Oxidative Stress in Female B6C3F1 Mice following Acute and Subchronic Exposure to 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)

B. P. Slezak*, G. E. Hatch{dagger}, M. J. DeVito{dagger},1, J. J. Diliberto{dagger}, R. Slade{dagger}, K. Crissman{dagger}, E. Hassoun{ddagger} and L. S. Birnbaum{dagger}

* Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; {dagger} Experimental Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711; and {ddagger} College of Pharmacy, University of Toledo, Toledo, Ohio

Received September 3, 1999; accepted November 11, 1999


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly persistent trace environmental contaminant and is one of the most potent toxicants known to man. Hassoun et al. (1998, Toxicol. Sci. 42, 23–27) reported an increase in the production of reactive oxygen species (ROS) in the brain of female B6C3F1 mice following subchronic exposure to TCDD at doses as low as 0.45 ng/kg/day. In the present study, oxidative stress was characterized in liver, spleen, lung, and kidney following subchronic (0.15–150 ng/kg; 5 days/week for 13 weeks, po) or acute exposure (0.001–100 µg/kg, po) to TCDD in order to investigate the interaction between tissue concentration and time for production of ROS. Seven days following acute administration of TCDD, mice were sacrificed; they demonstrated increases in liver superoxide anion production (SOAP) and thiobarbituric acid reactive substances (TBARS) at doses of 10 and 100 µg/kg, associated with hepatic TCDD concentrations of 55 and 321 ng/g, respectively. Liver obtained from mice following subchronic TCDD exposure demonstrated an increase in SOAP and TBARS above controls at doses of 150 ng/kg/day with liver TCDD concentration of only 12 ng/g. Interestingly, glutathione (GSH) levels in lung and kidney following subchronic TCDD exposure were decreased at the low dose of 0.15 ng/kg/day. This effect disappeared at higher TCDD doses. The data suggest that higher tissue TCDD concentrations are required to elicit oxidative stress following acute dosing than with subchronic TCDD exposure. Therefore, the mechanism of ROS production following TCDD exposure does not appear to be solely dependent upon the concentration of TCDD within the tissue. In addition, very low doses of TCDD that result in tissue concentrations similar to the background levels found in the human population produced an effect on an oxidative stress endogenous defense system. The role of this effect in TCDD-mediated toxicity is not known and warrants further investigation.

Key Words: oxidative stress; TCDD; reactive oxygen species (ROS).


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Polyhalogenated aromatic hydrocarbons (PHAHs) are highly persistent environmental contaminants that pose a potential risk to human health. The prototypical representative of these substances is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which is one of the most potent man-made toxicants known. Because of this demonstrated toxic potency and its environmental persistence, TCDD is also the most extensively studied halogenated aromatic hydrocarbon. TCDD exposure in experimental animals results in an array of tissue and species-specific responses, including the following: dermal toxicity, immunotoxicity, hepatotoxicity, carcinogenicity, teratogenicity, and neurobehavioral, endocrine, and numerous other biochemical alterations (Birnbaum, 1994Go; DeVito et al., 1995Go; Safe, 1994Go; Viluksela et al., 1995Go). An additional response under investigation is oxidative stress following exposure to TCDD.

Oxidative stress can be viewed as the disturbance in the oxidant-antioxidant balance in favor of the former. It is well established that acute high-dose exposure to TCDD results in oxidative stress in multiple tissues and species (reviewed in Stohs, 1990). Oxidative stress from TCDD exposure in laboratory animals increases the production of reactive oxygen species (ROS), lipid peroxidation, and DNA damage (Alsharif et al., 1994bGo, Muhammadpour et al., 1988Go). Oxidative stress following TCDD administration is aryl hydrocarbon (Ah) receptor-mediated (Alsharif et al., 1994aGo). A recent study demonstrated that TCDD causes a sustained oxidative stress response in female C57BL/6J mice that persists as long as 8 weeks following administration of 5 µg/kg on 3 successive days (Shertzer et al., 1998Go). In addition, recent studies reported an elevation of oxidative stress in the brain following subchronic doses as low as 0.45 ng TCDD/kg/day (Hassoun et al., 1998Go). However, oxidative stress resulting from low-dose subchronic TCDD exposure has not been fully characterized, and a relationship between TCDD tissue concentration and the corresponding oxidative stress response has not been clearly defined.

One possible mechanism of TCDD-mediated ROS production has been proposed to involve the cytochrome P450s (Park et al., 1996Go). Due to their membership in the Ah gene battery, CYP1A1 and CYP1A2 have been suggested to be associated with TCDD-mediated oxidative stress (Nebert, 1993). One proposed pathway is through the metabolic activation of estrogen via TCDD-induced cytochrome P450 enzymes (Tritscher et al., 1996Go). The involvement of cytochrome P450s in the production of ROS during normal enzyme function has long been known (Bondy and Naderi, 1994Go; Ingelman-Sundberg and Johansson, 1984Go; Kuthan and Ullrich, 1982Go; Morehouse and Aust, 1988Go) and may also contribute to TCDD-induced ROS. Although water is a normal product of the electron transfer in the CYP450 catalytic cycle, hydrogen peroxide can be produced by specific isoforms in addition to other ROS. The physiologic consequence of this free radical formation has not been thoroughly investigated. The large induction of CYP1A1, 1A2, and 1B1 by TCDD may result in increased free radical formation.

Oxidative stress following exposure to TCDD has been clearly demonstrated following high-dose acute exposure. Although results of previous studies on oxidative stress are beneficial to the understanding of the wide range of effects following exposure to TCDD, the doses used result in body burdens that are not physiologically relevant in humans following environmental exposure to TCDD. The objective of this study was to investigate whether tissue concentration accurately predicts the magnitude of TCDD-mediated oxidative stress through a direct comparison of oxidative stress resulting from two different TCDD exposure regimens. Acute and subchronic administration of TCDD across wide dose ranges in female B6C3F1 mice resulted in similar TCDD tissue concentrations, enabling a comparison of oxidative stress parameters. These data suggest that oxidation response to TCDD following subchronic exposure is qualitatively different than that observed following acute exposure. Because of these differences, tissue concentration is not a useful dose metric to describe oxidative damage from TCDD.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was obtained from Radian Corp. (Austin, TX) with stated purity of > 98% as determined by gas chromatography/mass spectrometry. [1,6-3H]TCDD was synthesized by Chemsyn Science Laboratories (Lenexa, TX) with a specific activity of 39.5 Ci/mmol and purity of > 98%. Radiochemical purity was verified by reverse-phase high-pressure liquid chromatography (HPLC) (System Gold, Beckman Instruments, Inc., Fullerton, CA) using a C18 µBondapak stainless steel column with a Guard-PAK precolumn insert (Waters, Milford, MA) and an isocratic solvent system of 85% methanol/15% water. A radioactive flow detector (Beckman Model 171, Beckman Instruments, Tampa, FL) was used to monitor radioactivity. Additionally, a bioassay of rat biliary excretion of TCDD-derived radioactivity was used to verify the purity of > 98% for the radiochemical (Jackson et al., 1998Go). All other chemicals used in the study were of highest grade commercially available. Sonicating a suspension of TCDD (1 mg) in acetone (10 ml) until the TCDD was dissolved provided a stock solution of unlabeled TCDD. Corn oil (10 ml) (Sigma Chemical Co., St. Louis, MO) was then added to the solution. The acetone was evaporated using a Speed-Vac (Savant Speed-Vac, Savant Instruments, Inc., Farmingdale, NY). Dosing solutions were prepared by adding the stock of cold TCDD and [3H]TCDD to corn oil.

Animals.
Female B6C3F1 mice 8–10 weeks of age were obtained from Jackson Laboratories (Raleigh, NC). Animals were maintained and treated at the U.S. Environmental Protection Agency (Research Triangle Park, NC). The animals were housed in an AAALAC-approved animal facility and maintained according to the National Institutes of Health Guideline on the Care and Use of Laboratory Animals. Animals were randomly separated and housed in groups of 10 in clear polycarbonate cages with bedding of pine shavings (North Eastern Products Inc., Warrensburg, NY) under controlled conditions of temperature (22 ± 1°C), humidity (40–60%), and light (12/12 h light/dark cycle). Animals were provided with Rodent Chow (Purina, St. Louis, MO) and tap water ad libitum throughout the study.

Treatments
Acute.
Each animal received a single dose of 0, 0.001, 0.01, 0.1, 1, 10, or 100 µg TCDD/kg body weight by oral gavage with corn oil as vehicle at a dosing volume of 10 ml/kg. Seven days following treatment, animals were euthanized using carbon dioxide asphyxiation. Organs were removed, snap- frozen in liquid nitrogen, and stored at –80°C until measurement of oxidative stress indicators and tissue TCDD concentration.

Subchronic.
TCDD solutions with corn oil as vehicle were administered by oral gavage at doses of 0, 0.15, 0.45, 1.5, 15, and 150 ng TCDD/kg for 13 weeks, 5 days/week (Monday–Friday) at a dosing volume of 10 ml/kg. Three days after the last treatment, animals were euthanized using carbon dioxide asphyxiation. Organs were removed, snap-frozen in liquid nitrogen, and stored at –80°C until measurement of oxidative stress indicators and tissue TCDD concentration.

Pre-exposure.
TCDD was administered by oral gavage on day 1 and 6 days later (day 7 of the study). Control group received corn oil vehicle only on both day 1 and day 7. Treatment group 1 received 0.1 µg TCDD/kg on day 1 and corn oil on day 7. Treatment group 2 was administered corn oil on day 1 and 25 µg TCDD/kg on day 7. Treatment group 3 received 0.1 µg TCDD/kg on day 1 and 25 µg TCDD/kg on day 7. All animals were euthanized using carbon dioxide asphyxiation 13 days after the initial treatment (day 14 of study). Organs were removed, snap-frozen in liquid nitrogen, and stored at –80°C until measurement of oxidative stress indicators and tissue TCDD concentrations.

Cytochrome C Reduction Assay
Superoxide anion production (SOAP) in tissue homogenates was measured by the method of Babior et al. (1973), which is based on the reduction of cytochrome c collected for subsequent spectrophotometric measurement on a Pharmacia Biotech, Novaspec II spectrophotometer (Cambridge, England). Although not entirely specific, other intracellular substances do reduce cytochrome c. This method, however, has been often used to estimate SOAP (Hassoun et al., 1998Go). A 10% (w/v) tissue homogenate was prepared in Tris-KCl buffer (0.05 M Tris and 1.15% w/v KCl, pH 7.4). Each sample for analysis contained the following: 2 ml PBS buffer (pH 7.2), 45 nmol cytochrome c, and 20 µl tissue homogenate (stored on ice). Samples were incubated for 15 min at 37°C; the reaction was terminated by placing the tubes on ice. Absorbance was determined at 550 nm and converted to nanomoles of cytochrome c reduced/min, using an extinction coefficient of 2.1 x 104 M–1 cm–1.

Thiobarbituric Acid Reactive Substances (TBARS)
Lipid peroxidation was determined in tissue homogenates by measuring the formation of thiobarbituric acid-reactive substances (TBARS), as previously described by Ghio et al. (1991) and Hassoun et al. (1998). A 10% (w/v) tissue homogenate was prepared in Tris-KCl buffer (0.05 M Tris and 1.15% KCl, pH 7.4). Each sample for analysis contained the following: 0.5 ml homogenate, 3 ml phosphoric acid (1% w/v solution), and 1 ml 6% thiobarbituric acid. Samples were incubated at 95°C for 1 h. Samples were then cooled on ice, and 4 ml 1-butanol were added. Following vortexing, the samples were centrifuged at 3000 rpm; the top layer from each sample was removed for analysis. 1,1,3,3-Tetramethoxypropane was employed as a standard. The TBARS concentration was determined on a Pharmacia Biotech, Novaspec II spectrophotometer at 535 nm and using the molar absorptivity constant of 1.56 x 10 M–1 cm–1.

Determination of Tissue TCDD Concentration
All tissues were oxidized using a Packard 307 Sample Oxidizer, followed by analysis in a Beckman Model LS6000 LL liquid scintillation spectrometer as described in Diliberto et al. (1995).

Determination of Ascorbic Acid
Ascorbic acid (AA) concentrations were determined as described by Ghio et al. (1998). Between 50 and 100 mg of tissue was homogenized/acidified with 3 ml 60% perchloric acid and centrifuged at 20,000 x g for 30 min at 4°C. The supernatant was assayed for ascorbate and urate using high-performance liquid chromatography (Waters RCM µBondaPak C18 column, Millipore Corporation, Marlborough, MA) with electrochemical detection (BAS model LC-4B, Bioanalytical Systems, W. Lafayette, IN).

Total Glutathione (GSH)
Total GSH was determined by a modification of 5–5'-dithiobis-2-nitrobenzoic acid (DNTB)-GSSG reductase recycling assay previously described by Anderson (1985). The assay reagent contained 10 mg NADPH, 6 mM DTNB, glutathione reductase in 143 µM sodium phosphate, 6.2 mM ethylenediamine tetra acetic acid (EDTA), and pH 7.4 buffer. Sample concentrations of GSH were determined from a standard curve. All chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). The assay was modified for use on the COBAS FARA II (Hoffman-La Roche, Branchbury, NJ) centrifugal spectrophotometer.

Protein
Protein concentration from GSH and AA samples was determined using Pierce Comassie Plus Protein assay reagent (Pierce, Rockford, IL). Sample protein concentration was determined from a standard curve using bovine serum albumin standards obtained from Sigma Chemical Co. (St. Louis, MO). Protein concentrations from TBARS and cytochrome c reduction assays were determined by the method of Bradford (1976). The assay was modified for use on the COBAS FARA II centrifugal spectrophotometer.

Data Analysis
Data are presented as mean ± standard deviation. Intergroup comparisons were performed by a one-way analysis of variance (ANOVA) using Sigma Stat (Jandel Scientific Company, San Rafael, CA). Using Bonferroni's method, differences between groups were considered statistically significant when p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
TCDD was administered to female B6C3F1 mice by oral gavage in two separate exposure regimens. One group received a single acute dose of 0.001, 0.01, 0.1, 1, 10, or 100 µg TCDD/kg (acute exposure) and was sacrificed 7 days later. Another group of mice received a dose of TCDD ranging from 0.15 up to 150 ng TCDD/kg/day for 5 days/week (Monday–Friday) for 13 weeks (subchronic exposure). The resulting TCDD tissue concentrations in liver, lung, kidney, and spleen following acute and subchronic exposure are listed in Tables 1–4GoGoGoGo, respectively. The TCDD dose range resulted in overlapping tissue concentrations following acute or subchronic administration for each respective tissue. For example, liver TCDD concentration following acute TCDD exposure ranged from 2 to 321,270 pg/g wet weight tissue and following TCDD subchronic exposure ranged from 2.5 to 11,959 pg/g tissue. This overlap in tissue TCDD concentration following acute and subchronic exposures enabled a comparison of the relationship between tissue concentration and oxidative stress. This information provides insight into whether tissue concentration is an appropriate dose metric for TCDD-mediated oxidative stress. Acute and subchronic exposure resulted in overlapping TCDD tissue concentrations in lung, kidney, and spleen. The highest TCDD concentrations were in liver, with each tissue demonstrating a dose-dependent increase in TCDD concentration (supralinear for liver and sublinear for all other organs examined).


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TABLE 1 Liver Oxidative Stress Parameters following Subchronic and Acute TCDD Exposure
 

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TABLE 2 Lung Oxidative Stress Parameters following Subchronic and Acute TCDD Exposure
 

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TABLE 3 Kidney Oxidative Stress Parameters following Subchronic and Acute Exposure to TCDD
 

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TABLE 4 Spleen Oxidative Stress Parameters following Subchronic and Acute TCDD Exposure
 
Table 1Go summarizes in liver the oxidative stress parameters assayed following acute or subchronic TCDD exposure. Hepatic production of superoxide anion (SOAP), measured as the reduction of cytochrome c, demonstrated a significant increase above control levels in mice receiving an acute exposure of 10 or 100 µg/kg. The level of SOAP following subchronic exposure was significantly decreased at 0.15 ng/kg/day and was significantly increased above controls at 15 and 150 ng/kg/day. Significant increases in SOAP occurred following acute exposure at doses that resulted in hepatic TCDD tissue concentrations that ranged from 55 to 321 ng/g. In comparison, statistically significant increases above controls occurred following subchronic exposures, resulting in hepatic TCDD tissue concentrations that ranged from 1.1 to 12.0 ng/g. A lower TCDD tissue concentration was required to elicit a significant increase in SOAP following subchronic exposure than acute exposure.

Hepatic lipid peroxidation (TBARS) following acute and subchronic exposure to TCDD also demonstrated that tissue TCDD concentration at the time of sacrifice was not predictive of oxidative response. Significant increases in TBARS compared to controls were observed at the highest doses following acute (100 µg/kg) and subchronic (and 150 ng/kg/day) exposures. These doses resulted in hepatic TCDD concentrations of 321 ng/g (acute exposure) and 12 ng/g (subchronic exposure). As with superoxide anion production, significant increases in TBARS were observed at lower TCDD concentrations in liver after subchronic than after acute dosing.

Elevation of ascorbic acid (AA) in the liver also followed a similar pattern to SOAP and TBARS. Significant increases in AA occurred at acute exposures of 1, 10, and 100 µg/kg and at subchronic exposures of 15 and 150 ng/kg/day. A comparison of tissue concentrations demonstrated that a lower hepatic TCDD concentration resulted in significant AA increases following subchronic exposure than after acute exposure.

Hepatic total glutathione (GSH) levels demonstrated a somewhat varied response. Following subchronic exposure to TCDD, total GSH decreased at 0.15 ng/kg/day, increased at 0.45 and 150 ng/kg/day, and did not change at 1.5 or 15 ng/kg/day. Following acute TCDD exposure to TCDD, total GSH increased at 10 and 100 µg/kg.

Table 2Go summarizes the oxidative stress parameters assayed in lung following acute or subchronic TCDD exposure. No significant increase in SOAP was observed at any dose tested. Interestingly, total GSH was increased at the acute doses of 0.001, 0.1, 1, and 10 µg/kg and decreased at the subchronic dose of 0.15 ng/kg/day. After acute exposure, AA decreased at 0.001 µg/kg and increased at 0.1, 1, and 10 µg/kg. After subchronic exposure, AA decreased at 0.15 ng/kg/day and significantly increased at 15 and 150 ng/kg/day.

Table 3Go summarizes the oxidative stress parameters assayed in kidney following acute or subchronic TCDD exposure. Kidney SOAP demonstrated no statistical differences at any acute dose and a statistical increase above controls only at the subchronic doses of 15 and 150 ng/kg/day. After acute exposure, total GSH did not significantly change from control levels. After subchronic exposure, total GSH, much like lung and liver, exhibited a decrease following 0.15 ng/kg/day. This total GSH decrease was maintained at subchronic doses of 0.45 and 1.5 ng/kg/day. Kidney AA levels exhibited significant increases following acute doses of 1, 10, and 100 µg/kg and significant decreases following all subchronic doses except 1.5 ng/kg/day.

Table 4Go summarizes the oxidative stress parameters assayed in spleen following acute or subchronic TCDD exposure. Spleen SOAP did not deviate from controls at any dose or dosing paradigm tested. Total GSH was elevated only after subchronic dosing of 150 ng/kg/day. Ascorbic acid levels were significantly decreased following subchronic exposures of 0.15, 1.5, and 150 ng/kg/day. However, this response was quite varied, with no other dose producing a significant change.

In addition, we conducted preliminary experiments that demonstrated a significant increase in superoxide anion production (SOAP) (Fig. 1Go). This was in response to a high-dose challenge of TCDD (25 µg/kg) given to female B6C3F1 mice. These mice were either pretreated with corn oil (controls) or with a low dose of TCDD (0.1 µg/kg) that produced no observable oxidative stress.



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FIG. 1. Liver superoxide anion production (SOAP) following acute exposure to TCDD: effect of TCDD pretreatment. A high dose challenge of TCDD (25 µg/kg) was given by oral gavage to female B6C3F1 mice. These mice were either pretreated with corn oil (controls) or to a low dose of TCDD (0.1mg/kg) that does not produce observable oxidative stress. Each point represents the mean ± standard deviation. n = 4 for each group. T-1 represents the group of animals that received 0.1 µg TCDD/kg on day 7 and corn oil on day 14. T-2 represents the group of animals that received corn oil on day 7 and 25 µg TCDD/kg on day 14. T-3 represents the group of animals that received 0.1 µg TCDD/kg on day 7 and 25 µg TCDD/kg on day 14. All groups were sacrificed on day 14. (a) Represents a statistically significant difference from control group; (b) represents a statistically significant difference from T-1 group, and (c) represents a statistically significant difference from T-2 group (ANOVA; p < 0.05).

 
The possibility of a rebound effect occurring 7 days after exposure to TCDD was investigated by employing a time-course study to identify the SOAP from 8 h up to 35 days postexposure. At all time points tested, there was an elevated response compared to controls, demonstrating a sustained response to TCDD. In this study, we demonstrated an oxidative stress response (depletion of total GSH) resulting from TCDD exposures to doses that produced tissue concentrations near background levels.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study, we demonstrated that following acute and subchronic exposure different patterns of oxidative stress occurred, and that tissue concentration does not reliably predict this response. Tissue concentration has been demonstrated to be predictive of many responses to TCDD exposure. Previous studies have demonstrated correlation between hepatic concentration of TCDD and TCDD-induced CYP1A1 and CYP1A2 protein expression (Tritscher et al., 1992Go; Diliberto et al., 1995Go; Santostefano et al., 1998Go; Walker et al., 1999Go), antibody plaque-forming cell (PFC) response to sheep red blood cells (Narasimhan et al., 1994Go), and alterations in the reproductive system of developing pups (Hurst et al., 1998Go).

The mechanism by which TCDD produces oxidative stress is unknown. The importance of CYP1A1 and CYP1A2 in TCDD-mediated oxidative stress response may be associated with the production of hydrogen peroxide during cytochrome P450 metabolic cycling. Cytochrome P450s transfer two electrons to molecular oxygen (donated by NADPH) (Guengerich and Liebler, 1985Go; Poulos and Raag, 1992Go). Park et al. (1996) indicated that an increase in Ah receptor or CYP1A1 could be associated with the TCDD-mediated oxidative stress response. CYP1A1 induction may result in an increased production of ROS, perhaps due to futile cycling of the P450 in its effort to unsuccessfully metabolize TCDD. In addition, earlier studies have shown that oxidative stress resulting from TCDD exposure appears to be Ah receptor-mediated (Alsharif et al., 1994aGo, 1994bGo; Ashida et al., 1996Go; Hassoun et al., 1996Go; Smith et al., 1998Go). A recent report by Shertzer et al. (1998) demonstrated that TCDD exposure produced a sustained oxidative stress response in female C57BL/6J mice. Hepatic glutathione levels were reported to increase 2-fold and persist for at least 8 weeks following a dose of 5 µg TCDD/kg for 3 consecutive days. Our data demonstrated a similar increase 7 days after a single acute exposure of 10 µg TCDD/kg. In addition, SOAP in female B6C3F1 mice was found to be significantly elevated from controls as early as 8 h following exposure and continued to demonstrate a similar level of increase above controls up to 35 days post-treatment. This dose-dependent response to TCDD exposure requires further characterization. In addition, our data is in agreement with the proposed sustained oxidative stress response seen in the C57BL/6J mice used by Shertzer et al. (1998). The sustained oxidative stress response may be related to the elevated CYP1A1 persisting after high-dose TCDD administration, as shown by Diliberto et al. (1995).

In the present study, low-dose subchronic exposure to TCDD (0.15 ng TCDD/kg/day) resulted in a depletion of GSH compared to control animals. This response was observed in liver (16.8% decrease), lung (29.0% decrease), and kidney (45.9% decrease). In liver, the response was reversed with an increased dose (0.45 ng TCDD/kg/day). In lung and kidney, the increase in subchronic dose did not elevate GSH concentration significantly higher than controls at any dose examined (up to 150 ng TCDD/kg/day). Diliberto et al. (1995) previously reported that a subchronic TCDD exposure of 0.15 ng TCDD/kg/day resulted in a significant induction of CYP1A1. In contrast, no induction was detected in lung (unpublished observation). This may indicate that GSH depletion seen in lung is a very sensitive response to low-dose subchronic TCDD exposure.

GSH plays a fundamental role in the antioxidant biology of mammals. Severe GSH depletion is associated with pathologic consequences including, but not limited to, susceptibility to the development of lipid peroxidation (Anundi et al., 1979Go; Gillette et al., 1974; Mitchell and Jollows, 1975Go; ). In addition, the loss of GSH has an effect upon other antioxidant systems due to its role in the regeneration of ascorbic acid (vitamin C) and reduction of the oxidized form of tocopherol (vitamin E) (Comporti et al., 1991Go). The depletion of GSH in lung and kidney may in part contribute to the decrease in ascorbic acid observed following 0.15 ng TCDD/kg/day. In liver, the levels of ascorbic acid were not significantly reduced. However, this is not surprising, as the liver is a major site of storage and synthesis of ascorbic acid. The overall effect of GSH depletion in extrahepatic tissues following TCDD exposure is not well understood and warrants further investigation. The loss of GSH may leave an individual vulnerable to oxidant challenge or less able to handle the ROS produced through normal metabolism. The role of GSH depletion following TCDD exposure is not understood. Moreover, it is of concern that the GSH depletion occurs in multiple organs after an exposure that results in body burdens of TCDD near background body burdens in the human population (DeVito et al., 1995Go). Previous studies have indicated that TCDD inhibits the enzyme glutathione peroxidase, which is involved in the reduction of oxidized GSH (GSSG) (Hassan, et al., 1983Go, 1985Go; Stohs et al., 1984aGo, 1984bGo). However, these studies were based on high-dose acute TCDD exposure (40 µg/kg for 3 consecutive days) and do not explain the loss of total GSH.

SOAP, total GSH, and AA all appear to be more sensitive markers of TCDD-mediated oxidative stress in female B6C3F1 mice than TBARS. In addition to the difference in sensitivity, much smaller quantities of tissue are required to perform these assays as compared to TBARS.

In this study, we demonstrated that a high acute dose of TCDD that is well above that encountered in the environment results in alterations in oxidative stress indicators. It is noteworthy that low-dose subchronic TCDD exposure similar to that experienced by the human population might be expected to produce a depletion in GSH. Therefore, background TCDD exposure may leave the exposed population more vulnerable to future challenge. Figure 1Go demonstrates hepatic superoxide anion production was elevated in both pretreated groups following a challenge dose of 25 µg TCDD/kg, as expected. However, SOAP (Fig. 1Go) in the group of mice receiving TCDD pretreatment was significantly greater than in the group of mice receiving corn oil pretreatment. This preliminary data suggests that low-dose TCDD exposure may result in a condition where an individual is less prepared to respond to a subsequent oxidative stress challenge. Further attempts to characterize this response in hepatic and extrahepatic tissues are warranted, including a study of the factors that affect GSH synthesis, degradation, metabolism, and transport. Moreover, the data indicate that TCDD tissue concentration at time of sacrifice does not appear to be the appropriate dose metric to predict or to characterize the oxidative stress response. Because of this, characterization of the oxidative stress response warrants careful consideration.

In summary, extensive research has investigated the acute effects of TCDD upon oxidative stress in the liver. However, the sensitivity and responsiveness of extrahepatic tissues such as lung and kidney to TCDD in terms of oxidative stress have been less characterized. This study demonstrated that oxidative stress following TCDD exposure is not solely dependent upon TCDD tissue concentration. In addition, subchronic exposure to low doses of TCDD resulted in a significant depletion of GSH as well as effects on other antioxidants in extrahepatic tissues. The result of this depletion may lead to enhanced vulnerability to further oxidative challenge.


    ACKNOWLEDGMENTS
 
This project could not have been completed without the assistance of Frances McQuaid, Judy Richards, David Ross (U.S. Environmental Protection Agency, Research Triangle Park, NC), Dr. Michael Santostefano, and Dr. Jon Hamm (Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, NC). The authors would like to acknowledge their excellent technical assistance during tissue collection. In addition, the authors would like to thank Dr. Nigel Walker of the National Institute of Environmental Health Sciences, Research Triangle Park, NC, and Chris Hurst of the Curriculum in Toxicology, University of North Carolina at Chapel Hill, Chapel Hill, NC for their comments during preparation of the manuscript prior to submission. B.P.S. was supported in this research by UNC/EPA cooperative training agreement CT 902908.


    NOTES
 
This document has been reviewed in accordance with U.S. Environmental Protection Agency policy and approved for publication. Approval does not signify that the contents necessarily reflect the view and policies of the agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

1 To whom correspondence should be addressed at U.S. Environmental Protection Agency (USEPA), Experimental Toxicology Division (ETD), Pharmacokinetics Branch, Mail Drop-74, Research Triangle Park, NC 27711. Fax (919) 541-4284. E-mail: devito.mike{at}epa.gov. Back


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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