Natural selection in the water: freshwater invasion and adaptation by water colour in the Amazonian pufferfish.

Natural selection and ecological adaptation are ultimately responsible for much of the origin of biodiversity. Yet, the identification of divergent natural selection has been hindered by the spatial complexity of natural systems, the difficulty in identifying genes under selection and their relationship to environment, and the confounding genomic effects of time. Here, we employed genome scans, population genetics and sequence-based phylogeographic methods to identify divergent natural selection on population boundaries in a freshwater invader, the Amazonian pufferfish, Colomesus asellus. We sampled extensively across markedly different hydrochemical settings in the Amazon Basin and use 'water colour' to test for ecological isolation. We distinguish the relative contribution of natural selection across hydrochemical gradients from biogeographic history in the origin and maintenance of population boundaries within a single species and across a complex ecosystem. We show that spatially distinct population structure generated by multiple forces (i.e. water colour and vicariant biogeographic history) can be identified if the confounding effects of genetic drift have not accumulated between selective populations. Our findings have repercussions for studies aimed at identifying engines of biodiversity and assessing their temporal progression in understudied and ecologically complex tropical ecosystems.

Subchronic oral toxicity study of furan in B6C3F1 Mice.

Furan is a heterocyclic organic compound formed during heat treatment for processing and preservation of various types of food. Rodent studies have previously shown that furan is a hepatocarcinogen. Those studies were conducted over a high dose range, which induced tumors at nearly 100% incidence at all doses. This ninety-day gavage study in mice was conducted to extend the dose to a lower range (0.0, 0.03, 0.12, 0.5, 2.0, and 8.0 mg/kg body weight [bw] per day) to identify a no-observed adverse effect level for hepatotoxicity and to characterize non-neoplastic effects, including those affecting clinical biochemistry, hematology, tissue morphology, and histopathology. The liver was the primary target organ with dose-dependent toxicity. Liver weights were increased at the 8.0 mg/kg bw dose in females only. Levels of the serum enzyme alanine transaminase, representative of liver damage, were increased three-fold at the highest dose. Histological changes in the liver were observed at 2.0 and 8.0 mg/kg bw in both sexes. Although clinical parameters were also altered for the kidney, these differences were not accompanied by histological changes. Based on these clinical biochemical and histological changes, a no-observed adverse effect level of 0.12 mg/kg bw per day of furan in mice is suggested.

Subchronic oral toxicity study of furan in Fischer-344 rats.

Rodent studies have shown that furan is a hepatocarcinogen. Previous studies conducted with high doses showed tumors at nearly 100% incidence at all doses. In this paper, a ninety-day gavage experiment conducted with lower doses (0.0, 0.03, 0.12, 0.5, 2.0, and 8.0 mg/kg bw) to identify a no-observed adverse effect level for hepatotoxicity and to characterize non-neoplastic effects including gross changes and histopathology, clinical biochemistry, hematology, and immunotoxicology is reported. As indicated by changes in serum biomarkers, increased liver weights and gross and histological lesions, the liver is the major target organ affected by furan. There were no changes in body weights, food consumption, or histology in other organs. Some of the serum electrolyte markers, including phosphorus, were altered. There was a significant increase in serum thyroxine and triidothyronine in males. This increase was not accompanied by histological thyroid changes. Immunophenotypic analysis showed that thymic lymphocyte maturation was altered in male rats. Although altered clinical biochemistry and hematological parameters were observed at a dose of > 0.5 mg/kg bw, mild histological lesions in the liver were observed at > 0.12 mg/kg bw. Based on this finding, a furan dose of 0.03 mg/kg bw was proposed as the no-observed adverse effect level for hepatic toxicity.

Toxicokinetics of p-tert-octylphenol in male and female Sprague-Dawley rats after intravenous, oral, or subcutaneous exposures.

This study was undertaken to characterize the toxicokinetics of p-tert-octylphenol (OP), a weak estrogenic compound, in male and female rats. Male and female Sprague-Dawley rats were given a single dose of OP either by oral gavage (50, 125 or 250 mg/kg), by intravenous (iv) injection (2, 4, or 8 mg/kg), or by subcutaneous (sc) injection (125 mg/kg). In a repeated dosing experiment, rats were given OP (oral) daily (25, 50, or 125 mg/kg) for 35 d (female) or 60 d (male). Blood and tissue samples were collected and analyzed for OP content using gas chromatography with detection by mass spectrometry. Blood OP concentrations were generally higher in female than male rats following a single oral or sc administration but were similar following a single iv injection. Tissue OP concentrations were also higher in female than male rats following oral exposure, consistent with the faster metabolism of OP observed in male rat liver microsomes. After subchronic administration, blood OP concentrations were higher at the end of exposure for female (33 d) (2.26-fold, not significant) and male (57 d) (3.47-fold) rats than single dosing but there was no change in the tissue OP concentrations. Gender differences in tissue OP concentrations may contribute, in part, to gender differences in the toxicity of OP in rats. The fact that OP was found in all reproductive tissues confirms its potential for direct endocrine-like effects.

Determination of p-tert-octylphenol in blood and tissues by gas chromatography coupled with mass spectrometry.

A sensitive and reproducible procedure using gas chromatography coupled with mass spectrometry is described for the determination of p-tert-octylphenol (OP), a persistent degradation product of alkylphenol ethoxylates that binds to the estrogen receptor in blood and tissues. The first step involved the extraction of blood (200 microL) or tissue homogenate (400 microL) with methyl tert-butyl ether, including p-tert-butylphenol (BP) as internal standard. After extraction, the sample was evaporated to dryness with a gentle stream of nitrogen at 45 degrees C, and OP and BP were derivatized with an acetylation reaction involving acetic anhydride and catalyzed by pyridine. Samples were then analyzed by a gas chromatograph equipped with a mass spectrometer (single ion monitoring) with a Varian VF-5ms capillary column. The limit of detection and the limit of quantification of the method in blood were 4.6 and 15.5 ng/mL, respectively. The linearity and reproducibility of the method were acceptable, with coefficients of variation of approximately 10% for blood and ranging between 9% and 27% for tissues. This method was applied to the determination of unchanged OP in blood and tissues obtained from Sprague-Dawley rats after oral and IV OP administration.

The submicrosomal distribution in rat and boar testis of some enzymes involved in androgen and 16-androstene biosynthesis.

The submicrosomal localization of enzymes involved in androgen and 16-androstene biosynthesis in rat and boar testis tissue has been studied. 16-androstene production was not evident in the rat testis but two enzymes concerned with androgen generation (17 alpha-hydroxy C21 steroid C-17,20 lyase; 17 beta-hydroxysteroid dehydrogenase, were found predominantly in agranular microsomes. In the boar testis the C-17,10 lyase had a similar distribution to the rat enzyme but the 17 beta-hydroxysteroid dehydrogenase was found to be evenly distributed between the two microsomal types. The enzyme system, "andien-betha synthetase", involved in the conversion of pregnenolone to 5,16-androstadien-3 beta-ol, was found mainly (66%) in the agranular microsomes but had a lower specific activity than those of the two enzymes of androgen biosynthesis that have been studied.

Improved expression cloning using reporter genes and Epstein-Barr virus ori-containing vectors.

Levels of expression of two reporter genes cloned into SV40 or Epstein-Barr virus (EBV) ori-containing plasmids were measured following transient transfection of cell lines constitutively expressing T-antigen or EBV nuclear antigen 1 (EBNA1). The TSA201 and COS7 cell lines stably produce T-antigen and support replication of the SV40 ori-containing constructs while the 293EBNA cell line produces EBNA1 and supports replication of EBV ori-containing plasmids. We found that 293EBNA cells express > 25-fold more beta-galactosidase (beta Gal) per mg protein than COS7 cells and 11-fold more beta Gal than TSA201 cells. We also demonstrate that 293EBNA cells are able to express 70-100-fold more angiotensin II type-1 receptor (AT1) per mg protein than COS7 or TSA201 cells. We examined the suitability of each cell line for use in expression cloning using a NaOH 'scrape' method as an improvement over emulsion autoradiography for detection. Measurable AT1 signals can be detected when reporter plasmids are diluted up to 1000-fold for COS7 and TSA201 cells, and up to 80,000-fold for 293EBNA cells. These data demonstrate that 293EBNA cells offer a significant improvement in expression cloning technology as compared to the conventionally used T-antigen-based cell lines.

Cloning, expression and tissue distribution of the gene encoding rat fibroblast growth factor receptor subtype 4.

The rat homologue of the gene encoding the fibroblast growth factor receptor subtype 4 (FGFR4) was cloned from rat lung mRNA, and the cDNA sequence was found to be 95% similar and 92% identical to the human homologue. Northern blot analysis of adult rat tissues demonstrated that a 3.1-kb mRNA encoding FGFR4 is detectable only in the lung and kidney. The receptor variant described here encodes two potential immunoglobulin-like domains, 21 hydrophobic amino acids encoding a potential transmembrane domain, and a split tyrosine kinase motif. However, the acidic box and hydrophobic signal peptide domains are not present in this cDNA isolate.

Sulphoconjugation of steroids in porcine liver. Partial purification of the cytosolic sulphotransferases for pregnenolone and 5 alpha-androst-16-en-3 beta-ol.

Steroid sulphotransferase activities for 5 alpha-androst-16-en-3 beta-ol and pregnenolone in porcine liver cytosol have been assayed using 3'-phosphoadenosine-5'-phospho[35S]sulphate as sulphate donor. 5 alpha-Androst-16-en-3 beta-ol sulphotransferase activity was obtained from porcine liver cytosol by gel filtration chromatography; activity was linear with time up to about 5 min., the optimum pH was near 8.0 and optimum temperature 37 degrees C. Pregnenolone sulphotransferase activity was partially purified from porcine liver cytosol using DEAE-cellulose chromatography with an ionic gradient of KC1. This enzyme activity was linear with time up to 10 min and had optimum pH and temperature of 8.0 and 37 degrees C, respectively.

Investigations into the possible effects of naturally occurring steroids on biosynthesis of 16-androstenes and androgens in microsomes of boar testis.

The possible interactions between naturally occurring steroids and three enzymes of the biosynthetic pathways of 16-androstenes and androgens in the boar testis were investigated. High concentrations (1 mmol/1) of several steroids reduced the production of 5,16-androstadien-3 beta-ol when a microsomal fraction of boar testis was incubated with pregnenolone at pH 7.0 (the pH optimum of this reaction was found to be 6.6). When some of these inhibitors were investigated in more detail using Lineweaver-Burk analyses, the apparent inhibition constants, Kv increased in value with increasing concentrations of inhibitors. When testosterone was added to 5,16-androstadien-3 beta-ol synthetase assays, the apparent Ki for 0.1 mumol testosterone/1 was 0.165 mumol/1 whereas those for 1.0, 10.0 and 100.0 mumol testosterone/1 were 1.65, 16.5 and 48.7 mumol/1 respectively. The apparent Michaelis constant, Km, of the reaction was 0.6 mumol/1. Similar results were obtained when oestrone, 17 alpha-hydroxyprogesterone and 4,16-androstadien-3-one were added as effectors. At physiological concentrations, these steroids would not affect the biosynthesis of 5,16-androstadien-3 beta-ol in vivo. Similarly, both 5 alpha-androst-16-en-3 beta-ol, quantitatively the most important 16-androstene in the boar testis and 5,16-androstadien-3 beta-ol were examined for their effects on the 17 alpha-hydroxy-C21-steroid, C-17,20 lyase (C-17,20 lyase) and 17 beta-hydroxysteroid dehydrogenase (17 beta-OHSDH) enzymes of androgen biosynthesis. Neither of these enzymes was affected by the 16-androstene steroids even at concentrations of 100 mumol/1, and the apparent Km values were 3.3 and 26.0 mumol/1 for C-17,20 lyase and 17 beta-OHSDH respectively. This lack of interaction between these pathways implies that the high levels of 16-androstene steroids produced by the testis will not interfere with androgen production, and also that the two side-chain cleavage steps from C21 precursors to C19 steroids are catalysed by independent systems.

Effects of PNU157706, a dual 5alpha-reductase inhibitor, on gene expression in the rat epididymis.

The epididymis is the site of sperm maturation and storage. 5alpha-Reductases (types 1 and 2) are key enzymes in this tissue because they convert testosterone to dihydrotestosterone (DHT), the main androgen regulating epididymal functions. Examining the consequences of inhibiting DHT formation is likely to provide important information regarding the regulation of epididymal functions, yet few inhibitor studies have focused on this tissue. To understand better DHT-mediated regulation of epididymal gene expression, we employed a dual 5alpha-reductase inhibitor and cDNA microarrays to examine the effects of 5alpha-reductase inhibition on gene expression in the initial segment, caput, corpus, and cauda epididymidis. Inhibition of epididymal 5alpha-reductase activity by PNU157706 was confirmed by in vitro enzyme assays. Rats were treated with 0, 0.1, 1.0 or 10 mg/kg per day PNU157706 for 28 days. The weights of DHT-dependent tissues, including the epididymis, were decreased following treatment. The effect of treatment on gene expression was dose-dependent and highly segment-specific. The initial segment responded uniquely in that a similar number of genes increased and decreased in expression compared with the other segments where the majority of affected genes decreased in expression. Some of the more dramatically affected genes were involved in signal transduction as well as fatty acid and lipid metabolism, regulation of ion and fluid transport, luminal acidification, oxidative defense and protein processing and degradation. These are essential processes contributing to the formation of an optimal luminal microenvironment required for proper sperm maturation. These results provide a novel insight into the DHT-dependent mechanisms that control epididymal functions.

The role of pregnenolone-metabolizing enzymes in the regulation of oestradiol biosynthesis during development of the first wave dominant follicle in the cow.

During the luteal phase in the cow, a first-wave dominant follicle grows to reach ovulatory size, but then ceases to grow, becomes no longer dominant and enters a phase of slow regression. During this growth transition, the concentration of oestradiol has been shown to decrease in follicular fluid. The objective of this study was to determine if follicular fluid oestradiol concentrations are regulated by the activity of three major steroidogenic enzymes, namely P450-aromatase (P450-arom), 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) and 17 alpha-hydroxylase C-17,20 lyase cytochrome P450 enzyme (P450-17 alpha) measured in granulosa and theca cells isolated from individual first-wave dominant follicles. Follicle growth and state of dominance was assessed by ultrasonography and follicles were classified as growing-dominant (GD, n = 6), non-growing-dominant (NGD, n = 8) or non-growing-non-dominant (NGD, n = 6). Mean follicular fluid concentrations of oestradiol were higher in GD than in NGD or NGND follicles (511 +/- 98 versus 136 +/- 16 and 20 +/- 11 nmol/l respectively). Oestradiol was not correlated with P450-arom in any of the three groups. In GD follicles, oestradiol was positively correlated with pregnenolone concentration but neither was correlated with granulosa or theca 3 beta-HSD activity or with theca P450-17 alpha activity. In NGD follicles, oestradiol was negatively correlated with theca 3 beta-HSD activity and pregnenolone was negatively correlated with granulosa 3 beta-HSD activity. In NGND follicles, oestradiol was positively correlated, and pregnenolone was negatively correlated with theca 3 beta-HSD and P450-17 alpha activities. These studies demonstrated that pregnenolone supply is the principal regulating factor of oestradiol output during follicle dominance and during the loss of dominance but that the levels of P450-17 alpha and 3 beta-HSD activity become rate-limiting when the follicle is no longer dominant.

Differential effects of trilostane and cyanoketone on the 3 beta-hydroxysteroid dehydrogenase-isomerase reactions in androgen and 16-androstene biosynthetic pathways in the pig testis.

3 beta-Hydroxysteroid dehydrogenase-isomerase (3 beta-HSD-I) activity in the pig testis is responsible for the conversion of dehydroepiandrosterone (DHA) to 4-androstenedione and also for the conversion of 5,16-androstadien-3 beta-ol (andien-beta) to 4, 16-androstadien-3-one (dienone). Therefore, 3 beta-HSD-I plays an essential role in the biosynthesis of hormonally and pheromonally active steroids. Previous studies from this laboratory have suggested that the 3 beta-HSD-I reactions in the androgen and 16-androstene biosynthetic pathways may be catalysed by different enzymes with selective substrate specificities [3, 4]. The aim of the present studies was to investigate the reactions further by examining the effects of two classical steroidal inhibitors of 3 beta-HSD-I, trilostane (WIN 24540) and cyanoketone (WIN 19578), on the kinetic parameters of the 3 beta-HSD-I reactions in immature (< 3 weeks) pig testis microsomes. In kinetic analyses of the conversion of DHA to 4-androstenedione, both trilostane and cyanoketone caused increases in the Km(app) for DHA which at the highest concentration used, were 15-fold the control Km(app) of 1.4 mumol/l. No effect on the Vmax(app) (6.55 +/- 0.74 nmol/h/mg protein) was observed, demonstrating that competitive inhibition was evident. Slope and intercept replots confirmed the competitive nature of the inhibition and Ki(app) values of 0.16 mumol/l for trilostane and 0.20 mumol/l for cyanoketone were respectively 9 and 7-fold lower than the Km(app) value. In contrast, trilostane and cyanoketone had no effect on the Km(app) for andien-beta (0.26 mumol/l). The Vmax(app) (1.12 nmol/h/mg protein) was decreased by 40-50% only by trilostane at the highest concentration used, demonstrating a very low affinity for the andien-beta active site. Ki(app) values for trilostane and cyanoketone, obtained from slope and intercept replots were, respectively 1.1 and 1.6 mumol/l, which were 4 and 6-fold greater than the Km(app) for andien-beta. Therefore, trilostane and cyanoketone were powerful competitive inhibitors of the conversion of DHA to 4-androstenedione but were weak non-competitive inhibitors of the conversion of andien-beta to dienone. The selective effects of trilostane and cyanoketone on the 3 beta-HSD-Is involved in the androgen and 16-androstene biosynthetic pathways strongly suggest that the reactions are catalysed by separate enzymes, or at least separate, non-interacting active sites on a single enzyme.

Phospholipases modulate immature pig testicular androgen and 16-androstene biosynthetic pathways in vitro.

The role of membrane phospholipids in porcine testicular androgen and 16-androstene biosynthesis was examined by monitoring the effects of phospholipase treatments on the activities of the steroid transforming enzymes. Untreated (control) microsomes from immature pig testes converted pregnenolone to 17-hydroxypregnenolone and DHA to 5,16-androstadien-3 beta-ol (andien-beta) and 4,16-androstadien-3-one (dienone) in the 16-androstene pathway, these metabolites accounting for most (65%) of the pregnenolone converted. The 4-ene steroids in the androgen pathway (progesterone, 17-hydroxyprogesterone, androstenedione and testosterone) totalled less than 10% of the pregnenolone metabolites. No estrogens or 5 alpha-reduced metabolites were detected. Treatment with phospholipase A2 or C, decreased the conversion of pregnenolone to 4-ene-3-oxo steroids but did not decrease the quantities of 5-ene-3 beta-hydroxysteroids. Confirmation of these findings was obtained by measuring the individual enzymatic steps. Phospholipases A2 and C significantly reduced the conversion of DHA to androstenedione and andien-beta to dienone but did not affect 17-hydroxylase or 'andien-beta-synthetase'. However, when the C-17, 20 lyase step was measured alone, phospholipase C decreased the quantity of androstenedione produced indicating that the side-chain cleavage reaction may involve a lipid component. The different effects of phospholipases on these enzymes suggests that pregnenolone metabolism may be regulated by alterations in the membrane microenvironment.

Identification of phospholipids capable of modulating the activities of some enzymes involved in androgen and 16-androstene biosynthesis in the immature pig testis.

Testicular steroidogenic enzymes in the microsomal fraction from immature pigs were investigated for the effects of phospholipids of known structure on androgen and 16-androstene biosynthesis. Untreated (control) microsomes metabolized pregnenolone to 17-hydroxypregnenolone, DHA and small quantities of progesterone, 17-hydroxyprogesterone, androstenedione and testosterone; and to 5,16-androstadien-3 beta-ol (andien-beta) and 4,16-androstadienone (dienone) in the 16-androstene pathway. Phosphatidyl(P)-serine, P-glycerol, P-ethanolamine, P-inositol, P-choline and phosphatidic acid did not significantly alter the 17-hydroxylase/C-17,20 lyase or "andien-beta-synthetase" activities. Thus, the C21 side-chain cleavage reactions appeared not to be dependent upon phospholipids for optimal activity. The conversion of pregnenolone to 4-ene steroids (progesterone, 17-hydroxyprogesterone, androstenedione and testosterone) was inhibited by dilinoleoyl-phosphatidyl-choline, but other phospholipids tested were without effect. On the other hand, the conversion of andien-beta to dienone was inhibited by P-serine, P-inositol and P-cholines with short saturated or long polyunsaturated acyl chains. Therefore, the presence of these phospholipids in pregnenolone incubations had different consequences for 3 beta-hydroxysteroid dehydrogenase-isomerase activities. It is concluded that substrate specific 3 beta-HSD-isomerases exist for androgen and 16-androstene biosynthesis and that phospholipids may play an intrinsic role in their catalytic activity.

17-Hydroxylase and andien-beta synthetase activities in immature pig testis microsomal fraction: kinetic studies of the pregnenolone binding site and possible intermediates of the reactions.

The microsomal fraction from the testes of immature pigs can convert pregnenolone to 17-hydroxypregnenolone and also to 5,16-androstadien-3 beta-ol (andien-beta). The available evidence supports the hypothesis that both these reactions are catalysed by one enzyme, cytochrome-P450(17 alpha). In the absence of cytochrome b5, 17-hydroxypregnenolone will be the major product but that if cytochrome b5 is present in sufficient quantity, andien-beta becomes a major product. The point of divergence between the conversion of pregnenolone to either 17-hydroxypregnenolone or andien-beta was investigated using enzyme kinetic analysis to determine whether 16 alpha-hydroxypregnenolone, 20 beta-hydroxypregnenolone or 16-dehydropregnenolone could be specific intermediates to one reaction or the other. Product inhibition by 17-hydroxypregnenolone and andien-beta was competitive for both 17-hydroxylase and "andien-beta synthetase" supporting the current view of a common active site for both reactions. 16 alpha-Hydroxypregnenolone was a very poor competitive inhibitor of 17-hydroxylase and andien-beta synthetase with Ki(app) values many fold greater than the Km(app) for pregnenolone or the Ki(app) for reaction product, rendering it unlikely that 16 alpha hydroxylation is a key intermediary step in either pathway. 20 beta-Hydroxypregnenolone was a more potent inhibitor of andien-beta synthetase than of 17-hydroxylase and for the latter enzyme activity, the Ki(app) was lower than that for 17-hydroxypregnenolone itself. However, for andien-beta synthetase, 20 beta-hydroxypregnenolone may be an early intermediate as the Ki(app) was consistent with the affinity for the active site being intermediate between the Km(app) for pregnenolone and the Ki(app) for andien-beta. 16-Dehydropregnenolone was equipotent at inhibiting 17-hydroxylase and andien-beta synthetase activities suggesting that 16-dehydropregnenes may be involved in the stages immediately prior to C21 side-chain cleavage.

Kinetic evidence for separate 3 beta-hydroxysteroid dehydrogenase-isomerases in androgen and 16-androstene biosynthetic pathways in the pig testis.

The microsomal fraction from the testes of immature pigs (less than 1 week old) contains 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD-isomerase) activities that convert dehydroepiandrosterone (DHA) to 4-androstenedione and 5,16-androstadien-3 beta-ol (andien-beta) to 4,16-androstadien-3-one (dienone). These reactions are necessary for the biosynthesis of hormonally and pheromonally active steroids. Kinetic analyses of these activities were done to determine whether they are catalysed by a single enzyme or if there is any interaction between the substrates and products of one reaction on the activity of the other enzyme. Kinetic parameters were determined and the affinities for steroid substrate were similar (7-9 mumol/l) but the Vmaxapp value for the conversion of andien-beta to dienone was 10-fold that of the DHA to 4-androstenedione reaction. In analyses of the conversion of DHA to 4-androstenedione, neither andien-beta nor dienone inhibited the reaction and especially, no effect on the Kmapp for DHA was observed which would have indicated competition between DHA and andien-beta for the same active site (Kiapp from slope and intercept replots were between 3 and 80 times the values of the kinetic constants). Similarly, DHA and 4-androstenedione had minor or negligible effects on the conversion of andien-beta to dienone (Kiapp from slope replots were the same as the Kmapp but the Kiapp from the intercept replot was 12 to 25% of the Vmaxapp). It is concluded that substrate specific 3 beta-HSD-isomerases for andien-beta and DHA exist in the immature pig testis and there is little, if any interaction between these enzymes.

The effects of progesterone, 4,16-androstadien-3-one and MK-434 on the kinetics of pig testis microsomal testosterone-4-ene-5alpha-reductase activity.

The enzyme 3-oxo-steroid: NADP+ 4-oxidoreductase (EC 1.3.1.22; 5alpha-reductase) was assayed in testicular microsomes of pigs of 3, 20 and 24 weeks of age. The activity was very low in 3-week-old animals and approximately 10-fold higher in 5- and 6-month-old pigs. The pH optimum was 6.3 in 6-month-old animals, 5.7 in 5-month-old animals, but could not be reliably determined in 3-week-old animals. The kinetic parameters for 5alpha-reductase in testis microsomes from 6-month-old animals were; K((m)(app)), 8.0 micromol/l, V((max)(app)), 6.7 nmoles/90 min/mg protein. Progesterone was a competitive inhibitor of testosterone 5alpha-reduction with an apparent K((i)(app)) of 0.86 micromol/l. However, 4,16-androstadien-3-one (dienone), which undergoes 5alpha-reduction in the biosynthesis of the pheromonally active 16-androstenes, was a comparatively poor inhibitor with a K((i)(app)) of 4.9 micromol/l. Similarly, MK434, which is a selective inhibitor of the human type 2 5alpha-reductase, but which inhibits both types 1 and 2 in the rat, was also a poor competitive inhibitor of testosterone 5alpha-reductase in the pig testis (K((i)(app)), 3.1 micromol/l). It would appear from these studies that the pig testis microsomal 5alpha-reductase corresponds to a type 1 isozyme that is not capable of reducing dienone other than under conditions where the dienone concentration would be in considerable excess of testosterone. It is, therefore, probable that substrate-specific 5alpha-reductases exist in the pig testis for the 5alpha-reduction of testosterone and dienone.

Effects of in utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on serum androgens and steroidogenic enzyme activities in the male rat reproductive tract.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been shown to impair reproductive function of males in animal models, possibly due to a reduction in serum androgen levels. Thus, TCDD may alter the testosterone biosynthetic pathway in the testis or the conversion of testosterone to 5alpha-dihydrotestosterone (DHT) in androgen target tissues. Pregnant Sprague Dawley rats were gavaged with TCDD (0, 0.2 or 1.0 microg/kg) on day 15 of gestation only. TCDD caused a reduction in the body weight gain of the dams in both dose groups and a significant reduction in litter size in the higher dose group. Litters delivered normally and TCDD exposed male offspring grew at the same rate as controls. Males were sacrificed at 15, 30, 45, 60, 90 and 120 d of age. Steroidogenic enzyme activities were determined in testicular microsomes and androgen target tissue nuclear fractions. Serum androgens were measured by radioimmunoassay (RIA). At 30 d of age, rats exposed to 1.0 microg/kg TCDD exhibited lower 17-hydroxylase activity (P < 0.05) and lower caput-corpus epididymal weights (P < 0.05). At 45 d of age, the same treatment resulted in testicular 3beta-HSD, 17beta-HSD and 5alpha-reductase activities that were significantly greater (P < 0.05) but, conversely, serum androgens were one quarter the values evident in controls (P < 0.05). At the other ages, no differences were observed in serum androgens and, with the exception of lower 17beta-HSD activity at 90 d of age (P < 0.05), no other differences in testicular steroidogenic enzyme activities were found. 5Alpha-reductase activities in the androgen target tissues were also unchanged. Histological examination of testes showed that the spermatogenic profile was identical to controls at all ages.

Examination of selected food additives and organochlorine food contaminants for androgenic activity in vitro.

In order to produce a reporter gene assay for androgenic chemicals, a constitutive expression vector coding for the human androgen receptor and a reporter construct containing the firefly luciferase coding sequence under transcriptional control of the androgen responsive MMTV promoter were cotransfected into the androgen-insensitive human PC-3 prostate carcinoma cell line and stable transfectants selected. One colony of transfectants, PC-3 LUCAR+, was characterized further. 5alpha-Dihydrotestosterone (DHT) enhanced luciferase activity in a linear fashion for up to 3 days of culture. The Kd for DHT activation was within the range of 25.0-60.0 pM (r2 values >0.95). Flutamide competitively inhibited DHT activation (mean Ki value of 0.89 microM). Progesterone, estradiol, dexamethasone, and hydrocortisone were weak agonists (100-fold less effective than DHT) and diethylstilbestrol was without effect. The effects of organochlorine food contaminants (0, 0.1, 1.0, and 10.0 microM) on luciferase activity in PC-3 LUCAR+ cells were determined after exposure to the chemical for 18 h in the presence and absence of DHT (50 pM). 1,1-dichloro-2,2-bis(p-chlorophenyl)-ethylene (p,p'-DDE) induced luciferase activity in the absence of DHT (100 microM p,p'-DDE equivalent to 50 pM DHT), but in the presence of DHT (50 pM), p,p'-DDE acted antagonistically. 2,3,7,8-Tetrachlorodibenzo-p-dioxin, kepone, butylated hydroxyanisole, and butylated hydroxytoluene all partially inhibited activation by DHT (50 pM) but alone had little or no effect. Toxaphene at 10 microM induced luciferase activity in the absence of DHT but decreased cell viability. Alpha- and delta-Hexachlorocyclohexanes (HCH) at 10 microM antagonized the DHT effect, but beta-HCH and gamma-HCH mirex, photomirex, oxychlordane, cis- and trans-nonachlor were without effect. Thus, of the chemicals tested, some interact with the human androgen receptor in vitro as agonists, others as antagonists, and some as partial agonists/antagonists.