ISSN:0960-314X    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

The increased risk of rapid acetylator humans for the development of colorectal cancer has created interest in experimental animal models to study the relationship of N-acetyltransferase phenotype to colon cancer. Colon cytosols from inbred mouse lines were assayed for the ability to N-acetylate 2-aminofluorene to determine if the mouse model of the N-acetyltransferase polymorphism could be used to study this relationship. The results indicate that the colon acetylcoenzyme A: 2-aminofluorene- N-acetyltransferase activity parallels that of the liver. Colon activity from slow acetylator (A and B6.A) mouse lines is significantly lower than that of rapid acetylator (B6, B6.D, and A.B6) lines. p-Aminobenzoic acid N-acetyltransferase activity also differed between colon cytosols from rapid and slow acetylator strains. Isoniazid acetylation in colon and in liver did not differ between phenotypes. Northern blot analysis demonstrated the presence of mRNA for both NAT-1 and NAT-2 in mouse colon as well as in mouse liver. These results indicate that the N-acetyltransferase polymorphism is expressed in mouse colon when 2-aminofluorene or p-aminobenzoic acid is used as substrate and therefore the mouse may be a model for study of the effect of acetylator phenotype on development of colorectal cancer in humans.

ISSN:0960-314X    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

Microsomal hydroxylation of tolbutamide in Japanese livers was studied in vitro to ascertain the enzyme catalysing this reaction. Rates of tolbutamide hydroxylation differed individually 33-fold and 42-fold at 0.1 mM and 2.4 mM tolbutamide concentrations, respectively, and were segregated into two groups, rapid and slow metabolizers. An antibody raised against P450 human-2 (a form of CYP2C9) strongly inhibited the hydroxylation in livers of rapid metabolizers but only weakly inhibited in the slow metabolizer. Kinetic experiments further demonstrated a clear distinction in tolbutamide hydroxylation between two groups; the mean of apparent Km values for tolbutamide was 0.25 mM (n =3) in the rapid group and 2.58 mM (n =2) in the slow, respectively. These data suggest that different enzymes are involved in the hydroxylation in both metabolizer groups. Furthermore, CYP2C9 produced by cDNA expression in yeasts, catalysed tolbutamide hydroxylation at rates similar to the rapid metabolizer group at both the 0.1 mM and 2.4 mM concentrations. The apparent Km value of the expressed protein for tolbutamide, 0.26 mM, was similar to that determined for the rapid group of microsomal samples. Clear correlations were observed between the rate of microsomal tolbutamide hydroxylation at 0.1 mM and CYP2C9 protein content or the rate of S-mephenytoin 4'-hydroxylation in human liver. These results indicate that considerable portions of microsomal tolbutamide hydroxylation are catalysed by CYP2C9 or the closely related form in the rapid metabolizers.

ISSN:0960-314X    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

The tolbutamide hydroxylation capacity was studied in 106 healthy unrelated volunteers from the Australian population. Following a 500 mg oral dose of tolbutamide, the ratio of metabolites (hydroxytolbutamide plus carboxytolbutamide) to unchanged tolbutamide excreted in urine from 6 to 12 h post-dose (urinary metabolic ratio, MR) was determined. Metabolic ratio values did not appear bimodally distributed, even following various transformations of the data (i.e. Log10, inverse, Log10inverse). A poor metabolizer (PM) subject from a previous clinical study, however, could be distinguished (MR value 159) from the above subjects (MR value range 324-3033), particularly from the histogram plot of inverse tolbutamide metabolic ratio. The poor metabolizer's parents had metabolic ratio values (526 and 478) that were at the lower end of the range of metabolic ratios obtained from the population study, and may indicate that they both have a heterozygous genotype and that a recessive form of inheritance is most likely. As the hydroxylations of tolbutamide and phenytoin are closely linked, the incidence of slow tolbutamide metabolizers is likely to be similar to that for phenytoin (about 1:500) and this is consistent with the failure to detect a single poor tolbutamide metabolizer in our random sample of 106 individuals.

ISSN:0960-314X    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

Pronounced differences in the CYP2D6 gene between Chinese and Caucasians have previously been described. There was a low frequency of detrimental mutations in the Chinese CYP2D6 gene causing the poor metabolizer (PM) phenotype. In contrast to Caucasians where the Xba 144 kb allele is almost always associated with the PM phenotype, Chinese with the 44/44 kb RFLP pattern are extensive metabolizers (EM). In order to evaluate whether the debrisoquine hydroxylation seen in subjects with this haplotype is catalysed by a functionally similar enzyme to CYP2D6 or is catalysed by another type of P450 isozyme, product selectivity of the 4-hydroxylation was studied in 27 Chinese. The inhibition of CYP2D6 by quinidine was also investigated.In the 26 Chinese EM the S( + )-4-hydroxy enantiomer was found to be the major urinary metabolite of debrisoquine with an enantiomeric excess of 96.8-100%, which is similar to that in Caucasians. A correlation between the amount of S( + )-4-hydroxy and the minor 7-hydroxy metabolites excreted in urine (r =0.72; ρ<0.001) was seen. The amount of these two metabolites excreted was less in Chinese EM of debrisoquine with the 44/44 kb RFLP pattern, than in those with the wild type 29/29 kb pattern (ρ <0.01). The stereoselectivity was very high in both groups. All Chinese homozygous for the 44 kb fragment (n=5) were transformed to apparent PM after a single 100 mg dose of quinidine similarly to five Caucasian EM. Both the S( + )-4- and 7-hydroxylations of debrisoquine were inhibited by quinidine in both populations.This study shows that the cytochrome P450 catalysing the 4- and 7-hydroxylations of debrisoquine in Chinese EM has the same properties (product stereoselectivity and inhibition by quinidine) as the CYP2D6 in Caucasian EM.

ISSN:0960-314X    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

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 ah, 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. We are proposing that the heterogeneity observed with TI-1 and TI-2 represent two unique subtypes of the Ah receptor, possibly resulting from a single ligand-binding subunit in association with different partner proteins.

ISSN:0960-314X    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

The enzyme family carbonyl reductase, which catalyses the reduction of xenobiotic as well as endogenous ketones and aldehydes, has not been very well studied in terms of its biological functions and structural aspects. The aim of the present study was to check for the occurrence of inter-individual variability of carbonyl reductase activity in human liver. In vitro metabolism of p-nitroacetophenone (PNAP, a prototype substrate) indicated the presence of a high- and low-affinity enzyme site. The reductase activity of 17 kidney donor livers was screened at two concentrations (0.05 and 0.5 mM PNAP, below and above Km). The rates of reductase activity at 0.05 mM suggested a normal distribution. In contrast, at 0.5 mM the rates indicated a non-normal distribution, i.e. bi- or tri-modality. As an index of variability of enzyme affinity, ratios of velocities at 0.5 to 0.05 mM PNAP were calculated in order to check their frequency distributions. Three out of 17 kidney donor livers showed an atypical ratio. In these three cases, the high ratio was due to the low activity of the high affinity form of carbonyl reductase. Autopsy livers are a more readily available tissue source and about half the activity of the kidney donor livers was found in 43 autopsy livers indicating that they are a useful source of human tissue for studies of carbonyl reductase.

ISSN:0960-314X    

出版商:OVID    

年代:1993

数据来源: OVID

摘要:

The level of catechol O-methyltransferase (COMT) activity and COMT thermal stability in human tissue are controlled by a common genetic polymorphism. We studied individual hepatic biopsy samples shown previously to have phenotypically high, low or intermediate COMT activities and thermal stabilities to test the hypothesis that the molecular mass (Mr) and/or isoelectric point (pI) of the enzyme might differ in tissue from subjects with different presumed genotypes for the COMT genetic polymorphism. COMT was partially purified from each hepatic tissue sample by sequential ion exchange and gel filtration chromatography, and photoaffinity labelling was performed with [3H-methyl]- S-adenosyl-L-methionine ([3H-methyl]-Ado-Met), the methyl donor for the COMT enzymatic reaction. Two-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE) analysis of individual samples consistently showed the presence of three [3H-methyl]-Ado-Met photoaffinity labelled proteins with pI values of 5.4, 5.5 and 5.7, all three of which had Mrvalues of approximately 27.1 kDa. The same pattern was observed in all samples irrespective of COMT phenotype. Western blot analysis of 2-D SDS-PAGE gels performed with rabbit polyclonal antibodies to partially purified human kidney COMT showed a pattern similar to that found during photoaffinity labelling. Once again, the same pattern was found in all samples irrespective of COMT phenotype. Therefore, neither photoaffinity labelling nor Western blot analysis revealed differences in either Mror pI of cytoplasmic COMT in hepatic tissue from subjects selected on the basis of different phenotypic expression of the COMT genetic polymorphism.

ISSN:0960-314X    

出版商:OVID    

年代:1993

数据来源: OVID