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Genetic heterogeneity among slow acetylator N-acetyltransferase 2 phenotypes in cryopreserved human hepatocytes.

ABSTRACT: Genetic polymorphisms in human N-acetyltransferase 2 (NAT2) modify the metabolism of numerous drugs and carcinogens. These genetic polymorphisms modify both drug efficacy and toxicity and cancer risk associated with carcinogen exposure. Previous studies have suggested phenotypic heterogeneity among different NAT2 slow acetylator genotypes. NAT2 phenotype was investigated in vitro and in situ in samples of human hepatocytes obtained from various NAT2 slow and intermediate NAT2 acetylator genotypes. NAT2 gene dose response (NAT2*5B/*5B > NAT2*5B/*6A > NAT2*6A/*6A) was observed towards the N-acetylation of the NAT2-specific drug sulfamethazine by human hepatocytes both in vitro and in situ. N-acetylation of 4-aminobiphenyl, an arylamine carcinogen substrate for both N-acetyltransferase 1 and NAT2, showed the same trend both in vitro and in situ although the differences were not significant (p > 0.05). The N-acetylation of the N-acetyltransferase 1-specific substrate p-aminobenzoic acid did not follow this trend. In comparisons of NAT2 intermediate acetylator genotypes, differences in N-acetylation between NAT2*4/*5B and NAT2*4/*6B hepatocytes were not observed in vitro or in situ towards any of these substrates. These results further support phenotypic heterogeneity among NAT2 slow acetylator genotypes, consistent with differential risks of drug failure or toxicity and cancer associated with carcinogen exposure.

SUBMITTER: Doll MA

PROVIDER: S-EPMC5713641 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Genetic heterogeneity among slow acetylator N-acetyltransferase 2 phenotypes in cryopreserved human hepatocytes.

Doll Mark A MA Hein David W DW

Archives of toxicology 20170517 7

Genetic polymorphisms in human N-acetyltransferase 2 (NAT2) modify the metabolism of numerous drugs and carcinogens. These genetic polymorphisms modify both drug efficacy and toxicity and cancer risk associated with carcinogen exposure. Previous studies have suggested phenotypic heterogeneity among different NAT2 slow acetylator genotypes. NAT2 phenotype was investigated in vitro and in situ in samples of human hepatocytes obtained from various NAT2 slow and intermediate NAT2 acetylator genotype ...[more]

PMID: 28516247

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Project description:Isolated human hepatocytes are used in all facets of liver research, from the clinical management of liver failure to in vitro studies of drug disposition. Cryopreservation provides a reliable source of hepatocytes, but the associated cellular stress causes a highly variable and heterogeneous loss of a differentiated phenotype, manifested by a decreased ability to form cell-matrix and cell-cell interactions. We reasoned that this problem could be mitigated at the post-thawing stage, which would increase the availability of well-functioning human hepatocytes. We applied quantitative global proteomics to analyze the differences between attached and non-attached fractions of cryopreserved human hepatocyte batches. Hepatocytes that were unable to attach to a collagen matrix showed many signs of cellular stress, including a glycolytic phenotype and activation of the heat shock response, with increased apoptosis activation as the ultimate consequence. Further analysis of the activated stress pathways revealed an increase in early apoptosis immediately after thawing, hinting at the possibility of stress reversal. Therefore, we transiently treated the cells with compounds aimed at decreasing cellular stress via different mechanisms. We found that brief exposure to the pan-caspase apoptosis inhibitor Z-VAD-FMK restored the ability to attach to collagen, and promoted a differentiated morphology with increased metabolic function. Further, Z-VAD-FMK treatment did not alter hepatocyte protein expression, suggesting that these ‘rescued’ cells would be suitable for applications where hepatocytes of high quality are required. Conclusion: Cryopreserved human hepatocytes are affected by considerable amounts of cellular stress, often resulting in loss of differentiation with a decreased ability to form cell-matrix and cell-cell interactions. This can be alleviated by brief apoptosis inhibition post-thawing, substantially improving key morphological and functional properties of these cells.

Dataset Information

Genetic heterogeneity among slow acetylator N-acetyltransferase 2 phenotypes in cryopreserved human hepatocytes.

Publications

Genetic heterogeneity among slow acetylator N-acetyltransferase 2 phenotypes in cryopreserved human hepatocytes.

Similar Datasets

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