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Nicofluprole is a novel insecticide of the phenylpyrazole class conferring selective antagonistic activity on insect GABA receptors. After repeated daily dietary administration to Wistar rats for 28/90 days, Nicofluprole induced increases in thyroid (and liver) weight, associated with histopathology changes. Nicofluprole did not inhibit thyroid peroxydase nor sodium/iodide symporter, two key players in the biosynthesis of thyroid hormones, indicating the absence of a direct thyroid effect. The results seen in rats suggested a mode of action of Nicofluprole driven by the molecular initiating event of CAR/PXR nuclear receptor activation in livers, with key events of increases in liver weight and hypertrophy, decreasing circulatory thyroid hormones, a compensatory increase in TSH release and follicular cell hypertrophy. To explore the relevance of these changes to humans, well established in vitro rat and human sandwich-cultured hepatocytes were exposed to Nicofluprole up to 7 days. A concentration-dependent CYP3A induction (PXR-activation), an increase in T4-glucuronoconjugation accompanied by UGT1A/2B inductions was observed in rat but not in human hepatocytes. The inductions seen with Nicofluprole in rat (in vivo and in vitro in hepatocytes) that were absent in human hepatocytes represent another example of species-selectivity of nuclear CAR/PXR receptor activators. Importantly, the different pattern observed in rat and human models demonstrate that Nicofluprole-related thyroid effects observed in the rat are with no human relevance.

Polychlorinated biphenyls (PCBs) are endocrine disrupting chemicals with documented, though mechanistically ill-defined, reproductive toxicity. The toxicity of dioxin-like PCBs, such as PCB126, is mediated via the aryl hydrocarbon receptor (AHR) in non-ovarian tissues. The goal of this study was to examine the uterine and ovarian effects of PCB126 and test the hypothesis that the AHR is required for PCB126-induced reproductive toxicity. Female Holzman-Sprague Dawley wild type (n = 14; WT) and Ahr knock out (n = 11; AHR(−/−)) rats received a single intraperitoneal injection of either corn oil vehicle (5 ml/kg: WT_O and AHR(−/−)_O) or PCB126 (1.63 mg/kg in corn oil: WT_PCB and AHR(−/−)_PCB) at four weeks of age. The estrous cycle was synchronized and ovary and uterus were collected 28 days after exposure. In WT rats, PCB126 exposure reduced (P < 0.05) body and ovary weight, uterine gland number, uterine area, progesterone, 17β-estradiol and anti-Müllerian hormone level, secondary and antral follicle and corpora lutea number but follicle stimulating hormone level increased (P < 0.05). In AHR(−/−) rats, PCB126 exposure increased (P ≤ 0.05) circulating luteinizing hormone level. Ovarian or uterine mRNA abundance of biotransformation, and inflammation genes were altered (P < 0.05) in WT rats due to PCB126 exposure. In AHR(−/−) rats, the transcriptional effects of PCB126 were restricted to reductions (P < 0.05) in three inflammatory genes. These findings support a functional role for AHR in the female reproductive tract, illustrate AHR’s requirement in PCB126-induced reprotoxicity, and highlight the potential risk of dioxin-like compounds on female reproduction.

Glyphosate (GLY) usage for weed control is extensive. To investigate ovarian impacts of chronic GLY exposure, female C57BL6 mice were orally administered saline as vehicle control (CT) or GLY at 0.25 (G0.25), 0.5 (G0.5), 1.0 (G1.0), 1.5 (G1.5), or 2 (G2.0) mg/kg for five days per wk. for 20 wks. Feed intake increased (P < .05) in G1.5 and G2.0 mice and body weight increased (P < .05) in G1.0 mice. There was no impact of GLY on estrous cyclicity, nor did GLY affect circulating levels of 17β-estradiol or progesterone. Exposure to GLY did not impact heart, liver, spleen, kidney or uterus weight. Both ovarian weight and follicle number were increased (P < .05) by G2.0 but not affected at lower GLY concentrations. There were no detectable effects of GLY on ovarian protein abundance of pAKT, AKT, pAKT:AKT, γH2AX, STAR, CYP11A1, HSD3B, CYP19A, ERA or ERB. Increased (P < .05) abundance of ATM protein was observed at G0.25 but not higher GLY doses. A dose-dependent effect (P < .10) of GLY exposure on ovarian protein abundance as quantified by LC-MS/MS was observed (G0.25–4 increased, 19 decreased; G0.5–5 increased, 25 decreased; G1.0–65 increased, 7 decreased; G1.5–145 increased, 2 decreased; G2.0–159 increased, 4 decreased). Pathway analysis was performed using DAVID and identified glutathione metabolism, metabolic and proteasome pathways as GLY exposure targets. These data indicate that chronic low-level exposure to GLY alters the ovarian proteome and may ultimately impact ovarian function.

Arsenic induces reproductive disorders in pubertal males after prepubertal exposure. However, it is unclear the extent to which those effects remain in testis and epididymis of sexually mature rats after arsenic insult. This study evaluated the effects of prepubertal arsenic exposure in male organs of pubertal rats, and their reversibility in adult rats. Male pups of Wistar rats on postnatal day (PND) 21 were divided into two groups (n = 20/group): Control animals received filtered water and exposed rats received 10 mg L‐−1 arsenic from PND 21 to PND 51. At PND 52, testis and epididymis of ten animals per group were examined for toxic effects under morphological, functional, and molecular approaches. The other animals were kept alive under free arsenic conditions until PND 82, and further analyzed for the same parameters. Pubertal rats overexpressed mRNA levels of SOD1, SOD2, CAT, GSTK1, and MT1 in their testis and SOD1, CAT, and GSTK1 in their epididymis. In those organs, catalase activity was altered, generating byproducts of oxidative stress. The antioxidant gene expression was unchanged in adult rats in contrast to the altered activity of antioxidant enzymes. Histological alterations of testis and epididymis tissues were observed in pubertal and adult rats. Interestingly, only adult rats exhibited a remarkable decrease in serum testosterone levels. Prepubertal exposure to arsenic caused morphological and functional alterations in male reproductive organs of pubertal rats. In adult rats, these damages disappeared, remained, get worsened, or recovered depending on the parameter analyzed, indicating potential male fertility disorders during adulthood.

Chemicals induce liver cancer in rodents through well characterized adverse outcome pathways (AOPs). We hypothesized that measurement of molecular initiating events (MIEs) and downstream key events (KEs) in liver cancer AOPs in short-term assays will allow early identification of chemicals and their associated doses that are likely to be tumorigenic in the liver in two-year bioassays. We tested this hypothesis using the rat in vivo TG-GATES study data to measure MIEs (genotoxicity, cytotoxicity, AhR, CAR, ER, PPARα) and associated KEs (oxidative stress, cell proliferation, liver to body weights) across 77 chemicals that could be linked to doses with previously established effects on rat liver tumor induction. Gene expression biomarkers for MIEs generally considered to be rodent specific and human irrelevant (CAR, PPARα) and for MIEs that would be considered of greater risk at human relevant exposures (ER, AhR) were built using microarray comparisons from the livers of rats treated with prototypical activators of the receptors. The genotoxicity biomarker, also a potentially human relevant MIE, was comprised of 7 p53-responsive genes known to be induced upon DNA damage. The ability of the biomarkers to accurately predict MIE activation ranged from 91% to 98%. The Toxicological Priority Index (ToxPi) was used to rank chemicals based on their ability to activate MIEs/KEs. Chemicals administered at tumorigenic doses clearly gave the highest ranked scores. Our AOP-directed approach could be used in short term assays to identify chemicals and their doses that would be predicted to cause liver tumors in rats.

Per- and polyfluoroalkyl substances (PFAS) are pervasive in the environment resulting in nearly universal detection in people. Human serum PFAS concentrations are strongly associated with increased serum low-density lipoprotein cholesterol (LDL-C), and growing evidence suggests an association with serum triacylglycerides (TG). Here, we tested the hypothesis that perfluorooctanoic acid (PFOA) dysregulates liver and serum triacylglycerides in human peroxisome proliferator activated receptor α (hPPARα)-expressing mice fed an American diet. Mice were exposed to PFOA (3.3 mg/l) in drinking water for 6 weeks resulting in a serum concentration of 48 ± 9 μg/ml. In male and female hPPARα mice, PFOA increased total liver TG and TG substituted with saturated and monounsaturated fatty acids. Lack of expression of hPPARα alone also increased total liver TG, and PFOA treatment had little effect on liver TG in null mice. In hPPARα mice, PFOA neither significantly increased nor decreased serum TG; however, there was a modest increase in TG associated with very low-density cholesterol particles in both sexes. Across studies, a non-monotonic effect of PFOA on serum TG is evident, with the serum PFOA concentration in this study falling near the “null point” between increasing and decreasing serum TG. Intriguingly, in female PPARα null mice, PFOA significantly increased serum TG, with a similar trend in males. PFOA also modified fatty acid and TG homeostasis-related gene expression in liver, in a hPPARα-dependent manner, but not in adipose. The results reveal the importance of context (serum concentration and genotype) in determining the effect of PFOA on lipid homeostasis.

High-fat diet (HFD)-induced obesity is implicated in diabetic nephropathy (DN). EX-527, a selective Sirtuin 1 (SIRT1) inhibitor, has multiple biological functions; however, its protective effect against DN is yet to be properly understood. This study was aimed to explore the protective effect of EX-527 against DN in HFD-induced diabetic Zucker (ZDF) rats. After 21 weeks of continually feeding HFD to the rats, the apparent characteristics of progressive DN were observed, which included an increase in kidney weight (~160%), hyperglycemia, oxidative stress, and inflammatory cytokines, and subsequent renal cell damage. However, the administration of EX-527 for 10 weeks significantly reduced the blood glucose concentration and kidney weight (~59%). Furthermore, EX-527 significantly reduced the serum concentration of transforming growth factor-β1 (49%), interleukin (IL)-1β (52%), and IL-6 in the HFD-fed rats. Overall, the antioxidant activities significantly increased, and oxidative damage to lipids or DNA was suppressed. Particularly, EX-527 significantly reduced blood urea nitrogen (81%), serum creatinine (71%), microalbumin (43%), and urinary excretion of protein-based biomarkers. Histopathological examination revealed expansion of the extracellular mesangial matrix and suppression of glomerulosclerosis following EX-527 administration. EX-527 downregulated the expression of α-SMA (~64%), TGF-β (25%), vimentin, α-tubulin, fibronectin, and collagen-1 in the kidneys of the HFD-fed rats. Additionally, EX-527 substantially reduced claudin-1 and SIRT1 expression, but increased the expression of SIRT3 in the kidneys of the HFD-fed rats. EX-527 also inhibited the growth factor receptors, including EGFR, PDGFR-β, and STAT3, which are responsible for the anti-fibrotic effect of SIRT-1. Therefore, the administration of EX-527 protects against HFD-induced DN.

Serotonin level plays a role in suppressing the pathological findings of benign prostatic hyperplasia (BPH). Thus a new selective serotonin reuptake inhibitor, dapoxetine was used to test its ability to ameliorate the pathological changes in the rat prostate. A dose response curve was constructed between the dose of dapoxetine and prostate weight as well as relative prostate weight, then a 5mg/kg dose was used as a representative dose for dapoxetine administration. Rats were divided into four groups; the control group that received the vehicle; the BPH-induced group received daily s.c injection of 3mg/kg testosterone propionate dissolved in olive oil for four weeks; BPH-induced group treated with finasteride 5mg/kg/day p.o and BPH-induced group treated with dapoxetine 5mg/kg/day p.o. Injection of testosterone increased prostate weight and relative prostate weight which were both returned back to the normal value after treatment with dapoxetine as well as finasteride. Testosterone also upregulated androgen receptor (AR) and proliferating cell nuclear antigen gene expression. Furthermore, testosterone injection elevated cyclooxygenase-II (COX II), inducible nitric oxide synthase (iNOS), B-cell lymphoma-2 (Bcl2) expression and tumor necrosis factor alpha content and reduced caspase-3 activity, Bcl-2-associated X protein (Bax) expression and Bax/Bcl2 ratio. Dapoxetine and finasteride administration reverted most of the changes made by testosterone injection. In conclusion, the current study provides an evidence for the protective effects of dapoxetine against testosterone-induced BPH in rats. This can be attributed, at least in part, to decreasing AR expression, and the anti-proliferative, anti-inflammatory and pro-apoptotic activities of dapoxetine in BPH.

Bromo and extra terminal (BET) proteins (BRD2, BRD3, BRD4 and BRDT) are epigenetic transcriptional regulators required for efficient expression of growth promoting, cell cycle progression and antiapoptotic genes. Through their bromodomain, these proteins bind to acetylated lysine residues of histones and are recruited to transcriptionally active chromatin. Inhibition of the BET-histone interaction provides a tractable therapeutic strategy to treat diseases that may have epigenetic dysregulation. JQ1 is a small molecule that blocks BET interaction with histones. It has been shown to decrease proliferation of patient-derived multiple myeloma in vitro and to decrease tumor burden in vivo in xenograft mouse models. While targeting BET appears to be a viable and efficacious approach, the nonclinical safety profile of BET inhibition remains to be well-defined. We report that mice dosed with JQ1 at efficacious exposures demonstrate dose-dependent decreases in their lymphoid and immune cell compartments. At higher doses, JQ1 was not tolerated and due to induction of significant body weight loss led to early euthanasia. Flow cytometry analysis of lymphoid tissues showed a decrease in both B- and T-lymphocytes with a concomitant decrease in peripheral white blood cells that was confirmed by hematology. Further investigation with the inactive enantiomer of JQ1 showed that these in vivo effects were on-target mediated and not elicited through secondary pharmacology due to chemical structure.

Studies demonstrated that perfluorooctane sulfonate (PFOS) tends to accumulate in the liver and is capable to cause hepatomegaly. In the present study, we investigated the roles of miR-155 in PFOS-induced hepatotoxicity in SD rats and HepG2 cells. Male SD rats were orally administrated with PFOS at 1 or 10mg/kg/day for 28 days while HepG2 cells were treated with 0-50 μM of PFOS for 24h or 50 μM of PFOS for 1, 3, 6, 12 or 24h, respectively. We found that PFOS significantly increased the liver weight and serum alanine transaminase (ALT) and aspartate amino transferase (AST) levels in rats. Morphologically, PFOS caused actin filament remodeling and endothelial permeability changes in the liver. Moreover, PFOS triggered reactive oxygen species (ROS) generation and induced apoptosis in both in vivo and in vitro assays. Immunoblotting data showed that NF-E2-related factor-2 (Nrf2) expression and activation and its target genes were all suppressed by PFOS in the liver and HepG2 cells. However, PFOS significantly increased miR-155 expression. Further studies showed that pretreatment of HepG2 cells with catalase significantly decreased miR-155 expression and substantially increased Nrf2 expression and activation, resulting in reduction of PFOS-induced cytotoxicity and oxidative stress. Taken together, these results indicated that miR-155 plays an important role in the PFOS-induced hepatotoxicity by disrupting Nrf2/ARE signaling pathway.

Subchronic and chronic studies in rats of the gasoline oxygenates ethyl tert-butyl ether (ETBE) and tert-butanol (TBA) report similar noncancer kidney and liver effects but differing results with respect to kidney and liver tumors. Because TBA is a major metabolite of ETBE, it is possible that TBA is the active toxic moiety in all these studies, with reported differences due simply to differences in the internal dose. To test this hypothesis, a physiologically-based pharmacokinetic (PBPK) model was developed for ETBE and TBA to calculate internal dosimetrics of TBA following either TBA or ETBE exposure. This model, based on earlier PBPK models of methyl tert-butyl ether (MTBE), was used to evaluate whether kidney and liver effects are consistent across routes of exposure, as well as between ETBE and TBA studies, on the basis of estimated internal dose. The results demonstrate that noncancer kidney effects, including kidney weight changes, urothelial hyperplasia, and chronic progressive nephropathy (CPN), yielded consistent dose-response relationships across routes of exposure and across ETBE and TBA studies using TBA blood concentration as the dose metric. Relative liver weights were also consistent across studies on the basis of TBA metabolism, which is proportional to TBA liver concentrations. However, kidney and liver tumors were not consistent using any dose metric. These results support the hypothesis that TBA mediates the noncancer kidney and liver effects following ETBE administration; however, additional factors besides internal dose are necessary to explain the induction of liver and kidney tumors.

The somatostatin analog pasireotide and the 11β-hydroxylase inhibitor osilodrostat (LCI699) reduce cortisol levels by distinct mechanisms of action. There exists a scientific rationale to investigate the clinical efficacy of these two agents in combination. This manuscript reports the results of a toxicology study in rats, evaluating different doses of osilodrostat and pasireotide alone and in combination. Sixty male and 60 female rats were randomized into single-sex groups to receive daily doses of pasireotide (0.3mg/kg/day, subcutaneously), osilodrostat (20mg/kg/day, orally), osilodrostat/pasireotide in combination (low dose, 1.5/0.03mg/kg/day; mid-dose, 5/0.1mg/kg/day; or high dose, 20/0.3mg/kg/day), or vehicle for 13weeks. Mean body-weight gains from baseline to Week 13 were significantly lower in the pasireotide-alone and combined-treatment groups compared to controls, and were significantly higher in female rats receiving osilodrostat monotherapy. Osilodrostat and pasireotide monotherapies were associated with significant changes in the histology and mean weights of the pituitary and adrenal glands, liver, and ovary/oviduct. Osilodrostat alone was associated with adrenocortical hypertrophy and hepatocellular hypertrophy. In combination, osilodrostat/pasireotide did not exacerbate any target organ changes and ameliorated the liver and adrenal gland changes observed with monotherapy. Cmax and AUC0–24h of osilodrostat and pasireotide increased in an approximately dose-proportional manner.In conclusion, the pasireotide and osilodrostat combination did not exacerbate changes in target organ weight or toxicity compared with either monotherapy, and had an acceptable safety profile; addition of pasireotide to the osilodrostat regimen may attenuate potential adrenal gland hyperactivation and hepatocellular hypertrophy, which are potential side effects of osilodrostat monotherapy.

Arsenic (As) tops the ATSDR list of hazardous environmental chemicals and is known to cause liver injury. Although the concentrations of As found in the US water supply are generally too low to directly damage the liver, subhepatotoxic doses of As sensitize the liver to experimental NAFLD. It is now suspected that GI microbiome dysbiosis plays an important role in development of NALFD. Importantly, arsenic has also been shown to alter the microbiome. The purpose of the current study was to test the hypothesis that the prebiotic oligofructose (OFC) protects against enhanced liver injury caused by As in experimental NAFLD. Male C57Bl6/J mice were fed low fat diet (LFD), high fat diet (HFD), or HFD containing oligofructose (OFC) during concomitant exposure to either tap water or As-containing water (4.9 ppm as sodium arsenite) for 10 wks. HFD significantly increased body mass and caused fatty liver injury, as characterized by an increased liver weight-to-body weight ratio, histologic changes and transaminases. As observed previously, As enhanced HFD-induced liver damage, which was characterized by enhanced inflammation. OFC supplementation protected against the enhanced liver damage caused by As in the presence of HFD. Interestingly, arsenic, HFD and OFC all caused unique changes to the gut flora. These data support previous findings that low concentrations of As enhance liver damage caused by high fat diet. Furthermore, these results indicate that these effects of arsenic may be mediated, at least in part, by GI tract dysbiosis and that prebiotic supplementation may confer significant protective effects.

This project developed from studies demonstrating that Libby Amphibole Asbestos (LAA) causes a non-typical set of health outcomes not generally reported for asbestos, including systemic autoimmunity and an unusual and devastating lamellar pleural thickening that progresses to severe pulmonary dysfunction and death. Further, mineral fiber mixtures with some similarities to LAA have recently been discovered in southern Nevada and northwestern Arizona, where the material exists in extensive recreational areas and is present in yards, roads, parking lots and school yards. The objective was to compare the health outcomes in mice exposed to either LAA or the fibrous amphiboles collected in Arizona at the Lake Mead National Recreational Area at very low doses to represent environmental exposures. In this study, the fibrous amphibole asbestos sample from Arizona (AzA) is composed of winchite (69%), actinolite (22%), and non-amphibole minerals (9%) and has a mean aspect ratio of 16.7±0.9. Fibrous amphibole asbestos from Libby (LAA) is composed of winchite (70%), richterite (9%), tremolite (5%), and non-amphibole minerals (16%) with a mean aspect ratio of 8.4±0.7. C57BL/6 mice were exposed by oropharyngeal aspiration to fiber suspensions at a very low dose of 3μg/mouse. After seven months, both LAA- and AzA-exposed mice had indices of chronic immune dysfunction related to a TH17 cytokine profile, with B cell activation, autoantibody production and proteinuria, suggesting kidney involvement. In addition, both exposures led to significant lung and pleural fibrosis. These data suggest that there is risk of pulmonary disease and autoimmune outcomes with environmental exposure to amphibole asbestos, and that this is not limited to Libby, Montana.

The renin-angiotensin system (RAS) in the brain is involved in the pathogenesis of hypertension. We hypothesized that inhibition of angiotensin-converting enzyme (ACE) in the hypothalamic paraventricular nucleus (PVN) attenuates angiotensin II (ANG II)-induced hypertension via restoring neurotransmitters and cytokines. Rats underwent subcutaneous infusions of ANG II or saline and bilateral PVN infusions of ACE inhibitor enalaprilat (ENL, 2.5μg/h) or vehicle for 4weeks. ANG II infusion resulted in higher mean arterial pressure and cardiac hypertrophy as indicated by increased whole heart weight/body weight ratio, whole heart weight/tibia length ratio, left ventricular weight/tibia length ratio, and mRNA expressions of cardiac atrial natriuretic peptide and beta-myosin heavy chain. These ANG II-infused rats had higher PVN levels of glutamate, norepinephrine, tyrosine hydroxylase, pro-inflammatory cytokines (PICs) and the chemokine monocyte chemoattractant protein-1, and lower PVN levels of gamma-aminobutyric acid, interleukin (IL)-10 and the 67-kDa isoform of glutamate decarboxylase (GAD67), and higher plasma levels of PICs, norepinephrine and aldosterone, and lower plasma IL-10, and higher renal sympathetic nerve activity. However, PVN treatment with ENL attenuated these changes. PVN microinjection of ANG II induced increases in IL-1β and IL-6, and a decrease in IL-10 in the PVN, and pretreatment with angiotensin II type 1 receptor (AT1-R) antagonist losartan attenuated these changes. These findings suggest that ANG II infusion induces an imbalance between excitatory and inhibitory neurotransmitters and an imbalance between pro- and anti-inflammatory cytokines in the PVN, and PVN inhibition of the RAS restores neurotransmitters and cytokines in the PVN, thereby attenuating ANG II-induced hypertension and cardiac hypertrophy.

Life threatening complications from chemotherapy occur frequently in cancer survivors, however little is known about genetic risk factors. We treated male normotensive rats (WKY) and strains with hypertension (SHR) and hypertension with cardiomyopathy (SHHF) with 8 weekly doses of doxorubicin (DOX) followed by 12weeks of observation to test the hypothesis that genetic cardiovascular disease would worsen delayed cardiotoxicity. Compared with WKY, SHR demonstrated weight loss, decreased systolic blood pressure, increased kidney weights, greater cardiac and renal histopathologic lesions and greater mortality. SHHF showed growth restriction, increased kidney weights and renal histopathology but no effect on systolic blood pressure or mortality. SHHF had less severe cardiac lesions than SHR. We evaluated cardiac soluble epoxide hydrolase (sEH) content and arachidonic acid metabolites after acute DOX exposure as potential mediators of genetic risk. Before DOX, SHHF and SHR had significantly greater cardiac sEH and decreased epoxyeicosatrienoic acid (EET) (4 of 4 isomers in SHHF and 2 of 4 isomers in SHR) than WKY. After DOX, sEH was unchanged in all strains, but SHHF and SHR rats increased EETs to a level similar to WKY. Leukotriene D4 increased after treatment in SHR. Genetic predisposition to heart failure superimposed on genetic hypertension failed to generate greater toxicity compared with hypertension alone. The relative resistance of DOX-treated SHHF males to the cardiotoxic effects of DOX in the delayed phase despite progression of genetic disease was unexpected and a key finding. Strain differences in arachidonic acid metabolism may contribute to variation in response to DOX toxicity.

Serum levels of cardiac troponins serve as biomarkers of myocardial injury. However, troponins are released into the serum only after damage to cardiac tissue has occurred. Here, we report development of a mouse model of doxorubicin (DOX)-induced chronic cardiotoxicity to aid in the identification of predictive biomarkers of early events of cardiac tissue injury. Male B6C3F(1) mice were administered intravenous DOX at 3mg/kg body weight, or an equivalent volume of saline, once a week for 4, 6, 8, 10, 12, and 14weeks, resulting in cumulative DOX doses of 12, 18, 24, 30, 36, and 42mg/kg, respectively. Mice were sacrificed a week following the last dose. A significant reduction in body weight gain was observed in mice following exposure to a weekly DOX dose for 1week and longer compared to saline-treated controls. DOX treatment also resulted in declines in red blood cell count, hemoglobin level, and hematocrit compared to saline-treated controls after the 2nd weekly dose until the 8th and 9th doses, followed by a modest recovery. All DOX-treated mice had significant elevations in cardiac troponin T concentrations in plasma compared to saline-treated controls, indicating cardiac tissue injury. Also, a dose-related increase in the severity of cardiac lesions was seen in mice exposed to 24mg/kg DOX and higher cumulative doses. Mice treated with cumulative DOX doses of 30mg/kg and higher showed a significant decline in heart rate, suggesting drug-induced cardiac dysfunction. Altogether, these findings demonstrate the development of DOX-induced chronic cardiotoxicity in B6C3F(1) mice.

The human testis is sensitive to toxicant-induced injury but current methods for detecting adverse effects are limited, insensitive and unreliable. Animal studies use sensitive histopathological endpoints to assess toxicity, but require testicular tissue that is not available during human clinical trials. More sensitive and reliable molecular biomarkers of testicular injury are needed to better monitor testicular toxicity in both clinical and preclinical. Adult male Wistar Han rats were exposed for 4weeks to compounds previously associated with testicular injury, including cisplatin (0, 0.2, 0.3, or 0.4mg/kg/day), BI665915 (0, 20, 70, 100mg/kg/d), BI665636 (0, 20, 100mg/kg/d) or BI163538 (0, 70, 150, 300mg/kg/d) to evaluate reproductive toxicity and assess changes in sperm mRNA levels. None of the compounds resulted in any significant changes in body, testis or epididymis weights, nor were there decreases in testicular homogenization resistant spermatid head counts. Histopathological evaluation found that only BI665915 treatment caused any testicular effects, including minor germ cell loss and disorganization of the seminiferous tubule epithelium, and an increase in the number of retained spermatid heads. A custom PCR-array panel was used to assess induced changes in sperm mRNA. BI665915 treatment resulted in a significant increase in clusterin (Clu) levels and decreases in GTPase, IMAP family member 4 (Gimap4), prostaglandin D2 synthase (Ptgds) and transmembrane protein with EGF like and two follistatin like domains 1 (Tmeff1) levels. Correlation analysis between transcript levels and quantitative histopathological endpoints found a modest association between Clu with retained spermatid heads. These results demonstrate that sperm mRNA levels are sensitive molecular indicators of testicular injury that can potentially be translated into a clinical setting.

Sex is a risk factor for development of cardiotoxicity, induced by the anti-cancer drug, doxorubicin (DOX), in humans. To explore potential mechanisms underlying differential susceptibility to DOX between sexes, 8-week old male and female B6C3F

Geogenic dust from arid environments is a possible inhalation hazard for humans, especially when using off-road vehicles that generate significant dust. This study focused on immunotoxicological and neurotoxicological effects following subacute exposure to geogenic dust generated from sediments in the Nellis Dunes Recreation Area near Las Vegas, Nevada that are particularly high in arsenic; the naturally-occurring arsenic concentrations in these surficial sediments ranged from 4.8 to 346μg/g. Dust samples from sediments used in this study had a median diameter of 4.5μm and also were a complex mixture of naturally-occurring metals, including aluminum, vanadium, chromium, manganese, iron, cobalt, copper, zinc, strontium, cesium, lead, uranium, and arsenic. Adult female B6C3F1 mice exposed via oropharyngeal aspiration to 0.01 to 100mg dust/kg body weight, four times, a week apart, for 28days, were evaluated 24h after the last exposure. Peripheral eosinophils were increased at all concentrations, serum creatinine was dose responsively increased beginning at 1.0mg/kg/day, and blood urea nitrogen was decreased at 10 and 100mg/kg/day. Antigen-specific IgM responses and natural killer cell activity were dose-responsively suppressed at 0.1mg/kg/day and above. Splenic CD4+CD25+ T cells were decreased at 0.01, 0.1, 10, and 100mg/kg/day. Antibodies against MBP, NF-68, and GFAP were selectively reduced. A no observed adverse effect level of 0.01mg/kg/day and a lowest observed adverse effect level of 0.1mg/kg/day were determined from IgM responses and natural killer cell activity, indicating that exposure to this dust, under conditions similar to our design, could affect these responses.

The mechanism by which nonylphenol (NP) interferes with male reproduction is not fully elucidated. Therefore, the present study was conducted to evaluate the effect of NP on male reproductive organ's weight, sperm characteristics, and to elucidate the nature and mechanism of action of NP on the epididymis. Adult male Wistar rats were gavaged with NP, dissolved in corn oil, at 0, 100, 200 or 300mg/kg/day for 30 consecutive days. Control rats were gavaged with vehicle (corn oil) alone. Body weight did not show any significant change while, absolute testes and epididymides weights were significantly decreased. Sperm count in cauda and caput/corpus epididymides, and sperm motility was significantly decreased. Daily sperm production was significantly decreased in a dose-related manner. Sperm transit time in cauda epididymis was significantly decreased by 300mg/kg, while in the caput/corpus epididymis it was significantly decreased by 200 and 300mg/kg of NP. Plasma LDH was significantly increased while; plasma testosterone was significantly decreased in a dose-related pattern. In the epididymal sperm, NP decreased acrosome integrity, Δψm and 5'-nucleotidase activity. Hydrogen peroxide (H(2)O(2)) production and LPO were significantly increased in a dose-related pattern. The activities of SOD, CAT and GPx were significantly decreased in the epididymal sperm. In conclusion, this study revealed that NP treatment impairs spermatogenesis and has a cytotoxic effect on epididymal sperm. It disrupts the prooxidant and antioxidant balance. This leads oxidative stress in epididymal sperms of rat. Moreover, the reduction in sperm transit time may affect sperm quality and fertility potential.

The developing immune system displays a relatively high sensitivity as compared to both general toxicity parameters and to the adult immune system. In this study we have performed such comparisons using di(2-ethylhexyl) phthalate (DEHP) as a model compound. DEHP is the most abundant phthalate in the environment and perinatal exposure to DEHP has been shown to disrupt male sexual differentiation. In addition, phthalate exposure has been associated with immune dysfunction as evidenced by effects on the expression of allergy. Male wistar rats were dosed with corn oil or DEHP by gavage from postnatal day (PND) 10-50 or PND 50-90 at doses between 1 and 1000 mg/kg/day. Androgen-dependent organ weights showed effects at lower dose levels in juvenile versus adult animals. Immune parameters affected included TDAR parameters in both age groups, NK activity in juvenile animals and TNF-alpha production by adherent splenocytes in adult animals. Immune parameters were affected at lower dose levels compared to developmental parameters. Overall, more immune parameters were affected in juvenile animals compared to adult animals and effects were observed at lower dose levels. The results of this study show a relatively higher sensitivity of juvenile versus adult rats. Furthermore, they illustrate the relative sensitivity of the developing immune system in juvenile animals as compared to general toxicity and developmental parameters. This study therefore provides further argumentation for performing dedicated developmental immune toxicity testing as a default in regulatory toxicology.

Alcoholic liver disease (ALD) is a serious health problem with significant morbidity and mortality. In this study we examined the progression of ALD along with lipidomic changes in rats fed ethanol for 2 and 3 months to understand the mechanism, and identify possible biomarkers. Male Fischer 344 rats were fed 5% ethanol or caloric equivalent of maltose–dextrin in a Lieber-DeCarli diet. Animals were killed at the end of 2 and 3 months and plasma and livers were collected. Portions of the liver were fixed for histological and immunohistological studies. Plasma and the liver lipids were extracted and analyzed by nuclear magnetic resonance (NMR) spectroscopy. A time dependent fatty infiltration was observed in the livers of ethanol-fed rats. Mild inflammation and oxidative stress were observed in some ethanol-fed rats at 3 months. The multivariate and principal component analysis of proton and phosphorus NMR spectroscopy data of extracted lipids from the plasma and livers showed segregation of ethanol-fed groups from the pair-fed controls. Significant hepatic lipids that were increased by ethanol exposure included fatty acids and triglycerides, whereas phosphatidylcholine (PC) decreased. However, both free fatty acids and PC decreased in the plasma. In liver lipids unsaturation of fatty acyl chains increased, contrary to plasma, where it decreased. Our studies confirm that over-accumulation of lipids in ethanol-induced liver steatosis accompanied by mild inflammation on long duration of ethanol exposure. Identified metabolic profile using NMR lipidomics could be further explored to establish biomarker signatures representing the etiopathogenesis, progression and/or severity of ALD.

Flavonoids possess strong anti-oxidant and cancer chemopreventive activities. Chrysin (5,7-dihydroxyflavone) occurs naturally in many plants, honey, and propolis. In vitro, chrysin acts as a general anti-oxidant, causes cell cycle arrest and promotes cell death. However, the mechanism by which chrysin inhibits cancer cell growth and the subcellular pathways activated remains poorly understood. Effect of dietary supplementation with chrysin on proliferation and apoptosis during diethylnitrosamine (DEN)-induced early hepatocarcinogenesis was investigated in male Wistar rats. To induce hepatocarcinogenesis, rats were given DEN injections (i.p., 200 mg/kg) three times at a 15 day interval. An oral dose of chrysin (250 mg/kg bodyweight) was given three times weekly for 3 weeks, commencing 1 week after the last dose of DEN. Changes in the mRNA expression of COX-2, NFkB p65, p53, Bcl-xL and β-arrestin-2 were assessed by quantitative real-time PCR. Changes in the protein levels were measured by western blotting. Chrysin administration significantly (P

Oxidative stress has been proposed as an important promoter of the progression of fatty liver diseases. The current study investigates the potential functions of the Nrf2-Keap1 signaling pathway, an important hepatic oxidative stress sensor, in a rodent fatty liver model. Mice with no (Nrf2-null), normal (wild type, WT), and enhanced (Keap1 knockdown, K1-kd) expression of Nrf2 were fed a methionine- and choline-deficient (MCD) diet or a control diet for 5 days. Compared to WT mice, the MCD diet-caused hepatosteatosis was more severe in the Nrf2-null mice and less in the K1-kd mice. The Nrf2-null mice had lower hepatic glutathione and exhibited more lipid peroxidation, whereas the K1-kd mice had the highest amount of glutathione in the liver and developed the least lipid peroxidation among the three genotypes fed the MCD diet. The Nrf2 signaling pathway was activated by the MCD diet, and the Nrf2-targeted cytoprotective genes Nqo1 and Gstalpha1/2 were induced in WT and even more in K1-kd mice. In addition, Nrf2-null mice on both control and MCD diets exhibited altered expression profiles of fatty acid metabolism genes, indicating Nrf2 may influence lipid metabolism in liver. For example, mRNA levels of long chain fatty acid translocase CD36 and the endocrine hormone Fgf21 were higher in livers of Nrf2-null mice and lower in the K1-kd mice than WT mice fed the MCD diet. Taken together, these observations indicate that Nrf2 could decelerate the onset of fatty livers caused by the MCD diet by increasing hepatic antioxidant and detoxification capabilities.

Polychlorinated biphenyls (PCBs) are ubiquitous contaminants found as complex mixtures of coplanar and non-coplanar congeners. The hepatic temporal and dose-dependent effects of the most abundant non-dioxin-like congener, 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153), were examined in immature, ovariectomized C57BL/6 mice, and compared to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the prototypical aryl hydrocarbon receptor (AhR) ligand. Animals were gavaged once with 300 mg/kg PCB153 or sesame oil vehicle and sacrificed 4, 12, 24, 72 or 168 h post dose. In the dose-response study, mice were gavaged with 1, 3, 10, 30, 100 or 300 mg/kg PCB153 or sesame oil for 24 h. Significant increases in relative liver weights were induced with 300 mg/kg PCB153 between 24 and 168 h, accompanied by slight vacuolization and hepatocellular hypertrophy. The hepatic differential expression of 186 and 177 genes was detected using Agilent 4 x 44 K microarrays in the time course (|fold change|> or =1.5, P1(t)> or =0.999) and dose-response (|fold change|> or =1.5, P1(t)> or =0.985) studies, respectively. Comparative analysis with TCDD suggests that the differential gene expression elicited by PCB153 was not mediated by the AhR. Furthermore, constitutive androstane and pregnane X receptor (CAR/PXR) regulated genes including Cyp2b10, Cyp3a11, Ces2, Insig2 and Abcc3 were dose-dependently induced by PCB153. Collectively, these results suggest that the hepatocellular effects elicited by PCB153 are qualitatively and quantitatively different from TCDD and suggestive of CAR/PXR regulation.

Statistical comparison of organ weights between treated and untreated animals have traditionally been used to predict potential toxicity for patients. The manner of presentation of organ weight data, and the value of statistical analyses have been topics of discussion. Historically, a decision tree approach has been applied for statistical comparison of organ weights which does not control the overall error rate and can lead to different statistical tests being used by chance for identical settings causing confusion. This paper proposes a simple nonparametric approach for assessing treatment effects on organ weights in terms of ratios based on the Hodges-Lehmann estimator. This allows for simple interpretation of results and aids in the identification of potential target organs as the evaluation is based on effect sizes and not on p-values allowing a robust proof of effect as well as a robust proof of no effect. The proposed estimate and the corresponding nonparametric confidence interval applied to a rank-sum score can be used as a confirmatory test for difference and as a confirmatory test for equivalence. Exploratory analyses can be performed calculating the proposed estimates for each organ separately to be summarized graphically in a confidence interval plot.

We evaluated the protective efficacy of nasal atropine methyl bromide (AMB) which does not cross the blood-brain barrier against sarin inhalation exposure. Age and weight matched male guinea pigs were exposed to 846.5 mg/m(3) sarin using a microinstillation inhalation exposure technique for 4 min. The survival rate at this dose was 20%. Post-exposure treatment with nasal AMB (2.5 mg/kg, 1 min) completely protected against sarin induced toxicity (100% survival). Development of muscular tremors was decreased in animals treated with nasal AMB. Post-exposure treatment with nasal AMB also normalized acute decrease in blood oxygen saturation and heart rate following sarin exposure. Inhibition of blood AChE and BChE activities following sarin exposure was reduced in animals treated with nasal AMB, indicating that survival increases the metabolism of sarin or expression of AChE. The body weight loss of animals exposed to sarin and treated with nasal AMB was similar to saline controls. No differences were observed in lung accessory lobe or tracheal edema following exposure to sarin and subsequent treatment with nasal AMB. Total bronchoalveolar lavage fluid (BALF) protein, a biomarker of lung injury, showed trends similar to saline controls. Surfactant levels post-exposure treatment with nasal AMB returned to normal, similar to saline controls. Alkaline phosphatase levels post-exposure treatment with nasal AMB were decreased. Taken together, these data suggest that nasal AMB blocks the copious airway secretion and peripheral cholinergic effects and protects against lethal inhalation exposure to sarin thus increasing survival.

Trichloroethylene (TCE) is a lipophilic solvent rapidly absorbed and metabolized via oxidation and conjugation to a variety of metabolites that cause toxicity to several internal targets. Increases in liver weight (hepatomegaly) have been reported to occur quickly in rodents after TCE exposure, with liver tumor induction reported in mice after long-term exposure. An integrated dataset for gavage and inhalation TCE exposure and oral data for exposure to two of its oxidative metabolites (TCA and DCA) was used, in combination with an updated and more accurate physiologically-based pharmacokinetic (PBPK) model, to examine the question as to whether the presence of TCA in the liver is responsible for TCE-induced hepatomegaly in mice. The updated PBPK model was used to help discern the quantitative contribution of metabolites to this effect. The update of the model was based on a detailed evaluation of predictions from previously published models and additional preliminary analyses based on gas uptake inhalation data in mice. The parameters of the updated model were calibrated using Bayesian methods with an expanded pharmacokinetic database consisting of oral, inhalation, and iv studies of TCE administration as well as studies of TCE metabolites in mice. The dose-response relationships for hepatomegaly derived from the multi-study database showed that the proportionality of dose to response for TCE- and DCA-induced hepatomegaly is not observed for administered doses of TCA in the studied range. The updated PBPK model was used to make a quantitative comparison of internal dose of metabolized and administered TCA. While the internal dose of TCA predicted by modeling of TCE exposure (i.e., mg TCA/kg-d) showed a linear relationship with hepatomegaly, the slope of the relationship was much greater than that for directly administered TCA. Thus, the degree of hepatomegaly induced per unit of TCA produced through TCE oxidation is greater than that expected per unit of TCA administered directly, which is inconsistent with the hypothesis that TCA alone accounts for TCE-induced hepatomegaly. In addition, TCE-induced hepatomegaly showed a much more consistent relationship with PBPK model predictions of total oxidative metabolism than with predictions of TCE area-under-the-curve in blood, consistent with toxicity being induced by oxidative metabolites rather than the parent compound. Therefore, these results strongly suggest that oxidative metabolites in addition to TCA are necessary contributors to TCE-induced liver weight changes in mice.

The mastic (Pistacia lentiscus var. chia) tree is native throughout the Mediterranean region and has long proved a source of food additives and medical treatments. To investigate the modifying effects of Chios Mastic Gum on rat liver carcinogenesis, 6-week-old male F344 rats were subjected to the established rat liver medium-term carcinogenesis bioassay (Ito-test). At the commencement, rats (groups 1-4) were intraperitoneally injected with 200 mg/kg body weight of diethylnitrosamine (DEN). After two weeks, mastic was added to CRF (Charles River Formula)-1 powdered basal diet at doses of 0, 0.01, 0.1 and 1% in groups 1-4, respectively. At week 3, all rats were underwent two-thirds partial hepatectomy. The experiment was terminated at week 8. As results show, liver weights were significantly increased in a mastic dose-dependent manner among groups 1-4. The numbers (/cm(2)) and the areas (mm(2)/cm(2)) of glutathione S-transferase placental form (GST-P)-positive cell foci (>or=0.2 mm in diameter) were significantly increased in the DEN-1% group compared to the DEN-alone group, along with the average areas per foci and larger-sized foci (>or=0.4 mm). 5-Bromo-2'-deoxyuridine (BrdU)+GST-P double-immunohistochemistry showed the highest BrdU-labeling indices within GST-P foci in the DEN-1% group. 8-hydroxydeoxyguanosine (8-OHdG) levels in liver DNA did not vary, while real-time quantitative polymerase chain reaction (PCR) analysis of livers revealed many up- or down-regulated genes in the DEN-1% group. In conclusion, this is the first report to display a promotion potential of Chios Mastic Gum on the formation of preneoplastic lesions in the established rat liver medium-term carcinogenesis bioassay.

Because of their persistence in the environment dioxins are one of the most concerned classes of carcinogens. Displaying both pro- and anti-agonistic properties to some hormone receptors, the pollutants are also known to be endocrine disruptors. Humans can be exposed to this pollutant through contaminated food, air, drinking water, etc. The female hormone estrogen may initiate various physiological functions, and excessive exposure to this hormone is a documented risk factor for carcinogenesis. Cyp19 (aromatase) catalyses the last step of estrogen biosynthesis, while cyp1a1 can hydroxylate and deactivate the hormone. In the present study, we investigated the effect of 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD) on aromatase expression in the brain and adipose tissue in ovariectomized Sprague Dawley rats. Female rats were given 2.5 microg/kg TCDD p.o. before and after ovariectomy. Real-time PCR and western blot analysis indicated that pre-ovariectomy administration of TCDD could significantly reduce aromatase expression in the brain but increase the expression in the adipose tissue. In addition, increased plasma estrogen level and uterine weight were observed in these rats. These parameters did not change in rats with post-ovariectomy TCDD treatment. Our results suggested that the timing of exposure to the toxicant could determine the estrogenicity of TCDD. No correlation between cyp1a1 and cyp19 expression was observed.

The toxic equivalency factor (TEF) approach was employed to compare immunotoxic potency of mixtures containing polychlorinated dibenzo- p -dioxins, polychlorinated dibenzofurans and polychlorinated biphenyls relative to 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD), using the antibody response to sheep erythrocytes (SRBC). Mixture-1 (MIX-1) contained TCDD, 1,2,3,7,8-pentachlorodibenzo- p -dioxin (PeCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-pentachlorodibenzofuran (1-PeCDF), 2,3,4,7,8-pentachlorodibenzofuran (4-PeCDF), and 1,2,3,4,6,7,8,9-octachlorodibenzofuran (OCDF). Mixture-2 (MIX-2) contained MIX-1 and the following PCBs, 3,3′,4,4′-tetrachlorobiphenyl (IUPAC No. 77), 3,3′,4,4′,5-pentachlorobiphenyl (126), 3,3′,4,4′,5,5 N -hexachlorobiphenyl (169), 2,3,3′,4,4′-pentachlorobiphenyl (105), 2,3′,4,4′,5-pentachlorobiphenyl (118), and 2,3,3′,4,4′,5-hexachlorobiphenyl (156). The mixture compositions were based on relative chemical concentrations in food and human tissues. TCDD equivalents (TEQ) of the mixture were estimated using relative potency factors from hepatic enzyme induction in mice [DeVito, M.J., Diliberto, J.J., Ross, D.G., Menache, M.G., Birnbaum, L.S., 1997. Dose–response relationships for polyhalogenated dioxins and dibenzofurans following subchronic treatment in mice. I .CYP1A1 and CYP1A2 enzyme activity in liver, lung and skin. Toxicol. Appl. Pharmacol. 130, 197–208; DeVito, M.J., Menache, G., Diliberto, J.J., Ross, D.G., Birnbaum L.S., 2000. Dose–response relationships for induction of CYP1A1 and CYP1A2 enzyme activity in liver, lung, and skin in female mice following subchronic exposure to polychlorinated biphenyls. Toxicol. Appl. Pharmacol. 167, 157–172] Female mice received 0, 1.5, 15, 150 or 450 ng TCDD/kg/day or approximately 0, 1.5, 15, 150 or 450 ng TEQ/kg/day of MIX-1 or MIX-2 by gavage 5 days per week for 13 weeks. Mice were immunized 3 days after the last exposure and 4 days later, body, spleen, thymus, and liver weights were measured, and antibody response to SRBCs was observed. Exposure to TCDD, MIX-1, and MIX-2 suppressed the antibody response in a dose-dependent manner. Two-way ANOVA indicated no differences in the response between TCDD and the mixtures for body weight, spleen/body weight and decreased antibody responses. The results support the use of the TEF methodology and suggest that immune suppression by dioxin-like chemicals may be of concern at or near background human exposures.

We studied the effect of administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by i.p. injection once every 2 weeks in combination with a high-fat (HF) diet for 8 or 16 weeks on the body and organ weight changes as well as on the hepatic enzyme activity for estrogen metabolism in C3H/HeN female mice. Administration of TCDD at 100 microg/kg b.w. once every 2 weeks for 8 weeks increased the body weight by 46% in the HF diet-fed animals, but not in the regular diet-fed animals. This is the first observation suggesting that TCDD at a high dose (100 microg/kg b.w.), but not at lower doses (1 or 10 microg/kg b.w.), may have a strong obesity-inducing effect in C3H/HeN mice fed an HF diet. While TCDD increased liver weight and decreased thymus weight in animals, these effects were enhanced by feeding animals an HF diet. Metabolism studies showed that TCDD administration for 8 or 16 weeks increased the liver microsomal activity for the 2- and 4-hydroxylation of 17 beta-estradiol in animals fed a control diet, but surprisingly not in animals fed an HF diet. Treatment with TCDD dose-dependently increased the hepatic activity for the O-methylation of catechol estrogens in both control and HF diet-fed animals, and it also decreased the levels of liver microsomal sulfatase activity for hydrolysis of estrone-3-sulfate. TCDD did not significantly affect the hepatic enzyme activity for the glucuronidation or esterification of endogenous estrogens. It is suggested that enhanced metabolic inactivation of endogenous estrogens by hepatic estrogen-metabolizing enzymes in TCDD-treated, control diet-fed animals contributes importantly to the reduced incidence of estrogen-associated tumors in animals treated with TCDD.

Endocrine-disrupting chemicals (EDC) are linked to human health and diseases as they mimic or block the normal functioning of endogenous hormones. The present work dealt with a comparative study of the androgenic potential of wastewater treatment plant (WWTP) influents and effluents in Northern region of India, well known for its polluted water. Water samples were screened for their androgenic potential using the Hershberger assay and when they were found positive for androgenicity, we studied their mode of action in intact rats. The data showed a significant change in the weight and structure of sex accessory tissues (SATs) of castrated and intact rats. Reverse transcriptase polymerase chain reaction (RT–PCR) analysis demonstrated a significant change in the expression patterns of the major steroidogenic enzymes in adrenal and testis: cytochrome P 450 SCC , cytochrome P 450 C17 , 3β-hydroxysteroid dehydrogenase, 17β-hydroxysteroid dehydrogenase. This was further supported by increased enzymatic activities measured in vitro spectrophotometrically. Serum hormone profile showed a decreased level of gonadotrophic hormones and increased testosterone level. Further, increase in the serum level of alkaline phosphatase, SGPT and SGOT and histopathological changes in kidney and liver of treated animals, confirmed the toxic effects of contaminating chemicals. Analysis of water samples using HPLC and GC–MS showed the presence of various compounds and from them, four prominent aromatic compounds viz. nonylphenol, hexachlorobenzene and two testosterone equivalents, were identified. Our data suggest that despite rigorous treatment, the final treated effluent from WWTP still has enough androgenic and toxic compounds to affect general health.

The effects on the hypothalamus–pituitary–testicular axis of the well-known antispermatogenic drug lonidamine (LND) has not been elucidated so far. In the present study, the possible changes of the testicular steroid hormones were evaluated in immature mice for a better characterization of the LND adverse effects both in its use as antitumoral agent and male contraceptive. Male CD1 mice were orally treated on postnatal day 28 (PND28) with LND single doses (0 or 100 mg/kg b.w.) and euthanized every 24 h from PND29 to PND32, on PND35 and on PND42 (1 and 2 weeks after the administration, respectively). Severe testicular effects were evidenced in the LND treated groups, including: a) significant testis weight increase, 24 h and 48 h after dosing; b) sperm head counts decrease (more than 50% of the control) on PND29–32; c) damage of the tubule morphology primarily on the Sertoli cell structure and germ cell exfoliation. All these reproductive endpoints were recovered on PND42. At the same time, a significant impairment of the testicular steroid balance was observed in the treated mice, as evidenced by the decrease of testosterone (T) and androstenedione (ADIONE) and the increase of 17OH-progesterone (17OH-P4) on the first days after dosing, while the testicular content of 17β-estradiol (E2) was unchanged. The hormonal balance was not completely restored afterwards, as levels of T, ADIONE and 17OH-P4 tended to be higher in the treated mice than in the controls, on PND35 and PND42. These data showed for the first time that LND affects intratesticular steroids in experimental animals. However further data are needed both to elucidate the mechanism responsible for the impairment of these metabolic pathways and to understand if the androgens decrease observed after LND administration could be partially involved in the testicular damage.

Studies showed that early postnatal exposure to the herbicide atrazine (ATR) delayed preputial separation (PPS) and increased incidence of prostate inflammation in adult Wistar rats. A cross-fostering paradigm was used in this study to determine if gestational exposure to ATR would also result in altered puberty and reproductive tissue effects in the male rat. Timed-pregnant Long-Evans (LE) rats were dosed by gavage on gestational days (GD) 15-19 with 100 mg ATR/kg body weight (BW) or 1% methylcellulose (controls, C). On postnatal day (PND)1, half litters were cross-fostered, creating 4 treatment groups; C-C, ATR-C, C-ATR, and ATR-ATR (transplacental-milk as source, respectively). On PND4, male offspring in the ATR-ATR group weighed significantly less than the C-C males. ATR-ATR male pups had significantly delayed preputial separation (PPS). BWs at PPS for C-ATR and ATR-ATR males were reduced by 6% and 9%, respectively, from that of C-C. On PND120, lateral prostate weights of males in the ATR-ATR group were significantly increased over C-C. Histological examination of lateral and ventral prostates identified an increased distribution of inflammation in the lateral prostates of C-ATR males. By PND220, lateral prostate weights were significantly increased for ATR-C and ATR-ATR, but there were no significant changes in inflammation in either the lateral or ventral prostate. These results suggest that in LE rats, gestational ATR exposure delays PPS when male offspring suckle an ATR dam, but leads to increased lateral prostate weight via transplacental exposure alone. Inflammation present at PND120 does not increase in severity with time.

Oxidative stress has been suggested to be a major cause of male reproductive failure. Here, we investigated whether arsenic, which impairs male reproductive functions in rodent models, acts by inducing oxidative stress. Male 8-week-old ICR mice were given drinking water containing 20 or 40 mg/l sodium arsenite with or without 0.75 or 1.5 g/l of the antioxidant ascorbic acid for 5 weeks. The arsenic-treated mice showed decreased epididymidal sperm counts and testicular weights compared to untreated mice. These effects were reversed in mice that were co-treated with ascorbic acid. Similarly, arsenic treatment lowered the activities of testicular 3beta-hydroxysteroid dehydrogenase (HSD) and 17beta-HSD, which play important roles in steroidogenesis, and this was reversed by co-treatment with ascorbic acid. The testicles of arsenic-treated mice had decreased glutathione (GSH) levels (which correlate inversely with the degree of cellular oxidative stress) and elevated levels of protein carbonyl (a marker of oxidative damage to tissue proteins). Ascorbic acid co-treatment reversed both of these effects. Thus, ascorbic acid blocks both the adverse effects of arsenic on male reproductive functions and the arsenic-induced testicular oxidative changes. These observations support the notion that arsenic impairs male reproductive function by inducing oxidative stress.

Early life exposure to endocrine disrupting compounds has been linked to chronic diseases later in life, like obesity and related metabolic disorders. We exposed C57BL/6JxFVB hybrid mice to the aryl hydrocarbon receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the constitutive androstane receptor/pregnane X receptor agonist polychlorinated biphenyl 153 (PCB 153) in an experimental design relevant for human exposure. Exposure occurred during gestation and lactation via maternal feed to a wide dose range (TCDD: 10-10,000 pg/kg body weight/day; PCB 153: 0.09-1406 μg/kg body weight/d). Then exposure was ceased and offspring were followed up to 1 year of age. Metabolic parameters like body weight, fat pad weights, glucose tolerance, endocrine serum profile, and neurobehavioral and immunological parameters were determined. Body weight was transiently affected by both compounds throughout the follow-up. TCDD-exposed males showed decreased fat pad and spleen weights and an increase in IL-4 production of splenic immune cells. In contrast, females showed increased fat pad weights and production of IFNγ. PCB 153-exposed males showed an increase in glucose, whereas females showed an increase in glucagon, a decrease in pancreas weight, and an increase in thymus weight. In conclusion, early life exposure to TCDD appears to affect programming of energy and immune homeostasis in offspring, whereas the effects of perinatal PCB 153 were mainly on programming of glucose homeostasis. Both compounds act sex-specifically. Lowest derived BMDLs (lower bounds of the (two sided) 90%-confidence interval for the benchmark dose) for both compounds are not lower than current tolerable daily intakes.

Ancestral TCDD exposure could induce epigenetic transgenerational phenotypes, which may be mediated in part by imprinted gene inheritance. The aim of our study was to evaluate the transgenerational effects of ancestral TCDD exposure on the imprinted gene insulin-like growth factor-2 (Igf2) in rat somatic tissue. TCDD was administered daily by oral gavage to groups of F0 pregnant SD rats at dose levels of 0 (control), 200 or 800 ng/kg bw during gestation day 8-14. Animal transgenerational model of ancestral exposure to TCDD was carefully built, avoiding sibling inbreeding. Hepatic Igf2 expression of the TCDD male progeny was decreased concomitantly with hepatic damage and increased activities of serum hepatic enzymes both in the F1 and F3 generation. Imprinted Control Region (ICR) of Igf2 manifested a hypermethylated pattern, whereas methylation status in the Differentially Methylated Region 2 (DMR2) showed a hypomethylated manner in the F1 generation. These epigenetic alterations in these two regions maintained similar trends in the F3 generation. Meanwhile, the expressions of DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) changed in a non-monotonic manner both in the F1 and F3 generation. This study provides evidence that ancestral TCDD exposure may promote epigenetic transgenerational alterations of imprinted gene Igf2 in adult somatic tissue.

A 4-nitrophenol (PNP) isolated from diesel exhaust particles (DEP) has been identified as a vasodilator. PNP is also a known degradation product of the insecticide parathion. We used uterotrophic and Hershberger assays to study the estrogenic and anti-androgenic activities of PNP in-vivo. In ovariectomized immature female rats injected subcutaneously with 1, 10, or 100 mg/kg PNP daily for 7 days, significant (P

To understand the effect of thuringiensin on the lungs tissues, male Sprague-Dawley rats were administrated with thuringiensin by intratracheal instillation at doses 0.8, 1.6 and 3.2 mg/kg of body weight, respectively. The rats were sacrificed 4 h after treatment, and lungs were isolated and examined. Subsequently, an effective dose of 1.6 mg/kg was selected for the time course study (4, 8, 12, and 24 h). Intratracheal instillation of thuringiensin resulted in lung damage, as evidenced by increase in lung weight and decrease in alkaline phosphatase (10-54%), an enzyme localized primarily in pulmonary alveolar type II epithelial cells. Furthermore, the administration of thuringiensin caused increases in lipid peroxidation (21-105%), the indices of lung injury. In addition, the superoxide dismutase (SOD) and glutathione (GSH) activities of lung tissue extracts were measured to evaluate the effect of thuringiensin on antioxidant defense system. The SOD activity and GSH content in lung showed significant decreases in a dose-related manner with 11-21% and 15-37%, respectively. Those were further supported by the release of proinflammatory cytokines, as indicated by increases in IL-1beta (229-1017%) and TNF-alpha (234%) levels. Therefore, the results demonstrated that changes in the pulmonary oxidative-antioxidative status might play an important role in the thuringiensin-induced lung injury.

Chronic exposure to marijuana produces adverse effects on the endocrine and reproductive systems in humans; however, the experimental evidence for this presented thus far has not been without controversy. In this study, the estrogenic effect of marijuana smoke condensate (MSC) was evaluated using in vitro bioassays, viz., the cell proliferation assay, the reporter gene assay, and the ER competitive binding assay. The results of these assays were compared with those of three major cannabinoids, i.e., THC, CBD, and CBN. The estrogenic effect of MSC was further confirmed by the immature female rat uterotrophic assay. MSC stimulated the estrogenicity related to the ER-mediated pathway, while neither THC, CBD, nor CBN did. Moreover, treatment with 10 and 25 mg/kg MSC induced significant uterine response, and 10 mg/kg MSC resulted in an obvious change in the uterine epithelial cell appearance. MSC also enhanced the IGFBP-1 gene expression in a dose-dependent manner. To identify the constituents of MSC responsible for its estrogenicity, the MSC fractionated samples were examined using another cell proliferation assay, and the estrogenic active fraction was analyzed using GC-MS. In the organic acid fraction that showed the strongest estrogenic activity among the seven fractions of MSC, phenols were identified. Our results suggest that marijuana abuse is considered an endocrine-disrupting factor. Furthermore, these results suggest that the phenolic compounds contained in MSC play a role in its estrogenic effect.

Fenbuconazole, a triazole fungicide, has been associated with an increase in the incidence of liver adenomas in female mice following long-term dietary exposure. The aim of this study was to evaluate whether the mode of action for liver tumor formation by fenbuconazole is similar to that of phenobarbital. Treatment of CD1 mice with 0, 20, 60, 180 or 1300 ppm fenbuconazole for up to 4 weeks caused a dose-dependent increase in liver weight that was associated with centrilobular hepatocellular hypertrophy, cytoplasmic eosinophilia and panlobular hepatocellular vacuolation, as well as an initial increase in the cell proliferation labeling index. Fenbuconazole also caused a dose-dependent increase in liver microsomal cytochromes b 5 and P450 and the levels of immunoreactive CYP2B10 and its associated activity 7-pentoxyresorufin O -dealkylation (PROD). Treatment of mice with 1000 ppm phenobarbital elicited the same effects as treatment of mice with 1300 ppm fenbuconazole, except that phenobarbital was more effective than fenbuconazole at inducing PROD activity, even though fenbuconazole induced CYP2B10 to the same extent as did phenobarbital. This difference was attributed to the ability of fenbuconazole to bind tightly to CYP2B10 and partially mask its catalytic activity in liver microsomes, which is characteristic of several azole-containing drugs. All hepatocellular changes and induced enzyme activity returned to control levels within 4 weeks of discontinuing treatment with fenbuconazole or phenobarbital, indicating that the observed changes were fully reversible. We conclude that fenbuconazole is a phenobarbital-type inducer of mouse liver cytochrome P450, and the mode of action by which fenbuconazole induces liver adenomas in mice is similar to that of phenobarbital.

Indole-3-carbinol (I-3-C) and 3,3'-diindolylmethane (DIM) have been shown to reduce the incidence and multiplicity of cancers in laboratory animal models. Based on the observation that I-3-C induced hepatocyte hypertrophy when administered orally for 13 weeks to rats, a treatment and recovery study was undertaken to test the hypothesis that the induction of hepatocyte hypertrophy and cytochrome P450 (CYP) activity by I-3-C are adaptive, reversible responses. Additionally, we directly compared the effects of I-3-C to those of its principle metabolite DIM. Rats were treated orally for 28 days with 2 doses of I-3-C (5 and 50 mg I-3-C/kg body weight/day) and DIM (7.5 and 75 mg DIM/kg body weight/day) and then one-half of the animals were not treated for an additional 28 days. Organ weights, histopathology, and the CYP enzyme activities of 1A1/2, 2B1/2, 2C9, 2D6, 2E1, 3A4, and 19 A were measured both after treatment and after recovery. Oral administration of 50 mg I-3-C/kg body weight/day to rats for 28 days significantly increased liver weights and CYP enzyme activities. The effects in males were more pronounced and persistent after recovery than the effects in females. The increased organ weights returned to control values after treatment. Conversely, DIM did not alter liver weights and had no effect on CYP activities after the 28-day treatment. Some changes in CYP activities were measured after the DIM recovery period but the magnitudes of the changes were considered biologically insignificant. The results show that I-3-C, but not DIM, induces reversible adaptive responses in the liver.

In a 2-year NTP bioassay, Bromoethane (BE) was found to induce endometrial neoplasms in the uterus of B6C3F1 mice [; ]. In women, hormonal influences, such as "unopposed" estrogenic stimulus, have been implicated as important etiologic factors in uterine cancer. BE, however, does not affect the serum concentrations of sex hormones in female B6C3F1 mice [] and the mechanism of BE-induced uterine carcinogenesis still remains unclear. In the present study, we examined the estrogenic effects of BE on the uterus of ovariectomized B6C3F1 mice and on Ishikawa cells. Groups of 6 mice were given daily s.c. injections of 0, 100, 500 or 1000 mg BE/kg for 3 consecutive days. Mice treated with 17beta-estradiol served as positive controls. Mice were necropsied 24 h after the final injection, and uteri were weighed and examined histologically and immunohistochemically along with the vagina. Changes observed in the estrogen-treated mice included increased uterine weights, edema and inflammation of the endometrium, increased epithelial layers of the uterine and vaginal lumens and keratinization of the vaginal epithelium. In the BE-treated mice, no such changes occurred; however, immunohistochemical staining of the uterus revealed a significant increase in immunoexpression of the estrogen receptor alpha (ERalpha) in the two higher dose groups. Analysis of mRNA also showed slightly increased uterine ERalpha expression in these groups. Upregulated expression of ERalpha was confirmed in BE-treated Ishikawa cells, in which Western blotting analyses identified an intense signal at approximately 66 kDa, which is consistent with ERalpha. These data suggest that upregulated expression of ERalpha may be important in the induction of endometrial neoplasms in BE-treated mice.

The molecular pathology of cardiac hypertrophy is multifactorial with transcript regulation of ion channels, ion exchangers and Ca(2+)-handling proteins being speculative. We therefore investigated disease-associated changes in gene expression of various ion channels and their receptors as well as ion exchangers, cytoskeletal proteins and Ca(2+)-handling proteins in normotensive and spontaneously hypertensive (SHR) rats. We also compared experimental findings with results from hypertrophic human hearts, previously published (Borlak, J., and Thum, T., 2003. Hallmarks of ion channel gene expression in end-stage heart failure. FASEB J. 17, 1592-1608). We observed significant (P < 0.05) induction in transcript level of ATP-driven ion exchangers (Atp1A1, NCX-1, SERCA2a), ion channels (L-type Ca(2+)-channel, K(ir)3.4, Na(v)1.5) and RyR-2 in hypertrophic hearts, while gene expression was repressed in diseased human hearts. Further, the genes coding for calreticulin and calmodulin, PMCA 1 and 4 as well as alpha-skeletal actin were significantly (P < 0.05) changed in hypertrophic human heart, but were unchanged in hypertrophic left ventricles of the rat heart. Notably, transcript level of alpha- and beta-MHC, calsequestrin, K(ir)6.1 (in the right ventricle only), phospholamban as well as troponin T were repressed in both diseased human and rat hearts. Our study enabled an identification of disease-associated candidate genes. Their regulation is likely to be the result of an imbalance between pressure load/stretch force and vascular tonus and the observed changes may provide a rational for the rhythm disturbances observed in patients with cardiac hypertrophy.

Although benomyl and its metabolite carbendazim have been shown to adversely affect male reproduction, the mechanisms of action do not appear to involve the endocrine system. However, few studies have been conducted using currently proposed tests specifically focused on endocrine disruption. Here, potential estrogen- and androgen-mediated activity of benomyl was therefore investigated in vitro and in vivo. Benomyl and carbendazim proved negative for agonistic and antagonistic activity in reporter gene assays for the human estrogen receptor alpha and androgen receptor. In uterotrophic and Hershberger assays using Crj:CD(SD)IGS rats, benomyl (100, 300 or 1000 mg/kg/day, p.o., N = 6) did not exert agonistic effects. However, the highest dose decreased uterine weights in the uterotrophic assay, and decreased weights of some androgen-related tissues of castrated rats receiving a testosterone propionate (TP, 0.2 mg/kg) injection in the Hershberger assay; the effects were less severe than those with p,p'-DDE (100 mg/kg/day). When 4 mg/kg/day of TP was injected, decrease of organ weights due to benomyl was attenuated but still observed. Thus, its influence in some tissues was more potent than that of p,p'-DDE. Benomyl had no apparent effects on serum androgen levels. Microarray analysis of the gene expression profile in the ventral prostate of TP-injected castrated rats treated with benomyl indicated clear differences from the patterns observed with p,p'-DDE and flutamide. Taken together, these findings suggest the decreased organ weights observed in vivo to be caused by mechanisms that are not steroid-receptor-mediated, such as interfering with assembly of microtubules by benomyl. The study furthermore suggests that functional genomics may provide a reliable evidence for accurate categorization of test chemicals.