A duplicated HNF-3 binding site in the CYP2H2 promoter underlies the weak phenobarbital induction response.

We are investigating induction of chicken cytochrome P450 genes by the sedative phenobarbital in chick embryo hepatocytes. The steady-state level of induced mRNA for the gene CYP2H1 is about 10-fold higher than that of a second gene, CYP2H2. Here, we show that a difference in drug-responsive enhancer activity does not underlie the differential response of these genes to phenobarbital since upstream enhancer regions are identical in these genes. The first 198 bp of CYP2H2 promoter sequence is identical to the CYP2H1 gene promoter, except that the functional HNF-3 binding site in the CYP2H1 promoter is replaced with a duplicated HNF-3 sequence in the CYP2H2 promoter. Transient expression analysis established that the promoter activity of the CYP2H2 gene was about ninefold lower than the CYP2H1 gene. Mutagenesis of either of the partially overlapping HNF-3 sites in the CYP2H2 gene substantially induced drug induction. Gel-shift analysis established that each of these HNF-3 sites bound HNF-3, most likely HNF-3beta. In-vitro footprint analysis demonstrated that all the identified sites in the CYP2H2 promoter bound protein except the duplicated HNF-3 region. However, protein binding was observed by in-vitro footprint analysis if either of the HNF-3 sites was mutated in the CYP2H2 promoter. Hence, duplication of the HNF-3 site in the CYP2H2 promoter does not allow binding of HNF-3 in the promoter context and may be predominantly, if not exclusively, responsible for the poor response of the CYP2H2 gene to phenobarbital.

The antiglucocorticoid RU486 inhibits phenobarbital induction of the chicken CYP2H1 gene in primary hepatocytes.

The cytochrome P450 gene CYP2H1 is highly induced by phenobarbital in chick embryo hepatocytes. Recent studies have established that the orphan nuclear receptor CAR plays a critical role in the induction mechanism. Here, we show that a high concentration of the potent glucocorticoid and progesterone receptor antagonist RU486 almost completely blocks phenobarbital-induced accumulation of CYP2H1 mRNA in hepatocytes yet has no effect on basal expression. In marked contrast, CYP2H1 mRNA induced by the phenobarbital-type inducers glutethimide and 2-allylisopropylacetamide is not affected by RU486. RU486 inhibition is not mediated through the glucocorticoid or progesterone receptors. Transient transfection studies showed that RU486 does not repress through activation of the orphan receptor PXR and subsequent competition with CAR for binding to the upstream drug-responsive 556-base-pair enhancer. Additionally, none of the known functional transcription factor binding sites found in the enhancer region was a target of RU486 inhibition. Using an artificial construct containing multiple CAR binding sites, we also established that RU486 has no direct effect on the activity of exogenously expressed CAR. There is no evidence that phenobarbital binds to CAR; we propose that RU486 inhibits phenobarbital induction, either by interfering with a phenobarbital-dependent mechanism responsible for nuclear import of CAR or with the metabolism of phenobarbital to the true inducer. Whether a novel nuclear receptor that binds RU486 at high concentrations plays a role in the inhibitory action of RU486 is an interesting possibility.

The prothrombin gene is expressed in the rat kidney: Implications for urolithiasis research.

There is considerable interest in determining the role of prothrombin fragments, especially urinary prothrombin fragment 1 (UPTF1), in the pathogenesis of calcium oxalate (CaOx) urinary calculi. This fragment is present in abundance in the matrix of CaOx crystals generated in human urine in vitro and has also been detected in human urinary stones containing calcium. More recently, prothrombin gene expression has been reported in the human kidney. However, studies examining the renal biosynthesis of prothrombin or perhaps only its fragments during experimental lithogenesis, and in consequence, the role of UPTF1 in stone formation, cannot be carried out in humans. The aim of this investigation therefore was to determine whether prothrombin gene expression is present in the rat kidney. Total RNA was isolated from the kidneys and livers of 12 rats. Using reverse transcriptase PCR, mRNAs corresponding to the thrombin and fragment 1 + 2 (F1+2) regions of prothrombin were analysed by agarose gel electrophoresis. The expression of glyceraldehyde 3-phosphate dehydrogenase was also examined to determine whether the quality of the tissue mRNAs was adequate for analyses. The amplified products were identified by sequence analysis. All kidneys displayed evidence of expression of the thrombin and F1+2 domains of the prothrombin gene. Furthermore, the sequences of these PCR-derived products from kidney were identical to those from liver. This suggests that the prothrombins secreted by these two organs are identical. The fact that prothrombin biosynthesis occurs in both the human and rat kidney presents an opportunity for using established rat models of stone disease to evaluate the influence of lithogenic conditions on prothrombin gene expression, and the potential role of UPTF1 in vivo.

Analysis of a phenobarbital-responsive enhancer sequence located in the 5' flanking region of the chicken CYP2H1 gene: identification and characterization of functional protein-binding sites.

We previously identified in the chicken CYP2H1 gene an upstream enhancer domain (-5900/-1100) that responds to phenobarbital. Deletion and restriction enzyme analyses of this domain have now identified two separate enhancer regions that respond to phenobarbital (from -5900 to -4550 and from -1956 to -1400). We have focused here on the latter and in particular a resident 240-base pair (bp) restriction enzyme fragment that retains drug responsiveness. Using deletion analysis and in vitro DNase I footprinting, transcription factor binding sites have been located in the 240-bp fragment. The sites identified are an E-box-like element, a consensus hepatocyte nuclear factor 1 site, a CCAAT box motif, and a novel site. Mutagenesis demonstrated that each site contributed to enhancer activity, although there was a weaker contribution from the CCAAT box, and that no individual site was critical for responsiveness. In keeping with the tissue-restricted expression of the CYP2H1 gene, gel shift experiments established that the proteins binding to these enhancer sites are enriched in chicken liver, kidney, and small intestine. In vitro footprint experiments showed a stronger protection with liver nuclear extracts from drug-treated chickens compared with control extracts on the E-box-like element, the CCAAT box motif, and the novel binding site; however, the basis for this apparent increase in binding remains to be determined. The proteins binding to the 240-bp fragment are different from those recently reported to be required for the activity of the phenobarbital responsive enhancer domains of rodent CYP2 genes.

Transcriptional activation of cytochrome P450 genes by different classes of chemical inducers.

1. We review here the molecular mechanisms underlying the xenobiotic induction of genes encoding cytochrome P450 (CYP) enzymes in the liver and other tissues. We will focus on four major families of CYP genes. 2. Members of the CYP1 gene family are induced by polycyclic aromatic hydrocarbons and this process is mediated by the basic helix-loop-helix proteins: the Ah receptor and its heterodimeric partner Arnt. Considerable progress has been made in elucidating the molecular details of this induction process. 3. CYP4 genes are activated by peroxisomal proliferators, a group of structurally diverse chemicals that also induce peroxisome proliferation. The transcriptional response is dependent on the peroxisome proliferator-activated receptor and its partner RXR, both members of the nuclear receptor superfamily; their role in the induction process has been well characterized at the molecular level. 4. In contrast, the mechanism of gene induction of CYP2 genes by phenobarbital and other structurally diverse inducers is not well understood and a specific phenobarbital-responsive receptor has not been identified. 5. Induction of the CYP3 gene family by the glucocorticoid dexamethasone appears to involve the glucocorticoid receptor, but this receptor is not apparently required for induction by metapyrone and a complete molecular understanding of the induction processes is lacking at present.

Liver-enriched transcription factors, HNF-1, HNF-3, and C/EBP, are major contributors to the strong activity of the chicken CYP2H1 promoter in chick embryo hepatocytes.

Chicken CYP2H1 promoter constructs express strongly in chick embryo hepatocytes at a level comparable with that of Rous sarcoma viral promoter. We have identified the transcription factors responsible for the active CYP2H1 promoter. Binding sites for transcription factors were located within the first 160 bp of promoter sequence using promoter deletion experiments and DNase I footprint analysis. Sequence analysis revealed characteristic sites for the liver-enriched transcription factors of the HNF-1, HNF-3, and C/EBP families and for the ubiquitous factor, USF. Protein binding to these sites was established by gel mobility shift assays. Mutagenesis and transient transfection experiments demonstrated that these sites, in combination, were responsible for the strong promoter activity with a substantial contribution from HNF-1 and HNF-3. The promoter was also active in mammalian HepG2 and COS-1 cell lines where expression was dependent on the identified transcription factor binding sites but promoter activity in the HeLa cells was low. Transactivation experiments revealed that promoter expression could be activated through the appropriate binding sites by exogenously expressed rat HNF-1alpha or HNF-1beta, rat HNF-3alpha or HNF-3beta and chicken C/EBP alpha. Transcriptional synergism between HNF-1 and C/EBP was observed in these transactivation experiments. A Barbie box-like sequence overlapped the USF element but was not functional. The results demonstrate that liver-enriched transcription factors and USF direct strong expression of the CYP2H1 promoter in transiently transfected cells. By comparison, in vivo expression of this gene in uninduced chick embryo hepatocytes is low but markedly increased by phenobarbital. Drug induction may therefore substantially reflect derepression of this inherently active promoter.

Phenobarbital-induced activation of CYP2H1 and 5-aminolevulinate synthase genes in chick embryo hepatocytes is blocked by an inhibitor of protein phosphorylation.

The phenobarbital-induced activation of cytochrome P4502H1 (CYP2H1) and 5-aminolevulinate synthase (ALAS-1) genes in chick embryo hepatocytes occurs at the level of gene transcription, but the molecular mechanism underlying this induction is not understood in detail. In the present study, we report that the protein kinase inhibitor 2-aminopurine markedly inhibits the phenobarbital-induced activation of CYP2H1 and ALAS-1 genes as measured by Northern blot analysis, but does not alter the basal expression of these genes in the absence of drug. Transient expression studies confirmed these findings. The construct pCATBg4.8 contains a 4.8-kb drug-responsive domain of the CYP2H1 gene fused to the enhancerless SV40 promoter and the drug-induced expression of this construct in chick embryo hepatocytes was inhibited by 2-aminopurine. Another construct pCAT, with the first 547 bp of 5' flanking region of the CYP2H1 gene, is not responsive to drug and basal expression of this construct was not altered by the addition of 2-aminopurine. The evidence presented here demonstrates that the inhibitory action of 2-aminopurine on drug-induction is not due to a toxic effect on the cells. The induction of the CYP2H1 gene by phenobarbital was not altered by treating cells with the specific inhibitors for protein kinase C (GF 109203X and Ro 31-8220) or prolonged exposure to 12-O-tetradecanoyl-phorbol 13-acetate (TPA) or treatment with the specific inhibitors for tyrosine kinase (genistein and tyrphostin A25). Overall, the data indicate that a 2-amino-purine-sensitive protein kinase activity is required for the phenobarbital-induction mechanism but this is unlikely to be a protein kinase C or tyrosine kinase. It can be postulated that phosphorylation of a drug receptor protein may be an important step in the drug-induction process.

Superinduction by cycloheximide of cytochrome P4502H1 and 5-aminolevulinate synthase gene transcription in chick embryo liver.

The present study examines the effect of inhibiting protein synthesis with cycloheximide on the induction of the genes for cytochrome P4502H1 (CYP2H1) and 5-aminolevulinate synthase (ALAS) in phenobarbital-treated chick embryo livers. Phenobarbital administration caused a 10- to 15-fold increase in the levels of mRNAs for both CYP2H1 and ALAS. Cycloheximide treatment alone also induced the levels of mRNA for CYP2H1 and ALAS by 7- and 3-fold, respectively, but in combination, cycloheximide and phenobarbital elicited an additional effect resulting in a 33- and 40-fold increase, respectively. To investigate whether these effects were due to transcriptional activation or a post-transcriptional mechanism, nuclear transcription run-on experiments were conducted. The observed changes in mRNA levels for CYP2H1 and ALAS were shown to be predominantly due to changes in the rate of transcription of the respective genes. These findings establish that drug induction of the CYP2H1 and ALAS genes can proceed in the almost complete absence of protein synthesis and also imply that a labile repressor protein may be involved in modulating expression of these genes. In addition, these results indicate that drug induction of ALAS does not require concomitant synthesis of P450 apoprotein.

Prevalence of Campylobacter infections in animals and children in Haryana, India.

To study the prevalence of infections with Campylobacter spp in Haryana, India, a stool sample was collected using a rectal swab from 30 buffaloes, 62 cattle, 95 pigs and 94 children and was bacteriologically cultured. The subjects were either apparently healthy or had diarrhoea. The organisms were isolated in a medium containing reducing agents and antibiotics, and culture plates were incubated in a candle jar at 42 degrees C. 63% of all thermophilic Campylobacter were cultured from rectal swabs taken from young livestock and children with diarrhoea. Of 32 isolates fully identified, 23 were C. jejuni, 8 were C. coli and 1 was C. laridis. The C. jejuni isolates belonged to the Lior's biotype II.

Vitamin K1 amplification of benzo(a)pyrene metabolism in chick embryos.

Seventeen-day-old chick embryos were used as a test system to assess the effect of vitamin K1(K1) on benzo(a)pyrene (BP) metabolism as measured by the induction of arylhydrocarbon hydroxylase (AHH) and cytochrome P-450 and the levels of glutathione (GSH) and glutathione S-transferase (GST) in liver. Twenty-four hours after injection of BP into the air sac there was a sharp rise in AHH and P-450 and a drop in GSH. When K1 was injected 24 hr prior to BP there was a decrease in GST activity as compared with the control plus an augmented increase in AHH induction. This augmentation in BP metabolism (Phase I) together with a concomitant decrease in at least one mechanism of Phase II conjugation is in keeping with other evidence that K1 can play an adjuvant role in BP induced mutagenicity and carcinogenicity. Ubiquinone has a much lesser effect on BP metabolism than does K1 in equimolar concentration.

Effect of vitamin A deficiency on pulmonary and hepatic in vitro metabolism of benzo(a)pyrene in rat.

The metabolism of (3H)-benzo(a)pyrene and the activities of enzymes involved in its metabolism were studied in rat lung and liver in vitamin A deficiency. Deficiency of vitamin A resulted a significant decrease in the overall metabolism of benzo(a)pyrene in the liver in vitro, whereas no significant difference was evident in the lung. The ethyl acetate-soluble metabolites of benzo(a)pyrene formed by lung and liver preparations were unaltered qualitatively by vitamin A deficiency. However, quantitative analysis revealed that vitamin A deficiency decreased the yield of dihydrodiols, quinones and phenols in liver, and dihydrodiols in lung. The hepatic cytochrome P-450 content, arylhydrocarbon hydroxylase and uridine diphosphate-glucuronosyl transferase activities were reduced, whereas glutathione S-transferase activity was increased in the vitamin A deficient animals. Contrary to this, pulmonary cytochrome P-450 content was above the control values (p less than 0.01) and no alteration in pulmonary arylhydrocarbon hydroxylase activity was observed in vitamin A deficient rats. Uridine diphosphate-glucuronosyltransferase and glutathione S-transferase activities were impaired in lung by inducing vitamin A deficiency. However, no significant difference was evident in the overall metabolism of benzo(a)pyrene by lung supernatants from the two groups.

The effect of vitamin A deficiency on the initiation and postinitiation phases of benzo(a)pyrene-induced lung tumourigenesis in rats.

The present investigation shows the effect of vitamin A deficiency on the initiation and postinitiation phases of benzo(a)pyrene-induced lung carcinogenesis in male Wistar rats. Lung tumours were induced by giving three intratracheal instillations, one week apart, of 10 mg benzo(a)pyrene per instillation. Maximum tumour incidence (100%) and tumour burden per rat was found in rats which were kept on vitamin A deficient diet for 4 weeks prior to the first administration and 8 weeks after the last administration of benzo(a)pyrene. Rats in which vitamin A deficiency was terminated after the last administration of the carcinogen had 83% tumour incidence, whereas corresponding control pairfed animals had 39% incidence of tumours. These data represent the values obtained 32 weeks after the last administered dose of the carcinogen and indicate the role of vitamin A, both in the initiation as well as in the postinitiation phases of lung carcinogenesis.

The effect of medroxyprogesterone acetate on the hepatic drug-metabolizing enzymes in normal and protein-deficient female rats.

Effect of depot medroxyprogesterone acetate on the hepatic drug-metabolizing enzymes was studied in female protein-deficient and normal pair-fed rats. Treatment with this drug did not cause any change in organ weight, microsomal protein, and soluble protein yield per gram of tissue in both groups. MPA administration resulted in significant increases in the content of cytochrome P-450 and b5, and activities of benzo[a]pyrene hydroxylase, UDP-glucuronosyltransferase, and NADPH-Cyt c reductase in both pair-fed control and protein-deficient rats. However, the content of glutathione and activity of glutathione-S-transferase were not affected appreciably. The present study suggests that MPA treatment induces drug-metabolizing enzymes in liver to almost the same extent in both protein-deficient and normal pair-fed rats.

Effect of intratracheally instilled benzo(a)pyrene on the pulmonary and hepatic drug-metabolizing enzymes in normal and vitamin A deficient rats.

The effect of intratracheal instillation of different doses of benzo(a)pyrene (0.1, 1.0 and 2.0 mg) on the drug metabolizing enzymes of lung and liver was analysed in rats fed diet with or without vitamin A for 5-6 weeks. Benzo(a)pyrene exposure at 2.0 mg dose only elevated the level of cytochrome P-450 and b5, and activity of benzopyrene hydroxylase in liver, and extent of increase was similar in normal and vitamin A deficient groups. Contrary to this, pulmonary contents of cytochrome P-450 and b5, and benzopyrene hydroxylase activity increased over control values in both the groups even at lower doses of benzo(a)pyrene. Moreover, their values were higher in vitamin A deficient-treated groups compared to normal-treated controls. Increase in these parameters was greater in lung as compared to increase in liver. NADPH cytochrome C-reductase in lung and liver was not affected either by inducing vitamin A deficiency or exposing these rats further to benzo(a)pyrene. Uridine-diphospho-glucuronosyl-transferase (UDP-GT) activity in normal and vitamin A deficient groups was enhanced following exposure to benzo(a)pyrene both in lung and liver. However, activity of this enzyme remained impaired in vitamin A deficient groups, benzo(a)pyrene exposed or not exposed when compared to respective normal controls. Glutathione S-transferase activity remained unchanged following exposure to benzo(a)pyrene both in lung and liver. The apparent increase in hepatic glutathione S-transferase and decrease in pulmonary glutathione S-transferase activity in vitamin A deficiency was only due to vitamin A deficient status of rats with no further effect of benzo(a)pyrene.