Contributions of DNA repair and damage response pathways to the non-linear genotoxic responses of alkylating agents

Abstract

From a risk assessment perspective, DNA-reactive agents are conventionally assumed to have genotoxic risks at all exposure levels, thus applying a linear extrapolation for low-dose responses. New approaches discussed here, including more diverse and sensitive methods for assessing DNA damage and DNA repair, strongly support the existence of measurable regions where genotoxic responses with increasing doses are insignificant relative to control. Model monofunctional alkylating agents have in vitro and in vivo datasets amenable to determination of points of departure (PoDs) for genotoxic effects. A session at the 2013 Society of Toxicology meeting provided an opportunity to survey the progress in understanding the biological basis of empirically-observed PoDs for DNA alkylating agents. Together with the literature published since, this review discusses cellular pathways activated by endogenous and exogenous alkylation DNA damage. Cells have evolved conserved processes that monitor and counteract a spontaneous steady-state level of DNA damage. The ubiquitous network of DNA repair pathways serves as the first line of defense for clearing of the DNA damage and preventing mutation. Other biological pathways discussed here that are activated by genotoxic stress include post-translational activation of cell cycle networks and transcriptional networks for apoptosis/cell death. The interactions of various DNA repair and DNA damage response pathways provide biological bases for the observed PoD behaviors seen with genotoxic compounds. Thus, after formation of DNA adducts, the activation of cellular pathways can lead to the avoidance of a mutagenic outcome. The understanding of the cellular mechanisms acting within the low-dose region will serve to better characterize risks from exposures to DNA-reactive agents at environmentally-relevant concentrations.

Keywords: Biological pathways; DNA damage; DNA repair; Dose–response; Low-dose; Points of departure.

Figure 1. Mutant cells accumulate with age in tissues under normal conditions

Wiktor-Brown et al. [19] investigated the effects of aging on the frequency of HR events in the FYDR mice. In pancreas, 23-fold increase in recombinant cell frequency with age was noted in vivo.

Figure 2. Dose-response for endogenous and exogenous methylating adducts

Endogenous (unlabeled) and exogenous (D₃-labelled) N7-MeG and O⁶-MeG were measured after treatment with D₃-MNU in AHH-1 cells (from Sharma et al. [5]). Exogenous N7-MeG adducts did not significantly contribute to the total N7-MeG adduct load under low-dose treatment conditions.

Figure 3. Overlapping DNA repair systems are involved in removal of alkylation DNA adducts

BER and MGMT substrates are also handled by NER. Unrepaired damage or repair intermediates can be funneled to tolerance mechanisms HR, NHEJ or TLS. Black arrows indicate adducts induced in significant proportion by S_N1 alkylating agents only. White arrows indicate adducts induced by both S_N1 and S_N2 alkylating agents. Arrow thickness correlates with the frequency of induced adducts. Adapted from Wyatt and Pittmann [69].

Figure 4. Dose-response curves for mutation and DNA adducts

Exogenous N7-MeG and O⁶-MeG adducts were measured following 24 hour treatment with ¹³C-labelled MMS [62]. Adducts demonstrated linear dose response. Mutation induction at HPRT locus, demonstrating bilinear dose-response, with similarly treated AHH-1 cells was taken from Doak et al. [56].

Figure 5. Repair of alkylated DNA bases by the BER repair pathway and channeling of DNA repair intermediates into the HR pathway during DNA replication

Unrepaired DNA adducts, abasic sites, gaps and DNA nicks in S-phase are handled by the HR pathway.

Figure 6. No detected changes in DNA damage or responses after repeated exposure to low level radiation

After continuous exposure over 5 weeks to 0.0002 cGy/min radiation (400-fold over background radiation), the exposed mice did not demonstrate increased levels of DNA nucleobase damage (hypoxanthine, 8oxoG, 1,N6-ethenoadenine, or 3,N4-ethenocytosine) or DNA fragmentation (MNT assay and double strand break–induced HR) above background levels. In addition, low dose-rate radiation did not induce Cdkn1a, Gadd45a, Mdm2, Atm, or Dbd2 gene expression before and after irradiation (from Olipitz et al. [93]). The same total dose delivered acutely induced both MN and transcriptional responses.

Figure 7. Cellular processing and repair of O⁶-MeG adducts in DNA

MGMT directly repairs O⁶-MeG adducts. If unrepaired, O⁶-MeG preferably mispairs with T during DNA replication leading to G:C-to-A:T transitions. Alternatively, O⁶-MeG lesion induces apoptosis via an MMR-dependent pathway. O⁶-MeG/MMR-dependent DNA damage response includes multi-pathway, multi-time scale signaling network activation led by early ATM, H2AX, ATR-CHK1, and p53 phosphorylation, then followed by late phosphorylation of ATM-CHK2 and JNK kinase, as well as dramatic increases in p53 levels and p53 transcriptional targets [85]. Sister chromatid exchanges (SCE) and chromosomal aberrations are induced by O⁶-MeG lesions via an MMR-dependent pathway in the second cell cycle [102]. The gaps and nicks present during this phase can form DSB that are handled by HR. With loss of MMR, cells become “methylation-tolerant” accumulating mutations and escaping cell death in the presence of unrepaired O⁶-MeG.

Figure 8. Comparison of responses across endpoints for DNA damage response for three DNA-damaging compounds

Values shown are for BMDLs (lower 95% confidence limit for BMD). For each chemical, MN induction (purple) occurred at lower doses than gene transcription changes (orange). With MMS, the BMDL for the transcriptional activation was closest to the MN-BMDL for any of the compounds, but was still greater than the MN-BMDL.

Figure 9. Organization of gene transcriptional changes 24 hr after treating HT-1080 cells with various concentrations of MMS

Cells were treated with up to 500 μM MMS. Responses at key doses are presented. The union of all significantly changed genes was used to assess all GO ontology categories that were significantly enriched at any treatment. This organization provided the structure of categories shown by the various encircled patterns. The colors, green (up-regulated) and purple (down-regulated), show the groupings that were significantly changed at each treatment and the size of circles represents the numbers of genes changed in particular GO-categories. At 100 μM, the transcriptional changes were minimal even though this concentration was 10-fold above the MN-BMDL. The visualization tools were developed in work with nuclear receptors [133, 134].

Figure 10. Computational Model for Threshold Response

A working model explaining threshold responses, demonstrated for MN formation with increasing exposures, has two response pathways – a fast acting, post-translational pathway that works to maintain perfect control (and threshold behaviors) and a transcriptional pathway with p53 tetramer that contributes at much higher levels of damage (thus higher doses), after there is an increase in MN formation.

Figure 11. Time-Course Behaviors of DNA-Repair Centers (DRCs) at low and higher doses

Top: Images of DRC foci in control nuclei. Middle section: images of DRCs following treatment with very high doses of NCS. The foci in individual nuclei show the co-location of two repair proteins – p53 binding protein and γ-H2AX. Lower left: dose- and time-response for DRCs (as foci per nucleus) following treatment with NCS. At lower concentrations (left), foci resolve quickly. At higher concentrations, DRCs persist out beyond 24 hrs. The lower doses are in the sub-threshold region for MN-formation and the higher doses are those with increased MN frequencies. Plots are representative of studies reported in other work from the Hamner-Unilever collaboration [135].