Molecular Characterization of Mitomycin C-Induced Large Deletions and Tandem-Base Substitutions in the Bone Marrow of gpt delta Transgenic Mice

Abstract

Deletion mutations constitute an important class of mutations that may result in a variety of human diseases, including cancer. Although many chemicals and ionizing radiations induce deletions, this class of mutation has been poorly characterized at the molecular level, particularly in vivo. Here we report the molecular nature of deletions as well as base substitutions induced by antitumor antibiotic mitomycin C (MMC) in the bone marrow using a novel transgenic mouse, gpt delta. In this mouse model, deletions and point mutations in lambda DNA integrated in the chromosome are individually selected as Spi(-) (sensitive to P2 interference) phages and 6-thioguanine-resistant bacterial colonies, respectively. The mice were treated with MMC (1 mg/kg/day) for five consecutive days. One week after the last treatment, lambda phage was rescued from the genomic DNA of the bone marrow by in vitro packaging reactions and subjected to Spi(-) and 6-thioguanine selections. The mutant frequency of Spi(-) with large deletions increased more than 20-fold over that of the control. Molecular sizes of the large deletions were mostly more than 2,000 base pairs. The large deletions frequently occurred between two short direct repeat sequences from 2 to 6 base pairs, suggesting that they are generated during the end-joining repair of double-strand breaks induced by interstrand cross-links in DNA. In 6-thioguanine selection, tandem-base substitutions, such as 5'-GG-3' to 5'-AT-3', were induced. It highlights the relevance of intrastrand cross-links as genotoxic lesions. Previous in vitro studies report the induction of single-base substitutions and single-base deletions by MMC. However, no such mutations were identified in vivo. Thus, our results strongly caution that in vitro mutation spectra do not necessarily reflect genotoxic events in vivo and emphasize the importance of transgenic rodent genotoxicity assays to examine the roles of DNA adducts in mutagenesis and carcinogenesis.

Thioredoxin-like domains required for glucose regulatory protein 58–mediated reductive activation of mitomycin C leading to DNA cross-linking

“…It is known that MMC is metabolized by reductases such as NAD(P)H:quinone oxidoreductase 1 to 2,7-diaminomitosene, which has optimal absorption spectrum at 554 nm (16). The absorption maximum for MMC is 365 nm (16).…”

“…The absorption maximum for MMC is 365 nm (16). Therefore, we analyzed the capacity of CHO cell lysates expressing endogenous alone or endogenous plus cDNA derived WT and mutant GRP58 to metabolize MMC to 2,7-diaminomitosene.…”

Novel DNA Polymerases and Novel Genotoxicity Assays

“…MMC induces tandem-base substitutions, mainly in 5?-GG-3? in the bone marrow (73). This might be resulted from the presence of intra-strand cross link induced by MMC.…”

“…We suggest that heavy-particle irradiation induces double-strand breaks (DSBs) in DNA through direct deposition of energy on DNA and through oxidation of DNA. The large deletions are also induced by UVB and MMC, which induce lesions that block progression of DNA replication, thereby inducing DSBs in DNA (73,78). Middle size deletions of more than one bp but less than one kilo bps are induced by high dose (50 Gy) radiation of gray (67).…”

An Assessment of Mutagenic Effect of 3, 3', 4, 4', 5 Pentachlorobiphenyl (PCB126) in Muta Mouse Fetuses

“…Therefore, PCB is accumulated all over the environment, and it can become concentrated in a biota in a food chain such as fish, birds and mammals. Thus, further studies by use of other detecting system of gene mutagenicity such as gpt-delta transgenic mice carrying lambda EG10 DNA [27], or Big Blue mice carrying bacterial lacI gene [5] are necessary. …”