The gene expression profile of a drug metabolism system and signal transduction pathways in the liver of mice treated with tert-butylhydroquinone or 3-(3'-tert-butyl-4'-hydroxyphenyl)propylthiosulfonate of sodium.

Tert-butylhydroquinone (tBHQ) is a highly effective phenolic antioxidant used in edible oils and fats in foods as well as in medicines and cosmetics. TBHQ has been shown to have both chemoprotective and carcinogenic effects. Furthermore, it has potential anti-inflammatory, antiatherogenic, and neuroprotective activities. TBHQ induces phase II detoxification enzymes via the Keap1/Nrf2/ARE mechanism, which contributes to its chemopreventive functions. Nonetheless, there is growing evidence that biological effects of tBHQ may be mediated by Nrf2-independent mechanisms related to various signaling cascades. Here, we studied changes in gene expression of phase I, II, and III drug metabolizing enzymes/transporters as well as protein levels and activities of cytochromes P450 (CYPs) elicited by tBHQ and its structural homolog TS-13 in the mouse liver. Next, we carried out gene expression analysis to identify signal transduction pathways modulated by the antioxidants. Mice received 100 mg/kg tBHQ or TS-13 per day or only vehicle. The liver was collected at 12 hours and after 7 days of the treatment. Protein and total RNA were extracted. Gene expression was analyzed using Mouse Drug Metabolism and Signal Transduction PathwayFinder RT2Profiler™PCR Arrays. A western blot analysis was used to measure protein levels and a fluorometric assay was employed to study activities of CYPs. Genes that were affected more than 1.5-fold by tBHQ or TS-13 treatment compared with vehicle were identified. Analysis of the gene expression data revealed changes in various genes that are important for drug metabolism, cellular defense mechanisms, inflammation, apoptosis, and cell cycle regulation. Novel target genes were identified, including xenobiotic metabolism genes encoding CYPs, phase II/III drug metabolizing enzymes/transporters. For Cyp1a2 and Cyp2b, we observed an increase in protein levels and activities during tBHQ or TS-13 treatment. Changes were found in the gene expression regulated by NFκB, androgen, retinoic acid, PI3K/AKT, Wnt, Hedgehog and other pathways

Cyp1a1, Cyp1a2, and Cyp2b protein expression changes after 7 days of tBHQ or TS-13 treatment.

<p>(A) Western blot analysis. The number of animals in control and tBHQ groups was 3; in the TS-13 group– 4. (B) Data were densitometrically analyzed and normalized to β-actin. The results are shown as mean ± SD; *p < 0.05.</p

A summary of genes modulated (more than 1.5-fold) by tBHQ or TS-13 in the mouse liver.

<p>Hepatic gene expression patterns were analyzed after 12 h or 7 days of administration of tBHQ or TS-13.</p

Mouse drug metabolism PCR array.

<p>Mouse drug metabolism PCR array.</p

Effects of tBHQ and TS-13 on Cyp1a1 (A), Cyp1a2 (B), and Cyp2b (C) enzymatic activities in pmol × min^-1 × mg^-1 protein; *p < 0.05.

<p>Effects of tBHQ and TS-13 on Cyp1a1 (A), Cyp1a2 (B), and Cyp2b (C) enzymatic activities in pmol × min^-1 × mg^-1 protein; *p < 0.05.</p

Mouse signal transduction PathwayFinder PCR array.

<p>Mouse signal transduction PathwayFinder PCR array.</p

Chemical structures of tBHQ and TS-13.

<p>Chemical structures of tBHQ and TS-13.</p

Functional Analysis of the Unique Cytochrome P450 of the Liver Fluke <i>Opisthorchis felineus</i>

<div><p>The basic metabolic cytochrome P450 (CYP) system is essential for biotransformation of sterols and xenobiotics including drugs, for synthesis and degradation of signaling molecules in all living organisms. Most eukaryotes including free-living flatworms have numerous paralogues of the CYP gene encoding heme monooxygenases with specific substrate range. Notably, by contrast, the parasitic flatworms have only one CYP gene. The role of this enzyme in the physiology and biochemistry of helminths is not known. The flukes and tapeworms are the etiologic agents of major neglected tropical diseases of humanity. Three helminth infections (<i>Opisthorchis viverrini</i>, <i>Clonorchis sinensis</i> and <i>Schistosoma haematobium</i>) are considered by the International Agency for Research on Cancer (IARC) as definite causes of cancer. We focused our research on the human liver fluke <i>Opisthorchis felineus</i>, an emerging source of biliary tract disease including bile duct cancer in Russia and central Europe. The aims of this study were (i) to determine the significance of the CYP activity for the morphology and survival of the liver fluke, (ii) to assess CYP ability to metabolize xenobiotics, and (iii) to localize the CYP activity in <i>O</i>. <i>felineus</i> tissues. We observed high constitutive expression of CYP mRNA (Real-time PCR) in <i>O</i>. <i>felineus</i>. This enzyme metabolized xenobiotics selective for mammalian CYP2E1, CYP2B, CYP3A, but not CYP1A, as determined by liquid chromatography and imaging analyses. Tissue localization studies revealed the CYP activity in excretory channels, while suppression of CYP mRNA by RNA interference was accompanied by morphological changes of the excretory system and increased mortality rates of the worms. These results suggest that the CYP function is linked to worm metabolism and detoxification. The findings also suggest that the CYP enzyme is involved in vitally important processes in the organism of parasites and is a potential drug target.</p></div

Transcriptional responses for mRNA encoding CYP following <i>in vitro</i> exposure of <i>Opisthorchis felineus</i> to xenobiotics.

<p>Analysis by real-time PCR with normalization based on the expression stability value (M-value); normalization was undertaken using <i>Ub</i> and <i>MrpL16</i> genes as endogenous internal controls (M < 1.2). Triplicate real-time PCRs were run for each sample. <b>A</b>. An adult worm; <b>B</b>. Newly excysted metacercariae (NEM). <b>C.</b> CYP mRNA level after <i>in vitro</i> treatment of <i>O</i>. <i>felineus</i> for 20 h with hemoglobin (DDPCR). CYP gene expression levels were quantified and values were simultaneously normalized to reference gene <i>MrpL16</i> expression using QuantaLife (Bio-Rad, USA). The data are shown as the normalized ratio of CYP to <i>MrpL16</i> ± S.D. Each run was made in duplex. Results of three independent experiments are presented.</p

<i>In situ</i> activity of CYP.

<p>Fluorescence micrographs of <i>Opisthrochis felineus</i> after exposure <i>in vitro</i> for 20 h to several substances. <b>A.</b> A control fluke (multiple fluorescence filters: FITC, rhodamine, DAPI). <b>B.</b> Adult worms were treated with methoxyresorufin. <b>C.</b> Adult worms were treated with benzoxyresorufin. <b>D.</b> Adult worms were treated with pentoxyresorufin (PR). The resorufin (<b>R</b>) that was formed in fluke tissues is indicated with an arrow (FITC, rhodamine fluorescence filters). <b>E.</b> Adult worms were treated with penzoxyresorufin (rhodamine). <b>F.</b> Excretory granules of the resorufin formed in <i>O</i>. <i>felineus</i> treated with pentoxyresorufin. Monochrome images were acquired using a rhodamine filter (5F is a part of image 5E). <b>G.</b> A worm was treated with PR and ketoconazole (rhodamine filter).</p