In vitro bioactivation of N-hydroxy-2-amino-α-carboline

Abstract

2-Amino-alpha-carboline (A alpha C) is a mutagenic and carcinogenic heterocyclic amine present in foods cooked at high temperature and in cigarette smoke. The mutagenic activity of A alpha C is dependent upon metabolic activation to N-hydroxy-A alpha C (N-OH-A alpha C); however, the metabolism of N-OH-A alpha C has not been studied. We have synthesized 2-nitro-alpha-carboline and N-OH-A alpha C and have examined in vitro bioactivation of N-OH-A alpha C by human and rodent liver cytosolic sulfotransferase(s) and acetyltransferase(s) and by recombinant human N-acetyltransferases, NAT1 and NAT2. The sulfotransferase-dependent bioactivation of N-OH-A alpha C by human liver cytosol exhibited large inter-individual variation (0.5-75, n = 14) and was significantly higher than bioactivation of N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (N-OH-PhIP). Correlation and inhibition studies suggested that the isoform of sulfotransferase primarily responsible for bioactivation of N-OH-A alpha C in human liver cytosol is SULT1A1. O-Acetyltransferase-dependent bioactivation of N-OH-A alpha C by human liver cytosol also exhibited large inter-individual variation (16-192, n = 18). In contrast to other N-hydroxy heterocyclic amines, which are primarily substrates only for NAT2, both NAT1 and NAT2 catalyzed bioactivation of N-OH-A alpha C. The rate of bioactivation of N-OH-A alpha C by both NAT1 and NAT2 was significantly higher than that for N-OH-PhIP. In rat and mouse liver cytosols, the level of sulfotransferase-dependent bioactivation of N-OH-A alpha C was similar to the level in the high sulfotransferase activity human liver cytosol. The level of O-acetyltransferase-dependent bioactivation of N-OH-A alpha C in rat liver cytosol was also comparable with that in the high acetyltransferase activity human liver cytosol. However, the level of O-acetyltransferase-dependent bioactivation of N-OH-A alpha C in mouse liver cytosol was comparable with that in the low acetyltransferase activity human liver cytosol. In contrast to N-OH-PhIP, bioactivation of N-OH-A alpha C was not inhibited by glutathione S-transferase activity; however, DNA binding of N-acetoxy-A alpha C was inhibited 20% in the presence of GSH. These results suggest that bioactivation of N-OH-A alpha C may be a significant source of DNA damage in human tissues after dietary exposure to AalphaC and that the relative contribution of each pathway to bioactivation or detoxification of N-OH-A alpha C differs significantly from other N-hydroxy heterocyclic or aromatic amines.

Investigation of the genotoxic effects of 2-amino-9H-pyrido[2,3-b]indole in different organs of rodents and in human derived cells

“…The reason for this discrepancy might be that the human lymphoblastoid MCL-5 line is genetically engineered and expresses high levels of various cytochrom P-450 enzymes only [37], whereas HepG2 cells have retained the activities of many different xenobiotic drug metabolizing enzymes, including those involved in the activation and detoxification of HAs (for review see [38]). King et al [19] proposed that sulfotransferase and N-acetyltransferase (NAT) play a key role in the activation of A␣C to DNA-reactive metabolites. However, Glatt and co-workers [39,40] recently showed by use of transformed V79 cells, which constitutively expressed CYP1A2 and human sulfotransferases, that A␣C is converted to genotoxic metabolites whereas negligible effects were observed in lines co-expressing CYP1A2 and NAT.…”

“…To find out if the compound is genotoxic in human derived cells, micronucleus (MN) and SCGE experiments were conducted with two human derived hepatoma cell lines (HepG2 and Hep3B) and with peripheral lymphocytes, respectively. Additionally, the activities of sulfotransferase (SULT) and cytochrome P4501A (CYP1A) were measured in the human hepatoma cell lines, two enzymes which play a key role in the activation of A␣C [17][18][19]. Fig.…”

Sulfation of indoxyl by human and rat aryl (phenol) sulfotransferases to form indoxyl sulfate

“…Sprague-Dawley rats (male) were from Charles River (Wilmington, MA, USA) and handled with IACUC approval. Human and rat liver cytosolic fractions (1OOOOOg supernatant) were prepared by differential centrifugation in our laboratory as described previously (4). Cytosolic fractions were stored in 1 ml aliquots at -80°C and, after thawing, were kept at 4°C.…”

“…These metabolic reactions are catalyzed by sulfotransferases that involve the transfer of a sulfuryl group from 3' -phosphoadenosine 5' -phosphosulfate (PAPS) to acceptor molecules, thereby producing more water soluble and often less toxic metabolites. Although it is generally true for the sulfation of molecules to aid excretion from the body, sulfonation of certain allylic alcohols and polycyclic aromatic hydrocarbons bearing benzylic alcohol groups and sulfonation of N-hydroxy arylamines may produce metabolites with increased toxicity that cause mutagenic and carcinogenic responses (2)(3)(4). Therefore, sulfate conjugation may act as an important metabolic activation reaction for certain molecules, as well as a detoxication reaction.…”

Blue cotton, Blue Rayon and Blue Chitin in the analysis of heterocyclic aromatic amines—a review

“…Epidemiological studies point to a relationship between the intake of foods containing HCAs and cancer, but there are also studies showing no correlation [7]. HCA metabolites can bind to DNA, RNA and proteins by covalent bonding, and such adducts have been found in almost all kinds of tissue in studies on rodents and non-human primates [8], and even if only a small proportion of the HCA metabolites form adducts, this is presumably enough to induce tumours or other toxic effects.…”