Quantitative dose–response analysis of ethyl methanesulfonate genotoxicity in adult gpt‐delta transgenic mice

Abstract

The assumption that mutagens have linear dose-responses recently has been challenged. In particular, ethyl methanesulfonate (EMS), a DNA-reactive mutagen and carcinogen, exhibited sublinear or thresholded dose-responses for LacZ mutation in transgenic Muta™Mouse and for micronucleus (MN) frequency in CD1 mice (Gocke E and Müller L [2009]: Mutat Res 678:101-107). In order to explore variables in establishing genotoxicity dose-responses, we characterized the genotoxicity of EMS using gene mutation assays anticipated to have lower spontaneous mutant frequencies (MFs) than Muta™Mouse. Male gpt-delta transgenic mice were treated daily for 28 days with 5 to 100 mg/kg EMS, and measurements were made on: (i) gpt MFs in liver, lung, bone marrow, kidney, small intestine, and spleen; and (ii) Pig-a MFs in peripheral blood reticulocytes (RETs) and total red blood cells. MN induction also was measured in peripheral blood RETs. These data were used to calculate Points of Departure (PoDs) for the dose responses, i.e., no-observed-genotoxic-effect-levels (NOGELs), lower confidence limits of threshold effect levels (Td-LCIs), and lower confidence limits of 10% benchmark response rates (BMDL10 s). Similar PoDs were calculated from the published EMS dose-responses for LacZ mutation and CD1 MN induction. Vehicle control gpt and Pig-a MFs were 13-40-fold lower than published vehicle control LacZ MFs. In general, the EMS genotoxicity dose-responses in gpt-delta mice had lower PoDs than those calculated from the Muta™Mouse and CD1 mouse data. Our results indicate that the magnitude and possibly the shape of mutagenicity dose responses differ between in vivo models, with lower PoDs generally detected by gene mutation assays with lower backgrounds.

IWGT report on quantitative approaches to genotoxicity risk assessment II. Use of point-of-departure (PoD) metrics in defining acceptable exposure limits and assessing human risk

“…More recently, Cao et al [113] have conducted experiments in which male gpt-delta transgenic mice were treated daily for 28 days with 5-100 mg/kg EMS, and measurements were made on: (i) gpt mutant frequencies in liver, lung, bone marrow, kidney, small intestine, and spleen; and (ii) Pig-a mutant frequencies in peripheral blood reticulocytes (RETs) and total red blood cells. MN induction also was measured in peripheral blood RETs.…”

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

“…For alkylating agents, representative studies include three in vitro [56-58] and three in vivo [2, 3, 138] low-dose dose-response genotoxic datasets (an example is presented in Figure 4). The genotoxic chemicals used in these studies included direct-acting, monofunctional ethylmethanesulfonate (EMS), MMS, ethylnitrosourea (ENU), and MNU, although not all of the studies used all four chemicals.…”

“…The data clearly demonstrated an increasing level of hydroxyethylvaline hemoglobin adducts corresponding with increasing administered dose of EMS, but with no increase in corresponding MN induced until the administered dose exceeded the 80 mg/kg bw/day dose in mouse. Mutational in vivo dose-response with EMS was also recently investigated in adult gpt-delta transgenic mice within different tissues at the gpt transgene and at the Pig-a endogenous gene [2]. In the study, measured mutation NOGELs were identified at below 13 mg/kg/day and the lowest calculated PoD value, BMDL 10 , of 0.038 mg/kg/day was found in the lung of gpt-delta mice.…”

“…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 [2-6]. 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.…”

Assessment of mechanisms driving non-linear dose–response relationships in genotoxicity testing

“…This led researchers to 48 question the reliability of the general assumption of dose-49 response linearity at low doses for numerous genotoxicants with 50 diverse modes of action (MoA). In this context, the term MoA can 51 be defined as the sequence of key events and processes involved in 52 the interaction of an agent with DNA or other cellular target 53 structures that result in specific DNA damage [10]. 54 Non-linear dose-responses have been reported for low doses of 55 compounds with diverse MoA such as aneugens topoisomerase 56 inhibitors, alkylating agents and oxidants by evaluating different 57 genotoxic endpoints (e.g.…”