Search

The gut microbiome regulates important host metabolic pathways including xenobiotic metabolism and intermediary metabolism, such as the conversion of primary bile acids (BAs) into secondary BAs. The nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are well-known regulators for xenobiotic biotransformation in liver. However, little is known regarding the potential effects of PXR and CAR on the composition and function of the gut microbiome. To test our hypothesis that activation of PXR and CAR regulates gut microbiota and secondary BA synthesis, 9-week-old male conventional and germ-free mice were orally gavaged with corn oil, PXR agonist PCN (75 mg/kg), or CAR agonist TCPOBOP (3 mg/kg) once daily for 4 days. PCN and TCPOBOP decreased two taxa in the Bifidobacterium genus, which corresponded with decreased gene abundance of the BA-deconjugating enzyme bile salt hydrolase. In liver and small intestinal content of germ-free mice, there was a TCPOBOP-mediated increase in total, primary, and conjugated BAs corresponding with increased Cyp7a1 mRNA. Bifidobacterium, Dorea, Peptociccaceae, Anaeroplasma, and Ruminococcus positively correlated with T-UDCA in LIC, but negatively correlated with T-CDCA in serum. In conclusion, PXR and CAR activation downregulates BA-metabolizing bacteria in the intestine and modulates BA homeostasis in a gut microbiota-dependent manner.

Microsomal enzyme inducers (MEI) that increase UDP-glucuronosyltransferases (UGTs) can impact thyroid hormone homeostasis in rodents. Increased glucuronidation can result in reduction of serum thyroid hormone and a concomitant increase in thyroid-stimulating hormone (TSH). UGT2B2 is thought to glucuronidate triiodothyronine (T(3)). The purposes of this study were to determine the role of UGT2B2 in T(3) glucuronidation and whether increased T(3) glucuronidation mediates the increased TSH observed after MEI treatment. Sprague Dawley (SD) and UGT2B2-deficient Fischer 344 (F344) rats were fed a control diet or diet containing pregnenolone-16alpha-carbonitrile (PCN; 800 ppm), 3-methylcholanthrene (3-MC; 200 ppm), or Aroclor 1254 (PCB; 100 ppm) for 7 days. Serum thyroxine (T(4)), T(3), and TSH concentrations, hepatic androsterone/T(4)/T(3) glucuronidation, and thyroid follicular cell proliferation were determined. In both SD and F344 rats, MEI treatments decreased serum T(4), whereas serum T(3) was maintained (except with PCB treatment). Hepatic T(4) glucuronidation increased significantly after MEI in both rat strains. Compared with the other MEI, only PCN treatment significantly increased T(3) glucuronidation (281 and 497%) in both SD and UGT2B2-deficient F344 rats, respectively, and increased both serum TSH and thyroid follicular cell proliferation. These data demonstrate an association among increases in T(3) glucuronidation, TSH, and follicular cell proliferation after PCN treatment, suggesting that T(3) is glucuronidated by other PCN-inducible UGTs in addition to UGT2B2. These data also suggest that PCN (rather than 3-MC or PCB) promotes thyroid tumors through excessive TSH stimulation of the thyroid gland.

Murine double minute 2 (Mdm2) negatively regulates p53 by mediating its ubiquitination and proteosomal degradation, and Mdm2 is recognized as a proto-oncogene. In the present study, hepatic gene expression patterns induced by phenobarbital (PB; 100 mg/kg) and pregnenolone 16alpha-carbonitrile (PCN, 100 mg/kg) were evaluated in male and female Sprague-Dawley rats using Affymetrix Rat Genome U34A gene arrays. In addition to changes in the hepatic expression of well-characterized drug-metabolizing enzymes, an increase in Mdm2 mRNA was observed with both compounds after single or repeat dosing (5 days). However, gene array analyses did not reveal changes in other p53-dependent genes, suggesting that induction of Mdm2 occurred in a p53-independent manner. Real-time polymerase chain reaction confirmed the microarray results, as PB increased Mdm2 mRNA approximately twofold after single or repeat doses in male and female rats. PCN treatment increased Mdm2 mRNA levels up to 5- and 12-fold in male and female rats, respectively, after 5 days of dosing. Hepatic Mdm2 protein levels were increased, and immunohistochemical evaluation of rat liver demonstrated nuclear localization of Mdm2, suggesting an interaction with p53. Consequently, p53 protein levels were also decreased by approximately 35 and 50% after 5 days of PB and PCN treatment, respectively. In direct contrast to rats, PB and PCN (100 mg/kg) did not induce Mdm2 mRNA in mouse liver after 5 days of dosing. Finally, although Mdm2 in mice and humans is reported to migrate electrophoretically as two proteins with molecular weights of 76 and 90 kDa, rat Mdm2 protein was detected primarily as a 120-kDa species. Follow-up experiments indicated that rat hepatic Mdm2 was subject to posttranslational modification with small ubiquitin-modifying (SUMO) proteins. Although the molecular mechanisms controlling Mdm2 induction by PB and PCN in rats have not yet been determined, these results suggest that early effects on cell cycle regulation, response to DNA damage or cell transformation may contribute to liver tumor development.

Mammalian liver contains a pregnane X receptor (PXR, NR1I2), which binds drugs and other xenobiotics, and stimulates (or suppresses) expression of numerous genes involved in the metabolic elimination of foreign compounds and some toxic endogenous substances. In the present study, we used microarray analysis to identify genes whose expression in rat liver was significantly altered by pregnenolone 16alpha-carbonitrile (PCN) treatment. PCN is a synthetic steroid that induces cytochrome P4503A expression and is hepatoprotective by increasing resistance to subsequent stressful insults. Significant induction was seen for 138 genes while expression of 82 genes was significantly repressed. We found induction of genes known to be induced by PCN, such as enzymes involved in drug metabolism and transport. In addition, many genes were differentially expressed whose functions concerned intracellular metabolism, transport of essential small molecules, cell cycle, and redox balance. Our results support the idea that the domain of PXR-controlled gene networks may be even more extensive than currently thought and may extend to functions apart from xenobiotic metabolism.

Microsomal enzyme inducers that increase UDP-glucuronosyltransferase (UDP-GT) activity are suspected to affect the thyroid gland by increasing the glucuronidation of T(4), which reduces serum thyroxine (T(4)). In response to reduced serum T(4), serum thyroid-stimulating hormone (TSH) increases. However, not all microsomal enzyme inducers that reduce serum T(4) produce an increase in serum TSH. We have shown that serum TSH is increased the most in rats treated with the microsomal enzyme inducers phenobarbital (PB) or pregnenolone-16alpha-carbonitrile (PCN), whereas TSH is affected less in rats treated with 3-methylcholanthrene (3MC) and Aroclor 1254 (PCB). It is unclear why serum TSH is differentially affected by various microsomal enzyme inducers. We propose that the glucuronidation of T(3) might be the reason serum TSH is increased by some microsomal enzyme inducers but not by others. Male Sprague-Dawley rats were fed either a basal diet or a diet containing PB (at 300, 600, 1200, or 2400 ppm), PCN (at 200, 400, 800, or 1600 ppm), 3MC (at 50, 100, 200, or 400 ppm), or PCB (at 25, 50, 100, or 200 ppm) for 7 days; and T(4) and T(3) UDP-GT activities were then determined. T(4) UDP-GT activity was increased in rats treated with PB (120%), PCN (250 to 400%), 3MC (400 to 600%), or PCB (300 to 430%). In contrast, T(3) UDP-GT activity was increased in rats treated with PB (90%) or PCN (120 to 200%), whereas 3MC and PCB treatments did not have an appreciable effect. In conclusion, differential effects on T(3) glucuronosyltransferase activity were found in rats treated with microsomal enzyme inducers.

Reduced thyroid hormone concentrations (T4 and/or T3) and increased thyroid-stimulating hormone (TSH) have been proposed to mediate the thyroid tumor promoting effects of hepatic microsomal enzyme inducers (MEI) and antithyroid drugs. TSH is known to stimulate thyroid gland function and growth, as well as neoplasia. Thyroid weight has been used as an indicator of thyroid gland growth in MEI studies, but little is known about the effects of these inducers on thyroid cell proliferation. Therefore, we determined the time-course of thyroid cell proliferation of rats treated with MEI, and with the antithyroid drug propylthiouracil (PTU). Male Sprague-Dawley rats were fed either a basal diet or a diet containing phenobarbitol (PB) (1200 ppm), PCN (500 ppm), or PTU (30 ppm) for 3, 7, 14, 21, 30, 45, 60, or 90 days. PB and PCN treatments did not affect T3, but PTU reduced T3 60%. PB and PCN treatments reduced T4 25%, whereas PTU treatment reduced T4 90%. PB and PCN treatments increased thyroid weight 80%, and PTU increased thyroid weight 500%. TSH was not appreciably altered in PB-treated rats, but was increased 75% and 830% in PCN- and PTU-treated rats, respectively. Thyroid cell proliferation was increased 260, 330, and 850% in rats treated with PB, PCN, or PTU, respectively, for 7 days, but returned to control levels by the 45th treatment day. In conclusion, treatment with MEI that produced mild increases in TSH resulted in dramatic increases in thyroid cell proliferation, which peaked after 7 days of treatment and then returned to control values. This result is similar to that of antithyroid drugs, which produce large increases in TSH. These findings may have important implications for the role thyroid follicular cell proliferation has in mediating the thyroid tumor promoting effects of MEI.

The purpose of this study was to evaluate the effect of pregnenolone 16α-carbonitrile (PCN) on the interconversion pharmacokinetics and metabolism of dapsone. To determine microsomal CYP3A activity and protein, eight rats (4 PCN, 4 corn oil) received a 1mg kg−1 intravenous bolus dose of dapsone, followed by blood and urine sampling. The formation clearance of dapsone hydroxylamine (CLf DDS-NOH) was calculated from the obtained samples. Interconversion pharmacokinetics estimates were obtained after 10 rats (5 PCN, 5 control) received 1mg kg−1 dapsone or 1.17 mg kg−1 monoacetyldapsone, with a 24-h wash-out. Results from the interconversion analysis demonstrated that PCN significantly increased systemic clearance (CLs) of dapsone, but not its interconversion. The in-vivo/in-vitro correlation study demonstrated that PCN significantly increased CLs of dapsone (8.55 to 16.39mL min−1; P We conclude that PCN treatment produces significant increases in CLs (dapsone) and CLf (DDS-NOH) in rats. These changes were not due to changes in the reversible metabolism of dapsone. These results suggest that the formation clearance of dapsone hydroxylamine reflects alterations in CYP3A activity, despite the fact that it accounted for a small part of the systemic clearance of dapsone.

The microsomal enzyme inducers 3-methylcholanthrene, phenobarbitone and pregnenolone-16α-carbonitrile (PCN) are known to affect other aspects of hepato-biliary disposition in addition to metabolism. This study was designed to determine if presystemic elimination of the non-metabolized xenobiotic ouabain could be altered by these inducers. Male Sprague-Dawley rats were pretreated with inducers or saline for four days. A day later, ouabain (0.5 mg kg−1) was administered into either the ileocolic vein (portal administration) or the femoral vein (systemic administration). Blood and bile samples were collected for up to 90 min after ouabain administration. Biliary excretion rate and cumulative biliary excretion of ouabain were increased by pretreatment with PCN (75 mg kg−1 day−1) relative to controls. Phenobarbitone pretreatment (75 mg kg−1 day−1) also increased these parameters, but to a lesser extent than PCN. In contrast, 3-methylcholanthrene pretreatment (20 mg kg−1 day−1) had no effect on biliary excretion. Plasma concentrations of ouabain were much lower after PCN pretreatment relative to controls, whereas neither phenobarbitone nor 3-methylcholanthrene had any effect. Similarly, clearance (both biliary and total) and volume of distribution were increased by PCN, but not by phenobarbitone or 3-methylcholanthrene pretreatment. Interestingly, the magnitude of biliary and plasma effects induced by PCN appeared to be comparable whether ouabain was administered portally or systemically. Pretreatment of rats with PCN, but not phenobarbitone or 3-methylcholanthrene was shown to increase total clearance of ouabain, mainly via an increase in biliary clearance. Furthermore, because the enhanced clearance occurs after systemic as well as after portal administration of ouabain, a significant change in hepatic presystemic elimination was not detected.

Tamoxifen is the major therapeutic agent for the treatment of hormone-dependent breast cancer. Tamoxifen treatment appears to be associated with an increased incidence of endometrial carcinoma in humans and hepatocellular carcinoma in rats. These carcinogenic effects of tamoxifen might be induced by the formation of a tamoxifen reactive intermediate that binds covalently to macromolecules. Liver microsomal cytochrome P450s (CYPs) catalyze the metabolism of tamoxifen, forming a reactive intermediate that binds irreversibly to microsomal proteins, primarily to a 54 kDa protein (Mani, C. and Kupfer, D., Cancer Res., 51, 6052-6058, 1991). The current study identifies the P450 enzymes that catalyze the activation of tamoxifen to a reactive intermediate in rats and humans. Among the species examined, rats, chickens and humans demonstrate low tamoxifen binding activity, ranging from 0.1 to 0.4 nmol bound/mg protein/h. In contrast, hamsters and mice exhibit high binding, 1.2 and 1.6 nmol/mg protein/h respectively. Treatment of male rats with phenobarbital or pregnenolone-16 alpha-carbonitrile (PCN) markedly elevated the binding of tamoxifen to liver microsomal proteins. Methylcholanthrene treatment had no effect on binding. These findings suggested the involvement of CYP3A in catalysis of the covalent binding. Alternate substrates of CYP3A, cortisol and erythromycin, inhibited tamoxifen binding in liver microsomes from PCN- and phenobarbital-treated rats. Treatment of rats with troleandomycin (TAO), an inducer of CYP3A, followed by the dissociation of the TAO-CYP3A complex, elevated the covalent binding to liver microsomes approximately 3-fold. Antibodies against rat CYP3A1 strongly inhibited tamoxifen binding to liver microsomes from PCN- and phenobarbital-treated rats, whereas the antibodies anti-CYP2B1/2B2 did not inhibit binding. In humans, tamoxifen binding was inhibited by the anti-rat CYP3A1 IgG and also by alternate substrates of CYP3A. These results indicate that the activation of tamoxifen to a reactive intermediate by rat and human liver microsomes is principally catalyzed by CYP3A enzymes.

I-compounds are covalent DNA modifications presumably derived from endogenous electrophiles. To investigate the possible role of cytochrome P450 in I-compound metabolism, groups of female Sprague-Dawley rats (225-250 g) were treated i.p. with vehicle or cytochrome P450 inducers, i.e. 80 mg/kg phenobarbital (PB), 20 mg/kg 3-methylcholanthrene (MC) or 50 mg/kg pregnenolone-16 alpha-carbonitrile (PCN), once daily for 4 days. DNA synthesis rate was measured via [3H]methylthymidine incorporation. DNA adducts and I-compounds in liver and kidney were analyzed 1 and 8 days after the last treatment. Total liver and kidney microsomal cytochrome P450 content and activities of representative drug-metabolizing enzymes for PB, MC and PCN, i.e. benzphetamine N-demethylase, ethoxycoumarin O-deethylase (ECD) and erythromycin N-demethylase, were also determined in all groups. PCN caused significant depletion of total non-polar I-compounds at 1 day, compared to controls. Levels of several individual I-spots in liver were differentially reduced by each of the three inducers at 1 day. Most I-spots were restored to control levels at 8 days. Kidney I-compounds were not affected by PB or PCN, but MC reduced the level of one non-polar individual I-compound at 1 day. Except for the expected DNA adduct formation from MC, there were no qualitative changes in profiles of postlabeled modified nucleotides. Total cytochrome P450 content in liver microsomes and activities of individual P450 enzymes were significantly increased by treatment with each of the inducers at 1 day. This was, however, not the case at 8 days in PB- and PCN-treated livers. MC-treated rats, on the other hand, displayed elevated levels of liver cytochrome P450 and ECD at 8 days. In kidney, PB and PCN did not elicit induction of P450 and individual enzymes, but MC increased total P450 content and ECD activity at 1 day, and ECD activity alone at 8 days. These results suggest a major role for cytochrome P450 enzymes in the metabolism of I-compounds.

The present studies were undertaken to resolve conflicting reports on the inducibility of liver microsomal benzo[a]pyrene hydroxylase activity in rats treated with pregnenolone-16 alpha-carbonitrile (PCN). Several studies have shown that treatment of Long Evans rats with PCN causes a 5- to 10-fold induction of benzo[a]pyrene hydroxylase activity, whereas little or no induction occurs in Sprague-Dawley or Wistar rats. Studies with one-month-old-male Long Evans, Sprague-Dawley, Wistar and Holtzman rats failed to reveal an anticipated strain difference in the inducibility of benzo[a]pyrene hydroxylase activity by PCN. Studies with immature and mature male and female Long Evans rats revealed that the inducibility of benzo[a]pyrene hydroxylase activity decreases with age in male but not female rats, i.e., PCN induced benzo[a]Pyrene hydroxylase activity 5- to 8-fold in immature male, immature female and mature female rats but only 2-fold in mature male rats. The age-dependent decrease in inducibility by PCN in male rats coincided with an age-dependent increase (2.4-fold) in the basal activity of benzo[a]pyrene hydroxylase. These sex-dependent developmental changes can be explained by an age-dependent increase in the constitutive levels of the major PCN-inducible form of cytochrome P-450 (cytochrome P-450-PCN) in male but not female rats. Electrophoresis of liver microsomes and studies on the binding of metyrapone to dithionite-reduced cytochrome P-450 provided additional evidence for age- and sex-dependent differences in the levels of microsomal cytochrome P-450-PCN. In addition to reconciling the conflicting literature reports, the age- and sex-dependent differences in cytochrome P-450-PCN levels account, at least in part, for age and sex differences in certain liver microsomal enzyme activities, including benzo[a]pyrene hydroxylase activity.

The effects of treating rats with various pregnenolone-16 alpha-carbonitrile (PCN)-type inducers of cytochrome P-450p on the liver microsomal metabolism of aflatoxin B1 (AFB1) were investigated. Treatment of male rats with PCN resulted in a 6-fold increase in the 9-hydroxylation of AFB1 to aflatoxin Q1 (AFQ1). Treatment of female rats with PCN resulted in a 16-fold increase in the formation of AFQ1. The age-dependent decline in constitutive cytochrome P-450p levels in female but not male rats resulted in a sex difference in the formation of AFQ1 in liver microsomes from untreated rats (male:female approximately 3:1). The formation of AFQ1 was stimulated up to 5.4-fold when liver microsomes from triacetyloleandomycin (TAO)-treated rats were treated with potassium ferricyanide, which dissociates the complex between cytochrome P-450p and TAO. Treatment of male rats with the cytochrome P-450p inducer, dexamethasone, increased (approximately 7-fold) the 9-hydroxylation of AFB1 to AFQ1 by liver microsomes, and also enhanced (approximately 2-fold) the microsomal activation of AFB1 to metabolites that were mutagenic to Salmonella typhimurium TA98 and TA100. These results indicate that the 9-hydroxylation of AFB1 to AFQ1 is catalyzed by rat liver microsomal cytochrome P-450p.