Mutagenesis associated with nitric oxide production in transgenic SJL mice

We recently reported development of an experimental model for the study of nitric oxide (NO·) toxicology in vivo. SJL mice were injected with superantigen-bearing RcsX (pre-B-cell lymphoma) cells, which migrated to the spleen and lymph nodes, where their rapid growth induced activation of macrophages to produce large amounts of NO· over a period of several weeks. In the experiments described here, we used this model to investigate mutagenesis in splenocytes exposed to NO· during RcsX cell growth. Transgenic mice were produced by crossbreeding animals of the pUR288 transgenic C57BL/6 and SJL strains. RcsX cells were injected into F1 mice and NO· production was confirmed by quantification of urinary nitrate, the ultimate metabolite of NO·. Mutant frequency in the lacZ gene of the pUR288 plasmid was determined in DNA isolated from spleen (target) and kidney (nontarget) tissues. A significant elevation in mutant frequency was found in the spleen, but not in the kidney, of tumor-bearing mice. Furthermore, increases in mutant frequency in the spleen as well as NO· production were abrogated by administration of N-methylarginine, a NO· inhibitor, to mice following injection of RcsX cells. These results indicate that NO· had mutagenic activity in RcsX tumor-bearing mice and thus support a possible role for its involvement in the carcinogenic process.

Mutational properties of the primary aflatoxin B1-DNA adduct.

The mutagenic activity of the major DNA adduct formed by the liver carcinogen aflatoxin B1 (AFB1) was investigated in vivo. An oligonucleotide containing a single 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) adduct was inserted into the single-stranded genome of bacteriophage M13. Replication in SOS-induced Escherichia coli yielded a mutation frequency for AFB1-N7-Gua of 4%. The predominant mutation was G --> T, identical to the principal mutation in human liver tumors believed to be induced by aflatoxin. The G --> T mutations of AFB1-N7-Gua, unlike those (if the AFB1-N7-Gua-derived apurinic site, were much more strongly dependent on MucAB than UmuDC, a pattern matching that in intact cells treated with the toxin. It is concluded that the AFB1-N7-Gua adduct, and not the apurinic site, has genetic requirements for mutagenesis that best explain mutations in aflatoxin-treated cells. While most mutations were targeted to the site of the lesion, a significant fraction (13%) occurred at the base 5' to the modified guanine. In contrast, the apurinic site-containing genome gave rise only to targeted mutations. The mutational asymmetry observed for AFB1-N7-Gua is consistent with structural models indicating that the aflatoxin moiety of the aflatoxin guanine adduct is covalently intercalated on the 5' face of the guanine residue. These results suggest a molecular mechanism that could explain an important step in the carcinogenicity of aflatoxin B1.

Transient excess of MYC activity can elicit genomic instability and tumorigenesis

Overexpression of the MYC protooncogene has been implicated in the genesis of diverse human tumors. Tumorigenesis induced by MYC has been attributed to sustained effects on proliferation and differentiation. Here we report that MYC may also contribute to tumorigenesis by destabilizing the cellular genome. A transient excess of MYC activity increased tumorigenicity of Rat1A cells by at least 50-fold. The increase persisted for >30 days after the return of MYC activity to normal levels. The brief surfeit of MYC activity was accompanied by evidence of genomic instability, including karyotypic abnormalities, gene amplification, and hypersensitivity to DNA-damaging agents. MYC also induced genomic destabilization in normal human fibroblasts, although these cells did not become tumorigenic. Stimulation of Rat1A cells with MYC accelerated their passage through G1/S. Moreover, MYC could force normal human fibroblasts to transit G1 and S after treatment with N-(phosphonoacetyl)-l-aspartate (PALA) at concentrations that normally lead to arrest in S phase by checkpoint mechanisms. Instead, the cells subsequently appeared to arrest in G2. We suggest that the accelerated passage through G1 was mutagenic but that the effect of MYC permitted a checkpoint response only after G2 had been reached. Thus, MYC may contribute to tumorigenesis through a dominant mutator effect.

Mechanism of stimulation of the DNA glycosylase activity of hOGG1 by the major human AP endonuclease: bypass of the AP lyase activity step

The generation of reactive oxygen species in the cell provokes, among other lesions, the formation of 8-oxo-7,8-dihydroguanine (8-oxoG) in DNA. Due to mispairing with adenine during replication, 8-oxoG is highly mutagenic. To minimise the mutagenic potential of this oxidised purine, human cells have a specific 8-oxoG DNA glycosylase/AP lyase (hOGG1) that initiates the base excision repair (BER) of 8-oxoG. We show here that in vitro this first enzyme of the BER pathway is relatively inefficient because of a high affinity for the product of the reaction it catalyses (half-life of the complex is >2 h), leading to a lack of hOGG1 turnover. However, the glycosylase activity of hOGG1 is stimulated by the major human AP endonuclease, HAP1 (APE1), the enzyme that performs the subsequent step in BER, as well as by a catalytically inactive mutant (HAP1-D210N). In the presence of HAP1, the AP sites generated by the hOGG1 DNA glycosylase can be occupied by the endonuclease, avoiding the re-association of hOGG1. Moreover, the glycosylase has a higher affinity for a non-cleaved AP site than for the cleaved DNA product generated by HAP1. This would shift the equilibrium towards the free glycosylase, making it available to initiate new catalytic cycles. In contrast, HAP1 does not affect the AP lyase activity of hOGG1. This stimulation of only the hOGG1 glycosylase reaction accentuates the uncoupling of its glycosylase and AP lyase activities. These data indicate that, in the presence of HAP1, the BER of 8-oxoG residues can be highly efficient by bypassing the AP lyase activity of hOGG1 and thus excluding a potentially rate limiting step.

Nucleotide excision repair in rat male germ cells: low level of repair in intact cells contrasts with high dual incision activity in vitro

The acquisition of genotoxin-induced mutations in the mammalian germline is detrimental to the stable transfer of genomic information. In somatic cells, nucleotide excision repair (NER) is a major pathway to counteract the mutagenic effects of DNA damage. Two NER subpathways have been identified, global genome repair (GGR) and transcription-coupled repair (TCR). In contrast to somatic cells, little is known regarding the expression of these pathways in germ cells. To address this basic question, we have studied NER in rat spermatogenic cells in crude cell suspension, in enriched cell stages and within seminiferous tubules after exposure to UV or N-acetoxy-2-acetylaminofluorene. Surprisingly, repair in spermatogenic cells was inefficient in the genome overall and in transcriptionally active genes indicating non-functional GGR and TCR. In contrast, extracts from early/mid pachytene cells displayed dual incision activity in vitro as high as extracts from somatic cells, demonstrating that the proteins involved in incision are present and functional in premeiotic cells. However, incision activities of extracts from diplotene cells and round spermatids were low, indicating a stage-dependent expression of incision activity. We hypothesize that sequestering of NER proteins by mispaired regions in DNA involved in synapsis and recombination may underlie the lack of NER activity in premeiotic cells.