. Fifty adult male SD rats received 80mg/kg sodium nitrite and treated with either 25 or 50mg/kg TQ daily by oral-gavage for twelve weeks. Testis were removed for sperms’ count. Testicular tissue homogenates were used for assessment of protein and gene expression of IL-1β, IL-6, TNF-α, Nrf2 and caspase-3. Serum samples were used for measurement of testosterone, LH, FSH and prolactin. Moreover, all the parameters were measured in human normal testis cell-lines, CRL-7002. Sodium nitrite produced significant decrease in serum testosterone associated with raised FSH, LH and prolactin. Moreover, sodium nitrite significantly elevated TNF-α, IL-1β, IL-6, caspase-3 and reduced Nrf2. TQ significantly reversed all these effects both in vivo and in vitro. In conclusion, TQ ameliorated testicular tissue inflammation and restored the normal balance of sex hormones induced by sodium nitrite both in vivo and in vitro.

Co-Formulants in Glyphosate-Based Herbicides Disrupt Aromatase Activity in Human Cells below Toxic Levels.

Pesticide formulations contain declared active ingredients and co-formulants presented as inert and confidential compounds. We tested the endocrine disruption of co-formulants in six glyphosate-based herbicides (GBH), the most used pesticides worldwide. All co-formulants and formulations were comparably cytotoxic well below the agricultural dilution of 1% (18–2000 times for co-formulants, 8–141 times for formulations), and not the declared active ingredient glyphosate (G) alone. The endocrine-disrupting effects of all these compounds were measured on aromatase activity, a key enzyme in the balance of sex hormones, below the toxicity threshold. Aromatase activity was decreased both by the co-formulants alone (polyethoxylated tallow amine—POEA and alkyl polyglucoside—APG) and by the formulations, from concentrations 800 times lower than the agricultural dilutions; while G exerted an effect only at 1/3 of the agricultural dilution. It was demonstrated for the first time that endocrine disruption by GBH could not only be due to the declared active ingredient but also to co-formulants. These results could explain numerous in vivo results with GBHs not seen with G alone; moreover, they challenge the relevance of the acceptable daily intake (ADI) value for GBHs exposures, currently calculated from toxicity tests of the declared active ingredient alone.

Exposure to the mixture of organophosphorus pesticides is embryotoxic and teratogenic on gestational rats during the sensitive period

Mixture of organophosphorus pesticides (MOPs) has been used worldwide to increase food production. The MOPs are harmful, and the exposure to them is both agricultural and nonagricultural through contaminated food. The neurotoxicity of MOPs has received more consideration recently due to the increased cases of malformed fetuses suspected to be caused by the MOPs exposure during gestation; however, relevant studies in animal model are rare. In this study, we performed a comprehensive analysis and demonstrated potential perinatal embryotoxicity and teratogenicity of MOPs exposure. As results, we found that MOPs decreased in utero fetal growth and alter the ratio of organs to whole body weight of the pregnant rats. MOPs also had been shown to disturb the balance of sex hormones and affect the reproduction of rats. Furthermore, we found various significantly elevated deformities in MOPs exposed embryos, confirming the embryotoxic and teratogenic effects of maternal exposure to MOPs. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 139-146, 2017.

Effect of chronic exposure to two components of Tritan copolyester on Daphnia magna, Moina macrocopa, and Oryzias latipes, and potential mechanisms of endocrine disruption using H295R cells.

Tritan copolyester is a novel plastic form from Eastman Company utilizing three main monomers, 1,4-cyclohexanedimethanol (CHDM), dimethyl terephthalate (DMT), and 2,2,4,4-tetramethyl-1,3-cyclobutanediol. Despite Tritan has been widely applied for plastic bottles, the effects of long-term exposure to these compounds have seldom been investigated. We investigated chronic effects and endocrine disruption potential of CHDM and terephthalic acid (TPA), main mammalian metabolite formed from DMT, using crustacean Daphnia magna and Moina macrocopa, and freshwater fish (Oryzias latipes). The effects on sex hormone balance and the associated mechanisms were also investigated by use of H295R cells. In chronic toxicity test, D. magna showed significant decrease in reproduction (number of young per female) after exposure to 10 mg/L TPA. In early life stage exposure using O. latipes, significant decrease of juvenile survival and weight were observed in fish exposed to 10 mg/L and ≥1 mg/L CHDM, respectively. Expressions of vtg2 mRNA in fish exposed to CHDM and those of cyp19b, star, cyp17, and cyp19a mRNAs in fish exposed to TPA were significantly up-regulated. The results of H295R cell assay also showed that both chemicals at high concentrations could alter sex hormone production in steroidogenic pathway. The effective concentrations of the tested compounds were several orders of magnitude greater than the concentrations can be detected in ambient waters. Further in vivo and in vitro studies will be needed to investigate the effect of co-polymer on endocrine disruption.

Digit ratio (2D:4D) does not correlate with daily 17β-estradiol and progesterone concentrations in healthy women of reproductive age.

Second-to-fourth digit ratio (2D:4D) is proposed as a proxy for the prenatal balance of sex hormones, is related to hormone-dependent characteristics in adult life, and is a possible predictor of disease later in life. Here, we studied the relationship between 2D:4D and ovarian steroid hormones (17β-estradiol and progesterone) among women of reproductive age. From 186 healthy premenopausal women, aged 24-37 years, we collected saliva samples daily during the entire menstrual cycle. Data on reproductive and lifestyle characteristics were collected via questionnaires, and anthropometric measurements were performed. No statistically significant relationships were detected between adult women's sex hormone concentrations (17β-estradiol and progesterone) during the menstrual cycle and 2D:4D, in either left or right hand, when controlling for size at birth, body mass index, and physical activity. This study shows, for the first time in a large sample of women of reproductive age, that 2D:4D is not a predictor of adult women's sex hormone concentration. The lack of relationship may be because 2D:4D might be genetically determined and is not related to maternal nutritional environment during fetal development. These results support the hypothesis that, in contrast to the nutritional quality of the fetal environment, the fetal hormonal environment (reflected by 2D:4D) does not determine reproductive physiology in later life.

Minimising menopausal side effects whilst treating endometriosis and fibroids.

Medical management of endometriosis and fibroids involves manipulation of the hypothalamic-pituitary-gonadal axis to alter the balance of sex hormones thereby inhibiting disease progression and ameliorate symptoms. Unfortunately, resultant menopausal symptoms sometimes limit the tolerability and duration of such treatment. The use of gonadotrophin-releasing hormone agonists to treat these diseases can result in short-term hypoestrogenic and vasomotor side effects as well as long-term impacts on bone health and cardiovascular risk. The routine use of add-back hormone replacement has reduced these risks and increased patient compliance, making this group of drugs more useful as a medium-term treatment option. The estrogen threshold hypothesis highlights the concept of a 'therapeutic window' in which bone loss is minimal but the primary disease is not aggravated. It explains why add-back therapy is appropriate for such patients and helps to explain the basis behind new developments in the treatment of hormonally responsive gynaecological conditions such as gonadotrophin-releasing hormone antagonists and progesterone receptor modulators.

Hormonal support of menstrual function and features of the balance of sex hormones in women of childbearing age with changes of reproductive health and autonomic homeostasis disorders

The results of the study of hormonal menstrual function in women of childbearing age with autonomic homeostasis disorders and various changes in reproductive health are shown. Studies concluded 360 women with disorders of autonomic homeostasis such as autonomic dysfunction syndrome and somatoform dysfunction of the autonomic nervous system. Hormonal support of menstrual function and features a balance of sex hormones was studied in women with disorders of autonomic homeostasis and various changes in reproductive health (women after artificial abortion, infertility women and women with uterine leiomyomas).It was found that the combination of changes in reproductive health with such disorders of autonomic homeostasis syndrome as somatoform autonomic dysfunction and autonomic nervous system dysfunction is accompanied by significant changes in the regulatory mechanisms of menstrual function of women and relationships of stress-associated hormones, that is enhances the reproductive health of surveyed women.

Investigating the Periocular-Based Face Recognition Across Gender Transformation

This paper introduces a novel face recognition problem domain: the medically altered face for gender transformation. A data set of >1.2 million face images was constructed from wild videos obtained from You Tube of 38 subjects undergoing hormone replacement therapy (HRT) for gender transformation over a period of several months to three years. The HRT achieves gender transformation by severely altering the balance of sex hormones, which causes changes in the physical appearance of the face and body. This paper explores that the impact of face changes due to hormone manipulation and its ability to disguise the face and hence, its ability to effect match rates. Face disguise is achieved organically as hormone manipulation causes pathological changes to the body resulting in a modification of face appearance. This paper analyzes and evaluates face components versus full face algorithms in an attempt to identify regions of the face that are resilient to the HRT process. The experiments reveal that periocular face components using simple texture-based face matchers, local binary patterns, histogram of gradients, and patch-based local binary patterns out performs matching against the full face. Furthermore, the experiments reveal that a fusion of the periocular using one of the simple texture-based approaches (patched-based local binary patterns) out performs two Commercial Off The Shelf Systems full face systems: 1) PittPatt SDK and 2) Cognetic FaceVACs v8.5. The evaluated periocular-fused patch-based face matcher outperforms PittPatt SDK v5.2.2 by 76.83% and Cognetic FaceVACS v8.5 by 56.23% for rank-1 accuracy.

Vitamin D as a regulator of steroidogenic enzymes

During the last decades, the outlook on vitamin D has widened, from being a vitamin solely involved in bone metabolism and calcium homeostasis, to being a multifunctional hormone known to affect a broad range of physiological processes. The aim of this review is to summarize the research on vitamin D as a regulator of steroidogenic enzymes. Steroid hormones exert a wide range of physiological responses, including functions in the immune system, protein and carbohydrate metabolism, water and salt balance, reproductive system and development of sexual characteristics. The balance of sex hormones is also of importance in the context of breast and prostate cancer. Steroid hormones are synthesized in steroidogenic tissues such as the adrenal cortex, breast, ovaries, prostate and testis, either from cholesterol or from steroidogenic precursors secreted from other steroidogenic tissues. The hormonally active form of vitamin D has been reported to act as a regulator of a number of enzymes involved in the regulation of steroid hormon production, and thereby the production of both adrenal steroid hormones and sex hormones. The research reviewed in the article has in large part been performed in cell culture based experiments and laboratory animal experiments, and the physiological role of the vitamin D mediated regulation of steroidogenic enzyme need to be further investigated.

Republished study: long-term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modified maize.

BackgroundThe health effects of a Roundup-tolerant NK603 genetically modified (GM) maize (from 11% in the diet), cultivated with or without Roundup application and Roundup alone (from 0.1 ppb of the full pesticide containing glyphosate and adjuvants) in drinking water, were evaluated for 2 years in rats. This study constitutes a follow-up investigation of a 90-day feeding study conducted by Monsanto in order to obtain commercial release of this GMO, employing the same rat strain and analyzing biochemical parameters on the same number of animals per group as our investigation. Our research represents the first chronic study on these substances, in which all observations including tumors are reported chronologically. Thus, it was not designed as a carcinogenicity study. We report the major findings with 34 organs observed and 56 parameters analyzed at 11 time points for most organs.ResultsBiochemical analyses confirmed very significant chronic kidney deficiencies, for all treatments and both sexes; 76% of the altered parameters were kidney-related. In treated males, liver congestions and necrosis were 2.5 to 5.5 times higher. Marked and severe nephropathies were also generally 1.3 to 2.3 times greater. In females, all treatment groups showed a two- to threefold increase in mortality, and deaths were earlier. This difference was also evident in three male groups fed with GM maize. All results were hormone- and sex-dependent, and the pathological profiles were comparable. Females developed large mammary tumors more frequently and before controls; the pituitary was the second most disabled organ; the sex hormonal balance was modified by consumption of GM maize and Roundup treatments. Males presented up to four times more large palpable tumors starting 600 days earlier than in the control group, in which only one tumor was noted. These results may be explained by not only the non-linear endocrine-disrupting effects of Roundup but also by the overexpression of the EPSPS transgene or other mutational effects in the GM maize and their metabolic consequences.ConclusionOur findings imply that long-term (2 year) feeding trials need to be conducted to thoroughly evaluate the safety of GM foods and pesticides in their full commercial formulations.

Aromatase and regulation of the estrogen‐to‐androgen ratio in synovial tissue inflammation: common pathway in both sexes

Sex hormones play an active role in inflammatory responses, with androgens being anti-inflammatory, whereas estrogens have both pro- and anti-inflammatory effects. In rheumatoid arthritis (RA) patients, low levels of androgens and high levels of estrone are found in the synovial fluid. Aromatase is the key enzyme for the conversion of androgens into estrogens. Proinflammatory cytokines stimulate aromatase activity so that the inflammatory milieu can induce conversion of androgens to estrogens. Moreover, testosterone inhibits aromatase activity. As local androgen levels are low in RA, this can contribute to high aromatase activity in the synovium. Importantly, aromatase-converted estrogens are converted into proproliferative and proinflammatory 16-hydroxylated estrogens. A hormone involved in aromatase activity is vitamin D, which downregulates aromatase in human RA macrophages. Collectively, evidence suggests a key role of aromatase in sex hormone balance during chronic inflammation and points to the importance of vitamin D as a possible new tool for aromatase modulation.

Polyfluorinated Alkyl Phosphate Ester Surfactants – Current Knowledge and Knowledge Gaps

Fluorochemicals are a diverse group of synthetically produced compounds with the unique ability to repel water as well as oil. This property makes them ideal for multiple purposes in a variety of consumer and industrial products. Fluorochemicals have been detected in the environment, as well as in human blood, urine and milk. Due to their long half-life in human beings, there is an increased risk that exposure to these compounds can cause adverse effects. However, except for perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS), there is a large data gap regarding toxicological information on fluorochemicals. Polyfluorinated alkyl phosphate ester surfactants (PAPs) belong to the group of polyfluorinated alkyl surfactants. They have been detected in indoor dust and are widely used in food-contact materials, from which they have the ability to migrate into food. Toxicological data on PAPs are very limited, but current studies indicate that some PAPs have the potential to interfere with sex hormone synthesis in vitro. Disturbance of the sex hormone balance in foetal life has been suggested to be an important mechanism involved in adverse effects on, for example, male reproductive health and development. The current lack of toxicological data on PAPs impairs the risk assessment of this group of compounds. However, until more toxicological data on PAPs are available, the limited data currently accessible give reason to believe that these compounds might have the ability to cause potentially adverse effects, as seen for other perfluorinated chemicals, including some metabolic products of PAPs.

4-Nitrophenol induces Leydig cells hyperplasia, which may contribute to the differential modulation of the androgen receptor and estrogen receptor-α and -β expression in male rat testes

4-Nitrophenol (PNP) is generally regarded as an environmental endocrine disruptor capable of estrogenic and anti-androgenic activities. To investigate PNP-induced reproductive effects, immature male rats were injected subcutaneously with PNP (0.1, 1, 10mg/kg body weight or vehicle) daily for 4 weeks. We assessed reproductive tract alterations, sex hormone balance in the serum and estrogen receptor (ER)-α, -β and androgen receptor (AR) expression in testes. Although no significant difference was observed in body weight or testes weights of PNP-treated rats compared with the controls, the serum concentrations of testosterone in the 10mg/kg PNP-treated group were significantly elevated. This effect was accompanied by Leydig cells hyperplasia in the testes. Conversely, there was a significant decrease in estradiol concentration and aromatase expression in the testes of the 10mg/kg PNP-treated group. Furthermore, we observed a significant increase in ERα expression in the testes of the 10mg/kg PNP-treated group compared with the control group. Conversely, ERβ expression displayed a significant reduction. Moreover, AR expression was significantly increased in the 10mg/kg PNP-treated group compared with the control group. The existence of AR, ER-α and -β in the testes suggests that estradiol and testosterone directly affect germ cells and that differential modulation of AR, ER-α and -β in the testis may be involved in the direct effects of PNP or either the indirect effects of PNP-induced disruption of the estradiol-to-testosterone balance or the Leydig cells hyperplasia. Thus, the measurement of many endpoints is necessary for good risk assessment.

Unmasking the potential of the angiotensin AT2 receptor as a therapeutic target in hypertension in men and women: What we know and what we still need to find out

Major sex differences exist in the development and progression of hypertension and cardiovascular disease. Prior to menopause, women have lower arterial pressure and, furthermore, are protected from hypertension and cardiovascular disease relative to age-matched men. However, after menopause this cardiovascular protection in women is lost. These sex differences have been linked to sexual dimorphism in the physiological mechanisms that regulate arterial pressure, including the renin-angiotensin system (RAS), which can also impact on the male and female response to different therapeutic approaches. This suggests that antihypertensive regimens need to be tailored according to sex. Newly discovered components of the RAS have emerged in recent years, allowing us to look beyond the classical RAS for novel therapeutic targets for hypertension. In this context, it is now well established that the angiotensin AT2 receptor (AT2 R) elicits depressor and natriuretic effects and that these effects are greater in females due to enhanced AT2 R levels modulated by oestrogen. In light of knowledge that AT2 R expression is regulated by oestrogen and that the prevalence of hypertension and cardiovascular risk is greater in women after menopause, AT2 R agonist therapy may represent an innovative therapeutic approach to treat hypertension. Consequently, understanding how ageing and changes in the sex hormone balance influence the RAS is vital if we are to evaluate the potential of the AT2 R as a therapeutic target in women and also in men.

Effects of TDCPP or TPP on gene transcriptions and hormones of HPG axis, and their consequences on reproduction in adult zebrafish (Danio rerio)

Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) and triphenyl phosphate (TPP) belong to the group of triester organophosphate flame retardants (OPFRs), which have been used in a wide range of consumer products. These chemicals have been frequently detected in effluents, surface water, and fish, and hence their potential adverse effects on aquatic ecosystem are of concern. The present study was conducted to investigate the reproduction-related effects and possible molecular mechanisms of TDCPP and TPP using a 21 day reproduction test employing adult zebrafish (Danio rerio). After 21 d of exposure to TDCPP or TPP, significant decrease in fecundity along with significant increases of plasma 17β-estradiol (E2) concentrations, vitellogenin (VTG) levels, and E2/testosterone (T) and E2/11-ketotestosterone (11-KT) ratios were observed. The transcriptional profiles of several genes of the hypothalamus–pituitary–gonad (HPG) axis changed as well after the exposure, but the trend was sex-dependent. In male fish, gonadotropin-releasing hormone2 (GnRH2), GnRHR3, cytochrome P450 (CYP) 19B, estrogen receptor α (ERα), ER2 β1, and follicle stimulating hormone β (FSHβ) were upregulated in the brain, while luteinizing hormone β (LHβ) and androgen receptor (AR) were downregulated. Corresponding to the upregulation of FSHβ and downregulation of LHβ in the brain, FSHR was upregulated and LHR was downregulated in the testis. Among the genes that regulate the steroidogenesis pathway, transcription of hydroxyl methyl glutaryl CoA reductase (HMGRA), steroidogenic acute regulatory protein (StAR), and 17β-hydroxysteroid dehydrogenase (17βHSD) decreased, while transcription of CYP11A, CYP17, and CYP19A increased. In female fish, transcription ofGnRH2 and GnRHR3 decreased, but FSHβ, LHβ, CYP19B, ERα, ER2β1, and AR transcription increased in the brain. In the ovary, FSHR and LHR were significantly upregulated, and most steroidogenic genes were significantly upregulated. The observed disruption of GnRH and GtHs could be further related to subsequent disruption in both sex steroid hormone balance and plasma VTG levels, as well as reproductive performance. Overall, our observation indicates that both TDCPP and TPP could disturb the sex hormone balance by altering regulatory mechanisms of the HPG axis, eventually leading to disruption of reproductive performance in fish.

RETRACTED: Long term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modified maize

The health effects of a Roundup-tolerant genetically modified maize (from 11% in the diet), cultivated with or without Roundup, and Roundup alone (from 0.1 ppb in water), were studied 2 years in rats. In females, all treated groups died 2-3 times more than controls, and more rapidly. This difference was visible in 3 male groups fed GMOs. All results were hormone and sex dependent, and the pathological profiles were comparable. Females developed large mammary tumors almost always more often than and before controls, the pituitary was the second most disabled organ; the sex hormonal balance was modified by GMO and Roundup treatments. In treated males, liver congestions and necrosis were 2.5-5.5 times higher. This pathology was confirmed by optic and transmission electron microscopy. Marked and severe kidney nephropathies were also generally 1.3-2.3 greater. Males presented 4 times more large palpable tumors than controls which occurred up to 600 days earlier. Biochemistry data confirmed very significant kidney chronic deficiencies; for all treatments and both sexes, 76% of the altered parameters were kidney related. These results can be explained by the non linear endocrine-disrupting effects of Roundup, but also by the overexpression of the transgene in the GMO and its metabolic consequences.

Hepatitis B virus surface antigen‐negative and hepatitis C virus antibody‐negative hepatocellular carcinoma

Although the incidence of hepatitis B virus surface antigen (HBsAg)-negative/hepatitis C virus antibody (HCVAb)-negative hepatocellular carcinoma (NBNC-HCC) is gradually increasing, it has been mostly ignored in previous studies. The objective of this exploratory study was to investigate the clinicopathologic characteristics and prognostic factors that influence recurrence and survival in patients with NBNC-HCC. A retrospective analysis was performed of 675 patients with NBNC-HCC and 3529 patients with HBsAg-positive/HCVAb-negative HCC (BNC-HCC) who underwent curative resection between 1997 and 2009. Intrahepatic recurrences were classified into early (≤1 year) and late (>1 year) recurrences. Multivariate competing risks analyses with Bonferroni correction were used to evaluate independent prognostic factors. There were no significant differences between the NBNC-HCC and BNC-HCC groups regarding overall survival, cumulative incidence of HCC-specific death, and recurrence. However, the patients with NBNC-HCC were much older (P < .001), were associated less often with cirrhosis or elevated α-fetoprotein levels (P < .001), and had a much lower ratio of men to women (P < .001). NBNC-HCC tumors were larger (P < .001), but were involved less often with vascular invasion (P = .004). Women, serum γ-glutamyl transpeptidase level, tumor size, tumor capsule, and tumor differentiation were identified as independent risk factors for HCC-specific survival in patients with NBNC-HCC. The cumulative incidence of HCC-specific death for women with NBNC-HCC was significantly greater than for men with NBNC-HCC (P < .001).Tumor capsule and vascular invasion were identified as independent risk factors for early recurrence of NBNC-HCC, whereas tumor differentiation was identified as the only significant risk factor for late recurrence. Patients who had NBNC-HCC had characteristics and prognostic factors that differed from those in patients who had BNC-HCC. Women with NBNC-HCC should be more closely monitored, and it may be worthwhile to evaluate estrogen administration for the maintenance of sex hormone balance and to improve these poor outcomes.

Quantification of steroids and endocrine disrupting chemicals in rat ovaries by LC-MS/MS for reproductive toxicology assessment

Reproductive function is controlled by a finely tuned balance of androgens and estrogens. Environmental toxicants, notably endocrine disrupting chemicals (EDCs), appear to be involved in the disruption of hormonal balance in several studies. To further describe the effects of selected EDCs on steroid secretion in female rats, we aim to simultaneously investigate the EDC concentration and the sex hormone balance in the ovaries. Therefore, an effective method has been developed for the quantification of the sex steroid hormones (testosterone, androstenedione, estradiol, and estrone) and four endocrine disrupting chemicals (bisphenol A, atrazine, and the active metabolites of methoxychlor and vinclozolin) in rat ovaries. The sample preparation procedure is based on the so-called "quick, easy, cheap, effective, rugged, and safe" approach, and an analytical method was developed to quantify these compounds with low detection limits by liquid chromatography coupled with a tandem mass spectrometer. This analytical method, applied to rat ovary samples following subacute EDC exposure, revealed some new findings for toxicological evaluation. In particular, we showed that EDCs with the same described in vitro mechanisms of action have different effects on the gonadal steroid balance. These results highlight the need to develop an integrative evaluation with the simultaneous measurement of EDCs and numerous steroids for good risk assessment.