dextromethorphan and 4-nitrophenol

The aim was to characterize mouse gender and strain differences in the metabolism of commonly used human cytochrome (CYP) P450 probe substrates. Thirteen human CYP probe substrates (phenacetin, coumarin, 7-ethoxy-4-trifluoromethyl coumarin, amiodarone, paclitaxel, diclofenac, S-mephenytoin, bufuralol, dextromethorphan, chlorzoxazone, p-nitrophenol, testosterone and lauric acid) were used in activity measurements. The metabolism of the probe substrates was compared in liver microsomes from male and female NMRI, CBA, C57bl/6, 129/SvJ and CD1 strains. The expression of proteins identified on Western blots with commonly available antibodies selective for specific human and rat CYP enzymes were compared in the different mouse strains. Males had higher metabolism than corresponding females for phenacetin O-deethylation (human marker for CYP1A2 activity), and a high correlation was found between phenacetin activity and immunoreactivity in Western blots produced with rat CYP1A2 antibodies. Protein detected by antibodies cross-reacting with human CYP2B6 and rat CYP2B1/2 antibodies was female specific except for the 129/SvJ strain, where it was absent in both genders. Females generally had a higher metabolism of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation (human markers for CYP2D activity). Bufuralol 1'-hydroxylation correlated with a female-dominant mouse CYP, which was detected with antibodies against rat CYP2D4. p-Nitrophenol 2-hydroxylation correlated better than chlorzoxazone 6-hydroxylation with the protein detected with antibodies against rat CYP2E1, indicating that p-nitrophenol is a more specific substrate for mouse CYP2E1.

Topics: Amiodarone; Animals; Aryl Hydrocarbon Hydroxylases; Blotting, Western; Chlorzoxazone; Coumarins; Cytochrome P-450 CYP1A2; Cytochrome P-450 CYP2B6; Cytochrome P-450 Enzyme System; Dextromethorphan; Diclofenac; DNA, Complementary; Ethanolamines; Female; Humans; Kinetics; Lauric Acids; Male; Mephenytoin; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Microsomes, Liver; Mixed Function Oxygenases; NADP; Nitrophenols; Oxidoreductases, N-Demethylating; Oxygen; Paclitaxel; Phenacetin; Rats; Sex Factors; Species Specificity; Substrate Specificity; Testosterone

In this study, we performed a screening of the specificities of rat cytochrome P450 (CYP) isoforms for metabolic reactions known as the specific probes of human CYP isoforms, using 13 rat CYP isoforms expressed in baculovirus-infected insect cells or B-lymphoblastoid cells. Among the metabolic reactions studied, diclofenac 4-hydroxylation (DFH), dextromethorphan O-demethylation (DMOD) and midazolam 4-hydroxylation were specifically catalyzed by CYP2C6, CYP2D2 and CYP3A1/3A2, respectively. These results suggest that diclofenac 4-hydroxylation, dextromethorphan O-demethylation and midazolam 4-hydroxylation are useful as catalytic markers of CYP2C6, CYP2D2 and CYP3A1/3A2, respectively. On the other hand, phenacetin O-deethylation and 7-ethoxyresorufin O-deethylation were catalyzed both by CYP1A2 and by CYP2C6. Benzyloxyresorufin O-dealkylation and pentoxyresorufin O-dealkylation were also catalyzed by CYP1A2 in addition to CYP2B1. Bufuralol 1'-hydroxylation was extensively catalyzed by CYP2D2 but also by CYP2C6 and CYP2C11. p-Nitrophenol 2-hydroxylation and chlorzoxazone 6-hydroxylation were extensively catalyzed by CYP2E1 but also by CYP1A2 and CYP3A1. Therefore, it is necessary to conduct further study to clarify whether these activities in rat liver microsomes are useful as probes of rat CYP isoforms. In contrast, coumarin 7-hydroxylation and S- and R-mephenytoin 4'-hydroxylation did not show selectivity toward any isoforms of rat CYP studied. Therefore, activities of coumarin 7-hydroxylation and S- and R-mephenytoin 4'-hydroxylation are not able to be used as catalytic probes of CYP isoforms in rat liver microsomes. These results may provide useful information regarding catalytic probes of rat CYPs for studies using rat liver microsomal samples.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Baculoviridae; Cells, Cultured; Chlorzoxazone; Coumarins; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Dextromethorphan; Diclofenac; DNA, Complementary; Ethanolamines; Gene Expression; Humans; Insecta; Isoenzymes; Mephenytoin; Midazolam; Nitrophenols; Pharmaceutical Preparations; Phenacetin; Rats; Substrate Specificity; Testosterone; Tumor Cells, Cultured