Purification and characterization of mycoferritin from Aspergillus parasiticus (255)

As intracellular iron storage molecules, only hydroxymate type siderophores have been reported in ascomycetes and basidiomycetes. This is the first report documenting the presence of mycoferritin in ascomycetes. The fungus, Aspergillus parasiticus (255), is capable of producing mycoferritin only upon induction with iron in yeast extract sucrose (YES) medium. The same has been purified from Aspergillus sps by application of conventional biochemical techniques. The molecular mass, yield, iron and carbohydrate contents of the HPLC purified protein were 460kDa, 0.012mg/g of wet mycelia, 1.6% and 6.0%, respectively. The iron content was much lower than Mortierella alpina mycoferritin (17%). Native PAGE revealed the presence of trimeric and monomeric forms of ferritin. Subunit analysis by SDS-PAGE showed a single protein subunit of approximately 20kDa suggesting structural simplicity of the apoferritin shell. Variation in amino acid composition was noted upon comparison with ferritins of other species. Interestingly, no phenylalanine could be detected in the mycoferritin of Aspergillus sps. The acidic amino acid content was 1.5-1.6 fold higher than mammalian and fish ferritins. The spectral characteristics (UV/VIS and fluorescence) of mycoferritin were akin to equine spleen ferritin. However, circular dichroic spectra revealed a lower degree of helicity.     

Purification and characterization of mycoferritin from Fusarium verticillioides MRC 826

The fungus Fusarium verticillioides MRC 826 (ascomycetes species), a toxigenic isolate is capable of synthesizing mycoferritin only upon induction with iron in yeast extract sucrose medium. The molecular mass, yield, iron and carbohydrate contents of the purified mycoferritin were 460 kDa, 0.010 mg/g of wet mycelia, 1.0 and 40.2%, respectively. Native gel electrophoresis of the mycoferritin revealed two bands possibly representing isoforms of ferritin. Subunit analysis by SDS–PAGE showed a single protein subunit of ~24 kDa suggesting similar sized subunits in the structure of apoferritin shell. Immunological cross reactivity was observed with the anti-fish liver ferritin. Transmission electron microscopy revealed an apparent particle size of 100 Å. N-terminal amino acid sequencing of mycoferritin showed identities with other eukaryotic ferritin sequences. The spectral characteristics were similar to equine spleen ferritin. However, circular dichroic spectra revealed a higher degree of helicity. Functionally, induction of mycoferritin minimizes the pro-oxidant role of iron.     

The role of oxidative stress in the toxicity of pyridoxal isonicotinoyl hydrazone (PIH) analogues

Pyridoxal isonicotinoyl hydrazone (PIH) analogues are effective iron chelators in vivo and in vitro, and may be of value for the treatment of secondary iron overload. The sensitivity of Jurkat cells to Fe-chelator complexes was enhanced several-fold by the depletion of the antioxidant glutathione, indicating the role of oxidative stress in their toxicity. K562 cells loaded with eicosapentaenoic acid, a fatty acid particularly susceptible to oxidation, were also more sensitive to the toxic effects of the Fe complexes, and toxicity was proportional to lipid peroxidation. Thus Fe-chelator complexes cause oxidative stress, which may be a major component of their toxicity. As was the case for their Fe complexes, the toxicity of PIH analogues was enhanced by glutathione depletion of Jurkat cells and eicosapentaenoic acid-loading of K562 cells. Thus the toxicity of the chelators themselves is also enhanced by compromised cellular redox status. In addition, the toxicity of the chelators was diminished by culturing Jurkat cells under hypoxic conditions, which may limit the production of the reactive oxygen species that initiate oxidative stress. A significant part of the toxicity of the chelators may be due to intracellular formation of Fe-chelator complexes, which oxidatively destroy the cell.     

Antioxidant actions of nitroxyl (HNO)

Nitrogen oxides are endogenously produced signaling/effector molecules that have the potential to both cause and ameliorate oxidative stress. Whether nitrogen oxides behave as oxidants or antioxidants is dependent on many factors including the cellular environment, the concentration, and the presence of other reactive species. To date, the nitrogen oxide nitroxyl (HNO) has only been reported to possess prooxidant properties. However, some of its chemical properties would predict that it could also serve as an antioxidant. In this study, the possible antioxidant actions of HNO were examined using the yeast Saccharomyces cerevisiae model system. The effect of HNO on membrane lipid peroxidation was examined and HNO was determined to act solely as an antioxidant in this system. In the presence of glutathione, a thiol-containing peptide that scavenges HNO, the antioxidant action was decreased. In addition, the antioxidant properties of HNO were not due to the conversion of HNO to NO. These results were also confirmed with in vitro assays of oxidative stress. Thus, HNO has the potential to preserve lipid membrane integrity by its antioxidant actions.     

Acrolein toxicity involves oxidative stress caused by glutathione depletion in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Exposure of yeast cells to allyl alcohol results in intracellular production of acrolein. The toxicity of so formed acrolein involves oxidative stress, as (1) strains deficient in antioxidant defense are hypersensitive to allyl alcohol, (2) exposure to allyl alcohol increases the level of thiobarbituric-acid-reactive substances and decreases glutathione level in the cells, (3) hypoxic and anoxic atmosphere and antioxidants protect against allyl alcohol toxicity, and (4) allyl alcohol causes activation of Yap1p. No increased formation of reactive oxygen species was detected in cells exposed to allyl alcohol, so oxidative stress is due to depletion of cellular thiols and thus alteration in the redox state of yeast cells.     

Prevention of selenite induced oxidative stress and cataractogenesis by luteolin isolated from Vitex negundo

Free radical mediated oxidative stress plays a crucial role in the pathogenesis of cataract and the present study was to determine the efficacy of luteolin in preventing selenite induced oxidative stress and cataractogenesis in vitro. Luteolin is a bioactive flavonoid, isolated and characterized from the leaves of Vitex negundo. Lenses were extracted from Sprague-Dawley strain rats and were organ cultured in DMEM medium. They were divided into three groups with eight lenses in each group as follows: lenses cultured in normal medium (G I), supplemented with 0.1mM sodium selenite (G II) and sodium selenite and 2 μg/ml luteolin (G III). Treatment was from the second to fifth day, while selenite administration was done on the third day. After the experimental period, lenses were taken out and various parameters were studied. The antioxidant potential of luteolin was assessed by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. In the selenite induced group, morphological examination of the lenses showed dense cortical opacification and vacuolization. Biochemical examinations revealed a significant decrease in activities of antioxidant enzymes and enzymes of the glutathione system. Additionally decreased glutathione level and increased reactive oxygen species (ROS) and thiobarbituric acid reactive substances (TBARS) were observed. Luteolin treatment abated selenite induced oxidative stress and cataractogenesis by maintaining antioxidant status, reducing ROS generation and lipid peroxidation in the lens. These finding demonstrated the anticataractogenic effect of luteolin by virtue of its antioxidant property, which has been reported in this paper for the first time.     

Oxidative stress and iron are implicated in fragmenting vacuoles of Saccharomyces cerevisiae lacking Cu,Zn-superoxide dismutase.

The absence of the antioxidant enzyme Cu,Zn-superoxide dismutase (SOD1) is shown here to cause vacuolar fragmentation in Saccharomyces cerevisiae. Wild-type yeast have 1-3 large vacuoles whereas the sod1Delta yeast have as many as 50 smaller vacuoles. Evidence that this fragmentation is oxygen-mediated includes the findings that aerobically (but not anaerobically) grown sod1Delta yeast exhibit aberrant vacuoles and genetic suppressors of other oxygen-dependent sod1 null phenotypes rescue the vacuole defect. Surprisingly, iron also is implicated in the fragmentation process as iron addition exacerbates the sod1Delta vacuole defect while iron starvation ameliorates it. Because the vacuole is reported to be a site of iron storage and iron reacts avidly with reactive oxygen species to generate toxic side products, we propose that vacuole damage in sod1Delta cells arises from an elevation of iron-mediated oxidation within the vacuole or from elevated pools of "free" iron that may bind nonproductively to vacuolar ligands. Furthermore, additional pleiotropic phenotypes of sod1Delta cells (including increased sensitivity to pH, nutrient deprivation, and metals) may be secondary to vacuolar compromise. Our findings support the hypothesis that oxidative stress alters cellular iron homeostasis which in turn increases oxidative damage. Thus, our findings may have medical relevance as both oxidative stress and alterations in iron homeostasis have been implicated in diverse human disease processes. Our findings suggest that strategies to decrease intracellular iron may significantly reduce oxidatively induced cellular damage.     

Glutathione is an important antioxidant molecule in the yeast Saccharomyces cerevisiae

Abstract The tripeptide γ-l-glutamyl-l-cystinylglycine (glutathione) is one of the major antioxidant molecules of cells and is thought to play a vital role in buffering the cell against reactive oxygen species and toxic electrophiles. We wished to determine the role of glutathione in the protection of the yeast Saccharomyces cerevisiae against oxidative stress. This study shows that glutathione is an important antioxidant molecule in yeast, with γ-glutamylcysteine synthetase ( gshI ) mutants, deficient in glutathione synthesis, being hypersensitive to H₂O₂ and Superoxide anions in both exponential- and stationary-phase cultures. Despite this, these mutants are still able to induce adaptive stress responses to oxidants.     

Anti-oxidative stress effects of Herba leonuri on ischemic rat hearts

Our current study was to test the hypothesis that the extract of Herba leonuri (HL) would have antioxidant and cardioprotective effects on ischemic myocardium. The extract of HL (400 mg/kg/day) was administered orally (daily) starting from 1 week before and continuing until 3 weeks after myocardial infarction (MI). Surviving rats were sacrificed at different time points to obtain left ventricles for biochemical assays. Our study demonstrates for the first time that HL does have antioxidant effects both in vitro and in vivo. The antioxidant effects of HL are exerted only under the condition of oxidative stress, by selectively preserving the activities of superoxide dismutase and glutathione peroxidase, as well as depressing the formation of malondialdehyde, especially in the acute phase of acute MI. Its effects of scavenging free radicals and inhibiting the formation of reactive oxygen species may play a key role in protecting the endogenous antioxidant system from oxidative stress in vivo.     

Protective effect of Didymocarpus pedicellata on ferric nitrilotriacetate (Fe-NTA) induced renal oxidative stress and hyperproliferative response

Didymocarpus pedicellata R. Br. (Gesneriaceae) is widely used in traditional Indian medicines against renal afflictions. In the present study, we have revealed ethanolic extract of aerial parts of D. pedicellata to possess significant antioxidant activity and protect against ferric nitrilotriacetate (Fe-NTA) mediated renal oxidative stress, nephrotoxicity and tumor promotion response. D. pedicellata extract was found to possess a high content of total polyphenolics, exhibit potent reducing power and significantly scavenge free radicals including several reactive oxygen species (ROS) and reactive nitrogen species (RNS). The extract also significantly and dose-dependently protected against Fe-NTA plus H(2)O(2)-mediated damage to lipids and DNA. Protective efficacy of the extract was also tested in vivo against Fe-NTA mediated nephrotoxicity and tumor promotion response. Administration of Fe-NTA (9 mg/kg body weight, i.p.) to Swiss albino mice depleted renal glutathione content and activities of antioxidant and phase II metabolizing enzymes with concomitant induction of oxidative damage. Fe-NTA also incited hyperproliferation response elevating ornithine decarboxylase activity and [(3)H]-thymidine incorporation into DNA. Elevation in serum creatinine (SCr) and blood urea nitrogen (BUN), and histopathological changes were also evident and suggested Fe-NTA to afflict damage to kidney. Pretreatment of mice with D. pedicellata extract (100-200 mg/kg body weight) for 7 days not only restored antioxidant armory near normal values but also significantly protected against renal oxidative stress and damage restoring normal renal architecture and levels of renal damage markers, viz., BUN and SCr. The results of the present study indicate D. pedicellata to possess potent antioxidant and free radical scavenging activities and preclude oxidative damage and hyperproliferation in renal tissues.     

Reactive oxygen species,antioxidant mechanisms and serum cytokine levels in cancer patients: impact of an antioxidant treatment

Objective. So far, it is not well established whether oxidative stress found in cancer patients results from an increased production of oxidants in the body or from a failure of physiological antioxidant systems. To further investigate this question we have assessed the blood levels of reactive oxygen species as a marker of free radicals producing oxidative stress and the most relevant of the physiological body enzymes counteracting reactive oxygen species, namely glutathione peroxidase and superoxide dismutase. Serum levels of proinflammatory cytokines and IL‐2 were also investigated. All these parameters were studied in relation to the clinically most important index of disease progression, namely Performance Status (ECOG PS). We also tested the reducing ability of different antioxidant agents on reactive oxygen species levels by measuring the increase in glutathione peroxidase activity, and the reduction of serum levels of IL‐6 and TNF. Design, setting and subjects. We carried out an open non randomized study on 28 advanced stage cancer patients (stage III, 10.7%, and stage IV, 89.3%) with tumours at different (8) sites: all were hospitalized in the Medical Oncology Dept, University of Cagliari Interventions. The patients were divided into 5 groups and a different antioxidant treatment was administered to each group. The selected antioxidants were: alpha lipoic acid 200 mg/day orally, N‐acetylcysteine 1800 mg/day i.v. or carboxycysteine‐lysine salt 2.7 g/day orally, amifostine 375 mg/day i.v., reduced glutathione 600 mg/day i.v., vitamin A 30000 IU/day orally plus vitamin E 70 mg/day orally plus Vitamin C 500 mg/day orally. The antioxidant treatment was administered for 10 consecutive days. Results. Our results show that all but one of the antioxidants tested were effective in reducing reactive oxygen species levels and 2 of them (cysteine‐containing compounds and amifostine) had the additional effect of increasing glutathione peroxidase activity. Comprehensively, the “antioxidant treatment” was found to have an effect both on reactive oxygen species levels and glutathione peroxidase activity. The antioxidant treatment also reduced serum levels of IL‐6 and TNF. Patients in both ECOG PS 0‐1 and ECOG PS 2‐3 responded to antioxidant treatment.     

Antioxidant action of <Emphasis Type="Italic">Andrographis paniculata</Emphasis> on lymphoma

Regulation of the balance between production of reactive oxygen species (ROS) by cellular processes and its removal by antioxidant defense system maintains normal physiological processes. Any condition leading to increased ROS results in oxidative stress which has been related with a number of diseases including cancer. Improvement in antioxidant defense system is required to overcome the damaging effects of oxidative stress. Therefore in the present study, effect of the aqueous extract of a medicinal plant Andrographis paniculata (AP) on antioxidant defense system in liver is investigated in lymphoma bearing AKR mice. Estimating catalase, superoxide dismutase and glutathione S transferase monitored the antioxidant action. Oral administration of the aqueous extract of A. paniculata in different doses causes a significant elevation of catalase, superoxide dismutase and glutathione S transferase activities. It reveals the antioxidant action of the aqueous extract of AP, which may play a role in the anticarcinogenic activity by reducing the oxidative stress. LDH activity is known to increase in various cancers due to hypoxic condition. Lactate dehydrogenase is used as tumor marker. We find a significant decrease in LDH activity on treatment with AP, which indicates a decrease in carcinogenic activity. A comparison with Doxorubicin (DOX), an anticancerous drug, indicates that the aqueous extract of AP is more effective than DOX with respect to its effect on catalase, superoxide dismutase, glutathione S transferase as well as on lactate dehydrogenase activities in liver of lymphoma bearing mice.     

Decreasing peroxiredoxin II expression decreases glutathione, alters cell cycle distribution, and sensitizes glioma cells to ionizing radiation and H(2)O(2)

Glioblastomas are notorious for their resistance to ionizing radiation and chemotherapy. We hypothesize that this resistance to ionizing radiation is due, in part, to alterations in antioxidant enzymes. Here, we show that rat and human glioma cells overexpress the antioxidant enzyme peroxiredoxin II (Prx II). Glioma cells in which Prx II is decreased using shRNA exhibit increased hyperoxidation of the remaining cellular Prxs, suggesting that the redox environment is more oxidizing. Of interest, decreasing Prx II does not alter other antioxidant enzymes (i.e., catalase, GPx, Prx I, Prx III, CuZnSOD, and MnSOD). Analysis of the redox environment revealed that decreasing Prx II increased intracellular reactive oxygen species in 36B10 cells; extracellular levels of H(2)O(2) were also increased in both C6 and 36B10 cells. Treatment with H(2)O(2) led to a further elevation in intracellular reactive oxygen species in cells where Prx II was decreased. Decreasing Prx II expression in glioma cells also reduced clonogenic cell survival following exposure to ionizing radiation and H(2)O(2). Furthermore, lowering Prx II expression decreased intracellular glutathione and resulted in a significant decline in glutathione reductase activity, suggesting a possible mechanism for the observed increased sensitivity to oxidative insults. Additionally, decreasing Prx II expression increased cell cycle doubling times, with fewer cells distributed to S phase in C6 glioma cells and more cells redistributed to the most radiosensitive phase of the cell cycle, G2/M, in 36B10 glioma cells. These findings support the hypothesis that inhibiting Prx II sensitizes glioma cells to oxidative stress, presenting Prxs as potential therapeutic targets.     

REACTIVE OXYGEN METABOLISM IN INTERTIDAL FUCUS SPP. (PHAEOPHYCEAE)

Our previous research suggests that interspecific variation in stress tolerance in intertidal Fucus spp. (Phaeophyceae) is partially mediated by differences in the production of, or ability to detoxify, reactive oxygen. Here we report on the content of antioxidants (ascorbate, glutathione, carotenoids, and tocopherols) and protective enzymes (catalase, superoxide dismutase, ascorbate peroxidase, and glutathione reductase) involved in reactive oxygen metabolism in three species of intertidal brown algae— Fucus spiralis L., F. evanescens C. Ag., and F. distichus L.—that differ in stress tolerance and position in the intertidal zone. Contents of the major antioxidants were similar in the three species and were not correlated with stress tolerance. The least stress tolerant species, F. distichus, had the lowest activity of reactive-oxygen-scavenging enzymes, although F. spiralis, the species with the highest stress tolerance, and F. evanescens contained similar activities of antioxidant enzymes on a fresh-weight basis. However, the activities of superoxide dismutase and ascorbate peroxidase in F. evanescens are lower than those of F. spiralis when expressed on the basis of chlorophyll. These data show that the ratio between reactive oxygen protection and production might be more important than the absolute content of antioxidants and protective enzymes. It also shows the importance of localization of detoxifying mechanisms and avoidance of oxidative stress.     

Increased glutathione content in yeastSaccharomyces cerevisiae exposed to NaCl

Glutathione is one of the major antioxidant molecules of cells and is thought to play a vital role in buffering the cell against reactive oxygen species and toxic electrophiles. Since an overlap between osmotic and oxidative stress response is already know, the aim of the work was to determine the role of glutathione in yeast stress response to NaCl. YeastSaccharomyces cerevisiae ZIM 2155 was exposed to NaCl in concentration of 1–8% (w/v). Measuring cell cultivability showed a significant decrease appeared in cell exposed to 5–8% NaCl for 1 h. Cultivable cells were about 50% of control. Increased production of reactive oxygen species in cells exposed to 6, 7 and 8% NaCl for 1 h (1.3-fold, 1.9-fold and 2.8-fold increase, respectively) led to elevated glutathione content in reduce d form (119.1%, 122.6%, 141.5%, respectively). Two hours from NaCl addition intracellular oxidant level was slightly elevated compared to 1-h exposure, while glutathione content in reduced form was almost the same. We demonstrated that glutathione plays an important role in yeast stress response to NaCl.     

The effect ofGongronema latifolium extracts on serum lipid profile and oxidative stress in hepatocytes of diabetic rats

Diabetes is known to involve oxidative stress and changes in lipid metabolism. Many secondary plant metabolites have been shown to possess antioxidant activities, improving the effects of oxidative stress on diabetes. This study evaluated the effects of extracts from Gongronema latifolium leaves on antioxidant enzymes and lipid profile in a rat model of non insulin dependent diabetes mellitus (NIDDM). The results confirmed that the untreated diabetic rats were subjected to oxidative stress as indicated by significantly abnormal activities of their scavenging enzymes (low superoxide dismutase and glutathione peroxide activities), compared to treated diabetic rats, and in the extent of lipid peroxidation (high malondialdehyde levels) present in the hepatocytes. The ethanolic extract of G. latifolium leaves possessed antioxidant activity as shown by increased superoxide dismutase and glutathione peroxidase activities and decreases in malondialdehyde levels. High levels of triglycerides and total cholesterol, which are typical of the diabetic condition, were also found in our rat models of diabetes. The ethanolic extract also significantly decreased triglyceride levels and normalized total cholesterol concentration.     

Response of the antioxidant defense system to tert-butyl hydroperoxide and hydrogen peroxide in a human hepatoma cell line (HepG2)

The aim of this work was to investigate the response of the antioxidant defense system to two oxidative stressors, hydrogen peroxide and tert-butyl hydroperoxide, in HepG2 cells in culture. The parameters evaluated included enzyme activity and gene expression of superoxide dismutase, catalase, glutathione peroxidase, and activity of glutathione reductase. Besides, markers of the cell damage and oxidative stress evoked by the stressors such as cell viability, intracellular reactive oxygen species generation, malondialdehyde levels, and reduced glutathione concentration were evaluated. Both stressors, hydrogen peroxide and tert-butyl hydroperoxide, enhanced cell damage and reactive oxygen species generation at doses above 50 microM. The concentration of reduced glutathione decreased, and levels of malondialdehyde and activity of the antioxidant enzymes consistently increased only when HepG2 cells were treated with tert-butyl hydroperoxide but not when hydrogen peroxide was used. A slight increase in the gene expression of Cu/Zn superoxide dismutase and catalase with 500 microM tert-butyl hydroperoxide and of catalase with 200 microM hydrogen peroxide was observed. The response of the components of the antioxidant defense system evaluated in this study indicates that tert-butyl hydroperoxide evokes a consistent cellular stress in HepG2.     

Oxidative stress as a prerequisite for aflatoxin production by Aspergillus parasiticus

The relevance of free radical generation and oxidative stress with regard to aflatoxin production was examined by comparing the oxygen requirement and antioxidant status of a toxigenic strain of Aspergillus parasiticus with that of a nontoxigenic strain at early (trophophase) and late logarithmic (idiophase) growth phases. In comparison to the nontoxigenic strain, wherein the oxygen requirements were relatively unaltered at various growth phases, the toxigenic strain exhibited greater oxygen requirements at trophophase coinciding with onset of aflatoxin production. The activities of antioxidant enzymes such as xanthine oxidase, superoxide dismutase, and glutathione peroxidase and the mycelial contents of thiobarbituric acid-reactive substances as well as of reduced glutathione were all enhanced during the progression of toxigenic strain from trophophase to idiophase. The combined results suggest that aflatoxin production by the toxigenic strain may be a consequence of increased oxidative stress leading to enhanced lipid peroxidation and free radical generation.     

Antioxidant activities of seabuckthorn (Hippophae rhamnoides) during hypoxia induced oxidative stress in glial cells

The present study reports the cytoprotective and antioxidant properties of alcoholic leaf extract of seabuckthorn (SBT) against hypoxia induced oxidative stress in C-6 glioma cells. Exposure of cells to hypoxia for 12 h resulted in a significant increase in cytotoxicity and decrease in mitochondrial transmembrane potential compared to the controls. Further an appreciable increase in nitric oxide and reactive oxygen species (ROS) production was noted which in turn was responsible for fall in intracellular antioxidant levels and GSH/GSSG ratio. There was a significant increase in DNA damage during hypoxia as revealed by comet assay. Pretreatment of cells with alcoholic leaf extract of SBT at 200 μg/ml significantly inhibited cytotoxicity, ROS production and maintained antioxidant levels similar to that of control cells. Further, the leaf extract restored the mitochondrial integrity and prevented the DNA damage induced by hypoxia. These results indicate that the leaf extract of SBT has strong antioxidant and cytoprotective activity against hypoxia induced oxidative injury. (Mol Cell Biochem 278: 9–14, 2005)     

2,3,7,8-Tetrachlorodibenzo-p-dioxin-mediated oxidative stress in CYP1A2 knockout (CYP1A2-/-) mice.

The objective of the study was to compare alterations in indicators of oxidative stress following 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in cytochrome P4501A2 (CYP1A2) knockout mice and their parental lineage strains (C57BL/6N and 129/Sv). This study will aid in determining the role, if any, of CYP1A2 in TCDD-mediated oxidative stress. Formation of thiobarbituric acid-reactive substances (TBARS) as a measurement of lipid peroxidation, production of reactive oxygen species (ROS) via the in vitro reduction of cytochrome c in tissue homogenate, and changes in the biochemical antioxidant glutathione were monitored to determine oxidative stress 7 days following a single oral dose of 25 microg TCDD/kg. TBARS, reduction of cytochrome c, and changes in glutathione demonstrated a similar response in CYP1A2 knockout and parental strains. These data suggest that CYP1A2 does not play a critical role in the acute oxidative stress response following TCDD exposure.