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

Abstract

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 (AαC), 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 (MeAαC), 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 α (ERα), androgen receptor (AR), glucocorticoid receptor (GR), peroxisome proliferator-activated receptor γ2 (PPARγ2), polycyclic aromatic hydrocarbons (PAH), Nrf2, and p53 CALUX® reporter gene assays. Trp-P-1 was the only HCA that led to a positive response in the ERα, PPARγ2, and Nrf2 CALUX® assays. In the PAH CALUX® assay, Trp-P-2, MeAαC, and AαC induced luciferase activity to a greater extent than MeIQ and PhIP. In the p53 CALUX® 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 CALUX® assay, Trp-P-1, Glu-P-2, MeIQ, MeIQx, and PhIP induced a positive response. No HCA was positive in the AR and GR CALUX® assays. Taken together, the results obtained show that the battery of CALUX® assays performed in the present study can successfully be used to screen for molecular cell targets of carcinogenic compounds such as HCAs.

Combining Different In Vitro Bioassays to Evaluate Genotoxicity of Water-Accommodated Fractions from Petroleum Products

“…Additionally, petroleum WAFs and relevant compounds (PAHs) have induced strong alterations of antioxidant enzyme activities like catalase or superoxide dismutase [53,[56][57][58]. As previously discussed for other mechanism-specific endpoints using in vitro-based assays [35,42], it has to be considered that the U2-OS cells do have limited capability for metabolization [51]. Thus, the toxicity of many petroleum constituents like phenanthrene or benzo[a]pyrene, which occurs after bioactivation during xenobiotic biotransformation [5,6], were not addressed with this transgenic cell line.…”

“…Cytotoxicity was excluded by pretests (MTT assay, see SI, Section S1). Material adaptions of commercial CALUX ® assay procedures [41,42] to WAF sample testing included the usage of glass-coated 96-well plates (WebSeal Plate+, VWR, Darmstadt, Germany) and glass plate covers (details in [35]). 3×-concentrated assay medium prepared from cell culture medium powder (Sigma, D2902) was supplemented with FCS (charcoal stripped, Biowest, Cholet, France), non-essential amino acids, and penicillin-streptomycin as described in ISO guideline no 1904-3 [43].…”

Chromogenic Escherichia coli reporter strain for screening DNA damaging agents

“…Additionally, single-cell strategies cannot differentiate damage from xenobiotic agents that are not available due to their association with organic matter. The toolbox for assessing genotoxicity in different samples is ample (Johann et al 2020 ), such as cell viability by MTT assays (Mosmann 1983 ), oxidative stressors by Nrf2-CALUX (Steinberg et al 2016 ), micronucleus assay for chromosomal aberrations (ISO 21427-2) and the Ames mutagenicity assay (ISO 11,350).…”

Determination of 14 heterocyclic aromatic amines in meat products using solid‐phase extraction and supercritical fluid chromatography coupled to triple quadrupole mass spectrometry

“…Different conditions can affect the formation of HAs during cooking, such as pH, the cooking time, temperature, content of water, and water‐soluble materials . In recent years, the harm of HAs to human health is a great concern of issue . These kinds of substances have long been associated with cancer morbidity, both in rodents and nonhuman primates .…”