* ManTech Environmental Technology, Inc., Dayton, Ohio 45437; and
Operational Toxicology Branch (AFRL/HEST), Air Force Research Laboratory, Wright-Patterson Air Force Base, Ohio 45433-7400
Received January 9, 2002; accepted June 10, 2002
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ABSTRACT |
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Key Words: hydrazine; in vitro; hepatocytes; oxidative stress; catalase; glutathione; reactive oxygen species.
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INTRODUCTION |
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The primary source of exposure to U.S. Department of Defense personnel occurs from the fueling of rockets and aircraft propulsion systems. To protect personnel, a full-body protection suit is required when working with HzN propellants. Because of the highly toxic nature of HzN, novel candidate chemicals are being investigated by the U.S. Air Force Research Laboratories as possible replacements that would have equivalent performance characteristics but less toxicity (Hussain and Frazier, 2001). Therefore, understanding the biochemical basis of HzN toxicity is an essential starting point for developing replacement chemicals.
Previous reports have identified several toxic effects associated with exposure to HzN compounds, e.g., liver damage, hyperglycemia, neurodegeneration, and cancer (Kenyon et al., 1999; Moloney and Prough, 1983
; Petersen et al., 1970
; Wald et al., 1984
). Experimentally in rats, HzN causes depletion of glutathione (GSH) and the accumulation of triglycerides in the liver (Jenner and Timbrell, 1994
). In addition, HzN interferes with the urea cycle of the rat liver (Roberge et al., 1971
). HzN has been reported to induce methylation of DNA (Bosan et al., 1987
). HzN exposure leads to adenosine triphosphate (ATP) depletion and megamitochondria formation in vivo (Kerai and Timbrell, 1997
; Preece et al., 1990
; Wakabayashi et al., 2000
). HzN inhibits the mitochondrial enzyme succinate dehydrogenase (Ghatineh et al., 1992
), which subsequently reduces mitochondrial function. Further, HzN produces toxicity by interfering with a number of metabolic processes such as gluconeogenesis (Kleineke et al., 1979
) and glutamine synthetase (Kaneo et al., 1984
; Noda et al., 1987
; Sendo et al., 1984
; Willis, 1966
).
Oxidative stress plays a role in the mechanisms of toxicity of a number of compounds, whether by production of free radicals or by depletion of cellular antioxidant capacity. Cellular integrity is affected by oxidative stress when the production of reactive oxidants overwhelms antioxidant defense mechanisms (Halliwell et al., 1992; Yu, 1994
). The metabolism of HzN and its derivatives is thought to involve the production of free radicals that may induce cellular toxicity either by covalent binding to tissue macromolecules or by initiating an autoxidative process such as lipid peroxidation in vivo (Choudhary and Hansen, 1998
; Preece and Timbrell, 1989
). Evidence for the production of radicals, including methyl, acetyl, hydroxyl, and hydrogen radicals, has been observed during the metabolism of HzN (Ito et al., 1992
; Noda et al., 1988
; Runge-Morris et al., 1988
; Sinha, 1987
). Thus, studies show that multiple pathways, both enzymatic and nonenzymatic, appear to be involved in free radical generation. Free radicals have been implicated in protein (hemoglobin) damage associated with HzN in human erythrocytes, suggesting that free radicals may be involved in the anemic effects of HzN observed in animals in vivo (Runge-Morris et al., 1988
). Therefore, the formation of free radicals during the metabolism of HzN may be important to the mechanism of action of HzN toxicity.
It is known from the literature that HzN interferes in a broad range of physiological reactions. However, a complete toxicological profile and the mechanism of HzN toxicity are not yet fully understood. No studies are available on the acute lethality of HzN in vivo. Previous in vitro studies are based on 24-h exposures at minimally lethal doses of HzN. Therefore, the objective of this study was to investigate the acute cytotoxicity of HzN exposure in primary rat hepatocytes following short (4-h) exposures. The present study describes the acute cytolethality of HzN, and some aspects of its mechanism of toxicity in primary rat hepatocytes with reference to oxidative stress.
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MATERIALS AND METHODS |
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Animals.
Male Fischer 344 rats (225300 g) were obtained from Charles River Laboratory (Raleigh, NC). Rats were anesthetized with 1 ml/kg of a mixture of ketamine (70 mg/ml; Parke-Davis, Morris Plains, NJ) and xylazine (6 mg/ml; Mobay Corp., Shawnee, KS) prior to undergoing liver perfusion. All animals used in this study were handled in accordance with the principles stated in the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, and the Animal Welfare Act of 1966, as amended (National Research Council, National Academy Press, 1996).
Liver perfusion, hepatocytes enrichment, and culture.
Rat livers were perfused, and hepatocytes were isolated and enriched by the two-step Seglen procedure (Seglen, 1976) with minor modifications as previously described (DelRaso and Frazier, 1999
). For all perfusions, Chee medium (pH 7.2) was supplemented with 10 mM HEPES. Washout medium was further supplemented with heparin (2.0 U/ml) and EGTA (0.5 mM), and digestion medium was supplemented with 500 mg/l collagenase. Viable primary rat hepatocytes were enriched by low-speed centrifugation (500 x g) for 3 min. Typically, the yield of isolated hepatocytes was from 300 to 400 million cells, with viability ranging from 85 to 95% as determined by trypan blue dye exclusion. For cell culture studies, suspensions of primary hepatocytes were adjusted to a cell density of 1.0 x 106 cell/ml in Chee culture medium (pH 7.2) supplemented with HEPES (10 mM), insulin/transferrin/sodium selenite (5 mg/l, 5 mg/l, 5 µg/l), gentamicin (50 mg/l), and dexamethasone (0.4 mg/ml). Cells were seeded in either 96-well (4 x 104 cells/well) or 6-well (1.0 x 106 cells/well) culture plates previously coated with rat tail collagen (1.0 µg/well or 25 µg/well, respectively). After 4-h incubation in a 5% CO2 incubator at 37°C to allow for attachment, hepatocytes were re-fed with fresh Chee culture medium lacking dexamethasone. Hepatocytes were cultured for an additional 20 h before treatment.
Treatment.
In all studies, primary rat hepatocytes were treated for 4 h with various concentrations of HzN dissolved in Chee culture media. At the end of the exposure period, toxicity end points were evaluated.
LDH leakage.
Lactose dehydrogenase (LDH) leakage was assessed by spectrophotometrically measuring the oxidation of NADH at 340 nm in both the cells and media, as described by Moldeus et al., (1978). The percent of activity in the media was then calculated by dividing the amount of activity in the media by the total activity (medium plus cell lysate).
Mitochondrial function.
Mitochondrial function was determined spectrophotometrically by measuring the reduction of the tetrazolium salt MTT to formazan by succinic dehydrogenase as previously described (Carmichael et al., 1987).
Reduced and oxidized GSH.
Reduced GSH and oxidized glutathione (GSSG) were measured in 96-well plates using a SpectraMAX Plus 190 microplate reader (Molecular Devices, Sunnyvale, CA.) according to the procedures described in the Glutathione Assay Kit (Cayman Chemical Company, Ann Arbor, MI). The assay is based on the enzymatic recycling method, using glutathione reductase (GR) and 5,5'-dithiobis-2-nitrobenzoic acid (Ellmans reagent; Tietze, 1969). Data are reported as percent of control.
Reactive oxygen species (ROS) generation.
ROS generation was determined using the method described by Wang and Joseph (1999). Fluorescence was detected using an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Data are reported as fold increase in fluorescence intensity relative to control.
Lipid peroxidation.
The extent of lipid peroxidation in control or HzN-exposed hepatocytes was determined by measuring the thiobarbituric acid reactive substances (TBARS). TBARS were determined according to the procedures of Ohkawa et al.(1979) with minor modifications. At the end of exposure, cells were washed and scraped into 1 ml phosphate-buffered saline (PBS). This was followed by addition of 100 µl of 10% sodium dodecyl sulfate for solubilization. Then 650 µl of 0.5% thiobarbituric acid in 20% (v/v) glacial acetic acid (pH 3.5) were added and incubated at 80°C for 30 min. After incubation, the samples were cooled and the absorbance was measured at 532 nm in a SpectraMAX Plus 190 microplate reader (Molecular Devices, Sunnyvale, CA). Data are expressed as nmol TBARS per mg cellular protein.
Determination of mitochondrial membrane potential.
Mitochondrial membrane potential was determined by the uptake of rhodamine 123 according to the method of Wu et al.(1990). After treatment, hepatocytes cultured in 96-well plates were washed with PBS and incubated with 10 µg/ml rhodamine 123 (Molecular Probes, Inc. Eugene, OR) in Chee medium for 30 min at 37°C. After further washing with PBS, the hepatocytes were incubated in Chee media for 30 min. After removing the media, 0.2 ml ethanol/water solution (1 part ethanol:1 part water) was added to extract the dye retained by the cells. Fluorescence was measured with a SpectraMAX Gemini-XS fluorescence plate reader (Molecular Devices, Sunnyvale, CA) with an excitation wavelength of 485 nm and an emission wavelength of 530 nm. Data are expressed as percent of control uptake.
Antioxidant enzymes.
Treated hepatocytes were washed with PBS and scraped into 1 ml PBS. A cell pellet was obtained by centrifugation at 500 x g for 5 min. The pellet was resuspended in potassium phosphate buffer (pH 7.2), placed on ice, and sonicated three times (10 s each) with an Ultrasonic Homogenizer (Cole-Pharmer Instrument Company, Chicago, IL). The resulting homogenates were centrifuged for 30 min at 18,000 x g (4°C) and the supernatant was collected in a SpectraMAX Plus 190 microplate reader (Molecular Devices, Sunnyvale, CA) to measure enzyme activities. Catalase activity was assayed in the supernatant by the method of Aebi (1984), which involves monitoring the disappearance of hydrogen peroxide (H2O2) at 240 nm. The enzymatic activity was expressed in Units/mg cellular protein. Glutathione peroxidase (GPx) activity in the supernatant was estimated according to the method of Flohe and Gunzler (1984). GR was measured according to the method of Carlberg and Mannervik (1985). GPx and GR activities were expressed in mUnits/mg cellular protein (one mUnit of activity represents one nmol NADPH oxidized per minute).
Determination of protein concentration.
Total protein concentration in cell lysate was determined using an ESL Protein Assay Kit according to manufacturers instructions (Boehringer-Mannheim Biochemicals, Indianapolis, IN). This method is an optimized procedure that combines the biuret reaction and the copper (I)-bathocuproine chelate reaction, as described by Matsushita et al. (1993).
Statistical analysis.
The data are expressed as means ± SD of three independent experiments with hepatocytes from three different rats. The data were subjected to statistical analysis by one-way analysis of variance (ANOVA) followed by Dunnetts method for multiple comparisons. A value of p < 0.05 was considered significant. The SigmaStat for Windows version 2.03 software is used for the statistical analysis.
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RESULTS |
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DCFH-DA is widely used to measure ROS generation in cells. ROS generation following 4-h exposure to HzN is shown in Figure 2A. The levels of ROS in hepatocytes increased in a concentration-dependent manner and were statistically increased (p < 0.05) at the lowest concentration studied (25 mM). HzN treatment at 100 and 150 mM resulted in an approximately 8-fold increase in ROS over control levels. Measurement of lipid peroxidation is used to investigate the process of cellular damage induced by free radicals. There was no significant increase in the level of TBARS at 10, 25, and 50 mM HzN (Fig. 2B
). However, the variability between the responses of the three separate hepatocyte preparations, as indicated by the standard deviation, increased significantly at the 50 mM concentration. At the higher exposure concentrations (100 and 150 mM), TBARS levels in cells were significantly elevated, approximately 2-fold, over control levels. These results indicate lipid peroxidation is observed at higher HzN exposures.
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DISCUSSION |
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To put this into perspective, Preece et al. (1992) investigated the hepatotoxic effects of a single oral dose of HzN in rats. At the highest dose studied (81 mg/kg), they observed minor liver pathology, i.e., intracellular fat droplets in two of three rats, but no deaths at 4 days postexposure. The maximum plasma concentration observed following the 81 mg/kg dose was slightly over 1 mM (at 90 min postdosing) and the area-under-the-plasma-curve (AUC) was roughly 1 mM-h. The lowest concentration found to be cytolethal to isolated rat hepatocytes after 4 h of exposure in our study, 25 mM, results in an AUC of about 100 mM-h. Thus, there is an approximately 100-fold difference in the AUC between accumulation of lipid droplets in vivo at 4 days and detectable cytolethality in vitro at 4 h. The two end points are quite different, making a meaningful comparison difficult. However, considering the differences in the magnitude of toxicity, subclinical/morbidity versus lethality, and the differences in time frames, 4 days versus 4 h, the observations on the mechanisms of action of HzN presented here are probably relevant to the issue of acute lethality.
The results observed for biochemical end points described in this paper demonstrate that HzN increased LDH leakage and reduced MTT reduction in a dose-dependent manner over a wide range of exposure durations. However, mitochondria appear to be more vulnerable to HzN exposure at shorter exposure times, as indicated by the differences in EC50s, e.g., MTT reduction (EC50 [4h]: 37 mM) compared with LDH leakage (EC50 [4h]: 78 mM). Although the molecular target for HzN in the mitochondria is not known with certainty, it appears that mitochondrial dysfunction plays an important a role in HzN toxicity.
Another interesting observation relating to the time-course studies is the dependence of the CT product on exposure time. The fact that the CT product is not constant implies that the mechanisms of toxicity are highly nonlinear with dose. This suggests that acute cytolethality may be a consequence of a different mechanism of action than is operative at lower doses and longer exposure times.
Cell integrity is affected by oxidative stress when the production of active oxidants overwhelms antioxidant defense mechanisms. Hepatocytes live in a balance of free radical production, free radical scavenging, and repair of damage caused by free radicals (Cai et al., 1995). The addition of HzN can upset this balance by inducing increased formation of ROS through mitochondria dysfunction and/or depleting or inhibiting antioxidant systems. The ROS increase following exposure to any chemical depends on the balance between oxidative and antioxidant cellular systems. ROS are by-products of biological redox reactions and are involved in various pathological conditions (Farber et al., 1990
). The results here show that there was a significant increase in ROS at the lowest concentration of HzN studied (25 mM). ROS generation increased with exposure concentration up to 100 mM, with no further increase at 150 mM. The apparent lack of increase in ROS at the higher exposure concentrations may be a consequence of the leakage of fluorescent product from the cell, as evidence of significance membrane damage (LDH leakage) was apparent at the highest concentration. Increased generation of ROS by HzN is likely to contribute to oxidative stress that may ultimately lead to the observed cytotoxicity (Loft and Poulsen. 1999
; Preece and Timbrell, 1989
). Another index of oxidative stress is lipid peroxidation, an important organic biomarker of oxidative stress induced by reactive free radicals (Duthie, 1993
; Kappus, 1987
). The toxicity of HzN, as indicated by LDH release and MTT reduction, is strongly correlated to ROS generation and lipid peroxidation, indicating induction of marked oxidative stress in primary culture of rat hepatocytes following HzN exposure.
Mitochondria are vulnerable targets for toxic injury by a variety of compounds because of their crucial role in maintaining cellular structure and function via aerobic ATP production. Our results indicate that the mitochondrial membrane potential is reduced with increasing concentration of HzN. It has been suggested that mitochondrial membrane disruption due to altered membrane potential contributes to release of the apoptotic factor cytochrome c. (Kluck et al., 1997; Liu et al., 1996
). Thus, shutdown of mitochondrial function under conditions of oxidative stress may induce apoptosis at exposure concentrations that do not lead directly to cytolethality.
GSH is a ubiquitous sulfhydryl-containing molecule in cells that is responsible for maintaining cellular oxidation-reduction homeostasis (Sies, 1999). GSH protects cells against damage by scavenging highly reactive free radicals that otherwise would interact with critical cellular components (Hayes and McLellan, 1999
). Therefore, changes in GSH homeostasis can be monitored as an indication of cell damage. It is interesting to note that the lowest concentration of HzN tested (25 mM) depleted GSH significantly. It is not known how HzN depletes GSH levelswhether it binds directly to GSH, inhibits enzymes involved in GSH synthesis, or increases GSH consumption in secondary enzymatic reactions. Colvin et al. (1969) reported that GSH and cysteine adducts have been observed upon incubation of isopropylhydrazine or acetylhydrazine with microsomes in the presence of NADPH. Experimentally in rats, HzN causes depletion of GSH and the accumulation of triglycerides in the liver (Jenner and Timbrell, 1994
). ). It is likely that GSH depletion may be due to its reaction with HzN or its metabolites. GSH depletion in primary culture of rat hepatocytes exposed to HzN is strongly correlated to increased ROS generation. It has been postulated that the loss of GSH may compromise cellular antioxidant defenses and lead to the accumulation of ROS that are generated as by-products of normal cellular function. Previously, it was shown that the depression of GSH concentration increased endogeneous ROS to toxic levels in hepatocytes (Anundi et al., 1979
). As demonstrated in our study, depletion of GSH with BSO resulted in an increased susceptibility to HzN. The EC50 when normal hepatocytes were exposed to HzN was 65 mM, and this measure of the toxicity was reduced to 18 mM in GSH-depleted hepatocytes that was a 4-fold reduction in EC50 (Fig. 5
). Studies with cultured cells have demonstrated that upon depletion of antioxidant resources such as GSH and catalase, cells become susceptible to chemicals known to generate ROS (Hussain et al., 1999
; Jones et al., 1978
). The results of this study strongly suggest that GSH is playing a role in protecting cells from HzN toxicity.
Cellular defenses also include antioxidant enzymes, i.e., catalase, GPx, GR, and superoxide dismutase (SOD), that prevent oxidative damage from ROS. Catalase is one of the major antioxidant enzymes involved in the detoxification of H2O2, which is produced as a result of the dismutase of superoxide catalyzed by SOD (Harris, 1992). It is apparent from the data presented here that inhibition of catalase increases HzN-induced oxidative stress. Studies with isolated hepatocytes have demonstrated that under conditions of GSH depletion, catalase functions in the catalysis of H2O2 produced by the cytochrome P450-linked monooxygenase system (Jones et al., 1978
). Thus, under the condition of extreme oxidative stress, catalase may become important in providing cytoprotection. For example, doxorubicin-induced cardiotoxicity is suppressed by overexpression of catalase in the heart of transgenic mice (Kang et al., 1986
). Experimental studies described here were conducted to ascertain the effects of HzN on the enzyme activities of catalase, GPx, and GR. The data show that the activity of catalase decreases markedly in HzN-treated cells. However, GPx and GR activities remain relatively unaffected. To further evaluate the role of catalase, 3-amino triazole was used to inhibit the catalase activity in hepatocytes that were then exposed to HzN. The inhibition of catalase dramatically increased the susceptibility of cells to HzN. These results support the hypothesis that catalase is a key factor in protecting cells against HzN toxicity.
In summary, this study has shown that HzN causes increased generation of ROS, the depletion of GSH, marked inhibition of catalase activity, increased reduction of MMP, and the increased sensitivity of hepatocytes following GSH depletion and catalase inhibition. The rapid depletion of GSH by acute HzN toxicity may impair the cells defense against oxidative stress. There is a strong correlation between GSH depletion and ROS generation that suggests induction of oxidative stress. An elevated lipid peroxide profile also is indicative of oxidative stress. In conclusion, our data provide evidence that acute cytotoxicity of HzN is primarily the result of induction of oxidative stress.
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ACKNOWLEDGMENTS |
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NOTES |
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