Hepatic Steroid Metabolism Research Articles

Inhibition of Ileal Bile Acid Transport is Protective Against Cholestatic Liver Injury in Cyp2c70‐/‐Mice with Humanized Bile Acid Composition

Cyp2c70 is the key murine liver enzyme responsible for the synthesis of 6‐hydroxylated muricholates. Cyp2c70 knockout (2c70‐/‐) mice present with a more human‐like bile acid (BA) composition devoid of protective, hydrophilic muricholic acids and with features of cholestatic liver injury. Pharmacological inhibition of the ileal BA transporter (IBAT) has been shown to be protective in cholestatic models such as the Mdr2‐/‐ mouse. The aims of this study were to determine if IBAT inhibition is protective in 2c70‐/‐ mice and identify the underlying molecular mechanisms.Methods2c70‐/‐ mice (C57Bl/6J) were generated using CRISPR/Cas9 technology. Male and female wild type (WT) and 2c70‐/‐ mice were fed chow or chow containing 0.006% SC‐435 for 9 weeks following weaning at 3 weeks of age. Standard growth parameters were recorded; organs, gallbladder bile and blood were collected at necropsy for histologic, RNA and biochemical analyses.ResultsMale and female 2c70‐/‐ mice maintained on chow diet exhibited increased levels of serum liver injury markers ALT, AST, and BAs compared to WT mice. Evaluation of 2c70‐/‐ liver histology revealed increased hepatic inflammation, macrophage infiltration and bilary cell proliferation, which were markedly reduced with SC‐435 treatment. IBAT inhibition in 2c70‐/‐ mice improved liver histology, and reduced hepatic inflammation and bilary cell proliferation to levels indistinguishable from WT. The liver BA content was increased by 53% in 2c70‐/‐ mice, and was enriched in more hydrophobic BA species with an overall hydrophobicity similar that of humans (Figure 1). Treating 2c70‐/‐ mice with SC‐435 reduced the hepatic BA burden by 61%, but did not affect the overall hydrophobicity. RNA‐Seq and KEGG pathway analysis performed using liver RNA revealed genes involved in hepatic steroid and lipid metabolism (Sult2a1, Sult1e1, Hsd17b7), and detoxification (Cyp2c39, Cyp2c54, Cyp2c59, Cyp3a41a/b) were down‐regulated in 2c70‐/‐ mice and their expression were rescued with SC‐435 treatment. Conversely, genes in pathways related to immune cell activation and inflammation were significantly up‐regulated in 2c70‐/‐ mice and reduced to WT levels with IBAT inhibition (Figure 2).ConclusionsOur findings indicate that reducing the hepatic BA burden without altering the hydrophobic compostion is sufficient to protect 2c70‐/‐ mice from hepatic injury. The findings suggest that there may be a threshold in liver for pathological accretion of hydrophobic BAs and reducing the hepatic BA pool is sufficient to alleviate liver injury, independent of hydrophobicity. Moreover, these data support the inhibition IBAT activity, and other approaches to reduce hepatic return of BAs in the enterohepatic circulation, as therapeutic targets to treat cholestasis.

Modifications of Western-type diet regarding protein, fat and sucrose levels as modulators of steroid metabolism and activity in liver

The aim of the study was to evaluate whether the modification of the Western-type diet (high-fat, high-sucrose diet rich in saturated fatty acids) considering macronutrients content would influence hepatic metabolism and activity of steroids. For 3 weeks Wistar rat were fed the Western-type diet (21% fat, 35% sucrose, 19% protein, lard) and its modifications regarding dietary protein (10 and 19%), fat (5 and 21%) and sucrose (0 and 35%) levels. The steroid 5α-reductase type 1 (Srd5a1) and androgen receptor (Ar) gene expression as well as testosterone (T) conversion towards 5α-reduced derivatives in liver were positively correlated with body weight gain. The Western-type diets with decreased protein content regardless of the sucrose level exerted the most negative effect on the antioxidant system decreasing catalase (Cat), sodium dismutase (Sod1) and glutathione peroxidase (Gpx1) gene expression as well as Cat and Gpx activity and total antioxidant status, simultaneously intensifying lipid peroxidation. The impaired antioxidant system was accompanied by decreased level of hepatic T metabolism towards estrogens: 17β-estradiol (E2) and estriol, and increased estrogen receptor type 1 (Esr1) gene expression. Liver Esr1 mRNA level was differently correlated with T (positively) and E2 (negatively) plasma levels. Whereas the fat reduction in Western-type diet restored the plasma proportion between T and E2. In conclusion it could be stated that Western-type diet modification relating to protein, sucrose and fat content can influence hepatic steroid metabolism and activity; however the estrogens and androgens metabolism in liver would be connected with impairment of liver function or catabolic activity, respectively.

3,5-T2 alters murine genes relevant for xenobiotic, steroid, and thyroid hormone metabolism

The endogenous thyroid hormone (TH) metabolite 3,5-diiodo-l-thyronine (3,5-T2) acts as a metabolically active substance affecting whole-body energy metabolism and hepatic lipid handling in a desirable manner. Considering possible adverse effects regarding thyromimetic action of 3,5-T2 treatment in rodents, the current literature remains largely controversial. To obtain further insights into molecular mechanisms and to identify novel target genes of 3,5-T2 in liver, we performed a microarray-based liver tissue transcriptome analysis of male lean and diet-induced obese euthyroid mice treated for 4 weeks with a dose of 2.5 µg/g bw 3,5-T2 Our results revealed that 3,5-T2 modulates the expression of genes encoding Phase I and Phase II enzymes as well as Phase III transporters, which play central roles in metabolism and detoxification of xenobiotics. Additionally, 3,5-T2 changes the expression of TH responsive genes, suggesting a thyromimetic action of 3,5-T2 in mouse liver. Interestingly, 3,5-T2 in obese but not in lean mice influences the expression of genes relevant for cholesterol and steroid biosynthesis, suggesting a novel role of 3,5-T2 in steroid metabolism of obese mice. We concluded that treatment with 3,5-T2 in lean and diet-induced obese male mice alters the expression of genes encoding hepatic xenobiotic-metabolizing enzymes that play a substantial role in catabolism and inactivation of xenobiotics and TH and are also involved in hepatic steroid and lipid metabolism. The administration of this high dose of 3,5-T2 might exert adverse hepatic effects. Accordingly, the conceivable use of 3,5-T2 as pharmacological hypolipidemic agent should be considered with caution.

Distinguishing primary from secondary Δ(4) -3-oxosteroid 5β-reductase (SRD5B1, AKR1D1) deficiency by urinary steroid analysis.

Deficiency of Δ(4) -3-oxosteroid 5β-reductase (5β-reductase), a bile acid synthesis disorder, presents findings of neonatal cholestasis and hyper-3-oxo-Δ(4) bile aciduria. The 5β-reductase enzyme participates in not only bile acid synthesis but also hepatic steroid metabolism. Deficiency of 5β-reductase includes 2 types: primary deficiency, with an SRD5B1 gene mutation; and secondary deficiency, lacking a mutation. Secondary deficiency is caused by fulminant liver failure from various aetiologies including neonatal hemochromatosis (NH). Distinguishing primary from secondary deficiency based on γ-glutamyltransferase (GGT), serum total bile acids (TBA), and urinary bile acid analysis using gas chromatography-mass spectroscopy (GC-MS) is very difficult. SRD5B1 gene analysis is the only reliable method. We examined urinary steroid analysis as a way to distinguish primary from secondary 5β-reductase deficiency. We examined 12 patients with cholestatic jaundice, normal or slightly elevated GGT, and hyper-3-oxo-Δ(4) bile aciduria using urinary steroid analysis by GC-MS of both cortisol and cortisone compounds, such as 5β-tetrahydrocortisol (5β-THF) and 5β-tetrahydrocortisone (5β-THE). Patients previously were diagnosed with primary 5β-reductase deficiency (n = 3), deficiency secondary to NH (n = 3) and deficiency secondary to other liver disorders (n = 6). Urinary steroid analysis in 3 primary deficiency and 3 NH patients showed low 5β-THE and elevated 5α/5β-THE ratios, making distinction difficult without also considering the clinical course and abdominal magnetic resonance imaging (MRI) findings, such as a very low signal intensity in liver and/or pancreas, especially in T2 -weighted images. In the six patients with other secondary deficiencies, urinary 5β-THF and 5α/5β-THF differed from those in primary deficiency (P < 0·05). Urinary steroid analysis can distinguish primary and NH-related deficiencies from other secondary deficiencies.

Investigation of Testosterone, Androstenone, and Estradiol Metabolism in HepG2 Cells and Primary Culture Pig Hepatocytes and Their Effects on 17βHSD7 Gene Expression

Steroid metabolism is important in various species. The accumulation of androgen metabolite, androstenone, in pig adipose tissue is negatively associated with pork flavor, odour and makes the meat unfit for human consumption. The 17β-hydroxysteroid dehydrogenase type 7 (17βHSD7) expressed abundantly in porcine liver, and it was previously suggested to be associated with androstenone levels. Understanding the enzymes and metabolic pathways responsible for androstenone as well as other steroids metabolism is important for improving the meat quality. At the same time, metabolism of steroids is known to be species- and tissue-specific. Therefore it is important to investigate between-species variations in the hepatic steroid metabolism and to elucidate the role of 17βHSD7 in this process. Here we used an effective methodological approach, liquid chromatography coupled with mass spectrometry, to investigate species-specific metabolism of androstenone, testosterone and beta-estradiol in HepG2 cell line, and pig cultured hepatocytes. Species- and concentration-depended effect of steroids on 17βHSD7 gene expression was also investigated. It was demonstrated that the investigated steroids can regulate the 17βHSD7 gene expression in HepG2 and primary cultured porcine hepatocytes in a concentration-dependent and species-dependent pattern. Investigation of steroid metabolites demonstrated that androstenone formed a 3′-hydroxy compound 3β-hydroxy-5α-androst-16-ene. Testosterone was metabolized to 4-androstene-3,17-dione. Estrone was found as the metabolite for β-estradiol. Inhibition study with 17βHSD inhibitor apigenin showed that apigenin didn’t affect androstenone metabolism. Apigenin at high concentration (50 µM) tends to inhibit testosterone metabolism but this inhibition effect was negligible. Beta-estradiol metabolism was notably inhibited with apigenin at high concentration. The study also established that the level of testosterone and β-estradiol metabolites was markedly increased after co-incubation with high concentration of apigenin. This study established that 17βHSD7 is not the key enzyme responsible for androstenone and testosterone metabolism in porcine liver cells.

Diverse effects of oats on cholesterol metabolism in C57BL/6 mice correlate with expression of hepatic bile acid-producing enzymes

We previously reported that two substrains of C57BL/6 mice respond differently to oats with respect to reduction in plasma cholesterol. Analysis of this difference might offer clues to mechanisms behind the cholesterol-lowering effect of oats. Here, we address the possible roles of hepatic steroid metabolism and the intestinal microbiota in this respect. Female C57BL/6 mice were fed an atherogenic diet with oat bran (27 %) or control fibres for 4 weeks. C57BL/6 NCrl mice responded to oat bran with 19 ± 1 % (P < 0.001) lower plasma cholesterol, 40 ± 5% (P < 0.01) higher excretion of bile acids and increased expression of the bile acid-producing hepatic enzymes CYP7A1 and CYP8B1, but none of these effects were found in C57BL/6JBomTac mice. However, on control diet, C57BL/6JBomTac had tenfold higher expression of CYP7A1 and levels of hepatic cholesterol esters than C57BL/6NCrl mice. Plasma levels of fructosamine indicated improved glycemic control by oat bran in C57BL/6NCrl but not in C57BL/6JBomTac. C57BL/6JBomTac had higher intestinal microbiota diversity, but lower numbers of Enterobacteriaceae, Akkermansia and Bacteroides Fragilis than C57BL/6NCrl mice. Oat bran increased bacterial numbers in both substrains. Microbiota diversity was reduced by oats in C57BL/6JBomTac, but unaffected in C57BL/6NCrl. Our data do not support a connection between altered microbiota diversity and reduced plasma cholesterol, but the bacterial composition in the intestine may influence the effects of added fibres. The cholesterol-lowering properties of oats involve increased production of bile acids via the classical pathway with up-regulation of CYP7A1 and CYP8B1. Altered cholesterol or bile acid metabolism may interfere with the potential of oats to reduce plasma cholesterol.

Expression Analysis of Hepatic Mitochondria-Related Genes in Mice Exposed to Acrylamide and Glycidamide

Acrylamide (AA) is an industrial chemical that has been extensively investigated for central nervous system (CNS), reproductive, and genetic toxicity. However, AA effects on the liver, a major organ of drug metabolism, have not been adequately explored. In addition, the role of mitochondria in AA-mediated toxicity is still unclear. Changes in expression levels of genes associated with hepatic mitochondrial function of male transgenic Big Blue (BB) mice administered 500 mg/L AA or an equimolar concentration (600 mg/L) of its reactive metabolite glycidamide (GA) in drinking water for 3 and 4 wk, respectively, were examined. Transcriptional profiling of 542 mitochondria-related genes indicated a significant downregulation of genes associated with the 3-beta-hydroxysteroid dehydrogenase family in AA- and GA-treated mice, suggesting a possible role of both chemicals in altering hepatic steroid metabolism in BB mice. In addition, genes associated with lipid metabolism were altered by both treatments. Interestingly, only the parental compound (AA) significantly induced expression levels of genes associated with oxidative phosphorylation, in particular ATP synthase, which correlated with elevated ATP levels, indicating an increased energy demand in liver during AA exposure. Acrylamide-treated mice also showed significantly higher activity of glutathione S-transferase in association with decreased levels of reduced glutathione (GSH), which may imply an enhanced rate of conjugation of AA with GSH in liver. These results suggest different hepatic mechanisms of action of AA and GA and provide important insights into the involvement of mitochondria during their exposures.

Lead and Cadmium Co-exposure Mediated Toxic Insults on Hepatic Steroid Metabolism and Antioxidant System of Adult Male Rats

The redox status and steroid metabolism of liver of adult male rat exposed to lead (Pb) and cadmium (Cd) either alone or in co-exposure (0.025 mg/kg body weight intraperitoneally/15 days) was studied. Pb and Cd significantly accumulated in the liver. The activity of steroid metabolizing enzymes 17-betahydroxysteroid oxidoreductase and uridine diphosphate-glucuronyltransferase were decreased in experimental animals. 17-beta-Hydroxysteroid dehydrogenase was reduced to 33%, 38%, and 24% on treatment of Pb, Cd, and co-exposure (Pb + Cd). Furthermore, the activity of uridine diphosphate-glucuronosyltransferase was significantly reduced to 27% (Pb exposure), 36% (Cd exposure), and 25% (co-exposure of Pb + Cd). Cd exposure exhibited more toxic effect than Pb, while co-exposure demonstrated the least. The activities of antioxidant enzymes, superoxide dismutase, catalase, glutathione reductase, and glucose-6-phosphate dehydrogenase decreased and glutathione peroxidase increased in mitochondrial and post-mitochondrial fractions. The level of lipid peroxidation increased, and cellular glutathione concentration decreased. Hepatic DNA was decreased, whereas RNA content and the activity of alanine transaminase remained unchanged. Histological studies revealed that only Cd-exposed groups exhibited cytotoxic effect. These results suggest that when Pb and Cd are present together in similar concentrations, they exhibited relatively decreased toxic effect when compared to lead and cadmium in isolation with regard to decreased steroid metabolizing and antioxidant enzyme activities. This seems that the toxic effect of these metals is antagonized by co-exposure due to possible competition amongst Pb and Cd for hepatic accumulation.

Antiandrogenic effects of oestradiol on enzyme activities of hepatic steroid metabolism.

Intact female rats and gonadectomized rats of both sexes were administered 0.5 mg 5 alpha-dihydrotestosterone s.c. daily for 15 days. On the 16th day the activities of hepatic microsomal 3 alpha- and 3 beta-hydroxysteroid dehydrogenase and 5 alpha-reductase were determined. Androgen treatment increased the activity of both hydroxysteroid dehydrogenases and decreased the activity of the 5 alpha-reductase in all animal models. Simultaneous administration of a steroidal antiandrogen, cyproterone (5 mg/day; 15 days s.c.) did not prevent this androgenic effect, whereas oestradiol (5 micrograms/day; 15 days s.c.) completely reversed the action of 5 alpha-dihydrotestosterone. The nonsteroidal antiandrogen, flutamide, also exhibited a potent antiandrogenic influence on these enzyme activities in female rats, whereas its antagonizing action in males was limited. The effects of cyproterone, oestradiol and flutamide on enzyme activities of hepatic steroid metabolism do not reflect the biological activity of these compounds on the weights of target organs (uterus, seminal vesicle, levator ani).

Contradictory evidence concerning the "universal" role of growth hormone in the regulation of the enzyme activities of hepatic steroid metabolism.

Recent publications suggest that the sexual dimorphism observed in the activities of enzymes involved in drug and steroid metabolism in rat liver are due to sex-specific differences in the rate of growth hormone release. In this paper we set out to demonstrate that this hypothesis cannot be generalized, but has its limitations. Prepuberal hypophysectomy led to the expected "masculinization" of the activities of cytoplasmic 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSDH), microsomal 3 alpha-HSDH and microsomal 5 alpha-reductase which could be reversed by continuous infusion of human growth hormone (hGH). However, one activity did not conform to this pattern: cytoplasmic 17 beta-HSDH activity reacted to hypophysectomy with a "feminization" and was completely unaffected by hGH infusion. Moreover, microsomal 3 alpha-HSDH in hypophysectomized rats was "feminized" as efficiently by infusion of ovine prolactin (oPRL) as by hGH. Ablation of the pituitary caused loss of measurable cytoplasmic receptor oestrogen concentrations. The inability of either hypophyseal hormone to cause consistent and significant elevation of oestrogen receptor concentrations is probably due to the early age at which the animals were hypophysectomized.

Age- and sex-dependent expression of multiple murine hepatic hydroxysteroid sulfotransferase (SULT2A) genes

Hydroxysteroid sulfotransferase (SULT2A) enzymes play important roles in hepatic steroid and xenobiotic metabolism. Unlike humans, which express one SULT2A, inspection of mouse genome information indicated the presence of seven SULT2A genes within a cluster on chromosome 7. The age- and sex-dependent expressions of the seven murine SULT2A family members were characterized in the livers of C57BL/6 mice using real-time RT-PCR. The transcripts for three of the SULT2A forms (NCBI reference/model sequences XM_001471624, NM_009286 and NM_001111296) were abundant in pre-pubertal male and female mouse liver but were essentially silenced in the livers of adult male mice. The mRNAs of three other SULT2A forms ( NM_001101534, XM_894052 and NM_001081325) were also expressed in pre-pubertal male and female mouse liver, but at markedly reduced levels relative to those of the abundant forms. The mRNA levels of these lower-abundance forms were further suppressed in adult animals. A seventh SULT2A mRNA ( XM_983034) was expressed in adult male and female mouse liver, but was not detected in pre-pubertal mouse liver of either sex. Full-length amplifications with primers targeting untranslated regions confirmed that all SULT2A forms were expressed. However, while the XM_001471624, NM_001111296, NM_001101534, XM_894052 and NM_001081325 transcripts were detected at their predicted sizes, the NM_009286 and XM_983034 transcripts each lacked two predicted exons. These results demonstrate that seven murine SULT2As display different profiles of age- and sex-dependent hepatic expression.

Growth Hormone Regulation of Sex-Dependent Liver Gene Expression

The liver is a primary target for the action of GH, a pituitary protein hormone that regulates a broad range of physiological processes, including long bone growth, fatty acid oxidation, glucose uptake, and hepatic steroid and foreign compound metabolism. GH exerts sex-dependent effects on the liver in many species, with many hepatic genes, most notably genes coding for cytochrome P450 (CYP) enzymes, being transcribed in a sex-dependent manner. Sex differences in CYP expression are most striking in rats and mice (up to 500-fold male-female differences), but are also seen, albeit to a much smaller degree, in humans, where they are an important determinant of the sex dependence of hepatic drug and steroid metabolism. This article examines the mechanisms whereby GH, via its sex-dependent temporal patterns of pituitary release, activates intracellular signaling leading to the sexually dimorphic transcription of CYPs and other liver-expressed genes. Recent findings implicating the GH-regulated transcription factor STAT5b (signal transducer and activator of transcription 5b), hepatocyte nuclear factors 3beta, 4alpha and 6, and sex differences in DNA methylation and chromatin structure in the sex-dependent actions of GH are reviewed, and current mechanistic models are evaluated.

Sex and the Liver – A Journey Through Five Decades

Metabolism of steroids and drugs in rodents is sexually differentiated. The reason for this turned out to be the sexually differentiated growth hormone (GH) secretory pattern regulating the expression of a number of hepatic cytochrome P-450 genes. Although not fully resolved, it is clear that several signaling pathways and transcription factors are involved in mediating the effects of GH. It may be argued that such a well-controlled physiological system should have an important biological role and we speculate that the demands of a robust hepatic steroid metabolism during pregnancy has led to the development of this sexually differentiated hypothalamo-pituitary-liver axis.

Gender‐specific expression of liver organic anion transporters in rat

Sex differences in drug pharmacokinetics have been well recognized and gender has been considered a risk factor for adverse events to medications. The aim of this study was to investigate the effect of gender on the expression of hepatocellular transport proteins involved in uptake and secretion of organic anions in rat. Expression of the rat liver organic anion transporting polypeptides (Oatps) and multidrug resistance proteins (Mrps) was analysed by reverse transcription polymerase chain reaction (RT-PCR), immunoblot analysis and immunofluorescence microscopy in male and female rats. Regulation of these transport proteins in response to the steroid dehydroepiandrosterone (DHEA) was investigated. In untreated rats, protein expression significantly differed between genders being higher (Mrp2, Mrp3), comparable [Oatp1a1 (Oatp1); Oatp1b2 (Oatp4)] or lower [Oatp1a4 (Oatp2)] in female than in male rat. DHEA treatment over 3 days (100 mg d(-1)) led to a further increase in Mrp3 expression only in female rats. Mrp2 expression was not influenced by DHEA treatment. Oatp1a1 and Oatp1b2 were significantly down-regulated after DHEA treatment in both male and female rats. In contrast, Oatp1a4 was down-regulated in male rats only. In rat, liver transport proteins of the Oatp and Mrp family are expressed and regulated in a gender-specific manner according to sexual differences in the hepatic metabolism of steroids and drugs. These findings may partly explain the well-known sex differences in hepatic handling of organic anions.

Steroids and the Scientist

Our interest in nuclear receptors (NRs) originated from early studies on hepatic steroid metabolism. We discovered a new hypothalamo-pituitary-liver axis, imprinted neonatally by androgens and operating through sexually differentiated GH secretory patterns. Male and female patterns have opposite effects on sexually differentiated hepatic genes, explaining sexually dimorphic liver patterns. To further understand steroid action, we purified the glucocorticoid receptor (GR) leading to our discovery of the NR three-domain structure, with separable DNA binding domain and ligand binding domains and a third domain now known to have transcriptional regulatory properties. Knowledge of this domain structure has been immensely important for deciphering NR actions. Using this first purified NR, we collaborated with Keith Yamamoto and first demonstrated specific NR binding to DNA. This also was the first demonstration of a mammalian transcription factor, a breakthrough that led to discovery of NR response elements. In further collaboration with Yamamoto, we cloned the first NR cDNA sequences, leading to cloning of the superfamily of NR genes. With Yamamoto and Kaptein, we determined the first three-dimensional NR structure, that of DNA binding domain. Later work on orphan receptors resulted in the first discovery of: 1) endogenous ligands for an orphan receptor (fatty acids as activators of peroxisomal proliferator-activated receptor alpha); 2) liver X receptor beta (OR-1) and its role in central nervous system cholesterol homeostasis; and 3) estrogen receptor beta, leading to a paradigm shift in understanding of estrogen signaling, of importance in endocrinology, immunology, and oncology and to development of estrogen receptor beta agonists for treatment of autoimmune diseases, prostate disease, depression, and ovulatory dysfunction.

Metabolism of the 16-androstene steroids in primary cultured porcine hepatocytes

The hepatic metabolism of the 16-androstene steroids was investigated using isolated porcine hepatocytes. This study demonstrated that the liver is capable of producing both phase I and phase II steroid metabolites from 16-androstene steroid precursors. 16-Androstene metabolites were recovered by solid-phase extraction and identified by gas chromatography–mass spectrometry (GC–MS). When 5α-androstenone was provided as a substrate, both 3β- and 3α-androstenol were produced as well as a metabolite that showed evidence of hydroxylation. Incubations with the various 16-androstene steroids produced metabolic profiles which suggested that the major role of the liver is phase II conjugation. Sulfoconjugated 16-androstene steroids included androstadienol, 5α-androstenone, 3β-, 3α-androstenol, and possibly the hydroxylated metabolite of 5α-androstenone. It was determined that hydroxysteroid sulfotransferase (HST) is the likely candidate for the sulfoconjugation of the 16-androstene steroids within the liver. Despite the capacity of the hepatocytes to sulfoconjugate the 16-androstene steroids, the principle metabolites produced from incubations with 5α-androstenone, 3β-, and 3α-androstenol were glucuronide conjugates, accounting for approximately 68% of all phase II metabolism. These findings underline the importance of steroid conjugation and suggest that hepatic metabolism of the 16-androstene steroids may influence the levels of 5α-androstenone present in the circulation, and thus, capable of accumulating in fat.

The contribution of hepatic steroid metabolism to serum estradiol and estriol concentrations in nonylphenol treated MMTVneu mice and its potential effects on breast cancer incidence and latency

The two major pathways for the metabolism of estradiol-17beta (E2) are the 2- and 16-hydroxylase pathways. Research has suggested that the increased production of the estrogenically active 16-hydroxy products such as estriol (E3) may be involved in increased susceptibility to breast cancer. 4-Nonylphenol (4-NP) is an environmental estrogen that also can activate the pregnane-X receptor (PXR) and induce P-450 enzymes responsible for the production of E3. It is hypothesized that 4-NP may act in part as an environmental estrogen by increasing E3 production. Based on its affinity for the estrogen receptor (ER) alone, 4-NP may be more potent than predicted at increasing mammary cancer incidence in the MMTVneu mouse. Female mice were treated per os for 7 days at 0, 25, 50 or 75 mg kg(-1) day(-1) 4-NP to investigate the effects of 4-NP on hepatic estrogen metabolism after an acute treatment. 4-Nonylphenol increased the hepatic formation of E3 in a dose-dependent manner. However, serum E3 concentrations were only increased at 25 mg kg(-1) day(-1) presumably due to direct inhibition of E3 formation by 4-NP. MMTVneu mice were then treated for 32 weeks at 0, 30 or 45 mg kg(-1) day(-1) 4-NP to determine its effects on mammary cancer formation and estrogen metabolism. 4-Nonylphenol increased mammary cancer formation in the MMTVneu mice at 45 mg kg(-1) day(-1) but not at 30 mg kg(-1) day(-1). Mice treated with an equipotent dose of E2, 10 microg kg(-1) day(-1), based on the relative binding affinities of nonylphenol and estradiol for ER alpha, did not develop mammary cancer. This suggests that nonylphenol is more potent than predicted based on its affinity for the estrogen receptor. However, no changes in serum E3 concentrations or hepatic E3 production were measured after the chronic treatment. Changes in E3 formation were correlated with increased CYP2B levels after the 7 day 4-NP treatment, and repression of CYP2B and CYP3A after 32 weeks of 4-NP treatment. Microarray analysis and Q-PCR of liver mRNA from the mice treated for 32 weeks demonstrated a decrease in RXR alpha, the heterodimeric partner of the PXR, which may in part explain the repressed transcription of the P450s measured. In conclusion, 4-NP treatment for 32 weeks increased mammary cancer formation at a dose of 45 mg kg(-1) day(-1). However, chronic treatment with 4-NP did not increase hepatic E3 formation or serum E3 concentrations. The transient induction by 4-NP of hepatic E3 formation and serum concentrations is most likely not involved in the increased incidence of mammary cancer in MMTVneu mice since E3 serum concentrations were only increased at 25 mg kg(-1) day(-1), a dose that was not sufficient to induce mammary tumor formation. Nevertheless, the induced hepatic E3 production in the acute exposures to 4-NP was indicative of an increase in mammary cancer incidence after the chronic exposure.

Estrogenic effects of silymarin in ovariectomized rats

The objective of this study was to test whether silymarin induces changes indicative of estrogenic effects in gonadal organs of ovariectomized (OVX) rats. Silymarin was administered in two experimental groups of OVX rats (n&amp;nbsp;= 7 + 7) for 30 days at the doses of 25 or 50 mg per animal per day. OVX rats (n&amp;nbsp;= 7) receiving 5&amp;nbsp;&amp;micro;g of 17b-estradiol (E2) for the last three days before killing and untreated OVX rats (n&amp;nbsp;= 7) were used as the positive and the negative controls, respectively. Uterine and blood samples were collected immediately after killing. Compared with the negative controls, total and normalized uterine weights were significantly higher in the two experimental groups (P&amp;nbsp;&amp;lt; 0.01 and&amp;nbsp;P&amp;nbsp;&amp;lt;&amp;nbsp;0.05, respectively). Uterotrophic effects of silymarin were also evident from increased heights of the luminal epithelium (P&amp;nbsp;&amp;lt; 0.01) and the endometrium (P&amp;nbsp;&amp;lt; 0.05). The response was dose-independent within the tested range. The strongest uterine response was observed in the OVX rats treated with E2. A highly significant decrease in mean density of estrogen receptor (ERa) immunostaining in the luminal and the glandular endometrial epithelia (P&amp;nbsp;&amp;lt; 0.01) and a stronger ERa immunostaining in stromal cells were observed in the two experimental and the positive control groups. The activities of alkaline and acid phosphatases were significantly increased in the luminal (P&amp;nbsp;&amp;lt; 0.05) and the glandular (P&amp;nbsp;&amp;lt; 0.01) epithelia only in the rats treated with E2. Both silymarin and E2&amp;nbsp;induced an increase in thyroid hormone concentrations in blood serum. The rises of free T3&amp;nbsp;and T4&amp;nbsp;were significant (P&amp;nbsp;&amp;lt; 0.05) in the group receiving 50 mg of silymarin per day. Hepatic oxidative metabolism of steroids was assumed to be another target of the action of silymarin. The mitochondrial cytochrome&amp;nbsp;P450-dependent testosterone hydroxylase activity and the cytochrome&amp;nbsp;P4501A-dependent 7-ethoxyresorufin O-deethylase activity were significantly increased (P&amp;nbsp;&amp;lt; 0.05) in the group receiving 50&amp;nbsp;mg of silymarin per day and in the E2-treated control group, respectively. However, the modulations of the CYP enzymes played only a minor role in the overall estrogenic effect of silymarin. Histological and functional alterations in the OVX rats treated orally with silymarin for 30 days were consistent with those seen in E2-treated rats and were indicative of estrogenic effects of silymarin.

Altered hepatic cytochrome P450-mediated steroid metabolism in environmentally-exposed seals

Altered hepatic cytochrome P450-mediated steroid metabolism in environmentally-exposed seals

17Beta-hydroxysteroid dehydrogenase type 2 and dehydroepiandrosterone sulfotransferase in the human liver.

The liver plays important roles in the clearance and metabolism of sex steroids. Its dysfunction is considered to influence the metabolic pathways of sex steroids, and to result in gynecomastia and other abnormalities of sex steroids. However, the details of its mechanism have not been well-characterized. We therefore examined the enzymes involved in the hepatic clearance and/or metabolism of sex steroids in human liver and its disorders using immunohistochemistry to determine whether there are any abnormalities of expression of these enzymes in human liver disorders. These enzymes are 17beta-hydroxysteroid dehydrogenase (17beta-HSD) type 2, an enzyme that catalyzes the biologically active estrogen, estradiol (E2), to inactive estrogen, estrone (El), and dehydroepiandrosterone sulfotransferase (DHEA-ST), which catalyzes sulfonation of dehydroepiandrosterone (DHEA) to form biologically inactive DHEA-S. A total of 162 cases including normal liver (n=31), chronic hepatitis (n=41), liver cirrhosis (n = 21), hepatocellular carcinoma (n = 47), cholangiocellular carcinoma (n = 22) and fetal liver (n = 4) were examined by immunohistochemistry. Both enzymes were expressed in the hepatocytes around portal area and central vein in normal liver. Immunopositive area for DHEA-ST was significantly larger in chronic hepatitis than in normal liver, but that of 17beta-HSD type 2 in chronic hepatitis was not different from normal liver. There were no significant differences in the immunopositive area for both enzymes between liver cirrhosis and normal liver. In hepatocellular carcinoma, immunoreactivity for both enzymes were categorized into Group A, or low positive group, and Group B, or high positive group. The latter tended to be poorly differentiated carcinoma. In cholangiocellular carcinoma, immunopositive areas of both enzymes were significantly smaller than those of normal liver. These findings indicate that the amount of expression of the enzymes involved in metabolism and/or clearance of sex steroids per hepatocyte did not decrease in liver cirrhosis. Therefore, sex steroids' abnormalities may be due to the decreased quantity of hepatocytes associated with liver cirrhosis. In hepatocellular carcinoma, some poorly differentiated cases were associated with increased expression of 17beta-HSD type 2 but its biological significance needs to be determined by further studies.