Hepatic Testosterone Metabolism Research Articles

Developmental alterations and endocrine-disruptive responses in farmed Nile crocodiles (Crocodylus niloticus) exposed to contaminants from the Crocodile River, South Africa

In the present study, the developmental (including fertility) and endocrine-disruptive effects in relation to chemical burden in male and female Nile crocodiles (Crocodylus niloticus), from a commercial crocodile farm in the Brits district, South Africa, exposed to various anthropogenic aquatic contaminants from the natural environment was investigated. Hepatic transcript levels for vitellogenin (Vtg), zona pellucida (ZP) and ERα (also in gonads) were analyzed using real-time PCR. Plasma estradiol-17β (E2), testosterone (T) and 11-ketotestosterone (11-KT) were analyzed using enzyme immunoassay. Gonadal aromatase and hepatic testosterone metabolism (6β-hydroxylase (6β-OHase)) were analyzed using biochemical methods. Overall, there is high and abnormal number (%) of infertile and banded eggs during the studied reproductive seasons, showing up to 57 and 34% of infertile eggs in the 2009/2010 and 2013/2014 seasons, respectively. In addition, the percentage of banded eggs ranged between 10 and 19% during the period of 2009–2014 seasons. While hepatic ERα, Vtg, ZP mRNA and testosterone 6β-OHase, were equally expressed in female and male crocodiles, gonadal ERα mRNA and aromatase activity were significantly higher in females compared to male crocodiles. On the other hand, plasma T and 11-KT levels were significantly higher in males, compared to female crocodiles. Principal component analysis (PCA) produced significant grouping that revealed correlative relationships between reproductive/endocrine-disruptive variables and liver contaminant burden, that further relates to measured contaminants in the natural environment. The overall results suggest that these captive pre-slaughter farm crocodiles exhibited responses to anthropogenic aquatic contaminants with potentially relevant consequences on key reproductive and endocrine pathways and these responses may be established as relevant species endocrine disruptor biomarkers of exposure and effects in this threatened species.

Early exposure to low doses of atrazine affects behavior in juvenile and adult CD1 mice

Environmental exposure to endocrine disrupting chemicals is receiving increasing attention, with particular regard to distinct periods of development where neuroendocrine circuitries are critical for shaping the mammalian brain. Atrazine (ATZ), a widely used herbicide, has been reported to affect steroid hormones and interfere with pathways critical for sex-specific physiological and behavioral development. Aim of the present study was to evaluate effects of perinatal exposure to environmentally relevant subtoxic doses of ATZ, on neurobehavioral development in mice and investigate possible alterations in steroid hormone metabolism. Neurobehavioral development of female and male mice delivered from CD1 dams, and daily exposed from Gestational Day 14 until Postnatal Day 21 (PND 21) to 1 or 100 μg/kg bw ATZ, was investigated. Specifically, locomotor and exploratory activity, social interactions and cognitive performance were evaluated at PND 16, 31 and 60, respectively. Moreover, general toxicity clinical signs, testicular parameters, rate of testosterone metabolism and aromatase activity in F1 male liver were analyzed at adulthood. Changes in exploratory profile and in affiliative/investigative behavior were observed, revealing a feminization of behavioral profile in ATZ-exposed males. Alteration in learning performance at adulthood was also evident. A limited decreased sperm count and concentration, as well as some slight impairment in hepatic testosterone metabolism and in aromatase activity (slightly but not significantly decreased) were observed in both low and high dose exposed animals. In conclusion developmental exposure to non-toxic, environmentally relevant doses of ATZ can produce subtle functional alterations, detectable in juvenile rodents by a detailed behavioral analysis. Behavioral disturbances appeared mainly related with neurodevelopmental disorder affecting the social domain and the emotional/affective repertoire, although further research is needed to elucidate the mechanism through which the effects are induced.

Inhibition of male rat hepatic testosterone metabolism by modulation of androgenic signalling and castration

Inhibition of male rat hepatic testosterone metabolism by modulation of androgenic signalling and castration

Effects of nonylphenol on hepatic testosterone metabolism and the expression of acute phase proteins in winter flounder ( Pleuronectes americanus): Comparison to the effects of Saint John's Wort

4-Nonylphenol (4-NP), a major by-product of alkylphenol ethoxylates, is used in several industries and as a consequence is quite common in rivers, estuaries and other aquatic environments that receive sewage discharges or are near offshore oil platforms. 4-NP is an environmental estrogen that also binds human and rodent Pregnane X-receptor (PXR), the orphan nuclear receptor that controls the expression of several detoxication genes in mammals, including several CYP3A and CYP2B family members. These P450s preferentially hydroxylate testosterone in the 6β- and 16β-positions, respectively. In this study, the effects of 4-NP on testosterone metabolism and hepatic CYP3A induction were compared to the effects of St. John's Wort (SJW), a well established mammalian PXR agonist, in winter flounder. Male winter flounder ( Pleuronectes americanus) were injected with 100 mg/kg/day 4-NP or 500 mg/kg/day SJW or both (S and N) every 24 h. Forty-eight hours after the initial injections, flounder were euthanized. Western blots and testosterone 6β-hydroxylation indicated that CYP3A was increased 50% by 4-NP, but was not affected by SJW. Testosterone 16β-hydroxylase activity was also significantly increased in flounder treated with 4-NP (2.8×), but not with SJW. This is not consistent with our hypothesis that both SJW and 4-NP would induce CYP3A. Subtractive hybridization was performed between control and 4-NP treated hepatic mRNA samples to isolate differentially expressed genes. Subtractive hybridization indicated that several acute phase proteins were altered by 4-NP. Quantitative real-time PCR (Q-PCR) confirmed 4-NP altered the expression of complement components C8b, cathepsin L, C-type lectin domain, FK506 binding protein 2 precursor (FKBP2) and an EST (expressed sequence tag). SJW and 4-NP treated flounder demonstrated similar induction profiles for the EST, cathepsin L and FKBP2, suggesting that SJW was at a sufficient dose to alter gene expression but not induce P450s. In conclusion, testosterone hydroxylase activity and Western blots indicate that SJW did not activate detoxication pathways in a similar manner to 4-NP.

DDT increases hepatic testosterone metabolism in rats.

DDT and its metabolites are considered as endocrine disruptors able to promote hormone-dependent pathologies. We studied the effects of technical-grade DDT on hepatic testosterone metabolism and testosterone hydroxylase activity ratios in the rat. Male and female Wistar rats were treated by gavage with a single dose of technical-grade DDT (0, 0.1, 1, 10, and 100 mg/kg body weight) and killed 24 h later. Hepatic microsomes were incubated with [4-14C]-testosterone and the metabolites were separated by thin-layer chromatography and quantified by radio scanning. DDT increased testosterone biotransformation and modified the profile of metabolites produced in a sex-dependent manner. Males treated with a representative dose (10 mg/kg) produced relatively less androstenedione (AD), 2alpha-hydroxytestosterone (OHT), and 16alpha-OHT but higher 6beta-OHT whereas treated females produced less 7alpha-OHT and AD but higher 6beta-OHT and 6alpha-OHT than their respective controls. In both sexes DDT decreased the relative proportion of AD and increased that of 6beta-OHT suggesting that the androgen-saving pathway was affected. The testosterone 6alpha-/15alpha-OHT ratio, a proposed indicator of demasculinization, was increased in treated males. This effect was in agreement with the demasculinizing ability proposed for DDT. The effects on 6alpha-/16alpha-OHT and 6-dehydrotestosterone/16alpha-OHT ratios followed a similar tendency, with the ratio 6alpha-/16alpha-OHT being the most sensitive marker. Interestingly, these ratios were reduced in treated females suggesting that technical-grade DDT shifted testosterone hydroxylations toward a more masculine pattern. Thus, technical-grade DDT altered the hepatic sexual dimorphism in testosterone metabolism and decreased the metabolic differences between male and female rats.

Effect of dietary administration of genistein, nonylphenol or ethinyl estradiol on hepatic testosterone metabolism, cytochrome P-450 enzymes, and estrogen receptor alpha expression

The objective of this study was to examine effects of estrogenic agents of varying potencies (genistein, p-nonylphenol, and ethinyl estradiol) on hepatic testosterone metabolism, cytochrome P-450 (CYP450) enzymes, and ERα expression. These endpoints were examined as potential biomarkers of, and contributors to, endocrine disruptive activity. Exposure occurred during critical developmental periods, from gestational day 7 through weaning via the mothers' diet. Thereafter, rats were exposed via their diet to the compounds until puberty (postnatal day 50). Testosterone hydroxylase and 5α-reductase activities, CYP2C and CYP3A levels were determined. In general, the compounds were more active in male rats than female rats. The only effect observed in female rats was at the 250 ppm genistein dose, in which an approximately 40% increase in 5α-reductase activity was observed. In male rats, genistein treatment had mixed effects on testosterone metabolism. The 1250 ppm dose decreased both CYP2C and CYP3A protein levels. Nonylphenol had the most profound effects on testosterone metabolism and CYP450 expression in male rats, with effects occurring at doses as low as 25 ppm. An increase in 5α-reductase activity and a decrease in the formation of 16α-OH-, 2α-OH-testosterone metabolites, CYP2C and CYP3A protein were observed. EE2 decreased the formation of several testosterone metabolites and CYP2C protein. All compounds had some effect on hepatic ERα expression, although a consistent effect was not observed. This study demonstrates that the test compounds can influence hepatic testosterone hydroxylase activity and CYP450 expression, as well as ERα expression, although these activities cannot be directly related to estrogenic activity.

Developmental effects and health aspects of soy protein isolate, casein, and whey in male and female rats.

Dietary factors other than the traditional nutrients are found in the so-called functional foods. They are becoming increasingly recognized as potentially important for maintaining good health. Soybeans are rich in such factors thought to help prevent certain chronic diseases. Soy protein isolate (SPI) is one of the three major proteins used in infant formulas sold in the United States, with casein (CAS) and whey (WPH) proteins being the others. We have been studying the health effects of these proteins. Safety concerns have developed over the consumption of soy-based infant formula, partly because of the high circulating levels of the total isoflavones (phytoestrogens) during "critical periods of infant development." There is a paucity of data on developmental, physiological, neurophysiological, behavioral, metabolic, or molecular effects of soy phytochemicals in humans, especially during pregnancy and infancy. We have studied the effects of CAS, SPI, and WPH in short-term, long-term, and multigenerational studies in rats. Aside from minor differences in body weight gain profiles, CAS-, SPI- or WPH-fed rats did not differ in development, organ weights, in vitro hepatic metabolism of testosterone (T), or reproductive performance. However, some endocrine-related functions differed between rats fed these proteins. We found that SPI accelerated puberty in female rats (p < .05) and WPH delayed puberty in males and females, as compared with CAS (p < .05). Gender differences were also found in gonadectomy-induced steroid responses. Male rats had normal serum T levels, but female rats fed SPI had reduced serum 17beta-estradiol concentrations and a blunted 17beta-estradiol response to ovariectomy, as compared to rats fed CAS or WHP (p < .05). Female rats fed SPI or WHP or treated with genistein had reduced incidence of chemically induced mammary cancers (p < .05) compared to CAS controls, with WHP reducing tumor incidence by as much as 50%, findings that replicate previous results from our laboratory. Together, these results suggest gender-specific differences in development and certain endocrine responses among rats fed diets composed of a single protein source such as those used in infant formulas. Whether similar developmental effects occur in human infants is unknown, but unlikely because (1) most infants do not consume such diets throughout life as these rats did, and (2) no such effects have been reported in millions of American infants fed infant formula containing these proteins. The long-term health consequence implications of early diet exposure to SPI and WPH, such as reduced breast cancer incidence, are likely to be very positive.

Different Inactivation Mechanisms of NADPH- and NADH-Dependent Chains of Rat Liver Endoplasmic Reticulum in Tetrachloromethane

Cobalt-protoporphyrin (CoPP) administration to adult male rats results in a profound reduction in hepatic cytochrome P-450 concentration and activity, and decreased plasma concentrations of testosterone and luteinizing hormone (LH). The metabolism of progesterone by rat testicular microsomes isolated 48 h after treatment in vivo with CoPP was compared to that in microsomes from control rats. The conversion of progesterone to 17α-hydroxyprogesterone and 4-androstenedione, which is NADPH-dependent, was reduced by approximately 40% in testicular microsomes following treatment with CoPP (50 μmol/kg body weight) and this inhibition was dose-dependent. The concentration of cytochrome P-450 in testicular microsomes and the activity of 7-ethoxycoumarin de-ethylase (a cytochrome P-450 dependent function) were also reduced following treatment with CoPP in contrast to two other functional assays of cytochrome P-450, aryl hydrocarbon hydroxylase and ethylmorphine demethylase, which were unaffected by treatment with CoPP. Thus, the profound effect of CoPP on androgen homeostasis has been extended to include decreased testicular synthesis of 4-androstenedione in addition to increased hepatic metabolism of testosterone, attenuated pituitary LH release in response to luteinizing hormone-releasing hormone, and failure of testicular response to LH.

Cadmium-induced sexual dysfunction does not involve increased hepatic metabolism of testosterone nor increased circulating levels of corticosterone

Sexually experienced male rats were injected IP with 0, 0.3, 1.5, 3.0, or 6.0 mg/kg cadmium chloride. The highest dose was fatal within 48 h of injection. A dose-related deficit in erectile function was observed in ex copula tests 48 h after injection. Copulatory dysfunction was evident in mating tests 72 h after injection. Hepatic mixed function oxidase activity after 0.3 mg/kg injections was not different from controls, but was diminished by 50% in rats treated with 3.0 mg/kg. The higher cadmium doses (1.5 and 3.0 mg/kg) were associated with equivalent and marked reductions in circulating testosterone levels, and lesser decrements in circulating corticosterone levels. The rats treated with 1.5 or 3.0 mg/kg cadmium chloride lost weight (32 ± 12 and 39 ± 9 g, respectively). Relative decapsulated adrenal gland weights were markedly increased in rats treated with 3.0 mg/kg. These data indicate that exposure to cadmium is associated with alterations in sexual, hepatic, and adrenal function, with erectile dysfunction occurring at the lowest dose. The effects on copulatory behavior are especially striking because it requires some weeks for copulatory behavior to wane following castration.

Selective inhibition of cytochrome P-450 in rat testicular microsomes: Effect of cobalt-protoporphyrin on progesterone metabolism

Cobalt-protoporphyrin (CoPP) administration to adult male rats results in a profound reduction in hepatic cytochrome P-450 concentration and activity, and decreased plasma concentrations of testosterone and luteinizing hormone (LH). The metabolism of progesterone by rat testicular microsomes isolated 48 h after treatment in vivo with CoPP was compared to that in microsomes from control rats. The conversion of progesterone to 17 alpha-hydroxyprogesterone and 4-androstenedione, which is NADPH-dependent, was reduced by approximately 40% in testicular microsomes following treatment with CoPP (50 mumol/kg body weight) and this inhibition was dose-dependent. The concentration of cytochrome P-450 in testicular microsomes and the activity of 7-ethoxycoumarin de-ethylase (a cytochrome P-450 dependent function) were also reduced following treatment with CoPP in contrast to two other functional assays of cytochrome P-450, aryl hydrocarbon hydroxylase and ethylmorphine demethylase, which were unaffected by treatment with CoPP. Thus, the profound effect of CoPP on androgen homeostasis has been extended to include decreased testicular synthesis of 4-androstenedione in addition to increased hepatic metabolism of testosterone, attenuated pituitary LH release in response to luteinizing hormone-releasing hormone, and failure of testicular response to LH.

Effects of aging and caloric restriction on hepatic drug metabolizing enzymes in the Fischer 344 rat. I: The cytochrome P-450 dependent monooxygenase system

The effects of long-term caloric restriction on the hepatic cytochrome P-450 dependent monooxygenase system were investigated in the 22-month-old Fischer 344 rat. Caloric restriction decreased the age-related changes in hepatic testosterone metabolism, which are associated with demasculinization of the liver. Caloric restriction also increased hepatic microsomal testosterone 6β-hydroxylase, lauric acid 12-hydroxylase and 4-nitrophenol hydroxylase activities over corresponding values in both ad libitum fed 22-month and 60-day-old control male rats. This suggests that cytochrome P-450 isozymes, P-450 pcn 1&2, P-452 and P450 j may be induced by caloric restriction. Such changes in cytochrome P-450 isozyme profiles could result in altered carcinogen activation, radical formation or drug detoxication in the calorically restricted rat.

Hepatic testosterone metabolism in male rats with portal bypass

This study was performed to ascertain whether testosterone metabolism is altered in male rats with portal bypass, and whether such changes could contribute to the reduction in serum testosterone concentration and raised serum estrogen levels that are observed in this situation. The metabolic clearance rate of testosterone was determined by a prime-dose constant-infusion technique in male rats subjected to complete portal vein ligation and in sham-operated controls. Testosterone clearance was similar in rats with portal vein ligation and control rats (9.01 ± 2.29 and 8.26 ± 2.83 ml/min, respectively) but the clearance per gram of liver was greater in rats with portal vein ligation than in controls (1.18 ± 0.18 versus 0.68 ± 0.24 ml/min · g liver, p < 0.0001). After 180 min of [3H]testosterone infusion, [3H]estradiol comprised 1.2% of plasma total radioactivity in male controls but was increased to 11% in rats with portal vein ligation (p < 0.005). Similarly, biliary excretion of [3H]estradiol was eightfold greater in male rats with portal vein ligation compared with controls (p < 0.001). In control male rats, the major metabolites of testosterone present in bile were 2α-hydroxytestosterone, 16α-hydroxytestosterone, and 7α-hydroxytestosterone. Portal bypass was associated with reduced biliary excretion of 2α-hydroxytestosterone and 16α-hydroxytestosterone to ~50% of control, but there was no change in the excretion of 7α-hydroxytestosterone. Conversely, portal bypass was associated with increased formation of dihydrotestosterone, indicating stimulated activity of testosterone 5α-reductase. It is concluded that portal bypass in male rats is associated with altered pathways of testosterone metabolism and, in particular, with increased aromatization of testosterone to estradiol. The site of such estradiol formation has not been determined by this in vivo study. However, selective changes occurred in the regiospecific and stereospecific hydroxylation pathways of testosterone and in 5α-reductase activity after portal bypass in male rats. It is concluded that portal bypass, in the absence of parenchymal liver damage, results in demasculinization and feminization of C19 steroid metabolism in the male rat liver. These metabolic changes could be revelant to the pathogenesis of changes in sexual characteristics in cirrhosis.

Effects of chronic smoking on testosterone metabolism in dogs

The effects of long-term cigarette smoking on androgen hydroxylases and peripheral hormones were studied in male beagles. In the testis, chronic smoking of high nicotine/tar cigarettes was associated with decreased activity of the 7α-hydroxylase active on testosterone (68% of control, P < 0.05). Testicular 6β and 16α-hydroxylases were not altered. The hepatic androgen 6β-hydroxylase activity in control animals was approximately 6 times the testis levels and was stimulated markedly by smoking. This increase ranged from 221% in the low nicotine/tar group ( P < 0.02) to 304% in the high nicotine/tar group ( P < 0.006). Serum testosterone levels were reduced to 54% of control ( P < 0.02) and prostate size to 44% ( P < 0.001) of control with heavy smoking. Serum LH levels were elevated with smoking. These results suggest that chronic cigarette smoking increased hepatic metabolism of testosterone. In addition, serum testosterone levels and prostate size decreased and LH levels increased. Whether the hepatic and the endocrine effects are causally related cannot be determined from this preliminary study.

The influence of fetal and neonatal exposure to dichlorodiphenyltrichloroethane (DDT) on the testosterone status of neonatal male rat

The testicular synthesis and hepatic metabolism of testosterone and the plasma concentrations of androgens were measured in 2-, 4-, 6-, 8-, or 10-day-old male rats after a 10-day prenatal and variable postnatal exposure to dichlorodiphenyltrichloroethane (DDT) via maternal milk secretion. While there were significant elevations in the metabolism of testosterone and aminopyrine by liver microsomes from DDT-exposed rats there were no compensatory changes in either serum androgens or testicular synthesis of testosterone. The failure of DDT exposure to change plasma concentrations of androgens in spite of substantial increase in hepatic metabolism was further confirmed in male rats treated with human chorionic gonadotrophin to produce elevated testosterone concentrations. Tissue analysis showed 14C-labeled DDT and/or its metabolites crossed the placenta and was present in fetal rats (Day 21), but cross fostering of litters from treated and control rats indicated that the major response to DDT occurs upon exposure after birth. It is suggested that hepatic enzyme induction by DDT is essentially a postnatal phenomenon and the DDT is transferred in substantial amounts from the milk of lactating rats to their offspring.

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the hepatic metabolism of testosterone in the rat

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the hepatic metabolism of testosterone in the rat

Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the hepatic metabolism of testosterone in the rat

A dose of 20 micrograms/kg of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was given to adult male rats intragastrically and the metabolism of [4-14C]-testosterone by liver homogenate was examined 1 week later. The overall catabolism of testosterone was significantly reduced by the TCDD treatment. Dihydroxysteroids (intermediate reduced metabolites) were increased, while the production of polar non-conjugated metabolites (end products) was diminished.

Enhanced hepatic metabolism of testosterone, 4-androstene-3, 17-dione, and estradiol-17β in chickens pretreated with ddt or PCB

Recently there has been a large amount of interest in chlorinated hydrocarbons as mediators of adverse ecological changes in the biosphere. It has been suggested R. Kuntzman et al . Advance in Enzyme Regulation 4 , 249 (1966) that DDT induces changes in the metabolism of several steroid sex hormones possibly leading to the thining of eggshells and reproductive failure. Also, it has been reported that polychlorinated biphenyls (PCB's) are present in the tissues of various forms, of wildlife. Since the chemical formulae of PCB's are similar to that of DDT it has been suggested that their interactions in biological systems may be similar. A post-mitochondrial hepatic fraction (microsomes + cytosol) from untreated cockerels and pullets was shown to metabolize testosterone (I), estradiol-17β (II) and 4-androstene-3, 17-dione (III) in an in vitro incubation system when fortified with a TPNH-generating system. Cockerels had a higher metabolic rate of metabolism of I, II, or III compared to corresponding pullets. Treatment of either sex with o,p'-DDT or p,p'-DDT increased the metabolic rate of metabolism of I, II, or III approximately 3-fold over untreated cockerels or pullets. The PCB's mimicked the action of DDT in that they also enhanced the hepatic metabolism of natural steroid hormones.