Two Structurally Similar Maize Cytosolic Superoxide Dismutase Genes, Sod4 and Sod4A, Respond Differentially to Abscisic Acid and High Osmoticum1

The maize (Zea mays) superoxide dismutase genes Sod4 and Sod4A are highly similar in structure but each responds differentially to environmental signals. We examined the effects of the hormone abscisic acid (ABA) on the developmental response of Sod4 and Sod4A. Although both Sod4 and Sod4A transcripts accumulate during late embryogenesis, only Sod4 is up-regulated by ABA and osmotic stress. Accumulation of Sod4 transcript in response to osmotic stress is a consequence of increased endogenous ABA levels in developing embryos. Sod4 mRNA is up-regulated by ABA in viviparous-1 mutant embryos. Sod4 transcript increases within 4 h with ABA not only in developing embryos but also in mature embryos and in young leaves. Sod4A transcript is up-regulated by ABA only in young leaves, but neither Sod4 nor Sod4A transcripts changed in response to osmotic stress. Our data suggest that in leaves Sod4 and Sod4A may respond to ABA and osmotic stress via alternate pathways. Since the Sod genes have a known function, we hypothesize that the increase in Sod mRNA in response to ABA is due in part to ABA-mediated metabolic changes leading to changes in oxygen free radical levels, which in turn lead to the induction of the antioxidant defense system.

Glutathione-mediated destabilization in vitro of [2Fe-2S] centers in the SoxR regulatory protein.

SoxR is a transcription factor that governs a global defense against the oxidative stress caused by nitric oxide or excess superoxide in Escherichia coli. SoxR is a homodimer containing a pair of [2Fe-2S] clusters essential for its transcriptional activity, and changes in the stability of these metal centers could contribute to the activation or inactivation of SoxR in vivo. Herein we show that reduced glutathione (GSH) in aerobic solution disrupts the SoxR [2Fe-2S] clusters, releasing Fe from the protein and eliminating SoxR transcriptional activity. This disassembly process evidently involves oxygen-derived free radicals. The loss of [2Fe-2S] clusters does not occur in anaerobic solution and is blocked in aerobic solution by the addition of superoxide dismutase and catalase. Although H2O2 or xanthine oxidase and hypoxanthine (to generate superoxide) were insufficient on their own to cause [2Fe-2S] cluster loss, they did accelerate the rate of disassembly after GSH addition. Oxidized GSH alone was ineffective in disrupting the clusters, but the rate of [2Fe-2S] cluster disassembly was maximal when reduced and oxidized GSH were present at a ratio of approximately 1:3, which suggests the critical involvement of a GSH-based free radical in the disassembly process. Such a reaction might occur in vivo: we found that the induction by paraquat of SoxR-dependent soxS transcription was much higher in a GSH-deficient E. coli strain than in its GSH-containing parent. The results imply that GSH may play a significant role during the deactivation process of SoxR in vivo. Ironically, superoxide production seems both to activate SoxR and, in the GSH-dependent disassembly process, to switch off this transcription factor.

Fluorescent light-induced chromatid breaks distinguish Alzheimer disease cells from normal cells in tissue culture.

The neurodegeneration and amyloid deposition of sporadic Alzheimer disease (AD) also occur in familial AD and in all trisomy-21 Down syndrome (DS) patients, suggesting a common pathogenetic mechanism. We investigated whether defective processing of damaged DNA might be that mechanism, as postulated for the neurodegeneration in xeroderma pigmentosum, a disease with defective repair not only of UV radiation-induced, but also of some oxygen free radical-induced, DNA lesions. We irradiated AD and DS skin fibroblasts or blood lymphocytes with fluorescent light, which is known to cause free radical-induced DNA damage. The cells were then treated with either beta-cytosine arabinoside (araC) or caffeine, and chromatid breaks were quantified. At least 28 of 31 normal donors and 10 of 11 donors with nonamyloid neurodegenerations gave normal test results. All 12 DS, 11 sporadic AD, and 16 familial AD patients tested had abnormal araC and caffeine tests, as did XP-A cells. In one of our four AD families, an abnormal caffeine test was found in all 10 afflicted individuals (including 3 asymptomatic when their skin biopsies were obtained) and in 8 of 11 offspring at a 50% risk for AD. Our tests could prove useful in predicting inheritance of familial AD and in supporting, or rendering unlikely, the diagnosis of sporadic AD in patients suspected of having the disease.

The role of DT-diaphorase in the maintenance of the reduced antioxidant form of coenzyme Q in membrane systems.

The experiments reported here were designed to test the hypothesis that the two-electron quinone reductase DT-diaphorase [NAD(P)H:(quinone-acceptor) oxidoreductase, EC 1.6.99.2] functions to maintain membrane-bound coenzyme Q (CoQ) in its reduced antioxidant state, thereby providing protection from free radical damage. DT-diaphorase was isolated and purified from rat liver cytosol, and its ability to reduce several CoQ homologs incorporated into large unilamellar vesicles was demonstrated. Addition of NADH and DT-diaphorase to either large unilamellar or multilamellar vesicles containing homologs of CoQ, including CoQ9 and CoQ10, resulted in the essentially complete reduction of the CoQ. The ability of DT-diaphorase to maintain the reduced state of CoQ and protect membrane components from free radical damage as lipid peroxidation was tested by incorporating either reduced CoQ9 or CoQ10 and the lipophylic azoinitiator 2,2'-azobis(2,4-dimethylvaleronitrile) into multilamellar vesicles in the presence of NADH and DT-diaphorase. The presence of DT-diaphorase prevented the oxidation of reduced CoQ and inhibited lipid peroxidation. The interaction between DT-diaphorase and CoQ was also demonstrated in an isolated rat liver hepatocyte system. Incubation with adriamycin resulted in mitochondrial membrane damage as measured by membrane potential and the release of hydrogen peroxide. Incorporation of CoQ10 provided protection from adriamycin-induced mitochondrial membrane damage. The incorporation of dicoumarol, a potent inhibitor of DT-diaphorase, interfered with the protection provided by CoQ. The results of these experiments provide support for the hypothesis that DT-diaphorase functions as an antioxidant in both artificial membrane and natural membrane systems by acting as a two-electron CoQ reductase that forms and maintains the antioxidant form of CoQ. The suggestion is offered that DT-diaphorase was selected during evolution to perform this role and that its conversion of xenobiotics and other synthetic molecules is secondary and coincidental.

Susceptibility of cloned K+ channels to reactive oxygen species.

Free radical-induced oxidant stress has been implicated in a number of physiological and pathophysiological states including ischemia and reperfusion-induced dysrhythmia in the heart, apoptosis of T lymphocytes, phagocytosis, and neurodegeneration. We have studied the effects of oxidant stress on the native K+ channel from T lymphocytes and on K+ channels cloned from cardiac, brain, and T-lymphocyte cells and expressed in Xenopus oocytes. The activity of three Shaker K+ channels (Kv1.3, Kv1.4, and Kv1.5), one Shaw channel (Kv3.4), and one inward rectifier K+ channel (IRK3) was drastically inhibited by photoactivation of rose bengal, a classical generator of reactive oxygen species. Other channel types (such as Shaker K+ channel Kv1.2, Shab channels Kv2.1 and Kv2.2, Shal channel Kv4.1, inward rectifiers IRK1 and ROMK1, and hIsK) were completely resistant to this treatment. On the other hand tert-butyl hydroperoxide, another generator of reactive oxygen species, removed the fast inactivation processes of Kv1.4 and Kv3.4 but did not alter other channels. Xanthine/xanthine oxidase system had no effect on all channels studied. Thus, we show that different types of K+ channels are differently modified by reactive oxygen species, an observation that might be of importance in disease states.

Dietary lipids and calorie restriction affect mammary tumor incidence and gene expression in mouse mammary tumor virus/v-Ha-ras transgenic mice.

We have studied the effects of food restriction (FR) and substitution of fish oil (FO; omega 3) for corn oil (CO; omega 6) on breast tumor incidence and survival in mouse mammary tumor virus/v-Ha-ras transgenic (Onco) mice. The diets were as follows: group 1, 5% (wt/wt) CO fed ad libitum (AL); group 2, 5% CO, restricted calories (40% fewer calories than AL; FR); group 3, 20% CO fed AL; and group 4, 20% FO fed AL. After 3 years, 40% of FR Onco (group 2) mice were alive, whereas there were no survivors in the other three groups. Similarly, tumor incidence was reduced to 27% (5 out of 18) in FR animals (group 2), whereas it was 83% (11 out of 13) in group 1 mice, 89% (16 out of 18) in group 3 mice, and 71% (10 out of 14) in group 4 mice. These protective effects of FR on survival and tumor incidence were paralleled by higher expression of the tumor suppressor gene p53 (wild type) and free-radical scavenging enzymes (catalase and superoxide dismutase) in breast tumors. Immunoblotting showed less ras gene product, p21, and increased p53 levels in the tumors of FR mice. In addition, FR decreased RNA levels of c-erbB-2, interleukin 6, and the transgene v-Ha-ras in tumors. In contrast, analysis of hepatic mRNA from tumor-bearing FR mice revealed higher expression of catalase, glutathione peroxidase, and superoxide dismutase. Survival and tumor incidence were not influenced significantly by dietary supplementation with FO in place of CO. Taken together, our studies suggest that moderate restriction of energy intake significantly inhibited the development of mammary tumors and altered expression of cytokines, oncogenes, and free-radical scavenging enzymes.

Heritable, population-wide damage to cells as the driving force of neoplastic transformation.

Prolonged incubation of NIH 3T3 cells under the growth constraint of confluence results in the death of some cells in a manner suggestive of apoptosis. Successive rounds of prolonged incubation at confluence of the surviving cells produce increasing neoplastic transformation in the form of increments in saturation density and transformed focus formation. Cells from the postconfluent cultures are given a recovery period of various lengths to remove the direct inhibitory effect of confluence before their growth properties are studied. It is found that with each round of confluence the exponential growth rate of the cells at low densities gets lower and the size of isolated colonies of the same cells shows a similar progressive reduction. The decreased growth rate of cells from the third round of confluence persists for > 60 generations of growth at low density. The proportion of colonies containing giant cells is much higher after a 2-day recovery from confluence than after a 7-day recovery. Retardation of growth at low density and increased saturation density appear to be two sides of the same coin: both occur in the entire population of cells and precede the formation of transformed foci. We propose that the slowdown in growth and the formation of giant cells result from heritable damage to the cells, which in turn drives their transformation. Similar results have been reported for the survivors of x-irradiation and of treatment with chemical carcinogens and are associated with the aging process in animals. We suggest that these changes result from free radical damage to membrane lipids with particular damage to lysosomes. Proteases and nucleases would then be released to progressively modify the growth behavior and genetic stability of the cells toward autonomous proliferation.

Coenzyme Q reductase from liver plasma membrane: purification and role in trans-plasma-membrane electron transport.

A specific requirement for coenzyme Q in the maintenance of trans-plasma-membrane redox activity is demonstrated. Extraction of coenzyme Q from membranes resulted in inhibition of NADH-ascorbate free radical reductase (trans electron transport), and addition of coenzyme Q10 restored the activity. NADH-cytochrome c oxidoreductase (cis electron transport) did not respond to the coenzyme Q status. Quinone analogs inhibited trans-plasma-membrane redox activity, and the inhibition was reversed by coenzyme Q. A 34-kDa coenzyme Q reductase (p34) has been purified from pig-liver plasma membranes. The isolated enzyme was sensitive to quinone-site inhibitors. p34 catalyzed the NADH-dependent reduction of coenzyme Q10 after reconstitution in phospholipid liposomes. When plasma membranes were supplemented with extra p34, NADH-ascorbate free radical reductase was activated but NADH-cytochrome c oxidoreductase was not. These results support the involvement of p34 as a source of electrons for the trans-plasma-membrane redox system oxidizing NADH and support coenzyme Q as an intermediate electron carrier between NADH and the external acceptor ascorbate free radical.