HEPATOTOXICITY OF CHRONIC RIFAXIMIN ADMINISTRATION: Rifaximin, a nonsystemic antibiotic that exhibits low gastrointestinal absorption, is a potent agonist of human PXR, which contributes to its therapeutic efficacy in inflammatory bowel disease, which is a risk factor for colon cancer To investigate the effects of long-term administration of rifaximin on the liver, PXR-humanized mice were administered rifaximin for six months, with wild-type and Pxr-null mice treated in parallel as controls. There was time-dependent intense hepatocellular fatty degeneration and increased hepatic triglycerides in PXR-humanized mice and not in wild-type and Pxr-null mice. After long-term treatment, PXR target genes were induced in small intestine and liver, with significant up-regulation in the expression of hepatic genes related to triglyceride synthesis and lipid accumulation. However, no significant hepatic accumulation of rifaximin was found, even after six months of treatment, in PXR-humanized mice. Genes in the small intestine that are involved in the uptake of fatty acids and triglycerides were induced along with increased triglyceride accumulation in intestinal epithelial cells of PXR-humanized mice;this was not observed in wild-type and Pxr-null mice. These findings suggest that long-term administration of rifaximin could lead to PXR-dependent hepatocellular fatty degeneration as a result of activation of genes involved in lipid uptake, thus indicating a potential adverse effect of rifaximin on liver function after long-term exposure. MECHANISM OF HEPATOTOXICITY OF THE DUAL DRUG COMBINATORIAL THERAPY WITH RIFAXIMIN AND ISONIAZID. Tuberculosis is a global health problem, and chemotherapy is the most effective method for its control. A major issue during tuberculosis treatment is drug-induced liver injury. Once liver injury occurs, the antituberculosis regimen must be altered or discontinued, which can result in relapse, drug resistance and tuberculosis-related death. Both rifampicin and isoniazid are first-line antituberculosis drugs. The combination of these two drugs is highly effective. However, this combination frequently causes liver injury or even liver failure. Extensive studies have been conducted to investigate liver injury caused by rifampicin and isoniazid in mice and rats, but these studies poorly mimicked hepatotoxicity in humans. Species differences between rodents and humans in responding to rifampicin and isoniazid are expected. PXR is a major xenobiotic receptor that controls drug metabolism through its induction of drug transporters and enzymes that metabolize drugs such as CYP3A4. The human PXR, but not its mouse counterpart, is activated by rifampicin. Using the PXR-humanized mouse model, co-treatment with rifampicin and isoniazid was found to cause liver toxicity through PXR-mediated alteration of the heme biosynthesis pathway. In particular, PXR induced the gene encoding protoporphyrin IX synthase leading to an accumulation of protoporphyrin IX in the liver and gall bladder. This accumulation causes liver toxicity due to cholestasis. Isoniazid is thought to potentiate this accumulation through inhibition of enzymes downstream of protoporphyrin IX synthase. These results provide insight into the mechanism of liver injury induced by co-treatment with these compounds and may lead to their safer use in the clinic. ROLE OF PXR IN ALPH-TOCOPHEROL METABOLISM: PXR has been postulated to play a role in the metabolism of alpha-tocopherol owing to the up-regulation of hepatic CYP3A4 in human cell lines and murine models after alpha-tocopherol treatment. However, in vivo studies confirming the role of PXR in alpha-tocopherol metabolism in humans presents significant difficulties and has not been performed. PXR-humanized, wild-type, and Pxr-null mouse models were used to determine whether alpha-tocopherol metabolism is influenced by species-specific differences in PXR function in vivo. No significant difference in the concentration of the major alpha-tocopherol metabolites was observed among the PXR-humanized, wild-type, and Pxr-null mice through mass spectrometry-based metabolomics. Gene expression analysis revealed significantly increased expression of Cyp3a11 as well as several other P450s only in wild-type mice, suggesting species-specificity for alpha-tocopherol activation of PXR. Luciferase reporter assay confirmed activation of mouse PXR by alpha-tocopherol. Analysis of the Cyp2c family of genes revealed increased expression of Cyp2c29, Cyp2c37, and Cyp2c55 in wild-type, PXR-humanized, and Pxr-null mice, which suggests PXR-independent induction of Cyp2c gene expression. This study revealed that alpha-tocopherol is a partial agonist of PXR and that PXR is necessary for Cyp3a induction by alpha-tocopherol. ROLE OF HYPOXIA_INDUCIBLE FACTOR 1ALPHA IN OBESITY: Obesity has been identified as a major risk factor for type 2 diabetes, characterized by insulin resistance in insulin target tissues. Hypoxia-inducible factor 1alpha (HIF1alpha) regulates pathways in energy metabolism that become dysregulated in obesity. Earlier studies revealed that HIF1alpha in adipose tissue is markedly elevated in high-fat diet-fed mice that are obese and insulin-resistant. Genetic ablation of HIF1alpha in adipose tissue decreased insulin resistance and obesity, accompanied by increased serum adiponectin levels. However, the exact mechanism whereby HIF1alpha regulates adiponectin remains unclear. Here, acriflavine, an inhibitor of HIF1alpha, induced the expression of adiponectin and reduced the expression of SOCS3 in cultured 3T3-L1 adipocytes. Mechanistic studies revealed that HIF1alpha suppressed the expression of adiponectin through a SOCS3-STAT3 pathway. Socs3 was identified as a novel HIF1alpha target gene based on chromatin immunoprecipitation and luciferase assays. STAT3 directly regulated adiponectin in vitro in cultured 3T3-L1 adipocytes. Acriflavine was found to prevent diet-induced obesity and insulin resistance. In vivo, acriflavine also regulated the SOCS3-STAT3-adiponectin pathway, and inhibition of HIF1alpha in adipose tissue was essential for acriflavine to improve the SOCS3-STAT3-adiponectin pathway to counteract insulin resistance. This study provides evidence for a novel target gene and signal transduction pathway in adipocytes and indicates that inhibitors of HIF1alpha have potential utility for the treatment of obesity and type 2 diabetes. ROLE OF VITAMIN D RECEPTOR IN INFLAMMATORY BOWEL DISEASE: Inflammatory bowel disease (IBD) was investigated in intestine-specific Vdr knockout mice. Mice treated with a time-course of 3% dextran sulfate sodium (DSS) to induce colitis diagnosed by evaluating clinical symptoms and intestinal histopathology. Intestine-specific Vdr knockout mice showed abnormal body size, colon structures and feces color. Calcium, collagen, and intestinal proliferation-related gene expression were all decreased, and serum alkaline phosphatase was highly increased. DSS-treated intestine-specific Vdr knockout mice showed a high score of IBD symptoms including enlarged mucosal layer and damaged muscularis layer as compared to wild-type mice. They also had increased expression of genes encoding proinflammatory cytokines. Feces metabolomics revealed decreased concentrations of taurine, taurocholic acid, taurodeoxycholic acid and cholic acid in DSS-treated intestine-specific Vdr knockout mice. These results suggest that VDR may play an important role in IBD in agreement with human GWAS studies revealing that VDR could have a role in IBD. An understanding of the etiology of IBD and for development of diagnostic biomarkers for IBD could be obtained using the intestine-specific Vdr knockout mouse model.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC005708-22
Application #
8763002
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
22
Fiscal Year
2013
Total Cost
$988,205
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
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