In vitro studies suggest that reactive oxygen species contribute to the cardiopulmonary toxicity of particulate air pollution. To evaluate the ability of particulate air pollution to promote oxidative stress and tissue damage in vivo, we studied a rat model of short-term exposure to concentrated ambient particles (CAPs). We exposed adult Sprague-Dawley rats to either CAPs aerosols (group 1; average CAPs mass concentration, 300 +/- 60 micro g/m3) or filtered air (sham controls) for periods of 1-5 hr. Rats breathing CAPs aerosols for 5 hr showed significant oxidative stress, determined as in situ chemiluminescence in the lung [group 1, 41 +/- 4; sham, 24 +/- 1 counts per second (cps)/cm2] and heart (group 1, 45 +/- 4; sham, 24 +/- 2 cps/cm2) but not liver (group 1, 10 +/- 3; sham, 13 +/- 3 cps/cm2). Increases in oxidant levels were also triggered by highly toxic residual oil fly ash particles (lung chemiluminescence, 90 +/- 10 cps/cm2; heart chemiluminescence, 50 +/- 3 cps/cm2) but not by particle-free air or by inert carbon black aerosols (control particles). Increases in chemiluminescence showed strong associations with the CAPs content of iron, manganese, copper, and zinc in the lung and with Fe, aluminum, silicon, and titanium in the heart. The oxidant stress imposed by 5-hr exposure to CAPs was associated with slight but significant increases in the lung and heart water content (approximately 5% in both tissues, p < 0.05) and with increased serum levels of lactate dehydrogenase (approximately 80%), indicating mild damage to both tissues. Strikingly, CAPs inhalation also led to tissue-specific increases in the activities of the antioxidant enzymes superoxide dismutase and catalase, suggesting that episodes of increased particulate air pollution not only have potential for oxidant injurious effects but may also trigger adaptive responses.

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The time course of oxidative stress and tissue damage in zonal liver ischemia-reperfusion in rat liver in vivo was evaluated. After 180 min of ischemia, surface chemiluminescence decreased to zero, state 3 mitochondrial respiration decreased by 70-80%, and xanthine oxidase activity increased by 26% without change in the water content and in the activities of superoxide dismutase, catalase, and glutathione peroxidase. After reperfusion, marked increases in oxyradical production and tissue damage were detected. Mitochondrial oxygen uptake in state 3 and respiratory control as well as the activities of superoxide dismutase, catalase, and glutathione peroxidase and the level of nonenzymatic antioxidants (evaluated by the hydroperoxide-initiated chemiluminescence) were decreased. The severity of the post-reperfusion changes correlated with the time of ischemia. Morphologically, hepatocytes appeared swollen with zonal cord disarrangement which ranged from mild to severe for the tissue reperfused after 60-180 min of ischemia. Neutrophil infiltration was observed after 180 min of ischemia and 30 min of reperfusion. Mitochondria appear as the major source of hydrogen peroxide in control and in reperfused liver, as indicated by the almost complete inhibition of hydrogen peroxide production exerted by the uncoupler carbonylcyanide p-(trifluoromethoxy) phenylhydrazone. Additionally, inhibition of mitochondrial electron transfer by antimycin in liver slices reproduced the inhibition of state 3 mitochondrial respiration and the increase in hydrogen peroxide steady-state concentration found in reperfused liver. Increased rates of oxyradical production by inhibited mitochondria appear as the initial cause of oxidative stress and liver damage during early reperfusion in rat liver. (J. Clin. Invest. 1993.91:456-464.) Key words: chemiluminescence * hydrogen peroxide -mitochondrial damage * reperfusion injury. xanthine oxidase

Exposure of cells to hydrogen peroxide (H2O2) mediates adaptive responses or oxidative damage, depending on the magnitude of the challenge. Determining the threshold for peroxide-mediated oxidative stress thus requires quantitation of the changes in endogenous H2O2 production. The intracellular steady-state concentrations of H2O2 were measured in intact Escherichia coli under different conditions. Compounds that block electron transport at NADH dehydrogenase (rotenone) or between ubiquinone and cytochrome b (antimycin) showed that univalent reduction of O2 can occur at these sites in vivo to form superoxide anion (O2-), in agreement with reports for mammalian mitochondria. Mutational inactivation of different components of the respiratory chain showed that H2O2 production also depended on the energy status of the cell and on the arrangement of respiratory chain components corresponding to particular growth conditions. Production rates for O2- and H2O2 were linearly related to the number of active respiratory chains that reached maximal values during exponential growth. In the strains defective in respiratory chain components, catalase activity was regulated to compensate for changes in the H2O2 production rates, which maintained intracellular H2O2 at 0.1-0.2 microM during aerobic growth over a wide range of cell densities. The expression of a katG'::lacZ fusion (reporting transcriptional control of the catalase-hydroperoxidase I gene) was increased by H2O2 given either as a pulse or as a steady production. This response not only depended on the type and severity of the stimulus but was also strongly influenced by the growth phase of the cells.

The exponential phase of aerobic growth is associated with risk of endogenous oxidative stress in which cells need to cope with an ϳ10-fold increase in the rate of H 2 O 2 generation. We addressed this issue by studying the regulation of the intracellular concentration of H 2 O 2 in aerobically growing Escherichia coli. Intracellular H 2 O 2 was kept at an almost constant steady-state value of ϳ0.2 M (variation, less than twofold) over a broad range of cell densities in rich medium. This regulation was achieved in part by a transient increase in the OxyRdependent transcription of the catalase gene katG (monitored by using a katG::lacZ operon fusion) during exponential growth, directly correlated with the increased rate of H 2 O 2 generation. The OxyR-regulated alkyl hydroperoxide reductase encoded by ahpFC did not detectably affect H 2 O 2 or catalase activity levels. Induction of katG, ahpFC, and perhaps other genes prevented the accumulation of oxidatively modified lipids but may not have protected DNA: the spontaneous mutation rate was significantly increased in both wild-type and ⌬oxyR strains during exponential growth compared to that in these strains during lag or stationary phases. Strains lacking oxyR showed throughout growth an 8-to 10-fold-higher frequency of spontaneous mutation than was seen for wild-type bacteria. The ahp⌬5 allele also had a mutator effect half of that of ⌬oxyR in exponential and stationary phases and equal to that of ⌬oxyR in lag phase, perhaps by affecting organic peroxide levels. These results show that oxyR-regulated catalase expression is not solely an emergency response of E. coli to environmental oxidative stress, but also that it mediates a homeostatic regulation of the H 2 O 2 produced by normal aerobic metabolism. The activation of the oxyR regulon in this process occurs at much lower levels of H 2 O 2 (ϳ10 ؊7 M) than those reported for oxyR activation by exogenous H 2 O 2 (ϳ10 ؊5 M).