All-trans retinoic acid inhibits the recruitment of ARNT to DNA, resulting in the decrease of CYP1A1 mRNA expression in HepG2 cells

Aryl hydrocarbon receptor (AHR) and AHR nuclear translocator (ARNT) are well-conserved transcription factors among species. However, there are a very limited number of reports on the physiological function of AHR, particularly on the regulation of AHR by endogenous compounds. We hence investigated the effects of all-trans retinoic acid (atRA) on cytochrome P450 (CYP)1A1 gene transcription as a model of AHR-regulated transcription mechanisms in HepG2 cells, a human hepatoma cell line. Treatment with atRA significantly reduced transactivation and expression of CYP1A1 mRNA to less than half of its control value, and this inhibitory effect was mediated by RARα. The result of chromatin immunoprecipitation assay indicated that treatment with atRA at 1-100 nM drastically inhibited the recruitment of ARNT to DNA regions containing xenobiotic responsive elements. In conclusion, atRA at physiological concentrations could reduce AHR-mediated gene transcription via the inhibition of recruitment of ARNT to relevant DNA regions

Astaxanthin can alter CYP1A-dependent activities via two different mechanisms : Induction of protein expression and inhibition of NADPH P450 reductase dependent electron transfer

Astaxanthin (Ax), a xanthophyll carotenoid, is reported to induce cytochrome P450 (CYP) 1A-dependent activity. CYP1A is one of the most important enzymes participating in phase I metabolism for chemicals, and it can activate various mutagens. To investigate the effect of Ax on the metabolic activation of a typical promutagen, benzo[a]pyrene by CYP1A, we orally administrated Ax (100 mg / kg body weight / day for 3 days) to male Wistar rats. In the treated rat liver, expression of CYP1A1 mRNA, protein, and its activity were significantly increased (5.5-fold, 8.5-fold, and 2.5-fold, respectively). In contrast, the activities of phase II enzymes (glutathione S-transferase and glucuronosyl-transferase) were not modulated by Ax. As a consequence, the mutagenicity of benzo[a]pyrene was more enhanced in Ax-treated rats, compared with controls in the Ames assay. On the other hand, NADPH P450 reductase activity was decreased in liver microsomes from the treated group. This result suggests the possibility that Ax inhibits the electron supply necessary for CYP catalytic activities and decreases CYP1A activity indirectly. In conclusion, Ax intake can alter CYP1A-dependent activities through two different mechanisms: 1) induction of CYP1A1 mRNA, protein expression, and activity; and 2) inhibition of the electron supply for the enzyme

Metabolic Activation of Heterocyclic Amines and Expression of CYP1A1 in the Tongue

Xenobiotic metabolism in oral tissues, especially in the tongue, has never been reported. In the present study, the metabolic activation/detoxification ability of promutagens in the tongue and the expression levels of related enzymes were investigated. Quantitative PCR analysis of rat tongue demonstrated constitutive mRNA expression of numerous drug-metabolizing enzymes. In particular, we detected mRNA, protein expression and enzymatic activity of cytochrome P450 (CYP)1A1 in the tongue tissue. Metabolic activation of promutagens in the tongue was estimated using benzo[a]pyrene or heterocyclic amines (HCAs), found in cooked meat and tobacco products. Metabolic activation levels of HCAs in the tongue were comparable to those in the liver. In contrast, the expression levels of glutathione S-transferase (GST) and UDP-glucuronosyltransferase (UGT) in the tongue were considerably lower compared with those in the liver and, as a result, the mutagenic activity in the tongue was not decreased by GST- or UGT-dependent conjugation. Treatment of rats with sudan III, a typical inducer of CYP1A1, resulted in markedly increased CYP1A1 mRNA, protein expressions, CYP1A-dependent enzymatic and mutagenic activities. In addition, CYP1A1 mRNA expression in carcinoma cells (SAS) was induced by sudan III exposure. In conclusion, mutagenic activation of xenobiotics and an increased risk of cancer in the tongue were observed in this study. Furthermore, ingestion of drug-metabolizing enzyme inducers has the potential to increase the metabolic activation in the tongue tissue and increase the risk of biomolecular attack by promutagens

Carotenoids as regulators for inter-species difference in Cytochrome P450 1A expression and activity in ungulates and rats

Ungulates (deer, cattle and horses) are reported as animal species which show extreme-accelerated metabolism of CYP1A substrates, such as ethoxyresorufin compared to rats. This study was undertaken to investigate whether accumulation of carotenoids is a possible cause for inter-species difference in CYP1A-dependent activity in this group of animals. The relationship between inter-species differences in CYP1A-dependent activity and the accumulated carotenoids and retinoids as candidates of dietary CYP1A inducers in ungulate species was clarified. Interestingly, there were positive correlations between the accumulated carotenoids, such as β-carotene, with both EROD activity and CYP1A protein expression. These correlations were negative with the accumulated retinoids, such as retinol. The β-carotene was major component of carotenoids in ungulates, and known as an inducer of CYP1A. On the other hand, the retinol is reported as the inhibitor of CYP1A. Other factors which affect CYP1A1 expression, such as polycyclic aromatic hydrocarbons, were also analyzed. To cancel the effects of inter-species difference in CYP1A induction signal cascade among these animals, the rat cell line (H4-II-cells) was treated with the extracted carotenoids from the examined animals. In conclusion, carotenoids and retinoids may have direct effects on the inter-species differences in CYP1A-dependent activity and protein expression

Metabolic Activation of Heterocyclic Amines and Expression of Xenobiotic-Metabolizing Enzymes in the Gastrointestinal Tract of Rats

Heterocyclic amines get entry into human body mainly through ingestion of pan-fried meats cooked at high temperatures. Exposure of the gastrointestinal tract (GIT) to ingested xenobiotics prior to delivery to the liver may lead to metabolic activation, which may explain the high incidence of GIT carcinogenesis. Therefore, this study investigated the mutagenic activation of 2 heterocyclic amines, 2-aminoanthracene (2-AA) and 3-amino-1-methyl-5H-prydo[4,3-b]indole (Trp-P-2), in the GIT of rats. In addition, the constitutive mRNA expression profiles of xenobiotic-metabolizing enzymes (XMEs) in the GIT of rats were examined. Metabolic activation of 2-AA was detected in all GIT tissues except the duodenum and rectum, and it was detected at high levels in the ileum and cecum. Furthermore, we revealed high metabolic activation of 2-AA and Trp-P-2 in the jejunum. The mRNA expression of phase I and II enzymes in rat GIT corresponded with their mutagenic activation ability. In conclusion, our results suggest that different expression levels of XME among GIT tissues may contribute to the tissue-specific differences in metabolic activation of xenobiotics such as heterocyclic amines in rats. Practical Application This study declares mutagenic activation of 2 heterocyclic amines namely 2-aminoanthracene (2-AA) and 3-amino-1-methyl-5H-prydo[4,3-b]indole (Trp-P-2), in the gastrointestinal tract (GIT) of rats. In addition, results obtained in this study suggest that GIT tissue-specific expression of xenobiotic metabolizing enzymes may contribute to the tissue-specific mutagenesis/carcinogenesis

Strain differences in cytochrome P450 mRNA and protein expression, and enzymatic activity among Sprague Dawley, Wistar, Brown Norway and Dark Agouti rats

Rat cytochrome P450 (CYP) exhibits inter-strain differences, but their analysis has been scattered across studies under different conditions. To identify these strain differences in CYP more comprehensively, mRNA expression, protein expression and metabolic activity among Wistar (WI), Sprague Dawley (SD), Dark Agouti (DA) and Brown Norway (BN) rats were compared. The mRNA level and enzymatic activity of CYP1A1 were highest in SD rats. The rank order of Cyp3a2 mRNA expression mirrored its protein expression, i.e., DA>BN>SD>WI, and was similar to the CYP3A2-dependent warfarin metabolic activity, i.e., DA>SD>BN>WI. These results suggest that the strain differences in CYP3A2 enzymatic activity are caused by differences in mRNA expression. Cyp2b1 mRNA levels, which were higher in DA rats, did not correlate with its protein expression or enzymatic activity. This suggests that the strain differences in enzymatic activity are not related to Cyp2b1 mRNA expression. In conclusion, WI rats tended to have the lowest CYP1A1, 2B1 and 3A2 mRNA expression, protein expression and enzymatic activity among the strains. In addition, SD rats had the highest CYP1A1 mRNA expression and activity, while DA rats had higher CYP2B1 and CYP3A2 mRNA and protein expression. These inter-strain differences in CYP could influence pharmacokinetic considerations in preclinical toxicological studies

Influenza virus infection affects insulin signaling, fatty acid-metabolizing enzyme expressions, and the tricarboxylic acid cycle in mice

Although the severity of influenza virus infections has been associated with host energy metabolism, the related mechanisms have not yet been clarified. Here we examined the effects of influenza virus infection on host energy metabolism in mice. After infecting mice with intranasal applications of 500 plaque-forming units of A/Puerto Rico/8/34 (H1N1; PR8) virus, the serum levels of most intermediates in the tricarboxylic acid (TCA) cycle and related metabolic pathways were significantly reduced. These data suggest that substrate supply to the TCA cycle is reduced under these conditions, rather than specific metabolic reactions being inhibited. Then, we focused on glucose and fatty acid metabolism that supply substrates to the TCA cycle. Akt phosphorylation following insulin injections was attenuated in the livers of PR8 virus-infected mice. Furthermore, glucose tolerance tests revealed that the PR8 virus-infected mice showed higher blood glucose levels than the vehicle-inoculated control mice. These results suggest that influenza virus infection impairs insulin signaling, which regulates glucose uptake. However, increases in the hepatic expressions of fatty acid-metabolizing enzymes suggest that fatty acids accumulate in liver cells of infected mice. Collectively, our data indicate that influenza virus infection dysregulates host energy metabolism. This line of investigation provides novel insights into the pathogenesis of influenza