Chicken Fetal Liver DNA Damage and Adduct Formation by Activation-Dependent DNA-Reactive Carcinogens and Related Compounds of Several Structural Classes

Abstract

The chicken egg genotoxicity assay (CEGA), which utilizes the liver of an intact and aseptic embryo-fetal test organism, was evaluated using four activation-dependent DNA-reactive carcinogens and four structurally related less potent carcinogens or non-carcinogens. In the assay, three daily doses of test substances were administered to eggs containing 9-11-day-old fetuses and the fetal livers were assessed for two endpoints, DNA breaks using the alkaline single cell gel electrophoresis (comet) assay and DNA adducts using the (32)P-nucleotide postlabeling (NPL) assay. The effects of four carcinogens of different structures requiring distinct pathways of bioactivation, i.e., 2-acetylaminofluorene (AAF), aflatoxin B1 (AFB1), benzo[a]pyrene (B[a]P), and diethylnitrosamine (DEN), were compared with structurally related non-carcinogens fluorene (FLU) and benzo[e]pyrene (B[e]P) or weak carcinogens, aflatoxin B2 (AFB2) and N-nitrosodiethanolamine (NDELA). The four carcinogens all produced DNA breaks at microgram or low milligram total doses, whereas less potent carcinogens and non-carcinogens yielded borderline or negative results, respectively, at higher doses. AAF and B[a]P produced DNA adducts, whereas none was found with the related comparators FLU or B[e]P, consistent with comet results. DEN and NDELA were also negative for adducts, as expected in the case of DEN for an alkylating agent in the standard NPL assay. Also, AFB1 and AFB2 were negative in NPL, as expected, due to the nature of ring opened aflatoxin adducts, which are resistant to enzymatic digestion. Thus, the CEGA, using comet and NPL, is capable of detection of the genotoxicity of diverse DNA-reactive carcinogens, while not yielding false positives for non-carcinogens.

Modelling foetal exposure to maternal smoking using hepatoblasts from pluripotent stem cells

“…To date, the effect of maternal smoking on the foetal liver has been studied in vitro using cell lines, primary tissue and animal models (Neumann 1986 ; Rao et al 1988 ; Cho et al 2006 , Choi et al 2015 ; Baxter 2009 ; Sanchez et al 2011 ; Van Kesteren et al 2013 ; Williams et al 2014 ). While these models have proven to be informative, the scarcity of human tissue, the rapid loss of cell phenotype, batch-to-batch variation and species differences have led to difficulties in data extrapolation toward the human.…”

The urinary bladder carcinogen propoxur does not produce genotoxic effects in the urinary bladder of Wistar male rats

“…Based on the obtained mean comet value from each of the dose groups was then compared with corresponding negative (vehicle) control group, and a ratio value was obtained, which reflected the fold increase over the control value. Presently, within a limited data set of comet values, a working ratio value threshold for positive response was found to be >1.7-fold increase of the high dose percent DNA in the comet tail over the ratio value of the vehicle control (Williams et al, 2014).…”

Structure-Activity Relationships for DNA Damage by Alkenylbenzenes in Turkey Egg Fetal Liver

“…The standard comet assay as previously reported by Tice and Strauss (1995) was performed to assess DNA strand breaks according to OECD guidelines (OECD, 2014). The protocol previously described in detail (Williams et al, 2014) was used. DNA migration was analyzed by fluorescence microscopy using a Nikon OPTIPHOT microscope.…”

“…The endpoints of the TEGA include the alkaline single cell gel electrophoresis (comet) assay which detects DNA strand breaks and the nucleotide 32 P-postlabeling (NPL) assay which detects DNA adducts. This model is similar to the Chicken Egg Genotoxicity Assay which has been successfully used for the evaluation of chemical-induced genotoxic events and proven to be a reliable tool, capable of detection of the genotoxicity of diverse DNA-reactive carcinogens, while not yielding false-positive results for noncarcinogens (Williams et al, 2014). The detection of various DNA-reactive carcinogens that require bioactivation is possible in chicken and turkey fetal and 1 0 -hydroxylation and epoxidation of the allyl side-chain become major routs of metabolism.…”