PubMed · 8753629
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M Koizumi, K Kimura, T Toyota. 1995. [BT-PABA test].. https://pubmed.ncbi.nlm.nih.gov/8753629/
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Skin is the major target of allergic reactions to paraphenylenediamine (PPD). Such small molecules require activation to become immunogenic. The balance between activation and/or detoxification processes is critical for immunogenic potentials of compounds. Therefore, we investigated N-acetylation (NAT) capacities of human skin for PPD to gain a better understanding of its mechanisms of action. PPD is acetylated to monoacetyl-PPD (MAPPD), which in turn is acetylated to N,N'-diacetyl-PPD (DAPPD). This was found using cytosolic fractions from human skin (n = 9) and cultured normal human epidermal keratinocytes (n = 7). The cutaneous activities for MAPPD formation ranged from 0.41 to 3.68 nmol/mg/min (9-fold variation) and DAPPD formation from 0.65 to 3.25 nmol/mg protein/min (5-fold), respectively. Similar results were obtained with keratinocytes. NAT activities toward both substrates, PPD and MAPPD, were correlated in keratinocytes (r = 0.930), suggesting that the reactions were catalyzed by the same enzyme. Formation of MAPPD and DAPPD was competitively inhibited in the presence of p-aminobenzoic acid (300 microM), a typical NAT1 substrate, but not by sulfamethazine. These kinetic characteristics suggest that the acetylation of PPD in human skin and keratinocytes is predominantly attributable to the polymorphic NAT1, although both mRNAs (NAT1 and NAT2) are synthesized in human skin and keratinocytes. The metabolism of PPD by NAT1 in human skin and keratinocytes as well as the virtual absence of NAT2 activity may have important toxicological implications. In the case of PPD, our results emphasize that N-acetylation status may be a susceptibility factor for the development of an allergy to PPD.
Arylamine N-acetyltransferase 1 (NAT1) conjugates several aromatic amines and their N-hydroxylated metabolites by N- or O-acetylation. NAT1 genotype and phenotype is known to be variable in human populations. In this study, we set out to measure the functional relevance of the frequent NAT1 gene variants for the activity in human red blood cells. Healthy German volunteers (N = 314) were genotyped for NAT1 alleles *3, *4, *10, *11, *14, and *15 using polymerase chain reactions and restriction fragment length pattern analysis, and NAT1 enzyme kinetic parameters were measured in a subset of 105 individuals using p-aminobenzoic acid as specific substrate. There was no functional difference between NAT1 alleles *4 and *10. In particular, there was no trend of increasing activity from NAT1*4/*4 to *4/*10 and *10/*10. Carriers of the NAT1 *11 and *14 alleles had a statistically significant lower enzyme activity compared with carriers of the *3, *4, or *10 alleles. Compared with the wild-type genotype NAT1*4/*4, activity of the NAT1*11/*11, NAT1*11/*10, and NAT1*11/*4 genotypes was reduced by 20.7%, 35.7%, and 31.5%, respectively. Activity of the NAT1*10/*14 and NAT1*4/*14 genotypes was reduced by 49.8% and 55.6%, respectively. The difference in NAT1 activity between the *4/*11 and *4/*14 genotypes was also significant (P < 0.01). The carrier of the NAT1*15/*15 genotype had no detectable enzyme activity. In conclusion, functional consequences of NAT1 mutations were tested in a large population. Activity in carriers of NAT1 alleles *3, *4, and *10 did not differ, alleles NAT1*11 and *14 appeared to be low activity alleles, and allele NAT1*15 had no activity.
Mice have three arylamine N-acetyltransferase (NAT) isoenzymes (NAT1, NAT2, and NAT3) of which NAT2 is known to be polymorphic. Humans have two polymorphic isoenzymes, NAT1 and NAT2. The isoenzymes mouse NAT1 and human NAT2 are expressed predominantly in the liver and intestine and are involved in drug and xenobiotic metabolism. Mouse NAT2 and human NAT1 have a widespread tissue distribution and the folate catabolite p-aminobenzoylglutamate (pAB-Glu) has been proposed as a candidate endogenous substrate. All mice have detectable NAT2 activity, although inbred mouse strains have either a fast or slow acetylator phenotype conferred by the presence of either NAT2*8 (fast) or NAT2*9 (slow) alleles at the NAT2 locus. In this report, we describe a simple method for distinguishing these murine alleles by polymerase chain reaction followed by restriction fragment length polymorphism analysis. We compared the tissue distribution of the acetylation activity found in both fast (C57BL/6J) and slow (A/J) acetylating strains of mice using pAB-Glu and p-aminobenzoic acid as probe substrates. It has previously been demonstrated that murine NAT2 is expressed in the neural tube prior to closure (Stanley L, Copp A, Rolls S, Smelt V, Perry VH and Sim E, Teratology 58: 174-182, 1998). We demonstrate here that murine NAT2 is expressed in preimplantation embryonic stem cells. Murine NAT2 is likely to be expressed prior to neurulation and this may be important in view of the protective role of folate in neural tube development.