Expression and distribution of CYP3A genes, CYP2B22, and MDR1, MRP1, MRP2, LRP efflux transporters in brain of control and rifampicin-treated pigs.

The in vivo effect of rifampicin, a potent ligand of PXR, on gene expression of CYP2B22, 3A22, 3A29, 3A46, CAR, PXR and MDR1, MRP1, MRP2, LRP transporters in liver and cortex, cerebellum, midbrain, hippocampus, meninges and brain capillaries of pig was investigated. Animals were treated i.p. with four daily doses of rifampicin (40 mg/kg). The basal mRNA expressions of the individual CYP3As, CYP2B22, CAR, and PXR in various brain regions, except meninges, were about or below 10% of the corresponding hepatic mRNA values, whereas the mRNAs of brain transporters were closer or comparable to those in liver. After pig treatment with rifampicin, the mRNA expression of CYPs and transporters from brain regions did not appear to change, except CYP3A22 and 3A29 in cortex and hippocampus, CYP2B22 in meninges. An enzymatic analysis for CYP3As and CYP2B, in microsomes and mitochondria from liver and brain tissues using the marker activities 7-benzyloxyquinoline O-debenzylase and the anthraldehyde oxidase, showed the lack of rifampicin induction in all the brain regions, unlike liver. Taken together, our results demonstrate that CYP2B22, CYP3As, and MDR1, MRP1, MRP2, and LRP transporters are all expressed, although at different extent, in the brain regions but, despite the presence of PXR and CAR, are resistant to induction indicating that the regulation of these proteins is more complex in brain than in liver. These data obtained in vivo in the brain regions and liver of pig may be of interest to human metabolism in CNS.

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.

Expression, microsomal and mitochondrial activities of cytochrome P450 enzymes in brain regions from control and phenobarbital-treated rabbits.

Expression and monooxygenase activity of various cytochrome P450 (CYP) enzymes along with constitutive androstane (CAR) and the pregnane X (PXR) receptors were investigated in the brain of control and phenobarbital-treated rabbits (80 mg/kg for 4 days). RT-PCR analysis, using specific primers, demonstrated that in control rabbits mRNAs of CYP 2A10, 2B4/5 and 3A6 were expressed, though to a different extent, in the liver, as well as in brain cortex, midbrain, cerebellum, striatum, hippocampus and hypothalamus, whilst CYP2A11 and 4B1 were not expressed in the hypothalamus. CAR was expressed in liver and all the brain regions examined, whereas the PXR was expressed only in liver and cortex. Real time RT-PCR analysis demonstrated that in vivo treatment with phenobarbital, in contrast with what happened in liver, did not induce the expression of CYP 2B4/5 mRNA in cortex, midbrain and cerebellum. NADPH cytochrome c reductase and some other enzymatic activities markers of CYP 2A, 2B, 3A and 4B activities were studied in liver microsomes as well as in microsomes and mitochondria of brain cortex, midbrain and cerebellum of control and phenobarbital-treated rabbits. In contrast to what was observed in liver, phenobarbital treatment did not induce the aforementioned monooxygenase activities in brain. However, we cannot exclude that a longer phenobarbital treatment may lead to a significant induction of CYP activities in brain. These findings indicated that brain CYPs, despite the presence of CAR, were resistant to phenobarbital induction, indicating a possible different regulation of these enzymes between brain and liver.

Effect of beta-naphthoflavone on AhR-regulated genes (CYP1A1, 1A2, 1B1, 2S1, Nrf2, and GST) and antioxidant enzymes in various brain regions of pig.

The constitutive and inducible expression of aryl hydrocarbon receptor (AhR) and of the AhR-regulated genes coding for CYP1A1, CYP1A2, CYP1B1, CYP2S1, and Nrf2 was investigated by real-time or traditional PCR in cerebral areas (cortex, cerebellum, midbrain, and hippocampus), blood-brain interfaces (meninges and brain microvessels) and liver obtained from control pigs and from pigs treated with beta-naphthoflavone (betaNF), a potent AhR agonist. The enzymatic activities of ethoxyresorufin-O-deethylase (EROD), and methoxyresorufin-O-deethylase (MEROD), marker for CYP1A1 and CYP1A2, the GST and various antioxidant enzymes (catalase, superoxide dismutase, GSSG-reductase, and GSH-peroxidase) were also determined in the same CNS regions. The AhR, CYP1A1, CYP1A2, CYP1B1, Nrf2 mRNAs were detected, although at different extent, in all the CNS regions, while CYP2S1 mRNA was detected only in midbrain. In the blood-brain interfaces, the constitutive basal expression of AhR and CYP1A1 was comparable to the hepatic one and even higher for CYP1B1 and Nrf2. The treatment with betaNF determined the induction of CYP1A1 and 1B1 (but not of AhR, CYP1A2, and Nrf2) mRNA levels in various CNS areas; notably, CYP1A1 mRNA was increased to about 300-fold in the microvessels. The analysis of enzymatic activities revealed that EROD, but not MEROD, was induced in microsomes but not in mitochondria of all the CNS areas. However, the mitochondrial EROD activities were comparable (in midbrain, meninges) or higher (in cortex, cerebellum, hippocampus) than the microsomal ones, suggesting an important metabolic function of CYP1A1 in this subcellular localization. The activities of GST and antioxidant enzymes were detected in all CNS tissues, with levels lower than the hepatic ones, but found quite evenly distributed and marginally affected by betaNF treatment. The high expression of metabolic enzymes found in blood-brain interfaces could represent a very important defence toward toxins of CNS.

Effects of the anticancer dehydrotarplatin on cytochrome P450 and antioxidant enzymes in male rat tissues.

The effect of dehydrotarplatin (DTP), a new antineoplastic drug analogous to cisplatin, and its metabolite (Triacid) on the hepatic, renal and testicular CYP and antioxidant enzymes of male rats was investigated. The rats were treated i.p. with a single dose of DTP (25 mg kg(-1) day(-1)) or Triacid (17.5 mg kg(-1) day(-1)) and analysed 3 or 7 days post treatment. Three days after treatment, both drugs reduced body and liver weights, which partially recovered the control level after 7 days. DTP and, to a less extent, Triacid caused a depletion of plasmatic testosterone content and a down regulation in the liver of androgen dependent male specific CYP 2C11, but not of CYP 1A and 2E1, as determined by a significant decrease of 2alpha- and 16alpha-testosterone hydroxylase activities (markers for CYP 2C11) and of apoprotein immunoreactive with anti-rat CYP 2C11 antibodies. However, the activity of testicular 17alpha-progesterone hydroxylase, a key reaction in steroidogenesis, was not altered by these drugs. The DTP and Triacid administration did not cause any alteration of the plasmatic urea nitrogen and creatinine, known as markers of kidney toxicity. However, treatment with DTP, not Triacid, either 3 and 7 days post treatment, caused in the kidney microsomes a significant increase of the total CYP content, the CYP 4A-dependent (omega)- and (omega - 1)-lauric acid hydroxylase activities and apoprotein immunoreactive with anti-rat CYP 4A1. The present study also examined the enzymatic antioxidant status of kidney and liver. Neither DTP nor Triacid administration induced, with respect to control values, any alteration of hepatic and renal glutathione reductase, glutathione S-transferase, catalase, superoxide dismutase activities, hepatic GSH level and renal microsomal lipid peroxidation level. Among the antioxidant enzymes assayed, only the renal activity of glutathione peroxidase was significantly increased after DTP but not Triacid treatment. These results indicate that DTP at a dose of 25 mg/kg and Triacid cause a feminization of the CYP enzymes in male rat liver similar to that reported for cisplatin when administered at a low dose (5 mg/kg). However, unlike cisplatin, DTP and its metabolite were unable to enhance BUN and creatinine and cause any depression of CYP activities and antioxidant enzymes in the kidney, suggesting that DTP may have low or even no potential in inducing nephrotoxicity.