Biomedical subjects

Julian B S Leathart

Publications and source records attributed to Julian B S Leathart.

5 recordsLinked to original sources

PubMed2006

Genotyping for cytochrome P450 polymorphisms.

Protocols for the extraction of DNA from human blood and for genotyping for a number of common cytochrome P450 polymorphisms using either polymerase chain reaction (PCR)-restriction fragment length polymorphism or PCR-single-strand conformational polymorphism (SSCP) analysis are described. Rapid high-throughput techniques are also available for analyses of this type, but they require access to specialized equipment and are not considered here. General guidelines for performing amplification using PCR are described together with electrophoresis protocols for analysis of restriction digests of PCR products with agarose and polyacrylamide gels including the use of polyacrylamide-based gels for SSCP analysis. Protocols for the following specific isoforms and alleles are also provided: CYP1A1 (*2B and *4 alleles), CYP2C8 (*3 and *4 alleles), CYP2C9 (*2, *3, and *11 alleles), CYP2C19 (*2 and *3 alleles), CYP2D6 (*3, *4, *5, and *6 alleles), CYP2E1 (*5A, *5B, and *6 alleles), and CYP3A5 (*3 allele).

Base Sequence↗

PubMed2004

Hepatic adducts, circulating antibodies, and cytokine polymorphisms in patients with diclofenac hepatotoxicity.

Diclofenac is a nonsteroidal anti-inflammatory drug that causes rare but serious hepatotoxicity, the mechanism of which is unclear. The purpose of the present study was to explore the potential role played by the immune processes. Antibodies to diclofenac metabolite-modified liver protein adducts were detected in the sera of seven out of seven patients with diclofenac-induced hepatotoxicity, 12 of 20 subjects on diclofenac without hepatotoxicity, and none of four healthy controls. The antibodies recognized adducts expressed in livers from rats treated with multiple doses of diclofenac, but not in those given single doses. In addition, several potential diclofenac adducts were identified in the liver of a patient with diclofenac-induced hepatic failure, but not from a normal human donor liver, by immunoblotting with an adduct-selective rabbit antiserum. To determine whether or not polymorphisms in genes encoding cytokine-related proteins influence susceptibility to hepatotoxicity, genotyping for the polymorphisms -627 in the interleukin (IL)-10 gene, -590 in the IL-4 gene, and codon 551 in the IL-4 receptor (IL-4R) were performed on DNA from 24 patients on diclofenac with hepatotoxicity, 48 subjects on diclofenac without hepatotoxicity, and healthy controls. The frequencies of the variant alleles for IL-10 and IL-4 were higher in patients (OR [odds ratio]: 2.8 for IL-10; 2.6 for IL-4; 5.3 for IL-10 + IL-4) compared with healthy controls and subjects on diclofenac without hepatotoxicity (OR: 2.8 for IL-10; 1.2 for IL-4; 5.0 for IL-10 + IL-4). In conclusion, the observed polymorphisms, resulting in low IL-10 and high IL-4 gene transcription, could favor a T helper (Th)-2 mediated antibody response to neoantigenic stimulation associated with disease susceptibility.

Adult↗

PubMed2003

Do multiple cytochrome P450 isoforms contribute to parathion metabolism in man?

Phosphorothioate compounds are widely used in agriculture and public health for the control of unwanted pests. The phosphorothioate parathion was metabolised to the toxic metabolite paraoxon (0.038-0.683 nmol/min per mg protein) and p-nitrophenol (0.023-2.10 nmol/min per mg protein) by human liver microsomes ( n=27) in an NADPH-dependent reaction. There was a significant correlation ( P<0.02) between nifedipine oxidation and paraoxon formation from parathion (200 micro M) by human liver microsomes and with cytochrome P450 (CYP) 3A4/5 expression ( P<0.05), although not with midazolam 1'-hydroxylation or testosterone 6beta-hydroxylation. Paclitaxel 6'-hydroxylation and CYP2C8 expression correlated with paraoxon formation ( P<0.01), indicating CYP2C8 involvement. Of nine recombinant P450 isoforms, CYPs 3A4, 3A5, 1A2 and 2D6 activated parathion to paraoxon at the highest rates (6.5, 8.5, 5.7 and 6.2 pmol/pmol P450 per h) with K(m) values of 12.6, 2.7, 1.5 and 9.2 micro M, respectively. Similar K(m) values were seen with the human liver microsomes. These data indicate that CYP3A4/5 and CYP2C8, which constitute up to 40% of human liver P450s, are the most significant participants in the metabolism of parathion. However, several other isoforms could play an important role when CYP3A and CYP2C8 are poorly expressed due to environmental factors or the presence of a genetic polymorphism.

Animals↗

PubMed2003

CYP3A5 phenotype-genotype correlations in a British population.

AIMS: To develop a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP)-based assay to genotype for hepatic CYP3A5 expression and to use this assay to study a British population. METHODS: CYP3A5-specific primers were designed with one including a base-pair mismatch to create a RsaI site in samples positive for G6986 (CYP3A5*3 allele) [correction]. Following PCR and RsaI digestion, different band patterns on electrophoresis were predicted for individuals positive for CYP3A5 (CYP3A5*1 allele) compared with those who do not express the gene (CYP3A5*3 homozygotes). The assay was validated by DNA sequencing. DNA samples from a human liver bank consisting of 22 livers whose CYP3A5 expression had been determined by immunoblotting and a group of random individuals (n = 100) from the North-east of England were genotyped by the new assay. RESULTS: In the liver bank, five out of 22 samples expressed CYP3A5 at significant levels (>20 pmol mg-1 protein) and were found to have the genotype CYP3A5*1/CYP3A5*3 by the PCR-RFLP assay. All other liver DNA samples were CYP3A5*3 homozygotes. In the group of 100 random individuals, 13 had the genotype CYP3A5*1/CYP3A5*3 and all others were CYP3A5*3 homozygotes, predicting that 13% (95% confidence interval (CI) 6%, 20%) would show significant hepatic CYP3A5 expression. The frequency for the CYP3A5*1 allele was 0.065 (95% CI 0.032, 0.097). CONCLUSIONS: We have developed a simple assay for the detection of the CYP3A5*1/CYP3A5*3 alleles and shown that in a British population their frequency is similar to that reported previously. We have also shown a good correlation between hepatic CYP3A5 expression and genotype for a British Caucasian liver bank.

Adult↗

PubMed2002

CYP2C8 polymorphisms in Caucasians and their relationship with paclitaxel 6alpha-hydroxylase activity in human liver microsomes.

Published cDNA sequences suggest the existence of non-synonymous single nucleotide polymorphisms in the cytochrome P450 CYP2C8. To determine whether these polymorphisms could be confirmed in a Caucasian population and to investigate whether additional polymorphisms occur in the coding and upstream regions of this gene, we screened for previously described and for novel polymorphisms using PCR-RFLP and SSCP analysis. We confirmed the existence of two of the previously detected polymorphisms which give rise to the amino acid substitutions I264M and K399R, respectively, but failed to detect three others in our population. We also confirmed that a recently identified polymorphism (R139K) is linked to K399R (CYP2C8*3) in our study population. The allele frequencies for the I264M (CYP2C8*4 allele) and the CYP2C8*3 allele were 0.075 and 0.15, respectively. Three novel polymorphisms (T-370G, C-271A and T1196C/L390S) were also detected with the upstream polymorphisms showing allele frequencies of 0.061 and 0.196, respectively, but the L390S polymorphism detected only in a single subject. An additional single subject was heterozygous for a polymorphism recently described in African-Americans (A805T; CYP2C8*2 allele). The functional significance of the two upstream polymorphisms and the CYP2C8*3 and CYP2C8*4 alleles was investigated in human liver microsomes. Samples heterozygous for CYP2C8*3 showed significantly lower paclitaxel 6alpha-hydroxylase activity compared with wild-type samples. Median activity associated with CYP2C8*4 also appeared lower than the wild-type but the difference was not significant. There was no evidence that either upstream polymorphism gave rise to altered CYP2C8 expression.

Amino Acid Sequence↗