Deficient DNA repair exacerbates ethanol-initiated DNA oxidation and embryopathies in ogg1 knockout mice: gender risk and protection by a free radical spin trapping agent.

Reactive oxygen species (ROS) have been implicated in the teratogenicity of alcohol (ethanol, EtOH). To determine the involvement of embryonic oxidative DNA damage, DNA repair-deficient oxoguanine glycosylase 1 (ogg1) knockout embryos were exposed in culture to EtOH (2 or 4 mg/ml), with or without pretreatment with the free radical spin trap phenylbutylnitrone (PBN) (0.125 mM). Visceral yolk sacs were used to genotype embryos for DNA repair status and gender. EtOH caused a concentration-dependent decrease in anterior neuropore closure (ANPC), somite development, turning, crown-rump length (CRL), yolk sac diameter (YSD) and head length (HL) (p < 0.001) in all 3 ogg1 genotypes. There was a further ogg1 gene dose-dependent decrease from +/+ to -/- embryos in ANPC, somite development, turning, CRL and HL (p < 0.05), and a gene-dependent correlation between HL and ANPC (p < 0.01). Female embryos exhibited lesser ANPC and turning than males (p < 0.05), suggesting underlying gender-dependent target-specific determinants. PBN pretreatment increased ANPC, somite development, turning, CRL, YSD and HL (p < 0.001), although this protection against EtOH was slightly less effective in -/- embryos. Oxidatively damaged DNA determined as 8-oxo-2'-deoxyguanosine (8-oxodGuo), which is repaired by OGG1, was measured in single embryos in vivo after maternal EtOH treatment (4 g/kg i.p). EtOH increased embryonic 8-oxodGuo in an ogg1 gene-dependent fashion, with the highest levels in -/- embryos. These results show that embryonic DNA repair status and gender are determinants of risk. ROS-initiated embryonic DNA oxidation is involved in EtOH embryopathies.

Hypersensitivity phenotypes associated with genetic and synthetic inhibitor-induced base excision repair deficiency.

Single-base lesions in DNA are repaired predominantly by base excision repair (BER). DNA polymerase beta (pol beta) is the polymerase of choice in the preferred single-nucleotide BER pathway. The characteristic phenotype of mouse fibroblasts with a deletion of the pol beta gene is moderate hypersensitivity to monofunctional alkylating agents, e.g., methyl methanesulfonate (MMS). Increased sensitivity to MMS is also seen in the absence of pol beta partner proteins XRCC1 and PARP-1, and under conditions where BER efficiency is reduced by synthetic inhibitors. PARP activity plays a major role in protection against MMS-induced cytotoxicity, and cells treated with a combination of non-toxic concentrations of MMS and a PARP inhibitor undergo cell cycle arrest and die by a Chk1-dependent apoptotic pathway. Since BER-deficient cells and tumors are similarly hypersensitive to the clinically used chemotherapeutic methylating agent temozolomide, modulation of DNA damage-induced cell signaling pathways, as well as BER, are attractive targets for potentiating chemotherapy.

CsA can induce DNA double-strand breaks: implications for BMT regimens particularly for individuals with defective DNA repair.

Several human disorders mutated in core components of the major DNA double-strand break (DSB) repair pathway, non-homologous end joining (NHEJ), have been described. Cell lines from these patients are characterized by sensitivity to DSB-inducing agents. DNA ligase IV syndrome (LIG4) patients specifically, for unknown reasons, respond particularly badly following treatment for malignancy or BMT. We report the first systematic evaluation of the response of LIG4 syndrome to compounds routinely employed for BMT conditioning. We found human pre-B lymphocytes, a key target population for BMT conditioning, when deficient for DNA ligase IV, unexpectedly exhibit significant sensitivity to CsA the principal prophylaxis for GVHD. Furthermore, we found that CsA treatment alone or in combination with BU and fludarabine resulted in increased levels of DSBs specifically in LIG4 syndrome cells compared to wild-type or Artemis-deficient cells. Our study shows that CsA can induce DSBs and that LIG4 syndrome patient's fail to adequately repair this damage. These DSBs likely arise as a consequence of DNA replication in the presence of CsA. This work has implications for BMT and GVHD management in general and specifically for LIG4 syndrome.

Evidence for exposure-induced DNA repair abnormality is indicative of health and genetic risk.

A recent focus has been targeted toward the development of functional biomarkers that can be used to predict disease more reliably. One such biomarker is the challenge assay for DNA repair deficiency. Briefly, the assay involves challenging lymphocytes in culture to a DNA damaging agent in vitro and determining the repair outcome in chromosome aberrations and/or DNA strand breaks. The aim is to show that individuals who have chronic exposure to toxic substances will develop exposure-induced DNA repair deficiencies. Many studies around the world have shown that the assay detects DNA repair deficiency in environmentally/occupationally exposed populations and with significant exposure dose-response relationship. The prediction of health risk was also validated. In addition, exposure-induced repair deficiency which was apparently passed through the germ cells had caused genetic consequences in a 3-generation population. The assay is simple to conduct and is more sensitive than some traditional biomarker assays. Together with the functional significance of the assay, the challenge assay can be used with confidence in population studies for health risk assessment.