Impact of Five Cytochrome P450 Enzymes on the Metabolism of two Heterocyclic Aromatic Amines, 2‐Amino‐9<i>H</i>‐pyrido[2,3‐<i>b</i>]indole (AαC) and 2‐Amino‐3‐methyl‐9<i>H</i>‐pyrido[2,3‐<i>b</i>]indole (MeAαC)

2-Amino-9H-pyrido[2,3-b]indole (AαC) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAαC) are two mutagenic and carcinogenic heterocyclic aromatic amines formed during ordinary cooking. AαC and MeAαC are formed as pyrolysis products of tryptophan-rich compounds and are found in cooked food such as meat, chicken and fish and in cigarette smoke condensates (Felton et al. 2002). We have previously studied the in vitro metabolism of tritium labelled AαC and MeAαC in hepatic microsomes from human pools, rats induced with polychlorinated biphenyl (PCB) (Aroclor 1254) and control rats. Both detoxified and activated metabolites of AαC and MeAαC were separated and characterised. AαC is metabolised to two major and three minor detoxified metabolites, while MeAαC is metabolised to three major and one minor detoxified metabolites. An amount of both AαC and MeAαC are activated, by oxidation to the reactive metabolites N2-OH-AαC and N2-OH-MeAαC, respectively. These reactive N2-OH-metabolites reacts partially in the incubation system with the formation of protein adducts, dimers and the parent compound by reduction of the N2-OH-metabolites. The distribution between the detoxified and activated metabolites in the different types of hepatic microsomes showed same pattern for both AαC and MeAαC. In PCB-induced rat microsomes, the major part of the metabolites are detoxified (about 90%); only a little amount is activated. In the control rat microsomes there are a fifty, fifty distribution between detoxification and activation, while the major part (about 60%) of the metabolites from the human microsomes are activated and reacts reacted to form dimers and protein adducts (Frederiksen & Frandsen 2002). In the present study we have investigated the impact of five cytochrome P450 enzymes on the metabolism of AαC and MeAαC. Five different types of supersomes from GENTEST Corporation (BD Biosciences, USA) were used instead of microsomes. The expressed enzymes in the supersomes were human CYP1A1, human CYP1A2, human CYP3A4, rat CYP1A1 and rat CYP1A2. AαC and MeAαC were obtained from Toronto Research Chemicals (Toronto, Canada). Tritiation of AαC and MeAαC and enzymatic incubations were performed as previously described (Frandsen et al. 1998). The incubation mixture consisted of 2 mg/ml supersome protein, 1 U/ml DL-isocitrate dehydrogenase, 0.5 mM NADP+, 10 mM sodium DL-isocitrate and 5 mM MgCl2 in 50 mM MOPS, 0.15 mM KCl, pH 7.4. One hundred and fourty μl mixture was preincubated for 5 min. at 37 ° before addition of 10 μl test substance ([3H]-AαC or [3H]-MeAαC, 40 μg/ml dissolved in DMF). Aliquots of 10 μl were collected after 2, 5, 10, 15, 20 and 30 min. incubation and the reactions were terminated by addition of 10 μl ice-cold argon-purged ethanol. After centrifugation the supernatants were isolated and analysed by HPLC-MS. Pellet from the last part of the incubation mixture were analysed by liquid scintillation counting. The analytical methods as well as the syntheses of N2-OH-derivatives used for comparison are previously described (Frederiksen & Frandsen 2002). Assays were repeated at least three times with similar results. The impact of the cytochrome P450 enzymes towards AαC and MeAαC and the product distribution of AαC- and MeAαC-metabolites after 30 min. incubation are shown in table 1. Human CYP3A4 showed no catalytic activity towards AαC and MeAαC and the level of unmetabolised compound was constant in all collected aliquots (data not shown). Human CYP1A1 showed very low enzyme activity. Human CYP1A2 and rat CYP1A2 showed 2–5 times higher activity in the AαC incubations, than in the MeAαC incubations, on the contrary rat CYP1A1 showed highest activity towards MeAαC. In the assays containing human CYP1A1, only 2% of both AαC and MeAαC were metabolised and activated metabolites were not detected in these incubations. The major metabolites of AαC and MeAαC were 6-OH-AαC and 3-CH2OH-AαC, respectively. In assays with AαC incubated with human CYP1A2, there was a high degree of total metabolism (63.5%). Detoxified metabolites, 3-OH-AαC and 6-OH-AαC were found in equal amounts. About 27% of AαC was activated, 25% was detected as the very reactive N2-OH-AαC and 2% was bound to the protein pellet in the assays. Fig. 1 shows the time course of AαC metabolism over 30 min. catalysed by human CYP1A2. Human CYP1A2 showed a much lower metabolic activity towards MeAαC, only 12.5% was metabolised during 30 min. incubation. Activation seemed to be a minor metabolic pathway; the major metabolite was 3-CH2OH-AαC. AαC metabolised by human CYP1A2. Human CYP1A1 and human CYP3A4 showed very little or no catalytic activity towards AαC, this is in accordance with previous published results (Shimada & Guengerich 1991). In the contrary human CYP1A2 efficiently metabolised AαC mainly to detoxified products, only 25% was activated. None of these three human enzymes were responsible for activation of MeAαC; only 1% was activated by human 1A2. Previously we found that human hepatic microsomes mainly activated AαC and MeAαC (60%) (Frederiksen & Frandsen 2002) indicating that other microsomal enzymes may be involved in the activation of AαC and particularly MeAαC. Rat CYP1A1 efficiently metabolised AαC (45.3%), the major product was the reactive N2-OH-AαC, minor products were the detoxified metabolites 3-OH-AαC and 6-OH-AαC. Rat CYP1A2 metabolised AαC less efficiently (16.0%), major products were the detoxified metabolites 3-OH-AαC and 6-OH-AαC, only 16.0% was metabolised to the reactive N2-OH-AαC. Rat CYP1A1 also efficiently metabolised MeAαC (56.5%), the major product being the detoxified metabolite 3-CH2OH-AαC. Rat CYP1A2 showed low activity towards MeAαC, only 6.5% was metabolised, major products were detoxified metabolites (table 1). Previously, we found a fifty-fifty distribution in activation/detoxification of AαC and MeAαC in rat hepatic microsomes (Frederiksen & Frandsen 2002). The present results show that rat CYP1A1 was the enzyme mainly catalysing the activation and rat CYP1A2 was the enzyme mainly catalysing the detoxification, and rat CYP1A1 has 3–9 times higher catalytic activity than rat CYP1A2. Untreated rats expressed low levels of CYP1A1, <1 pmol/mg microsomal protein, compared with rat CYP1A2, 5–35 pmol/mg microsomal protein (Turesky et al. 1998), which indicates that also in rat microsomes other enzymes than CYP1A are involved in the metabolism. The activation of MeAαC catalysed by human and rat CYP1A was much lower than the activation of AαC, suggesting that the methyl group sterically hinders activation of MeAαC. Other microsomal enzymes than CYP1A may be involved in the activation AαC and particularly MeAαC. This study has been carried out with financial support from the commission of the European communities, specific RTD programme “Quality of life and management of living resources” QLK1-CT99–01197, Heterocyclic amines in cooked foods – Role in human health. It does not necessarily reflect its views and in no way anticipates the commission's future policy in this area.

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.