Debrisoquine polymorphism: novel CYP2D6 gene Bam HI restriction fragment length polymorphism in the Ngawbé Guaymí Indian of Panama.

Markedly decreased cytochrome P450-mediated metabolism of debrisoquine, sparteine, and more than two dozen additional commonly prescribed drugs is an autosomal recessive trait that has been associated with several RFLP patterns involving the CYP2D6 gene. In Caucasians there are at least six variant alleles known to be correlated with the 'poor metabolizer' (PM) phenotype. We examined debrisoquine and sparteine metabolism and CYP2D6 RFLP patterns in 22 Ngawbé Guaymí Indians of Panama. We studied a two-generation family, a three-generation family, and three other unrelated PM individuals. Digestion of all 22 DNA samples with Xba I or Hind III did not produce the same varying CYP2D6 RFLP patterns as those commonly seen in at least two-thirds of all Northern European Caucasians and Chinese so far screened. In contrast, we found a single heretofore undescribed Bam HI polymorphism that was correlated with the PM phenotype among all Ngawbé Guaymí individuals examined. It is possible that this novel RFLP might represent a recent founder effect that has occurred in this unadmixed Amerindian tribe within the past 20,000-30,000 years.

Human AH locus polymorphism and cancer: inducibility of CYP1A1 and other genes by combustion products and dioxin.

The polymorphism of mammalian aromatic hydrocarbon (Ah) responsiveness appears to be correlated with genetic differences in risk of bronchogenic carcinoma caused by cigarette smoking. The human polymorphism has been uncovered, largely as the result of corresponding genetic differences characterized first in the mouse. The murine Ah locus has been defined as the gene encoding the aromatic hydrocarbon-responsive (Ah) receptor, responsible for the inducibility of a battery of at least six genes, two of which encode P450 enzymes. The high-affinity receptor and, hence, more highly induced levels of P450, can result in greater concentrations of polycyclic aromatic reactive intermediates that form DNA adducts and, ultimately, mutation fixation (tumour initiation). The Ah receptor is also likely to participate in growth and differentiation signal transduction pathways (tumour promotion). Positive and negative control regions flanking the murine Cyp 1a-1 and human CYP1A1 (cytochrome P(1)450) genes have been identified. A DNA motif approximately 1 kb upstream of the transcription start site appears to affect the translatability of the CYP1A1 mRNA and activity of the enzyme. Expression of the CYP1A1 or CYP1A2 enzyme in mouse hepatoma Hepa-1 cells lacking endogenous CYP1A1 activity represses constitutive transcription of not only the endogenous Cyp1a-1 gene but other genes in the dioxin-inducible [Ah] battery. Human polymorphisms involving a Msp I site 450 bp downstream from the last CYP1A1 exon have been described in Japan, the Eastern Mediterranean, Norway and the USA. The '1.9 allele' is associated with an increased incidence of Kreyberg Type I bronchogenic carcinomas in Japan and has recently been correlated with a valine-to-isoleucine substitution at position 462 in the haeme-binding region. This allele is about 3 times more frequent in Japan than in Caucasians of Norway and the USA, in which no correlation has been found between this allele and lung cancer. More work is needed to clarify these findings. Isolation and sequencing of the human Ah receptor cDNA, and the subsequent screening of populations for polymorphisms, hold great promise for predicting interindividual risk of cancer caused by smoking and other environmental pollutants.

Isolation, sequence, and developmental expression of rat UGT2B2: the gene encoding a constitutive UDP glucuronosyltransferase that metabolizes etiocholanolone and androsterone.

The UDP glucuronosyltransferase gene UGT2B2 is constitutively expressed in rat liver, and the enzyme has been shown to conjugate glucuronic acid with various endogenous steroids, especially etiocholanolone and androsterone. We have cloned and sequenced much of the UGT2B2 gene and 5'-flanking (247 bp) and 3'-flanking (734 bp) regions. The gene contains six exons spanning about 15.3 kb. Translation begins at nucleotide 36 of exon 1 and terminates with 280 coding nucleotides into exon 6, encoding a protein of 530 amino acids (calculated Mr of the unmodified chain = 60,913). We have determined that the UGT2B2 full-length cDNA is 1,974 bp. Northern hybridization revealed that the hepatic UGT2B2 transcript is detectable 4 days before birth, becomes markedly elevated in the neonate, and is even further increased at 3 and 12 weeks of age in the liver of both male and female rats.

Murine mdr-1, mdr-2, and mdr-3 gene expression: no coinduction with the Cyp1a-1 and Nmo-1 genes in liver by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Multidrug-resistance (MDR) genes are induced in the liver of rodents treated with a variety of foreign chemicals and hepatocarcinogens. It has been reported that 2,3,6,7-tetrachlorodibenzo-p-dioxin (TCDD) might increase hepatic MDR transcripts in the Fischer rat and the C57BL/6 (B6) inbred mouse strain having the high-affinity aromatic hydrocarbon (Ah) receptor, but not in the DBA/2 (D2) strain having the low-affinity Ah receptor. These intriguing results suggest that TCDD might activate MDR gene expression by way of an Ah receptor-mediated signal transduction pathway. We have attempted to confirm these data in four inbred mouse strains: two (B6 and BALB/c) having the high-affinity Ah receptor, and two (D2 and AKR) having the low-affinity Ah receptor. The RNase protection assay was used to distinguish between the MDR1, MDR2, and MDR3 mRNAs. TCDD treatment at high (100 micrograms/kg) and low (1 mu/kg) doses, a time course from 6 to 96 hr of TCDD treatment, progeny from the B6D2F1 x D2 backcross, and transcriptional run-on experiments were performed. The Cyp1a-1 (cytochrome P1450) and Nmo-1 [NAD(P)H:menadione oxidoreductase] genes, two members of the TCDD-inducible [Ah] battery, were used as positive controls. We were unable to detect significant coinduction of MDR1, MDR2, or MDR3 mRNA with CYP1A1 mRNA or with Cyp1a-1 or Nmo-1 transcription under any conditions. Therefore, we conclude that any effects that TCDD might have on MDR expression must be substantially different from TCDD effects on genes known to be induced via the Ah receptor.

Human CYP1A1 gene: cosegregation of the enzyme inducibility phenotype and an RFLP.

The human CYP1A1 (cytochrome P1450) gene encodes an enzyme involved in the activation of procarcinogens, such as benzo[a]pyrene, to the ultimate reactive intermediate. Approximately 10% of the human population exhibit high CYP1A1 inducibility, and Kouri et al. reported that the high-inducibility phenotype might be at greater risk than low-inducibility individuals for cigarette smoke-induced bronchogenic carcinoma. In one 3-generation family of 15 individuals, we show here that the high-CYP1A1-inducibility phenotype segregates concordantly with an infrequent polymorphic site located 450 bases downstream from the CYP1A1 gene. Our findings are consistent with the study of Kawajiri et al., who demonstrated an association between this polymorphism and an increased incidence of squamous-cell lung cancer. Our data suggest that the CYP1A1 structural gene, or a region near this gene, might be correlated with the inducibility phenotype.

Cellular responses to oxidative stress: the [Ah] gene battery as a paradigm.

A major source of oxidative stress in animals is plant stress metabolites, also termed phytoalexins. The aromatic hydrocarbon-responsive [Ah] gene battery is considered here as a model system in which we can study metabolically coordinated enzymes that respond to phytoalexin-induced oxidative stress. In the mouse, the [Ah] battery comprises at least six genes: two Phase I genes, CYP1A1 and CYP1A2; and four Phase II genes, Nmo-1, Aldh-1, Ugt-1, and Gt-1. All six genes appear to be regulated positively by inducers such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and other ligands of the Ah receptor. In the absence of foreign inducer, the control of Nmo-1 gene expression is independent of the control of CYP1A1 and CYP1A2 gene expression. The radiation deletion homozygote c14CoS/c14CoS mouse is lacking about 1.1 centiMorgans of chromosome 7. Although having no detectable CYP1A1 or CYP1A2 activation, the untreated c14CoS/c14CoS mouse exhibits markedly elevated transcripts of the Nmo-1 gene and three growth arrest- and DNA damage-inducible (gadd) genes. These data suggest that the missing region on chromosome 7 in the c14CoS/c14CoS mouse contains a gene(s), which we propose to call Nmo-1n, encoding a trans-acting factor(s) that is a negative effector of the Nmo-1 and gadd genes. The three other [Ah] battery Phase II genes behave similarly to Nmo-1 in the c14CoS/c14CoS mouse. This coordinated response to oxidative stress and DNA damage, by way of the release of a mammalian battery of genes from negative control, bears an interesting resemblance to the SOS response in bacteria.

3' noncoding region of phosphoenolpyruvate carboxykinase mRNA contains a glucocorticoid-responsive mRNA-stabilizing element.

The stabilization of phosphoenolpyruvate carboxykinase mRNA by glucocorticoids appears to result from the interaction of an induced factor with an RNA element located in the 3' noncoding sequence of the mRNA. This element can confer glucocorticoid-dependent stabilization upon a heterologous mRNA, and thus strategies developed to investigate the control of mRNA transcription can now be applied to the analysis of hormone-regulated mRNA stabilization.

Marked increases in hepatic NAD(P)H:oxidoreductase gene transcription and mRNA levels correlated with a mouse chromosome 7 deletion.

The NAD(P)H:menadione oxidoreductase gene (Nmo-1) codes for a quinone reductase (also called DT diaphorase; EC 1.6.99.2) believed to play a central role in protection against oxidative stress. We have studied mice with a radiation-induced chromosomal deletion involving the albino locus (c) on chromosome 7 and found that Nmo-1 mRNA levels and the rate of Nmo-1 gene transcription are markedly increased (greater than 100-fold and greater than 12-fold, respectively) in the untreated c14CoS/c14CoS deletion homozygote, compared with the untreated Cch/Cch wild-type and the Cch/C14CoS heterozygote. These data suggest that a gene located on chromosome 7 encodes a trans-acting regulatory factor that might be a negative effector of the Nmo-1 gene, which we show here is located on chromosome 8 approximately 1.4 centimorgans (about 1000 kilobase pairs) from the Es-2 gene. Conversely, there are no detectable basal levels of cytochrome P1450 (Cyp1a1 gene) or cytochrome P3450 (Cyp1a2 gene) mRNA, indicating that the regulation of basal expression of the Cyp1a1 and Cyp1a2 genes is distinct from that of the Nmo-1 gene. Moreover, the Cyp1a1 and Cyp1a2 genes and the Nmo-1 gene are induced by tetrachlorodibenzo-p-dioxin in the cch/cch, cch/c14CoS, and c14CoS/c14CoS mice. The mechanism of tetrachlorodibenzo-p-dioxin inducibility of the Cyp1a1, Cyp1a2, and Nmo-1 genes is, therefore, independent of the mechanism of Nmo-1 gene activation in untreated c14CoS/c14CoS mice.

Location and characterization of two widely separated glucocorticoid response elements in the phosphoenolpyruvate carboxykinase gene.

Chimeric genes were constructed by fusion of various regions of the 5'-flanking sequence from the phosphoenolpyruvate carboxykinase (GTP) (PEPCK) gene to the chloramphenicol acetyltransferase-coding sequence and to simian virus 40 splice and polyadenylation sequences. These were used to demonstrate that two glucocorticoid regulatory elements (GREs) combine to confer glucocorticoid responsiveness upon the PEPCK gene in H4IIE hepatoma cells. Both elements, a distal one whose 5' boundary is located between -1264 and -1111 base pairs and a proximal one located between -468 and -420 base pairs relative to the transcription initiation site, act independently, in various positions and orientations, and upon the heterologous thymidine kinase promoter. Each element accounts for half of the maximal response of the chimeric genes. Therefore, two widely separated enhancerlike elements contribute equally to the response of the PEPCK gene to glucocorticoid hormones. Neither of the PEPCK GREs contains the TGTTCT consensus sequence associated with most other GREs.

Multihormonal regulation of phosphoenolpyruvate carboxykinase gene transcription. The dominant role of insulin.

We used a nuclear RNA transcript elongation assay to show that cAMP analogs and dexamethasone cause a selective increase of transcription of the P-enolpyruvate carboxykinase gene in H4IIE hepatoma cells. 8-(4-chlorophenylthio)-cAMP increased transcription within 5 min and the maximal rate, generally 10-15-fold above the basal rate, was attained by 30 min. This increase was of sufficient magnitude to account for the effect on mRNAPEPCK (for example, where PEPCK is phosphoenolpyruvate carboxykinase) accumulation. After the initial increase, and with continued presence of cAMP, transcription of this gene declined to a new steady-state level which was 2-3 times the basal value. The effect of cAMP analogs on P-enolpyruvate carboxykinase gene transcription was obtained in the absence of protein synthesis. This, and the rapidity of the response, indicates that the effect of cAMP is exerted directly on the P-enolpyruvate carboxykinase gene. Dexamethasone results in a specific, 6-fold increase of transcription, sufficient to account for the increase of mRNAPEPCK which follows treatment of H4IIE cells with this glucocorticoid. When 1 nM insulin was added to either untreated H4IIE cells, or cells first treated with a cAMP analog or dexamethasone, there was a marked reduction of cytoplasmic mRNAPEPCK. The inhibitory effect of insulin was readily reversible, as cells regained the basal level of mRNAPEPCK and full responsiveness to cAMP within 1 h after removing insulin. The transcript elongation assay was used to show that insulin inhibits transcription of the gene coding for mRNAPEPCK. The concentration of insulin required for 50% inhibition was 2-5 pM, whereas approximately 200 pM of proinsulin was required to achieve the same inhibition of transcription. This effect was specific, since insulin did not affect the synthesis of total RNA; it was rapid, as 5 nM insulin decreased the rate of P-enolpyruvate carboxykinase gene transcription by 50% within 15 min; and it also does not require ongoing protein synthesis. The magnitude and kinetics of the response suggest that the primary action of insulin in the regulation of P-enolpyruvate carboxykinase synthesis is exerted at the level of mRNAPEPCK transcription. The insulin-mediated inhibition of mRNAPEPCK transcription was noted in untreated cells and in cells first treated with 8-(4-chlorophenylthio)-cAMP, dexamethasone, or both of these agents. Hence, among these compounds, insulin is the dominant regulatory molecule.