We demonstrate that alpha-ketoacids reduce and, in some instances, abrogate menadione-induced DNA damage and cytotoxicity in the human breast cancer cell line, MCF7. We confirm that alpha-ketoacids quench the copious amounts of H2O2 generated by menadione while these alpha-ketoacids undergo nonenzymatic oxidative decarboxylation; our data thus support enhanced H2O2 production as an important pathway for menadione-induced DNA damage and cytotoxicity. We also demonstrate that alpha-ketoacids scavenge H2O2 generated by mitochondria and microsomes when these organelles are exposed to menadione; additionally, alpha-ketoacids protect oxidant-vulnerable enzymes against functional impairment induced by H2O2. Finally, we provide the first in vivo demonstration that acute elevations in concentrations of alpha-ketoacids in rat tissues and urine scavenge H2O2. We conclude that enhanced H2O2 production is a major pathway for menadione-induced DNA damage and cytotoxicity and that the diverse alpha-ketoacids present within the cell must be considered, along with glutathione peroxidase and catalase, as part of the intracellular antioxidant defense mechanisms that regulate the ambient levels of H2O2.

The o-quinone forms of 2,3- and 3,4-catechol estrogens have been implicated in the carcinogenicity of these hormones. The concomitant production of reactive oxygen species during reduction of the o-quinone estrogens has been inferred to play a mechanistic role in their mutagenic potential. Conclusive evidence documenting the production of hydrogen peroxide, the hydroxyl radical, and the estrone 3,4-semiquinone in estrone 3,4-quinone (3,4-EQ)-treated human breast cancer subcellular fractions was demonstrated in the absence of exogenously added catalysts. Subcellular fractions of MCF-7 cells treated with 3,4-EQ and NADPH, including nuclei, mitochondria, and microsomes, were shown to support significant amounts of hydrogen peroxide production. Hydrogen peroxide production in 3,4-EQ-treated cellular fractions and the chromosomal DNA damage induced in 3,4-EQ-treated MCF-7 cells were abolished by the addition of catalase. A significant and potentially physiologically relevant spontaneous reduction of 3,4-EQ by NADPH resulting in hydrogen peroxide production was demonstrated. The results unequivocally demonstrate that free radicals are produced during the metabolism of estrone 3,4-quinone in human cells.

In the present study, we have generated an antisense NTP RNA construct in which the 3-untranslated region is replaced by a hammerhead ribozyme. The constitutive synthesis of the chimeric antisense RNA-ribozyme construct in parasites that were stably transfected with this construct resulted in a dramatic reduction in the steady-state levels of NTPase. This inhibition was accompanied by a decrease in the capacity of the parasites to replicate. The reduction in parasite proliferation was due to a specific effect of antisense NTP RNA, since a drastic inhibition of hypoxanthinexanthine-guanine phosphoribosyl transferase (HXG-PRT) expression by a chimeric antisense HXGPRT RNAribozyme construct did not alter NTPase expression nor compromise parasite replication. These data implicate NTPase in an essential parasite function and suggest that NTPase may have more than one function in vivo. These results also establish that it is possible to study gene function in apicomplexan parasites using antisense RNA coupled to ribozymes.

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