Toxicological and chemical investigation of untreated municipal wastewater: Fraction- and species-specific toxicity.

Absence of a municipal wastewater (WW) treatment plant results in the untreated WW discharge into the recipient. The present study investigated toxic effects and chemical composition of water extracts and fractions from untreated WW and recipient Danube River (DR). Samples were prepared by solid-phase extraction and silica gel fractionation and screened for EROD activity and cytotoxicity using aquatic models, comprising of fish liver cells (PLHC-1) and a model of the early development of zebrafish embryos, while rat (H4IIE) and human (HepG2) hepatoma cells served as mammalian models. Polar fraction caused cytotoxicity and increased the EROD activity in PLHC-1 cells, and increased mortality and developmental abnormalities in developing zebrafish embryos. In H4IIE, polar fraction induced inhibition of cell growth and increased EROD activity, whereas HepG2 exerted low or no response to the exposure. Non-polar and medium-polar fractions were ineffective. Tentative identification by GC/MS showed that WW is characterized by the hydrocarbons, alkylphenols, plasticizers, and a certain number of benzene derivatives and organic acids. In DR, smaller number of organic compounds was identified and toxicity was less pronounced than in WW treatments. The present study revealed the potent toxic effect of polar fraction of untreated WW, with biological responses varying in sensitivity across organisms. Obtained results confirmed that fraction- and species-specific toxicity should be considered when assessing health risk of environmental pollution.

Transcriptional profiles of glutathione-S-Transferase isoforms, Cyp, and AOE genes in atrazine-exposed zebrafish embryos.

Glutathione-S-transferase (GST) superfamily consists of multiple members involved in xenobiotic metabolism. Expressional pattern of the GST isoforms in adult fish has been used as a biomarker of exposure to environmental chemicals. However, GST transcriptional responses vary across organs, thus requiring a cross-tissue examination of multiple mRNAs for GST profiling in an animal after chemical exposure. Zebrafish embryos express all GST isoforms as adult fish and could therefore represent an alternative model for identification of biomarkers of exposure. To evaluate such a possibility, we studied a set of cytosolic and microsomal GST isoform-specific expression profiles in the zebrafish embryos after exposure to atrazine, a widely used herbicide. Expression of the GST isoforms was compared with that of CYP genes involved in the phase I of xenobiotic metabolism and antioxidant enzyme (AOE) genes. Using quantitative real-time PCR, we showed dynamic changes in the expressional pattern of twenty GST isoforms, cyp1a, cyp3a65, ahr2, and four AOEs in early development of zebrafish. Acute (48 and 72 h) exposure of 24 h-old embryos to atrazine, from environmentally relevant (0.005 mg/L) to high (40 mg/L) concentrations, caused a variety of transient, albeit minor changes (<2.5-fold) in the GST isoforms, ahr2 and AOE genes response. However, expression of cyp1a and cyp3a65 mRNA was markedly and consistently induced by high doses of atrazine (5 and 40 mg/L). In summary, an analysis of the response of multiple systems in the zebrafish embryos provided a comprehensive understanding of atrazine toxicity and its potential impact on biological processes.

HBCDD-induced sustained reduction in mitochondrial membrane potential, ATP and steroidogenesis in peripubertal rat Leydig cells.

Hexabromocyclododecane (HBCDD), a brominated flame retardant added to various consumer products, is a ubiquitous environmental contaminant. We have previously shown that 6-hour exposure to HBCDD disturbs basal and human chorionic gonadotropin (hCG)-induced steroidogenesis in rat Leydig cells. Reduction in mitochondrial membrane potential (ΔΨm) and cAMP production was also observed. Here, we further expanded research on the effect of HBCDD on Leydig cells by using a prolonged exposure scenario. Cells were incubated in the presence of HBCDD during 24h and then treated with HBCDD+hCG for additional 2h. Results showed that HBCDD caused a sustained reduction in ATP level after 24h of exposure, which persisted after additional 2-hour treatment with HBCDD+hCG. cAMP and androgen accumulations measured after 2h of HBCDD+hCG treatment were also inhibited. Real-time PCR analysis showed significant inhibition in the expression of genes for steroidogenic enzymes, luteinizing hormone receptor, regulatory and transport proteins, and several transcription factors under both treatment conditions. Western blot analysis revealed a decreased level of 30kDa steroidogenic acute regulatory protein (StAR) after HBCDD+hCG treatment. In addition, HBCDD decreased the conversion of 22-OH cholesterol to pregnenolone and androstenedione to testosterone, indicating loss of the activity of cytochrome P450C11A1 (CYP11A1) and 17ß-hydroxysteroid dehydrogenase (HSD17ß). Cell survival was not affected, as confirmed by cytotoxicity and trypan blue tests or DNA fragmentation analysis. In summary, our data showed that HBCDD inhibits ATP supply, most likely through a decrease in ΔΨm, and targets multiple sites in the steroidogenic pathway in Leydig cells.

Hexabromocyclododecane facilitates FSH activation of ERK1/2 and AKT through epidermal growth factor receptor in rat granulosa cells.

The toxicity of hexabromocyclododecane (HBCDD) has been extensively studied; however, the mechanism and the effects of HBCDD on female reproductive system have been less frequently reported. In this study, we exposed rat granulosa cells to HBCDD during in vitro follicle-stimulating hormone (FSH)-driven cell proliferation and differentiation. Here, we show that HBCDD affects the FSH-driven signal transduction and ovulatory competence of granulosa cells. We found that HBCDD over-activates the FSH-stimulated extracellular-regulated kinase 1/2 (ERK1/2) and protein kinase B (PKB, also known as AKT). Inactivation of the epidermal growth factor receptor (EGFR) kinase activity with AG1478 and the mitogen-regulated kinase activity with U0126 completely prevented ERK1/2 activation in the FSH-stimulated and HBCDD-exposed granulosa cells. Moreover, AG1478 restored the HBCDD-induced AKT activation to the level observed in the FSH-stimulated cells. Western blot shows that HBCDD potentiates FSH-stimulated EGFR phosphorylation in granulosa cells. Real-time PCR demonstrates that HBCDD decreases the FSH-induced luteinizing hormone receptor (Lhr) expression. Inadequate level of LHR in the HBCDD-exposed granulosa cells prevented human chorionic gonadotropin in stimulating expression of the ovulatory genes such as amphiregulin (Areg), epiregulin (Ereg), and progesterone receptor (Pgr). Addition of U0126 and AG1478 restored Lhr level in the FSH-stimulated and HBCDD-exposed granulosa cells. These results indicate a direct effect of HBCDD on EGFR activation, resulting in over-activation of ERK1/2 and AKT signal transduction pathways in the FSH-treated cells. Increased activity of the EGFR-ERK1/2 pathway above physiological level prevents sufficient acquisition of LHR in proliferating granulosa cells, thus compromising ovulation.

Involvement of ERK1/2 signaling pathway in atrazine action on FSH-stimulated LHR and CYP19A1 expression in rat granulosa cells.

Worldwide used herbicide atrazine is linked to reproductive dysfunction in females. In this study, we investigated the effects and the mechanism of atrazine action in the ovary using a primary culture of immature granulosa cells. In granulosa cells, follicle-stimulating hormone (FSH) activates both cyclic adenosine monophosphate (cAMP) and extracellular-regulated kinase 1/2 (ERK1/2) cascades, with cAMP pathway being more important for luteinizing hormone receptor (LHR) and aromatase (CYP19A1) mRNA expression. We report that 48h after atrazine exposure the FSH-stimulated LHR and CYP19A1 mRNA expression and estradiol synthesis were decreased, with LHR mRNA being more sensitive to atrazine than CYP19A1 mRNA. Inadequate acquisition of LHR in the FSH-stimulated and atrazine-exposed granulosa cells renders human chorionic gonadotropin (hCG) ineffective to stimulate amphiregulin (Areg), epiregulin (Ereg), and progesterone receptor (Pgr) mRNA expression, suggesting anti-ovulatory effect of atrazine. To dissect the signaling cascade involved in atrazine action in granulosa cells, we used U0126, a pharmacological inhibitor of ERK1/2. U0126 prevents atrazine-induced decrease in LHR and CYP19A1 mRNA levels and estradiol production in the FSH-stimulated granulosa cells. ERK1/2 inactivation restores the ability of hCG to induce expression of the ovulatory genes in atrazine-exposed granulosa cells. Cell-based ELISA assay revealed that atrazine does not change the FSH-stimulated ERK1/2 phosphorylation in granulosa cells. The results from this study reveal that atrazine does not affect but requires ERK1/2 phosphorylation to cause decrease in the FSH-induced LHR and CYP19A1 mRNA levels and estradiol production in immature granulosa cells, thus compromising ovulation and female fertility.

Toxicological profiles assessment of the water and sediments from the Krivaja and Jegricka Rivers, Serbia.

This study utilizes a combinatorial bio/chemical approach to assess the toxicological profiles of organic pollutants in water and sediment samples from two watercourses that are under significant anthropogenic pressure-the Krivaja and Jegricka rivers in Serbia. Sample preparation by solid-phase extraction and silica-gel fractionation followed by GC/MS analysis, allowed the tentative identification of a variety of non-target contaminants, divided into non-polar, medium-polar and polar fractions. The instrumental analysis revealed slightly different toxicological profiles for the water and sediment from both rivers, and confirmed the presence of various classes of organic contaminants, from non-polar hydrocarbons, to more polar compounds such as aldehydes, ketones and phenols. Polycyclic aromatic hydrocarbons and pesticides were identified, but below toxicologically relevant concentrations. The results of bioanalyses on H4IIE and PLHC-1 cells indicated that cytotoxic potential was pronounced in Jegricka water and sediment samples, and CYP1A inducing potential was observed in both Krivaja and Jegricka sediment samples, although they did not reflect high levels of contamination. Based on the overall data, the sediments of the Krivaja and Jegricka rivers are a more toxicologically relevant matrix than the water.

Atrazine acts as an endocrine disrupter by inhibiting cAMP-specific phosphodiesterase-4.

Atrazine, one of the most commonly used herbicides worldwide, acts as an endocrine disruptor, but the mechanism of its action has not been characterized. In this study, we show that atrazine rapidly increases cAMP levels in cultured rat pituitary and testicular Leydig cells in a concentration-dependent manner, but less effectively than 3-isobutyl-1-methylxanthine, a competitive non-specific inhibitor of phosphodiesterases (PDEs). In forskolin (an activator of adenylyl cyclase)- and probenecid (an inhibitor of cyclic nucleotide transporters)-treated cells, but not in 3-isobutyl-1-methylxanthine-treated cells, atrazine further increased cAMP levels, indicating that inhibition of PDEs accounts for accumulation of cAMP. In contrast to cAMP, atrazine did not alter cGMP levels, further indicating that it inhibits cAMP-specific PDEs. Atrazine-induced changes in cAMP levels were sufficient to stimulate prolactin release in pituitary cells and androgen production in Leydig cells, indicating that it acts as an endocrine disrupter both in cells that secrete by exocytosis of prestored hormones and in cells that secrete by de novo hormone synthesis. Rolipram abolished the stimulatory effect of atrazine on cAMP release in both cell types, suggesting that it acts as an inhibitor of PDE4s, isoforms whose mRNA transcripts dominate in pituitary and Leydig cells together with mRNA for PDE8A. In contrast, immortalized lacto-somatotrophs showed low expression of these mRNA transcripts and several fold higher cAMP levels compared to normal pituitary cells, and atrazine was unable to further increase cAMP levels. These results indicate that atrazine acts as a general endocrine disrupter by inhibiting cAMP-specific PDE4s.

Synthesis, structural studies and biological activity of a dioxovanadium(v) complex with pyridoxal semicarbazone.

Reaction between the NH4VO3 and pyridoxal semicarbazone (PLSC) in a methanol/ammonia solution forms an orange, diamagnetic, mononuclear NH4[VO2(PLSC-2H)] complex in which vanadium is in the oxidation state +5, and pyridoxal semicarbazone is coordinated in its dianionic form. The complex cocrystallizes with a neutral molecule of PLSC and two water molecules. The coordination environment around vanadium can be described as an almost ideal square-pyramid. The complex was characterized by elemental analysis, conductometric and magnetochemical measurements, IR spectra, X-ray diffraction, and in vitro cytotoxicity analysis.

Interaction of organotin compounds with three major glutathione S-transferases in zebrafish.

Glutathione S-transferases (GSTs) play an important role in cellular detoxification as enzymatic mediators of glutathione (GSH) conjugation with a wide range of deleterious compounds, enabling their easier extrusion out of the organism. GSTs are shown to interact with organotin compounds (OTCs), known environmental pollutants, either as substrates, serving as electrophilic targets to the nucleophilic attack of GSH, or as noncompetitive inhibitors by binding to GST active sites and disrupting their enzymatic functions. There is a wide range of deleterious biological effects caused by OTCs in low concentration range. Their environmental concentrations, further potentiated by bioaccumulation in aquatic organisms, correspond with inhibitory constants reported for Gsts in zebrafish, which implies their environmental significance. Therefore, our main goal in this study was to analyze interactions of three major zebrafish Gsts - Gstp1, Gstr1, and Gstt1a - with a series of ten environmentally relevant organotin compounds. Using previously developed Gst inhibition assay with recombinant Gst proteins and fluorescent monochlorobimane as a model substrate, we determined Gst inhibitory constants for all tested OCTs. Furthermore, in order to elucidate nature of Gst interactions with OTCs, we determined type of interactions between tested Gsts and the strongest OTC inhibitors. Our results showed that OTCs can interact with zebrafish Gsts as competitive, noncompetitive, or mixed-type inhibitors. Determined types of interactions were additionally confirmed in silico by molecular docking studies of tested OTCs with newly developed Gst models. In silico models were further used to reveal structures of tested Gsts in more detail and identify crucial amino acid residues which interact with OTCs within Gst active sites. Our results revealed more extensive involvement of Gstr1 and Gstp1 in detoxification of numerous tested OTCs, with low inhibitory constants in nanomolar to low micromolar range and different types of inhibition, whereas Gstt1a noncompetitively interacted with only two tested OTCs with significantly higher inhibitory constants.

Molecular characterization of zebrafish Gstr1, the only member of teleost-specific glutathione S- transferase class.

Glutathione S-transferases (GSTs) are multifunctional phase II detoxification enzymes with primary function of glutathione conjugation of various endogenous and exogenous compounds. Teleost-specific Gstr1 in zebrafish (Danio rerio) was previously shown to have high expression in toxicologically relevant tissues and high activity towards model substrates. The aim of this study was a detailed functional characterization of zebrafish Gstr1. Molecular docking analyses were used to get novel insight into structural characteristics of Gstr1 and elucidation of the mechanistic interactions with both GSH and various Gstr1 substrates or inhibitors. An initial screening inhibition assay performed using model fluorescence substrate monochlorobimane (MCB) revealed interactions of different endogenous compounds and environmentally relevant xenobiotics with zebrafish Gstr1. All interacting compounds were further analyzed to determine their inhibition type and Ki values. Our data revealed that pregnenolone, progesterone, testosterone, DHEAS and corticosterone competitively inhibited transformation of MCB by Gstr1 with the calculated Ki values in the range 14-26 µM, implying that these hormones are physiological substrates of zebrafish Gstr1. Estrogens had no effect on Gstr1 activity. Taurochenodeoxycholate (TCDC) expressed lower inhibition potency toward Gstr1 with the Ki value of 33 µM. Among tested xenobiotics tributyltin chloride and rifampicin non-enzymatically bound Gstr1 enzyme (the calculated Ki values are 0.26 µM and 65 µM, respectively) and inhibited its activity, showing that these compounds are reversible noncompetitive inhibitors of zebrafish Gstr1. Insecticide diazinon competitively inhibited Gstr1 activity with calculated Ki value of 27 µM, while other Gstr1-interacting insecticides, chlorpyrifos-methyl (CPF-methyl) and malathion, showed allosteric activation-like effect. Among tested pharmaceuticals, tetracycline, erythromycin and methotrexate demonstrated competitive type of inhibition with the calculated Ki values of 17.5, 36.5 and 29 µM, respectively. In summary, we suggest that zebrafish Gstr1 has an important role in steroidogenesis, metabolism and/or physiological actions of androgens, but not estrogens in fish. Finally, our results imply the role of Gstr1 in metabolism of xenobiotics and protection of fish against deleterious environmental contaminants such as organophosphate insecticides and pharmaceuticals.

Effect of a PCB-based transformer oil on testicular steroidogenesis and xenobiotic-metabolizing enzymes.

Pyralene is a PCB-based transformer oil with a unique PCB congener profile when compared to other mixtures. We studied the influence of Pyralene on testicular steroidogenesis and the status of xenobiotic-metabolizing enzymes in the testis and liver of rats during oral exposure (10 and 50 mg/kg body weight, p.o. daily for 1 week) and a 3-week post-treatment recovery period. As expected, Pyralene induced a rapid and sustained increase in mRNA transcripts for CYP1A1 and CYP2B1 in hepatocytes that was associated with a dramatic increase in ethoxyresorufin-O-deethylase (EROD) and pentoxyresorufin-O-deethylase (PROD) activities. Testicular androgenesis and the conversion of progesterone to testosterone in testicular microsomes were bidirectionally affected. An increase in these parameters was observed 24h after the initial administration of Pyralene, followed by inhibition that lasted until the fourth post-treatment day. Expression PCR analysis revealed a significant decrease in 17beta-hydroxysteroid dehydrogenase (17betaHSD) transcript abundance at 48 h after Pyralene administration. In contrast, transcripts for several other steroidogenic enzymes and for testicular CYP1A1, CYP1B1, and CYP2B1 were unaffected under the same conditions. These results in the rat indicate that a sub-chronic exposure to Pyralene disrupted testicular steroidogenesis and suggest the mechanism may involve direct action on the regulation of specific steroidogenic enzymes such as 17betaHSD.

Synthesis and anti-aromatase activity of some new steroidal D-lactones.

Starting from D-seco derivatives of 5-androstene 1-3, the D-homo lactones, 4 and 5, were synthesized. By the Oppenauer oxidation and/or by dehydration of 4 and 5 with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) or 2,3,5,6-tetrachloro-1,4-benzoquinone (chloranil), the corresponding D-lactones 6-12 were obtained. The structures of 6 and 10 were unambiguously proved by the appropriate X-ray structural analysis. Anti-aromatase assay showed that tested compounds possess inhibition potency, however, two to four times smaller (IC50 from 0.2 to 0.7 microM, respectively) in comparison to aminoglutethimide (AG).

Characterization of glutathione-S-transferases in zebrafish (Danio rerio).

Glutathione-S-transferases (GSTs) are one of the key enzymes that mediate phase II of cellular detoxification. The aim of our study was a comprehensive characterization of GSTs in zebrafish (Danio rerio) as an important vertebrate model species frequently used in environmental research. A detailed phylogenetic analysis of GST superfamily revealed 27 zebrafish gst genes. Further insights into the orthology relationships between human and zebrafish GSTs/Gsts were obtained by the conserved synteny analysis. Expression of gst genes in six tissues (liver, kidney, gills, intestine, brain and gonads) of adult male and female zebrafish was determined using qRT-PCR. Functional characterization was performed on 9 cytosolic Gst enzymes after overexpression in E. coli and subsequent protein purification. Enzyme kinetics was measured for GSH and a series of model substrates. Our data revealed ubiquitously high expression of gstp, gstm (except in liver), gstr1, mgst3a and mgst3b, high expression of gsto2 in gills and ovaries, gsta in intestine and testes, gstt1a in liver, and gstz1 in liver, kidney and brain. All zebrafish Gsts catalyzed the conjugation of GSH to model GST substrates 1-chloro-2,4-dinitrobenzene (CDNB) and monochlorobimane (MCB), apart from Gsto2 and Gstz1 that catalyzed GSH conjugation to dehydroascorbate (DHA) and dichloroacetic acid (DCA), respectively. Affinity toward CDNB varied from 0.28 mM (Gstp2) to 3.69 mM (Gstm3), while affinity toward MCB was in the range of 5 µM (Gstt1a) to 250 µM (Gstp1). Affinity toward GSH varied from 0.27 mM (Gstz1) to 4.45 mM (Gstt1a). Turnover number for CDNB varied from 5.25s(-1) (Gstt1a) to 112s(-1) (Gstp2). Only Gst Pi enzymes utilized ethacrynic acid (ETA). We suggest that Gstp1, Gstp2, Gstt1a, Gstz1, Gstr1, Mgst3a and Mgst3b have important role in the biotransformation of xenobiotics, while Gst Alpha, Mu, Pi, Zeta and Rho classes are involved in the crucial physiological processes. In summary, this study provides the first comprehensive analysis of GST superfamily in zebrafish, presents new insight into distinct functions of individual Gsts, and offers methodological protocols that can be used for further verification of interaction of environmental contaminants with fish Gsts.

Antihormonal potential of selected D-homo and D-seco estratriene derivatives.

Since many estrogen derivatives exhibit anti-hormone or enzyme inhibition potential, a large number of steroidal derivatives have been synthesised from appropriate precursors, in order to obtain potential therapeutics for the treatment of hormone-dependent cancers. In molecular docking studies, based on X-ray crystallographic analysis, selected D-homo and D-seco estratriene derivatives were predicted to bind strongly to estrogen receptor α (ERα), aromatase and 17,20 lyase, suggesting they could be good starting compounds for antihormonal studies. Test results in vivo suggest that these compounds do not possess estrogenic activity, while some of them showed weak anti-estrogenic properties. In vitro anti-aromatase and anti-lyase assays showed partial inhibition of these two enzymes, while some compounds activated aromatase. Aromatase activators are capable of promoting estrogen synthesis for treatment of pathological conditions caused by estrogen depletion, e.g. osteopenia or osteoporosis.

Differential expression of CYP1A1 and CYP1A2 genes in H4IIE rat hepatoma cells exposed to TCDD and PAHs.

Rat hepatoma cells H4IIE were treated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and polycyclic aromatic hydrocarbons (PAHs) (dibenz(a,h)anthracene, benzo(a)pyrene, benz(a)anthracene, chrysene), low-concentration mixtures of PAHs and TCDD, and environmental mixtures contaminated by PAHs and their derivatives. Expression of the gene battery comprising cytochrome P450 Cyp1a1, Cyp1a2, Cyp1b1, and glutathione-s-transferase Gsta2 and Gstp was investigated using quantitative real time polymerase chain reaction (qRT-PCR) analysis. The results revealed that TCDD induce Cyp1a1>Cyp1a2>Cyp1b1, while PAHs and PAH-containing environmental mixtures induce Cyp1a2>Cyp1a1>Cyp1b1 gene expression pattern. While low-concentration mixtures elicited a more pronounced response in comparison to single treatments, the typical gene expression patterns were not observed. In all samples, Gsta2 was predominantly expressed relative to Gstp. These findings indicate that differential Cyp1a1 and Cyp1a2 expression in the H4IIE cells might be used for detection of PAHs in highly contaminated environmental mixtures, but not in low-concentration mixtures of these compounds.

Parallelism and dissociation in the actions of an Aroclor 1260-based transformer fluid on testicular androgenesis and antioxidant enzymes.

The mechanism by which Aroclors and other polychlorinated biphenyls (PCBs) inhibit testicular androgenesis in vivo and in vitro has not been characterized. Here we studied in adult rats the effects of intratesticular (i.t.t.), intraperitoneal (i.p.) and by gavage (p.o.) administration of Pyralene, an Aroclor 1260-based transformer fluid, on testicular androgenesis and oxidative status in androgen-producing interstitial cells and liver. Pyralene markedly decreased in vitro agonist stimulated androgenesis 24 h after bilateral i.t.t.-injection (25 microg/testis), 24 h and 96 h after single i.p.-injection (10 and 50 mg/kg body weight), and 96 h after p.o.-administration (7x10 mg and 7x50 mg/kg body weight daily). Inhibited androgenesis was accompanied by changes in the activity of antioxidant enzymes (AOEs) in interstitial cells after local i.t.t.-treatment and occasionally after systemic Pyralene application. Among changes in the activity, glutathione peroxidase and catalase reflected relatively well the toxicity of Pyralene in these cells. In liver, glutathione-S-transferase (GST) and glutathione peroxidase activities were enhanced after p.o.-treatment and total glutathione (tGSH) content and lipid peroxidation (LP) were enhanced after i.p.-administration. These results indicate that Pyralene inhibits androgenesis independently of the method of its administration. The results also suggest that changes in the oxidative status in testicular milieu are not critical for Pyralene-induced inhibition of androgenesis.

Synthesis, X-ray crystal structures and biological activity of 16-amino-17-substituted-D-homo steroid derivatives.

D-Homo derivatives in the androstane and estrane series, 12-19, were synthesized by a fragmentation-cyclization reaction of 16-oximino-17-hydroxy-17-substituted derivatives 3-9, or by cyclization of the corresponding D-seco derivatives 20-26. The structures were confirmed by X-ray analysis of compounds 12 and 16. Preliminary assessment of inhibitory effects of D-homo derivatives from androstane series towards aromatase, 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), 17 alpha-hydroxylase/C17-20 lyase (P450c17) and 17 beta-HSD indicated much lower inhibitory potential compared to previously tested activity of another type of D-modified steroids, namely D-seco derivatives. Also, assessment of potential antiestrogenic activity of derivatives from estrane series showed absence of such an activity.

Troponin T levels in detection of perioperative myocardial infarction after coronary artery bypass surgery.

OBJECTIVES: The present study was designed to evaluate the clinical relevance of serum cardiac troponinT (cTnT) assay in detection of perioperative myocardial infarction (PMI) after coronary artery bypass grafting (CABG). MATERIALS AND METHODS: The clinical utility of cTnT was compared to that of total CK, CKMB mass, CKMB activity and myoglobin. Serial venous blood samples were obtained before surgery and 4, 8, 16, 24, 48 and 72 hours after aortic unclamping (AU) in 42 patients who underwent CABG. We had 6 PMI patients, 24 patients with minor myocardial damage (MMD) and 12 without ischemic myocardial changes (no IMC). RESULTS: In discriminating no IMC from PMI the diagnostic sensitivity, specificity and the predictive values of cTnT were superior to that of CKMB mass, CKMB activity, myoglobin and total CK during 72 hours after AU. In discriminating MMD from PMI the diagnostic performance for CKMB mass and CKMB activity was superior to that of cTnT during the first 24 hours. After 24 hours the diagnostic performance for cTnT was improved but began to decline for CKMB isoenzymes. The discriminatory power of myoglobin measurements was lower than that of cTnT and CKMB mass. CONCLUSION: Our results indicate that troponin T is an accurate marker for the detection and monitoring of perioperative myocardial damage, especially 24 hours after AU.

Acute effects of hexabromocyclododecane on Leydig cell cyclic nucleotide signaling and steroidogenesis in vitro.

Hexabromocyclododecane (HBCDD), an additive brominated flame retardant routinely added to various consumer products, was reported to have toxic effects upon biota, including endocrine disruption. In this study, the potential toxicity of HBCDD was tested in peripubertal rat Leydig cells in vitro during 6h exposure. HBCDD inhibited human chorionic gonadotropin- and forskolin-supported cAMP accumulation and steroidogenesis. It also inhibited basal cAMP production, but elevated basal steroidogenesis. The expression of several cAMP-dependent genes, including steroidogenic acute regulatory protein, cholesterol side chain cleavage enzyme, and 3ß-hydroxysteroid dehydrogenase, was also inhibited by HBCDD treatment. Nevertheless, this was not accompanied by a decrease in steroidogenic acute regulatory protein expression, as documented by western blot analysis, and activity of steroidogenic enzymes, as documented by unaffected steroidogenesis in the presence of permeable 22(R)-hydroxycholesterol. However, HBCDD caused significant decrease in mitochondrial membrane potential in untreated and human chorionic gonadotropin-treated cells. This indicates that HBCDD acute toxicity in Leydig cells reflects changes in mitochondrial membrane potential-dependent cAMP production and basal and cAMP-regulated cholesterol transport. This in turn facilitates basal but inhibits cAMP-dependent steroidogenesis. Acute effects of HBCDD treatment on transcription are also indicative of its sustained effects on Leydig cell function.

Atrazine effects on antioxidant status and xenobiotic metabolizing enzymes after oral administration in peripubertal male rat.

Previously, we reported that in vivo applied atrazine from postnatal day 23 to 50 induced strong inhibition of testicular steroidogenesis. Therefore, the aim of the present study was to investigate, in the same experimental model, the oxidative status in androgen-producing testicular interstitial compartment characterized by diminished steroidogenesis. In parallel, we determined activities of antioxidative and cytochrome P450 (CYP) xenobiotic-metabolizing enzymes in liver. To confirm the results on atrazine induced-inhibition of testicular androgenesis, we measured ex vivo production of androgen in Leydig cells. The results revealed decreased activity of antioxidant enzymes, especially glutathione S-transferase (GST), but also glutathione peroxidase (GSH-Px) and catalase (CAT) in testicular interstitial cells, in parallel with strongly diminished ex vivo basal and agonist-stimulated Leydig cell androgenesis. In liver, atrazine increased the activity of GSH-Px, GST, and CYP1A1/2 enzyme, but not lipid peroxidation. These results indicate that atrazine markedly affects both antioxidant status and androgenesis in peripubertal rats.