Understanding the importance of low-molecular weight (ethylene oxide- and propylene oxide-induced) DNA adducts and mutations in risk assessment: Insights from 15 years of research and collaborative discussions

Abstract

The interpretation and significance of DNA adduct data, their causal relationship to mutations, and their role in risk assessment have been debated for many years. An extended effort to identify key questions and collect relevant data to address them was focused on the ubiquitous low MW N7-alkyl/hydroxyalkylguanine adducts. Several academic, governmental, and industrial laboratories collaborated to gather new data aimed at better understanding the role and potential impact of these adducts in quantifiable genotoxic events (gene mutations/micronucleus). This review summarizes and evaluates the status of dose-response data for DNA adducts and mutations from recent experimental work with standard mutagenic agents and ethylene oxide and propylene oxide, and the importance for risk assessment. This body of evidence demonstrates that small N7-alkyl/hydroxyalkylguanine adducts are not pro-mutagenic and, therefore, adduct formation alone is not adequate evidence to support a mutagenic mode of action. Quantitative methods for dose-response analysis and derivation of thresholds, benchmark dose (BMD), or other points-of-departure (POD) for genotoxic events are now available. Integration of such analyses of genetox data is necessary to properly assess any role for DNA adducts in risk assessment. Regulatory acceptance and application of these insights remain key challenges that only the regulatory community can address by applying the many learnings from recent research. The necessary tools, such as BMDs and PODs, and the example datasets, are now available and sufficiently mature for use by the regulatory community. Environ. Mol. Mutagen. 60: 100-121, 2019. © 2018 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.

Keywords: DNA adducts; N7-alkyl/hydroxyalkylguanine adducts; dose-response; genotoxic effects; mutations.

Figure 1

Structures of guanine‐cytosine base pairs and ethylene oxide (EO)‐induced adducts: N7‐HEG and O⁶‐HEG (S_N2 reaction). Disrupted hydrogen bonding (− − ‐) shown for O⁶‐HEG, which is unaffected for N7‐HEG. N7‐HEG adducts comprise the majority (>95%) of EO‐induced adducts.

Figure 2

Schema of DNA damage types and corresponding repair pathways. The present paper focuses on alkylating damage: repair of alkyl lesions involves Direct Reversal (DR) pathways (via ABH (AlkB Homolog) or via methylguanine methyltransferase (MGMT)) and Base Excision Repair (BER; AAG glycosylase); the example of Guanine shows the positions repaired by Direct Reversal in red and the positions repaired by BER in blue. Pathways not classically involved in alkylating damage repair are shown to highlight overlap among pathways: for instance, mismatch repair (MMR) is involved in O⁶‐alkylguanine metabolism leading to toxic repair intermediates. Alkylating adducts bulkier than methyl groups can also be repaired by nucleotide excision repair (NER).

Figure 3

Reprinted (with permission) from Doak et al., 2007: “Figure 1. Influence of MMS (A), EMS (B), MNU (C), and ENU (D) dose upon micronucleus frequency in the AHH‐1cell line. Points, mean of treatments done in duplicate; bars, SD. *, the first statistically significant increases in chromosome damage at 0.85 μg/mL MMS (A), 1.40 μg/mL EMS (B), 0.15 μg/mL MNU (C), and 0.50 μg/mL ENU (D); %Mn/Bn, percentage of binucleated cells containing one or more micronuclei.”

Figure 4

Reprinted (with permission) from Swenberg et al., 2008: “Figure 8. Comparison of N7‐methyl guanine DNA adducts and HPRT mutations in AHH‐1 cells exposed to MMS for 24 h. The endogenous adducts are N‐7Me‐G (⋄), while the exogenous adducts are [¹³C²H₃]‐7Me‐G (♦). The Hprt mutant frequency is shown as (○).”

Figure 5

Reprinted (with permission) from Sharma et al., 2014: “Figure 1. Endogenous versus exogenous adducts in AHH‐1 cells exposed to D3‐MNU (0.0075 μM to 2.5 μM) for 1 h. The endogenous and exogenous O⁶‐me‐dG and N7‐me‐G adducts at each exposure concentration are plotted on a log versus log scale. Data represent the mean ± SD.”

Figure 6

Reprinted (with permission) from Tompkins et al., 2008. “Figure 4. HPLC chromatogram demonstrating the separation of hydroxyethyl (HE) nucleoside adducts from unmodified nucleosides, using the conditions employed for the isolation of nucleoside‐HE adducts. Synthetic adduct standards have been used to produce this trace for illustrative purposes; in a typical digested DNA sample HE adducts would not usually be present at detectable levels and fractions would be collected on the basis of retention time.”

Figure 7

Reprinted (with permission) from Marsden et al., 2009. “Figure 2. Contribution of endogenously and exogenously derived N7‐HEG to the total adduct level in tissues of [¹⁴C]EO‐treated rats. Endogenous adducts were determined by LC–MS/MS. Exogenous ¹⁴C‐labelled adducts were quantified by AMS and are shown as black bars on top of bars representing endogenous adduct levels. Columns, mean of three animals per group; bars, SD. *, P < 0.05, the level of endogenous N7‐HEG in tissues of [¹⁴C]EO‐treated rats is significantly higher than the corresponding background level in control animals; **, P < 0.05, the total level of adducts (endogenous plus exogenous) is significantly higher than the level of endogenous adducts alone in a particular tissue.”

Figure 8

Schematic representation of theoretical dose–response curves for Biomarkers of (A) Exposure (DNA adducts) and (B) (Genotoxic) Effect (Mutations). Hashed regions represent background levels. Exogenous Compounds Requiring Metabolic Activation. Exogenous Reactive Direct‐Acting Compounds. Endogenous Reactive Direct‐Acting Compounds.

Figure 9

Reprinted with permission from Gocke and Müller, 2009, “Fig. 3. Frequencies of micronuclei as function of ethylvaline levels. MN‐PCE values are plotted for each mouse against the ethylvaline levels. Independent linear regression lines were fitted for animals receiving ≤80 mg/(kg day) and for animals receiving ≥80 mg/(kg day).”