Exposure to Ethylating Agents: Where Do the Thresholds for Mutagenic/Clastogenic Effects Arise?

Abstract

The dose response relationships for induction of micronuclei in bone marrow and for induction of lacZ mutations in liver, large intestine, and bone marrow were assessed in mice after treatment with ethyl methanesulfonate (EMS). The animals were treated orally at doses between 1.25 to 260 mg/kg for up to 28 days. Statistical analysis indicated that the dose response curves were well compatible with a thresholded relationship with apparent thresholds AE25 mg/kg/day. In contrast, no threshold was apparent for the induction of alkylation-adducts in proteins and DNA. An approximately linear dose dependency was observed, indicating that the macromolecular targets were alkylated proportionally to the given dose. It is concluded that the cells have the capacity to repair large amounts of EMS-induced alkylations, up to the threshold dose, virtually error free (i.e., without adding to the background burden of clastogenic/mutagenic events). Since the statistical power to deˆne the`true' shape of the dose response curve is much more limited for in vivo studies compared to in vitro experiments with e.g., a bacterial reverse mutation system we assessed the dose response relationship for EMS induced mutations at the his G46 locus in alkylation repair proˆcient (TA1535, ogt ＋) and deˆcient (YG7104, Dogt) bacteria using as many as 23 dose levels. Clear sublinearity was apparent for TA1535 while linearity was obvious for YG7104. Applying curveˆtting with a`hockeystick' model a threshold dose of about 750 mg/plate was determined for TA1535. With curveˆtting using a benchmark model (PROAST) we estimated the e‹ciency of error-free repair by the ogt system at the diŠerent EMS exposures. The likelihood of erroneous repair approaches 0 with decreasing EMS concentrations. Together with recent studies on other alkylating agents our data argue for a change of paradigm concerning risk assessment of the exposure to simple DNA-alkylating genotoxins.

Quantitative assessment of the dose–response of alkylating agents in DNA repair proficient and deficient ames tester strains

“…Since all Salmonella strains used are NER deficient, the removal of alkylation lesions can therefore mainly be attributed to Ogt. Gocke et al have calculated the rate of successfully (error free) repaired DNA lesions by Ogt as a function of EMS concentration [Gocke et al, ]. In brief, following treatment with EMS dose–response curves for the Ames tester strains TA1535 (Ogt+/Ada+) and YG7104 (Ogt−/Ada+) were fitted by Gocke et al using a benchmark dose model (PROAST).…”

“…However, it has been demonstrated that the expression of Ada protein is weakly induced by alkylating agents [Vaughan and Sedgwick, ].Yamada et al have found that Ames tester strains lacking methyltransferases became highly sensitive towards mutagenic alkylating agents [Yamada et al, ]. Recently, Gocke et al calculated the rate of DNA lesions faithfully repaired by alkyltransferases in response to EMS treatment in S. typhimurium [Gocke et al, ]. The value of the calculated repair rate was approximately 100% at dose levels below the observed threshold.…”

“…Other studies investigating the mechanism of genotoxicity tolerance revealed that the activity of DNA repair enzymes (e.g. base excision repair enzymes, alkyltransferases) and protection mechanisms against oxidative stress are the major contributors to the observed thresholds [Doak et al, ; Gocke et al, ; Seager et al, ]. Alkyltransferases are able to repair certain pro‐mutagenic adducts with high fidelity and play a crucial role in the surveillance of the genomic integrity [Zhang et al, ].…”