Gene expression and enzyme function of two cytochrome P450 3A isoenzymes in rat and cattle precision cut liver slices (original) (raw)
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World Rabbit Science
As considerable inter-species differences exist in xenobiotic metabolism, developing new pharmaceutical therapies for use in different species is fraught with difficulties. For this reason, very few medicines have been registered for use in rabbits, despite their importance in inter alia meat and fur production. We have developed a rapid and sensitive screening system for drug safety in rabbits based on cytochrome P450 enzyme assays, specifically CYP1A1, CYP1A2 and CYP3A6, employing an adaptation of the luciferin-based clinical assay currently used in human drug screening. Short-term (4-h) cultured rabbit primary hepatocytes were treated with a cytochrome inducer (phenobarbital) and 2 inhibitors (alpha-naphthoflavone and ketoconazole). In parallel, and to provide verification, New Zealand white rabbits were dosed with 80 mg/kg phenobarbital or 40 mg/kg ketoconazole for 3 d. Ketoconazole significantly increased CYP3A6 gene expression and decreased CYP3A6 activity both in vitro and in...
Biochemical Pharmacology, 2001
The expression of xenobiotic-metabolising cytochrome P450 proteins in the liver of cattle was determined using substrate probes and immunologically by Western blot analysis. Compared to the rat, cattle displayed much higher coumarin 7-hydroxylase (CYP2A) and ethoxyresorufin O-deethylase (CYP1) activity but, in contrast, it exhibited much lower debrisoquine 4-hydroxylase (CYP2D) and lauric acid hydroxylase activities (CYP4A). The ethoxyresorufin O-deethylase activity was markedly inhibited by furafylline and ␣-naphthoflavone, and coumarin 7-hydroxylase by 8-methoxypsoralen. Immunoblot analysis employing antibodies to rat CYP1A1 recognised two immunorelated proteins in bovine liver whose expression appeared to be higher compared with rat. Kinetic studies indicated that a single enzyme is likely to be responsible for the O-deethylation of 7-ethoxyresorufin in bovine liver. When bovine microsomes were probed with antibodies to rat CYP2A2, a single protein was detected in cattle liver. Kinetic analysis followed by construction of Eadie-Hofstee plots indicated that more than one enzyme contributes to the 7-hydroxylation of coumarin. Immunoblot analysis employing antibodies to human CYP2D6 and rat CYP4A1 revealed in both cases a single, poorly expressed immunoreacting band in bovine microsomes. Similar immunoblot studies detected proteins in cattle liver immunorelated to the CYP2B, CYP2C, CYP2E, and CYP3A subfamilies. Bovine microsomes metabolised testosterone but, in contrast to the rat, failed to produce 2␣and 16␣-hydroxytestosterone. On the other hand, bovine microsomes produced levels of another hydroxylated metabolite, possibly 12-hydroxytestosterone. In conclusion, results emanating from this study indicate the presence of proteins in the cattle liver belonging to all the xenobiotic-metabolising families of cytochrome P450.
Journal of Veterinary Pharmacology and Therapeutics, 2011
The effects of repeated administrations of dexamethasone (DEX) (3 mg ⁄ kg ⁄ day by IM route for 7 days) on the gene expression profile of a cytochrome P450 (CYP) 3A28-like isoenzyme, on the expression of a CYP3A-immunoreactive protein and on CYP3A-dependent metabolic activities in sheep liver and small intestinal mucosa were evaluated in the current work. CYP 3A-dependent metabolic activities (erythromycin and triacetyl-oleandomycin N-demethylations) were assessed in microsomal fractions. The mRNA expression of CYP3A28-like, glucocorticoid receptor, constitutive androstane receptor, pregnane X receptor and retinoic X receptor alpha (RXRa) was determined by quantitative real-time PCR. The expression of a CYP3A-immunoreactive protein was measured by Western blot analyses. In the liver, DEX treatment increased CYP3A28-like mRNA levels (2.67-fold, P < 0.01) and CYP3A apoprotein expression (1.34-fold, P < 0.05) and stimulated CYP3A-dependent metabolism. High and significant correlation coefficients between CYP3Adependent activities and CYP3A28-like gene (r = 0.835-0.856, P < 0.01) or protein (r = 0.728-0.855, P < 0.05) expression profiles were observed. Among the transcriptional factors, DEX only stimulated (2.1-fold, P < 0.01) the mRNA expression of RXRa. In sheep small intestine, DEX caused a slight increment (34.6%, P < 0.05) in erythromycin N-demethylase activity in the jejunal mucosa and a significant enhancement (P < 0.05) of CYP3A apoprotein level in the duodenal mucosa.
Absolute quantification and modulation of cytochrome P450 3A isoforms in cattle liver
The Veterinary Journal, 2014
In humans and laboratory animals, knowledge about cytochrome P450 (CYP) regulation and function is detailed and very extensive. However, CYPs have still been incompletely characterized in veterinary species so far. In this study, mRNA levels of three CYP3A enzymes (CYP3A28, CYP3A38 and CYP3A48) were measured in cattle liver by using quantitative real-time RT-PCR (qPCR) assays and an absolute quantification approach. In particular, the possible presence of breed-differences in CYP3A expression was investigated in five different meat cattle breeds (Charolais, CH; Piedmontese, PM; Blonde d'Aquitaine, BA; Marchigiana, MA; Valdostana, VALD) and the potential transcriptional effect of the prototypical inducer phenobarbital (PB) upon the CYP3A isoforms was evaluated. Cytochrome P450 3A38 showed the highest amounts of gene copy numbers, followed by CYP3A48 and CYP3A28. Significant breed-differences in CYP3A gene abundances were found, and PB significantly up-regulated all the CYP3A isoforms. The data provide new information about CYP3A expression in cattle, particularly the heterogeneity in the pattern of expression of distinct hepatic CYP3As (CYP3A38 > 3A48 >> 3A28), the significant effect of breed, and their common up-regulation following the exposure to PB, although with different orders of magnitude.
Current Drug Metabolism
Numerous members of the cytochrome P450 (CYP) superfamily are induced after exposure to a variety of xenobiotics in human liver. We have gained considerable mechanistic insights into these processes in hepatocytes and multiple ligand-activated transcription factors have been identified over the past two decades. Families CYP1, CYP2 and CYP3 involved in xenobiotic metabolism are also expressed in a range of extrahepatic tissues (e.g. intestine, brain, kidney, placenta, lung, adrenal gland, pancreas, skin, mammary gland, uterus, ovary, testes and prostate). Since the expression of the majority of the isoforms appears to be very low in the extrahepatic tissues in comparison with predominant expression in adult liver, the role of the enzymes in overall biotransformation and total body clearance is minor. However, basal expression and up-regulation of extrahepatic CYP enzymes can significantly affect local disposition of xenobiotics or endogenous compounds in peripheral tissues and thus modify their pharmacological/toxicological effects or affect absorption of xenobiotics into systemic circulation. The goal of this review is to critically examine our current understanding of molecular mechanisms involved in induction of xenobiotic metabolizing CYP genes of human families CYP1, CYP2 and CYP3 by exogenous chemicals in extrahepatic tissues. We concentrate on organs such as the intestine, kidney, lung, placenta and skin, which are involved in drug distribution and clearance or are in direct contact with environmental xenobiotics. We also discuss single nucleotide polymorphisms (SNPs) of key CYPs, which at the level of transcription affect expression of the genes in the extrahepatic tissues or are associated with some pathophysiological stages or disorders in the organs.
Investigation of breed and sex effects on cytochrome P450 gene expression in cattle liver
Research in Veterinary Science, 2011
Many cytochrome P450 enzymes are involved in xenobiotic metabolism and elimination. In humans, genetic variation in some of these enzymes contributes to inter-individual drug responses, sometimes having significant clinical effects. Transcript levels of eight P450 genes were evaluated in liver to investigate potential differences in breed and sex in cattle. In Angus calves, heifers appeared to have higher gene expression than steers for two of the eight genes. In Angus and Simmental pregnant cows, Angus appeared to have higher gene expression for three of the eight genes. Transcript evaluation is just the first step toward determining if differences exist between breeds and sexes in enzyme catalytic activity. However, others (Giantin et al., 2008) have shown correlations between transcript levels and catalytic activity in other cattle breeds. Therefore breed and/or sex of an animal may need to be considered before administering a dose of a xenobiotic due to the potential for harmful drug residues in foodstuffs as well as improper treatment of disease conditions.
Effect of Breed upon Cytochromes P450 and Phase II Enzyme Expression in Cattle Liver
Drug Metabolism and Disposition, 2008
Cattle represent an important source of animal-derived foodproducts; nonetheless, our knowledge about the expression of drug-metabolizing enzymes (DMEs) in present and other foodproducing animals still remains superficial, despite the obvious toxicological consequences. Breed represents an internal factor that modulates DME expression and catalytic activity. In the present work, the effect of breed upon relevant phase I and phase II DMEs was investigated at the pretranscriptional and post-translational levels in male Charolais (CH), Piedmontese (PM) and Blonde d'Aquitaine (BA) cattle. Because specific substrates for cattle have not yet been identified, the breed effect upon specific cytochrome P450 (P450), UDP-glucuronosyltransferase (UGT), or glutathione S-transferase (GST) DMEs, in terms of catalytic activity, was determined by using human marker substrates. Among P450s, benzphetamine N-demethylase, 16-, 6-, and 2-testosterone hydroxylase, aniline and p-nitrophenol hydroxylase, and ␣-naphthol and p-nitrophenol UGT activities were significantly higher in CH; in contrast, lower levels of CYP1A1-, CYP1A2-, CYP2B6-, CYP2C9-, CYP2C18-, CYP3A4-, and UGT1A1-like mRNAs were noticed, with CH < PM < BA as a trend. CYP2B and CYP3A mRNA results were confirmed with immunoblotting, too. As regards conjugative DMEs, UGT1A6like mRNA levels were consistent with respective catalytic activities. Both 1-chloro-2,4-dinitrobenzene and 3,4-dichloronitrobenzene GST activities were higher in BA, and these results agreed with GSTA1-, GSTM1-, and GSTP1-like mRNA amounts. Correlation analysis between catalytic activities and mRNAs showed either significant or uneven results, depending on the substrate. These findings confirm previous data obtained in laboratory species; however, further studies are required to ascribe this behavior to pretranscriptional or post-translational phenomena.
Stability of cytochrome P450 proteins in cultured precision-cut rat liver slices
Toxicology, 2000
The objective of this study was to evaluate the stability of individual, xenobiotic-metabolising, cytochrome P450 proteins in precision-cut rat liver slices cultured for up to 72 h using the multiwell plate system. This was achieved using established diagnostic probes (O-dealkylation of methoxy-, ethoxy-and pentoxy-resorufin, testosterone 2a-hydroxylase, debrisoquine 4-hydroxylase, aniline p-hydroxylase and lauric acid hydroxylase) and immunologically using Western blotting. All cytochrome P450 activities declined in culture, the most rapid loss occurring at about 8-12 h of culture; in all cases no detectable activity was present in the 72-h cultured slices. Isoform-specific differences in the stability of various cytochrome P450 proteins were observed, with CYP2E1 being the most stable. When cytochrome P450 expression was determined immunologically, a different picture emerged. High levels of apoprotein were retained in the slices even when activity was very low. In the case of CYP2B, apoprotein levels even increased following the culture of hepatic slices. It is concluded, that for tissue slices to become an acceptable in vitro alternative system for long-term incubations, the culturing conditions must be improved to ensure that cytochrome P450 activities are better maintained.
animal, 2010
The objective of this study was to investigate the tissue-specific mRNA expression of different cytochrome P450 (CYP) isoforms, UDP glucuronsyl transferase 1A1 (UGT1A1) and glutathione-S-transferase (GSTA1) in the different tissues (liver, mammary gland, lungs, spleen, kidney cortex, heart, masseter muscle and tongue) of cattle, using quantitative real-time polymerase chain reaction (qPCR). CYP1A1-like mRNA was expressed in all of the tissues examined, including the liver, with the highest expression level in the kidney. CYP1A2-, 2E1-and 3A4-like mRNAs were only expressed hepatically. Interestingly, significant expression of CYP2B6-like mRNA was recorded in the lung tissue, while CYP2C9-like mRNA was expressed in the liver and kidney tissues of the cattle examined. UGT1A1-and GSTA1-like mRNAs were expressed in all of the examined tissues, except the mammary glands, and the highest expression levels were recorded in the kidney. The high expression of UGT1A1 in the lung tissue and GSTA1 in the liver tissue was unique to cattle; this has not been reported for rats or mice. The findings of this study strongly suggest that the liver, kidneys and lungs of cattle are the major organs contributing to xenobiotics metabolism.
Cytochrome P450 3A mRNA expression along goat and rat gastrointestinal tracts
The Japanese journal of veterinary research
The cytochrome P450 (CYP) 3A family is involved in the elimination processes of almost 50% of commonly used drugs. CYP3A mRNA expressions in goat and rat gastrointestinal tracts in comparison to the liver were investigated using real-time PCR. In goats, the expression of CYP3A-like mRNAs was comparatively higher in the liver than in the gastrointestinal tract. The intestinal expression of CYP3A-like mRNA showed a gradual decrease from the duodenum to the ileum. In rats, the highest CYP3A62 mRNA expression was found in the duodenum followed by the liver. This study provides insights into the contribution of CYP3A enzymes to xenobiotic metabolism, especially in small ruminants such as goats.