Effects of perinatal exposures to a TAML catalyst on the mammary gland and hormone-sensitive outcomes in male mice.

Estrogenic chemicals are common pollutants in wastewater and current effluent treatment processes are not typically effective in removing these compounds. Tetra-amido macrocyclic ligands (TAMLs) are catalysts that mimic endogenous peroxidases that may provide a solution to remove environmental pollutants including low concentrations of estrogenic compounds. Yet relatively little is known about the toxicity of TAMLs, and few studies have evaluated whether they may have endocrine disrupting properties. We administered one of three doses of a TAML, NT7, to mice via drinking water throughout pregnancy and lactation. Two pharmacologically active compounds, ethinyl estradiol (EE2) and flutamide were also included to give comparator data for estrogen receptor agonist and androgen receptor antagonist activities. Male pups were evaluated for several outcomes at weaning, puberty, and early adulthood. We found that EE2 exposures during gestation and the perinatal period induced numerous effects that were observed across the three ages including changes to spleen and testis weight and drastic effects on the morphology of the mammary gland. Flutamide had fewer effects but altered anogenital distance at weaning as well as spleen, liver, and kidney weight. In contrast, relatively few effects of NT7 were observed, but included alterations to spleen weight and modest changes to adult testis weight and morphology of the mammary gland at weaning. Collectively, these results provide some of the first evidence suggesting that NT7 may alter some hormone-sensitive outcomes, but that the effects were distinct from either EE2 or flutamide. Additional studies are needed to characterize the biological activity of this and other TAML catalysts.

Norethindrone suppress the germ cell development via androgen receptor resulting in male bias.

Progestins are widely used and detected in surface waters, and can affect gonad development and sexual differentiation in fish. However, the toxicological mechanisms of sexual differentiation induced by progestins are not well understood. Here, we investigated the effects of norethindrone (NET) and androgen receptor (AR) antagonist flutamide (FLU) on gonadal differentiation in zebrafish from 21 dpf (days post-fertilization) to 49 dpf. The results showed that NET caused male bias, while FLU resulted in female bias at 49 dpf. The NET and FLU mixtures significantly decreased the percentage of males compared to the NET single exposure. Molecular docking analysis showed that FLU and NET had similar docking pocket and docking posture with AR resulting in competitively forming the hydrogen bond with Thr334 of AR. These results suggested that binding to AR was the molecular initiating event of sex differentiation induced by NET. Moreover, NET strongly decreased transcription of biomarker genes (dnd1, ddx4, dazl, piwil1 and nanos1) involved in germ cell development, while FLU significantly increased transcription of these target genes. There was an increase in the number of juvenile oocytes, which was consistent with the female bias in the combined groups. The bliss independence model analysis further showed that NET and FLU had antagonistic effect on transcription and histology during gonadal differentiation. Thus, NET suppressed the germ cell development via AR, resulting in male bias. Understanding the molecular initiation of sex differentiation in progestins is essential to provide a comprehensive biological basis for ecological risk assessment.

Mechanism of non-steroidal anti-androgen-induced liver injury: Reactive metabolites of flutamide and bicalutamide activate inflammasomes.

Flutamide is a non-steroidal anti-androgen agent, which is mainly used for the treatment of prostate cancer. Flutamide is known to cause severe adverse events, which includes idiosyncratic liver injury. However, details of the mechanism of these adverse reactions have not been elucidated. We investigated whether flutamide induces the release of damage-associated molecular patterns (DAMPs) that activate inflammasomes. We also tested bicalutamide, enzalutamide, apalutamide, and darolutamide for their ability to activate inflammasomes in differentiated THP-1 cells. The supernatant from the incubation of flutamide and bicalutamide with human hepatocarcinoma functional liver cell-4 (FLC-4) cells increased caspase-1 activity and production of IL-1ß by differentiated THP-1 cells. In the supernatant of FLC-4 cells with flutamide and bicalutamide, the heat shock protein (HSP) 40 or 60 was significantly increased. Addition of a carboxylesterase or a CYP inhibitor to the FLC-4 cells prevented release of HSPs from the FLC-4 cells. These results suggested that the reactive metabolites of flutamide and bicalutamide can cause the release of DAMPs from hepatocytes and activate inflammasomes. Inflammasome activation may be an important step in the activation of the immune system by flutamide or bicalutamide, which in some patients, can cause immune-related adverse events.

Efficient in vivo and in silico assessments of antiandrogenic potential in zebrafish.

This study aimed to establish zebrafish-based in vivo and in silico assay systems to evaluate the antiandrogenic potential of environmental chemicals. Zebrafish embryos were exposed to 17α-methyltestosterone (TES) alone or coexposed to TES and representative antiandrogens including flutamide, p,p'-DDE, vinclozolin, fenitrothion, and linuron. We assessed the transcript expression of the androgen-responsive gene sulfotransferase family 2, cytosolic sulfotransferase 3 (sult2st3). The expression of sult2st3 was significantly induced by TES in the later stages of embryonic development. However, the TES-induced expression of sult2st3 was inhibited by flutamide in a concentration-dependent manner (IC50: 5.7 µM), suggesting that the androgen receptor (AR) plays a role in sult2st3 induction. Similarly, p,p'-DDE, vinclozolin, and linuron repressed the TES-induced expression of sult2st3 (IC50s: 0.35, 3.9, and 52 µM, respectively). At the highest concentration tested (100 µM), fenitrothion also suppressed sult2st3 expression almost completely. Notably, p,p'-DDE and linuron did not inhibit sult2st3 induction due to higher concentrations of TES; instead, they potentiated TES-induced sult2st3 expression. Fenitrothion and linuron, which had relatively low antiandrogenic potentials in terms of sult2st3 inhibition, induced broader toxicities in zebrafish embryos; thus, the relationship between developmental toxicities and antiandrogenic potency was unclear. Additionally, an in silico docking simulation showed that all five chemicals interact with the zebrafish AR at relatively low interaction energies and with Arg702 as a key amino acid in ligand binding. Our findings suggest that a combination of zebrafish-based in vivo and in silico assessments represents a promising tool to assess the antiandrogenic potentials of environmental chemicals.

QbD Approach towards Robust Design Space for Flutamide/PiperineSelf-Emulsifying Drug Delivery System with Reduced Liver Injury.

Flutamide which is used to treat prostate cancer and other diseases induces liver damage during and after the therapy. The aim of this study was to develop a flutamide/piperineco-loaded self-emulsifying drug delivery system (FPSEDDS) to inhibit flutamide-induced liver injury by utilizing piperine as a metabolic inhibitor. The development of SEDDS was carried out following a quality by design (QbD) approach. The risk assessment study was performed to identify critical quality attributes (CQAs) and critical material attributes (CMAs)/critical process parameters (CPPs). I-optimal mixture design was executed with three CMAs as the independent variables and CQAs as the dependable variables. The effectiveness of optimized SEDDS to circumvent flutamide-induced hepatotoxicity was assessed in mice. The numerical optimization suggested an optimal formulation with a desirability value of 0.621, using CQAs targets as optimization goals with 95% prediction intervals (α = 0.05). The optimal formulation exhibited the grade A SEDDS characteristics with the guarantee of high payloads in self-formed oily droplets. The design space was also obtained from the same optimization goals. All CQA responses of verification points were found within the 95% prediction intervals of the polynomial models, indicating a good agreement between actual versus predicted responses within the design space. These obtained responses also passed CQAs acceptance criteria. Finally, hematoxylin-eosin staining revealed the minimal flutamide-induced hepatotoxicity from the optimal SEDDS formulation as compared to the control and flutamide/piperine normal suspension. We demonstrate that the piperine containing optimized SEDDS formulation developed by QbD significantly reduces the flutamide-induced liver injury in mice.

Potentiation of flutamide-induced hepatotoxicity in mice by Xian-Ling-Gu-Bao through induction of CYP1A2.

ETHNOPHARMACOLOGICAL RELEVANCE: Xian-Ling-Gu-Bao (XLGB) Fufang is herbal formula widely used to treat osteoporosis and other bone disorders. Because of its commonality in the clinical use, there is a safety concern over the use of XLGB combined with other androgen deprivation therapy (ADT) drugs such as flutamide (FLU) that is associated with reduced bone density. To date, there have been no evaluations on the side effects of the drug-drug interaction between XLGB and FLU. AIM OF THE STUDY: The present study was designed to investigate the hepatotoxicity in the context of the combined treatment of XLGB and FLU in a mouse model, and to determine whether the metabolic activation of FLU through induction of CYP1A2 plays a role in the increased hepatoxicity caused by the combination of XLGB and FLU. MATERIALS AND METHODS: C57 mice were administered with either XLGB (6,160 mg/kg), FLU (300 mg/kg), or with the combination of the two drugs. Animals were treated with XLGB for 5 days before the combined administration of XLGB and FLU for another 4 days. The serum of mice from single or the combined administration groups was collected for biochemical analysis. The mouse liver was collected to examine liver morphological changes, evaluate liver coefficient, as well as determine the mRNA expression of P450 isozymes (Cyp1a2, Cyp3a11 and Cyp2c37). For metabolism analysis, mice were treated with XLGB, FLU, or the combination of XLGB and FLU for 24 h. The urine samples were collected for the analysis of FLU-NAC conjugate by UPLC-Q-Orbitrap MS. The liver microsomes were prepared from fresh livers to determine the activity of metabolizing enzyme CYP1A2. RESULTS: The combined treatment of XLGB and FLU caused loss of mice body weight and elicited significant liver toxicity as evidenced by an increased liver coefficient and serum lactate dehydrogenase (LDH) activity as well as pathological changes of fatty lesion of liver tissue. FLU increased hepatic expression of Cyp1a2 mRNA that was further elevated in the liver of mice when administered with both FLU and XLGB. Treatment of FLU resulted in an increase in the expression of Cyp3a11 mRNA that was negated when mice were co-treated with FLU and XLGB. No significant difference in Cyp2c37 mRNA expression was observed among the different treatment groups as compared to the control. Analysis of metabolic activity showed that the combined administration caused a synergic effect in elevating the activity of the CYP1A2 enzyme. Mass spectrometry analysis identified the presence of FLU reactive metabolite derived FLU-NAC conjugate in the urine of mice treated with FLU. Strikingly, about a two-fold increase of the FLU-NAC conjugate was detected when treated with both FLU and XLGB, indicating an elevated amount of toxic metabolite produced from FLU in the present of XLGB. CONCLUSION: FLU and XLGB co-treatment potentiated FLU-induced hepatoxicity. This increased hepatoxicity was mediated through the induction of CYP1A2 activity which in turn enhanced bioactivation of FLU leading to over production of FLU-NAC conjugate and oxidative stress. These results offer warnings about serious side effects of the FLU-XLGB interaction in the clinical practice.

Flutamide induces uterus and ovary damage in the mouse via apoptosis and excessive autophagy of cells following triggering of the unfolded protein response.

Intrauterine exposure to flutamide not only causes abnormal development of the reproductive organs in male offspring, but also damages ovaries and uteri. The unfolded protein response (UPR) is believed to play an important role in embryo development and teratogenic processes. In the present study, pregnant mice were administered either flutamide (300mg kg-1 day-1, p.o.) on an equivalent volume of soybean oil (control) on Days 12-18 of gestation. Eight weeks after birth, female offspring in the flutamide-treated group had a lower bodyweight and lower ovarian and uterine weights, but there was no significant difference in uterine and ovarian weights normalised by bodyweight between the flutamide-treated and control groups. Furthermore, histopathological changes were observed in all uteri and ovaries in the flutamide-treated group, with fewer and less-developed follicles in the ovaries. In both the uteri and ovaries, flutamide increased the expression of UPR members, although the expression of cell cycle-related genes remained unchanged compared with the control group. Flutamide increased the expression of all autophagy- and apoptosis-related genes evaluated in the uterus, as well as some in the ovary. The results suggest that the in utero exposure of mice to flutamide may contribute to uterine and ovarian damage in the offspring, with endoplasmic reticulum stress possibly triggered by the UPR leading to the induction of excessive autophagy and apoptosis.

Assessment of the impact of mitochondrial genotype upon drug-induced mitochondrial dysfunction in platelets derived from healthy volunteers.

Mitochondrial DNA (mtDNA) is highly polymorphic and encodes 13 proteins which are critical to the production of ATP via oxidative phosphorylation. As mtDNA is maternally inherited and undergoes negligible recombination, acquired mutations have subdivided the human population into several discrete haplogroups. Mitochondrial haplogroup has been found to significantly alter mitochondrial function and impact susceptibility to adverse drug reactions. Despite these findings, there are currently limited models to assess the effect of mtDNA variation upon susceptibility to adverse drug reactions. Platelets offer a potential personalised model of this variation, as their anucleate nature offers a source of mtDNA without interference from the nuclear genome. This study, therefore, aimed to determine the effect of mtDNA variation upon mitochondrial function and drug-induced mitochondrial dysfunction in a platelet model. The mtDNA haplogroup of 383 healthy volunteers was determined using next-generation mtDNA sequencing (Illumina MiSeq). Subsequently, 30 of these volunteers from mitochondrial haplogroups H, J, T and U were recalled to donate fresh, whole blood from which platelets were isolated. Platelet mitochondrial function was tested at basal state and upon treatment with compounds associated with both mitochondrial dysfunction and adverse drug reactions, flutamide, 2-hydroxyflutamide and tolcapone (10-250 µM) using extracellular flux analysis. This study has demonstrated that freshly-isolated platelets are a practical, primary cell model, which is amenable to the study of drug-induced mitochondrial dysfunction. Specifically, platelets from donors of haplogroup J have been found to have increased susceptibility to the inhibition of complex I-driven respiration by 2-hydroxyflutamide. At a time when individual susceptibility to adverse drug reactions is not fully understood, this study provides evidence that inter-individual variation in mitochondrial genotype could be a factor in determining sensitivity to mitochondrial toxicants associated with costly adverse drug reactions.

Evaluation of covalent binding of flutamide and its risk assessment using 19F-NMR.

The formation of reactive metabolites (RMs) is a problem in drug development that sometimes results in severe hepatotoxicity. As detecting RMs themselves is difficult, a covalent binding assay using expensive radiolabelled tracers is usually performed for candidate selection. This study aimed to provide a practical approach toward the risk assessment of hepatotoxicity induced by covalent binding before candidate selection. We focused on flutamide because it contains a trifluoromethyl group that shows a strong singlet peak by 19F nuclear magnetic resonance (NMR) spectrometry. The covalent binding of flutamide was evaluated using quantitative NMR and its risk for hepatotoxicity was assessed by estimating the RM burden, an index that reflects the body burden associated with RM exposure by determining the extent of covalent binding, clinical dose and in vivo clearance. The extent of covalent binding and RM burden was 296 pmol/mg/h and 37.9 mg/day, respectively. Flutamide was categorised as high risk with an RM burden >10 mg/day consistent with its clinical hepatotoxicity. These results indicate that a combination of covalent binding assay using 19F-NMR and RM burden is useful for the risk assessment of RMs without using radiolabelled compounds.

Evaluation of dietary dose administration as an alternative to oral gavage in the rodent uterotrophic and Hershberger assays.

The rodent uterotrophic and Hershberger assays evaluate potential estrogenic and (anti)-androgenic effects, respectively. Both US EPA and OECD guidelines specify that test substance is administered daily either by subcutaneous injection or oral gavage. However, dietary administration is a relevant exposure route for agrochemical regulatory toxicology studies due to potential human intake via crop residues. In this study, equivalent doses of positive control chemicals administered via dietary and gavage routes of administration were compared in the uterotrophic (17α-ethinyl estradiol) and Hershberger (flutamide, linuron, dichloro-2,2-bis(4-chlorophenyl) ethane; 4,4'-DDE) assays in ovariectomized and castrated rats, respectively. For all positive control chemicals tested, statistically significant changes in organ weights and decreases in food consumption were observed by both routes of test substance administration. Decreased body weight gain observed for dietary linuron and 4,4'-DDE indicated that the maximum tolerated dose was exceeded. Hershberger dietary administration resulted in a similar blood exposure (AUC24) for each positive control chemical when compared to gavage. Overall, the correlation in organ weight changes for both the uterotrophic and Hershberger assays suggest that dietary administration is an acceptable route of exposure with similar sensitivity to oral gavage dosing for evaluation of the endocrine potential of a test substance and represents a more appropriate route of test substance administration for most environmental exposure scenarios.

Maternal exposure to mixtures of dienestrol, linuron and flutamide. Part II: Endocrine-related gene expression assessment on male offspring rat testes.

Exposure to endocrine-disrupting compounds (EDCs) during pregnancy and early development can lead to adverse developmental outcomes in offspring. One of the endpoints of concern is feminization. The present study aimed to investigate for any possible correlations with endocrine sensitive parameters in the testes of male rat offspring following dam exposure to three EDCs by assessing the expression of endocrine-related genes. Dienestrol (DIES) [0.37-6.25 µg/kg bw/day], linuron (LIN) [1.5-50 mg/kg bw/day], flutamide (FLU) [3.5-50 mg/kg bw/day] as well as their binary mixtures were administered to sexually mature female rats from gestation day (GD) 6 until postnatal day (PND) 21. Gene expression analysis of Star, Cyp11a1, Cyp17a1, Hsd3b2, Pgr and Insl3 was performed by RT-qPCR. Administration of the anti-androgen FLU alone significantly upregulated Cyp11a1 and Cyp17a1 gene expression while administration of LIN and DIES alone did not alter significantly gene expression. The effects of the binary mixtures on gene expression were not as marked as those seen after single compound administrations. Deregulation of Cyp17a1 in rat pup testis, following administration of FLU alone or in mixtures to dams, was significantly correlated with the observed feminization endpoints in male pups.

Role of unfolded protein response in genital malformation/damage of male mice induced by flutamide.

The unfolded protein response (UPR) is one of a switch of autophagy and apoptosis, and the endoplasmic reticulum stress (ERS) which inducing UPR plays a role in the malformations caused by some genetic and environmental factors. Exposure to flutamide during pregnancy will also cause abnormalities in some male offspring reproductive organs such as cryptorchidism. In this study, after administered the pregnant mouse orally at a dose of 300 mg/kg body weight every day during gestational day (GD)12 to GD18, flutamide can not only caused hypospadias in the male mouse offspring but also damaged the morphology and function of their testis. And the expression of UPR-related genes and proteins, autophagy, apoptosis, and angiogenesis-related genes of the damaged/teratogenic testis and penis in the mice were investigated to determine the role of UPR in this model. It was found that flutamide activated maybe the Atg7-Atg3-Lc3 pathway through the UPR pathway, caused cells excessive autophagy and apoptosis, and inhibited the formation of penile and testicular blood vessels by activating UPR and affecting the messenger RNA level of vascular endothelial growth factor and hypoxia-inducible factor 1.

Maternal exposure to mixtures of dienestrol, linuron and flutamide. Part I: Feminization effects on male rat offspring.

Exposure to endocrine-disrupting compounds (EDCs) during pregnancy can result in negative health effects in later generations, including sex changes and feminization. The present study assessed the feminization effects on male offspring rats of three EDCs: Dienestrol (DIES), Linuron (LIN), and Flutamide (FLU). Sexually mature female rats were exposed from gestation day (GD) 6 until postnatal day (PND) 21 to: 0.37, 0.75, 1.5, 3.12 or 6.25 µg/kg/day of DIES, 1.5, 3, 6, 12.5, 25 or 50 mg/kg/day of LIN, 3.5, 6.7, 12.5, 25 or 50 mg/kg/day of FLU, and the following mixtures: FLU + DIES (mg/kg/day+µg/kg/day), 3.5 + 0.37, or 3.5 + 3, 25 + 0.37, or 25 + 3; FLU + LIN (mg/kg/day + mg/kg/day), 3.5 + 12.5, or 25 + 12.5; and DIES + LIN (µg/kg/day + mg/kg/day), 0.37 + 12.5, or 3 + 12.5. Anogenital distance (AGD), nipple retention (NR) and cryptorchidism were evaluated. FLU produced a decrease of AGD, an increase of NR, and an increase of cryptorchidism at the highest dose. None of these three endpoints were significantly affected by LIN or DIES treatments alone. Combinations of FLU + LIN and FLU + DIES increased NR, and decreased AGD, while DIES + LIN did not produce any effects in male pups. Results show that FLU is able to induce feminization in male pups, while binary combinations of LIN and DIES did not modify the effects produced by FLU.

Retardation of Preputial Wound Healing in Rats with Hypospadias Induced by Flutamide.

Objective: The aim of this study was to identify a theoretical support for the prevention of urethral fistula following hypospadias repair, by comparing the preputial wound healing process in Sprague-Dawley (SD) rats with and without hypospadias induced by flutamide. Methods: Fifteen pregnant SD rats were randomly divided into three groups. These rats in one group received the androgen receptor antagonist flutamide (25 mg/kg/day) from gestation days 11-17, to establish a rat model of hypospadias for further study of the molecular mechanisms of the hypospadias etiology. The pregnant rats in the control groups were not administered flutamide. The pups from the control and experiment groups underwent an incision on the dorsal prepuce on postnatal day 25 and were sacrificed on postoperative days 3, 7, and 14 to collect penis samples. The penis morphology was examined in all groups. Subsequently, transforming growth factor ß1 (TGF-ß1), α-smooth muscle actin (α-SMactin), and signal transducers and activators of the transcription 3 (STAT3) expression levels in the different groups were measured at the indicated time points postoperatively using qRT-PCR and Western blot. Results: There was less regeneration of the subcutaneous tissue in hypospadias rats than in the sham-operated group (P < 0.05) on postoperative day 3. No differences were found in the regeneration of the subcutaneous tissue between these groups on postoperative days 7 or 14. Additionally, there were no differences in the epithelial cell regeneration between the control and the hypospadias groups at any postoperative timepoint. Moreover, the expression levels of TGF-ß1, α-SMactin, and STAT3 were all significantly lower in hypospadias group than that in the sham-operated group (P < 0.05). Conclusion: The results from the present work suggest that preputial wound healing is retarded in rats with hypospadias induced by flutamide and that this retardation might result from multi-gene regulation.

Selenium and Glutathione-Depleted Rats as a Sensitive Animal Model to Predict Drug-Induced Liver Injury in Humans.

Drug-induced liver injury (DILI) is one of the most serious and frequent drug-related adverse events in humans. Selenium (Se) and glutathione (GSH) have a crucial role for the hepatoprotective effect against reactive metabolites or oxidative damage leading to DILI. The hepatoprotective capacity related to Se and GSH in rodents is considered to be superior compared to the capacity in humans. Therefore, we hypothesize that Se/GSH-depleted rats could be a sensitive animal model to predict DILI in humans. In this study, Se-deficiency is induced by feeding a Se-deficient diet and GSH-deficiency is induced by l-buthionine-S,R-sulfoxinine treatment via drinking water. The usefulness of this animal model is validated using flutamide, which is known to cause DILI in humans but not in intact rats. In the Se/GSH-depleted rats from the present study, decreases in glutathione peroxidase-1 protein expression and GSH levels and an increase in malondialdehyde levels in the liver are observed without any increase in plasma liver function parameters. Five-day repeated dosing of flutamide at 150 mg/kg causes hepatotoxicity in the Se/GSH-depleted rats but not in normal rats. In conclusion, Se/GSH-depleted rats are the most sensitive for detecting flutamide-induced hepatotoxicity in all the reported animal models.

[Effect of flutamide exposed during pregnancy on reproductive organs development and oxidative stress responsein in male mice offspring].

OBJECTIVE: To investigate the effects of flutamide on the reproductive organs development and oxidative stress response of male offspring after exposure to pregnant mice, and to provide further evidence for the application of the model and mechanism research. METHODS: Choose 7-week-old ICR mice, female mice were randomly divided into experimental group and control group after pregnancy, thenflutamide was orally administered to pregnant mice with dose 300 mg/( kg·d) during gestation days 12-18, while the control group was treated with equal volume of soybean oil. More than 7 weeks after birth, 40 male mice were randomly chosen to observe the urethral development and test the anogenital distance( AGD), testicular volume and penile length, and then some of these mice's reproductive organs were took for histopathological examination. After grind and centrifuge to obtain the supernatant, twelve penis and testes were randomly selected for examining the level of malondialdehyde( MDA), superoxide dismutase( SOD), and glutathioneperoxidase( GSH-Px) use corresponding kit and microplate reader. The abundance of oxidative stress related genes Noxo1, Sod1 and Gpx1 were detected by qPCR. RESULTS: The incidence of hypospadias in male mice in the treatment group was 100%( 91/91). AGD in the treatment group was( 10. 22 ± 1. 53) mm, which was significantly lower than the control( 17. 46 ± 1. 25) mm( P < 0. 05). The volume of testicles in the treatment group was( 166. 34 ± 26. 59) vs. ( 178. 25 ± 25. 77) mm~3 in the control group( P < 0. 05). The length of the penis in the treatment group was( 7. 46 ± 0. 76) vs. ( 12. 60 ± 0. 80) mm in the control group( P <0. 05). In the testicles, the level of SOD was( 171. 08 ± 57. 24) vs. ( 102. 79 ± 15. 31) U/mg pro, MDA was( 4. 45 ± 1. 67) vs. ( 2. 93 ± 1. 00) nmol/mg pro, GSH-Px was( 41. 55± 12. 15) vs. ( 78. 27 ± 7. 60) U/mg pro( P < 0. 05). The abundance of Noxo1 in the testis from the treatment group was higher than that in the control group, while the expressions of Sod1 and Gpx1 in testis from the treatment group were lower than those in the control group( P < 0. 05). There is no significant difference in SOD, MDA, GSH-Px levels and Sod1, Noxo1, and Gpx1 expressions were found in the penises of both groups. CONCLUSION: Prenatal exposure of flutamide may cause developmental abnormalities such as hypospadias and shortening of the penis in male offspring, and abnormal testicular function of oxidation and anti-oxidation. However, there is no significant effect on penis oxidative/anti-oxidant function.

The effects of flutamide on cell-cell junctions in the testis, epididymis, and prostate.

In this review, we summarize recent findings on the effect of the anti-androgen flutamide on cell-cell junctions in the male reproductive system. We outline developmental aspects of flutamide action on the testis, epididymis, and prostate, and describe changes in junction protein expression and organization of junctional complexes in the adult boar following prenatal and postnatal exposure. We also discuss findings on the mechanisms by which flutamide induces alterations in cell-cell junctions in reproductive tissues of adult males, with special emphasis on cytoplasmic effects. Based on the results from in vivo and in vitro studies in the rat, we propose that flutamide affects the expression of junction proteins and junction complex structure not only by inhibiting androgen receptor activity, but equally important by modulating protein kinase-dependent signaling in testicular cells. Additionally, results from studies on prostate cancer cell lines point to a role for the cellular molecular outfit in response to flutamide.

Testicular developmental impairment caused by flutamide-induced and DEHP-induced cryptorchid rat models is mediated by excessive apoptosis and deficient autophagy.

BACKGROUND: Cryptorchidism is a common condition of childhood, and it is known to impair fertility potential. However, the underlying mechanisms remain unclear. METHODS: This study constructed two cryptorchid rat models to investigate the roles of apoptosis and autophagy in testicular impairment induced by cryptorchidism. Pregnant rats were randomly divided into three groups. Group I: non-treated rats were used as controls. Group II: injected with drug Flutamide (Flu) 25 mg/kg/bw/d from gestation day (GD) 11-19. Group III: daily intragastric administration of 750 mg/kg/bw/d di-2-ethylhexylphosphate (DEHP) from GD 7-19. The cubs were feed normally and the testes were excised on postnatal day (PND) 30. RESULTS: Our results demonstrated cryptorchidism models induced noticeable decreased fertility, significantly reduced sperm count, increased sperm abnormality rate, decreased testosterone and severe testicular damage in histomorphology. Intriguingly, the level of apoptosis marker FAS, Cytochrome C and caspase-3 increased in Flu-induced and DEHP-induced groups. DEHP-induced treatment simultaneously increased the number of autophagosomes and the levels of autophagy marker LC3-II and p62. Significant decrease of autophagy gene (LC3-II and p62) expression is found in Flu-induced rats testes. CONCLUSION: Taken together, deficient autophagy is involved in testicular spermatogenesis damage of cryptorchidism rats. And this autophagy defect is caused by deficient degradation.

Investigations on the dose-response relationship of combined exposure to low doses of three anti-androgens in Wistar rats.

The current investigation examines whether combined exposure to three anti-androgens (flutamide, prochloraz, vinclozolin) result in interference with endocrine homeostasis when applied at very low dose levels, and whether the results of combined exposure are more pronounced than to the individual compounds. A pre-post-natal in vivo study design was chosen with more parameters than regulatory testing protocols require (additional endpoints addressing hormone levels, morphology and histopathological examinations). Dose levels were chosen to represent the lowest observed adverse effect level (LOAEL), the no observed adverse effect level (NOAEL), and the acceptable daily intake for each individual substance. Anti-androgenic changes were observable at the effect level (LOAEL) but not at lower exposures. Nipple/areola counts appeared to be a sensitive measure of effect, in addition to male sex organ weights at sexual maturation, and finally gross findings. The results indicate the absence of evidence for effects at low or very low dose levels. No (adverse) effects were seen at the NOAEL dose. A non-monotonic dose-response relationship was not evident. Combined exposure at LOAEL level resulted in enhanced responses for anogenital index, number of areolas/nipples, delayed preputial separation and reduced ventral prostate weight in comparison to the individual compounds.

[Hypospadias : Insights and challenges]. / Hypospadie : Erkenntnisse und Herausforderungen.

Disorders of the ventral tubularization of the urethra, such as the hypospadias, are among the second most frequent congenital childhood malformations. An increasing incidence has been observed suggesting a doubling in the US, which could not be documented for the European area. The underlying causes of this congenital defect remain unidentified. Genetic risk constellations or environmental influences, in particular by so-called endocrine disrupting chemicals (EDCs), are discussed as triggering factors. Boys after in vitro fertilization are more likely to have hypospadias than in nonreproductive-assisted pregnancies. Animal models (especially mice) elicited causal relationships between prenatal hormonal exposure (estrogens, progesterone) and antiandrogens such as flutamide, finasteride, antiandrogenic fungicides (vinclozolin) and phthalates and the formation of hypospadias. An aesthetic and/or functional deficit are indications for surgical correction. The indications and the complications of hypospadias surgery must be in detail and realistically discussed with the parents and patients. Recent publications demonstrated that the risk of complications increases with the increase of the follow-up time. High-volume centers with extensive experience have a positive effect on the complication rate. Competent follow-up to adult age should be ensured.