AB1253 Sigma-AldrichAnti-Cytochrome P450 Enzyme CYP3A1 Antibody

The aim of this study was to investigate the impact on expression of mRNA and protein by paradigm inducers/activators of nuclear receptors and their target genes in rat hepatic and intestinal cells. Furthermore, assess marked inter laboratory conflicting reports regarding species and tissue differences in expression to gain further insight and rationalise previously observed species differences between rodent and human based systems.Quantitative real time-polymerase chain reaction (QRT-PCR) and immunoblots were used to assess messenger RNA (mRNA) and protein expression for CYP2B2, CYP3A1, CYP3A2, CYP3A9, ABCB1a, ABCB1b, ABCC1, ABCC2, pregnane X receptor (PXR), farnesoid X receptor (FXR) and constituitive androstane receptor (CAR) in rat hepatoma cell line H411E, intestinal cells, Iec-6, and rat primary hepatocytes, in response to exposure for 18 h with prototypical inducers.Dexamethasone (DEX) and pregnenolone 16alpha carbonitrile (PCN) significantly induced PXR, CYP3A9, ABCB1a and ABCB1b. However, when co-incubated, DEX appeared to restrict PCN-dependent induction. Chenodeoxycholic acid (CDCA) was the only ligand to induce FXR in all three cell types. Despite previously reported species differences between PCN and rifampicin (RIF), both compounds exhibited a similar profile of induction.Data presented herein may explain some of the discrepancies previously reported with respect to species differences from different laboratories and have important implications for study design.

BACKGROUND: The available information about hepatic drug-metabolizing enzymes in the large-for-gestational-age (LGA) neonate is limited. OBJECTIVE: To determine whether LGA status alters expression of the cytochrome p450 enzymes, CYP3A23/1 and CYP3A2, and nuclear receptors PXR, CAR and HNF4alpha. METHODS: Appropriate-for-gestational-age (AGA) and LGA pups from 10 litters of rats (n=15 pups/group) were used. Hepatic expression levels of CYP3A23/1, CYP3A2, pregnane X receptor (PXR), constitutive androstane receptor (CAR), and HNF4alpha mRNA were detected by quantitative real-time RT-PCR. Protein expression was determined immunohistochemically and expression levels were evaluated using staining intensity scores and percentage of positive hepatocytes. RESULTS: The mean body weights of AGA and LGA rat pups were 6.20 +/- 0.49 and 7.77 +/- 0.70 g (mean +/- SD), respectively (P 0.001). Liver weight/body weight ratios trended higher in the LGA group (5.18 +/- 0.50%, mean +/- SD) than in the AGA group (4.90 +/- 0.01%, mean +/- SD), but the difference was not significant. CYP3A2 mRNA levels were higher in LGA than in AGA pups (P 0.05), but there were no group differences in CYP3A23/1 mRNA levels. HNF4alpha levels were also higher in the LGA group (P 0.05), but PXR and CAR mRNA levels did not differ between the groups. The staining intensity and frequency of CYP3A23/1-positive and HNF4alpha-positive hepatocytes differed between LGA and AGA pups, whereas no significant differences in CYP3A2 or CAR protein expression were observed between groups. CONCLUSIONS: LGA status affects the hepatic expression of CYP3A23/1, CYP3A2, and HNF4alpha, suggesting that further research on this issue is warranted.

Cytochrome P-450 3A (CYP3A) together with its nuclear receptors plays a critical role in drug metabolism. The present study investigated the effects of undernutrition in utero on hepatic mRNA and protein expression of the enzyme CYP3A23/3A1 and nuclear receptors including pregnane X receptor (PXR; NR1I2), constitutive androstane receptor (CAR; NR1I3) and nuclear factor-4alpha (HNF4alpha; HNF4A) in neonatal rats. At gestational day 2, pregnant rats were randomly divided into two groups: nourished (fed ad libitum) and undernourished (50% of nourished group). The pups delivered by nourished rats were designated as the normal-birth-weight group (NBW, n=15) and those delivered by undernourished rats were designated as the low-birth-weight group (LBW, n=15). Hepatic mRNA expression was detected by quantitative real-time PCR and the corresponding protein expression was examined by immunohistochemistry (IHC). Compared with NBW pups, LBW pups tended to have lower mRNA expression levels of CYP3A23/3A1, PXR and CAR but higher levels of HNF4alpha. Only the CAR mRNA expression differences were significant (p0.05). mRNA expression of CYP3A23/3A1 correlated with that of HNF4alpha in both the LBW(r=0.808, p=0.007) and NBW (r=0.452, p=0.012) groups. CYP3A23/3A1 and CAR protein expression differed between the two groups (CYP3A23/3A1, chi(2)=7.87, p=0.005; CAR, chi(2)=12.069, p=0.001). In conclusion, these findings suggest that undernutrition may influence the mRNA expression of CAR and protein expression of both CYP3A23/3A1 and CAR in neonatal rats. Since CYP3A23/3A1 and CAR are critically involved in drug metabolism, these results may have clinical implications for optimal medication in LBW children.

Assessment of cytochrome P450 (CYP) induction at the mRNA level in preclinical rodent studies has gained interest in recent years, but there are still concerns regarding correlations between the mRNA and the enzyme activity levels, especially in mice. The purpose of the present study was to systematically evaluate patterns of temporal changes of CYPs 1a1, 1a2, 2b10, 3a11, and 4a10 at mRNA, protein, and activity levels in order to determine to what extent mRNA levels could be used either qualitatively or quantitatively for the assessment of CYP enzyme induction. In this study, livers from male CD-1 mice treated daily with beta-naphthoflavone, phenobarbital, dexamethasone, clofibrate, and control vehicles were collected for RNA and microsomal analysis after 0.5, 1, 2, 4, and 8 days of daily dose. The results revealed a good correlation among mRNA, protein, and enzyme activity levels, with the best correlation at the time points between Days 2 and 8, suggesting that the appropriate time to monitor CYP mRNA may be beyond Day 2 of chemical treatments. Based on these results, we concluded that the mRNA approach is a useful tool to monitor CYP induction in mice, particularly when treatment duration is beyond 2 days.

A major cause of death and illness in children under the age of five, most living in polluted cities, is respiratory disease. Previous studies have shown that neonatal animals are more susceptible to bioactivated pulmonary cytotoxicants than adults, despite lower expression of the pulmonary cytochrome P-450s (CYP450s) thought to be involved in bioactivation. One CYP450 that is well documented in the bioactivation of many drugs and environmental toxicants in adult lung, but whose expression has not been evaluated during postnatal pulmonary development, is CYP450 3A (CYP3A). We compared age-specific expression of CYP3A1 in 7-day-old and adult male Sprague-Dawley rats. Unlike those shown for previously studied pulmonary CYP450s, expression levels for CYP3A1 mRNA in differentiating airway cells of postnatal rats are the same as in fully differentiated airway cells of adults. CYP3A1 protein expression (28%) and enzymatic activity (23%) were lower in postnatal airways compared with adults. Although other CYP450 immunoreactive proteins are primarily expressed in nonciliated cells, immunoreactive CYP3A1 protein was expressed in both ciliated and nonciliated cells in postnatal and adult rat proximal airways. CYP3A1 protein is detected diffusely throughout ciliated and nonciliated cells in 7-day-old rats, whereas it is only detected in the apex of these cells in adult rats. This study demonstrates that the lungs of postnatal rats have detectable levels of CYP3A1 and that CYP3A1 mRNA expression appears not to be age dependent, whereas steady-state CYP3A1 protein levels and enzyme activity show an age-dependent pattern.

PURPOSE: Paclitaxel (Taxol) is an effective agent against a broad range of human cancers. Studies on the metabolism and disposition of paclitaxel have shown that it is primarily eliminated via hepatic metabolism by P450 enzymes (2C8 and 3A4) to essentially inactive metabolites, and that biliary and gut transport by P-glycoprotein (PGP) as well as urinary elimination of the parent compound play relatively minor roles. Recent studies in vitro have shown that paclitaxel treatment increases the level of CYP2C8 and CYP3A4 in human hepatocytes as well as PGP in colon tumor cells. The data suggest that previous paclitaxel exposure may influence metabolism and elimination of subsequent doses. Further, since weekly paclitaxel dose schedules are becoming more common as opposed to the original every 21-day dosing, the likelihood of enzyme induction from previous doses impacting that from subsequent doses is increased. METHODS: To study the potential for such sequence-dependent alterations in paclitaxel pharmacokinetics, we carried out pharmacokinetic studies in mouse plasma and tissues following day 1 and days 1 and 5 dosing at 20 mg/kg. Paclitaxel concentrations were determined by a sensitive LC/MS/MS assay out to 16 h post-dosing in plasma, liver, kidney, gut and heart. The effect of paclitaxel treatment on hepatic expression of PGP and P450 isoforms (CYP2C and CYP3A) was determined to elucidate the mechanism by which paclitaxel disposition is altered by previous drug exposure. RESULTS: Pharmacokinetic analysis of the data showed that plasma and tissue AUC values after treatment on day 5 following a dose on day 1 were between 50% and 74% of those determined following a single dose on day 1. The terminal elimination half-life was not different. Activity and protein levels for CYP2C in liver were elevated at 24 and 96 h after paclitaxel dosing. Cremophor EL, a carrier solvent for paclitaxel, also caused elevated CYP2C activity. Neither CYP3A nor PGP levels in liver were altered by paclitaxel or Cremophor EL treatment at the 24-h and 96-h time points. The levels of 6alpha-OH-paclitaxel in feces were increased on day 5 as opposed to day 1 while paclitaxel levels in feces were unchanged. CONCLUSIONS: The results of our studies showed that paclitaxel pharmacokinetics are altered by previous paclitaxel exposure up to 96 h earlier.

The purpose of the studies presented here is to determine if alterations in doxorubicin (DOX) pharmacokinetics that seem to occur following multiple-dosing are due to changes in DOX elimination via P-glycoprotein (PGP) mediated transport in the liver, kidney and gut. A pharmacokinetic study in female Balb/c mice was carried out with blood and tissue DOX levels measured in animals following a single DOX treatment (6 mg/kg), and in animals following a second DOX treatment after receiving a DOX treatment a week earlier. The pharmacokinetics of DOX in blood and tissues was altered by earlier exposure to DOX, as the animals that were treated once a week for 2 weeks showed an increased rate of DOX elimination from blood and tissues following the second treatment. Immunoblot analysis of PGP expression in liver and kidney from naïve and DOX-treated mice showed an approximately 1.2-fold elevation of PGP protein in these tissues in response to DOX exposure. Immunohistochemical staining of liver and small intestine sections for PGP showed 1.6-fold and 1.9-fold increases, respectively, in the DOX-treated tissues. These results have implications both in multiple-dosing regimens, as well as multiple-drug regimens, where DOX is used in combination with other drugs that are substrates for PGP-mediated efflux. Increases in PGP expression in both hepatic and extrahepatic tissues can lead to changes in the pharmacokinetics of DOX, as well as other drugs that are transported by PGP.

Previously, we have shown that highly specific antibodies against cytochrome P450 enzymes can be produced by targeting a 5-amino acid sequence at the C-terminus. Although rat CYP3A1 and CYP3A2 share 89% amino acid sequence similarity, they differ by 3 out of 5 of their C-terminal residues. In an effort to produce antibodies specific to each form, rabbits were immunised with the peptides IITGS and VINGA, corresponding to the C-termini of CYP3A1 and CYP3A2, respectively. Both antibodies bound strongly to hepatic microsomal fraction from rats treated with pregnenolone 16 alpha-carbonitrile (PCN) in enzyme-linked immunosorbent assay. Binding of the anti-IITGS antibody was strongly inhibited by incubation with IITGS, but VINGA was 60 times less effective. Conversely, binding of the anti-VINGA antibody was inhibited by VINGA 100 times more effectively than IITGS. Similar inhibition of antibody binding was also found using immunoblotting. Immunoadsorption using the anti-IITGS antibody yielded a single protein from solubilised hepatic microsomal fraction from PCN-treated rats, which was recognised only by the anti-IITGS antibody. Both antibodies bound to single proteins in the liver which were increased following treatment with PCN, but only the anti-IITGS antibody recognised protein in the lung, small intestine, and kidney of untreated and PCN-treated rats. Also, the binding of the two antibodies to hepatic and extrahepatic microsomal fractions from uninduced and induced rats showed differences in the expression of proteins recognised by the two antibodies, providing further evidence of antibody specificity. Thus, the binding of anti-IITGS and anti-VINGA antibodies is mutually exclusive and consistent with specific binding to their target antigens, CYP3A1 and CYP3A2, respectively. Immunocytochemistry was used to determine the distribution of CYP3A1 and CYP3A2. In the liver of untreated animals, both CYP3A1 and CYP3A2 were found to be expressed in the centrilobular region. However, some CYP3A1 immunoreactivity was also detected in many, but not all, hepatocytes throughout the lobule. However, following treatment of rats with PCN, both CYP3A1 and CYP3A2 were found to be strongly expressed in hepatocytes throughout the lobule, although CYP3A2 showed greater expression in the centrilobular region. PCN treatment was also found to result in induction of CYP3A1 in specific regions of the small intestine, lung, and kidney.