* Institute of Molecular Biology and Biophysics, Novosibirsk 630127, Russia;
Institute of Bioorganic Chemistry, Novosibirsk 630090, Russia; and
Institute of Cytology and Genetics, Novosibirsk 630090, Russia
Received November 23, 2002; accepted January 27, 2003
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ABSTRACT |
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Key Words: liver; inbred mouse strains; cytochrome P4501A1 and 1A2; aryl hydrocarbon receptor; 3-methylcholanthrene; o-aminoazotoluene.
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INTRODUCTION |
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Genetic variations in the inducibility of CYP1A can modify the metabolism pathway of a carcinogen (Daly et al., 1994). The basal expression of CYP1A1 and CYP1A2 proteins in the liver is not high, and the amount of highly reactive metabolites is not significant. However, induction of CYP1A1 and CYP1A2 increasing both the mRNA levels and the enzyme activities, follows treatment by many chemical substances, including PAH, heterocyclic amines, aromatic amines, and azobenzenes. One such substance is o-aminoazotoluene (OAT), which is activated mainly by CYP1A2 through N-hydroxylation and induces CYP1A1 and CYP1A2 in the livers of rats (Cheung et al., 1994
) and mice (Timofeeva et al., 2000
). Furthermore, it was shown that repeated treatment of mice with OAT causes tumors in the liver of a number of mouse strains (C57BL, A/Sn, CBA, C3H/He, A/He, DBA, SWR, DD) but not in others (BALB/c, AKR; Kaledin et al., 1990
; Kaledin and Zakharova, 1984
). However, the effect of OAT on the mouse CYP1A induction is not described completely. Interestingly, in previous experiments with rats, it was shown that mutagenicity and CYP1A induction by azobenzenes, including OAT, correlates with their carcinogenicity (Cheung et al., 1994
). Therefore, one could expect the same effect in the studies with the different inbred mouse strains. Since the effect of MC on the mouse monooxygenases is well studied (Gonzalez et al., 1984
, 1993
; Nebert, 1989
), we have chosen this compound as the standard for comparison of CYP1A induction during treatment with OAT. CYP1A1 and CYP1A2 induction by OAT, as well as by PAH, is mediated by an aryl hydrocarbon receptor (AhR)-dependent mechanism (Cheung et al., 1994
; Dogra et al., 1998
; Gonzalez et al., 1993
).
Inbred mouse strains are widely used as a model for investigating mechanisms of chemical carcinogenesis. In the mouse, the strain-dependent variation in the response to aromatic hydrocarbon inducers of drug metabolism is well established as a polymorphism of the AhR locus (Chang et al., 1993; Gielen et al., 1972
). Considerable increase of CYP1A1 and CYP1A2 mRNAs and enzyme activities has been shown in the liver of mice with the AhbAhb or AhbAhd (PAH responsive mice, Ah+ genotype), but not with the AhdAhd (PAH nonresponsive mice, Ah- genotype; Nebert, 1989
).
For rat and human CYP1A1 7-ethoxyresorufin (ER) is considered to be a specific substrate, while 7-methoxyresorufin (MR) is specific for CYP1A2 (Burke et al., 1994). However, it was shown that microsomes from CYP1A2 (-/-) knockout mice did not have immunodetectable CYP1A2 protein, but methoxyresorufin-O-demethylase (MROD) activity was high after induction with 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD; Hamm et al., 1998
). Studies with cDNA-expressed microsomal and purified CYP1A1 and CYP1A2 proteins in C57BL mice showed that the substrate specificity of CYP1A1 overlaps CYP1A2 (Tsyrlov et al., 1993
). Thus, it is quite difficult to estimate the inducibility and activities of CYP1A isozymes. This difficulty can be alleviated by using highly specific antibodies.
The purpose of this study was to estimate and compare CYP1A1 and CYP1A2 gene expression in the liver of 3-methylcholanthrene- or o-aminoazotoluene-treated mice, differing in the Ah receptor genotype. We used C57BL, A/Sn (Ah+ genotype, aromatic hydrocarbon responsive) and AKR, DBA, SWR (Ah- genotype, aromatic hydrocarbon nonresponsive) mouse strains.
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MATERIALS AND METHODS |
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Enzyme assays.
The microsomal liver fraction was isolated from freshly excised livers by standard differential centrifugation (Burke et al., 1985). The protein concentrations in microsomes were measured according to Lowry et al.(1951)
. The cytochrome P450 content in the liver was determined as described by Omura and Sato (1964)
. The rates of O-dealkylation of 7-ethoxy- and 7-methoxyresorufins were assessed according to Burke and Mayer (1974)
.
Western blotting and immunodetection.
SDS-polyacrylamide gradient (7.515%) gel electrophoreses of microsomal proteins (60 µg) were carried out as described by Laemmli (1970). Electrophoretic transfer of the proteins to 0.45-µm nitrocellulose sheets (Schleicher & Schuell, Germany) was performed as described by Towbin et al.(1979)
. The immunoassay was normally done by blocking the transfer membrane with a 5% nonfat milk powder to prevent further nonspecific binding of proteins; this was followed by incubation of the membrane in a diluted 14H5 antibody solution (Grishanova and Lyakhovich, 1992
), washing of the membrane, incubation in diluted alkaline phosphatase-conjugated rabbit-anti-mouse IgG, further washing, and the colorimetric detection using bromo-chloro-indolyl phosphate (BCIP) and Nitro-blue-tetrazolium (NBT) salts.
RNA isolation and cDNA synthesis.
Total RNA was extracted from liver tissue by the SDS/phenol method (Chattopadhyay et al., 1993). Purity of the RNA was assessed by the ratio of the optical densities at 260 and 280 nm, and the integrity examined by electrophoresis on a 1% agarose gel containing of ethidium bromide (0.5 µg/ml). One µg of total RNA was used for synthesis of single-stranded cDNA. Briefly, RNA samples were reverse transcribed at 42°C for 1 h by incubation with 20 µl of a reverse transcription mixture containing the following constituents: 5 µM of oligo(dT)17 primer; 1 x reverse transcriptase buffer; 10 mM 1,4-dithiothreitol; 5 mM MgCl2; 20 units of RNasin; 0.5 mM deoxynucleotide triphosphates; and 200 units of Moloney Murine Leukemia Virus reverse transcriptase (Promega, USA). The resultant single-stranded cDNA was used for the PCR procedure.
Multiplex PCR.
The oligonucleotide primers for CYP1A1, CYP1A2, and RPL30 were synthesized according to sequences chosen using PCR primer design software GeneRunner (www.generunner.com) to ensure specific and efficient coamplification of target sequences. The sequences of oligonucleotide primers were:
A sequence of PCR reactions using each primer pair was performed initially to determine the number of cycles required to barely visualize these PCR products on ethidium-bromide-stained agarose gels and to remain within the exponential phase of the amplification curve (data not shown).
Multiplex PCRs (Wong et al., 1994) were performed in a total volume of 40 µl of buffer solution containing the following: 2 µl of RT mixture; 1x PCR buffer; 0.2 mM deoxynucleotide triphosphates; 20 pmol of each 5' and 3' starter primer pair (for CYP1A1 or CYP1A2); and 2 units of Taq DNA polymerase. The PCR reactions were conducted for 1 min at 94°C, 0.5 min at 55°C, and 1 min at 72°C.
Appropriate PCR cycle was determined by means of "primer-dropping" method (Wong et al., 1994). On the basis of this, amplification was performed for three or eight cycles with CYP1A2 or CYP1A1 primers, respectively, after which ribosomal protein L30 (RPL30) primers (20 pmol of each) were added to the reaction. Amplification was continued for 22 cycles with conditions described above. The "housekeeping" gene, RPL30, was chosen as an endogenous internal control to which other PCR amplification products were normalized.
The PCR products (10 µl) were separated by electrophoresis, illuminated with UV light, photographed, and analyzed by computerized densitometric scanning of the images using Scion Image program (www.scioncorp.com). The intensities of the ethidium bromide fluorescence signals were determined from the area under the curve for each peak and data were graphed.
Statistical analysis.
Statistical analysis was performed using Students t-test. The data are presented as mean ± SD. Each experimental group consisted of eight animals.
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RESULTS |
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Treatment with MC or OAT caused a 34-fold induction of the CYP1A2 mRNA level in the livers of A/Sn, C57BL, and AKR mice. There was no significant increase in CYP1A2 mRNA expression in SWR mice, but there was a modest, but statistically significant (p < 0.05) increase in the expression of CYP1A2 mRNA in DBA mice during treatment with MC and OAT.
After treatment with MC and OAT, the CYP1A1 mRNA level increased in all the strains examined. This finding does not correlate with the data on enzyme activity and was unexpected, since SWR, AKR, and DBA mice (AhdAhd genotype) are traditionally considered to be PAH noninducible (Nebert, 1989). Among the mouse strains examined, the CYP1A1 mRNA level was approximately two-fold higher in the livers of A/Sn and C57BL mice (Ah responsive), than in SWR, AKR, and DBA mice (Ah nonresponsive).
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DISCUSSION |
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Distinctions in the CYP1A1 and CYP1A2 inducibility between C57BL (Ah responsive) and DBA (Ah nonresponsive) mice during induction with MC have been demonstrated (Nebert and Gelboin, 1969). Further investigations have shown that PAH induction is mediated by AhR (Dogra et al., 1998
; Gonzalez et al., 1984
, 1993
). The AhR is a protein that positively regulates CYP1A1 and CYP1A2 gene expression. The mechanism underlying the difference in responsiveness has not been completely clarified. It has been reported, however, that the low responsiveness of DBA mice to aryl hydrocarbon is due to a low binding affinity of AhR to ligands because of amino acid substitution and elongation of the C-terminus in the AhR (Ema et al., 1994
).
Previously, it was shown by Gonzalez et al.(1984) that MC induces both proteins in C57BL/6N (Ah responsive) but not DBA/2N (Ah nonresponsive) mice, whereas sufficiently high doses of TCDD induce both CYP1A1 and CYP1A2 in both inbred mouse strains. In C57BL/6N mice, transcriptional rates of the CYP1A1 and CYP1A2 genes increased dramatically after MC treatment; in contrast, no increase in either gene is found in MC-treated DBA/2N mice. Following TCDD administration, both CYP1A1 and CYP1A2 gene transcription rates were elevated in DBA/2N mice (Gonzalez et al., 1984
). Authors have assumed that affinity of TCDD to the Ah-receptor is so high that, despite of defective receptor, the complex formed is stable enough to initiate transcription of CYP1A1 and CYP1A2 genes. One can notice these data contradict the results observed in our study, where treatment with MC or OAT increased the CYP1A1 mRNA level in all the strains examined, even in SWR, AKR, and DBA mice (Ah-nonresponsive). We suggest the possible reason for this could be the posttranscriptional mechanisms of CYP1A regulation and probably the defects in the protein translation but not the defects of the Ah receptor.
At present, many reports point to contradictions between the Ah-receptor genotype and the inducibility of CYP1A1 and CYP1A2. These results provided basis for hypothesizing AhR-independent mechanisms of induction for CYP1A1 and CYP1A2. Such cases are known for CYP1A2. For example, acenaphthylene and related tricyclic hydrocarbons induce CYP1A2 gene expression in B6C3F1 mice via an Ah receptor-independent pathway without the coinduction of CYP1A1 (Chaloupka et al., 1994). A similar effect was shown with the food mutagen 2-amino-3-methylimidazo[4,5-f]quinoline (Nerurkar et al., 1993
). Increases in CYP1A2 protein and mRNA level were observed for isosafrole- or piperonyl butoxide-treated mice in both Ah+- and Ah--strains, suggesting an Ah receptor-independent mechanism for induction of this enzyme (Adams et al., 1993
). The mechanism of this phenomenon remains unknown, but it is thought to exist due to the activation of posttranscriptional mechanisms of mRNA stabilization or posttranslational stabilization of the enzyme-inducer complex. Some xenobiotics can influence CYP1A1 and CYP1A2 gene expression without binding to the Ah-receptor as a ligand, confirming the presence of an AhR-independent pathway. For example, phenobarbital does not bind the Ah-receptor with high affinity but induces in liver cells the expression of cytochrome P4501A. Experiments using both wild type and AhR knock out C57BL mice (AhR-/- genotype) showed a lack of responsiveness to the classical Ah-receptor ligand MC of both CYP1A1 and CYP1A2 genes in the liver of knock out animals. However, phenobarbital induced hnRNA, mRNA, and protein for the CYP1A2 gene, irrespective of the presence of the Ah-receptor (Corcos et al., 1998
). It is appears that in the absence of the Ah-receptor, the other transcription factors are capable of binding with special DNA sequences (drug response elements) followed by the initiation of CYP1A2 gene transcription. However, such a mechanism for CYP1A1 has not been shown so far.
The discrepancies found in our study between the increase of mRNA level and the Ah-receptor genotype could be explained by activation of an AhR-independent signal transduction pathway. Other possible mechanisms of regulation can not be excluded; however, lack of protein accumulation during active transcription of the correspondent genes could indicate defects in translation of CYP1A1 and CYP1A2. To reveal the exact mechanism of this phenomenon, a more detailed study of the molecular genetics of Ah-receptor as well as further investigations of posttranscriptional mechanisms of CYP1A1 and CYP1A2 gene regulation in the liver of different inbred mouse strains are required.
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ACKNOWLEDGMENTS |
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NOTES |
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