Caucasian Liver Research Articles

Pharmacokinetics of a once-daily tacrolimus formulation in first nations and caucasian liver transplant recipients.

Patient ethnicity may influence the pharmacokinetics (PK) of tacrolimus. Because the Canadian First Nations (FN) constitute a large and increasing segment of the liver transplant population, we undertook to determine whether PK differences exist for a once-daily, extended release formulation of tacrolimus (Advagraf) in FN compared to Caucasian (CAUC) liver transplant recipients. Following achievement of a steady state with Advagraf, blood samples were drawn at 0, 1, 2, 4, 6, 8 and 24 hours for whole blood tacrolimus levels by commercial immunoassay and CYP3A4 and CYP3A5 allele analyses were performed by polymerase chain reactions. Nineteen subjects participated in the study (7 FN and 12 CAUC). The FN cohort had significantly higher AUC (214 ± 48 versus 168 ± 25, P < 0.05), Cmax (16.7 ± 4.4 ng/ml versus 11.3 ± 1.7 ng/ml, P < 0.05), Cmin (6.1 ± 1.0 ng/ml versus 4.7 ± 0.5 ng/ml, P < 0.05) and shorter Tmax (1.6 ± 0.2 hours versus 2.8 ± 0.3 hours, P < 0.05) values than CAUCs. CYP3A4 genotypes were C/C in both cohorts, while the CYP3A5 *1/*3 allele was present in 2/5 FN and 0/9 CAUC. The results of this study indicate that once-daily, extended release Advagraf results in higher blood tacrolimus levels and shorter times to Cmax in FN compared to CAUC liver transplant recipients.

A New Panel-Based Next-Generation Sequencing Method for ADME Genes Reveals Novel Associations of Common and Rare Variants With Expression in a Human Liver Cohort.

We developed a panel-based NGS pipeline for comprehensive analysis of 340 genes involved in absorption, distribution, metabolism and excretion (ADME) of drugs, other xenobiotics, and endogenous substances. The 340 genes comprised phase I and II enzymes, drug transporters and regulator/modifier genes within their entire coding regions, adjacent intron regions and 5′ and 3′UTR regions, resulting in a total panel size of 1,382 kbp. We applied the ADME NGS panel to sequence genomic DNA from 150 Caucasian liver donors with available comprehensive gene expression data. This revealed an average read-depth of 343 (range 27–811), while 99% of the 340 genes were covered on average at least 100-fold. Direct comparison of variant annotation with 363 available genotypes determined independently by other methods revealed an overall accuracy of >99%. Of 15,727 SNV and small INDEL variants, 12,022 had a minor allele frequency (MAF) below 2%, including 8,937 singletons. In total we found 7,273 novel variants. Functional predictions were computed for coding variants (n = 4,017) by three algorithms (Polyphen 2, Provean, and SIFT), resulting in 1,466 variants (36.5%) concordantly predicted to be damaging, while 1,019 variants (25.4%) were predicted to be tolerable. In agreement with other studies we found that less common variants were enriched for deleterious variants. Cis-eQTL analysis of variants with (MAF ≥ 2%) revealed significant associations for 90 variants in 31 genes after Bonferroni correction, most of which were located in non-coding regions. For less common variants (MAF < 2%), we applied the SKAT-O test and identified significant associations to gene expression for ADH1C and GSTO1. Moreover, our data allow comparison of functional predictions with additional phenotypic data to prioritize variants for further analysis.

Abstract A05: Genetic factors associated with racial disparities of liver cancer

Abstract Liver cancer (hepatocellular carcinoma [HCC]) is a leading cause of cancer-related morbidity and mortality worldwide. Racial disparities in HCC have been well-known among several racial and ethnic groups in the U.S. The incidence rates (per 100,000/year) for non-Hispanic White (NHW), non-Hispanic Black (NHB), Hispanics (HISP), and Asian/Pacific Islanders (API) are 7.0, 15.7, 11.8, and 18.6, respectively. The 5-year survival rates (%) for the four population groups are 18.2, 15.1, 19.2, and 24.4, respectively. NHWs have the lowest incidence rates, while APIs have the highest 5-year survival rate among the four racial and ethnic groups. Previous studies have indicated that socioeconomic status, in general, is associated with HCC health disparity; however, they alone cannot fully explain the disparity between different racial and ethnic groups. Few studies have investigated the biologic factors that may be associated with the health disparity in HCC. Using RNA-seq data from The Cancer Genome Atlas (TCGA), we have performed a comprehensive analysis of differentially expressed genes and pathways between Caucasian and Asian American liver cancer patients and identified hundreds of differentially expressed genes. We also identified a handful of genes that are differentially mutated between Caucasian American and Asian American HCC patients using the somatic mutation data from TCGA. We will discuss in details the differentially expressed genes and pathways, and their implications for the HCC health disparity. As far as we know, our study represents the largest-scale study on the biologic factors for HCC health disparity between Caucasian and Asian American liver cancer patients. Note: This abstract was not presented at the conference. Citation Format: Zihan Cui, Zhe Wang, Tingting Zhao, Qingxiang Amy Sang, Jinfeng Zhang. Genetic factors associated with racial disparities of liver cancer [abstract]. In: Proceedings of the Tenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2017 Sep 25-28; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2018;27(7 Suppl):Abstract nr A05.

Polymorphisms of the human platelet antigen-1, -2, -3, -5, and -15 systems and acute cellular liver transplant rejection

The human platelet antigen (HPA)-1, -2, -3, -5, and -15 systems are characterized as polymorphic alloantigens expressed on platelets and endothelial cells. In this retrospective study, we investigated, whether HPA-1, -2, -3, -5, and -15 incompatibilities are associated with acute cellular liver transplant rejection. A total of 96 Caucasian liver transplant recipients and corresponding donors were analyzed, 43 with biopsy proven acute cellular rejection (BPAR) and 53 without acute cellular rejection (No-BPAR). Polymorphisms of mentioned HPA systems were determined by polymerase chain reaction-sequence specific primers (PCR-SSP). Our data demonstrate that acute cellular rejection episodes were associated with HPA-3 incompatibility (58% HPA-3 incompatibility in BPAR group vs. 32% HPA-3 incompatibility in No-BPAR group, p=0.013). Furthermore, the frequency of HPA-3bb genotype was significantly higher in BPAR recipients as compared to No-BPAR recipients (30% vs 6%, p=0.002). On the other hand, there was no association between acute cellular rejection and the other tested HPA systems. We conclude that in the Caucasian population the HPA-3 system confers susceptibility to acute cellular rejection after liver transplantation.

Variability in hepatic expression of organic anion transporter 7/SLC22A9, a novel pravastatin uptake transporter: impact of genetic and regulatory factors

Human organic anion transporter 7 (OAT7, SLC22A9) is a hepatic transport protein poorly characterized so far. We therefore sought to identify novel OAT7 substrates and factors contributing to variable hepatic OAT7 expression. Using OAT7-expressing cells, pravastatin was identified as a substrate. Hepatic SLC22A9/OAT7 mRNA and protein expression varied 28-fold and 15-fold, respectively, in 126 Caucasian liver samples. Twenty-four variants in SLC22A9 were genotyped, including three rare missense variants (rs377211288, rs61742518, rs146027075), which occurred only heterozygously. No variant significantly affected hepatic SLC22A9/OAT7 expression. The three missense variants, however, showed functional consequences when expressed in vitro. Hepatic nuclear factor 4-alpha (HNF4α) emerged as a major transcriptional regulator of SLC22A9 by a series of in silico and in vitro analyses. In conclusion, pravastatin is the first identified OAT7 drug substrate. Substantial inter-individual variability in hepatic OAT7 expression, majorly driven by HNF4α, may contribute to pravastatin drug disposition and might affect response.The Pharmacogenomics Journal advance online publication, 4 August 2015; doi:10.1038/tpj.2015.55.

Genetics is a major determinant of expression of the human hepatic uptake transporter OATP1B1, but not of OATP1B3 and OATP2B1.

BackgroundOrganic anion transporting polypeptide (OATP) 1B1, OATP1B3, and OATP2B1 (encoded by SLCO1B1, SLCO1B3, SLCO2B1) mediate the hepatic uptake of endogenous compounds like bile acids and of drugs, for example, the lipid-lowering atorvastatin, thereby influencing hepatobiliary elimination. Here we systematically elucidated the contribution of SLCO variants on expression of the three hepatic OATPs under consideration of additional important covariates.MethodsExpression was quantified by RT-PCR and immunoblotting in 143 Caucasian liver samples. A total of 109 rare and common variants in the SLCO1B3-SLCO1B1 genomic region and the SLCO2B1 gene were genotyped by MALDI-TOF mass spectrometry and genome-wide SNP microarray technology. SLCO1B1 haplotypes affecting hepatic OATP1B1 expression were associated with pharmacokinetic data of the OATP1B1 substrate atorvastatin (n = 82).ResultsExpression of OATP1B1, OATP1B3, and OATP2B1 at the mRNA and protein levels showed marked interindividual variability. All three OATPs were expressed in a coordinated fashion. By a multivariate regression analysis adjusted for non-genetic and transcription covariates, increased OATP1B1 expression was associated with the coding SLCO1B1 variant c.388A > G (rs2306283) even after correction for multiple testing (P = 0.00034). This held true for haplotypes harboring c.388A > G but not the functional variant c.521T > C (rs4149056) associated with statin-related myopathy. c.388A > G also significantly affected atorvastatin pharmacokinetics. SLCO variants and non-genetic and regulatory covariates together accounted for 59% of variability of OATP1B1 expression.ConclusionsOur results show that expression of OATP1B1, but not of OATP1B3 and OATP2B1, is significantly affected by genetic variants. The SLCO1B1 variant c.388A > G is the major determinant with additional consequences on atorvastatin plasma levels.

Functional characterization of ABCC2 promoter polymorphisms and allele-specific expression

Multidrug resistance protein 2 (MRP2, ABCC2) is an efflux membrane transporter highly expressed in liver, kidney and intestine with important physiological and pharmacological roles. The goal of this study was to investigate the functional significance of promoter region polymorphisms in ABCC2 and potential allele specific expression. Twelve polymorphisms in the 1.6 kb region upstream of the translation start site were identified by resequencing 247 DNA samples from ethnically diverse individuals. Luciferase reporter gene assays showed that ABCC2 -24C>T both alone and as part of a common haplotype (-24C>T/-1019A>G/-1549G>A) increased promoter function 35% compared to the reference sequence (P < 0.0001). No other common variants or haplotypes affected ABCC2 promoter activity. Allele specific expression was also investigated as a mechanism to explain reported associations of the synonymous ABCC2 3972C>T variant with pharmacokinetic phenotypes. In Caucasian liver samples (n=41) heterozygous for the 3972C>T polymorphism, the 3972C allele was preferentially transcribed relative to the 3972T allele (P < 0.0001). This allelic imbalance was particularly apparent in samples with haplotypes containing two or three promoter/UTR variants (-1549G>A, -1019A>G and -24C>T). The observed allelic imbalance was not associated with hepatic or renal ABCC2 mRNA expression. Additional mechanisms will need to be explored to account for the interindividual variation in ABCC2 expression and MRP2 function.

Genetic and epigenetic regulation of the organic cation transporter 3, SLC22A3

Human organic cation transporter 3 (OCT3, SLC22A3) mediates the uptake of many important endogenous amines and basic drugs in a variety of tissues. OCT3 is identified as one of the important risk loci for prostate cancer and is markedly under-expressed in aggressive prostate cancers. The goal of this study was to identify genetic and epigenetic factors in the promoter region that influence the expression level of OCT3. Haplotypes that contained the common variants, g.-81G>delGA (rs60515630) (minor allele frequency (MAF) 11.5% in African American) and g.-2G>A (rs555754) (MAF>30% in all ethnic groups) showed significant increases in luciferase reporter activities and exhibited stronger transcription factor binding affinity than the haplotypes that contained the major alleles. Consistent with the reporter assays, OCT3 mRNA expression levels were significantly higher in Asian (P<0.001) and Caucasian (P<0.05) liver samples from individuals who were homozygous for g.-2A/A in comparison with those homozygous for the g.-2G/G allele. Studies revealed that the methylation level in the basal promoter region of OCT3 was associated with OCT3 expression level and tumorigenesis capability in various prostate cancer cell lines. The methylation level of the OCT3 promoter was higher in 62% of prostate tumor samples compared with matched normal samples. Our studies demonstrate that genetic polymorphisms in the proximal promoter region of OCT3 alter the transcription rate of the gene and may be associated with altered expression levels of OCT3 in human liver. Aberrant methylation contributes to the reduced expression of OCT3 in prostate cancer.

Metabolic capabilities of cytochrome P450 enzymes in Chinese liver microsomes compared with those in Caucasian liver microsomes

The most common causes of variability in drug response include differences in drug metabolism, especially when the hepatic cytochrome P450 (CYP) enzymes are involved. The current study was conducted to assess the differences in CYP activities in human liver microsomes (HLM) of Chinese or Caucasian origin. The metabolic capabilities of CYP enzymes in 30 Chinese liver microsomal samples were compared with those of 30 Caucasian samples utilizing enzyme kinetics. Phenacetin O-deethylation, coumarin 7-hydroxylation, bupropion hydroxylation, amodiaquine N-desethylation, diclofenac 4'-hydroxylation (S)-mephenytoin 4'-hydroxylation, dextromethorphan O-demethylation, chlorzoxazone 6-hydroxylation and midazolam 1'-hydroxylation/testosterone 6β-hydroxylation were used as probes for activities of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A, respectively. Mann-Whitney U test was used to assess the differences. The samples of the two ethnic groups were not significantly different in cytochrome-b(5) concentrations but were significantly different in total CYP concentrations and NADPH-P450 reductase activity (P < 0.05). Significant ethnic differences in intrinsic clearance were observed for CYP1A2, CYP2C9, CYP2C19 and CYP2E1; the median values of the Chinese group were 54, 58, 26, and 35% of the corresponding values of the Caucasian group, respectively. These differences were associated with differences in Michaelis constant or maximum velocity. Despite negligible difference in intrinsic clearance, the Michaelis constant of CYP2B6 appeared to have a significant ethnic difference. No ethnic difference was observed for CYP2A6, CYP2C8, CYP2D6 and CYP3A. These data extend our knowledge on the ethnic differences in CYP enzymes and will have implications for drug discovery and drug therapy for patients from different ethnic origins.

Influence of CYP3A5 and ABCB1 gene polymorphisms and other factors on tacrolimus dosing in Caucasian liver and kidney transplant patients

Tacrolimus is a substrate of cytochrome P4503A (CYP3A) enzymes as well as of the drug transporter ABCB1. We have investigated the possible influence of CYP3A5 and ABCB1 single nucleotide polymorphisms (SNPs) and other factors (e.g. albumin, hematocrit and steroids) on tacrolimus blood levels achieved in a population of Caucasian liver (n=51) and kidney (n=50) transplant recipients. At 1, 3 and 6 months after transplantation, tacrolimus doses (mg/kg/day) and trough blood levels (C0) were recorded and the weight-adjusted tacrolimus dosage (mg/kg/day) was calculated. Polymerase chain reaction followed by restriction fragment length polymorphism analysis was used for genotyping CYP3A5*1 and *3 [6986A>G] as well as ABCB1 at exons 21 [2677G>T/A] and 26 [3435C>T] in both liver transplant donors and recipients and in kidney transplant recipients. Of the 152 subjects studied, 84.9% showed a CYP3A5*3/*3 genotype. The total frequency of the allelic variant *3 was 93%. For the G2677T/A and C3435T polymorphisms the total frequencies of the allelic variants T/A and T were 44.7 and 46.7%, respectively. At 1, 3 and 6 months after transplantation the dose-adjusted C0 levels were significantly lower in patients with one copy of the *1 allele compared to those homozygous for the *3 allele. In the case of liver transplant patients the tacrolimus dose requirements were dominantly influenced by the polymorphisms of the CYP3A5 gene in the donors. With regard to the ABCB1 SNPs, in general they did not show any appreciable influence on tacrolimus dosing requirements; however, kidney transplant recipients carrying the 2677T/A allele required significantly higher daily tacrolimus doses than subjects homozygous for the wild-type allele. Identification of CYP3A5 single nucleotide polymorphisms prior to transplantation could contribute to evaluate the appropriate initial dosage of tacrolimus in the patients.

The UGT1A1∗28 polymorphism correlates with erlotinib’s effect on SN-38 glucuronidation

The combination of irinotecan and erlotinib has been evaluated in clinical trials, although toxicity has been significant. We aimed to investigate the effect of erlotinib on SN-38 glucuronidation and the association between UGT1A polymorphisms and SN-38 glucuronidation activity in the presence of erlotinib. The inhibitory effect of erlotinib on SN-38 glucuronidation was determined by measuring the formation rates for SN-38 glucuronide, using recombinant human UGT1A1, pooled human liver microsomes (HLMs) and 52 Caucasian liver microsomes in the absence or presence of erlotinib. Inhibition kinetic studies were conducted. AUC ratios were used to predict the risk of potential drug–drug interactions (DDI) in vivo. Our data showed that erlotinib exhibited potent non-competitive inhibition against SN-38 glucuronidation in pooled HLMs and UGT1A1. Using the physiological and pharmacokinetic parameters obtained from the literature, we estimated the in vivo concentrations of unbound erlotinib available for UGT1A1 active site and thus the AUC ratios of SN-38 were also quantitatively predicted. It is estimated that erlotinib administered at 50 mg/day or higher doses may result in at least a 24% increase in SN-38 AUC. Significant correlations were observed between SN-38 glucuronidation activity in the presence of erlotinib and UGT1A1∗28 in 52 Caucasian liver microsomes. Our results suggest that erlotinib is a potent inhibitor of SN-38 glucuronidation via UGT1A1 inhibition. The coadministration of erlotinib with irinotecan may result in clinically significant DDI. UGT1A1∗28 polymorphism correlates with erlotinib’s effect on SN-38 glucuronidation. The present findings shed light on the development and optimisation of combinations involving irinotecan and erlotinib.

Three haplotypes associated with CYP2A6 phenotypes in Caucasians

The human cytochrome P450 2A6 (CYP2A6) enzyme metabolizes several xenobiotic compounds of clinical or toxicological importance. We aimed to identify genetic variants and major CYP2A6 haplotypes associated with CYP2A6 phenotypic variation. CYP2A6 mRNA level, protein level, activity and haplotypes were determined in Caucasian liver samples via real-time polymerase chain reaction, Western blot, coumarin 7-hydroxylation, DNA sequencing and genotyping, respectively. Phenotypes were then analyzed for associations with haplotypes. CYP2A6 transcript, protein and activity levels were correlated among each other. In 45 African-American, 156 Caucasian, 47 Chinese, 50 Japanese and 47 Korean DNA samples, we detected 95 different polymorphisms in the CYP2A6 gene, 49 of which had not been described previously. Caucasian variants formed 33 haplotypes which built four clades. Allele *9B and the CYP2A7/2A6 partial deletion allele CYP2A6*12B were both associated with decreased expression. The latter haplotype extends at least over 147 kb up into the CYP2B6 gene. A haplotype almost identical to allele *1A was associated with decreased expression and activity of CYP2A6 compared to all other haplotypes. In summary A CYP2A6*1A-like allele, *9B and *12B are major genetic determinants of CYP2A6 phenotype variation in Caucasians.

Lack of Association of Recipient MCP-1 Gene Promoter Polymorphism With Acute Graft Rejection After Orthotopic Liver Transplantation

Acute graft rejection after orthotopic liver transplantation (OLT) is associated with leukocyte infiltration of the graft. Monocyte chemoattractant protein-1 (MCP-1) is a β-chemokine involved in the attraction and accumulation of mononuclear granulocytes toward sites of inflammation. A biallelic polymorphism (G/A) at position −2518 of the MCP-1 gene has been described. Cells obtained from individuals with the GG or AG genotypes have been found to produce more MCP-1 than those obtained from individuals with the AA genotype. The goal of this study was to assess the possible association between this polymorphism and susceptibility to acute graft rejection after OLT. One hundred fifty Caucasian liver transplant recipients from the South of Spain underwent genotyping using a polymerase chain reaction (PCR) amplification followed by restriction fragment length polymorphism (RFLP). No significant differences were observed when patients with versus without acute rejection episodes were compared for the distribution of −2518 MCP-1 genotypes. The present study supports the lack of involvement of polymorphism at position −2518 (A/G) of the MCP-1 gene on the susceptibility to acute allograft rejection among OLT recipients.

Generalized, pruritic, ulcerating maculopapular rash necessitating cessation of sirolimus in a liver transplantation patient

The use of sirolimus as an alternative to calcineurin antagonists has enabled the continuation of immunosuppression in patients with renal impairment with preservation of kidney function. Sirolimus is generally well tolerated, with the main causes of cessation of therapy related to its effect on blood lipid profile as well as leukopenia and thrombocytopenia. We report a case of a debilitating ulcerating maculopapular rash necessitating cessation of the drug in a liver transplantation patient. A 56-year-old Caucasian liver transplantation patient presented with a diffuse, debilitating rash attributed to sirolimus use. This ultimately necessitated cessation of the immunosuppressant with subsequent resolution of her symptoms. From a review of the current literature, this is a highly unusual adverse reaction to sirolimus.

CYP3A5 phenotype-genotype correlations in a British population.

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. 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. 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). 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.

The genetic determinants of the CYP3A5 polymorphism.

CYP3A proteins comprise a significant portion of the hepatic cytochrome P450 (CYP) protein and they metabolize around 50% of drugs currently in use. The dissection of the individual contributions of the four CYP3A genes identified in humans to overall hepatic CYP3A activity has been hampered by sequence and functional similarities. We have investigated the expression of CYP3A5 and its genetic determinants in a panel of 183 Caucasian liver samples. CYP3A5 expression is increased in 10% of livers in this ethnic group. Using a high density map of CYP3A5 variants, we searched for genetic markers of the increased CYP3A5 expression. In agreement with an independent, recent study, we report that a SNP within intron 3 (g.6986G>A) is the primary cause of the CYP3A5 protein polymorphism. The frequencies of the g.6986A variant which allow for normal splicing of CYP3A5 transcripts are 5% in Caucasians, 29% in Japanese, 27% in Chinese, 30% in Koreans and 73% in African-Americans. In the last ethnic group, the expression of CYP3A5 in some individuals who carry the g.6986A variant is affected adversely by a frame shift mutation (CYP3A5*7, D348., q = 0.10). In summary, these results should add to efforts to identify clinically relevant, CYP3A5-specific reactions and to further elucidate traits responsible for variable expression of the entire CYP3A family.

Cytotoxic T-lymphocyte antigen 4 gene polymorphisms and susceptibility to acute allograft rejection.

Cytotoxic T-lymphocyte antigen 4 (CTLA4) has been shown to play a critical role in the down-regulation of the immune response. We retrospectively examined the association between acute rejection and two polymorphisms in the CTLA4 gene, the dinucleotide (AT)n repeat polymorphism in exon 3 and the single nucleotide polymorphism A/G at position 49 in exon 1, in a cohort of liver and kidney transplant recipients. A total of 207 liver and 167 renal transplant recipients were analyzed. In the case of the (AT)n repeat polymorphism we found an increased incidence of acute rejection in association with allele 3 and 4 in both liver and kidney (P=0.002 and 0.05, respectively). In addition, in liver transplant recipients, allele 7 was associated with acute rejection independent of ethnicity (P<0.05). Allele 1 was less frequently observed in African American as compared with Caucasian liver and kidney transplant recipients, with a frequency of 33.8% and 69%, respectively (P<0.0001). Those patients with allele 1 had a tendency toward a lower rate of rejection at 42% versus 57.8% (P=0.058), suggesting a potential protective effect of allele 1. Analysis of the A/G single nucleotide polymorphism demonstrated no association between either allele and the incidence of acute rejection in the patients studied. These initial observations provide the necessary basis to further investigate the risk stratification of transplant recipients based on specific CTLA4 gene polymorphisms.

Trimethadione metabolism by human liver cytochrome P450: evidence for the involvement of CYP2E1

1. Caucasian liver samples were used in this study. N-demethylation of trimethadione (TMO) to dimethadione (DMO) was monitored in the presence of chemical inhibitors of CYPs, such as fluconazole, quinidine, dimethyl-nitrosamine, acetaminophen, phenacetin, chlorzoxazone and mephenytoin. Trimethadione N-demethylation was selectively inhibited by dimethylnitrosamine and chlorzoxazone (> 50%) and weakly inhibited by tolbutamide (12%) and fluconazole (22%), whereas other inhibitors showed no effect. This result suggested that TMO metabolism to DMO is mainly mediated by CYP2E1 and marginally by CYP2C and CYP3A4. 2. Fifteen human livers were screened and interindividual variability of TMO N-demethylation activity was 3-fold. Chlorzoxazone 6-hydroxylation activity was also measured and both activities were significantly correlated (r=0.735, p < 0.01). 3. DMO production by human cDNA expressed CYP enzymes was observed mainly for CYP2E1 (10.8 nmol/tube), marginally for CYP2C8 (0.22 nmol tube) and not detectable for other CYP enzymes. 4. These results indicate that TMO metabolism is primarily catalysed by CYP2E1 and that trimethadione would be a suitable selective probe drug for the estimation of human CYP2E1 activity in vivo.

A chemical and enzymological account of the multiple forms of human liver aldehyde dehydrogenase Implications for ethnic differences in alcohol metabolism

The major low-p I zones of aldehyde dehydrogenase (aldehyde: NAD + oxidoreductase, EC 1.2.1.3) may be visualized with specific histochemical staining after starch gel electrophoresis at pH 7.4 of Caucasian human liver extracts, whereas about 50% of Chinese human liver extracts show only two such zones. The three zones of activity were purified to apparent homogeneity from Caucasian liver. The substrate specificity of each form was investigated by double reciprocal plots using 13 aldehydes of various chemistries. The acetaldehyde-preferring isozyme I lacking in 50% of Chinese livers had a slightly lower native and subunit molecular weight than the ‘universal’ isozymes IIa and IIb. All forms were highly sensitive to disulfiram inbibition. this inhibition could be protected against, or reversed, by dithiothreitol. 2,2′-Dithiodipyridine was a slower inhibitor of isoenzyme I. All three purified forms of the enzyme, as well as crude extracts of normal and isozyme I-deficient Chinese livers, showed positive immunoreactivity to antibodies prepared in rabbits against type I enzyme. Tryptic peptide maps of forms IIa and IIb were almost identical, whereas that of form I, although showing some similarities, was clearly different. These results provide a consistent explanation for the acetaldehyde-mediated extreme sensitivity to moderate alcohol ingestion shown normally by abut 50% of oriental subjects and during disulfiram (Antabuse) therapy by all subjects.