Introduction

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CYP2A5¹² is a major P450 isoform in mouse olfactory mucosa (OM) and is also expressed in liver, kidney, and lung (Su et al., 1996). Previous studies have shown that hepatic CYP2A5 is inducible by a number of xenobiotic compounds, including phenobarbital (Honkakoski and Lang, 1989), pyrazole (Kojo et al., 1991), heavy metals, such as stannous chloride (Emde et al., 1996) and cobaltous chloride (Kocer et al., 1991), and porphyrinogenic agents, such as griseofulvin, thioacetamide, and aminotriazole (Salonpaa et al., 1995). The inducibility of CYP2A5 in extrahepatic tissues has not been studied extensively. CYP2A5 is inducible in the kidney by cerium chloride in DBA/2 but not C57BL/6 mice (Salonpaa et al., 1992) and by pyrazole and others in NMRI mice (Emde et al., 1996); however, it is not known whether CYP2A5 is also inducible in lung and OM and whether it is expressed in any other tissues. Induction of CYP2A5 in extrahepatic tissues may increase the rates of target tissue metabolic activation of toxic chemicals and potentially lead to increased sensitivity to xenobiotic toxicity.

In the present study, we examined the inducibility of CYP2A5 by pyrazole in liver and extrahepatic tissues of C57BL/6 mice. The cDNA probes and antibodies used for detecting CYP2A5 could not distinguish CYP2A4 from CYP2A5. Thus the combined levels of CYP2A4 and CYP2A5 (or CYP2A4/5) were determined on immunoblots and RNA blots. Microsomal activity toward coumarin, a preferred substrate for CYP2A5 but not a substrate for CYP2A4 (Negishi et al., 1989), was also determined to monitor specific induction of CYP2A5. In addition, the effects of several other known inducers of hepatic CYP2A5, including cobaltous chloride, stannous chloride, griseofulvin, thioacetamide, and aminotriazole, were also compared in liver and OM. Our results indicate that the mechanisms involved in the induction of hepatic and renal CYP2A5 by pyrazole and other xenobiotic compounds may be tissue-specific.

	Materials and Methods

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Animal Treatments. Two-month-old male C57BL/6 mice (about 20 g in body weight) were used in this study. Animals were treated essentially according to published protocols for induction of hepatic CYP2A5 with pyrazole (Kojo et al., 1991), stannous chloride (Emde et al., 1996), cobaltous chloride (Kocer et al., 1991), griseofulvin, thioacetamide, and aminotriazole (Salonpaa et al., 1995). Pyrazole was dissolved in PBS and was injected once daily (200 mg/kg body weight; ip) for 3 consecutive days. Tissues were collected on the fourth day. Stannous chloride was injected once daily (50 mg/kg; ip) for 2 days in 1.75% sodium citrate (pH 7.4), and tissues were collected 24 hr after the last injection. Cobaltous chloride was injected once daily (30 mg/kg; sc) for 2 days in saline, and tissues were collected 24 hr after the last injection. Griseofulvin (1000 mg/kg; suspended in corn oil) and thioacetamide (10 mg/kg; dissolved in saline) were injected only once (ip), and tissues were collected 48 hr after injection. Aminotriazole was also injected once (1000 mg/kg; ip; in saline), and tissues were collected 24 hr after injection. The control groups for each treatment received the corresponding vehicle only. All tissues were quick-frozen on dry ice and stored at 85^oC until use. Tissues from each treatment or control group of six mice were combined and used for the preparation of microsomes or RNAs.

RNA Blot and Immunoblot Analysis. Total RNA was prepared from frozen tissues with use of TRIZOL Reagent (Gibco BRL, Grand Island, NY). RNA concentration was determined spectrally. RNA blot analyses were performed as recently described (Su et al., 1996). CYP2A5 mRNA was detected with a 693-bp P-labeled PstI fragment of CYP2A5 cDNA (kindly provided by Dr. Masahiko Negishi, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC). The levels of -actin mRNA were determined with a ³²P-labeled -actin cDNA probe (Clontech, Palo Alto, CA) on the same RNA blots after removal of CYP2A5 cDNA probes. For quantifying the amounts of CYP2A5 mRNA in total RNA preparations from vehicle- and inducer-treated animals, the density of the hybridizing bands was determined with a LKB ImageMaster DTS densitometer (Pharmacia, Piscataway, NJ). The band intensities were within the linear range of the densitometric response, and the amounts of total RNA applied were corrected according to the amounts of -actin detected in RNA samples from pairs of vehicle- and inducer-treated animals. Immunoblot analyses were performed with an ECL detection system from Amersham (Arlington Heights, IL). The sources of the polyclonal antibody to rabbit CYP2A10/11 (Ding and Coon, 1990) and the monoclonal antibody (Mab 1-98-1) to rat CYP2E1 (Ding et al., 1991) have been described previously. Density of CYP2A5 or CYP2E1 immunoreactive bands were determined as described above. In all cases, the relative amounts of P450 proteins in samples from vehicle- and inducer-treated animals were determined and are shown in arbitrary units.

Other Methods and Materials. Microsomes were prepared from combined tissues of six mice in each group as previously described (Ding and Coon, 1990). Protein was determined using BCA reagent (Pierce, Rockford, IL), with bovine serum albumin as a standard. Microsomal coumarin 7-hydroxylation activity was measured by the method of Greenlee and Poland (Greenlee and Poland, 1978). The contents of reaction mixtures and incubation conditions are described in the legend to table 1.

	Results and Discussion

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Tissue-Selective Induction of CYP2A5 by Pyrazole. Animals were treated with pyrazole using conditions that are known to induce CYP2A5 in the liver and the levels of CYP2A4/5 mRNAs, microsomal CYP2A4/5 proteins, and microsomal coumarin 7-hydroxylation activities were determined, as shown in table 1. Representative immunoblots and RNA-blots are shown in fig. 1. The levels of the CYP2A mRNAs and proteins were elevated about 5- to 9-fold in livers and kidneys of pyrazole-treated mice, compared with those from vehicle-treated animals. These changes were accompanied by 10- to 16-fold increases in microsomal coumarin 7-hydroxylation activity. A marginal increase (30%) in CYP2A4/5 mRNAs was also observed in the OM but not in lung, and no increase in CYP2A4/5 proteins or microsomal coumarin 7-hydroxylation activity was observed in either OM or lung. In experiments not presented here, 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.

View larger version (75K): [in this window] [in a new window]   Fig. 1.   Tissue-selective induction of CYP2A5 protein and mRNA by pyrazole. Male C57BL/6 mice were treated (ip) with PBS (control) or pyrazole (200 mg/kg) in PBS once per day for 3 consecutive days and sacrificed on the fourth day. Microsomes or total RNA were prepared from the combined tissues of six mice in each group. A, Representative immunoblots with anti-CYP2A10/11 antibody and microsomal proteins from OM (0.2 µg, lanes 1 and 2), liver (2 µg, lanes 3 and 4), kidney (6 µg, lanes 5 and 6), and lung (6 µg, lanes 7 and 8) of PBS- (lanes 1, 3, 5, and 7, respectively) or pyrazole-treated mice (lanes 2, 4, 6, and 8, respectively). B, C, Representative RNA-blots with total RNAs from the OM (5 µg, lanes 1 and 2), liver (10 µg, lanes 3 and 4), kidney (10 µg, lanes 5 and 6), and lung (10 µg, lanes 7 and 8) of PBS- (lanes 1, 3, 5, and 7, respectively) or pyrazole-treated mice (lanes 2, 4, 6, and 8, respectively). The blots were hybridized either to a 32P-labeled CYP2A5 cDNA (B) or to a -actin cDNA (C) as described in "Materials and Methods."

Induction of CYP2E1 by Pyrazole. Immunoblot analyses were also conducted to determine the inducibility of CYP2E1 in liver, kidney, and OM by pyrazole treatment. As shown in table 1, the levels of CYP2E1 protein were elevated in the livers (2-fold), kidneys (9-fold), as well as the OM (3-fold) of pyrazole-treated mice, compared with those of vehicle-treated mice. Thus the concentration of pyrazole in the OM was sufficient to induce CYP2E1, indicating that the lack of induction of CYP2A5 in OM was not because pyrazole did not reach this tissue. However, we were unable to determine whether CYP2E1 protein was induced in the lung by pyrazole-treatment, because the antibody detected multiple bands in the P450 region in lung microsomes from both vehicle- and pyrazole-treated mice, which interfered with immunoblot quantitation (data not shown).

Differential Induction of CYP2A5 in Liver and OM by Other Xenobiotic Compounds. Since pyrazole did not induce CYP2A5 in the OM, we examined other known hepatic CYP2A5 inducers, including stannous chloride, cobaltous chloride, griseofulvin, thioacetamide, and aminotriazole, to see if they could induce the enzyme in the OM. As shown in table 2, all of the compounds induced CYP2A4/5 proteins in the liver, with the extent of induction varying between 2- and 14-fold. However, induction of CYP2A4/5 proteins was not observed in the OM with any of the compounds. In contrast, except for the experiments with stannous chloride, there was a general trend of decreased olfactory CYP2A4/5 protein levels after treatment with the hepatic inducers, with a statistically significant decrease in the experiments with griseofulvin (p < 0.05).