Gerard M. Cooke

Effect of organotins on human aromatase activity in vitro

The interaction between the human aromatase enzyme and some organotins was investigated. Tributyltin (TBT) at 12 and 59 microM and dibutyltin at 74 microM inhibited aromatase activity in vitro but monobutyltin and tri-, di- and monooctyltins were without effect. In four separate kinetic studies of aromatase, the K(m(app)) for testosterone was 0.24, 0.21, 0.16 and 0.24 microM. TBT inhibited aromatase activity by causing the K(m(app)) to be increased without affecting the V(max), indicative of competitive inhibition. Slope and intercept replots confirmed the effect of aromatase on the K(m(app)). Slope replots from three separate kinetic studies provided Ki values for TBT of 64.5, 40.9 and 37.3 microM. Consequently, TBT is a competitive inhibitor of human aromatase with a Ki approximately 300-fold the K(m(app)) value.

Persistent Organic Pollutant Residues in Human Fetal Liver and Placenta from Greater Montreal, Quebec: A Longitudinal Study from 1998 through 2006

Background There is general concern that persistent organic pollutants (POPs) found in the environment, wildlife, food, water, house dust, human tissues, and fluids may alter normal human physiologic activities (e.g., fetal development, immune and endocrine systems). Although the levels of some POPs [polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCs)] in these matrices have decreased after their ban, others [polybrominated diphenyl ethers (PBDEs)] have increased in recent years. Objective To determine the longitudinal trend of specific POPs in human fetal tissues for risk assessment purposes. Methods We analyzed early to mid-gestation fetal liver (n = 52) and placental (n = 60) tissues, obtained after elective abortions during 1998–2006, for selected PBDEs, PCBs, and OCs using gas chromatography–mass spectroscopy. Results Total PBDEs in fetal liver increased over time (mean ± SE: 1998, 284.4 ± 229.8 ng/g lipid; 2006, 1,607.7 ± 605.9; p < 0.03), whereas placental levels were generally lower, with no clear trend. Low levels of PCBs and OCs varied yearly, with no evident trend. The major analytes in 1998 were OCs (liver, 49%; placenta, 71%), whereas the major analytes in 2006 were PBDEs (liver, 89%; placenta, 98%). The 1998–2006 tissue PBDE congener profile is similar to that of DE-71, a commercial primarily pentabrominated diphenyl ether mixture manufactured in North America. Conclusions Although commercial production of penta- and octa-brominated diphenyl ethers in North America was halted in 2004, their concentrations in fetal liver and placenta are now greater than the tissue burdens for the analyzed OCs and PCBs. Our findings also demonstrate that PBDEs accumulate within the fetal compartment at a very early stage in gestation.

Low levels of the herbicide atrazine alter sex ratios and reduce metamorphic success in Rana pipiens tadpoles raised in outdoor mesocosms.

Background There are conflicting reports regarding the effects of atrazine (ATZ) on amphibian development. Therefore, further studies are needed to examine the potential mechanisms of action of ATZ in amphibians. Objectives Our aim in this study was to determine whether low concentrations of ATZ affect gonadal development and metamorphosis in the Northern leopard frog, Rana pipiens. Methods Tadpoles were exposed in outdoor mesocosms to nominal concentrations of 0.1 and 1.8 μg/L of formulated ATZ from Gosner stage 27 (G27) to metamorphic climax (G42). Exposure to 17α-ethinylestradiol (EE2; 1.5 μg/L) provided a positive control for induction of testicular oocytes in males. Endocrine-related gene expression and gonadal histopathology were examined at G42 and in a subset of premetamorphic G34 tadpoles that failed to metamorphose. Results Gonadal gross morphology revealed that the 1.8-μg/L ATZ treatment produced 20% more females compared with the control. Histologic analysis revealed that 22% of EE2-treated males had testicular oocytes, whereas none were observed in any animals from the control or either ATZ groups. ATZ increased brain estrogen receptor α mRNA to 2.5 times that of the control at premetamorphosis and altered liver levels of 5β-reductase activity at metamorphosis. In contrast, brain aromatase mRNA level and activity did not change. ATZ treatments significantly reduced metamorphic success (number of animals reaching metamorphosis) without affecting body weight, snout–vent length, or age at metamorphosis. Gene expression analysis indicated that ATZ decreased the expression of deiodinase type 3 in the tail at premetamorphosis. Conclusions Our study indicates that exposure to low concentrations of ATZ in experimental mesocosms alters gonadal differentiation and metamorphosis in developing R. pipiens.

Effects of Aroclors and individual PCB congeners on activation of the human androgen receptor in vitro

To investigate possible interactions between the human androgen receptor and PCBs in vitro, we have used a previously characterized human androgen receptor reporter gene assay in which PC-3 LUC(AR+) cells respond to 5alpha-dihydrotestosterone (DHT, 50 pM) with enhanced luciferase activity. The effects of Aroclors, commercial mixtures of PCBs, or polychlorinated terphenyls (PCTs) (0, 0.1, 1.0, and 10.0 microM) on luciferase activity in PC-3 LUC(AR+) cells were determined after exposure for 18 h in the presence and absence of DHT (50 pM). In the absence of DHT, none of the Aroclors induced luciferase activity but, in the presence of DHT (50 pM), Aroclors 1016, 1221, 1232, 1242, 1248, 1254, 1260, 5432, and 5442 acted antagonistically at concentrations that did not affect cell viability. Aroclor 5460 was without effect. Similarly, when PCBs found as human milk contaminants were assessed as individual congeners (each at 1 microM, where no cytotoxic effects were observed), none activated luciferase expression in the absence of DHT but PCBs 49, 66, 74, 105, and 118 completely antagonized the stimulation by DHT (50 pM) and PCBs 138, 153, and 156 were less effective antagonists, reducing the DHT stimulation by about 50%. Thus, 30% (by weight) of the PCBs in human milk are androgen antagonists (PCBs 66, 74, 105, and 118) and a further 25% are partial antagonists (PCBs 138, 153, and 156). A proportionally representative mixture of PCBs that contaminate human milk also caused the DHT-mediated activation of luciferase activity in PC-3 LUC(AR+) cells to be reduced by more than 50%.

Inhibition of rat testis microsomal 3β-hydroxysteroid dehydrogenase activity by tributyltin

In this study, we have examined the effects of a range of organotin compounds (mono-, di-, tributyltin, mono-, di-, trioctyltin) on the activities of rat testis microsomal 3beta-hydroxysteroid dehydrogenase (3beta-HSD), 17-hydroxylase (17-OHase) and 17beta-hydroxysteroid dehydrogenase (17beta-HSD). 17-OHase activity was inhibited by more than 50% compared with the control rate by 59 microM tributyltin (TBT) but other organotin compounds showed no inhibition. 17beta-HSD activity was unaffected by all organotins tested. 3beta-HSD was inhibited by monooctyltin (81 microM) and by TBT at all concentrations tested in a dose-dependent manner, with almost complete loss of activity at TBT concentrations of 12 microM. The mechanism of inhibition of 3beta-HSD was investigated in kinetic analysis with 0-12 microM TBT. Three rat testis microsomal preparations were incubated with dehydroepiandrosterone as the steroid substrate ranging from 1 to 10,000 nM. Tributyltin was primarily a competitive inhibitor of 3beta-HSD activity, causing an increase in the value of the K(m(app)). However, the mechanism was not entirely competitive as while there was an increase in K(m(app)), a decrease in the V(max(app)) was also observed with increasing concentrations of TBT. Slope and intercept replots demonstrated that the K(i)((app)) from slope replots was around 2.7 microM whereas the K(i)((app)) value from intercept replots was around 30 microM. When compared with the K(m(app)) for 3beta-HSD of around 0.42 microM, TBT could be an effective inhibitor of this enzyme.

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.

Differential effects of trilostane and cyanoketone on the 3β-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.

Altered testicular microsomal steroidogenic enzyme activities in rats with lifetime exposure to soy isoflavones.

Androgen production in the testis is carried out by the Leydig cells, which convert cholesterol into androgens. Previously, isoflavones have been shown to affect serum androgen levels and steroidogenic enzyme activities. In this study, the effects of lifelong exposure to dietary soy isoflavones on testicular microsomal steroidogenic enzyme activities were examined in the rat. F1 male rats were obtained from a multi-generational study where the parental generation was fed diets containing alcohol-washed soy protein supplemented with increasing amounts of Novasoy, a commercially available isoflavone supplement. A control group was maintained on a soy-free casein protein-based diet (AIN93G). The diets were designed to approximate human consumption levels and ranged from 0 to 1046.6 mg isoflavones/kg pelleted feed, encompassing exposures representative of North American and Asian diets as well as infant fed soy-based formula. Activities of testicular 3beta-hydroxysteroid dehydrogenase (3beta-HSD), P450c17 (CYP17), 17beta-hydroxysteroid dehydrogenase (17beta-HSD) were assayed on post natal day (PND) 28, 70, 120, 240 and 360 while 5alpha-reducatase was assayed on PND 28. At PND 28, 3beta-HSD activity was elevated by approximately 50% in rats receiving 1046.6 mg total isoflavones/kg feed compared to those on the casein only diet. A similar increase in activity was observed for CYP17 in rats receiving 235.6 mg total isoflavones/kg feed, a level representative of infant exposure through formula, compared to those receiving 0mg isoflavones from the casein diet. These results demonstrate that rats fed a mixture of dietary soy isoflavones showed significantly altered enzyme activity profiles during development at PND 28 as a result of early exposure to isoflavones at levels obtainable by humans.

The mammalian testis accumulates lower levels of organochlorine chemicals compared with other tissues.

Tissues were obtained from three separate experiments in order to quantify the tissue distribution of organochlorine chemicals that are thought to be potential reproductive toxicants in males: 1) Sprague Dawley rats received 1 microCi of 14C-Aldrin or 14C-Dieldrin (20.6 microCi/micromole) i.p. once a week for three weeks. One week and four weeks after the last injection, tissues were harvested and stored at -80 degrees C. Tissue 14C levels were quantified by scintillation spectrometry. 2) Cis- or trans-nonachlor (0, 0.25, 2.5, 25 mg/kg body weight) were administered daily in corn oil to male rats by gavage for 28 days. Tissues were harvested and frozen at -80 degrees C on the 29th day. Organochlorine residues were extracted and quantified by gas chromatography with electron capture detection. 3) Technical grade toxaphene (0, 0.1, 0.4 or 0.8 mg/kg body weight) was ingested daily by female cynomolgus monkeys of reproductive age for 18 months prior to being mated with control males. Dosing continued during pregnancy and lactation. Their infants received toxaphene via breast milk, and upon weaning, they ingested the same dose as their mothers for 48 to 49 weeks until, at 77 to 80 weeks of age, tissues were harvested and stored at -80 degrees C. Organochlorine residues were extracted and quantified as previously stated. In all three experiments, organochlorine residues in the testis were lower than in most of the other reproductive tract and nonreproductive tract tissues we examined. For example, testicular aldrin and dieldrin levels were <5% the epididymal content; testicular cis- and trans-nonachlor were <25% the epididymal content and, testicular toxaphene levels were <15% of the epididymal content. The reasons for the low degree of accumulation by the testis in comparison with other tissues are unknown. However, the lower testicular content may afford germ cells some protection from the potentially toxic effects of these chemicals.

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