Expression and induction by rifampicin of CAR- and PXR-regulated CYP2B and CYP3A in liver, kidney and airways of pig.

The transcript levels of CYP2B22, 3A22, 3A29, 3A46, CAR, PXR and HNF4alpha were investigated in liver, kidney and airways from control and rifampicin-treated male pigs. The presence and induction of CYP genes transcription were studied by RT-PCR, real-time PCR, Western blotting and enzymatic activity whereas the expression of receptors was studied by RT-PCR or real-time PCR. Pretreatment with rifampicin resulted in a transcriptional activation, although to different extents, of all the CYP3A genes in liver but not in kidney, lung, bronchi or trachea. In the hepatic microsomes, the induction of CYP3A genes was accompanied by an increase of CYP3As marker activities and of two protein bands immunoreactive with anti-human CYP3A4. The CYP2B22 transcript was found to be markedly induced only in liver and kidney. In parallel, a protein band immunoreactive with anti-rat CYP2B1 was elevated while enhanced CYP2B marker activities were observed in hepatic and renal microsomes. As expected, based on human data, the basal expression of CAR, PXR and HNF4alpha was found to be high in liver and low in airways and not susceptible to induction by rifampicin. A significant expression of these transcriptional factors was also demonstrated in kidney. Thus, it is likely that rifampicin induced CYP2B22 both in liver and kidney of pig, not via activation of CAR, but via PXR, through a cross-talk mechanism, as previously observed in human liver. Taken together, our results demonstrated a differential expression and regulation of three individual CYP3As, CYP2B22, CAR, PXR and HNF4alpha genes in liver, kidney and airways of pig.

Inducibility of AhR-regulated CYP genes by beta-naphthoflavone in the liver, lung, kidney and heart of the pig.

The presence and inducibility of CYP enzymes belonging to the family 1 (CYP 1A1, 1A2 and 1B1) and AhR have been studied in liver, lung, kidney and heart of control and beta-naphthoflavone (beta NF)-treated pigs. Segments of so far undescribed genes for porcine CYP 1A2, 1B1 and AhR were identified by RT-PCR and their sequences found to be highly homologous to those of the corresponding human genes. The mRNA level of CYP 1A1 was induced by beta NF, although to a different extent, in liver, lung, kidney and heart. This transcriptional activation of CYP 1A1 was accompanied in microsomes of all these organs by an induction of 7-ethoxyresorufin deethylase activity (a marker of this isoform) and an increase in a protein band immunoreactive with anti-rat CYP 1A1. An increase in CYP 1A2 transcription and in activity of microsomal 7-methoxyresorufin demethylase and acetanilide 4-hydroxylase (both markers of 1A2) was observed in the liver and, to a very small extent, in the lung but not in kidney and heart. As to CYP 1B1, its transcription was detected in liver, lung and heart only following the beta NF treatment; however this mRNA expression did result in any detectable microsomal 17beta-estradiol 4-hydroxylase activity (a marker of this isoform). The CYPs induced by beta NF were further investigated by using some other marker activities. It was found that porcine CYP 1A1 and 1A2, unlike the human counterparts, could only deethylate 7-ethoxycomarin to a very small extent, if at all, whereas 7-ethoxy 4-trifluoromethylcoumarin was a good substrate for pig CYP 1A1. Overall, our results demonstrated a differential expression and regulation of the AhR-mediated CYP genes in liver, lung, kidney and heart of the pig.naphthoflavone.

CAR and PXR expression and inducibility of CYP2B and CYP3A activities in rat and rabbit lungs.

Several CYP enzymes are expressed in the lung of mammals but studies on their regulation have been rather neglected. In this study, the CAR and PXR expression and the inducibility of CYP 2B and CYP 3A isoforms in the lung rats and rabbits were investigated. Rats were treated with phenobarbital, clotrimazole or a mixture of dexametasone plus pregnenolone 16alpha-carbonitrile, whereas rabbits were treated with phenobarbital or rifampicin. A low constitutive expression of CAR mRNA was demonstrated by RT-PCR analysis in the lung of rat but not in rabbit. Phenobarbital treatment did not change the CAR expression profiles and did not induce in either rats and rabbits the pulmonary CYP 2B isoforms, as judged by western blot analysis and the marker pentoxyresorufin O-dealkylase and 7-ethoxy-4-trifluoroethylcoumarin O-deethylase activities. On the contrary, these marker activities were strongly induced by phenobarbital in the liver of both species. A low constitutive level of PXR mRNA was also detected by RT-PCR in the lung of rabbit but not in rat. However, also in this case, their expressions were not altered by the administration of strong CYP 3A inducers such as clotrimazole or a mixture of dexametasone plus pregnenolone 16alpha-carbonitrile for the rat and rifampicin or phenobarbital for the rabbit. For the first time, it was demonstrated by RT-PCR that rat lung expresses CYP 3A2, 3A9, 3A18 and 3A23 whereas the rabbit lung expresses the CYP 3A6, the only CYP 3A isoform identified in the rabbit so far. However, notwithstanding the differences observed in the constitutive presence of PXR and CYP 3A transcripts in both species, the above mentioned treatments did not affect in their lungs, unlike their livers, neither the anti-rat 3A immunoreactive proteins nor the CYP 3A marker 7-benzyloxyquinoline O-debenzylase and the 6beta-testosterone hydroxylase activities. The results obtained indicate that the role of CAR and PXR in the lung of rat and rabbit is different from that observed in the liver or other extrahepatic tissues where the induction of the CYP 2B and CYP 3A isoforms is regulated by these receptors.

Cytochrome P450 2J2 polymorphism in healthy Caucasians and those with diabetes mellitus.

OBJECTIVE: Cytochrome P450 (CYP) 2J2 plays an important role in the biosynthesis of the biologically active cis-epoxyeicosatrienoic acids. An allelic variant named CYP2J2*6, which encodes an enzyme that is almost inactive in the metabolism of arachidonic acid, has recently been described. We investigated the frequency of the CYP2J2*6 variant in a Caucasian population and the relationship between this polymorphism and the development of micro- and macrovascular complications and hypertension in patients with type 1 or type 2 diabetes mellitus. METHODS: Genomic DNA was extracted from peripheral blood cells and the fragment containing the A/T single nucleotide polymorphism at position 25 661 in exon 8 of the CYP2J2 gene was amplified. The 532 bp amplified product was subsequently digested with Tsp509I and analyzed on 12% polyacrylamide gel electrophoresis. RESULTS: In the whole population, the frequency of the CYP2J2*6 allele was 0.0064 and the frequency of the CYP2J2*1 allele was 0.9936. Genotype distribution did not show significant differences between controls and patients with type 1 or type 2 diabetes. No homozygotes for CYP2J2*6 allele were found. No association was found between this allele and complications or hypertension in either type of diabetes. CONCLUSION: The CYP2J2*6 allele is rare in the Caucasian population, and no association is inferred between this allelic variant and diabetic complications.

Lisosan G, a powder of grain, does not interfere with the drug metabolizing enzymes and has a protective role on carbon tetrachloride-induced hepatotoxicity.

Lisosan G is a powder of grain registered as an alimentary integrator. The treatment of rats for 4 days with 0.5 g Lisosan G/kg had no effect on various drug metabolizing enzymes. Experiments in vitro showed that Lisosan G had radical scavenger activity. A confirmation of the antioxidative property of Lisosan G was also confirmed when it was administered in vivo to carbon tetrachloride (CCl(4))-intoxicated rats. The toxicity caused by CCl(4)-treatment of rats was restored to the control levels when the rats were given Lisosan G for 4 days before CCl(4). Lisosan G thus does not interfere with drug metabolizing system but has antioxidant properties and protects against CCl(4)-induced hepatotoxicity.

Expression and activity of CYP2E1 in circulating lymphocytes are not altered in diabetic individuals.

Cytochrome P4502E1 (CYP2E1) plays an important role in ROS production thus favouring accelerated membrane lipid peroxidation. This isoform is strongly expressed in the liver but it can be also found in lymphocytes. As such, lymphocyte may provide a non-invasive accessible pool for screening CYP2E1 expression in man. We have, therefore, analysed CYP2E1 expression and activity in lymphocyte microsomes from 12 healthy controls, 11 type 1 and 12 type 2 diabetic subjects by using Western blot and enzymatic activities. Immunoblotting did not show difference among CYP2E1 protein bands in controls, type 1 and type 2 diabetics. To assess CYP2E1 activity we used the 7-ethoxy-4-trifluoromethylcoumarin (7-EFC), as a fluorescent substrate. The rate of deethylation of 7-EFC from controls did not differ from type 1 and type 2 diabetic subjects. The lack of any difference in CYP2E1 activity also was confirmed by the NADPH-dependent microsomal lipid peroxidation CCL4-induced assay showing similar peroxidation rates among controls and diabetic subjects. The results show that CYP2E1 expression/activity in lymphocytes is not enhanced in diabetes.