Sex Differences in Diquat-Induced Hepatic Necrosis and DNA Fragmentation in Fischer 344 Rats

Sanjiv Gupta, Richard C. Husser, Robert S. Geske, Stephen E. Welty and Charles V. Smith1

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Redox cycling metabolism of diquat catalyzes generation of reactive oxygen species, and diquat-induced acute hepatic necrosis in male Fischer 344 (F344) rats has been studied as a model of oxidant mechanisms of cell killing in vivo. At equal doses of diquat, female F344 rats sustained less hepatic damage than did male rats, as estimated by plasma alanine aminotransferase (ALT) activities after 6 h. Biliary efflux of glutathione disulfide (GSSG) was greater in male than in female rats at each dose of diquat, but even comparable rates of GSSG excretion were associated with less hepatic injury in female rats. Hepatic activities of superoxide dismutase (SOD) and glutathione peroxidase (GPX) were similar in the two genders, and activities of glutathione reductase (GR) and glutathione S-transferase-{alpha} (GST-{alpha}) activities were higher in the male rats. Previous studies in male rats have implicated formation of 2,4-dinitrophenylhydrazine (DNPH)-reactive "protein carbonyls" and related iron chelate-catalyzed redox reactions as mechanisms critical to diquat-induced acute cell death in vivo. However, diquat-treated female rats showed higher levels of DNPH-reactive proteins in livers and in bile than did males, both at identical doses of diquat and at doses that produced similar elevations in plasma ALT activities. In female rats, fragmentation of hepatic deoxyribonucleic acids (DNA) was increased by doses of diquat that did not increase plasma ALT activities, and increased fragmentation was observed prior to elevation of plasma ALT activities. In the present studies, hepatic necrosis was most closely associated with DNA fragmentation, although additional studies are needed to determine the mechanisms responsible for and the pathophysiological consequences of the fragmentation.

Key Words: diquat; Fischer 344 rats; reactive oxygen species; hepatic necrosis; antioxidant defense mechanisms; 2,4-dinitrophenylhydrazine; protein carbonyls; DNA fragmentation.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reactive oxygen species and other oxidants have been implicated in many examples of cell and tissue injury (Smith, 1992Go; Stadtman and Berlett, 1997Go). The bipyridilium herbicides paraquat and diquat stimulate the generation of reactive oxygen species by redox cycling metabolism, and these agents have been studied extensively as models of oxidant-mediated cellular injury (Burk et al., 1980Go; Bus and Gibson, 1984Go; Nakagawa et al., 1992Go; Rikans and Cai, 1993Go; Smith et al., 1985Go). An important finding of these studies has been that increased production of glutathione disulfide (GSSG) did not correlate with initiation of injury (Gupta et al., 1994Go, 1997Go; Lauterburg et al., 1984Go; Smith, 1987aGo,bGo;). In addition, depletion of protein thiols (PSH) was not observed (Nakagawa et al., 1992Go; Smith et al., 1985Go), in contrast to interpretations generated from studies of other experimental models of oxidant injury in vitro (Di Monte et al., 1984Go; Jones et al., 1983Go). The initiation of injury by diquat has been more closely associated with iron chelate-catalyzed oxidations than with depletion of thiols or with thiol S-thiolations (Gupta et al., 1994Go, 1997Go; Smith, 1987aGo,bGo). The critical functions of redox-active iron chelates in oxidant injury have been implicated pharmacologically in potentiation of damage by administration of iron salts and by protection through administration of iron chelators such as desferrioxamine (Blakeman et al., 1995Go; Sandy et al., 1987Go; Shertzer et al., 1992Go; Smith, 1987bGo). In addition, the formation of DNPH-reactive carbonyl groups, principally aldehydes and ketones, has been observed in models of diquat toxicity. Studies to date suggest that these "protein carbonyls" may be useful biomarkers of critical mechanisms of injury (Davies and Delsignore, 1987Go; Dean et al., 1997Go; Stadtman and Berlett, 1997Go). The reactions through which these DNPH-reactive modifications are formed appear to depend upon both a source of oxidant, usually H2O2 or other hydroperoxide, and a redox-active transition metal catalyst, usually iron (Blakeman et al., 1995Go; Gupta et al., 1994Go; 1997Go; Rikans and Cai, 1993Go; Sandy et al., 1987Go; Shertzer et al., 1992Go).

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, 1999Go). 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
All chemicals used were analytical grade. Reagents for electrophoresis were obtained from Bio-Rad (Hercules, CA). Diquat was provided generously by Dr. Ian Wyatt of Zeneca (Macclesfield, Cheshire, UK).

Animals.
Adult (9–11 weeks-of-age) male (180–220 g) and female (160–180 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., 1983Go) 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 2–24 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 0–40 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, 1974Go). 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-{alpha}) activities.
GST-{alpha} activities were estimated by subtracting the GPX activities measured using H2O2 from the GPX activities measured using cumene hydroperoxide (Reddy et al., 1981Go).

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 SDS–polyacrylamide 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 Tris–HCl, 1 mM EDTA, pH 8.0. Next, 3 ml of lysis buffer containing 5 mM Tris–HCl, 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 Tris–HCl, 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, 1992Go). 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Administration of diquat caused acute hepatic necrosis in both male and female F344 rats, as indicated by elevations in plasma ALT activities (Fig. 1Go), although higher doses were required in the female rats than in the males. Significant increases in ALT activities were observed in the males with 0.1 mmol/kg, the lowest dose studied in the present series of experiments, whereas elevations were not observed in female rats at doses below 0.2 mmol/kg. Previous dose-response studies in male F344 rats have shown that doses of diquat of 0.05 or 0.075 mmol/kg did not normally cause observable hepatic injury except in F344 rats pretreated with ferrous sulfate or 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (Smith, 1987aGo,bGo). In the present studies, the time courses of the effects of diquat on female rats were not investigated extensively, but previous studies in males have shown that biomarkers of oxidant-stress responses, such as production of GSSG and expiration of ethane and pentane, are maximal within the first 3 h. Elevations in plasma ALT activities by 6 h after drug administration are reliable indicators of hepatic necrosis assessed histologically or with longer time-course data on plasma enzyme activities (Smith et al., 1985Go; Smith, 1987bGo). Histological examination of liver sections from animals in the present studies confirmed agreement between the plasma ALT activities measured and the extent of hepatic injury indicated by light microscopy (data not shown).



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FIG. 1. Dose-response effects of diquat on elevations of plasma ALT activities in male and female Fischer 344 rats. Plasma was collected from anesthetized animals 6 h after dosing, and ALT activities were determined as described in Methods. Data are means ± SEM, n = 4 per group. *Different from respective saline-treated (0 mmol/kg) control group by one-way ANOVA-Student-Newman-Keuls, p < 0.05. #Different from female rats given the same dose of diquat by 2-way ANOVA and modified t-tests on data log transformed to normalize variances, p < 0.05.

 
The simplest potential explanation for lower hepatic injury in diquat-treated female rats than in similarly treated males would be because of lower rates of intrahepatic generation of reactive oxygen species, which could arise from lower rates of redox cycling metabolism at comparable tissue concentrations of diquat or from lower intrahepatic concentrations of diquat in female rats than in male rats at similar doses (Madhu et al., 1992Go). We measured biliary GSSG efflux rates in male and female rats treated with diquat as a means of assessing the hepatocellular exposure to reactive oxygen species (Lauterburg et al., 1984Go). At each dose of diquat, male rats (Fig. 2AGo) showed greater increases in biliary efflux of GSSG than did females (Fig. 2BGo). However, the rates of diquat-stimulated efflux of GSSG in female rats treated with 0.15 or 0.2 mmol/kg of diquat were equivalent to the rates observed in males treated with 0.1 mmol/kg. This is a notable comparison because 0.15 or 0.2 mmol/kg of diquat do not elevate plasma ALT activities in female F344 rats, whereas 0.1 mmol/kg causes marked hepatic necrosis in male rats (Fig. 1Go). Hepatic diquat concentrations were uniformly higher in female rats than in males, whether compared at equal doses of diquat, extents of elevation of biliary GSSG efflux (Fig. 3AGo), or plasma ALT activities (Fig. 3BGo).



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FIG. 2. Time course on biliary efflux of total GR-reactive glutathione (GSH + GSSG + GSSX) in male (A) and female (B) F344 rats treated with diquat. Bile samples were collected in 0.1 M phosphoric acid for 30 min, immediately preceding (-30 to 0) and 30 min after administration of diquat, and every alternate 30 min thereafter. Concentrations of (GSH + GSSG + GSSX) were measured by the method of Tietze as modified by Smith (1987). *Different from respective samples before diquat by ANOVA-Newman-Keuls, p < 0.05.

 


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FIG. 3. Hepatic concentrations of diquat in male and female F344 rats. Male and female F344 rats were treated with 0 to 0.25 mmol/kg of diquat, and diquat concentrations were measured in livers and plasma ALT activities determined at 2 to 24 h after dosing. The data shown represent single animals following administration of 0.15 mmol/kg of diquat in male rats and 0.2 mmol/kg in female rats, which in the studies shown in Figure 2Go, gave comparable increases in biliary efflux of GSSG. The hepatic diquat concentrations (A) and plasma ALT activities (B) are from the same animals.

 
SOD activities were not different between male and female rats, other than in a statistical difference indicated in the animals treated with 0.2 mmol/kg (Fig. 4AGo). The SOD activities in the livers of the male rats treated with the higher doses of diquat are not statistically different from the activities in the other groups of males.



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FIG. 4. Dose-response effects of diquat on activities of antioxidants in livers of male and female F-344 rats. Livers were collected from anesthetized animals 6 h after dosing, and homogenized by Polytron, sonicated for 15 x 4 sec and centrifuged at 1000 rpm to remove cell debris. These supernatants were used for SOD enzyme-activity determinations (A). GPX (B), GR (C), and GST-{alpha} (D) activities were determined in 12,700 g supernatants as described in Materials and Methods. Data are means ± SEM, n = 4 per group. *Different from respective saline-treated (0 mmol/kg) control group by one-way ANOVA-Newman-Keuls, p < 0.05. #Different from female rats given the same dose of diquat by 2-way ANOVA and modified t-tests, p <0.05.

 
Hepatic GPX activities were also not different between male and female rats and were not affected in either sex by doses of diquat up to 0.2 mmol/kg (Fig. 4BGo). Female rats given higher doses of diquat showed increases in hepatic GPX activities. The effects of doses of diquat greater than 0.2 mmol/kg could not be studied in male rats because of animal mortality. The increases in hepatic GPX activities in the female rats were observed at doses that also enhanced plasma ALT activities (Fig. 1Go), but similar levels of hepatic injury in males, at doses of 0.1 to 0.2 mmol/kg, caused no changes in peroxidase activities. Hepatic GSH concentrations were not different in the male and female rats (data not shown)

GR activities were higher in the livers of male rats than of females (Fig. 4CGo). 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-{alpha} activities were much higher in the livers of male rats than of females (Fig. 4DGo). 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, 1990Go), was investigated by derivatizing the proteins with DNPH, separation by SDS–PAGE, and ECL detection using an anti-DNP antibody (Shacter et al., 1994Go). Diquat-induced oxidation of proteins was observed in both the 12,700 g supernatants (Fig. 5AGo) 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. 5AGo). Diquat-treated female rats showed reactive proteins in the range of 69–90 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. 5BGo). 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. 5BGo).



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FIG. 5. Dose response effects of diquat on DNPH-reactive protein carbonyls in livers and in bile of male and female F344 rats. (A) Six h after treatment with the dose of diquat indicated, livers were removed, homogenates prepared, and samples were centrifuged at 12,700 g for 20 min. Doses of diquat and plasma ALT activities in the individual animals were as indicated. (B) In a separate group of animals, bile samples were collected in 30-min increments, before and after treatment with diquat. Following derivatization with DNPH as described in Materials and Methods, 12 µg of protein from the supernatant fractions were separated by SDS–PAGE and DNPH-reactive bands detected by enhanced chemiluminescence (ECL). In B, lanes a through g are from sequential bile fractions collected from a male F344 rat before (a, b) and after (c–g) administration of 0.1 mmol/kg of diquat, ip. Lanes h–l are from a female F344 rat before (h, i) and after (j–m) 0.2 mmol/kg of diquat. The present comparison of different doses in the two sexes is viewed as more useful because of the greater comparability of plasma ALT activities (Fig. 1Go) and biliary GSSG efflux (Fig. 2Go).

 
DNA fragmentation, as measured by the centrifugal sedimentation of chromatin in detergent-treated tissue fractions, is increased by diquat treatment both in male and female F344 rats. Diquat caused more DNA fragmentation in male rats given 0.15 or 0.2 mmol/kg of diquat than in similarly treated female rats (Fig. 6Go). However, plasma ALT activities were elevated in male rats by even lower doses of diquat, whereas in female rats the elevation of ALT activities was observed only above 0.2 mmol/kg (Fig. 1Go). The possible relationship between DNA fragmentation and hepatic injury, as reflected by plasma ALT activities, was investigated further by examination of the early time-course responses in diquat-treated female rats. The results showed increases in hepatic DNA fragmentation occurring before even minor increases in plasma ALT activities (Fig. 7Go).



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FIG. 6. Dose-response effects of diquat on hepatic DNA fragmentation in male and female Fischer 344 rats. Rats were treated with the doses of diquat indicated or with equal volumes of saline, ip. At 6 h, the animals were anesthetized and livers freeze-clamped with liquid N2-cooled tongs. DNA fragmentation was estimated as described in Materials and Methods. Data are means ± SEM; n = 4 per group. *Different from respective saline-treated (0 mmol/kg) control group by one-way ANOVA-Newman-Keuls, p < 0.05. #Different from female rats given the same dose of diquat by 2-way ANOVA and modified t-tests, p < 0.05.

 


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FIG. 7. Time course of diquat-induced increases in plasma ALT activities and hepatic DNA fragmentation in female Fischer 344 rats after giving 0.2 mol/kg of diquat. Plasma ALT activities and hepatic DNA fragmentation were determined 2, 4, and 6 h after dosing, as described in Materials and Methods. Data are means ± SEM, n = 3 per group. *Different from respective saline-treated (0 mmol/kg) control group by 1-way ANOVA-Newman-Keuls, p < 0.05.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The greater resistance found to diquat-induced acute hepatic necrosis in female F344 rats than was observed in males (Fig. 1Go) may in part be due to differences in rates of generation of reactive oxygen species by redox cycling of diquat, as is indicated by the lower biliary efflux of GSSG noted in female rats at equivalent doses of diquat (Fig. 2Go). Biliary export is a major route of disposition of GSSG produced in response to exposure to oxidants, and biliary GSSG concentrations thus provide a useful index of the magnitude of the oxidant stress (Adams, et al., 1983Go; Lauterburg et al., 1984Go). GSH is the major species in bile in rats, under basal conditions, but the marked increases in efflux of total GR-reactive species that accompany strong intrahepatic oxidant stresses, such as are produced by diquat, are almost entirely in the form of GSSG. The much greater diquat concentrations in the livers of female rats than in male rats (Fig. 3AGo), compared both for dose and for comparable rates of biliary efflux of GSSG (Fig. 2Go), however, indicate that other factors contribute significantly to the gender differences in injury.

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-{alpha} 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. 4AGo) and GPX (Fig. 4BGo) 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., 1984Go). 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 SDS–PAGE, 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., 1997Go; Smith et al., 1985Go, 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 4CGoGo). 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-{alpha} 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., 1985Go, 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., 1995Go; Gupta et al., 1994Go; Rikans and Cai, 1992Go; Rikans et al., 1993Go; Sandy et al., 1987Go; Shertzer et al., 1992Go; Smith, 1987bGo). 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 5BGo 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. 5Go). 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. 5AGo, 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. 6Go) and the plasma ALT activities (Fig. 1Go) 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 7Go 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., 1997Go). 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 7Go, 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.


    ACKNOWLEDGMENTS
 
This work was supported by GM44263 from the National Institutes of Health.


    NOTES
 
1 To whom correspondence should be addressed at the Department of Pediatrics, Children's Research Institute, The Ohio State University, 700 Children's Drive, Columbus, OH 43205. Fax: (614) 722-3273. E-mail: smithcv{at}chi.osu.edu. Back


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