We performed a detailed analysis of mouse cytochrome P450 2A5 (CYP2A5) expression by in situ hybridization (ISH) and immunohistochemistry (IHC) in the respiratory tissues of mice. The CYP2A5 mRNA and the corresponding protein co-localized at most sites and were predominantly detected in the olfactory region, with an expression in sustentacular cells, Bowman's gland, and duct cells. In the respiratory and transitional epithelium there was no or only weak expression. The nasolacrimal duct and the excretory ducts of nasal and salivary glands displayed expression, whereas no expression occurred in the acini. There was decreasing expression along the epithelial linings of the trachea and lower respiratory tract, whereas no expression occurred in the alveoli. The hepatic CYP2A5 inducers pyrazole and phenobarbital neither changed the CYP2A5 expression pattern nor damaged the olfactory mucosa. In contrast, the olfactory toxicants dichlobenil and methimazole induced characteristic changes. The damaged Bowman's glands displayed no expression, whereas the damaged epithelium expressed the enzyme. The CYP2A5 expression pattern is in accordance with previously reported localization of protein and DNA adducts and the toxicity of some CYP2A5 substrates. This suggests that CYP2A5 is an important determinant for the susceptibility of the nasal and respiratory epithelia to protoxicants and procarcinogens.
Interaction of two members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family with the 3Јuntranslated region (UTR) of the murine inducible nitric-oxide synthase (iNOS) mRNA is demonstrated in this study. An iNOS RNA-protein complex is formed using protein extracts from untreated and septic shock treated mouse liver. UV cross-linking reveals that the complex consists of at least two proteins, with apparent molecular masses of 60 and 70 kDa, respectively. The 60-kDa protein binding site lies within a 112-nt pyrimidine-rich sequence, approximately 160 nt from the coding sequence, and the RNA-protein complex can be precipitated by a monoclonal antibody directed against hnRNP I [also named polypyrimidine tract binding protein (PTB)]. The 70-kDa protein binds a 43-nt sequence near the 3Јend of the 3ЈUTR and is immunoprecipitated by a monoclonal antibody against hnRNP L. A computersimulated conformation of the 3ЈUTR suggests that both binding sites reside in regions easily accessible for a protein.Supershifts of the native RNA-protein complex could only be achieved with anti-hnRNP L, suggesting that within this multiprotein RNA complex, only hnRNP L is exposed to the antibodies, whereas the hnRNP I/PTB is mainly responsible for its interaction with the mRNA. Up-regulation of iNOS by septic shock reduces the RNA-protein complex formation, thus showing that hnRNP I/PTB and hnRNP L binding to the iNOS mRNA is modulated by inflammation. This suggests a novel function for the two previously described proteins as regulators of the iNOS gene.
Artemisinin (a sesquiterpene lactone endoperoxide) has become important in multi-drug treatment of malaria. There is evidence that artemisinin induces drug metabolism which could result in drug-drug interactions. The objective of this study was to characterize the inductive properties of artemisinin on drug-metabolizing cytochrome P450 (CYP450) enzymes. The possibility of artemisinin to induce CYP450 was studied in artemisinin-treated (orally for four days) and vehicle-treated rats using reverse transcriptase polymerase chain reaction (RT-PCR). The effect on enzymatic activities in mouse microsomes from multiple artemisinin administration (intraperitonally) to mice were also studied as well as the effect on the expression in mouse primary hepatocytes and HEK293 cells. Increased CYP2B1 mRNA levels in rats could be seen after artemisinin treatment as well as a weak but reproducible increase in the intensity of CYP1A2. Administration of artemisinin to mice up-regulated hepatic CYP2B10-dependent, and to a lesser extent, CYP2A5-dependent enzyme activities. In primary hepatocyte culture, artemisinin significantly increased the CYP2B10 mRNA levels whereas the CYP2A5 mRNA levels were increased to a lesser extent. No significant changes were seen in the levels of other CYP enzymes. Artemisinin was an activator of constitutive androstane receptor (CAR) but not pregnane X receptor (PXR) in HEK293 cells. The results demonstrate that the drug exerts its effects on drug metabolism via the CAR receptor that results in up-regulation of genes such as the Cyp2b. The weaker up-regulation of CYP2A5 might also be CAR-dependent or alternatively, a consequence of artemisinin toxicity. The results of this study are of importance when predicting potential drug-drug interactions in multi-drug therapies with artemisinin.
hGSTA3-3 (human Alpha-class glutathione transferase 3-3) efficiently catalyses steroid Delta(5)-Delta(4) double-bond isomerization in vitro, using glutathione as a cofactor. This chemical transformation is an obligatory reaction in the biosynthesis of steroid hormones and follows the oxidation of 3beta-hydroxysteroids catalysed by 3beta-HSD (3beta-hydroxysteroid dehydrogenase). The isomerization has commonly been ascribed to a supplementary function of 3beta-HSD. The present study is the first to provide evidence that hGSTA3-3 contributes to this step in steroid hormone biosynthesis in complex cellular systems. First, we find glutathione-dependent Delta(5)-Delta(4) isomerase activity in whole-cell extracts prepared from human steroidogenic cells. Secondly, effective inhibitors of hGSTA3-3 dramatically decrease the conversion of Delta(5)-androstene-3,17-dione into Delta(4)-androstene-3,17-dione in cell lysates. Thirdly, we show that RNAi (RNA interference) targeting hGSTA3-3 expression decreases by 30% the forskolin-stimulated production of the steroid hormone progesterone in a human placental cell line. This effect is achieved at low concentrations of two small interfering RNAs directed against distinct regions of hGSTA3-3 mRNA, and is weaker in unstimulated cells, in which hGSTA3-3 expression is low. The results concordantly show that hGSTA3-3 makes a significant contribution to the double-bond isomerization necessary for steroid hormone biosynthesis and thereby complements the indispensable 3beta-hydroxysteroid oxidoreductase activity of 3beta-HSD. The results indicate that the lower isomerase activity of 3beta-HSD is insufficient for maximal rate of cellular sex hormone production and identify hGSTA3-3 as a possible target for pharmaceutical intervention in steroid hormone-dependent diseases.
The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) functions in the packaging of nascent RNA polymerase II transcripts and participates in a variety of nuclear and cytoplasmic processes that modulate gene expression. The RNA binding characteristics of hnRNP A1 suggest that it can modulate the expression of specific genes, but little is known about its possible targets in vivo. In this article, we show that hnRNP A1 interacts with the transcript of a cytochrome P450 gene, Cyp2a5, induced by xenobiotics and during liver damage. Binding of the hnRNP A1 to CYP2A5 mRNA was demonstrated by immunoprecipitation of the xenobiotic-stimulated (37/39 kDa) CYP2A5 mRNA-protein complex with a monoclonal antihnRNP A1 antibody, by partial trypsin digestion of the complex, and by showing that the RNA-protein complex is not formed with protein extracts from cells lacking the hnRNP A1. We also show that a specific hepatotoxic inducer of the Cyp2a5 gene, pyrazole, increases the cytoplasmic levels of hnRNP A1 in vivo. Finally, we show that hnRNP A1 can be overexpressed in mouse primary hepatocytes, leading to an accumulation of the CYP2A5 mRNA. Collectively, these results indicate that the hnRNP A1 is an important regulator of the Cyp2a5 gene.
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