High cell density attenuates reactive oxygen species: implications for in vitro assays.

In vitro cell-based assays are an essential and universally used step in elucidation of biological processes as well as in drug development. However, results obtained depend on the validity of protocols used. This statement certainly pertains to in vitro assays of oxidative stress. The holy grail of in vitro models is reliability and predictability of outcomes that relate to a single variable like addition of hydrogen peroxide or xanthine oxidase. Without such validated outcomes, comparison of results among different laboratories is not possible. Achieving this goal requires a thorough understanding of the complex interplay between the cells, their environment, and the experimental assays. Furthermore, as this knowledge is attained, it must be disseminated and used to update and standardize existing protocols. Here, we confirm and extend the effect of pyruvate and cell density on in vitro oxidative stress assays. Cell viability was assessed using a colorimetric assay measuring the reduction of a tetrazolium salt (XTT) into a colored formazan dye. Extracellular hydrogen peroxide concentrations were measured using the foxp3 assay. We confirmed a previously reported finding that pyruvate, a common ingredient in cell culture media, acts as an extracellular scavenger of reactive oxygen species. We also demonstrated that cell density directly correlates with resistance to oxidative stress in tissue culture. It is theorized that the protective effect due to cell density predominantly relates to intracellular factors such as reduced glutathione and extracellular factors such as catalase.

Stereoselective excision of thymine glycol lesions by mammalian cell extracts.

Thymine glycols (Tg) are major pyrimidine oxidation products produced by chemical agents and ionizing radiation. Recent improvements in purification procedures gave us the opportunity to examine the incision of DNA duplexes containing a single (5S,6R)- or (5R,6S)-Tg lesion by mouse NTH1 DNA glycosylase and mammalian cell nuclear extracts. Time course experiments and steady state enzyme kinetics indicated that mNTH1 discriminates between the cis-Tg isomers. In addition, a variety of mammalian cell nuclear extracts showed a similar discrimination between the cis-Tg isomers. Trapping of Schiff base intermediates with sodium borohydride demonstrated that a single protein-DNA complex was formed in the presence of the nuclear extracts. The electrophoretic mobility of trapped complexes formed with both Tg isomers was identical to one another and similar to that of the complex formed with recombinant mNTH1. These results suggest that among all Tg-active DNA glycosylases, NTH1 is the major enzyme in mammalian cell nuclear extracts responsible for incision of duplexes containing cis-Tg isomers.

Stereoselective excision of thymine glycol from oxidatively damaged DNA.

DNA damage created by reactive oxygen species includes the prototypic oxidized pyrimidine, thymine glycol (Tg), which exists in oxidatively damaged DNA as two diastereoisomeric pairs. In Escherichia coli, Saccharomyces cerevesiae and mice, Tg is preferentially excised by endonuclease III (Endo III) and endonuclease VIII (Endo VIII), yNTG1 and yNTG2, and mNTH and mNEIL1, respectively. We have explored the ability of these DNA glycosylases to discriminate between Tg stereoisomers. Oligonucleotides containing a single, chromatographically pure (5S,6R) or (5R,6S) stereoisomer of Tg were prepared by oxidation with osmium tetroxide. Steady-state kinetic analyses of the excision process revealed that Endo III, Endo VIII, yNTG1, mNTH and mNEIL1, but not yNTG2, excise Tg isomers from DNA in a stereoselective manner, as reflected in the parameter of catalytic efficiency (kcat/Km). When DNA glycosylases occur as complementary pairs, failure of one or both enzymes to excise their cognate Tg stereoisomer from oxidatively damaged DNA could have deleterious consequences for the cell.

Sorting the consequences of ionizing radiation: processing of 8-oxoguanine/abasic site lesions.

Clustered DNA damage is a hallmark of ionizing radiation. These complex lesions, composed of any combination of oxidized bases, abasic sites, or strand breaks within one helical turn, create a tremendous challenge for the base excision repair system, which must process the damage without generating cytotoxic double strand breaks (DSB). Clustered lesions affect the DNA incision activity of DNA glycosylases and AP endonucleases. Different levels of enzyme inhibition are dependent on lesion identity, orientation and separation. Very little is known about the simultaneous action of both classes of enzymes, which may lead to the creation of DSB. We have developed a novel substrate system of double-labeled hairpin duplexes, which allows the simultaneous determination of enzyme incision and formation of DBS. We use this system to study the processing of four clustered 8-oxoguanine/abasic site lesions by purified mouse Ogg1, human Ape1 and mouse embryonic stem cell nuclear extracts. Ape1 activity is least affected by the presence of a nearby oxidized base. In contrast, an abasic site inhibits the glycosylase and lyase activities of Ogg1 in an orientation-dependent manner. The combined action of both enzymes leads to the preferential formation of DSB with 5'-overhang ends. Processing of clusters by nuclear extracts displayed similar patter of enzyme inhibition and the same preference for avoiding double strand breaks with 3'-overhang ends.