Acute Dosing and p53-Deficiency Promote Cellular Sensitivity to DNA Methylating Agents

Abstract

Risk assessment of human exposure to chemicals is crucial for understanding whether such agents can cause cancer. The current emphasis on avoidance of animal testing has placed greater importance on in vitro tests for the identification of genotoxicants. Selection of an appropriate in vitro dosing regime is imperative in determining the genotoxic effects of test chemicals. Here, the issue of dosing approaches was addressed by comparing acute and chronic dosing, uniquely using low-dose experiments. Acute 24 h exposures were compared with equivalent dosing every 24 h over 5-day, fractionated treatment periods. The in vitro micronucleus assay was used to measure clastogenicity induced by methyl methanesulfonate (MMS) and N-methyl-N-nitrosourea (MNU) in human lymphoblastoid cell line, TK6. Quantitative real-time (qRT) PCR was used to measure mRNA level induction of DNA repair enzymes. Lowest observed genotoxic effect levels (LOGELs) for MMS were obtained at 0.7 µg/ml for the acute study and 1.0 µg/ml for the chronic study. For acute MNU dosing, a LOGEL was observed at 0.46 µg/ml, yet genotoxicity was completely removed following the chronic study. Interestingly, acute MNU dosing demonstrated a statistically significant decrease at 0.009 µg/ml. Levels of selected DNA repair enzymes did not change significantly following doses tested. However, p53 deficiency (using the TK6-isogenic cell line, NH32) increased sensitivity to MMS during chronic dosing, causing this LOGEL to equate to the acute treatment LOGEL. In the context of the present data for 2 alkylating agents, chronic dosing could be a valuable in vitro supplement to acute dosing and could contribute to reduction of unnecessary in vivo follow-up tests.

The in vitro ToxTracker and Aneugen Clastogen Evaluation extension assay as a tool in the assessment of relative genotoxic potential of e-liquids and their aerosols

“…As cigarette smoking is known to induce a number of (geno)toxic endpoints ( 29 , 31 , 66 , 70 ), the induction of the p53 response is unsurprising. This suggests that near basal p53 levels may be sufficient in the initial cellular response to the 1R6F-derived samples ( 71 ). Furthermore, activation of Ddit3, involved in the UPR, including endoplasmic reticulum stress, cell cycle arrest and apoptosis, may be caused by the increased oxidative stress-induced protein damage within the system.…”

Investigation of J-shaped dose-responses induced by exposure to the alkylating agent N -methyl- N -nitrosourea

“…The second J-shaped dose-response observed for MNU is presented here for the first time (Figure 2A), with the raw data in tabular form (Appendix A). The full dose-response for the higher dose region is included in a previous publication, with a LOEL at 0.46 µg/ml and a clear dose-dependent mutagenic effect at higher doses [31]. The full dose-response up to 1.1 µg/ml is also included here in a different form (Appendix B) for the purpose of illustrating that the hormetic dose belongs to a broader "J-shaped" dose-response.…”

“…Only the acute, 24h dosing revealed hormetic effects for MNU, whereas the chronic dosing for both MNU and MMS did not give evidence for hormesis [31]. Originally, it was hypothesized that if hormesis were to be identified it would more likely be observed following the chronic dosing.…”

“…The results suggested that functional p53 might be responsible for the J-shaped dose-response for micronucleus frequency observed in the latter cell line. Previous publications have also identified the involvement of p53 in genotoxicity dose-responses for TK6 cells [28,31]. It is unclear whether p53 was responsible for Thomas et al's dose-response; unlike TK6 cells, AHH-1 cells are heterozygous for p53 function due to a base pair substitution at codon 282, with consequences including loss of the G1 checkpoint and delayed apoptotic cell death [37].…”

Multiple-endpoint in vitro carcinogenicity test in human cell line TK6 distinguishes carcinogens from non-carcinogens and highlights mechanisms of action

“…Factors such as the choice of cell type and excessive toxicity from high doses can affect the frequency of misleading positive results from in vitro genotoxicity tests (Fowler et al 2012a(Fowler et al , b, 2014Shah et al 2016). The choice of treatment type, number of test concentrations and timescale in vitro will also impact on the outcome for certain endpoints at low doses (Chapman et al 2015(Chapman et al , 2020.…”