Human arylamine N-acetyltransferase 2 genotype-dependent protein expression in cryopreserved human hepatocytes
Open accessAbstract
Human N-acetyltransferases (NAT; EC 2.3.1.5) catalyze the N-acetylation of arylamine and hydrazine drugs and the O-acetylation of N-hydroxylated metabolites of aromatic and heterocyclic amines. Two different isoforms of this protein, N-acetyltransferase 1 (NAT1) and N-acetyltransferase 2 (NAT2), are expressed in human hepatocytes. Both are encoded by a single 870-bp open reading frame that exhibits genetic polymorphisms in human populations. NAT1 and NAT2 share more than 85% gene and protein sequence, making it challenging to produce antibodies with high specificity for NAT1 or NAT2. In the present study, we compared methods for the quantification of immunoreactive NAT1 and NAT2 with seven different antibodies and investigated the relationship of NAT2 genotype to NAT2 mRNA and protein expression in cryopreserved human hepatocytes. Sulfamethazine (NAT2-selective substrate) and NAT2 protein expression differed significantly with NAT2 acetylator genotype (p < 0.0001). NAT2 protein expression and sulfamethazine NAT2 catalytic activity correlated highly across the cryopreserved human hepatocytes of rapid, intermediate, and slow acetylator NAT2 genotypes. In conclusion, our data describe a specific analytical method for the quantification of NAT1 and NAT2 protein expression. We showed that the NAT2 activity in human hepatocytes is directly correlated to expression levels of NAT2 protein but not mRNA.
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This study included additional NAT2 variant alleles (*12, *13, *7) not previously examined.
“While previous studies have characterized differences between common genotypes, this study included additional variant alleles that have not been previously examined, such as *12, *13, *7 for NAT2.47”
From N-Acetyltransferase (NAT) 1 and NAT2 Enzyme Activities Drive Interindividual Variability in Sulfamethoxazole N-Acetylation (2025) · cites this paper as [47]
The Materials and methods section lists NAT2*12, *13 and *7 as alleles used to define acetylator phenotypes, but says the study selected only five haplotypes involving NAT2*4, *5 and *6. Thus this source did not include samples carrying the three additional alleles as the claim states. (LLM, conf=0.90)
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Experimental — accuracy not yet measuredNAT2 protein concentration has not been quantitated through targeted proteomics before.
“While NAT2 mRNA expression by qPCR and relative protein expression determined with specific antibodies and fluorescence assays have been previously described, the protein concentration has not been quantitated through targeted proteomics.47”
From N-Acetyltransferase (NAT) 1 and NAT2 Enzyme Activities Drive Interindividual Variability in Sulfamethoxazole N-Acetylation (2025) · cites this paper as [47]
The Discussion and Materials and methods describe NAT2 measurement by RT-qPCR and antibody-based fluorescence assays. Neither those passages nor searches for targeted proteomics, mass spectrometry, and prior protein-concentration measurements establish that NAT2 concentration had never been quantified by targeted proteomics. Page numbers were not available in our copy. (LLM, conf=0.72)
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- Authors
- Salazar-González, R. A., Doll, M. A., & Hein, D. W.
- Affiliations
- University of Louisville (US)
- Corresponding author
- Hein, David W.
- ORCID
- Hein, David W.
- Venue
- Scientific Reports
- Published
- 2020-05-05
- Volume
- 10
- Issue
- 1
- Pages
- 7566-7566
- ISSN
- 2045-2322 (ISSN-L)
- PMC
- PMC7200704
- OpenAlex
- W3023931426
- Cited by
- 21
- References
- 57
- Citations by year
- 2026: 4 · 2025: 3 · 2024: 3 · 2023: 1 · 2022: 8 · 2021: 2
- FWCI
- 2.43
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Scientific Reports (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Funders
- National Institute on Alcohol Abuse and Alcoholism (P50AA024337)
- National Institute of General Medical Sciences (P20GM113226)
- Topics
- Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmacology › Pharmacogenetics and Drug Metabolism
- Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmacology › Drug-Induced Hepatotoxicity and Protection
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Glutathione Transferases and Polymorphisms
- Keywords
- arylamine N-acetyltransferase 2, NAT2 genotype, NAT1, cryopreserved human hepatocytes, antibody specificity, sulfamethazine, acetylator phenotype, genetic polymorphism
- MeSH terms
- Arylamine N-Acetyltransferase, Cells, Cultured, Enzyme Activation, Genotype, Humans, Isoenzymes, RNA, Messenger, Gene Expression, Cryopreservation, Polymorphism, Single Nucleotide, Hepatocytes
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