Danny R. Moore

Extracellular superoxide production by Enterococcus faecalis requires demethylmenaquinone and is attenuated by functional terminal quinol oxidases

The intestinal commensal bacterium, Enterococcus faecalis, is unusual among prokaryotic organisms in its ability to produce substantial extracellular superoxide. Transposon mutagenesis, allelic replacement, and electron spin resonance (ESR)‐spin trapping showed that superoxide production and generation of derivative hydroxyl radical were dependent on membrane‐associated demethylmenaquinone. Extracellular superoxide was generated through univalent reduction of oxygen by reduced demethylmenaquinone. Moreover, extracellular superoxide production was inhibited by exogenous haematin, an essential cofactor for cytochrome bd, and by fumarate, a substrate for fumarate reductase. As integral membrane quinol oxidases, cytochrome bd and fumarate reductase redox cycle demethylmenaquinone, and are necessary for aerobic and anaerobic respiration respectively. A rat model of intestinal colonization demonstrated that conditions exist in the mammalian intestinal tract that permit a mode of respiration for E. faecalis that results in the formation of hydroxyl radical. These results identify and characterize the mechanism by which E. faecalis generates extracellular free radicals.

Sex-dependent differences in the effects of aging on antioxidant defense mechanisms of rat liver

Information about age-related factors that influence sensitivity to hepatotoxic injury is important to geriatric medicine and environmental health. The purpose of the present study was to determine whether age-associated changes occur in hepatic antioxidant defense mechanisms of male and female Fischer 344 rats. Liver homogenates and post-mitochondrial supernatant fractions from rats aged 4, 14, 24 and 29 months were analyzed for antioxidant enzyme activities and for vitamin E and malondialdehyde content. Age-associated changes in catalase and glutathione reductase activities were observed that could be described as sex-determined differences that disappeared in old age. Cytosolic superoxide dismutase and glutathione peroxidase activities displayed sex-dependent variations in activity but were unaffected by aging. Hepatic vitamin E concentrations were lower in male rats than in female malondialdehyde concentrations also were lower in males than in females; malondialdehyde content increased in old males and decreased in old females. The results indicate that age-associated changes in enzymatic and nonenzymatic antioxidant defense mechanisms of rat liver are sex-dependent. In addition, comparison with findings from other studies in rats suggests that the effects of aging may also depend on the strain of rat.

Effect of aging on aqueous-phase antioxidants in tissues of male Fischer rats.

The purpose of this study was to determine the influence of aging on concentrations of the important aqueous-phase antioxidants in rat tissues. Ascorbic acid, glutathione and uric acid were measured in tissues and organs of male Fischer 344 rats at 6, 15 and 26 months of age. Blood, liver, lungs, heart, kidneys, brain, testes and lenses were excised rapidly and were extracted with cold metaphosphoric acid. Aging diminished the concentration of ascorbic acid in liver, lung and lens; levels in 26-month-old rats were 40-60% of those in 6-month-old rats. Glutathione content was diminished only in lens, where it decreased almost 50% between 15 and 26 months. Some age-associated increases in antioxidant levels also were seen; testis ascorbic acid and kidney glutathione levels were elevated in the old compared with the younger rats. Uric acid concentrations were much lower than glutathione or ascorbic acid concentrations in every tissue except plasma. Old rats had lower levels of uric acid in liver but higher levels in heart, kidney and testis. These results demonstrate that aqueous-phase antioxidant levels are not uniformly diminished in tissues of old rats.

In vivo production of hydroxyl radical by Enterococcus faecalis colonizing the intestinal tract using aromatic hydroxylation

Enterococcus faecalis is an intestinal commensal that produces extracellular superoxide (O(2)(*-)) through autoxidation of membrane-associated demethylmenaquinone. To assess free radical production by E. faecalis in vivo, intestinal tracts of rats were colonized using wild-type E. faecalis or a mutant strain with attenuated O(2)(*-) production. Ex vivo electron paramagnetic resonance spin trapping study of colonic contents (mean +/- SD) showed 1.4 +/- 1.5 and 0.094 +/- 0.24 microM 5,5-dimethyl-1-pyrroline-N-oxide-hydroxyl radical adduct/gm stool for rats colonized with wild-type and mutant strains, respectively (p = .002). In vivo hydroxyl radical production was further assayed by aromatic hydroxylation using phenyl N-tert-butylnitrone (PBN) and D-phenylalanine. Hydroxylated PBN and D-phenylalanine products were recovered from stool (microM/gm colonic contents/10(9) colony forming units) and urine (microM/h/ml), respectively, and quantified using electrochemical detection. Hydroxylated (OH) PBNs and isomeric tyrosines (hydroxylated phenylalanine) were significantly increased (mean +/- SD) for rats colonized with wild-type E. faecalis (2-OH PBN, 63 +/- 58; 3-OH PBN, 63 +/- 84; ortho-tyrosine, 31 +/- 27; meta-tyrosine, 17 +/- 14) compared to the mutant strain (2-OH PBN, 2.5 +/- 7.3 (p < .001); 3-OH PBN, 3.9 +/- 12.3 (p = .01); ortho-tyrosine, 1.9 +/- 6.0 (p < .001); meta-tyrosine, 1.5 +/- 3.4 (p = .03)). Similar differences were observed following in vitro incubations of these bacteria with aromatic targets. These results confirm in vivo production of hydroxyl radical by E. faecalis colonizing the intestine, and indicate this bacterium may be a potent source of oxidative stress on the intestinal epithelium.

4-Hydroxy-2-Nonenal Mediates Genotoxicity and Bystander Effects Caused by Enterococcus faecalis–Infected Macrophages

BACKGROUND & AIMS Enterococcus faecalis is a human intestinal commensal that produces extracellular superoxide and promotes chromosome instability via macrophage-induced bystander effects. We investigated the ability of 4-hydroxy-2-nonenal (4-HNE), a diffusible breakdown product of ω-6 polyunsaturated fatty acids, to mediate these effects. METHODS 4-HNE was purified from E faecalis-infected macrophages; its genotoxicity was assessed in human colon cancer (HCT116) and primary murine colon epithelial (YAMC) cell lines. RESULTS 4-HNE induced G(2)-M cell cycle arrest, led to formation γH2AX foci, and disrupted the mitotic spindle in both cell lines. Binucleate tetraploid cells that formed after incubation with 4-HNE were associated with the activation of stathmin and microtubule catastrophe. Silencing glutathione S-transferase α4, a scavenger of 4-HNE, increased the susceptibility of epithelial cells to 4-HNE-induced genotoxicity. Interleukin-10 knockout mice colonized with superoxide-producing E faecalis developed inflammation and colorectal cancer, whereas colonization with a superoxide-deficient strain resulted in inflammation but not cancer. 4-HNE-protein adducts were found in the lamina propria and macrophages in areas of colorectal inflammation. CONCLUSIONS 4-HNE can act as an autochthonous mitotic spindle poison in normal colonic epithelial and colon cancer cells. This finding links the macrophage-induced bystander effects to colorectal carcinogenesis.

METABOLISM OF ETHANOL TO 1-HYDROXYETHYL RADICALS IN RAT LIVER MICROSOMES :COMPARATIVE STUDIES WITH THREE SPIN TRAPPING AGENTS

Metabolism of ethanol to 1-hydroxyethyl radicals by rat liver microsomes was studied with three nitrone spin trapping agents (POBN, PBN, and DMPO) under essentially comparable conditions. The data indicate that POBN was the superior spin trapping agent for 1-hydroxyethyl radicals, and that DMPO was least efficient. Addition of deferoxamine completely prevented detection of 1-hydroxyethyl radicals with PBN or DMPO, but caused only 50% decrease in EPR signals when POBN was the spin trap. However, superoxide dismutase only decreased 1-hydroxyethyl radical formation when POBN was the spin trap. Other experiments demonstrated that POBN was the most effective of these nitrones for reduction of Fe(III) in aqueous solutions. Furthermore, 1-hydroxyethyl radical adducts were formed when POBN was added to mixtures of ethanol, phosphate buffer, POBN and FeCl3, but this effect did not occur with either PBN or DMPO. Thus, these data indicate that undesirable effects of POBN on iron chemistry may influence results of spin trapping experiments, and complicate interpretation of the resulting data.

Aromatic hydroxylation in PBN spin trapping by hydroxyl radicals and cytochrome P-450.

Phenyl N-tert-butylnitrone (PBN) is widely used as a spin trapping agent, but is not useful detecting hydroxyl radicals because the resulting spin adduct is unstable. However, hydroxyl radicals could attack the phenyl ring to form stable phenolic products with no electron paramagnetic resonance signal, and this possibility was investigated in the present studies. When PBN was added to a Fenton reaction system composed of 25 mM H(2)O(2) and 0.1 mM FeSO(4), 4-hydroxyPBN was the primary product detected, and benzoic acid was a minor product. When the Fe(2+) concentration was increased to 1.0 mM, 4-hydroxyPBN concentrations increased dramatically, and smaller amounts of benzoic acid and 2-hydroxyPBN were also formed. Although PBN is extensively metabolized after administration to animals, its metabolites have not been identified. When PBN was incubated with rat liver microsomes and a reduced nicotinamide adenine dinculeotide phosphate (NADPH)-generating system, 4-hydroxyPBN was the only metabolite detected. When PBN was given to rats, both free and conjugated 4-hydroxyPBN were readily detected in liver extracts, bile, urine, and plasma. Because 4-hydroxyPBN is the major metabolite of PBN and circulates in body fluids, it may contribute to the pharmacological properties of PBN. But 4-hydroxyPBN formation cannot be used to demonstrate hydroxyl radical formation in vivo because of its enzymatic formation.

Effect of aging on enzymatic antioxidant defenses in rat liver mitochondria.

Antioxidant defenses within liver mitochondria are pivotal in preventing liver damage from oxidative toxicants. In this study we determined the activities of glutathione peroxidase (GPO), superoxide dismutase (SOD) and glutathione reductase (GRD) in mitochondria from livers of variously aged Fischer 344 rats. A mixed pattern of age-associated alterations in mitochondrial antioxidant activities was observed. In male rats, GRD activity decreased in old age, whereas GPO and SOD activities increased. In female rats, GPO activity decreased with age, but SOD activity increased and GRD activity was unchanged. Age-associated decreases in antioxidant protection from mitochondrial enzymes appeared to be counterbalanced by increases in protection from other enzymes.

Acetaminophen Hepatotoxicity in Aging Rats

Severity of liver damage 24 hr after i.p. administration of acetaminophen in doses of 0.4 and 0.8 g/kg was evaluated in male Fischer 344 rats at 4, 14 and 25 months of age. Both doses of acetaminophen produced significant elevations of serum alanine aminotransferase (ALT) and sorbitol dehydrogenase (SDH) activities in 4-month-old rats. Enzyme release was somewhat diminished in old age. Hepatic glutathione (GSH) and microsomal cytochrome P-450 concentrations were decreased in rats that received 0.8 g/kg of acetaminophen. The decreases occurred in young-adult and middle-aged rats, but not in old rats. The results demonstrated that old age does not enhance the hepatotoxic effects of acetaminophen in male Fischer 344 rats.

Free radical generation in the cochlea during combined exposure to noise and carbon monoxide: an electrophysiological and an EPR study.

Ototoxicity following combined exposure to noise and carbon monoxide (CO) is known to result in more severe permanent threshold shifts than exposure to noise alone. We have previously demonstrated that such potentiation of noise-induced auditory impairment by CO can be prevented by the administration of a nitrone spin-trapping agent. Although such protection implicates injury via free radical pathways, drug-induced protection does not provide direct evidence for the presence of free radicals in the cochlea. The objective of this study was to demonstrate the actual presence of nitrone spin adducts in the cochlea following simultaneous exposure to noise and CO. Using electrophysiological end-points, the protective effects of the nitrone spin-trapping agent alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN) were assessed following combined exposure of adult male Long Evans hooded rats to noise and CO. In addition, an ex-vivo evaluation of POBN spin adducts was done by electron paramagnetic resonance spectroscopy (EPR). The noise used was octave band noise with center frequency 13.6 kHz at 100 dB(Lin) for a duration of 2 h. The level of CO used was 1200 ppm. Electrophysiological results demonstrate that POBN protects against combined exposure to noise plus CO. The EPR study demonstrates POBN spin adducts in the cochleae of animals exposed to noise plus CO. Therefore, this study provides evidence to the hypothesis that ototoxicity due to noise plus CO exposure is mediated via free radicals.