Molecular Cloning of cDNA for Guinea Pig CYP1A2 Comparison with Guinea Pig CYP1A1

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS

Vol. 344, No. 1, August 1, pp. 11–17, 1997 Article No. BB970189

Molecular Cloning of cDNA for Guinea Pig CYP1A2 Comparison with Guinea Pig CYP1A1 Virginia H. Black,1 Ai-fei Wang, Michael Henry, and Peter Shaw Department of Cell Biology and Kaplan Cancer Center, New York University School of Medicine, New York, New York 10016

Received November 25, 1996, and in revised form May 6, 1997

Guinea pig CYP1A2 cDNA was isolated by RT-PCR from liver tissue of 3,3 *-methylcholanthrene-treated guinea pigs. It shares considerable sequence identity with guinea pig CYP1A1 (nt 77%, aa 65%), but differs in levels of constitutive expression, function, and inducibility. Western blot analysis of protein expressed by full-length cDNA in COS-1 cells identified CYP1A2 (56 kDa) and CYP1A1 (53 kDa) proteins in corun liver microsomes. CYP1A2 transfectants metabolized methoxyresorufin and ethoxyresorufin, while CYP1A1 transfectants metabolized only ethoxyresorufin. Constitutive expression of CYP1A2 mRNA (2.0 kb) and protein was much lower than that of CYP1A1 mRNA (2.6 kb) and protein, but the fold induction of CYP1A2 by 3,3 *-methylcholanthrene was greater than that of CYP1A1. Changes in splicing of CYP1A2 pre-mRNA occur upon treatment with 3,3 *-methylcholanthrene. q 1997 Academic Press

Key Words: guinea pig; cytochrome P450; CYP1A2; CYP1A1; cDNA; COS-1 cell expression; resorufin metabolism.

Cytochrome P450s (CYPs)2 comprise a gene superfamily encoding enzymes involved in synthesis and metabolism of endogenous compounds and the metabolism of foreign compounds (1). The two known members of the CYP1A family, CYP1A1 and CYP1A2, metabolize a variety of endogenous lipophilic substances, such as steroids and arachadonic acid (2– 7). They also 1 To whom correspondence should be addressed at Department of Cell Biology, New York University School of Medicine, 550 First Avenue, New York, NY 10016. Fax: (212) 263-8139. E-mail: blackv01 @mcrcr6.med.nyu.edu. 2 Abbreviations used: CYPs, cytochrome P450s; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; 3MC, 3,3*-methylcholanthrene; EROD, ethoxyresorufin O-deethylase; MROD, methoxyresorufin O-demethylase.

have a well-documented role in metabolism of aromatic hydrocarbons (8–10) and are induced by these exogenous compounds, e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3,3*-methylcholanthrene (3MC). The guinea pig has a higher sensitivity to TCDD than other species (11) and is reportedly refractory to induction of the CYP1A family members by aromatic hydrocarbons (12). Elucidating the mechanisms responsible has been hindered by lack of detailed information on the guinea pig CYP1A family members and their regulation. In this study, full-length cDNA for guinea pig CYP1A2 and CYP1A1 were isolated by RT-PCR. Precise identification of the proteins and analysis of their functions were facilitated by expression of full-length cDNA in COS-1 cells. This is the first report of a cDNA sequence for guinea pig CYP1A2 and the first comparative functional analysis of CYP1A1 and CYP1A2 in this species. Relative induction by 3MC was examined by Northern and Western blot, as well as enzymatic analyses. As opposed to other species, CYP1A1 is constitutively expressed at high levels, while the fold induction of CYP1A2 by 3MC is greater. Differential splicing of premRNA appears to be involved in the induction process for CYP1A2. Whether the greater sensitivity of guinea pigs to TCDD resides in differences in regulation and/ or function of guinea pig CYP1A family members remains to be determined. MATERIALS AND METHODS Animals, drug treatment, and subcellular fractionation. Male English short-haired guinea pigs (Hartley, Camm Research Laboratories, Wayne, NJ) (775–850 g) were fed standard laboratory chow ad libitum in a controlled lighting environment (lights on, 6 AM, lights off, 7 PM). They were injected ip daily for 2 days with 3MC (ICN Biochemicals, K / K Labs, Plainview, NY) (25 mg/kg BWt) suspended in corn oil or with the vehicle alone. At least three animals were included in each treatment group. On the third day, all animals were injected with sodium pentobarbital (Diabutal, Diamond Laboratories, Des Moines, IA) (60 mg/kg BWt) and liver tissue was quickly removed. Tissue for RNA preparation was immediately frozen in 11

0003-9861/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

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liquid nitrogen and stored in liquid nitrogen until use. Tissue for subcellular fractionation was placed on ice and immediately used to prepare microsomal fractions, as previously described (13). Protein analysis. Analysis of protein, SDS–PAGE, and immunoblotting were all conducted as previously described (13). Antibodies made by Dr. M. Negishi, in Dr. David Sabatini’s laboratory, against the major proteins induced by 3MC in rats, CYP1A1 and 1A2, were obtained from Drs. Milton Adesnik and Takashi Morimoto at NYU Medical Center (14). RT-PCR. RNA was prepared from liver tissue in the presence of guanidium thiocyanate (Fluka), according to methods described by Chirgwin and co-workers (15). M-MLV reverse transcriptase was then used to obtain cDNA for PCR screening. PCR employing Taq polymerase were carried out in a DNA thermal cycler (Perkin–Elmer Cetus, Emeryville, CA). Products were separated electrophoretically on 1% agarose gels and stained with ethidium bromide and their sizes deduced by comparison with a 1-kb DNA ladder (Gibco BRL Life Technologies, Grand Island, NY). Oligonucleotide primers used in the PCR for CYP1A1 corresponded to the 5* (nt 117–137, sense) and 3 * (nt 1661–1683, antisense) ends of the coding region of the guinea pig CYP1A1 sequence (GenBank, D11043) (16). PCR (denaturation, 947C, 1 min; annealing, 567C, 2 min; extension, 727C, 5 min; 35 cycles with additional extension at the end of the last cycle, 727C, 7 min) resulted in full-length cDNA for CYPA1. cDNA for guinea pig CYP1A1 was also received from Dr. Ishazaki (Sapporo, Japan) as two inserts subcloned in pUC19 (16). To obtain cDNA for CYP1A2, oligonucleotides were designed corresponding to regions of 90–95% homology between mammalian CYP1A1 and CYP1A2 sequences published in GenBank (nt 321– 342, sense, and nt 1052–1072, antisense). RT-PCR using liver RNA from 3MC-treated guinea pigs gave two products (200 and 400 pp) which showed high identity with CYP1A2 sequences from other species. Oligonucleotides, designed using this CYP1A2-like sequence, were employed in 5* and 3 * RACE (Gibco BRL Life Technologies) to obtain cDNA encompassing the entire coding region. Relative differences in RT-PCR products from control vs 3MCtreated liver RNA were determined by comparative kinetic analysis. Incorporation of 32P-end-labeled antisense primer into electrophoretically separated PCR products was examined as a function of cycle number. Bands visualized by ethidium bromide staining were excised and radioactivity was determined by liquid scintillation counting. The midpoint of the exponential amplification range was selected for comparison by inspection of semilogarithmic plots of cpm vs cycle number. PCR with primers for b-actin (Stratagen Cloning Systems, La Jolla, CA), which give a 661-bp PCR product, was run as a control for the RT reaction. Subcloning. Subcloning was performed by in-gel ligation–transformation (SeaPlaque GTG agarose, FMC BioProducts, Rockland, ME). Restriction enzyme sites incorporated into the 5* ends of the oligonucleotides (CYP1A1: XbaI, sense strand; HindIII, antisense strand; CYP1A2: KpnI, sense strand; SpeI or XbaI, antisense strand) facilitated ligation of the PCR products into the pBluescript IIKS(// 0) phagemid vector (Stratagene Cloning Systems) which was then used to transform competent RR1 Escherichia coli. Plasmid DNA was prepared by alkaline lysis (17). Following restriction enzyme digestion, the size of the insert was estimated on 1% agarose gels. Sequencing. Three cDNAs of the expected size for CYP1A1 (1.55 kb), obtained from independent RT-PCR, were sequenced by the dideoxy chain termination method using Sequenase Version 2.0 (United States Biochemical Corp., Cleveland, OH). Primers for sequencing CYP1A1 consisted of the standard M13 forward and reverse primers and five oligonucleotides corresponding to the published guinea pig CYP1A1 sequence (nt 141–161, 321–342, 530–548, 1020–1040, and 1261–1280, sense; nt 580–600 and 1052–1072, antisense). The initial sequence for CYP1A2 was obtained from overlapping 5* and 3* RACE PCR products, using standard M13 forward and

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reverse primers. The full-length sequence was obtained by ‘‘walking,’’ using oligonucleotides corresponding to the sequence as it became known (nt 74–88, 900–920, and 1220–1240, sense; nt 516–536 and 752–772, antisense). Oligonucleotides designed to the 5* and 3* ends of the coding region were then used in RT-PCR to obtain a full-length cDNA clone. Sequence was confirmed in cDNA from at least three independent PCR, using the same primers. Sequence comparisons were done using the Wisconsin Package, Version 7.3 (Program Manual for the GCG Package, Version 7, Genetics Computer Group, 1991, Madison, WI). Northern blot analysis. RNA was denatured, separated by electrophoresis on agarose/formaldehyde gels, and transferred to nitrocellulose sheets according to standard protocols (17). The RNA was fixed to the membrane by baking for 2 h at 807C or by uv irradiation for 30 s to 1 min. Following prehybridization (4 h) and hybridization (24 h), the blots were washed at high stringency (0.11 SSC/0.1% SDS) (20 min at room temperature, followed by 20 min at 557C) and exposed to X-ray film (X-OMAT, Eastman Kodak Co., Rochester, NY). Labeling of probes was done with [a-32P]dCTP using a nick translation kit from NEN (Biotechnology Research Systems, Boston, MA). Labeled probes were purified on Nacs Prepac cartridges (Gibco BRL, Gaithersberg, MD). Transfection experiments. Full-length CYP1A1 and CYP1A2 cDNA were subcloned into the mammalian expression vector pcDNA3 (Invitrogen Corp., San Diego, CA) and large-scale plasmid preparations were made. DNA was purified either on two successive cesium chloride gradients, followed by dialysis against 10 mM Tris– HCl containing 0.01 mM EDTA (17), or with the Qiagen Plasmid Kit, according to the instructions provided by the manufacturer (Qiagen Inc., Chatsworth, CA). COS-1 cells were grown in DMEM supplemented with 10% fetal calf serum, glutamine, penicillin, streptomycin, and fungizone. Transfection of cells 50–70% confluent with 9 mg DNA per 15-cm dish was performed using lipofectamine (Gibco Life Technologies) according to the manufacturer’s instructions. After 18–24 h the transfection medium was replaced with DMEM, supplemented as above. On the third day after transfection, the cells were scraped off the plates in lysis buffer (10 mM KCl, 10 mM Tris–HCl, 1 mM EDTA, pH 7.4) and homogenized in this buffer in a tight-fitting Dounce homogenizer. The homogenate was brought to 0.25 M sucrose and microsomal fractions were prepared as described above. Expression of proteins by the transfected cDNA was assessed by Western blot analysis and biochemical assay. Enzyme assays. Metabolism of resorufin substrates (ethoxy- and methoxyphenoxazones) was assayed as described by Burke and coworkers (18). Chemicals. M-MLV reverse transcriptase and Taq polymerase were obtained from Boehringer-Mannheim (Indianapolis, IN). Oligonucleotides were obtained from Operon Technologies (Alameda, CA) or synthesized on an Oligo 1000 DNA synthesizer (Beckman Instruments, Palo Alto, CA). Standard sequencing primers and restriction endonucleases were obtained from New England Biolabs (Beverly, MA). Resorufin substrates were obtained from Molecular Probes, Inc. (Eugene, OR). All other reagents were purchased as described previously (13).

RESULTS AND DISCUSSION

Cloning of cDNA for CYP1A2 and comparison with cDNA for CYP1A1. A 1757-bp cDNA for CYP1A2 was obtained by RT-PCR and the complete sequence data were submitted to GenBank under Accession No. U2350. CYP1A1 cDNA obtained by RT-PCR had a sequence identical to that previously published (16). The coding region of guinea pig CYP1A2 shares significant homology with guinea pig CYP1A1 (77% nt,

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FIG. 1. Comparison of cDNA for guinea pig CYP1A1 and CYP1A2 predicted amino acid sequence of the coding region. CYP1A1 sequence is shown in the top line and CYP1A2 sequence in the bottom line. Only amino acids which differ between the two are given for CYP1A2 (in bold typeface). The numbers at the side denote the relative positions of amino acids in each sequence. Gaps in predicted amino acid sequence alignment are represented by a bold hyphen. The region of the heme binding site is underlined. The conserved cysteinyl residue as well as other conserved amino acids known to be important in CYP function is double underlined. Helical regions are enclosed in boxes, labeled A–L, minus H, a helix not found in CYP1A1,2 (23).

65% aa) (Figs. 1 and 2). Although its amino terminus extends 5* beyond that of CYP1A1 by 6 aa, it terminates 7 aa earlier, resulting in proteins differing in predicted length by 1 aa. When compared to CYP1A family members across species, it shares higher iden-

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tity with sequences for CYP1A2 than with those for CYP1A1 (Fig. 2). This is particularly evident when predicted amino acid sequences are compared: 65 – 67% identity with CYP1A1, 73 – 77% identity with CYP1A2.

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FIG. 2. Comparison of the amino termini of guinea pig CYP1A1 and CYP1A2 with those of several other species. Conserved regions are shown in the boxes (I, II, III). Substitutions are unshaded. At the right the % identities of full-length guinea pig CYP1A2 nucleotide (NT) and predicted amino acid (AA) sequence with those of the other CYP1 family members are shown.

The greater identity of guinea pig CYP1A2 with human than with rodent CYP1A2 is of some interest. Analysis of cDNA sequences for several other proteins has shown a closer relationship of guinea pigs to primates than to rodents (19–21). Inclusion of CYP1A proteins in future analyses may add new information to the current debate of the evolution of guinea pigs and their proper phylogenetic position (19–22). A more detailed comparison of the predicted amino acid sequences of CYP1A1 and CYP1A2 across mammals revealed specific areas of interest which are highlighted in Figs. 1 and 2. Both have the 11 helices described for CYP1A proteins in other species (Fig. 1) (23) and their amino termini share three regions of homology with other CYP1A sequences (Fig. 2). Elsewhere in the protein, specific amino acids known to be important in the structure and function of CYPs are also conserved: the threonine in helix I which is thought to be responsible for deformation of this helix above the heme (24), the cysteine present in the hemebinding site, and several other invarient residues (25). Both proteins possess the ionic amino acids important for interaction with NADPH–cytochrome P450 reductase (26). Two substitutions of Arg for Lys, corresponding to positions 99 and 463 of rat CYP1A2, are seen in other species, but a similar substitution in position 94 occurs only in the guinea pig and human sequences. Differences in sequence are also notable (Fig. 1). The degree of identity between the guinea pig sequences for CYP1A1 and CYP1A2 varies among the helices (A– C, K, and L, 90–100%; E, I, and J, 67–70%; D, 58%; G, 42%; F, 38%), but in nine of these (A–C, E, G, I– L) they have few substitutions not found among the corresponding CYP1As. Helix G, a helix in which identity between the two guinea pig proteins is low (42%),

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has a high concentration of characteristic differences between CYP1A1 and CYP1A2 across species (11 of 24 aa) (see Ref. 23). The differences between guinea pig CYP1A1 and CYP1A2 in this region are usually identical to those in the corresponding human sequences. However, anti-peptide antibodies made against the Cterminus of this helix and the adjacent loop extending to helix I of rat CYP1A2 react with CYP1A2 proteins of many species, including human and guinea pig (27). In helices F and D the guinea pig proteins also have low identity (38–58%), but here they have several substitutions not found in other mammals (F: CYP1A1, 5, and CYP1A2, 3; D: CYP1A1, 2, and CYP1A2, 4). The majority of substitutions unique to the guinea pig, however, occurs in the nonhelical regions (CYP1A1, 46 of 65 or 11%; CYP1A2, 37 of 58 or 64%). Some of the substitutions are in positions where the amino acid is conserved in the corresponding sequences of other species (CYP1A1, 32; CYP1A2, 24); almost half of these occur in positions in which the amino acid is conserved in both CYP1A1 and CYP1A2; 4 substitutions for these highly conserved amino acids occur in the same position in the two guinea pig proteins and 3 are identical. Although their significance is not clear, these differences may contribute to distinctive functions of CYP1A1 vs CYP1A2 across species (e.g., helix G) or to functional distinctions between the guinea pig proteins and those of other species. Transfection experiments. Immunochemical analysis of guinea pig liver microsomes showed that two CYP1A proteins are present (53 and 56 kDa). However, it was not clear which was CYP1A1 and which was CYP1A2. Definitive identification came from transfection of COS-1 cells with full-length cDNA and comparison of the expressed microsomal proteins with those in

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GUINEA PIG CYP1A2, CLONING, AND COMPARISON WITH CYP1A1 TABLE I

Comparison of EROD and MROD Activity (pmol/mgrmin) in Microsomes from Transfected Cells and Guinea Pig Liver

FIG. 3. Western blot analysis of CYP1A proteins. Polyclonal antibodies made against rat CYP1A1,2 demonstrate two proteins in guinea pig liver microsomes. One, 53 kDa, comigrates with protein expressed by COS-1 cells transfected with CYP1A1 cDNA. It is present in liver microsomes of control animals (3MC0) and increases in treated animals (3MC/). The other, 56 kDa, comigrates with protein expressed by cells transfected with CYP1A2 cDNA. This protein is present in low to undetectable levels in control animals and is markedly increased following exposure to 3MC. Cells transfected with the vector (V) alone express no comigrating proteins. Equal amounts of liver microsomal protein (6 mg) were used for MC0 and MC/. The amounts of microsomal protein used for the COS-1 cells were greater and varied slightly (control, 25 mg; CYP1A1, 25 mg; CYP1A2, 20 mg).

liver microsomes (Fig. 3). Cells transfected with cDNA for CYP1A1 expressed a 53-kDa microsomal protein, while those transfected with cDNA for CYP1A2 expressed a 56-kDa microsomal protein. Neither protein was detectable in control cells transfected with plasmid DNA alone. The relative sizes of the two proteins (CYP1A1 õ CYP1A2) are the inverse of those in most other species. In view of their similarities in length and sequence, the reason for this difference in apparent size on SDS gels is not clear. The shorter N-terminus of CYP1A1 has been shown to be responsible, in part, for its faster mobility in SDS gels compared to other CYP1A1 proteins (16), but does not completely account for the difference. Hydrophobicity may also play a role. Although the total number of hydrophobic amino acids is less in CYP1A1 (263, 51%) than in CYP1A2 (273, 53%), Kyte– Doolittle plots show that the domains of hydrophobic amino acids in CYP1A1 are more pronounced. These may bind SDS to a greater degree, accounting, in part, for the faster mobility of CYP1A1 (28). The relative mobility of these proteins is also very sensitive to the ionic conditions of the gel (Black, unpublished observations). A contributing factor may be the differences between the isoelectric point for the two proteins. It is considerably lower for CYP1A1 (8.33) than for CYP1A2 (8.97). Expressing the proteins in host cells also allowed the functional specificity of guinea pig CYP1A1 and CYP1A2 for resorufin metabolism to be tested (Table I). Ethoxyresorufin O-deethylase (EROD) is usually attributed to CYP1A1, while methoxyresorufin O-demethylase (MROD) is associated with CYP1A2 (18).

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Microsomes

EROD

MROD

COS-1, pcDNA3 / CYP1A1 COS-1, pcDNA3 / CYP1A2 COS-1, pcDNA3 Liver, 0 3MC Liver, / 3MC

10.3 7.3 ND 39.9 209.7

ND 3.2 ND 19.2 132.5

Note. Values represent means of two experiments. ND, not detectable.

Cells transfected with cDNA for CYP1A1 had EROD, but not MROD, activity. MROD activity was detectable only in microsomes from cells transfected with CYP1A2 cDNA. However, CYP1A2 transfectant microsomes also possessed EROD activity. The exclusive capacity of CYP1A2 for MROD activity may be related to structural differences noted above. Induction of CYP1A proteins by 3MC in guinea pig liver. 3MC is an archetypal inducer of CYP1 family members in many species (2–5). CYP1A1 is prominent in liver microsomes of control guinea pigs, while CYP1A2 is barely detectable (Fig. 1). Following treatment with 3MC, levels of CYP1A1 increase, but the fold induction of CYP1A2 is much greater. Activity for EROD was two- to threefold greater than for MROD in liver microsomes of control guinea pigs (Table I). However, both increased five- to sixfold in 3MC-treated animals. The data obtained in transfected cells suggest that both CYP1A1 and CYP1A2 contribute to increased EROD activity, while CYP1A2 is solely responsible for the increase in MROD activity.

FIG. 4. Northern blot analysis. CYP1A1 and CYP1A2 cDNA detected two mRNA species (2.6 and 2.0 kb) in liver RNA from control (0) and 3MC-treated (/) guinea pigs. CYP1A1 cDNA hybridized predominantly with the 2.6-kb RNA species. CYP1A2 cDNA hybridized only with the 2.0-kb RNA. Gels were loaded with 6 mg RNA in all cases.

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Analysis of RNA by Northern blot and RT-PCR. In Northern blots, CYP1A1 cDNA hybridized with a 2.6kb RNA species present in control liver tissue which increased following 3MC treatment (Fig. 4). It also recognized a 2.0-kb species in liver tissue from 3MCtreated animals. CYP1A2 cDNA, on the other hand, recognized the 2.0-kb RNA species and detected it at significant levels only in 3MC-treated animals. It also hybridized less intensely with several smaller RNA species. In RT-PCR, amplification with oligonucleotide primers corresponding to a sequence adjacent to the ends of the coding region for CYP1A1 resulted in only one PCR product, the full-length cDNA (1.55 kb) (Fig. 5). This product increased dramatically following 3MC treatment. However, amplification using similar primers for CYP1A2 (5*, nt 1–20; 3*, nt 1597–1617, of the cDNA) resulted in the full-length product (1.55 kb) and a set of shorter products (Fig. 5). Following 3MC treatment, the full-length product increased relative to the smaller products (Figs. 5 and 6). The most prominent of the smaller products (Ç700 bp) was subcloned. Its nucleotide sequence was identical to that of nt 1–38 and 916–1617 of the CYP1A2 cDNA, but the intervening segment, almost half of the coding region, was missing. Comparison with the genomic sequence for rat CYP1A2 showed that this segment corresponded to the entire second exon. Comparative kinetic analysis (Fig. 6) revealed a change in this differential splicing of CYP1A2 premRNA following 3MC treatment. Incorporation of 32Pend-labeled antisense primer into the full-length PCR product increased 10-fold in treated animals, while incorporation into the 700-bp product increased only 5fold. Comparison of the 10-fold change in mRNA follow-

FIG. 5. RT-PCR analysis of CYP1A1 and CYP1A2 mRNA from control (0) and 3MC-treated (/) guinea pigs. The expected size of the full-length product of both CYP1A1 and CYP1A2 is Ç1.55 kb. The full-length product obtained by RT-PCR for CYP1A1 showed a dramatic increase following 3MC treatment. RT-PCR for CYP1A2 resulted in both the full-length product and a set of smaller products. Following 3MC treatment, the full-length product appeared to be relatively enhanced. b-Actin levels are shown for comparison.

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FIG. 6. Comparative kinetic RT-PCR analysis. CYP1A2 cDNA was synthesized from equivalent amounts of liver RNA of control (0) and 3MC-treated (/) guinea pigs and equal amounts were amplified for the number of cycles indicated. Samples of the PCR products were electrophoresed on 1% agarose gels and stained with ethidium bromide. Bands corresponding to the 1.55-kb and Ç700-bp PCR products were excised from the gel and the amount of 32P incorporated from the end-labeled antisense primer was quantitated by scintillation counting.

ing 3MC treatment with the greater increase in protein, shown in Fig. 3, suggests that posttranslational mechanisms may also contribute to regulation of CYP1A2 expression. It should be noted that the expression pattern of CYP1A1 and CYP1A2 mRNA and protein in the guinea pig is the reverse of that seen in the rat and most other species. In the guinea pig liver, CYP1A1 is constitutively expressed, while expression of CYP1A2 is dependent upon induction. In the rat, and in most other species, CYP1A2 is constitutive and CYP1A1 is more induction-dependent (29). Constitutive expression of CYP1A1 in guinea pig liver was reported by Ishizawa and co-workers (16), but information on induction relative to CYP1A2 was not presented. Furthermore, although the degree of induction by 3MC observed in this report is greater than that recorded by Thomas and coworkers of protein reacting with monoclonal antibodies to rat CYP1A (12), the fold induction of both proteins and their associated enzyme activities is lower than in the rat (6, 29–33). These differences in expression and inducibility may be related to the structure/function of the guinea pig CYP1A proteins. Nebert and co-workers demonstrated a link between regulation and structure/function in the CYP1A family (34). They showed that metabolism of endogenous substrates by murine Cyp1a-1 plays a direct role in controlling transcription of the gene that encodes it. Missense mutations in the Cyp1a-1 structural gene disrupted this autoregulatory loop, allowing high basal levels of Cyp1a-1 expression, not inducible by TCDD. Whether the greater susceptability of the guinea pig to TCDD resides in functional differences of their CYP1A family members and/or in differences in CYP1A regulation remains to be determined. CYP1A family

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members in other species are capable of metabolizing a variety of lipophilic substrates important for cell function, including steroid hormones and arachidonic acid, which may modulate and/or mediate their toxicity (2–7, 35). Having the cDNAs for guinea pig CYP1A1 and CYP1A2 will facilitate detailed analysis of these functions which may contribute to the impact of their induction in liver and in other tissues. ACKNOWLEDGMENTS This work was supported by NIH Research Grants DK39671 and HL 48476 to V.B. NIH training Grant 5T35 DK07421 sponsored M.H. The authors thank Douglas Weiner and Daniel Culliford for technical assistance, Heide Plesken for graphics, Dr. John Hill for ‘‘userfriendly’’ programs for sequence analysis available through MCCLBO, and Dr. Takashi Morimoto for his help and advice throughout these studies. Computing was supported in part by NSF BIR-93-18128.

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