Screening of molecular cell targets for carcinogenic heterocyclic aromatic amines by using CALUXA® reporter gene assays

Heterocyclic aromatic amines (HCAs) are compounds formed when meat or fish are cooked at high temperatures for a long time or over an open fire. To determine which pathways of toxicity are activated by HCAs, nine out of the ten HCAs known to be carcinogenic in rodents (2-amino-9H-pyrido[2,3-b]indole (A alpha C), 2-aminodipyrido[1,2-a:3',2-d]imidazole (Glu-P-2), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA alpha C), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2)) were tested in the estrogen receptor alpha (ER alpha), androgen receptor (AR), glucocorticoid receptor (GR), peroxisome proliferator-activated receptor gamma 2 (PPAR gamma 2), polycyclic aromatic hydrocarbons (PAH), Nrf2, and p53 CALUXA (R) reporter gene assays. Trp-P-1 was the only HCA that led to a positive response in the ER alpha, PPAR gamma 2, and Nrf2 CALUXA (R) assays. In the PAH CALUXA (R) assay, Trp-P-2, MeA alpha C, and A alpha C induced luciferase activity to a greater extent than MeIQ and PhIP. In the p53 CALUXA (R) assay without a coupled metabolic activation, only Trp-P-1 and Trp-P-2 enhanced luciferase expression; when a metabolic activation step was coupled to the p53 CALUXA (R) assay, Trp-P-1, Glu-P-2, MeIQ, MeIQx, and PhIP induced a positive response. No HCA was positive in the AR and GR CALUXA (R) assays. Taken together, the results obtained show that the battery of CALUXA (R) assays performed in the present study can successfully be used to screen for molecular cell targets of carcinogenic compounds such as HCAs.