ReviewContributions of DNA repair and damage response pathways to the non-linear genotoxic responses of alkylating agents
Introduction
Under current human health risk assessment practices, DNA-reactive agents are generally considered by regulatory agencies to have no thresholds for biological outcomes such as mutation and cancer [1]. The debate surrounding the linearity of low-dose effects related to genotoxicity and cancer has been on-going for decades. New understanding in biological mechanism and mode-of-action (MOA), along with new high-content and high-throughput approaches, and increasingly sensitive analytical methods, bring new evidence into this debate. New in vivo and in vitro data have demonstrated the existence of non-linear/bilinear dose–responses for genotoxic effects (i.e., a dose–response curve with a slope not significantly different from zero gradient below the estimated threshold or Break Point Dose (BPD)), where there is no significant difference in mutant frequency between the spontaneous background of control and the low-dose exposure region of DNA-reactive agents [2], [3], [4], [5], [6]. In recent years, new statistical approaches have also been developed and applied to analyze low-dose results to establish whether the dose–response is linear or non-linear/bilinear, derive a point of departure (PoD), and determine what impact the spontaneous background genotoxicity should have on risk assessment. These compelling, empirical dose–response data do not address the biological underpinnings of mutation at low-dose exposures per se and require focused investigations of the MOA behind these non-linear/bilinear dose–responses. For an expressed mutation, several key events must occur from the initial DNA adduct formation, including insufficient adduct repair, DNA replication and cell division. Moreover, endogenous DNA adducts are now recognized to be ubiquitously present at quantifiable levels in all living tissues. This new perception of the background exposome is shifting perspective on what is normal vs. adaptive vs. adverse [7], [8]. This review discusses the current understanding of biological, mechanistic processes that explain these PoDs, specifically DNA repair and DNA damage response, and complex interactions between these pathways. The detailed discussion presented here was initiated during a Society of Toxicology 2013 workshop entitled the Biology of the Low-Dose Response for DNA-Reactive Chemicals. A clear focus on molecular and biological approaches to defining and understanding consequences of DNA damage at the cellular level fits well with the 2007 NRC report, Toxicity Testing in the 21st Century: A Vision and A Strategy that envisions a future in which all routine testing will use cell-based in vitro assays of toxicity pathways [9], [10].
The genome continuously undergoes damage due to numerous stressors and to the limited DNA chemical stability. Even in the absence of any significant exogenous exposure, mammalian cells sustain thousands of pro-mutagenic DNA lesions every day. Normal metabolic processes are associated with hydrolysis, deamination, alkylation, and oxidation, resulting in base damage, single strand breaks (SSB), double strand breaks (DSB), and interstrand cross-links [20], [21], [22], [23]. Under normal conditions, the steady state level of endogenous DNA damage was recently estimated at ≥50,000 lesions per cell; the non-instructional and pro-mutagenic abasic sites are the most common DNA lesions, present daily at ∼30,000 nucleoside sites in DNA per cell [18], [22], [24]. DNA repair influences the outcome and dose–response of mutation and chromosome damage following exposure to DNA damaging agents at all exposure levels [11], [12], [13], [14], [15]. DNA repair is usually error-free, but there may be rare events where the mis-repair will result in genotoxic outcomes. Under certain conditions or at high exposure doses, DNA repair itself can increase mutation [89], [90], [91], [92]. Thousands of times per day, in every cell, DNA lesions are repaired by an integrated defense network that includes five major DNA repair arms: base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), non-homologous end-joining of double strand breaks (NHEJ) and homology-directed repair of DSB, cross-links and broken replication forks (homologous repair; HR). Among these repair activities, BER is considered to be one of the most active pathways, handling thousands of DNA lesions every day such as the major alkylation adducts, oxidized bases, deaminated bases, abasic sites, and SSB and nicks.
This significant and ubiquitous background of pro-mutagenic DNA damage is likely causative for the normal range of background mutations [5], [18]. Recent work has begun addressing the potential role of endogenous/background DNA damage in background mutagenesis [5], [6], [8], [16], [17], [18], [19]. The Engelward laboratory developed a sensitive mouse model in which HR events at an integrated Fluorescent Yellow Direct Repeat (FYDR) transgene give rise to a fluorescent signal. This model provided a clearer understanding of HR background activity, effects due to aging, and HR response after exposure to exogenous agents. This model demonstrated that background rearrangement events in mice accumulate with age at individual rates in different cells and within different tissues (Fig. 1) [17], [19].
Section snippets
New methods to investigate responses at low-dose exposures
New understanding of, and new techniques for measuring, the many ways in which normal cells handle DNA damage have led to consideration of the relationship between low-dose DNA damage and DNA repair, in an effort to understand how these processes contribute to cellular homeostasis. This revived interest has resulted in significant efforts to collect low-dose data on dose–response for genotoxic effects and to develop interpretive biological models for those observed dose–response behaviors for
Endogenous and exogenous sources
Alkylating agents are ubiquitous in the environment and within living cells. Endogenous DNA alkylation adducts are considered to be the major contributor to the total background levels of all DNA adducts present at steady-state levels in cells [5], [8], [18], [37]. Endogenous alkylating DNA adducts can arise from several different sources, for example from metabolic activity of gut bacteria, or as byproducts of lipid peroxidation, or reacting with cellular methyl donors such as S
DNA repair and break point doses
Manipulations of DNA repair activity have been known to influence genotoxicity and cancer predisposition. Two prototypical types of DNA-reactive agents are discussed below.
Interactions between DNA repair and DDR pathways
The realization that complex interactions exist between different DNA repair pathways is an important development. These complex biological relationships can combine to manifest as the non-linear dose–response for genotoxic effects.
Profiling of biological pathways and genotoxic dose–response relationships
High-throughput and high-content assays can inform on chemical-specific perturbations of toxicity pathways. Cells respond to physical and chemical stressors by activating signal transduction cascades that can lead to various cellular outcomes or even cell death. These biological outcomes can be analyzed together and modeled in order to predict the BPD for mutational outcomes in relation to activation of various biological pathways.
Conclusions
There is significant interest in understanding the contribution of biological mechanisms to the non-linear/bilinear dose–response curves for DNA-reactive agents. Model monofunctional alkylating agents have datasets amenable to PoD determination for genotoxic effects in both in vitro and in vivo tests systems; these findings were supported by robust statistical analysis of in vitro and in vivo datasets [27], [28], [29], [30]. Such new experimental and computational approaches will help develop
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
The authors are grateful to Dr. B.B. Gollapudi for his support and contributions during development of the workshop and this manuscript.
This work was supported by National Institutes of Health (NIH) (http://www.nih.gov) grant R01-CA079827; U01-ES016045 with partial support from R21-ES019498 (to B.P.E). The MIT Center for Environmental was funded by NIH grant P30-ES002109 (to B.P.E). This work was also supported by Unilever Safety & Environmental Assurance Centre, UK (to M.E.A, R.A.C, Y.A. and
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