Dose and temporal evaluation of ethylene oxide‐induced mutagenicity in the lungs of male big blue mice following inhalation exposure to carcinogenic concentrations

Abstract

Ethylene oxide (EO) is a direct acting alkylating agent; in vitro and in vivo studies indicate that it is both a mutagen and a carcinogen. However, it remains unclear whether the mode of action (MOA) for cancer for EO is a mutagenic MOA, specifically via point mutation. To investigate the MOA for EO-induced mouse lung tumors, male Big Blue (BB) B6C3F1 mice (10/group) were exposed to EO by inhalation, 6 hr/day, 5 days/week for 4 (0, 10, 50, 100, or 200 ppm EO), 8, or 12 weeks (0, 100, or 200 ppm EO). Lung DNA samples were analyzed for cII mutant frequency (MF) at 4, 8 and 12 weeks of exposure; the mutation spectrum was analyzed for mutants from control and 200 ppm EO treatments. Although EO-induced cII MFs were 1.5- to 2.7-fold higher than the concurrent controls at 4 weeks, statistically significant increases in the cII MF were found only after 8 and 12 weeks of exposure and only at 200 ppm EO (P ≤ 0.05), which is twice the highest concentration used in the cancer bioassay. Consistent with the positive response, DNA sequencing of cII mutants showed a significant shift in the mutational spectra between control and 200 ppm EO following 8 and 12 week exposures (P ≤ 0.035), but not at 4 weeks. Thus, EO mutagenic activity in vivo was relatively weak and required higher than tumorigenic concentrations and longer than 4 weeks exposure durations. These data do not follow the classical patterns for a MOA mediated by point mutations. Environ. Mol. Mutagen. 58:122-134, 2017. © 2017 Wiley Periodicals, Inc.

The Lambda Select <em>cII</em> Mutation Detection System

“…In conclusion, the λ Select cII assay in cultured cells of transgenic rodents treated with a test compound, or the corresponding animals treated with the tested chemical/agent, is a valuable approach for mutagenicity testing. We have successfully used the approach, as have other research groups throughout the world 4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,34,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75 . More recently, we have expanded the applications of this approach by developing a new technique in which a modification of the λ Select cII assay together with nextgeneration sequencing enables high throughput analysis of mutations in a time-, cost-, and labor-effective manner 23 .…”

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

“…Data on in vivo mutation from EO itself show that exposures at or below 10–50 ppm EO do not result in any quantifiable increases in the genotoxic effect markers evaluated in somatic cells (HPRT mutations, reciprocal translocations, micronucleus), but increases were quantifiable at higher doses (Sisk et al, , Walker et al, , van Sittert et al, , Recio et al, , Donner et al, ). More recently an extensive assessment of in vivo mutation induction in mouse lung by repeated exposures to EO utilized the Allele‐specific Competitive Blocker Polymerase Chain Reaction approach to quantify dose–response for mutations induced in a cancer‐relevant gene, Kras, along with measuring adducts and transgenic mutations in mouse lung (Haber et al, , Parsons et al, , Manjanatha et al, ). The investigators reported that, despite dose‐dependent increases in N 7‐HEG adducts, no statistically significant ( p < 0.05) increases were found for Kras (or cII ) mutations in mouse lung exposed to 10 ppm EO for 4 weeks of repeated exposure.…”

“…A thorough approach was described by an International Life Sciences Institute/Health and Environmental Sciences Institute (ILSI/HESI) Committee (Jarabek et al, ), which emphasized the role of DNA adducts as biomarkers of exposure. This and other approaches have been applied to some chemicals, offering case studies (Manjanatha et al, , Pottenger et al, ). Indeed, determination of a mutagenic MOA for cancer cannot depend solely on a positive in vitro genetox battery but requires a demonstrated causal link between adduct formation and induction of in vivo mutations in target tissue in cancer‐related genes (Moore et al, ).…”

Interplay between Cellular Metabolism and the DNA Damage Response in Cancer

“…Lipid peroxidation [60] Metabolite of N-nitrosopyrrolidine [102] Base substitutions [103] Frameshift mutations [103] Acrolein [60,107] Myeloperoxidation in neutrophils and monocytes [108] Base substitutions [109] Frameshift mutations Tandem bases substitutions [106] S-Adenosyl methionine (SAM) N7-methylguanine (7meG) N1-and N3-methyladenine (1meA and 3meA) O6-methylguanine (O6meG) O4-methylthymine O4meTO4-ethylthymine [110][111][112] Synthesized from ATP and methionine [113] 7meG: Harmless but can become an abasic site, promutagenic 3meA: DNA replication block, by-pass by error-prone TLS polymerase O-adducts: Bases mispairing [114] Ethylene oxide (EO) N7-(2-hydroxyethyl)dG (main product) N3-(2-hydroxyethyl)dA N3-(2-hydroxyethyl)dU O6-(2-hydroxyethyl)dG [115,116] Lipid peroxidation [60,117] Gut microflora [118] Mutagenic (unclear mode of action) [119,120] Different repair pathways are involved in protecting genome integrity following DNA adduct generation: base adducts are removed by BER or nucleotide excision repair (NER) in the case of bulky adducts. Intrastrand crosslinks are resolved by NER, while interstrand crosslinks are repaired by the Fanconi Anemia pathway and homologous recombination (HR).…”

“…7HEG adducts are not promutagenic alone but they lead to depurination of DNA and hence the resulting abasic site can generate mutations during DNA replication. Other minor adducts can also be mutagenic, but while EO has been proved to be genotoxic, the mechanisms of mutagenicity have not been fully described [120].…”