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R H Tukey

Publications and source records attributed to R H Tukey.

At least 55 records · Page 3Linked to original sources
PubMed1994

Cloning and characterization of cDNAs encoding mouse Ugt1.6 and rabbit UGT1.6: differential induction by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

In this report, cDNAs for mouse liver Ugt1.6 and rabbit liver UGT1.6 have been cloned and characterized. The predicted amino acid sequence of mouse Ugt1.6 is 93% and 78% similar to the rat and human UGT1.6 sequences, respectively, while the rabbit UGT1.6 is 79% and 83% similar to the rat and human UGT1.6 sequences, respectively. To examine the substrate specificities of the proteins encoded by the mouse Ugt1.6 and rabbit UGT1.6 cDNAs, the recombinants were expressed in monkey kidney COS-1 cells. Transfection of the mouse and rabbit recombinants allowed for the expression of the UGT1.6 proteins as determined by immunoprecipitation of newly synthesized protein. The expressed UGTs conjugated small planar phenolic molecules such as 4-nitrophenol, 1-naphthol, and 4-methylumbelliferone. While the bulky phenol 4-hydroxybiphenyl was not a substrate for the enzymes, 2-hydroxybiphenyl was an excellent substrate. Androgens and estrogens were not conjugated by either mouse Ugt1.6 or rabbit UGT1.6. In rodents, UGT1.6 mRNA is expressed constitutively and induced when the animals are treated with the Ah receptor ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Using wild-type mouse hepatoma cells and the Ah receptor deficient class II cells, it was demonstrated that induction of mouse Ugt1.6 was dependent upon a functional Ah receptor complex. However, when New Zealand white rabbits were treated with TCDD and liver mRNA was examined by Northern blot analysis, it was shown that TCDD had no effect on the induction of UGT1.6 mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Recognition of uridine diphosphate glucuronosyl transferases by LKM-3 antibodies in chronic hepatitis D.

Patients with chronic hepatitis D often have liver-kidney microsomal antibodies type 3 (LKM-3). These antibodies react with several microsomal antigens that have a molecular weight of 55 KDa and an isoelectric point of about 8. We studied the molecular nature of the antigen and, by immunoscreening a human liver cDNA expression library with KM-3 sera, found that uridine diphosphate glucuronosyl transferases (UGT) appeared as candidate antigens. We confirmed the identity of UGT as an antigen by reacting the sera with recombinant rabbit liver UGT proteins. Some sera reacted with rabbit UGT-2 proteins, but UGT-1 proteins were more sensitive and specific in detecting LKM-3 autoantibodies in patient sera. Anti-UGT-1 antibodies were detected in all LKM-3 positive sera from patients with hepatitis D and 1 out of 11 patients with autoimmune hepatitis type 2. Sera from patients who had hepatitis B only did not react with UGT proteins. The UGT proteins are part of the phase II enzymes of drug metabolism and are the first such enzymes to be identified as human autoantigens.

Antibody Specificity↗
PubMed1994

Expression of modified human cytochrome P450 1A1 in Escherichia coli: effects of 5' substitution, stabilization, purification, spectral characterization, and catalytic properties.

Human cytochrome P450 (P450) 1A1 is primarily an extrahepatic enzyme and is important because of its roles in the activation of polycyclic hydrocarbons and other xenobiotic chemicals. Purification of active enzyme from human tissues has not been successful. We report the expression and purification of the recombinant enzyme from Escherichia coli. A full-length cDNA of human cytochrome P450 1A1 and several modified constructs were engineered into a pCW vector and used to transform E. coli cells. Little expression was observed with the native sequence and several modified constructs, but successful expression (20-25 nmol membrane-bound P450 1A1 per liter of culture) was achieved with a construct in which the Ala codon GCT was placed in the second position and the 5'-terminal codons were maximized for AT content and minimized for the potential of secondary structure formation of the mRNA transcript. alpha-Naphthoflavone was found to protect against denaturation by detergents during solubilization and was added to buffers used for purification. The recombinant P450 1A1 was purified to electrophoretic homogeneity after two ion-exchange chromatography steps in approximately 50% yield. N-Terminal amino acid sequence analysis verified the expected first 21 residues, with the exception of the terminal Met. The isolated human ferric P450 1A1 was predominantly in the high spin state, in contrast to the orthologous rat and rabbit enzymes. Recombinant P450 1A1 catalyzed 7-ethoxyresorufin O-deethylation and benzo[a]pyrene 3-hydroxylation with Km values of 0.58 and 15 microM and Vmax values of 8.3 and 2.5 nmol min-1 (nmol P450 1A1)-1, respectively. The successful expression and purification of human P450 1A1 should increase the availability of this enzyme and the generation of antibodies for further biochemical and other biological studies.

Amino Acid Sequence↗
PubMed1994

Caffeine metabolism by human hepatic cytochromes P450: contributions of 1A2, 2E1 and 3A isoforms.

Caffeine (CA) N1-, N3- and N7-demethylase, CA 8-hydroxylase and phenacetin O-deethylase activities were measured in microsomes from 18 separate human livers which had been characterized previously for a range of cytochrome P450 (CYP) isoform-specific activities and immunoreactive CYP protein contents. Correlations between the high affinity components of the three separate CA N-demethylations were highly significant (r = 0.77-0.91, P < 0.001) and each of the three high affinity CA N-demethylations correlated significantly (r = 0.64-0.93, P < 0.05-0.001) with the high affinity phenacetin O-deethylase, 2-acetylaminofluorene N-hydroxylation and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) mutagenicity (all predominantly CYP1A2-mediated reactions). Consistent with these observations, cDNA-expressed human CYP1A2 catalyzed the N1-, N3- and N7-demethylation of CA and apparent Km values were similar (0.24-0.28 mM) for all three reactions and comparable to those observed previously with human liver microsomes. The low affinity components of CA N1- and N7-demethylation correlated significantly (r = 0.55-0.85, P < 0.05-0.001) with immunoreactive CYP2E1 content and the CYP2E1-specific activities 4-nitrophenol and chlorzoxazone hydroxylation. Diethyldithiocarbamate, a selective inhibitor of CYP2E1, inhibited the low affinity CA N1- and N7-demethylation, with IC50 values of 23 microM and 11 microM, respectively. The apparent Km values for CA N1- and N7-demethylation by cDNA-expressed CYP2E1 (namely 28 and 43 mM, respectively) were of a similar order to those calculated for the low affinity microsomal activities. Significant correlations (r = 0.87-0.97, P < 0.001) were observed between CA 8-hydroxylation and immunoreactive CYP3A content and the CYP3A-mediated reactions benzo(a)pyrene hydroxylation, omeprazole sulfoxidation and aflatoxin B1 mutagenesis. Effects of alpha-naphthoflavone, erythromycin, troleandomycin and nifedipine on microsomal CA 8-hydroxylation were generally consistent with CYP3A involvement. Taken together with previous data, the results indicate a major involvement of CYP1A2 in the high affinity component of all three human hepatic CA N-demethylations. In contrast, CYP2E1 appears to be the main enzyme involved in the low affinity components of CA N1- and N7-demethylation while CA 8-hydroxylation is catalysed predominantly by a CYP3A isoform(s).

Caffeine↗
PubMed1994

The human CYP1A2 gene and induction by 3-methylcholanthrene. A region of DNA that supports AH-receptor binding and promoter-specific induction.

The gene for cytochrome P4501A2 is constitutively expressed in the liver of vertebrates and shows induced expression when an organism is exposed to polycyclic aromatic hydrocarbons and halogenated hydrocarbons. To identify DNA elements regulating transcription of the human CYP1A2 gene, transient transfection experiments were conducted in the human hepatoma cell line HepG2. Dissection of the 5'-flanking portion of the CYP1A2 gene identified two regions that contributed to the overall induction by 3-methylcholanthrene. One region located at -2532/-2423 contains an xenobiotic-responsive element-like sequence, termed X1, that binds a nuclear 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible protein in HepG2 and wild type mouse Hepa-1 cells, but not in the Ah receptor nuclear translocation defective mouse C- mutant c4 cells. In addition, deletion of this region of the CYP1A2 gene reduces the 3-methylcholanthrene (3-MC)-initiated induction of chloramphenicol acetyltransferase activity in both promoter- and enhancer-specific constructs. The second responsive region is located at -2259/-1987. This region of the gene contains a second xenobiotic-responsive element-like element, but this element does not associate with the nuclear Ah receptor. However, there does exist several potential AP1 binding sites and a conserved TATA box. A DNA fragment from -2259/-1970 that contains these elements was shown to function as an efficient eukaryotic promoter, in addition to supporting 3-MC-induced promoter activity. These results suggest that Ah receptor-specific and promoter-specific elements regulate the expression of the human CYP1A2 gene.

Animals↗
PubMed1994

Expression of modified human cytochrome P450 1A2 in Escherichia coli: stabilization, purification, spectral characterization, and catalytic activities of the enzyme.

A full-length human cytochrome P450 (P450) 1A2 cDNA clone and four derivatives in which the 5'-terminus was modified were inserted into the pCW vector and used to transform Escherichia coli cells. Low levels of expression were seen with most of the constructs but high expression levels (245 nmol membrane-bound P450 recovered per liter culture) were achieved when the N-terminus was MALLLAVFL, as reported earlier by Fisher et al. (C. W. Fisher, D. L. Caudle, C. Martin-Wixtrom, L. C. Quattrochi, R. H. Tukey, M. R. Waterman, and R. W. Estabrook, 1992, FASEB J. 6, 759-764). The expressed human P450 1A2 in bacterial membranes was rapidly denatured to cytochrome P420 in the presence of detergents. This denaturation was blocked by the inhibitory ligand alpha-naphthoflavone (alpha NF, 7,8-benzoflavone). Human P450 1A2 was solubilized using high concentrations of sodium cholate and Triton N-101 and could be purified to near homogeneity in high yield in two steps. alpha NF was included in the buffer in the first step and then removed in the second chromatography step along with the detergent. The purified human P450 1A2 was found to be almost completely in the high spin iron configuration, in contrast to P450 1A2 enzymes isolated from rats and rabbits. The enzyme was catalytically active toward the known substrates 7-ethoxyresorufin and phenacetin. The N-terminal appears to be blocked, as is the case for other P450s we have expressed that contain the sequence MALLLAVFL in E. coli. Previously this human P450 has only been available in limited amounts; the methods presented here should facilitate further biochemical and practical studies on this interesting enzyme.

Amino Acid Sequence↗
PubMed1994

Activation of procarcinogens by human cytochrome P450 enzymes expressed in Escherichia coli. Simplified bacterial systems for genotoxicity assays.

Bacterial assays were used to examine the activation of 14 known procarcinogens by cytochrome P450 (P450) enzymes. Human P450s 1A1, 1A2 and 3A4 were expressed in Escherichia coli with slight modification of their N-terminal sequences. Genotoxicity was measured by the induction of the SOS response in Salmonella typhimurium NM2009 (TA1535/pSK1002/pNM12), which contains a umuC regulatory sequence attached to the lacZ reporter gene. Conditions for analysis were examined using E. coli membranes and purified enzymes. Membrane fractions, fortified with NADPH-P450 reductase, were found to be useful preparations for measuring activation of the procarcinogens. Conditions of linearity were established for these assays and the systems were applied to several particular problems related to bioactivation of procarcinogens by P450s. The patterns of activation of the 14 individual chemicals were consistent with the literature developed using human liver microsomes, purified liver P450s and other approaches. The P450s expressed in bacterial membranes could be inhibited by antibodies. 7,8-Benzoflavone inhibited P450s 1A1 and 1A2 and stimulated P450 3A4 in the membranes. The contributions of P450s 1A1 and 1A2 were distinguished with some of the arylamines and 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. Recombinant P450 3A4 was found to be more active than P450 1A2 in the activation of aflatoxin B1 at all substrate concentrations examined.

Amino Acid Sequence↗
PubMed1993

Cloning and characterization of rabbit liver UDP-glucuronosyltransferase cDNAs. Developmental and inducible expression of 4-hydroxybiphenyl UGT2B13.

A polyclonal antibody generated against rabbit liver p-nitrophenol UDP-glucuronosyltransferase (UGT) was used to screen a rabbit liver cDNA expression library constructed in lambda gt11. A 500-base pair cDNA clone, termed pPNP, generated a fusion protein that was antigenic with the antibody. Clone pPNP encoded the 3' region of a UGT. To identify larger recombinants, clone pPNP was used as a probe to screen a second cDNA library constructed in lambda ZAP. Two different cDNA clones were identified by DNA sequence analysis. Based upon their predicted amino acid sequence analysis, the clones encode transferases belonging to the UGT2 subfamily, and have been identified as UGT2B13 and UGT2B14. The predicted N-terminal sequence of UGT2B13 is identical to that determined for the purified rabbit liver estrone UGT. However, expression of the UGT2B13 cDNA in COS-1 cells displayed no activity in the presence of estrone but efficiently conjugated 4-hydroxybiphenyl. Results of Southern blot analysis using the 5' divergent region of the UGT2B13 cDNA that encodes exon 1 demonstrates that multiple genes share sequence homology to UGT2B13, an observation which indicates that the estrone UGT and UGT2B13 genes are encoded by separate alleles. When the 5' variable regions of the cDNAs where used in Northern blot analysis, the expression of UGT2B13 and UGT2B14 were shown to be expressed primarily in adult rabbits. However, when neonatal rabbits were treated with either dexamethasone or rifampicin, UGT2B13 mRNA levels were induced. The neonatal induction of UGT2B13 mRNA corresponded with similar increases in 4-hydroxybiphenyl UGT activity. The expression and induction of UGT2B13 paralleled that of the developmentally regulated rabbit liver progesterone 6 beta-hydroxylase P4503A6.

Amino Acid Sequence↗
PubMed1993

The catalytic activity of four expressed human cytochrome P450s towards benzo[a]pyrene and the isomers of its proximate carcinogen.

The catalytic properties of expressed human cytochromes P4501A1, 1A2, 3A4 and 3A5 with respect to the metabolite distribution for the carcinogen benzo[a]pyrene (BP) and the proximate carcinogen (-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene ((-)-BP-7,8-DHD) were determined. P4501A1 formed a higher proportion of the proximate carcinogen from BP than the other isozymes. Both P4501A1 and 1A2 were more selective for oxidation at the benzo ring of BP and showed a greater production of dihydrodiol metabolites than either P4503A4 or 3A5. The formation of diolepoxides from the individual enantiomers of BP-7,8-DHD occurred in a highly stereoselective fashion.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1993

Accumulation of the nuclear dioxin (Ah) receptor and transcriptional activation of the mouse Cyp1a-1 and Cyp1a-2 genes.

The treatment of C57BL/6 mice with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to the nuclear uptake of the arylhydrocarbon receptor (AhR) and transcriptional activation of Cyp1a-1 and Cyp1a-2 (S. T. Okino, et al., J. Biol. Chem. 267, 6991, 1992). In the present study, early nuclear uptake of the AhR and its role in transcriptional activation of the Cyp1 genes have been evaluated. After 30 min following a dose of TCDD to C57BL/6 mice, the AhR could be detected in liver nuclei. The effect of TCDD treatment within 30 min enhanced the transcriptional rate of the Cyp1a-2 gene to 70% of its maximal rate, with maximal levels of transcription occurring after 1 h. Early increases in 1a-2 mRNA were also observed by 30 min and increased to maximal levels by 12 h. In contrast, the levels of Cyp1a-1 transcription were 5 to 10% of maximal levels at 30 min, and gradually increased to maximal levels by 2 h. Concordant with the levels of transcription, 1a-1 mRNA was not detected until 1 h following TCDD treatment. While the AhR is responsible for transcriptional activation of the Cyp1a-1 gene, the concordant increase in the nuclear accumulation of the ligand-dependent AhR and Cyp1a-2 gene transcription suggests that the receptor plays an important role in the regulation of the Cyp1a-2 gene.

Animals↗
PubMed1993

Response of human CYP1-luciferase plasmids to 2,3,7,8-tetrachlorodibenzo-p-dioxin and polycyclic aromatic hydrocarbons.

The cytochrome P4501 gene family consists of two members, CYP1A1 and CYP1A2, that are induced by halogenated hydrocarbons and polycyclic aromatic hydrocarbons. The human CYP1 promoters and 5'-flanking sequences were cloned into luciferase expression vectors to develop cell lines that stably express luciferase activity in response to CYP1 gene induction. Plasmids were initially tested in transient transfection assays. Transient transfections resulted in high-level expression of luciferase by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) from both the CYP1 expression vectors, pLUC1A1 and pLUC1A2. In dose-response experiments, 10 nM TCDD caused a maximal induction of pLUC1A2-directed luciferase activity that was 10-fold over control. Maximal pLUC1A1-directed luciferase activity was 65-fold over control in cells treated with 10 nM TCDD. Stable integration of CYP1-luciferase-neo plasmids, pL1A1N and pL1A2N, into the human hepatoma cell line, HepG2, was achieved by selection with G418. G418-resistant colonies were isolated for both pL1A1N and pL1A2N plasmids. The pL1A2N transfectants showed basal-level luciferase activity, but were nonresponsive to treatment with 10 nM TCDD. These results are in contrast to the observed induction of pLUC1A2-mediated luciferase expression in transient transfection experiments. Stable integration of the human CYP1A2 gene sequences appears to silence the transcriptional activation by TCDD. The pL1A1N transfectants showed inducible luciferase activity and one cell line, referred to as 101L, was used to establish dose-response relationships for TCDD and various polycyclic aromatic hydrocarbons. Maximal induction occurred after treatment with 100 nM TCDD, 10 microM 3-methylcholanthrene, 50 microM benz[a]anthracene, and 50 microM benzo[a]pyrene. These studies illustrate the use of the CYP1A1-luciferase cell line for the study of structure-activity relationships.

Cell Line↗
PubMed1993

2,3,7,8-Tetrachlorodibenzo-p-dioxin induces the nuclear translocation of two XRE binding proteins in mice.

The administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene (3MC) to mice results in their binding to the ligand binding portion of the cytosolic dioxin-(Ah)-receptor, followed by translocation of the Ah receptor complex to the nucleus where the DNA binding form of the receptor can be measured by gel retardation analysis. In this report, extended electrophoresis of the nuclear DNA binding proteins isolated from liver demonstrate that TCDD and 3MC induce two nuclear DNA binding proteins in Ah-responsive C57BL/6 mice, while only TCDD induces these proteins in the Ah-nonresponsive DBA/2 mice. The two TCDD inducible (TI) nuclear DNA binding proteins, identified as TI-1 and TI-2, bind specifically to the Cypla-1 gene dioxin-(Ah)-receptor enhancer sequences (XREs) concordant with the properties of the Ah receptor. TI-1 is the predominant inducible form that is present in liver and extrahepatic tissues and most likely represents what is thought to be the Ah receptor, while TI-2 represents a minor form that is found only in liver. The nuclear induction of the Ah receptor by TCDD can be inhibited by phorbol esters such as TPA (Okino et al., 1992), but analysis of nuclear TI-1 and TI-2 shows that TPA can selectively inhibit the appearance of TI-1. The results of differential expression with regard to tissue and also inhibition by TPA suggests that TI-1 and TI-2 are under different modes of regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Nuclear uptake of the Ah (dioxin) receptor in response to omeprazole: transcriptional activation of the human CYP1A1 gene.

In the presence of halogenated and polycyclic aromatic hydrocarbons, the CYP1A1 gene is regulated through induction after ligand binding to the cytosolic Ah receptor (AhR). Ligand-dependent AhR activation leads to nuclear translocation and binding of the receptor to dioxin-responsive element (DRE) sequences, an event that initiates transcriptional activation of the CYP1A1 gene. We recently established a human hepatoma cell line stably integrated with the human CYP1A1 promoter and 5'-flanking enhancer sequences fused to the firefly luciferase gene. This cell line, 101L, was used to determine whether the induction of CYP1A1 by omeprazole, a gastric proton pump inhibitor, is AhR mediated. Treatment of 101L cells with either 50 microM omeprazole or 5 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin for 12-72 hr resulted in maximal activity at 24 hr for both inducers. A dose-response curve for omeprazole induction at 24 hr was determined and the EC50 for omeprazole induction of the human CYP1A1 gene was estimated to be 100 microM. The induction of the CYP1A1 gene by omeprazole corresponds to increases in CYP1A1 mRNA. To examine whether omeprazole-initiated transcriptional activation of the CYP1A1 gene correlates with nuclear accumulation of the AhR, binding of nuclear proteins to the DRE was examined. When gel mobility shift assays were performed using nuclear extracts isolated from 101L cells treated with omeprazole or 2,3,7,8-tetrachlorodibenzo-p-dioxin, specific binding of the AhR to the DRE was observed. These studies demonstrate that omeprazole initiates AhR activation and that induction of the human CYP1A1 gene by omeprazole is AhR dependent.

Base Sequence↗
PubMed1993

Specificity of substrate and inhibitor probes for human cytochromes P450 1A1 and 1A2.

Kinetic and inhibitor studies using cDNA-expressed enzymes and human liver microsomes have characterized the specificity of a range of cytochrome P450 (CYP) 1A substrate and inhibitor probes towards the two isoforms comprising this subfamily. Expressed CYP1A1 and CYP1A2 both catalyzed the O-deethylation of phenacetin, although the apparent Km was about 4-fold lower for CYP1A2 (25 vs. 108 microM). Phenacetin O-deethylation exhibited biphasic kinetics in human liver microsomes, and the apparent Km for the high-affinity component (9 +/- 6 microM) was consistent with the involvement of CYP1A2 in this reaction. The prototypic CYP1A xenobiotic inhibitor and substrate probes alpha-naphthoflavone, ellipticine, 7-ethoxycoumarin and 7-ethoxyresorufin all inhibited CYP1A1- and CYP1A2-mediated phenacetin O-deethylation as well as the high-affinity component of human liver phenacetin O-deethylase activity. alpha-Naphthoflavone and 7-ethoxycoumarin were, however, approximately 10-fold more potent as inhibitors of CYP1A2 than CYP1A1. Other putative human CYP1A xenobiotic substrates and inhibitors, including caffeine, 5- and 8-methoxypsoralen, nifedipine, paraxanthine, propranolol and theophylline similarly inhibited CYP1A1- and 1A2-catalyzed phenacetin O-deethylation and the high-affinity human liver phenacetin O-deethylase. In contrast, the monoclonal antibody MAb 1-7-1, raised against 3-methylcholanthrene-inducible rat cytochromes 450, almost abolished CYP1A1-mediated phenacetin O-deethylation, but had no effect on human liver microsomal- or CYP1A2-catalyzed phenacetin dealkylation. Together with previous data, the results indicate that the majority of human CYP1A xenobiotic inhibitor and substrate probes are nonspecific in their recognition of CYP1A1 and CYP1A2, although selectivity is apparent for some compounds.

Alkylation↗
PubMed1992

Characterization of the rabbit CYP1A1 and CYP1A2 genes: developmental and dioxin-inducible expression of rabbit liver P4501A1 and P4501A2.

In adult rabbits, the CYP1A1 and CYP1A2 genes are expressed constitutively. Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to elevations in both CYP1A1 and CYP1A2 gene products (S. T. Okino et al., 1985, Proc. Natl. Acad. Sci. USA 82, 5310-5314). In this report, we have characterized the rabbit CYP1A1 and CYP1A2 genes, and analyzed the pattern of expression of these genes in neonatal animals following exposure to TCDD. Genomic clones encoding the entire rabbit CYP1A1 and CYP1A2 genes were characterized. Restriction enzyme analysis and partial DNA sequence analysis identified the seven exons for the CYP1A1 and CYP1A2 genes. Primer extension analysis using mRNA from TCDD-treated neonatal rabbits helped confirm the start of transcription for the CYP1A genes. The length of the noncoding first exon of the CYP1A1 gene was 74 bases, compared to 90 and 88 bases for the human and rodent CYP1A1 genes. The length of the noncoding CYP1A2 gene first exon was 53 bases, similar to its counterpart in human and rodents. DNA sequence analysis of the 5' regulatory regions and comparison to the rodent and human CYP1 genes demonstrated that the rabbit CYP1A1 and CYP1A2 genes were most similar to their human orthologs. The 5' region of the CYP1A1 gene contained several consensus dioxin (Ah)-receptor responsive elements (XREs), while no functional XRE sequences were identified in the CYP1A2 gene. When expression of the two genes were monitored, a small amount of constitutive P4501A1 mRNA was detected in neonatal rabbits from the ages of 1 to 17 days, while P4501A2 mRNA levels could not be observed until 8-12 days postpartum. In response to TCDD treatment, P4501A1 mRNA levels were inducible at all neonatal time points, while P4501A2 mRNA levels could not be induced until the animals were 3-5 days postpartum. While the dioxin Ah-receptor most likely plays a major role in the induction of these genes by TCDD, early expression of the CYP1A1 and CYP1A2 genes is differentially regulated in a developmental fashion.

Aging↗
PubMed1992

Phorbol esters inhibit the dioxin receptor-mediated transcriptional activation of the mouse Cyp1a-1 and Cyp1a-2 genes by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Tetradecanoyl phorbol acetate (TPA) has been shown to inhibit 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced mouse P450IA1 benzo[a]pyrene hydroxylase activity (Raunio, H., and Pelkonen, O. (1983) Cancer Res. 43, 782-786). When we co-administered TPA and TCDD to C57BL/6 mice, the accumulation of TCDD-inducible liver P450IA1 and P450IA2 mRNA, as well as kidney P450IA1 mRNA, was greatly inhibited. When nuclear run-on assays were conducted, maximal levels of transcriptional activation were achieved for both liver Cyp1a-1 and Cyp1a-2 with 1 micrograms/kg (approximately equal to 3.0 nmol/kg) TCDD. TCDD elicited a dose-dependent increase in the rates of gene transcription, which paralleled the induction of P450IA1 and P450IA2 mRNA. Only Cyp1a-1 gene transcription was elevated in kidney. When these experiments were repeated following the co-administration of TPA with TCDD, the levels of TCDD-mediated transcriptional increases in liver Cyp1a-1 and Cyp1a-2 and P450IA1 and P450IA2 mRNAs were dramatically inhibited. The reduction in Cyp1a gene transcription by TPA could be accounted for by reduced DNA binding of the dioxin receptor to the xenobiotic-responsive element (XRE) sequences, as measured by gel-retardation analysis. Analysis of nuclear [3H]TCDD dioxin receptor by sucrose density gradients demonstrated that the inhibition of Cyp1a gene transcription and DNA binding by TPA resulted from a reduction in nuclear dioxin receptor concentration.

Animals↗
PubMed1992

Characterization of a rabbit factor IX cDNA.

A rabbit factor IX cDNA was isolated and characterized. The cDNA was 2,676 bp in length and contained the coding region for the leader peptide, the entire mature factor IX protein and a long 3' untranslated region. The deduced amino acid sequence shows a high degree of homology with the sequences of human factor IX and factor IX from other species. Northern blot analysis of liver RNA showed a single mRNA species of 2.8 kb for the rabbit factor IX.

Amino Acid Sequence↗