Differential xenobiotic induction of CYP2A5 in mouse liver, kidney, lung, and olfactory mucosa.

The effects of pyrazole, which is known to induce hepatic cytochrome P4502A5 (CYP2A5) through posttranscriptional mechanisms, on the level of CYP2A5 in liver and extrahepatic tissues were examined in this study. Intraperitoneal administration of pyrazole at 200 mg/kg for 3 days induced CYP2A4/5 mRNAs and proteins and microsomal coumarin 7-hydroxylation activity in liver and kidney of C57BL/6 mice. A marginal increase (30%) in CYP2A4/5 mRNAs was also observed in the olfactory mucosa but not in the lung, and no increase in CYP2A4/5 proteins or microsomal coumarin 7-hydroxylation activity was observed in either the olfactory mucosa or lung. CYP2A4/5 proteins were not detected on immunoblots in other tissues examined, including breast, bone marrow, testis, prostate, ovary, and uterus from control or pyrazole-treated mice. On the other hand, pyrazole treatment induced CYP2E1 in the olfactory mucosa as well as in liver and kidney, indicating that the olfactory mucosa was exposed to pyrazole. The lack of CYP2A inducibility in the olfactory mucosa was also observed for several other known inducers of hepatic CYP2A5, including cobaltous chloride, stannous chloride, griseofulvin, thioacetamide, and aminotriazole. These results suggest that the mechanisms involved in the induction of hepatic and renal CYP2A5 by pyrazole and other xenobiotic compounds may be tissue-specific.

Purification and characterization of heterologously expressed mouse CYP2A5 and CYP2G1: role in metabolic activation of acetaminophen and 2,6-dichlorobenzonitrile in mouse olfactory mucosal microsomes.

The metabolic activation of two known olfactory mucosal (OM) toxicants, acetaminophen (AP) and 2,6-dichlorobenzonitrile (DCBN), was examined with mouse liver and OM microsomes and purified, heterologously expressed mouse CYP2A5 and CYP2G1. In reconstituted systems, both isoforms were active in metabolizing DCBN and AP to metabolites that formed protein adducts. The formation of DCBN- or AP-protein adducts and other AP metabolites, including 3-hydroxy-AP and, in the presence of glutathione, AP-glutathione conjugate, was also detected in OM microsomal reactions and to a much greater extent than in liver microsomes. Evidence was obtained that CYP2A5 and CYP2G1 play major roles in mouse OM microsomal metabolic activation of DCBN and AP. Immunoblot analysis indicated that CYP2A5 and CYP2G1 are abundant P450 isoforms in OM microsomes. OM microsomal AP and DCBN metabolic activation was inhibited by 5- and 8-methoxsalen, which inhibit both CYP2A5 and CYP2G1, and by an inhibitory anti-CYP2A5 antibody that also inhibits CYP2G1. In addition, the roles of CYP1A2 and CYP2E1 in the OM bioactivation of AP and DCBN were ruled out by comparing activities of acetone-treated mice or Cyp1a2(-/-) mice with those of control mice. Thus, CYP2A5 and CYP2G1 may both contribute to the known OM-selective toxicity of AP and DCBN. Further analysis of the kinetics of AP and DCBN metabolism by the purified P450s suggested that CYP2A5 may play a greater role in OM microsomal metabolism of AP, whereas their relative roles in DCBN metabolism may be dose dependent, with CYP2G1 playing more important roles at low substrate concentrations.

Intraperitoneal administration of coumarin causes tissue-selective depletion of cytochromes P450 and cytotoxicity in the olfactory mucosa.

Coumarin is a naturally occurring fragrant compound widely used in consumer products and also as a therapeutic agent. The effects of intraperitoneal (ip) and oral administration of coumarin on cytochrome P450 (P450) expression in olfactory mucosa were examined. A single ip injection of coumarin at 50 mg/kg resulted in a significant reduction of levels of CYP2A and CYP2G in the olfactory mucosa of Wistar rats and C57BL/6 mice at 48 hr following injection. Dose-response analysis of coumarin effects indicated that Wistar rats were more sensitive than C57BL/6 mice. A significant suppression of nasal CYP2A levels was observed at 25 mg/kg in rats, but not in mice. Depletion of P450 content was not observed in liver of either rats or mice at 50 mg/kg, indicating tissue-selective effects. Decreased P450 levels were observed at 24 hr, 48 hr, and 7 days following treatment, with minimal levels seen at 48 hr. The decrease in P450 levels was accompanied by necrosis, cell loss, and basal cell metaplasia in the olfactory mucosa. Intraperitoneal injection of 7-hydroxycoumarin or 3,4-dihydrocoumarin at 50 mg/kg did not result in depletion of nasal P450, indicating that the toxicity is not mediated by P450-catalyzed coumarin 7-hydroxylation and supporting the hypothesis that the formation of coumarin 3,4-epoxide may be responsible for the toxicity. Oral treatment with coumarin in drinking water led to a small, yet significant induction of CYP2A protein and coumarin hydroxylase activity in the nasal mucosa of mice, but not rats. Thus, ip administration of coumarin causes tissue-selective depletion of P450 and cytotoxicity in the olfactory mucosa of Wistar rats and C57BL/6 mice. It remains to be determined whether similar toxicity occurs following coumarin administration by other routes.

cDNA cloning, heterologous expression, and characterization of mouse CYP2G1, an olfactory-specific steroid hydroxylase.

CYP2G1 is expressed specifically in the olfactory mucosa in rabbits and rats. In the present study, a full-length cDNA for mouse CYP2G1 was obtained using a PCR approach with RNA preparations from the olfactory mucosa of C57BL/6 mice. Sequence comparisons indicated that mouse CYP2G1 is highly homologous in deduced amino acid sequence to rabbit (82.4% identity) and rat CYP2G1 (94.9% identity). RNA blot and immunoblot analyses indicated that mouse CYP2G1 is expressed only in the olfactory mucosa. The coding region of the mouse CYP2G1 cDNA was cloned into a baculoviral expression vector for heterologous production of the enzyme in cultured insect cells. Heterologously expressed mouse CYP2G1 was active in a reconstituted system toward testosterone and progesterone, producing all the major metabolites detected in olfactory microsomal reactions, including 15 alpha-, 15 beta-, and 2 beta-hydroxytestosterone from testosterone and two unidentified metabolites from progesterone. Kinetic analysis indicated that mouse CYP2G1 has relatively high affinities toward the steroid substrates, with K(m) values in the micromolar range for both testosterone and progesterone. At a substrate concentration of 10 microM, microsomes of olfactory mucosa had much higher turnover numbers toward testosterone and progesterone than hepatic microsomes, consistent with the olfactory-specific expression of a high-affinity sex steroid hydroxylase. These findings will facilitate further molecular genetics studies on the biological function of CYP2G1 in a mouse model.

Expression of CYP2A genes in rodent and human nasal mucosa.

CYP2A10 and CYP2A11, which are abundant in olfactory microsomes from rabbits, are active in the metabolic activation of a number of nasal toxicants, such as hexamethylphosphoramide and N-nitrosodiethylamine. Previous immunohistochemical studies indicated that CYP2A-related cytochromes P450 may also be present in rodent and human olfactory tissue. In the present study, the expression of cytochromes P450 highly homologous to rabbit CYP2A10/11 in rat, mouse, and human nasal mucosa was studied. In Sprague-Dawley rats, CYP2A3 mRNA was detected in olfactory mucosa at levels much higher than those found in total RNA from lung. Similar observations were made for the level of microsomal CYP2A3 protein with the use of antibodies to rabbit CYP2A10/11. However, mRNAs for two other rat cytochrome P450 genes in the CYP2A subfamily, CYP2A1 and CYP2A2, were not detected in nasal tissue by RNA-polymerase chain reaction analysis. In C57BL/6 mice, both CYP2A4 and CYP2A5 mRNAs were detected in the olfactory mucosa by RNA-polymerase chain reaction, but the CYP2A5 transcript was present at a level much higher than that of CYP2A4. The expression of another mouse gene in CYP2A subfamily, CYP2A12, was not detected in nasal tissue. CYP2A5 protein was also detected in mouse olfactory microsomes at higher levels than in liver, lung, or kidney microsomes. However, no significant sex differences in the levels of CYP2A4/5 mRNA or microsomal coumarin 7-hydroxylase activity were found with the nasal tissue. In addition, consistent with previous immunohistochemical studies, the expression of CYP2A6 in human nasal mucosa was detected by RNA-polymerase chain reaction as well as RNA blot analysis. The identification of CYP2A6 in human nasal tissues may have important implications for risk assessment of potential nasal toxicants, and the abundant expression of the CYP2A genes in rat and mouse olfactory tissue suggests a molecular basis for the known tissue-specific toxicity of numerous inhaled compounds in rodents.