Metabolic activation of diesel exhaust carcinogens in primary and immortalized human TP53 knock‐in (Hupki) mouse embryo fibroblasts

Abstract

Approximately 50% of human tumors have a mutation in TP53. The pattern and spectra of TP53 mutations often differ between cancer types, perhaps due to different etiological factors. The Hupki (human TP53 knock-in) mouse embryo fibroblast (HUF) immortalization assay is useful for studying mutagenesis in the human TP53 gene by environmental carcinogens. Prior to initiating an immortalization assay, carcinogen treatment conditions must be optimized, which can require a large number of cells. As primary HUF cultures senesce within 2 weeks, restricting their use, we investigated whether immortalized HUFs retaining wild-type TP53 can be surrogates for primary HUFs in initial treatment optimization. DNA damage by eight compounds found in diesel exhaust, benzo[a]pyrene, 3-nitrobenzanthrone, 1-nitropyrene, 1,3-dinitropyrene, 1,6-dinitropyrene, 1,8-dinitropyrene, 6-nitrochrysene, and 3-nitrofluorene, was assessed by (32) P-postlabeling and the alkaline comet assay in primary HUFs and in an immortal HUF cell line J201. For most compounds, higher levels of DNA adducts accumulated in J201 cells than in primary HUFs. This difference was not reflected in the comet assay or by cell viability changes. Experiments in three additional immortal HUF cell lines (AAI49, U56, and E2-143) confirmed strong differences in DNA adduct levels compared with primary HUFs. However, these did not correlate with the protein expression of Nqo1 or Nat1/2, or with gene expression of Cyp1a1 or Cyp1b1. Our results show that using immortal HUFs as surrogates for primary HUFs in genotoxicity screening has limitations and that DNA adduct formation is the best measure of genotoxicity of the nitro-polycyclic aromatic hydrocarbons tested in HUFs.

Comparison of the metabolic activation of environmental carcinogens in mouse embryonic stem cells and mouse embryonic fibroblasts

“…We selected a variety of environmental carcinogens of different chemical classes where the metabolism is well studied and characterised. The cell culture test conditions were based on previous studies using these carcinogens in mammalian cells ( Arlt et al, 2007; Hockley et al, 2008; Kucab et al, 2012; Simoes et al, 2008 ). We used carcinogen-DNA adduct formation as a surrogate measure of the relevant XME activity as all tested environmental carcinogens induce specific and structurally-identified DNA adducts which can be detected by the 32 P-postlabelling assay ( Schmeiser et al, 2013 ).…”

“…Probes (Life Technologies, UK) used were Mm01253561_m1 ( Cyp1a1 ) and Mm00487218 ( Nqo1 ) and expression levels were normalised to Gapdh (4352341E). Relative gene expression was calculated using the comparative threshold cycle ( C T ) method ( Kucab et al, 2012 ).…”

Characterising Mutational Spectra of Carcinogens in the Tumour Suppressor Gene TP53 Using Human TP53 Knock-in (Hupki) Mouse Embryo Fibroblasts

“…All methods available for DNA adduct detection have their strengths and weaknesses, so the method of choice has to be decided on a case-by-case basis considering the carcinogen to be studied, prior knowledge of the carcinogen-induced DNA adducts formed and the infrastructure available in the host laboratory [26]. Alternative approaches to assess DNA damage (e.g., comet assay) have been considered in HUFs previously [34]. Again, only those concentrations that show a clear induction of DNA damage should be considered for the HIMA.…”

Nitroaromatic compounds: Environmental toxicity, carcinogenicity, mutagenicity, therapy and mechanism

“…DNA damage by eight compounds found in DE, benzo[a] pyrene, 3-NBA, 1-NP, 1,3-dinitropyrene, 1,6-dinitropyrene, 1,8dinitropyrene, 6-NC and 3-nitrofluorene, was assessed (Kucab , 2012). DNA adduct formation is the best measure of genotoxicity of the nitro-PAHs tested (Kucab et al, 2012). Day and night samples were taken at a roadside site and tunnel in China.…”