Abstract
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.
CYP2A51,2is 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 (Salonpaaet 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 (Salonpaaet al., 1992) and by pyrazole and others in NMRI mice (Emdeet 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
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 (Kojoet 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 −85oC 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 (Suet al., 1996). CYP2A5 mRNA was detected with a 693-bp32P-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 β-actin mRNA were determined with a 32P-labeled β-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 β-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
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.
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 table2, 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).
CYP2A5 has not been found to be inducible in the OM or lung in previous studies. Verschoyle et al. reported that treatment of DBA/2 and BALB/c mice with pyrazole did not affect pulmonary coumarin 7-hydroxylation activity (Verschoyle et al., 1997). Induction of a CYP2A-like P450 with coumarin 7-hydroxylation activity in the OM was reported in BDIV rats treated with coumarin in drinking water for 1 week (Bereziat et al., 1995). However, similar induction was not observed in Wistar rats or C57BL/6 mice (Gu et al., 1997). In contrast, a single ip injection of coumarin resulted in a tissue-selective reduction of the levels of CYP2A and other P450s as well as cytotoxicity in the OM at 48 hr after injection (Gu et al., 1997). Interestingly, significant reductions in P450 levels were also observed after griseofulvin treatment in the present study, possibly as a consequence of cytotoxicity resulting from the treatment. Thus, in addition to possible mechanistic differences in mRNA regulation between liver and the OM, the lack of olfactory induction of CYP2A5 by at least some of the inducers may also be due to tissue-selective toxicity.
The mechanism of hepatic CYP2A5 induction by pyrazole and possibly other compounds appears to involve posttranscriptional events (Aida and Negishi, 1991). A recent report indicated that pyrazole treatment induces hepatic proteins that are capable of binding to the 3′-untranslated region of CYP2A5 mRNA in vitro and, presumably, increasing its stability (Geneste et al., 1996). It remains to be determined whether the same proteins occur in other tissues and whether they play a role in the tissue-selective CYP2A5 induction in vivo.
Acknowledgments
We are grateful to Dr. Masahiko Negishi of the National Institute of Environmental Health Sciences, National Institutes of Health, for providing the CYP2A5 cDNA clone, and to Yali Zhou for technical assistance. The authors gratefully acknowledge the use of the Wadsworth Center’s Biochemistry Core facility. We would also like to thank Dr. Laurence Kaminsky of the Wadsworth Center for reading the manuscript.
Footnotes
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Send reprint requests to: Dr. Xinxin Ding, Wadsworth Center, New York State Department of Health, Empire State Plaza, Box 509, Albany, NY 12201-0509.
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This research was supported in part by Grant ES-07462 from the National Institute of Environmental Health Sciences, National Institutes of Health.
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↵2 The nomenclature used in this report is that of Nelson et al. (1996).
- Abbreviations used are::
- P450 or CYP
- cytochrome P450
- OM
- olfactory mucosa
- PBS
- phosphate-buffered saline, (2.7 mM KCl, 1.5 mM KH2PO4, 134 mM NaCl, and 8.2 mM Na2HPO4·7H2O)
- Received February 27, 1998.
- Accepted April 7, 1998.
- The American Society for Pharmacology and Experimental Therapeutics