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.

Immunosuppressive potential of several polycyclic aromatic hydrocarbons (PAHs) found at a Superfund site: new model used to evaluate additive interactions between benzo[a]pyrene and TCDD.

Exposure to environmental pollution is rarely limited to a single compound or even a single class of compounds. The Superfund site located in Massena, NY, is contaminated by both halogenated aromatic hydrocarbons (HAHs) and polycyclic aromatic hydrocarbons (PAHs). Since representatives of both HAHs and PAHs are capable of binding to the aromatic hydrocarbon receptor (AhR), two well-documented AhR-mediated effects, immunosuppression and induction of hepatic aryl hydrocarbon hydroxylase (AHH) activity, were used to evaluate the individual and interactive toxicity of these compounds. Fifteen PAHs were first screened for their ability to suppress the antibody response in C57BL/6 (Ah+/+) mice immunized 12 h after a single oral dose of 0.1, 1, 10, or 100 mg/kg. Acenaphthene, anthracene, benzo[g,h,i]perylene, fluoranthene, fluorene, naphthalene, phenanthrene, and pyrene had little or no effect. Seven PAHs caused > 50% suppression at 100 mg/kg. Listed in order of decreasing potency they were benzo[k]fluoranthene, benzo[b]fluoranthene, indeno[1,2,3,c,d]pyrene, benzo[a]pyrene, chrysene, dibenzo[a,h]anthracene, and benz[a]anthracene. Chrysene and benzo[a]pyrene (B[a]P), were further evaluated to determine the dependence of these effects on the Ah phenotype by comparing responses of C57BL/6 and congenic B6.D2 (Ah-/-) mouse strains. Chrysene immunosuppression was maximal at 0.1 mg/kg and was Ah phenotype-independent whereas chrysene AHH induction was Ah phenotype-dependent, but a 100-fold less sensitive indicator of exposure. In contrast, B[a]P immunosuppression and AHH induction were coincident in B6 mice and Ah phenotype-dependent. In the final phase, a new approach was used to evaluate toxic interactions. This approach considers the mechanism of action of each compound and accounts for the fact that the extent of increase in toxic response caused by an incremental change of dose is determined by its position on the dose-response curve rather than on the absolute amount of dose administered. Thus, the immunotoxic effects of combined exposure to B[a]P and the AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a representative HAH, were evaluated by combining the ED20 of B[a]P with the difference between the ED20 and ED40 of TCDD, and vice versa, to produce 40% suppression. The results of the combination were consistent with additivity regardless of the composite arrangement or phenotype although some antagonism could not be excluded with certainty.

Potentiation and antagonism of 2,3,7,8-tetrachlorodibenzo-p-dioxin effects in a complex environmental mixture.

There is increasing need to understand the toxicity of complex environmental mixtures. The organic phase of a leachate (OPL) from the Love Canal chemical dump site is a complex mixture that contains over 100 organic compounds, including 0.74 ppm 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Mice congenic at the Ah locus were used to evaluate several toxic effects of the OPL, including immune function and hepatic enzyme induction. OPL toxicity was compared with that of pure TCDD in both C57BL/6J Ahb/b and congenic C57BL/6 Ahd/d (B6.D2) mice. Mice were given single oral doses of up to 2 g OPL/kg or 100 micrograms TCDD/kg, immunized, and evaluated after 7 days. The TCDD equivalent of the OPL was determined to be 3.9 and 5.0 ppm in C57BL/6J and B6.D2 mice, respectively. This is six times the TCDD content. The Ah phenotype-dependent response ratio was calculated by dividing the dose required to cause an effect in the B6.D2 strain by the dose causing the same effect in the C57BL/6J strain. Ratios based on both ED50s and the lowest observed adverse effect levels were used to determine whether each adverse effect was Ah phenotype-dependent, the extent to which TCDD contributed to the effect, whether there were interactive effects between the AhR ligands and nonligands and if they were additive, antagonistic, or synergistic, and whether the response was predictable based on the known chemical composition of the mixture. It was concluded that the non-TCDD component potentiated TCDD immune suppression, and possibly thymic atrophy, through AhR mechanisms. In contrast, this analysis indicated that the non-TCDD component of the OPL antagonized the ability of the TCDD component to induce hepatic AHH activity whereas OPL hepatomegaly was caused primarily by the non-TCDD component of the OPL. This study demonstrates that the toxicity of mixtures containing TCDD may not be accurately predicted based on the TCDD content alone and that this approach could be useful in the toxicologic assessment and management of environmental contamination.