Oxidative stress and antioxidant status have been repeatedly investigated in animal models and in humans, but there is a considerable lack of studies validating whether antioxidants may be used as biomarkers to assess oxidative stress. This study was designed to continue to measure a series of oxidative stress biomarkers in blood and BALF collected from similarly exposed ozone rats. A strength of the design is that the experimental exposures and sample collections were all done in one laboratory under identical conditions. This sample collection design allows direct comparison of different methods using the same samples. Samples were sent in a blind manner to laboratories with extensive experience in respective analytical procedures. Thus, the design eliminates possible errors due to inexperience in sophisticated analytic techniques and also avoids any possible bias in sample analysis and reporting of data. However, in this design, there were logistical issues in collection of samples, e.g., order of collection and timing of processing, which could affect results of specific assays. These issues were addressed in the experimental design and have been previously discussed [17
Ozone exposure was chosen as a second rodent model of oxidative stress after CCl4
]. It is hypothesized that highly reactive ozone molecules react with unsaturated fatty acids at the air-tissue boundary to form lipid ozonation products, which, in turn, can activate membrane lipases and lead to the generation of proinflammatory mediators and free radicals [2
]. Epidemiological studies implicate ozone exposure as the most prominent environmental oxidant which has been linked to exacerbation of allergic airway diseases including asthma [24
Since antioxidants are known to scavenge free radicals [25
], our aim was to evaluate major antioxidant substances in blood plasma and BALF and to determine if loss of antioxidants could be used to assess the anticipated oxidative effects of ozone.
To gain insight into ozone toxicity under our experimental conditions we compared the histopathological changes that have occurred in the lungs of rats with and without ozone exposure. Results indicated that following both doses of ozone exposure, lung tissue changes consisted of necrosis of the epithelium lining in the bronchioles and accumulation of a mixture of exfoliated epithelial cells, a few PMNs, leucocytes, macrophages, cell debris, and various amounts of fibrin. In general, lung changes described in these studies are consistent with those reported in the literature although experimental protocols of the ozone inhalation exposure vary in animal models and in humans [26
]. Changes in the groups with the higher dose of 5 ppm were relatively more prominent in contrast to groups with the lower dose of 2ppm, where the changes were minimal. The severity of the changes induced in the lungs suggests that antioxidant concentrations in plasma and BALF might be altered in response to lung tissue damage caused by zone.
We found that ascorbic acid concentration in both plasma and BALF was significantly decreased below the control level by the higher dose of ozone 2 h after the exposure. At the higher dose of ozone, ascorbic acid in BALF was decreased at 7 h and 16 h after the inhalation exposure, but the differences were significant only at the 7 h time point. Ascorbic acid is a well studied antioxidant in plasma and tissues where it acts as a scavenger of free radicals and recycles other important antioxidant molecules [29
]. Although evidence from in vitro
studies and from some animal models has demonstrated the protective effects of ascorbic acid mediated by increases mainly in BALF, the clinical evidence is still controversial [33
]. In this study the ozone inhalation exposure of Fisher rats for 2 h determined that the distinct pathologic effects in the lung caused by the high dose of ozone 2 h and 7 h after the exposure were complemented with the loss of ascorbic acid in both plasma and BALF. Literature data show that ascorbate concentrations in the lung can be either increased or decreased depending on the length of exposure, sampling time, or age or dietary status of the animals studied. The results of the current study are in agreement with literature that reports in vitro depletion of ascorbic acid in BALF by ozone [37
] and in contradiction with others that show elevation of ascorbic acid in lungs and BALF after repeated ozone inhalation or diet restrictions [34
Another potential target of ozone oxidation could be α-tocopherol since it is quantitatively the major lipid-soluble antioxidant in plasma [39
], although under some circumstances α-tocopherol showed a pro- rather than anti-oxidant activity [40
]. Additional isomers of α-tocopherol are either present in very low concentrations or not detectable in plasma [42
]. Our time course measurements of α-tocopherol and its isomers in rat plasma revealed no significant changes after inhalation (2 h) of either low or high doses of ozone. In contrast, our studies showed no changes of α-tocopherol in BALF. Under different experimental conditions, other studies reported evidence of depletion of α-tocopherol in the lung and BALF following ozone exposure [26
]. Furthermore, ozone exposure had no significant effect on the plasma nitration product of γ-tocopherol, 5-nitro-γ-tocopherol or α-tocopheryl quinone concentrations. There are no literature data available for the effect of ozone on α-tocopheryl quinones although their role in modulating mitochondrial electron transfer and mitochondrial superoxide radical production has been reported [45
GSH has been documented and has been recognized for decades to play many roles in biological protective mechanisms and critical physiological functions. Conjugates, disulfides, and other GSH-derived products have also been studied as biomarkers of key metabolites of toxic agents. Despite the extensive evidence implicating the depletion and/or oxidation of GSH in a wide variety of human and experimental toxicities, critical examination of such studies frequently reveals that injury is not simply related to glutathione status [46
]. Indeed our studies show that neither dose of ozone exposure caused significant changes in plasma and BALF concentrations of either GSH or GSSG.
The reduction potentials (Eh) for the redox couples, GSH/GSSG and cysteine/cystine (Cys/CySS), in plasma are useful indicators of systemic oxidative stress and other medically relevant physiological states [21
]. Several lines of evidence indicate that perturbations in the extracellular thiol/disulfide redox environment correlate with the progression and severity of acute lung injury. Cysteine (Cys) and its disulfide Cystine (CySS) constitute the most abundant, low-molecular-weight thiol/disulfide redox couple in the plasma, and Cys homeostasis is adversely affected during the inflammatory response to infection and injury. While much emphasis has been placed on GSH and GSSG, little is known about the regulation of the Cys/CySS couple in acute lung injury [22
]. The calculated redox states of the GSH/GSSG and Cys/CySS couples showed no statistically significant differences between the experimental and control groups.
Uric acid is the metabolic degradation product of xanthine and a potent hydrophilic antioxidant that scavenges certain oxygen radicals [47
]. It has also been shown to stabilize ascorbic acid in human plasma at physiological concentrations [48
]. Several lines of evidence suggest that uric acid in upper airway secretions may play a significant role in removing inhaled ozone [37
]. Others have concluded that the reducing and acidic properties of urate are important in the effective scavenging of peroxynitrite and that Cys and ascorbic acid enhance urate’s antioxidant effect by reducing urate-derived radicals [49
]. Our findings in the present study that increases of uric acid in BALF by both doses of ozone in the early ozone post-exposure period and by the high dose 7 h after the exposure are surprising since BALF contains ascorbic acid, UA, and GSH all of which react easily with ozone [50
]. Among these three antioxidants, ascorbic acid decreased, GSH remained the same and uric acid increased. A mechanism which may explain the increased uric acid in BALF is enhanced permeability of the lung to blood plasma, which contains concentrations of uric acid commensurate with the increased BALF protein observed here (data not shown). The increase in uric acid might also be attributed to increased purine nucleotide catabolism in the lung with increased degradation of xanthine. Our histopathology findings and other studies have demonstrated that ozone could induce apoptosis and massive necrosis in lung tissue [51
]. Plasma uric acid concentration was increased in this study by only the high dose (5 ppm) of ozone inhalation 7 hours after the exposure and did not show any other time or dose dependence. Results from previous studies have demonstrated that ozone exposure could increase plasma uric acid concentration in humans and in guinea pigs [52
In summary, exposure of Fisher rats to ozone inhalation did not cause a significant decrease in plasma or BALF concentrations of various antioxidant substances in a time- and dose-dependent pattern. On the basis of this finding, we suggested that antioxidants measured in plasma or BALF do not play a major role in identifying early ozone-induced oxidative lung injury. We previously found that measurements of plasma antioxidants were unsuccessful biomarkers of oxidative damage by CCl4
]. Again, this continuation of the Biomarkers of Oxidative Stress Study (BOSS) found that rat plasma antioxidants - ascorbic acid, α-, δ-, γ-tocopherols, 5-nitro-γ-tocopherol, tocol, glutathione (GSH/GSSG), cysteine (Cys/CySS) and uric acid - were not further decreased by the ozone exposure and therefore did not constitute selective, sensitive and specific biomarkers for oxidative damage. We concluded that measurement of antioxidants in plasma is not a useful choice for the assessment of oxidative damage by ozone in vivo