DNA adducts, mutant frequencies, and mutation spectra in various organs of λlacZ mice exposed to ethylating agents

Abstract

To investigate tissue-specific relations between DNA adducts and mutagenesis in vivo, lambda lacZ transgenic mice were treated i.p. with N-ethyl-N-nitrosourea (ENU), diethylnitrosamine (DEN), and ethyl methanesulphonate (EMS). In liver, bone marrow, and brain DNA from mice sacrificed at several time points after treatment O6-ethylguanine (O6-EtG) and N7-ethylguanine (N7-EtG) levels were determined as well as the mutant frequency (MF) in lacZ. In liver DNA of ENU- and DEN-treated mice, the bulk of O6-EtG was removed at 3 days after treatment, while the MF continued to increase thereafter. This suggests that O6-EtG is not the major premutagenic lesion in the liver. Indeed, sequence analysis of mutants showed only 24% GC-->AT transitions, consistent with the O6-EtG lesion, and 28% TA-->AT transversions, expected from O2-ethylthymine. In bone marrow after ENU treatment, a maximum mutation induction occurred at 3 days post-treatment, of which 43% were GC-->AT mutations and 22% were TA-->AT mutations. This suggests that in bone marrow O6-EtG may be a major premutagenic lesion at the 3-day time point. In liver and bone marrow, EMS treatment gave rise to a high level of N7-EtG and a low level of O6-EtG but no increase in MF. No adducts or mutation induction were observed in bone marrow of DEN-treated mice. No MF increase was observed in the brain of either ENU- or EMS-treated mice, although O6- and N7-adducts were present.

Excision of mutagenic replication-blocking lesions suppresses cancer but promotes cytotoxicity and lethality in nitrosamine-exposed mice

“…Additionally, we have previously observed that knockout of Aag protects against methylationinduced immune cell infiltration and inflammation in the retina, whereas here we observed increased immune cell infiltration in the NDMA-treated liver of Aag À/À mice, pointing to tissue-specific differences. We have also shown that treatment of Aag À/À mice with the alkylating agent methyl nitrosourea (which causes the same lesions as NDMA) did not significantly induce sequence rearrangement mutations in the pancreas (Kiraly et al, 2014), suggesting tissue-specific differences in mutagenesis, as has previously been described (Loktionov et al, 1990;Schmezer et al, 1994;Mientjes et al, 1998;Wang et al, 1998). One possibility is that TLS is less efficient in the liver relative to the pancreas (exacerbating the consequences of blocking lesions).…”

Excision of mutagenic replication-blocking lesions suppresses cancer but promotes cytotoxicity and lethality in nitrosamine-exposed mice

“…For example, both overexpression and knockout of Aag have been associated with chromosomal aberrations in mammalian cell culture (Coquerelle et al, 1995; Engelward et al, 1998; Engelward et al, 1996; Ensminger et al, 2014; Ibeanu et al, 1992; Kaina et al, 1993). Furthermore, treatment of Aag −/− mice with the alkylating agent methyl nitrosourea (which causes the same lesions as NDMA) did not significantly induce sequence rearrangements in the pancreas (Kiraly et al, 2014), suggesting tissue-specific differences in mutagenesis (a phenomenon that has been demonstrated previously (Loktionov et al, 1990; Mientjes et al, 1998; Schmezer et al, 1994; Wang et al, 1998)).…”

Differential Gene Expression Induced by Two Genotoxic N-nitroso Carcinogens, Phenobarbital and Ethanol in Mouse Liver Examined with Oligonucleotide Microarray and Quantitative Real-time PCR

“…Regarding diŠerential gene expression in mouse liver 28 days after chemical exposure, it was previously reported that few DNA adducts were observed at this time after DEN administration but that mutations were observed (7). Our results showed that p21 retained high gene expression but was reduced to one-tenth compared to 4 h, and that Ccng1 and Snk showed minor increase in gene expression 28 days after administration of DEN and DPN.…”

“…In the present study we examined diŠerential gene expression in mouse liver 4 h and 28 days after administration of genotoxic N-nitroso carcinogens, DEN and DPN, compared to non-genotoxic carcinogen PB and non-carcinogenic toxin EtOH using in-house DNA microarray and qPCR. Four hours is a time at which genotoxic N-nitroso carcinogens induce DNA damage (7)(8)(9). We found diŠerential gene expression between the two N-nitroso carcinogens and PB and EtOH in 11 genes 4 h after administration.…”

“…It is known that genotoxic N-nitroso carcinogens induce DNA damage and repair in mouse liver in a matter of a few hours after their administration; DNA adducts (7), unscheduled DNA synthesis (8), DNA lesions (Comet assay) (9) and other lesions have been reported. It is also known that mutations are induced in transgenic mouse liver 28 days after genotoxic N-nitroso carcinogen administration (7,10,11). This coincides with the time at which chronic changes leading to carcinogenesis are known to begin.…”