8-oxoguanine (8-oxoG), ring-opened purines (formamidopyrimidines or Fapys), and other oxidized DNA base lesions generated by reactive oxygen species are often mutagenic and toxic, and have been implicated in the etiology of many diseases, including cancer, and in aging. Repair of these lesions in all organisms occurs primarily via the DNA base excision repair pathway, initiated with their excision by DNA glycosylase͞AP lyases, which are of two classes. One class utilizes an internal Lys residue as the active site nucleophile, and includes Escherichia coli Nth and both known mammalian DNA glycosylase͞AP lyases, namely, OGG1 and NTH1. E. coli MutM and its paralog Nei, which comprise the second class, use N-terminal Pro as the active site. Here, we report the presence of two human orthologs of E. coli mutM nei genes in the human genome database, and characterize one of their products. Based on the substrate preference, we have named it NEH1 (Nei homolog). The 44-kDa, wild-type recombinant NEH1, purified to homogeneity from E. coli, excises Fapys from damaged DNA, and oxidized pyrimidines and 8-oxoG from oligodeoxynucleotides. Inactivation of the enzyme because of either deletion of N-terminal Pro or Histag fusion at the N terminus supports the role of N-terminal Pro as its active site. The tissue-specific levels of NEH1 and OGG1 mRNAs are distinct, and S phase-specific increase in NEH1 at both RNA and protein levels suggests that NEH1 is involved in replicationassociated repair of oxidized bases.

Two candidate human orthologs of Escherichia coliMutM/Nei were recently identified in the human genome database, and one of these, NEH1, was characterized earlier (Hazra, T. K., Izumi, T., Boldogh, I., Imhoff, B., Kow, Y. W., Jaruga, P., and Dizdaroglu, M. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 3523-3528). Here we report characterization of the second protein, originally named NEH2 and now renamed NEIL2 (Nei-like). The 37-kDa wild-type NEIL2 expressed in and purified from E. coli has DNA glycosylase/AP lyase activity, primarily for excising oxidative products of cytosine, with highest activity for 5-hydroxyuracil, one of the most abundant and mutagenic lesions induced by reactive oxygen species, and with lower activity for 5,6-dihydrouracil and 5-hydroxycytosine. It has negligible or undetectable activity with 8-oxoguanine, thymine glycol, 2-hydroxyadenine, hypoxanthine, and xanthine. NEIL2 is similar to NEIL1 in having N-terminal Pro as the active site. However, unlike NEIL1, its expression was independent of the cell cycle stage in fibroblasts, and its highest expression was observed in the testes and skeletal muscle. Despite the absence of a putative nuclear localization signal, NEIL2 was predominantly localized in the nucleus. These results suggest that NEIL2 is involved in global genome repair mainly for removing oxidative products of cytosine.

Embryonic development involves dramatic changes in cell proliferation and differentiation that must be highly coordinated and tightly regulated. Cellular redox balance is critical for cell fate decisions, but it is susceptible to disruption by endogenous and exogenous sources of oxidative stress. The most abundant endogenous non-protein antioxidant defense molecule is the tri-peptide glutathione (γ-glutamyl-cysteinylglycine, GSH), but the ontogeny of GSH concentration and redox state during early life stages is poorly understood. Here, we describe the GSH redox dynamics during embryonic and early larval development (0–5 days post-fertilization) in the zebrafish (Danio rerio), a model vertebrate embryo. We measured reduced and oxidized glutathione (GSH, GSSG) using HPLC, and calculated the whole embryo total glutathione (GSHT) concentrations and redox potentials (Eh) over 0–120 hours of zebrafish development (including mature oocytes, fertilization, mid-blastula transition, gastrulation, somitogenesis, pharyngula, pre-hatch embryos, and hatched eleutheroembryos). GSHT concentration doubled between 12 hours post fertilization (hpf) and hatching. The GSH Eh increased, becoming more oxidizing during the first 12 h, and then oscillated around −190 mV through organogenesis, followed by a rapid change, associated with hatching, to a more negative (more reducing) Eh (−220 mV). After hatching, Eh stabilized and remained steady through 120 hpf. The dynamic changes in GSH redox status and concentration defined discrete windows of development: primary organogenesis, organ differentiation, and larval growth. We identified the set of zebrafish genes involved in the synthesis, utilization, and recycling of GSH, including several novel paralogs, and measured how expression of these genes changes during development. Ontogenic changes in the expression of GSH-related genes support the hypothesis that GSH redox state is tightly regulated early in development. This study provides a foundation for understanding the redox regulation of developmental signaling and investigating the effects of oxidative stress during embryogenesis.

The redox status of the extracellular compartment has only just been elucidated as a mechanism controlling intracellular signal transduction and correlates with aging, diabetes, heart disease and lung fibrosis. In the present paper, we describe a mechanism by which oxidizing extracellular environments, as maintained by the cysteine/cystine (Cys/CySS) redox couple, induce mitochondria-derived ROS (reactive oxygen species) generation and cause the activation of Nrf2 (nuclear factor-erythroid 2-related factor 2), inducing an antioxidant response. NIH 3T3 cells were cultured in medium with extracellular Cys/CySS redox potentials (Eh), ranging from 0 to -150 mV. Cellular and mitochondrial ROS production significantly increased in cells incubated under more oxidizing extracellular conditions (0 and -46 mV). Trx2 (thioredoxin-2) is a mitochondrial-specific oxidoreductase and antioxidant and became oxidized in cells incubated at 0 or -46 mV. MEFs (mouse embryonic fibroblasts) from Trx2-overexpressing transgenic (Trx2 Tg) mice produced less intracellular ROS compared with WT (wild-type) MEFs at the more oxidizing extracellular conditions. Nrf2 activity was increased in WT MEFs at the 0 or -46 mV conditions, but was inhibited in Trx2 Tg MEFs under the same conditions. Furthermore, Nrf2-regulated gene expression was significantly increased in the WT MEFs, but not in the Trx2 Tg MEFs. These results show that the Cys/CySS redox status in the extracellular compartment regulates intracellular ROS generated primarily in the mitochondria, which play an important role in the activation of Nrf2 and up-regulation of antioxidant and detoxification systems.