Heterologous expression of human N-acetyltransferases 1 and 2 and sulfotransferase 1A1 in Salmonella typhimurium for mutagenicity testing of heterocyclic amines.
A variety of carcinogenic heterocylic amines (HAs) are found in cooked food. They can be metabolised to reactive intermediates via N-hydroxylation catalysed by cytochrome P450 1A2, followed by conjugation of the resulting N-hydroxyl group by N-acetyltransferase (NAT) or sulfotransferase (SULT). In order to compare the role of O-acetylation and O-sulfonation by human enzymes in the activation of HAs, we have introduced the cDNAs for wild-type forms of human NAT1, NAT2 and SULT1A1 in the acetyltransferase-deficient Salmonella typhimurium strain TA1538/1,8-DNP. Functional expression of recombinant proteins was demonstrated using immunoblot analysis and determination of enzyme activity with characteristic substrates. The established strains were used to study the mutagenicity of the N-hydroxy derivatives of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). The results demonstrate that N-hydroxy-HAs are activated by different human enzymes. At the concentrations used in the mutagenicity assay, N-hydroxy-IQ was activated by human NAT2, but not by NAT1 or SULT1A1. In contrast, N-hydroxy-PhIP was activated specifically by human SULT1A1, but not by NAT1 or NAT2. Therefore, both O-acetylation and O-sulfonation by human enzymes have to be regarded as important determinants for HA genotoxicity in humans.
Muckel
E
E
German Institute of Human Nutrition, Department of Toxicology, Arthur-Scheunert-Allee 114-116, D-14558 Bergholz-Rehbrücke, Germany. muckel@www.dife.de
Frandsen
H
H
Glatt
H R
HR
D000123:Acetyltransferases / Q000235:genetics / Q000378:metabolism*; D000588:Amines / Q000493:pharmacokinetics*; D001191:Arylamine N-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D015239:Arylsulfotransferase; D001711:Biotransformation; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006571:Heterocyclic Compounds / Q000493:pharmacokinetics; D006801:Humans; D007093:Imidazoles / Q000378:metabolism; D015151:Immunoblotting; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D009152:Mutagenicity Tests; D009153:Mutagens / Q000032:analysis* / Q000378:metabolism; D011804:Quinolines / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics*; D015238:Sulfotransferases / Q000235:genetics / Q000378:metabolism*
D000588:Amines; D006571:Heterocyclic Compounds; D007093:Imidazoles; D007527:Isoenzymes; D009153:Mutagens; D011804:Quinolines; C029216:2-amino-3-methylimidazo(4,5-f)quinoline; C049584:2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine; D000123:Acetyltransferases; D001191:Arylamine N-Acetyltransferase; C089384:N-acetyltransferase 1; C478900:NAT2 protein, human; D015238:Sulfotransferases; D015239:Arylsulfotransferase; C117544:SULT1A1 protein, human