Combining Genomics To Identify the Pathways of Post-Transcriptional Nongenotoxic Signaling and Energy Homeostasis in Livers of Rats Treated with the Pregnane X Receptor Agonist, Pregnenolone Carbonitrile.

Transcriptomic, proteomic, phosphoproteomic, and metabolomic analyses were combined to determine the role of pregnane X receptor (PXR) in nongenotoxic signaling and energy homeostasis in liver after rats were repeatedly orally dosed with the PXR agonist pregnenolone carbonitrile (PCN) for 7 days. Analyses of mRNAs and proteins in the supernatant, membrane, and cytosolic fractions of enlarged liver homogenates showed diverse expression profiles. Gene set enrichment analysis showed that the synchronous increase in mRNAs and proteins involved in chemical carcinogenesis and the response to drug was possibly mediated by the PXR pathway and proteasome core complex assembly was possibly mediated by the Nrf2 pathway. In addition, levels of proteins in the endoplasmic reticulum lumen and involved in the acute-phase response showed specific increase with no change in mRNA level, and those composed of the mitochondrial inner membrane showed specific decrease. The analysis of phosphorylated peptides of poly(A) RNA binding proteins showed a decrease in phosphorylation, possibly by casein kinase 2, which may be related to the regulation of protein expression. Proteins involved in insulin signaling pathways showed an increase in phosphorylation, possibly by protein kinase A, and those involved in apoptosis showed a decrease. Metabolomic analysis suggested the activation of the pentose phosphate and anaerobic glycolysis pathways and the increase of amino acid and fatty acid levels, as occurs in the Warburg effect. In conclusion, the results of combined analyses suggest that PXR's effects are due to transcriptional and post-transcriptional regulation with alteration of nongenotoxic signaling pathways and energy homeostasis.

Sustained Isoprostane E2 Elevation, Inflammation and Fibrosis after Acute Ischaemia-Reperfusion Injury Are Reduced by Pregnane X Receptor Activation.

Liver grafts donated after cardiac death are increasingly used to expand the donor pool but are prone to ischaemic-type biliary lesions. The anti-inflammatory effects of the activated pregnane X receptor have previously been shown to be beneficial in a number of inflammatory liver conditions. However, its role in reducing peri-portal inflammation and fibrosis following ischaemia-reperfusion injury has not been investigated. Hepatic injury and its response to pregnane X receptor activation was examined after partial hepatic ischaemia-reperfusion injury induced by surgically clamping the left and middle lobar blood vessels in rats. Molecular and pathological changes in the liver were examined over the following 28 days. Ischaemia-reperfusion injury resulted in transient cholestasis associated with microvillar changes in biliary epithelial cell membranes and hepatocellular injury which resolved within days after reperfusion. However, in contrast to chemically-induced acute liver injuries, this was followed by sustained elevation in isoprostane E2, peri-portal inflammation and fibrosis that remained unresolved in the ischaemic reperfused lobe for at least 28 days after clamping. Administration of pregnenolone-16α-carbonitrile--a rodent-specific pregnane X receptor activator--resulted in significant reductions in cholestasis, hepatic injury, ischaemic lobe isoprostane E2 levels, peri-portal inflammation and fibrosis. Hepatic ischaemia-reperfusion injury therefore results in inflammatory and fibrotic changes that persist well beyond the initial ischaemic insult. Drug-mediated activation of the pregnane X receptor reduced these adverse changes in rats, suggesting that the pregnane X receptor is a viable drug target to reduce ischaemic-type biliary lesions in recipients of liver transplants donated after cardiac death.

Activation of pregnane X receptor by pregnenolone 16 α-carbonitrile prevents high-fat diet-induced obesity in AKR/J mice.

Pregnane X receptor (PXR) is known to function as a xenobiotic sensor to regulate xenobiotic metabolism through selective transcription of genes responsible for maintaining physiological homeostasis. Here we report that the activation of PXR by pregnenolone 16α-carbonitrile (PCN) in AKR/J mice can prevent the development of high-fat diet-induced obesity and insulin resistance. The beneficial effects of PCN treatment are seen with reduced lipogenesis and gluconeogenesis in the liver, and lack of hepatic accumulation of lipid and lipid storage in the adipose tissues. RT-PCR analysis of genes involved in gluconeogenesis, lipid metabolism and energy homeostasis reveal that PCN treatment on high-fat diet-fed mice reduces expression in the liver of G6Pase, Pepck, Cyp7a1, Cd36, L-Fabp, Srebp, and Fas genes and slightly enhances expression of Cyp27a1 and Abca1 genes. RT-PCR analysis of genes involved in adipocyte differentiation and lipid metabolism in white adipose tissue show that PCN treatment reduces expression of Pparγ2, Acc1, Cd36, but increases expression of Cpt1b and Pparα genes in mice fed with high-fat diet. Similarly, PCN treatment of animals on high-fat diet increases expression in brown adipose tissue of Pparα, Hsl, Cpt1b, and Cd36 genes, but reduces expression of Acc1 and Scd-1 genes. PXR activation by PCN in high-fat diet fed mice also increases expression of genes involved in thermogenesis in brown adipose tissue including Dio2, Pgc-1α, Pgc-1ß, Cidea, and Ucp-3. These results verify the important function of PXR in lipid and energy metabolism and suggest that PXR represents a novel therapeutic target for prevention and treatment of obesity and insulin resistance.

Microarray analysis on CYPs expression in pregnant rats after treatment with pregnenolone-16alpha-carbonitrile and phenobarbital.

We previously reported the protein expression profiles of nine cytochrome P450 isozymes (CYPs) in pregnant rat's liver, fetal liver, and placenta after treatment with pregnenolone-16alpha-carbonitrile (PCN), dexamethasone (DEX), or phenobarbital (PB). In this study, the gene expression of 40 CYPs and 2 orphan nuclear receptors for CYP inducers, that is, Nr1i2 (CYP3A subfamily inducible by PCN) and Nr1i3 (CYP2B subfamily inducible by PB), in pregnant rat's liver, fetal liver, and placenta was investigated at one time. Fischer 344 (F344) pregnant rats were daily treated intraperitoneally with 50 mg/kg of PCN or 80 mg/kg of PB from 13 to 16 days of gestation (DG). They were sacrificed on 17 DG, and microarray analysis using Affymetrix Rat Expression Array 230A was performed. Ten genes expression significantly increased in dam's liver in PCN group, and seven genes expression in PB group. On the other hand, four genes expression increased in fetal liver in PCN group, and three genes expression increased in PB group. Being common to dam's and fetal livers, the gene expression of Cyp3A1 (CYP3A subfamily) and cytochrome P-450e (CYP2B subfamily) increased in both PCN and PB groups. In placenta, the expression of Cyp3A1 gene was significantly induced in PB group, and it also showed a tendency to increase in PCN group. The expression of Nr1i2 gene was significantly elevated only in dam's liver of PCN group, while the expression of Nr1i3 gene showed no changes in all groups. The results of the present study of 40 CYPs gene expression mostly corresponded to our previous reports on 9 CYPs protein expression.

A transgenic mouse model with a luciferase reporter for studying in vivo transcriptional regulation of the human CYP3A4 gene.

Cytochrome p450 3A4 (CYP3A4) plays an important role in drug metabolism, and the enzymatic activity of CYP3A4 contributes to many adverse drug-drug interactions. Here we describe a transgenic mouse model that is useful in monitoring the in vivo transcriptional regulation of the human CYP3A4 gene. A reporter construct consisting of 13 kilobases of the human CYP3A4 promoter controlling the firefly luciferase gene was used to generate a transgenic mouse line [FVB/N-Tg(CYP3A4-luc)Xen]. Reporter gene expression was assessed using an in vivo imaging system (IVIS) in anesthetized mice. Basal expression of the reporter was highest in liver and kidney, and moderate in the duodenum in male transgenic mice, whereas the basal luciferase activity was highest in the duodenum and lower in kidney and liver in females. Injections of pregnenolone, phenobarbital, rifampicin, nifedipine, dexamethasone, 5-pregnen-3beta-ol-20-one-16alpha-carbonitrile (PCN), and clotrimazole resulted in a time-dependent induction of luciferase expression, primarily in liver, that peaked at 6 h post injection. The greatest induction was found with clotrimazole, dexamethasone, and PCN, whereas the lowest induction followed pregnenolone, phenobarbital, and rifampicin injection. In general, male mice responded to these drugs more strongly than did females. Our results suggest that the human CYP3A4 promoter functions in transgenic mice and that this in vivo model can be used to study transcriptional regulation of the CYP3A4 gene.

Nuclear receptor, pregname X receptor, is required for induction of UDP-glucuronosyltranferases in mouse liver by pregnenolone-16 alpha-carbonitrile.

The aim of this study was to determine the role of pregnane X receptor (PXR) in the induction of UDP-glucuronosyltransferases (UGTs) by pregnenolone-16 alpha-carbonitrile (PCN). Four- to six-month-old male wild-type and PXR-null mice received control or PCN-treated (1500 ppm) diet for 21 days. On day 22, livers were taken to prepare microsomes and total RNA to determine UGT activity and mRNA levels, respectively. In wild-type mice, PCN treatment significantly increased UGT activities toward bilirubin, 1-naphthol, chloramphenicol, thyroxine, and triiodothyronine. On control diet, the UGT activities toward the above substrates (except for 1-naphthol) in the PXR-null mice were significantly higher than those of wild-type mice. However, UGT activities in PXR-null mice were not increased by PCN. In agreement with the above findings, mRNA levels of mouse Ugt1a1 and Ugt1a9, which are involved in the glucuronidation of bilirubin and phenolic compounds, were increased about 100% in wild-type mice following PCN treatment, whereas the expression of Ugt1a2, 1a6, and 2b5 was not affected. In contrast, PCN treatment had no effect on the mRNA levels of these UGTs in PXR-null mice. Taken together, these results indicate that PCN treatment induces glucuronidation in mouse liver, and that PXR regulates constitutive and PCN-inducible expression of some UGTs.

Expression of cytochromes P450 3A in mouse lung: effects of dexamethasone and pregnenolone 16alpha-carbonitrile.

Expression of cytochromes P450 3A (CYP3A) has been reported in the lung, but its regulation has received little attention. In the present study, we assessed lung levels of Cyp3a mRNA, protein and activity in control mice and in mice treated with either dexamethasone (DEX), pregnenolone 16alpha-carbonitrile (PCN) or a mixture of DEX+PCN. Lung expression of the pregnane X receptor (PXR) was also investigated. Constitutive levels of Cyp3a mRNA were found in the lung from control mice by polymerase chain reaction after reverse transcription of total RNA (RT-PCR). These levels were significantly increased (2.0-fold, P<0.05) in mice treated with DEX and further enhanced (2.7-fold increase, P<0.01) in mice treated with DEX+PCN. In control mice, basal levels of Cyp3a protein and activity were also found, as assessed by western blot and measure of testosterone 6beta-hydroxylation, respectively. In mice treated with DEX or DEX+PCN, changes in Cyp3a protein and activity exhibited the same pattern as those in Cyp3a mRNA. In contrast, PCN alone failed to trigger consistent increases in lung Cyp3a mRNA, protein and activity. PXR mRNA was not detected in the lung from control or PCN-treated mice by RT-PCR, but was found at significant levels in the lungs from mice treated with DEX or DEX+PCN. Our results show that expression of Cyp3a is upregulated by glucocorticoids in mouse lung, and that this effect is potentiated by antiglucocorticoids. This potentiation may involve PXR, expression of which is induced in the lung of glucocorticoid-treated mice.

Effect of pregnenolone-16 alpha-carbonitrile and dexamethasone on acetaminophen-induced hepatotoxicity in mice.

Recently, we demonstrated that a microsomal enzyme inducer with a steroidal structure, pregnenolone-16 alpha-carbonitrile (PCN), markedly decreased the hepatotoxicity of acetaminophen (AA) in hamsters. Therefore, it was of interest to determine if PCN, as well as another steroid microsomal enzyme inducer, dexamethasone (DEX), would decrease the toxicity of AA in mice, another species sensitive to AA hepatotoxicity. Mice were pretreated with PCN or DEX (100 and 75 mg/kg, ip, for 4 days, respectively) and were given AA (300-500 mg/kg, ip). Twenty-four hours after AA administration, liver injury was assessed by measuring serum activities of sorbitol dehydrogenase and alanine aminotransferase and by histopathological examination. Neither PCN nor DEX protected markedly against AA hepatotoxicity in mice; PCN tended to decrease AA-induced hepatotoxicity, whereas DEX was found to enhance AA-induced hepatotoxicity and it produced some hepatotoxicity itself. DEX decreased the glutathione concentration (36%) in liver and increased the biliary excretion of AA-GSH, which reflects the activation of AA, whereas PCN produced neither effect. Thus, whereas PCN has been shown to markedly decrease the hepatotoxicity of AA in hamsters, apparently by decreasing the isoform of P450 responsible for activating AA to N-acetyl-p-benzoquinoneimine, this does not occur in mice after induction with either PCN or DEX. In contrast, DEX enhances AA hepatotoxicity apparently by decreasing liver GSH levels and increasing the activation of AA to a cytotoxic metabolite.

Effect of pregnenolone-16 alpha-carbonitrile on bilirubin-and sulfobromophthalein-binding to hepatic Y and Z proteins in the rat.

Pregnenolone-16 alpha-carbonitrile (PCN), administered twice daily p.o. for 3 days at a dose level of 20 mumol/100 g body weight, significantly enhances in vivo binding of 14C-bilirubin and sulfobromophthalein (BSP) to hepatic Y and Z proteins in female Charles River CD rats. 14C-bilirubin-binding to Y protein showed a 61% increase, while binding of the same moiety to the Z protein fraction was augmented by 59%. BSP-binding in vivo demonstrated rises of 114 and 71% in relation to Y and Z proteins, respectively. These data correlate well with previous investigations in which PCN was found to have a beneficial influence on experimentally induced hyperbilirubinemias and, furthermore, there is an indication that phenobarbital, another potent microsomal enzyme inducer, acts via a similar mechanism.

Effect of pregnenolone-16alpha-carbonitrile (PCN) on rat liver.

One time i.p. injections of 5 -20 mg Pregnenolone-16alpha-carbonitrile (PCN) effects the mitotic activity of rat liver cells during a time period of 24 hours to be 20 times higher than the normal mitotic rate. This, however, does not result in an measurable increase of cell numbers and cell volumes. Furthermore, total liver DNA, RNA, protein and dry-weight remain unchanged. The injection of 7 times 10 mg PCN also results in the elevation of the mitotic rate. Return of the elevated mitotic rate to base level takes 18 days from the time the last injection of PCN was administered. During this time the number and the average volume of liver cells increases by 28% and 30%, respectively. The dry weight of liver and tetraploid nuclei increase in number, whereas RNA- and protein content remains unchanged. All parameters have reached the base line after 4 - 6 weeks following the last injection of PCN. No histological changes were observed after multiple doses of PCN that cause hyperplasia and hypertrophy of liver tissue. Hypertrophy of liver cells follows the observed hyperplasia and therefore is not expected to be the cause of the hyperplastic processes.

Microsomal enzyme inducers and hypervitaminosis A in rats.

Pretreatment of rats with pregnenolone-16alpha-carbonitrile (PCN) completely prevented the osseous lesions produced by vitamin A overdosage, whereas pretreatment with phenobarbital or with phenytoin (diphenylhydantoin) provided only partial prophylaxis. These microsomal enzyme inducers also decreased vitamin A concentration in the liver and seemed to protect against hypervitaminosis A by enhancing the metabolism of the vitamin. Electron microscopy of the liver showed large, vitamin A-storing perisinusoidal cells. The number, size, and lipid content of these cells were decreased in rats treated simultaneously with vitamin A and PCN, but not in rats pretreated with phenobarbital or phenytoin. Vitamin A, given alone, produced moderate accumulation of smooth endoplasmic reticulum (SER) in hepatocytes, without accelerating microsomal drug-metabolizing enzyme activity; phenobarbital, phenytoin, and PCN also elicited SER accumulation, particularly when administered with vitamin A.

Regulation of resistance to various toxicants by PCN (pregnenolone-16alpha-carbonitrile) and thyroxine.

In rats, PCN (the most potent catatoxic steroid known to date) at the usual dose level powerfully inhibited the toxicity of antazoline, carbamazepine, cocaine, guanethidine, ibuprofen, ketamine, LSD, nembutal and reserpine, whereas (except in the case of nembutal) thyroxine sensitized the animals to intoxication with these same compounds. Even much lower doses of PCN or thyroxine exerted similar but weaker effects. PCN-induced resistance to the various substrates was generally not altered by concurrent administration of thyroxine but in a few cases its protective action was partially or totally inhibited, depending upon the respective dose levels of both compounds.

Effect of cycloheximide on pregnenolone-16alpha-carbonitrile (PCN)-induced ultrastructural changes in the rat liver.

Cycloheximide (10 mg/100 g body weight i.v. twice), an inhibitor of protein synthesis, caused smooth endoplasmic reticulum (SER) accumulation in the hepatocytes of female rats. Pregnenolone-16alpha-carbonitrile (PCN), given p.o., had a similar effect. When cycloheximide was administered to PCN-treated rats, SER proliferation as well as myelin figure formation were noted.