Genotype and allele frequencies of polymorphic cytochromes P450 CYP1A2 and CYP2E1 in Mexicans

CYP1A2 and CYP2E1 are two of the main cytochrome P450 isoforms involved in the metabolism of commonly used drugs and xenobiotic compounds considered to be responsible for or possible participants in the development of several human diseases. Individual susceptibility to developing these pathologies relies, among other factors, on genetic polymorphism which depends on ethnic differences, as the frequency of mutant genotypes varies in different human populations. Thus the aim of this study was to investigate the frequency of CYP1A2 5'-flanking region and CYP2E1 Rsa I/Pst I polymorphisms in Mexicans by PCR-RFLP methods. The DNA of 159 subjects was analysed and mutant allele frequencies of 30% for CYP2E1 Rsa I/Pst I sites and 43% for CYP1A2 5'-flanking region were found. These frequencies are higher than those previously reported for other human populations.     

High frequency of CYP1A1*2C allele in Brazilian populations

The genetic variability of the CYP1A1 I462V polymorphism (CYP1A1*2C) was investigated in four Brazilian populations: three groups of African descent and one group of European descent. The CYP1A1 polymorphism was analyzed by two different procedures, first by the allele-specific polymerase chain reaction (PCR) method and then by the PCR-restricted fragment length polymorphism (PCR-RFLP) method before digestion with BsrDI. The frequency of CYP1A1 *2C was 11% in Brazilians of European descent, a frequency that is slightly higher but not statistically different from that observed in European populations. In Brazilians of African ancestry this value was very high (12% to 15%). This allele was not observed in the only two African populations investigated thus far. By themselves, the two factors of interethnic admixture (with populations of European descent and/or Amerindian populations) and genetic drift cannot explain the high values observed here. Our findings suggest that the CYP1A1 *2C allele may possibly be present in Africa, but restricted to some ethnic groups not yet investigated. Environmental factors in South America might also have acted as selective factors increasing the CYP1A1 *2C gene frequency. Our data also suggest that the CYP1A1 *2C allele might possibly have originated in Africa.     

Molecular analysis of the ADH1-C m allele of maize

The Adh1-C ^mallele and each gene in the Adh1-FC ^mduplication have been cloned and restriction-mapped. Of the C ^mallele 6 kb was sequenced. A single amino acid substitution of aspartate for tyrosine at residue 52 accounts for the altered enzymatic properties of the C ^mprotein. Comparison of the nucleotide sequence to that of Adh1-1F and Adh1-1S shows structural and restriction site polymorphisms in the 3 flanking DNA. C ^mlacks the insertion sequence present in 1F and 1S and contains a complex sequence composed of two direct repeats and an inverted repeat. The two genes of the duplication allele have similar restriction maps to C ^mand each other.     

Polymorphism of CYP1A1 and CYP2D6 genes in tundra nentses and European populations of western Siberia

Data on the first examination of the CYP1A1 and CYP2D6 genes' polymorphism in the populations of Tundra Nentsis (Yamalo-Nenetskii Autonomous District) and migrant population of Western Siberia (Novosibirsk oblast and Altaiskii krai) are presented. The frequency of the 2D6*4 mutant allele in Tundra Nentsis, characterized by a two-component Caucasoid and Mongoloid origin, was shown to be intermediate in Caucasoid and Mongoloid populations. The frequencies of the 2D6*4 and 1A1Val* mutant alleles across migrant inhabitants of Western Siberia (Caucasoid populations) were similar to that reported for the Caucasoid populations overall. Distribution of the CYP1A1 genotypes (Ile/Ile, Ile/Val*, and Val*/Val*) in Tundra Nentsis was similar to that found in Mongoloid groups. However, the frequency of the 1A1Val* allele in Tundra Nentsis was 1.5 times higher than that in the Southern Mongoloid populations (Chinese, Koreans, and Japanese).     

A Genetic Polymorphism in Coumarin 7-Hydroxylation: Sequence of the Human CYP2A Gnes and Identification of Variant CYP2A6 Alleles

A group of human cytochrome P450 genes encompassing the CYP2A, CYP2B, and CYP2F subfamilies were cloned and assembled into a 350-kb contig localized on the long arm of chromosome 19. Three complete CYP2A genes—CYP2A6, CYP2A7, and CYP2A13—plus two pseudogenes truncated after exon 5, were identified and sequenced. A variant CYP2A6 allele that differed from the corresponding CYP2A6 and CYP2A7 cDNAs previously sequenced was found and was designated CYP2A6ν2. Sequence differences in the CYP2A6ν2 gene are restricted to regions encompassing exons 3, 6, and 8, which bear sequence relatedness with the corresponding exons of the CYP2A7 gene, located downstream and centromeric of CYP2A6ν2, suggesting recent gene-conversion events. The sequencing of all the CYP2A genes allowed the design of a PCR diagnostic test for the normal CYP2A6 allele, the CYP2A6ν2 allele, and a variant—designated CYP2A6ν1—that encodes an enzyme with a single inactivating amino acid change. These variant alleles were found in individuals who were deficient in their ability to metabolize the CYP2A6 probe drug coumarin. The allelic frequencies of CYP2A6ν1 and CYP2A6ν2 differed significantly between Caucasian, Asian, and African-American populations. These studies establish the existence of a new cytochrome P450 genetic polymorphism.     

CYP1A2 polymorphism (C > A at position -163) in Ovambos, Koreans and Mongolians

Cytochrome P450 1A2 (CYP1A2) plays an important role in metabolizing drugs and xenobiotics, and is a possible participant in the development of several human diseases. Recent studies have shown that genetic polymorphism of -163 C > A single nucleotide mutation of CYP1A2 increases the risk of myocardial infarction and modulates CYP1A2 activity. In this study, we investigated the frequency of the -163 C > A mutation in Ovambos (n = 177), Koreans (n = 250) and Mongolians (n = 153) and compared our results with other studies. Detection of this single nucleotide polymorphism was by polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP). The frequencies of mutation (CYP1A2*-163A) in the Ovambos, Koreans and Mongolians were 0.46, 0.32 and 0.21, respectively. Ovambos showed a relatively higher frequency of mutation, similar to that of Tanzanians, while the Mongolians showed the lowest frequency of all study groups, including those from previous studies. This study is the first to investigate the distribution of the CYP1A2 (-163 C > A single nucleotide polymorphism) mutant allele in Ovambo, Korean and Mongolian populations.     

A new allele at the Pgd locus in pigs

A new electrophoretic variant of porcine 6-phosphogluconate dehydrogenase (PGD) is described. The new variant, PGD C, has been shown to be controlled by a third allele, PgdC, at the Pgd locus.     

Polymorphism in Pg-1 (urinary pepsinogen-1) locus in the rat and its linkage analysis

Cramer (1981) reported that Pg-1 (urinary pepsinogen) in the rat was loosely linked with albinism in the linkage group I. We performed a three point test on the loci of pg-1, c, and Hbb. We could reconfirm that pg-1 was autosomal trait with two co-dominant alleles of pg-1a and pg-1b. But in progeny of ((WF X IS) X WF) backcross, pg-1 was linked to neither c nor Hbb, while a close linkage between c and Hbb was detected. Also pg-1 was not linked to Mup-1 (LG II), a (LG IV), Es-3 (LG V), and h (LG VI). pg-1 will be one of the most valuable genetic markers of the rats, since pg-1 was highly polymorphic among inbred strains of rats, and not linked to LG I, II, IV, V, and VI.     

A slow selection analysis of two locus, two allele traits.

A deterministic, continuous time model for the dynamics of two locus, two allele Mendelian traits in a large randomly mating diploid population is derived. The model allows for frequency and time dependent birth and death rates. It is analyzed under the assumption that the selective forces acting in the population are small. Slow selection approximations to the system's solution are then constructed. Two particular cases are studied. First, when linkage between loci is tight, the population is shown to rapidly approach Hardy-Weinberg proportions, which then may vary on a (slow) time scale determined by differential fitness. In the case of constant birth and death rates, a measure of the population's fitness is shown to increase on the slow time scale after an initial rapid adjustment. The second case considered is for loose linkage; a population near linkage equilibrium is studied. It is shown that the epistatic parameters cancel and that the results agree with the tight linkage case to leading order. The linkage disequlibrium is described in both cases.     

Cloning and characterization of a novel CYP3A1 allelic variant: analysis of CYP3A1 and CYP3A2 sex-hormone-dependent expression reveals that the CYP3A2 gene is regulated by testosterone.

A clone was isolated from a cDNA library constructed from phenobarbital-treated Wistar rat liver and proven to correspond to the full-length mRNA of a polymorphic variant of Sprague-Dawley CYP3A1. Eight nucleotide differences were detected in a single 76-nucleotide stretch and confirmed to be present in the genomic clone. They are seated in a region implicated in the definition of a substrate binding domain of the native P450. Three out of the eight nucleotide changes are nonconservative, implicating the replacement of Thr/Ala 207, Phe/Ile 213, and Ile/Val 232. This is the first report of an allelic variant of CYP3A1, a new example of interstrain P450 variability. The CYP3A subfamily is composed of several genes coding for active testosterone 6 beta-hydroxylases which are expressed in the liver. CYP3A genes are under strong and distinct developmental regulation. Conversely to CYP3A1, transiently expressed in immature animals, CYP3A2 is constitutively expressed in the liver early after birth and characterized by an extinction in the adult females. Castration of 90-day-old male rats causes a drastic reduction (80%) of CYP3A2 mRNA relative abundance. Administration of testosterone propionate restores the physiological levels of CYP3A2 mRNA characteristic of the male rat liver. Our results demonstrate the existence of a direct relationship between the male hormonal status and the constitutive expression of rat liver CYP3A2.     

CYP2D6 and CYP1A1 mutations in the Turkish population

Drugs and carcinogens are substrates of a group of metabolic enzymes including cytochrome p450 enzymes and gluthatione S-transferases. Many of the genes encoding these enzymes exhibit functional polymorphisms that contribute individual cancer susceptibility and drug response. Molecular studies based on these polymorphic enzymes also explain the aetiology of cancer and therapeutic management in clinics. We analysed the cytochrome p4501A1 (CYP1A1) and 2D6 (CYP2D6) variant genotype and allele frequencies by PCR-RFLP in Turkish individuals (n=140). The frequency of the CYP1A1*2A mutant allele was found to be 15.4%, and the CYP2D6*3 and *4 mutant allele (poor metabolizer) frequencies were 2.5% and 13.9%, respectively. This study presents the first results of CYP1A1 and CYP2D6 mutant allele distributions in the Turkish population and these data provide an understanding of epidemiological studies that correlate therapeutic approaches and aetiology of several types of malignancy in Turkish patients.     

Age-related changes in the mRNA levels of CYP1A1, CYP2B1/2 and CYP3A1 isoforms in rat small intestine

It has been established beyond doubt that, as well as the liver, the small intestine is an important site of first-pass metabolism of numerous drugs, food components and toxic xenobiotics. However, there is not much information available about age-dependent changes of intestinal biotransformation pathways. In the present paper, we evaluated the relationships between intestinal cytochrome P450 complex activity and the age of animals. The study was carried out on male Sprague–Dawley rats (n = 5) from 5 age series: 0.5-, 2-, 4-, 20-, and 28 months old. Animals at every age series were divided into 4 groups: control and three groups of rats treated with the CYP450 specific inducers: phenobarbital, β-naphtoflavone and dexamethasone, respectively. RNA was isolated from intestinal mucosa, and then standard RT-PCR was used for the analysis of CYP1A1, CYP2B1/2 and CYP3A1 mRNA expression. Additionally, the activities of NADPH-cytochrome P450 and NADH-cytochrome b₅ reductases in the microsomal fraction were biochemically estimated. The constitutive intestinal CYP1A1 mRNA expression changes during maturation and aging. Inducibility of CYP1A1 gene was evident in intestinal mucosa at 2-, 4- and 20-month-old rats. A similar pattern of changes was observed for CYP2B1/2 isoforms. CYP3A1 mRNA expression was not detected in small intestine of 2-week-old rats. In matured rats, constitutive intestinal CYP3A1 expression was low, although after induction, significant increases in CYP3A1 mRNA amount were noted in aged individuals. Intestinal activity of both analyzed reductases was lowest in immature rats and highest in 28-month-old animals. In conclusion, the activity of cytochrome P450 complex in rat small intestine was not decreased by the aging processes, so the high rate of oxidative metabolic reactions in intestinal mucosa can be maintained till the advanced life stage.     

Mitochondrial targeting of intact CYP2B1 and CYP2E1 and N-terminal truncated CYP1A1 proteins in Saccharomyces cerevisiae--role of protein kinase A in the mitochondrial targeting of CYP2E1

Previously we showed that intact rat cytochrome P450 2E1, cytochrome P450 2B1 and truncated cytochrome P450 1A1 are targeted to mitochondria in rat tissues and COS cells. However, some reports suggest that truncated cytochrome P450 2E1 is targeted to mitochondria. In this study, we used a heterologous yeast system to ascertain the conservation of targeting mechanisms and the nature of mitochondria-targeted proteins. Mitochondrial integrity and purity were established using electron microscopy, and treatment with digitonin and protease. Full-length cytochrome P450 2E1 and cytochrome P450 2B1 were targeted both to microsomes and mitochondria, whereas truncated cytochrome P450 1A1 (+ 5 and + 33/cytochrome P450 1A1) were targeted to mitochondria. Inability to target intact cytochrome P450 1A1 was probably due to lack of cytosolic endoprotease activity in yeast cells. Mitochondrial targeting of cytochrome P450 2E1 was severely impaired in protein kinase A-deficient cells. Similarly, a phosphorylation site mutant cytochrome P450 2E1 (Ser129A) was poorly targeted to the mitochondria, thus confirming the importance of protein kinase A-mediated protein phosphorylation in mitochondrial targeting. Mitochondria-targeted proteins were localized in the matrix compartment peripherally associated with the inner membrane and their ethoxyresorufin O-dealkylation, erythromycin N-demethylase, benzoxyresorufin O-dealkylation and nitrosodimethylamine N-demethylase activities were fully supported by yeast mitochondrial ferredoxin and ferredoxin reductase.