Departments of Pediatrics and Medicine and Center for Comparative Medicine, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030
Received January 14, 1999; accepted April 16, 1999
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ABSTRACT |
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Key Words: diquat; Fischer 344 rats; reactive oxygen species; hepatic necrosis; antioxidant defense mechanisms; 2,4-dinitrophenylhydrazine; protein carbonyls; DNA fragmentation.
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INTRODUCTION |
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The overall goal of our studies with diquat is to understand the fundamental mechanisms of critical cell damage by reactive oxygen species. The previous studies with diquat have provided interesting insights into the mechanisms of oxidant injury in vivo, but have employed male rats almost exclusively. To date, the effects on female rats have been largely unexplored. Prematurely born male human infants are significantly more susceptible than are female infants to development of chronic lung disease, for which similar oxidative mechanisms are significant contributors (Smith and Welty, 1999). Although fundamental mechanisms of cellular injury by reactive oxygen species should be common to both genders, findings of any differences between males and females could prove to be useful in distinguishing critical from non-critical oxidations in expression of oxidant cell injury. The initial purpose of the present studies was to investigate the effects of diquat on female Fischer 344 rats, which had proved to be more resistant than males to hepatic damage. Studies of possible antioxidant functions and biomarker responses were conducted in an effort to determine the factors responsible for this difference in susceptibilities and, more importantly, to attempt to employ this difference in distinguishing critical from non-critical pathways of oxidant-mediated hepatic necrosis in vivo.
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MATERIALS AND METHODS |
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Animals.
Adult (911 weeks-of-age) male (180220 g) and female (160180 g) Fischer 344 rats were purchased from Harlan Sprague-Dawley (Houston, TX) and were maintained by the Baylor Center for Comparative Medicine in an air conditioned room on a 12-h light/dark cycle. Food and drinking water were available ad libitum. The animals were adapted for at least 3 days before study. Diquat was administered to rats in normal saline, ip. Control animals received equal volumes of saline. At 2, 4, or 6 h post-dose, the animals were anesthetized with pentobarbital, blood was obtained by cardiac puncture, and plasma was isolated for the assay of ALT activities. Livers were removed, and animals were killed by exsanguination while under deep anesthesia. Liver homogenates (10% w/v) were prepared in a buffer containing 50-mM potassium phosphate (pH 7.6) containing 5-mM ethylenediaminetetraacetic acid (EDTA), and 0.02% Triton X-100. The homogenates were centrifuged at 12,700 g for 20 min at 4°C, and the supernatants were used for measurements of enzyme activities other than SOD. For the measurements of total SOD activities, liver homogenates (10% w/v) were prepared in 50-mM potassium phosphate buffer, pH 7.6, containing 1-mM diethylenetriaminepentaacetic acid (DETAPAC). These homogenates were sonicated for 45 s at 50 kilocycles/s and centrifuged at 100 g for 3 min. The supernatants were used for SOD-activity measurements.
Determination of plasma ALT activities.
Plasma ALT activities were determined using Sigma assay kit (Procedure No. 59-UV).
Bile duct cannulation and total glutathione (GSH + GSSG + GSSX) measurements.
For bile duct cannulation, the animals were anesthetized by administration of sodium pentobarbital (50 mg/kg, ip), and bile ducts were cannulated with polyethylene tubing (PE-10) for timed collections in tared tubes cooled on ice. Samples were collected at 30-min increments with time of administration of diquat or vehicle defined as 0 min. Samples collected from 30, 0, 0 to 30, 60 to 90, and 120 to 150 min in tubes containing 0.1 M phosphoric acid were used for the analysis of total glutathione reductase-reactive glutathione species (GSH + GSSG + GSSX) by the enzyme recycling method adopted (Adams et al., 1983) from Tietze (1969).
Measurements of tissue concentrations of diquat.
For measurements of diquat levels in livers, a separate series of rats were treated with 0.075 to 0.25 mmol/kg of diquat, or with saline alone, and animals were killed at time points from 224 h after dosing. Livers were freeze-clamped and stored at 80°C. For measurements of diquat levels, the methods of Madhu et al. (1995) and Fuke et al. (1996) were modified. A piece of frozen liver of about 0.1 g was weighed and added to 400 µl of 70% methanol containing 21 mM perchloric acid and homogenized with a Polytron for approximately 45 s, followed by centrifugation at 1000 g for 15 min. The resultant supernatants were analyzed by high performance liquid chromatography (HPLC) using a Zorbax ODS column (15 x 0.4 cm) eluted with 10% acetonitrile, 0.2 M ortho-phosphoric acid, 0.1 M diethylamine, and 7.5 mM octanesulfonic acid at a flow rate of 2 ml/min. Diquat was detected by absorbance at 310 nm, and concentrations were calculated from peak areas assessed by a Turbochrome data acquisition system and compared with experimentally derived standard curves prepared by adding 040 nmol of diquat to 70% methanol containing 21 mM perchloric acid and processed as above.
Determination of protein concentrations.
Protein concentrations were determined according to Lowry et al. (1951).
Determination of superoxide dismutase (SOD) activities.
SOD activities were measured by the method of Spitz and Oberly (1989) on a Beckman DU64 spectrophotometer using nitroblue tetrazolium (NBT) as an indicator for the generation of superoxide. Superoxide reduces NBT to form a blue product formazan that is measured at 560 nm.
Determination of glutathione peroxidase (GPX) activities.
GPX activities toward cumene hydroperoxide (CHP) and hydrogen peroxide (HP) were determined according to the method of Lawrence and Burk (1976). Briefly, the reaction mixture consisted of 50 mM potassium phosphate buffer (pH 7), 1 µmol EDTA, 1 µmol NaN3, 0.2 µmol nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), 1 U/ml GR, 1 µmol GSH, and 0.25 µmol H2O2 or 1.5 µmol cumene hydroperoxide, in a total volume of 1 ml. All ingredients except enzyme source and peroxide were combined prior to assay, and fresh solutions were prepared daily. Enzyme source (0.1 ml) was added to 0.8 ml of the above mixture and allowed to incubate for 5 min at room temperature before initiation of the reaction by the addition of 0.1 ml peroxide solution. Absorbances at 340 nm were recorded for 5 min, and the activities were calculated from the rates of change as µmol NADPH oxidized per min. Blank reactions with enzyme source replaced by distilled water were subtracted from each assay.
Determination of glutathione reductase (GR) activities.
GR activities were assayed according to the method of Horn (Bergmeyer, 1974). The assay mixtures consisted of 83 µmol tris(hydroxymethyl)aminomethane (Tris), pH 8.0, 0.8 µmol of EDTA, 5.70 µmol GSSG in 0.1 M Tris, pH 7.0, and 0.2 µmol NADPH. To the above mixture, 0.05 ml of sample was added, mixed rapidly, and the rates of change in absorbance at 340 nm were measured.
Determination of glutathione transferase (GST-) activities.
GST- activities were estimated by subtracting the GPX activities measured using H2O2 from the GPX activities measured using cumene hydroperoxide (Reddy et al., 1981
).
Electrophoresis and Western blotting.
Proteins were derivatized with DNPH by the method of Shacter et al. (1994) or with monobromobimane (mBBr) by the method of Weis et al. (1992). The derivatized samples were separated by SDSpolyacrylamide gel electrophoresis in 12.5% acrylamide gels according to the method of Laemmli (1970). The DNPH-derivatized proteins were transferred to polyvinylidene difluoride (PVDF) membranes, followed by Western blotting, as described by Towbin et al. (1979). Detection of DNPH-reactive proteins was accomplished by the use of monoclonal anti-dinitrophenyl-conjugated bovine serum albumin (DNP-BSA) as the primary antibody and horseradish peroxidase (HRP)-conjugated antiserum as the secondary antibody. The bands were visualized using a detection system based on enhanced chemiluminescence (ECL). The mBBr-derived fluorescence of the proteins was visualized with a TS-15 transilluminator (UVP Inc., San Gabriel, CA) equipped with a 254-nm light source and photographed with a Polaroid camera using Kodak Wratten gelatin filter No. 15.
Determination of DNA fragmentation.
Liver homogenates containing approximately 25 mg of protein were diluted to 2 ml with buffer containing 10 mM TrisHCl, 1 mM EDTA, pH 8.0. Next, 3 ml of lysis buffer containing 5 mM TrisHCl, 20 mM EDTA, 0.5% Triton X-100 (w/v), pH 8.0, were added. After 10 min, samples were centrifuged for 20 min at 27,000 g to separate the intact chromatin pellets from the fragmented DNA recovered in supernatants. The supernatants were decanted and saved, and the pellets were resuspended in 10 mM TrisHCl, 1 mM EDTA, pH 8.0. Pellets and supernatants were assayed for DNA contents by fluorometric measurement of dye binding (Hoechst 33258) using a TKO 100 Fluorometer (Hoefer Scientific Instruments, San Francisco, CA).
Statistics.
Data are expressed as means ± standard error of the means (SEM) and were assessed for statistical differences by 2-way analysis of variance (ANOVA), with modified t-tests as indicated, using the commercial software SPSS (Norusis, 1992). Dose-derived differences in each sex were assessed similarly by 1-way ANOVA, with Student-Newman-Keuls post-hoc tests. Plasma ALT activities were log transformed to normalize variances. Differences are attributed at p < 0.05.
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RESULTS |
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GR activities were higher in the livers of male rats than of females (Fig. 4C). In addition, GR activities were increased in male rats given 0.1 to 0.15 mmol/kg of diquat, but activities in female rats were uniformly unaffected by any dose of diquat studied.
GST- activities were much higher in the livers of male rats than of females (Fig. 4D
). No changes in activities in response to diquat administration were observed in the male rats, but the female rats showed modest increases in activities following doses of 0.225 mmol/kg.
Diquat-stimulated oxidation of hepatic proteins, as evidenced by increased levels of DNPH-reactive "protein carbonyls" (Stadtman, 1990), was investigated by derivatizing the proteins with DNPH, separation by SDSPAGE, and ECL detection using an anti-DNP antibody (Shacter et al., 1994
). Diquat-induced oxidation of proteins was observed in both the 12,700 g supernatants (Fig. 5A
) and pellets (data not shown) in both male and female F344 rats. The levels of immunoreactive proteins were consistently greater in female rats than in male rats, both at comparable doses and at comparable elevations in plasma ALT activities (Fig. 5A
). Diquat-treated female rats showed reactive proteins in the range of 6990 kDa that were not similarly oxidized in male rats. In contrast, male rats showed immunoreactive proteins at 23 kDa (arrow) that were not similarly responsive in female rats. Both male and female F344 rats showed diquat-induced increases in biliary efflux of oxidized proteins (Fig. 5B
). As in the tissues, the patterns of DNPH-reactive proteins in the bile of diquat-treated male rats were different from patterns in the female rats, with male rats showing more DNPH-reactive protein at 50 kDa after diquat, and female rats showing greater efflux of an oxidized protein at 70 kDa (Fig. 5B
).
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DISCUSSION |
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The gender differences in susceptibility to diquat-induced hepatic necrosis also are not explained by differences in hepatic activities of antioxidant enzymes and cofactors, which are comparable in both sexes for GSH, SOD, and GPX. Findings of higher hepatic GR and GST- activities in the male rats offer no explanation for greater oxidant sensitivity. Rikans et al. (1991) have reported higher hepatic activities of GR in male F344 rats than in females, in agreement with our data, but they observed lower activities of GPX and SOD in male rats, which contrasts with our studies. Differences in the methods used for tissue homogenization and enzyme assays may be responsible for differing data in the two studies; Rikans et al. measured enzyme activities at 37°C in post-mitochondrial supernatants that were treated with Triton X-100, then dialyzed. We regard the differences in the data as minor and unlikely to contain critical clues to the mechanisms of cell injury.
The comparable basal hepatic activities of SOD (Fig. 4A) and GPX (Fig. 4B
) in male and female F344 rats suggest that GSSG production rates are likely to reflect rates of generation of reactive oxygen species similarly in the two sexes, and GSSG does not accumulate appreciably in liver (Lauterburg et al., 1984
). The most likely mechanism by which increased intrahepatic GSSG levels might mediate cell injury would be by S-thiolation of protein thiols. However, by treatment of hepatic homogenates or subcellular fractions with monobromobimane, separation by SDSPAGE, and visualization of the fluorescent thioether derivatives, we observed no diquat-induced decreases in hepatic protein thiol contents in male or female rats (data not shown), which is consistent with our previous studies in male rats (Gupta et al., 1997
; Smith et al., 1985
, 1987a,b).
None of the enzymes studied shows significant diquat-dependent inactivation, as might be expected of events proximal to the initiation of cell injury. In fact, the increases in hepatic GR activities in male and GPX activities in female rats might be adaptive responses, but the increases are observed only with doses of diquat that increase plasma ALT activities (Figs. 1, 4B, and 4C). The GPX activities in livers of male rats and the GR activities in females are not increased by any of the doses of diquat studied, which is not consistent with the changes that are observed arising from either a response to oxidant stress or a response to acute lethal injury. The absence of diquat-driven responses of GPX in male rats, of GR in females, or of SOD or GST-
in either sex, also indicates that the apparent diquat-induced changes in the enzymes whose activities are altered are artifactual. Gallagher et al. (1995) have reported changes in hepatic messenger ribonucleic acid (mRNA) levels and enzyme activities of several cytochromes P450, GSTs, and selected other enzymes 24 h after administration of 0.1 mmol/kg of diquat to male Sprague-Dawley rats. However, Sprague-Dawley rats develop little, if any, hepatic necrosis from exposure to this dose of diquat (Smith et al., 1985
, 1987a). Gallagher et al. interpreted their data as suggesting pre-translational loss of mRNAs as a possible effect of increased cellular production of reactive oxygen species, but the late time point studied in an animal model that does not express necrosis limits the relevance of their data to the potential mechanisms of acute cell death by reactive oxygen species.
Several studies in vivo and in vitro have implicated metal-catalyzed oxidation reactions and production of DNPH-reactive modified protein as events tightly coupled with the acute lethal injury initiated by diquat and by other models of injury mediated by reactive oxygen species (Blakeman et al., 1995; Gupta et al., 1994
; Rikans and Cai, 1992
; Rikans et al., 1993
; Sandy et al., 1987
; Shertzer et al., 1992
; Smith, 1987b
). In the present studies, however, greater diquat-induced formation of total DNPH-reactive proteins is observed in samples from female rats than from males, at comparable doses, levels of GSSG efflux, or increases in plasma ALT activities. The results illustrated in Figures 5A and 5B
are not consistent with processes reflected by this biomarker of oxidative alterations contributing significantly to initiation of injury, unless the specific oxidations reflected by these analyses are markedly different in their respective biological effects. The higher levels of DNPH-reactive proteins, in samples from diquat-treated female rats, particularly at molecular weights greater than 60 kDa, are accompanied by greater band intensities in tissues and bile from male rats in the region of 50 kDa and at about 23 kDa (Fig. 5
). Enthusiasm for one or both of these proteins reflecting alterations critical to oxidant-induced cell death is diminished by the marked immunoreactivity noted in these proteins in the male rat showing normal ALT activities (48 IU/l, Fig. 5A
, lane 7), but further studies are needed of specificity in protein oxidation, including investigations of the high molecular weight DNPH-reactive proteins.
The similarities between the dose-response effects of diquat on hepatic DNA fragmentation (Fig. 6) and the plasma ALT activities (Fig. 1
) are striking. With the formal statistical analyses employed in the experimental design applied to the present data, increases in DNA fragmentation are not observed in male rats in the absence of diquat-induced hepatic damage, as evidenced by increased plasma ALT activities. In contrast, increased DNA fragmentation was observed in diquat-treated female rats at doses of diquat that did not elevate plasma ALT activities, which suggests DNA fragmentation occurring before or without other manifestations of hepatic injury. This distinction may arise more from the greater variances in the plasma ALT activities than in the DNA fragmentation measurements, rather than from any biologically relevant cause-effect relationship. However, the time course data shown in Figure 7
clearly indicate increases in fragmentation of hepatic DNA preceding even the minor increase in plasma ALT activities caused by the minimally hepatotoxic dose of diquat (0.2 mmol/kg in female rats) chosen for this study. In another recent report, we noted increased hepatic DNA fragmentation in male rats 2 h after 0.1 mmol/kg, in the absence of an increase in plasma ALT activities (Gupta et al., 1997
). However, in that study, the male rats treated with 0.1 mmol/kg of diquat showed marked hepatic damage by 6 h.
The more detailed time-course data, obtained with the very minimally hepatotoxic dose of diquat used for the data presented in Figure 7, offer much stronger evidence that DNA fragmentation is an early event in hepatic injury caused by diquat in vivo. The extent of DNA fragmentation estimated by the centrifugal sedimentation methods employed in the present studies exceeds the frequency of hepatocytes exhibiting histological changes (chromatin margination and fragmentation, cell shrinkage, and membrane-limited cell fragmentation) characteristic of apoptosis. Histochemical evidence of DNA fragmentation also was observed by TUNEL (TdT-mediated dUTP nick end labeling) analyses, although in the female rats less than 1% of TUNEL-positive hepatocytes were apoptotic as assessed by structural changes (data not shown, manuscript in preparation). We also have observed increases in oligonucleosomal fragmentation patterns ("DNA laddering") in livers of rats treated with hepatotoxic doses of diquat. Among the numerous biomarkers of oxidation and antioxidant enzyme activities investigated in the present studies, DNA fragmentation is most closely associated with diquat-induced hepatic necrosis. Additional studies are needed to determine the mechanisms responsible for the DNA fragmentation and the pathophysiological consequences of this effect. The precise nature of the alterations indicated as DNA fragmentation, the mechanisms responsible for those changes, and the pathophysiological consequences of these effects all may prove to be significant in defining the mechanisms of cell and tissue damage by reactive oxygen species.
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ACKNOWLEDGMENTS |
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NOTES |
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