- Email: [email protected]
Paraquat induces different pulmonary biochemical responses in Wistar rats and Swiss mice
Chemico-Biological Interactions 125 (2000) 79 – 91
www.elsevier.com/locate/chembiont
Paraquat induces different pulmonary biochemical responses in Wistar rats and Swiss mice S. Ali *, G. Diwakar, S. Pawa Department of Biochemistry, Faculty of Science, Jamia Hamdard (Hamdard Uni6ersity), New Delhi – 1100 62, India Received 4 August 1999; accepted 19 December 1999
Abstract The paper presents results showing differential response to paraquat toxicity in Wistar rats and Swiss strain of mice. Paraquat-induced pulmonary biochemical responses in the two animal species were studied at different time point after giving a single intraperitoneal injection of the respective LD10 doses of the herbicide paraquat to rats and mice. Paraquat induced different biochemical responses including different protective responses in the two animal species. As a protective response, NADPH-specific quinone reductase is induced in rats, while catalase is induced in mice. It is implied that an early induction of catalase in mice as opposed to rats may account for the resistance of Swiss mice to paraquat toxicity. Xanthine oxidase, which was induced in rats, remains unaffected in mice indicating that the enzyme contributes to paraquat toxicity only in Wistar rats. Time-course studies were also conducted to compare the differential responses of antioxidant enzymes and lipid peroxidation between the two species. The results of the study led us to suggest that the manifestation of paraquat toxicity involve distinct differences in early pulmonary biochemical responses in Wistar rats and Swiss mice. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Paraquat; Pulmonary biochemical responses; Wistar rats; Swiss mice
* Corresponding author. Fax: + 91-11-6988874. E-mail address: [email protected] (S. Ali) 0009-2797/00/$ - see front matter © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 2 7 9 7 ( 9 9 ) 0 0 1 6 7 - 2
80
S. Ali et al. / Chemico-Biological Interactions 125 (2000) 79–91
1. Introduction The herbicide paraquat (1,1%-dimethyl 4,4%-bipyridilium dichloride; PQ) facilitates the agricultural yields by killing weeds worldwide. First report of PQ poisoning in human appeared in 1966 [1]. Since then, a number of deaths have been reported as a result of occupational, accidental or suicidal ingestion of PQ. Although liver, kidney, heart and central nervous system are affected, irreversible lung damage is the usual cause of death [2–4]. Lungs appear to be the target organ as PQ selectively accumulates in type 11 pneumocytes by an active polyamine uptake process [5,6]. Saturation kinetics of PQ uptake by the human lung are very similar to that of rat lung with a Vmax of about 300 nmol accumulated/g lung per h, and a Km of about 4× 10 − 5 mol/l. The mechanism of PQ toxicity in various species has been investigated and is attributed to its redox cycling nature. Due to a lower redox potential of − 446 mV [7], PQ is easily reduce by one electron to form a relatively stable and toxic radical which can be re-oxidized back to its parent cationic form. One electron reduction activity of paraquat in subcellular fractions of various tissues in vitro has been compared using microsomal fraction [8]. Reduced PQ radical is immediately re-oxidized by molecular oxygen, resulting in the formation of superoxide anion radical which dismutates to hydrogen peroxide, thus initiating the process of tissue damage [9,10]. Beside this, polymorphonuclear cell migration and activation [11], NADPH-oxidation [12], and xanthine oxidase induction [13] have also been reported to contribute to PQ toxicity in different animal species. The results of the present study show that different pulmonary biochemical responses are elicited in Wistar rats and Swiss mice. Distinct differences in the induction of protective responses, antioxidant enzyme activity level and lipid peroxidation in the PQ treated rats and mice indicate toward the need for finding an animal species which show biochemical responses similar to those elicited in human tissue exposed to paraquat. This is expected to help produce a suitable animal model, which can be utilized for the development of an antidote against PQ.
2. Materials and methods
2.1. Materials Paraquat dichloride, nicotinamide adenine dinucleotide (NADPH) and flavin adenine dinucleotide (FAD) were purchased from Sigma (St Louis, MO, USA). All other chemicals were of highest purity grade commercially available in India. Specific activities of all the enzymes measured in this study were determined by monitoring the change in absorbence at their respective wavelength using a Milton Roy, Spectronic 21 D spectrophotometer.
S. Ali et al. / Chemico-Biological Interactions 125 (2000) 79–91
81
2.2. Treatment of animals Experiments were conducted on male Wistar rats (1209 15 g body weight) and male Swiss mice (22 9 2 g body weight). At these body weights the animals were 5–6 weeks old, and were in the similar developmental stage. All the animals were procured from Central Animal House Facility of Jamia Hamdard (New Delhi, India) and were kept in plastic cages in an environmentally controlled room, with each group containing six animals. Animals had free access to pellet diet (Lipton India Ltd) and water ad libitum. Animals were given a single intraperitoneal injection of paraquat. Respective LD10 dose of PQ was given to Wistar rats and Swiss mice; 30 mg/kg body weight to mice, and 20 mg/kg body weight to rats. The LD10 doses in the two animal species were determined by using probit test. The LD10 doses were chosen in order to have consistency in the doses used in the two animal species. The doses were given in such a way that the biochemical estimations could be performed at 12, 36 or 72 h interval after PQ administration. It was ensured that all the animals be treated in such a way that the time of killing should not differ in each case. The group of animals treated with saline served as control. Lungs were removed en bloc (i.e. along with the heart) immediately after killing. The lung tissue was then separated from the heart and washed in ice-cold saline solution. Tissue was blotted and weighed before subjecting it to homogenization.
2.3. Subcellular fractionation Accurately weighed pieces of tissue were minced and homogenized in nine volumes of ice cold 0.1 M phosphate buffer (pH 7.4), containing 1.15% KCl using a polytron homogenizer, giving five to six strokes at a speed of 2000 rpm for 30 s. The resulting 10% homogenate was subjected to differential centrifugation, first at a speed of 800×g for 10 min (to remove nuclei and unbroken cells) and then at 9500 × g for 20 min to get the post mitochondrial supernatant (PMS), which was used as a crude source of enzymes for assay purpose. Lipid peroxidation was measured using total lung homogenate. All subsequent operations were carried out at 4°C.
2.4. Biochemical estimations Measurement of the activity of xanthine oxidase (XOD) was based on the procedure described by Stirpe and Della Corte [14]. Reaction mixture consisting of 0.1 mM xanthine and 0.5 M Tris–HCl buffer (pH 8.1) was incubated with appropriate amount of enzyme source for 20 min at 37°C. The reaction was terminated by precipitating the enzyme using 10% perchloric acid. The mixture was then centrifuged at 4000 rpm for 10 min. Uric acid in the clear supernatant was determined at 290 nm. Results are expressed as mg of uric acid/mg protein. NAD(P)H:quinone reductase (QR) activity was determined according to the method of Ernster as modified by Benson et al. [15]. Reaction mixture in a volume
82
S. Ali et al. / Chemico-Biological Interactions 125 (2000) 79–91
of 3 ml consisted of 25 mM Tris –HCl (pH 7.4), 0.7 mg of crystalline bovine serum albumin, 0.01% Tween-20 (v/v), 5 mM flavin adenine nucleotide, 0.2 mM NADPH and an appropriate aliquot of the enzyme source. Electron acceptor dichlorophenol indophenol (DCPIP; 40 mM) in 20 ml of water was added to initiate the reaction and the initial velocity of the reduction of DCPIP was measured spectrophotometerically at 600 nm using a molar extinction coefficient of 2.1× 104 M − 1 cm − 1. Catalase activity was measured at 230 nm [16]. In brief, 50 ml of enzyme source was diluted to 2 ml with 50 mM potassium phosphate buffer at pH 7. The reaction was initiated in a quartz cuvette by addition of 1 ml of 0.34% H2O2 (v/v). Specific activity of the enzyme was calculated using a molar extinction coefficient of 81 M − 1 cm − 1. Results are expressed as mmol of H2O2 decomposed/min per mg protein. Glutathione reductase (GR) activity was measured according to the method described by Carlberg and Mannervik [17]. Reaction mixture consisted of 0.1 M phosphate buffer (pH 7.6), 0.1 mM NADPH, 0.5 mM ethylene diamine tetra acetate, and 1 mM glutathione (oxidized). To initiate the reaction, 50 ml of enzyme source was added. The oxidation of NADPH was measured at 340 nm. For calculations, an extinction coefficient of 6.3 M − 1 cm − 1 was used. Results are expressed as nmol NADPH oxidized/min per mg protein. Glucose 6-phosphate dehydrogenase (G6PD) activity was determined by the method described by Bergmeyer et al. [18]. Reduction of NADP to NADPH was followed at 340 nm using a molar extinction coefficient of 6.3 M − 1 cm − 1. Results are expressed as nmol of NADP reduced/min per mg protein. Glutathione S-transferase activity measurement was based on the spectrophotometeric determination of 1-chloro-2,4-dinitrobenzene (CDNB)-conjugate formed in a reduced glutathione (GSH) coupled reaction [19]. The reaction was initiated by adding 10 mM CDNB (in acetone) to a reaction mixture containing 1 mM GSH and 20 ml of PMS in 0.1 M phosphate buffer. A molar extinction coefficient of 9.6 M − 1 cm − 1 at 340 nm was used to calculate the specific activity. Malondialdehyde was measured as an index of oxidative tissue damage. It was measured as thiobarbituric acid reacting substances in the total lung homogenate [20]. Since the absorbence of the treated/control samples was less than 0.025 at time 0, the samples were incubated for 30 min in a water bath at 37°C to obtain measurable absorption values. The samples were then precipitated in a 1:1 (v/v) ratio with 20% trichloroacetic acid (w/v). The mixture was centrifuged at 3000 rpm for 10 min, and then the supernatant was heated with 0.67% thiobarbituric acid (w/v) at 100°C for 20 min in the presence of 100 mM butylhydroxyanisol to avoid further cleavage of hydroperoxides. After cooling, the absorbence of the supernatant was measured at 432 nm. Protein content was determined by the method of Lowry et al. [21] using bovine serum albumin as reference standard. The data are expressed as mean 9 S.E. of six animals. Values obtained in each experimental group of animals were individually compared with the respective normal control group using Student’s t-test.
S. Ali et al. / Chemico-Biological Interactions 125 (2000) 79–91
83
2.5. Sur6i6al studies Effect of catalase inhibitor hydroxylamine hydrochloride (HA-HCl) on the survival in PQ treated Swiss mice was studied. Mice were administered with HA-HCl (70 mg/kg body weight, i.p.) 30 min before injecting the LD10 dose of PQ. PQ alone, or HA-HCl alone treated group of mice served as positive controls. Each group consisted of ten mice. Survival in each group was monitored every 12 h for 6 days.
3. Results
3.1. Pulmonary XOD acti6ity in PQ treated Swiss mice and Wistar rats XOD contributes to tissue damage by generating oxygen free radicals as byproduct while catalyzing its reaction. As shown in Fig. 1, an increase in the activity of XOD is seen in rats sacrificed 36 h after PQ administration. When compared with the untreated group, the increase was 26% more in the PQ treated group of rats. However, in mice killed at the same time point no change in XOD activity is observed.
3.2. Pulmonary catalase and NAD(P)H-quinone reductase acti6ity in PQ treated animals Catalase and NAD(P)H:quinone reductase are the two defense system enzymes, which were studied in the present investigation. A marked increase in the activity of catalase with a peak at 12 h after PQ administration is observed in Swiss mice (Fig.
Fig. 1. Effect of paraquat on pulmonary XOD activity in mice and rats sacrificed 36 h after a single injection of the toxicant to each animal. Dose and the treatment protocol have been described in Section 2. Each value represents mean 9S.E. of six animals. *PB 0.05, when compared with the respective saline treated control group of animals. A significant increase in the activity of XOD suggests that the enzyme contributes to tissue damage only in Wistar rats.
84
S. Ali et al. / Chemico-Biological Interactions 125 (2000) 79–91
Fig. 2. Effect of paraquat on pulmonary catalase activity in Swiss mice and Wistar rats sacrificed at different time points after a single injection of the toxicant to each animal. Dose and the treatment protocol have been described in Section 2. Each value represents mean 9S.E. of six animals. *P B0.05, when compared with the respective saline treated control group of animals. Catalase is induced in paraquat treated Swiss mice only whereas the enzyme activity in rats remains unaffected.
2), while NAD(P)H:quinone reductase is induced in Wistar strain of rats with a peak value seen 72 h following the administration of PQ (Fig. 3). Pulmonary catalase activity in mice and rats was also studied at an earlier time point than 12 h. The results were consistent with the changes seen at 12 h time point; rats did not show any change in enzyme activity, while the activity in mice induced. The results are, however, not shown in order to keep consistency with the other enzymes, which were measured at 12-h time point onward.
Fig. 3. Effect of paraquat on pulmonary NAD(P)H:quinone reductase activity in Swiss mice and Wistar rats sacrificed at different time points after a single injection of the toxicant to each animal. Dose and the treatment protocol have been as described in Section 2. Each value represents mean 9S.E. (n =6). *P B 0.05, when compared with the respective saline treated control group of animals. The enzyme is induced in rats only and reaches a peak 3 days after the treatment. Mice show no change in enzyme activity.
S. Ali et al. / Chemico-Biological Interactions 125 (2000) 79–91
85
Fig. 4. Effect of paraquat on the activity of pulmonary glutathione reductase in Swiss mice and Wistar rats sacrificed at different time points after a single injection of the toxicant to each animal. Dose and the treatment protocol have been described in Section 2. Each value represents mean 9S.E. (n =6). *P B 0.05, when compared with the respective saline treated control group of animals. The enzyme activity is inhibited in rats whereas in mice the specific activity remains unaffected.
3.3. Acti6ities of GR, G6PD and glutathione S-transferase (GST) in PQ treated mice and rats The results of the effect of PQ on the activity of glutathione redox cycle enzyme GR in Swiss mice and Wistar rats are shown in Fig. 4. GR is involved in the regeneration of reduced glutathione from oxidized glutathione at the expense of NADPH. In Swiss mice, the activity of pulmonary GR remains unaffected. However, a decrease in GR activity with time is shown in Wistar rats. Since G6PD recycles NADP to NADPH, it was thought that the activity of G6PD would be perturbed only in Wistar rats. We observed a time-dependent increase in the activity of glucose-6 phosphate dehydrogenase in PQ treated Wistar rats reaching a maximum at 72 h (Fig. 5). As expected G6PD remained unperturbed in Swiss mice. The activity of a phase II drug metabolizing enzyme glutathione S-transferase (GST) was also determined in the two species of animals treated with PQ (Fig. 6). GST helps in detoxification by conjugate formation. In rats, GST activity level increased to its maximum by the 36th hour and then remains unchanged at this peak value in the 72-h group also. In mice, the activity could only be affected significantly (decrease) at 72 h time point.
3.4. Lipid peroxidation in PQ treated Swiss mice and Wistar rats The lipid peroxidation profile in PQ treated rats and mice is shown in Fig. 7. A significant increase is observed 72 h after the treatment. Before this time point, lipid peroxidation value does not differ from the control value. Interestingly, the results can be related to the activity of XOD, which show maximum induction following
86
S. Ali et al. / Chemico-Biological Interactions 125 (2000) 79–91
Fig. 5. Effect of paraquat on the activity of pulmonary G6PD in paraquat treated Swiss mice and Wistar rats. Animals were sacrificed at different time points after a single injection of the toxicant to each animal. Dose and the treatment protocol have been described in Section 2. Each value represents mean9 S.E. (n= 6). *PB 0.05, when compared with the respective saline treated control group of animals. Enzyme activity is induced in Wistar rats, whereas the G6PD measured in the tissue of Swiss mice shows no change when compared with the respective normal values.
36 h after PQ intoxication. This can be explained assuming that the oxidants generated as byproduct in XOD-catalyzed reaction damage the unsaturated lipids present in the cell. PQ treated mice, however, did not exhibit increase in lipid peroxidation (Table 1). Rather a decreasing trend in the lipid peroxidation is observed with time in mice. This may be attributed to an early increase in the activity of catalase, which seems to scavenge the hydrogen peroxide in PQ treated Swiss mice; thus making the hydrogen peroxide unavailable to induce tissue damage in mice.
Fig. 6. Changes in the activity of pulmonary GST in paraquat treated Swiss mice and Wistar rats. Animals were sacrificed at different time points after a single injection of the toxicant to each animal. Dose and the treatment protocol were the same as described in Section 2. Each value represents mean9 S.E. (n= 6). *PB 0.05, when compared with the respective saline treated control group of animals.
S. Ali et al. / Chemico-Biological Interactions 125 (2000) 79–91
87
Fig. 7. Comparison of changes in pulmonary lipid peroxidation and the activity of XOD in paraquat treated Wistar rats. Estimations were performed 12, 36, or 72 h after a single injection of the toxicant. Dose and treatment protocol has been described in Section 2. For comparison, values were calculated as % change with respect to the saline treated control group. Lipid peroxidation increases 72 h after paraquat treatment, i.e. only after the XOD has contributed to the toxicity of paraquat. Note the XOD peak is seen 36 h after paraquat treatment.
3.5. Effect on sur6i6al of catalase inhibitor in PQ treated Swiss mice Administration of HA-HCl, an in situ inhibitor of catalase, 30 min before the administration of PQ significantly affect survival in Swiss mice. When compared with the PQ alone treated group, a 40% decrease in survival was observed in HA-HCl pretreated group of mice (Fig. 8). This clearly suggests that induction of catalase constitute an important protective response against PQ-induced oxidative damage in mice. The experiment with the inhibitor was not performed with rats because the activity level of catalase remained unaffected in PQ treated Wistar rats. Table 1 Lipid peroxidation in the lungs of paraquat treated Wistar rats and Swiss albino strain of micea The nmol of malondialdehyde/mg protein Group
Swiss mice
Wistar rats
Control 12 h 36 h 72 h
13.39 1.27 12.89 1.23 10.99 0.89 09.39 2.3*
10.2 9 0.96 08.4 9 0.85 09.99 0.57 14.1 90.45*
a Dose and the treatment protocol have been described in Section 2. Animals were sacrificed at 12, 36, or 72 h after paraquat treatment and the whole tissue homogenate was used for the determination of lipid peroxidation. * PB0.05, when compared to the respective normal control group of animals. A decreasing trend in lipid peroxidation values in mice may be attributed to an early increase in the activity of catalase, which seem to scavenge the hydrogen peroxide moieties produced in paraquat treated mice. The removal of the toxic hydrogen peroxide by catalase at an earlier time point makes these species unavailable to induce tissue damage in Swiss mice.
88
S. Ali et al. / Chemico-Biological Interactions 125 (2000) 79–91
Fig. 8. Effect on survival of HA-HCl pretreatment on the manifestation of paraquat toxicity in Swiss mice. Dose and treatment protocol has been described in Section 2. Results are expressed as change in survival with time. Hydroxylamine hydrochloride is a known in vivo inhibitor of catalase. Its pretreatment is shown to result in a decrease in survival in paraquat treated group of mice. The dose of paraquat injected in mice caused 10% mortality. The group of mice treated with the inhibitor alone, however, shows no mortality. Wistar rats were not treated in the same way because the activity level of catalase remained unaffected in rats treated with paraquat.
4. Discussion Species variations in the LD50 doses of PQ treated animals have been reported [22]. However, little work has been done on the comparison of the biochemical responses elicited in different species of animals. In many species including plants, PQ redox cycling leading to the concomitant generation of reactive oxygen species (ROS) has been held responsible for mediating tissue damage [10,23]. In animal tissues, a variety of mechanisms including one electron reduction of PQ to PQ radical by microsomal NADPH-dependent cytochrome c reductase catalyzed reaction may be responsible for the generation of ROS. Abstraction of electron from PQ radical by molecular oxygen further leads to the formation of superoxide anion radical and other oxidants which subsequently attack various cell constituents including unsaturated lipids and DNA [10,24]. ESR spectrophotometeric data provide direct evidence for the formation of PQ radical as a tissue damaging species [26]. Recently, Cheng et al. [25] have used transgenic mice in order to establish the role of peroxide decomposing enzyme glutathione peroxidase against paraquat lethality. Importance of PQ reduction via xanthine-xanthine oxidase system as well as by NADPH-dependent reduction has also been emphasized in rat [13]. The present paper reports that XOD contributes to the PQ toxicity in Wistar rats only; PQ does not induce XOD in Swiss mice (Fig. 1). It is further reported here that different protective biochemical responses are induced in the two animal species treated with PQ. In Swiss mice, a several fold increase in the activity catalase is seen merely 12 h after PQ administration (Fig. 2). In Wistar rats, a time dependent increase in rat pulmonary NAD(P)H-specific quinone reductase (Fig. 3)
S. Ali et al. / Chemico-Biological Interactions 125 (2000) 79–91
89
is observed with the progression of time, reaching a peak at 72 h following PQ administration. No change in the activity of catalase is observed in rats. This finding suggests that H2O2 is probably the predominant oxidant moiety generated in Swiss mice as a result of PQ toxicity. An early induction of catalase in mice may explain why mice can tolerate relatively high doses of paraquat, which are otherwise lethal to rats. The observation also provides a reasonable explanation, as to why in mice, induction of any other protective response is not required. Whereas in rats, protective response (in the form of NAD(P)H-specific quinone reductase) is induced after the XOD has contributed to the toxicity. There are reports describing changes in the antioxidant status of the tissues of animals exposed to the redox cycling compounds. In one such study, an in situ inducer of reduced glutathione, L-2-oxothiazolidine 4-carboxylate has recently been reported to protect the tissue against PQ-mediated pulmonary damage [27]. We observe different responses in the activities of the enzymes related to glutathione redox cycle in PQ treated Wistar rats and Swiss mice. Change in the activities of pulmonary glutathione reductase and of G6PD is seen in PQ treated Wistar strain of rats; activity level of these enzymes in Swiss mice remain unaffected (Figs. 4 and 5). Activity of GR, which generates reduced glutathione at the expense of NADPH, decreases with time showing a maximum inhibition at 72 h after PQ administration. However, a time dependent increase in the activity of G6PD, which recycles NADP to NADPH in order to restore the reduced status of the cell via different mechanisms including acting as a substrate for GR, is observed in Wistar rats (Fig. 5). However, Swiss mice showed no significant change in either GR or G6PD activity. Further, GST, a phase II drug metabolizing enzyme which helps in detoxification by conjugate formation, showed a marked time-dependent increase in activity in paraquat treated rats (Fig. 6). On comparison of these responses of the rat tissue with that of mice, it is clear that the two animal species are responding to PQ in different ways. It should be noted that in Swiss strain of mice, neither of the antioxidant enzymes studied in this study is induced except catalase. However, in rats, induction in the activities of quinone reductase, glutathione reductase, G6PD, and GST is observed with time. This indicates towards the activation of different sets of biochemical responses against PQ mediated pulmonary damage in the two animal species. A several-fold increase in the activity of catalase during the early hours of PQ intoxication appears to be an important factor in providing protection against PQ toxicity in Swiss strain of mice. In Wistar rats the protective response (i.e. the induction of quinone reductase) appears late at a stage where XOD has already contributed to the tissue damage as can be seen in Fig. 1. Lipid peroxidation peak is seen only after the XOD has attained its peak value (Fig. 7). These findings and aforementioned changes in enzyme activities suggest that in PQ treated Wistar strain of rats, XOD-derived ROS may be responsible for enhanced lipid peroxidation and that a different protective response against PQ is elicited in rats. However, in Swiss mice, an early induction of catalase prevents the onset of damage at an early stage by scavenging the hydrogen peroxide before they can mediate the tissue damage. A decrease in the survival is reported in Swiss mice
90
S. Ali et al. / Chemico-Biological Interactions 125 (2000) 79–91
treated with the catalase inhibitor HA-HCl 30 min prior to PQ administration (Fig. 8). Hydroxylamine hydrochloride is a known in vivo inhibitor of catalase. Decrease in survival in PQ treated Swiss mice by this inhibitor clearly provides evidence supporting the protective role of catalase in Swiss mice. In conclusion, the results presented in the study show that different pulmonary biochemical responses are induced in paraquat treated Wistar strain of rats and Swiss strain of mice. Further, it is suggested that constitutive pulmonary catalase activity levels may dictate the potency for tissue injury by paraquat only in Swiss mice.
Acknowledgements Professor M. Athar, Department of Toxicology (Jamia Hamdard) is thanked for extending some of the necessary facilities to carry out the present research work. Shaukat Naqvi of Central Animal House Facility, Jamia Hamdard is acknowledged for her timely support in providing the animals. CSIR is acknowledged for providing grant in the form of fellowship.
References [1] C. Bullivant, Accidental poisoning by paraquat. Report of two cases in man, Br. Med. J. 1 (1966) 1272–1273. [2] R.D. Fairshter, A.F. Wilson, Paraquat poisoning manifestation and therapy, Am. J. Med. 59 (1975) 751–753. [3] T.J. Halley, Review of the toxicology of paraquat, J. Clin. Toxicol. 14 (1976) 1 – 46. [4] P. Smith, D. Heath, Paraquat, CRC Crit. Rev. Toxicol. 4 (1976) 411 – 445. [5] M.S. Rose, H.C. Crabtree, K. Fletcher, I. Wyatt, Biochemical effects of diquat and paraquat. Disturbance of control of corticosteroid synthesis in rat adrenal, and subsequent effects on the control of liver glycogen utilization, Biochem. J. (BIJONA) 138 (1974) 437 – 443. [6] H.J. Forman, T.K. Aldrich, M.A. Posner, A.B. Fisher, Differential paraquat uptake and redox kinetics of rat granular pneumocytes and alveolar macrophages, J. Pharmacol. Exp. Ther. 221 (1982) 428–433. [7] A.A. Akhavein, D.I. Linscott, The dipyridilium herbicides: paraquat and diquat, Residue Rev. 22 (1968) 97–145. [8] M. Tomita, Comparison of one electron reduction activity against bipyridilium herbicides, paraquat and diquat in microsomal and mitochondrial fractions of liver, lung and kidney in vitro, Biochem. Pharmacol. 42 (1991) 303–309. [9] J.S. Bus, S.D. Aust, J.E. Gibson, Superoxide- and singlet oxygen-catalyzed lipid peroxidation as a possible mechanism for paraquat (methyl viologen) toxicity, Biochem. Biophys. Res. Commun. 58 (1974) 749–755. [10] J.S. Bus, S.D. Aust, J.E. Gibson, Lipid peroxidation: a possible mechanism for paraquat toxicity, Res. Commun. Chem. Pathol. Pharmacol. 11 (1975) 31 – 38. [11] J.F. Turrens, C. Giulivi, C. Pinus, C. Lavagno, A. Boveris, Spontaneous lung chemiluminescence upon paraquat administration, Free Radic. Biol. Med. 5 (1988) 319 – 323. [12] H.K. Fisher, J.A. Clements, D.F. Tierney, R.R. Wright, Pulmonary effects of paraquat in the first day after injection, Am. J. Physiol. 228 (1975) 1217 – 1223. [13] Y. Kitazawa, M. Matsubara, N. Takayama, T. Tanaka, The role of xanthine oxidase in paraquat intoxication, Arch. Biochem. Biophys. 288 (1991) 220 – 224.
S. Ali et al. / Chemico-Biological Interactions 125 (2000) 79–91
91
[14] F. Stirpe, E. Della Corte, The regulation of rat liver xanthine oxidase. Involvement of thiol groups in the conversion of enzyme activity from dehydrogenase (Type D) into oxidase (Type O) and purification of the enzyme, Biochem. J. 126 (1972) 739 – 745. [15] A.M. Benson, M.J. Hunkeler, P. Talalay, Increase of NAD(P)H:quinone reductase by dietary antioxidants: possible role in protection against carcinogenesis and toxicity, Proc. Natl. Acad. Sci. USA 77 (1980) 5216–5220. [16] H. Luck, Catalase, in: H.U. Bergmeyer (Ed.), Methods in Enzymatic Analysis, Academic Press, New York, 1963, pp. 885–888. [17] I. Carlberg, B. Mannervik, Purification and characterization of the flavoenzyme glutathione reductase from rat liver, J. Biol. Chem. 250 (1975) 5475 – 5480. [18] H.U. Bergmeyer, K. Gawehn, M. Grassi, Glucose 6-phosphate dehydrogenase, in: H.U. Bergmeyer (Ed.), Methods der Enzymatischen Analyse, Verlag Chemie, Weinheim, 1970, pp. 417 – 418. [19] W.H. Habig, M.J. Pabst, W.B. Jakoby, Glutathione S-transferase. The first enzymatic step in mercapturic acid formation, J. Biol. Chem. 249 (1974) 7130 – 7139. [20] F. Bernheim, M.L.C. Bernheim, K.M. Wilburn, The reaction between thiobarbituric acid and the oxidation products of certain lipids, J. Biol. Chem. 174 (1948) 257 – 264. [21] O.H. Lowry, N.J. Rosenbrough, A.L. Farr, R.J. Randall, Protein measurement with Folin phenol reagent, J. Biol. Chem. 193 (1951) 265 – 275. [22] D.G. Clark, T.F. McElligott, E.W. Hurst, The toxicity of paraquat, Br. J. Ind. Med. 23 (1966) 126–132. [23] A.D. Dodge, N. Harns, B.C. Baldwin, The mode of action of paraquat, Biochem. J. 118 (1970) 43–44. [24] S. Ali, S.K. Jain, M. Abdulla, M. Athar, Paraquat induced DNA damage by reactive oxygen species, Biochem. Mol. Biol. Int. 39 (1996) 63 – 67. [25] W.-H. Cheng, Y.-S. Ho, B.A. Valentine, A.R. Deborah, G.F. Combs Jr, X.G. Lei, Cellular peroxidase is the mediator of body selenium to protect against paraquat lethality in transgenic mice, J. Nutr. 128 (1998) 1070–1076. [26] T. Sata, E. Kubota, H.P. Misra, M. Mojarad, H. Pakbaz, S.I. Said, Paraquat-induced lung injury: prevention by N-tert-butyl-a-phenylnitrone, a free radical spin trapping agent, Am. J. Physiol. 262 (1992) L147–L152. [27] S. Ali, M. Abdulla, M. Athar, L-2-Oxothiazolidine 4-carboxylate, an in situ inducer of glutathione protects against paraquat-mediated pulmonary damage in rat, Med. Sci. Res. 24 (1996) 699 – 701.
.
www.elsevier.com/locate/chembiont
Paraquat induces different pulmonary biochemical responses in Wistar rats and Swiss mice S. Ali *, G. Diwakar, S. Pawa Department of Biochemistry, Faculty of Science, Jamia Hamdard (Hamdard Uni6ersity), New Delhi – 1100 62, India Received 4 August 1999; accepted 19 December 1999
Abstract The paper presents results showing differential response to paraquat toxicity in Wistar rats and Swiss strain of mice. Paraquat-induced pulmonary biochemical responses in the two animal species were studied at different time point after giving a single intraperitoneal injection of the respective LD10 doses of the herbicide paraquat to rats and mice. Paraquat induced different biochemical responses including different protective responses in the two animal species. As a protective response, NADPH-specific quinone reductase is induced in rats, while catalase is induced in mice. It is implied that an early induction of catalase in mice as opposed to rats may account for the resistance of Swiss mice to paraquat toxicity. Xanthine oxidase, which was induced in rats, remains unaffected in mice indicating that the enzyme contributes to paraquat toxicity only in Wistar rats. Time-course studies were also conducted to compare the differential responses of antioxidant enzymes and lipid peroxidation between the two species. The results of the study led us to suggest that the manifestation of paraquat toxicity involve distinct differences in early pulmonary biochemical responses in Wistar rats and Swiss mice. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. Keywords: Paraquat; Pulmonary biochemical responses; Wistar rats; Swiss mice
* Corresponding author. Fax: + 91-11-6988874. E-mail address: [email protected] (S. Ali) 0009-2797/00/$ - see front matter © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 0 9 - 2 7 9 7 ( 9 9 ) 0 0 1 6 7 - 2
80
S. Ali et al. / Chemico-Biological Interactions 125 (2000) 79–91
1. Introduction The herbicide paraquat (1,1%-dimethyl 4,4%-bipyridilium dichloride; PQ) facilitates the agricultural yields by killing weeds worldwide. First report of PQ poisoning in human appeared in 1966 [1]. Since then, a number of deaths have been reported as a result of occupational, accidental or suicidal ingestion of PQ. Although liver, kidney, heart and central nervous system are affected, irreversible lung damage is the usual cause of death [2–4]. Lungs appear to be the target organ as PQ selectively accumulates in type 11 pneumocytes by an active polyamine uptake process [5,6]. Saturation kinetics of PQ uptake by the human lung are very similar to that of rat lung with a Vmax of about 300 nmol accumulated/g lung per h, and a Km of about 4× 10 − 5 mol/l. The mechanism of PQ toxicity in various species has been investigated and is attributed to its redox cycling nature. Due to a lower redox potential of − 446 mV [7], PQ is easily reduce by one electron to form a relatively stable and toxic radical which can be re-oxidized back to its parent cationic form. One electron reduction activity of paraquat in subcellular fractions of various tissues in vitro has been compared using microsomal fraction [8]. Reduced PQ radical is immediately re-oxidized by molecular oxygen, resulting in the formation of superoxide anion radical which dismutates to hydrogen peroxide, thus initiating the process of tissue damage [9,10]. Beside this, polymorphonuclear cell migration and activation [11], NADPH-oxidation [12], and xanthine oxidase induction [13] have also been reported to contribute to PQ toxicity in different animal species. The results of the present study show that different pulmonary biochemical responses are elicited in Wistar rats and Swiss mice. Distinct differences in the induction of protective responses, antioxidant enzyme activity level and lipid peroxidation in the PQ treated rats and mice indicate toward the need for finding an animal species which show biochemical responses similar to those elicited in human tissue exposed to paraquat. This is expected to help produce a suitable animal model, which can be utilized for the development of an antidote against PQ.
2. Materials and methods
2.1. Materials Paraquat dichloride, nicotinamide adenine dinucleotide (NADPH) and flavin adenine dinucleotide (FAD) were purchased from Sigma (St Louis, MO, USA). All other chemicals were of highest purity grade commercially available in India. Specific activities of all the enzymes measured in this study were determined by monitoring the change in absorbence at their respective wavelength using a Milton Roy, Spectronic 21 D spectrophotometer.
S. Ali et al. / Chemico-Biological Interactions 125 (2000) 79–91
81
2.2. Treatment of animals Experiments were conducted on male Wistar rats (1209 15 g body weight) and male Swiss mice (22 9 2 g body weight). At these body weights the animals were 5–6 weeks old, and were in the similar developmental stage. All the animals were procured from Central Animal House Facility of Jamia Hamdard (New Delhi, India) and were kept in plastic cages in an environmentally controlled room, with each group containing six animals. Animals had free access to pellet diet (Lipton India Ltd) and water ad libitum. Animals were given a single intraperitoneal injection of paraquat. Respective LD10 dose of PQ was given to Wistar rats and Swiss mice; 30 mg/kg body weight to mice, and 20 mg/kg body weight to rats. The LD10 doses in the two animal species were determined by using probit test. The LD10 doses were chosen in order to have consistency in the doses used in the two animal species. The doses were given in such a way that the biochemical estimations could be performed at 12, 36 or 72 h interval after PQ administration. It was ensured that all the animals be treated in such a way that the time of killing should not differ in each case. The group of animals treated with saline served as control. Lungs were removed en bloc (i.e. along with the heart) immediately after killing. The lung tissue was then separated from the heart and washed in ice-cold saline solution. Tissue was blotted and weighed before subjecting it to homogenization.
2.3. Subcellular fractionation Accurately weighed pieces of tissue were minced and homogenized in nine volumes of ice cold 0.1 M phosphate buffer (pH 7.4), containing 1.15% KCl using a polytron homogenizer, giving five to six strokes at a speed of 2000 rpm for 30 s. The resulting 10% homogenate was subjected to differential centrifugation, first at a speed of 800×g for 10 min (to remove nuclei and unbroken cells) and then at 9500 × g for 20 min to get the post mitochondrial supernatant (PMS), which was used as a crude source of enzymes for assay purpose. Lipid peroxidation was measured using total lung homogenate. All subsequent operations were carried out at 4°C.
2.4. Biochemical estimations Measurement of the activity of xanthine oxidase (XOD) was based on the procedure described by Stirpe and Della Corte [14]. Reaction mixture consisting of 0.1 mM xanthine and 0.5 M Tris–HCl buffer (pH 8.1) was incubated with appropriate amount of enzyme source for 20 min at 37°C. The reaction was terminated by precipitating the enzyme using 10% perchloric acid. The mixture was then centrifuged at 4000 rpm for 10 min. Uric acid in the clear supernatant was determined at 290 nm. Results are expressed as mg of uric acid/mg protein. NAD(P)H:quinone reductase (QR) activity was determined according to the method of Ernster as modified by Benson et al. [15]. Reaction mixture in a volume
82
S. Ali et al. / Chemico-Biological Interactions 125 (2000) 79–91
of 3 ml consisted of 25 mM Tris –HCl (pH 7.4), 0.7 mg of crystalline bovine serum albumin, 0.01% Tween-20 (v/v), 5 mM flavin adenine nucleotide, 0.2 mM NADPH and an appropriate aliquot of the enzyme source. Electron acceptor dichlorophenol indophenol (DCPIP; 40 mM) in 20 ml of water was added to initiate the reaction and the initial velocity of the reduction of DCPIP was measured spectrophotometerically at 600 nm using a molar extinction coefficient of 2.1× 104 M − 1 cm − 1. Catalase activity was measured at 230 nm [16]. In brief, 50 ml of enzyme source was diluted to 2 ml with 50 mM potassium phosphate buffer at pH 7. The reaction was initiated in a quartz cuvette by addition of 1 ml of 0.34% H2O2 (v/v). Specific activity of the enzyme was calculated using a molar extinction coefficient of 81 M − 1 cm − 1. Results are expressed as mmol of H2O2 decomposed/min per mg protein. Glutathione reductase (GR) activity was measured according to the method described by Carlberg and Mannervik [17]. Reaction mixture consisted of 0.1 M phosphate buffer (pH 7.6), 0.1 mM NADPH, 0.5 mM ethylene diamine tetra acetate, and 1 mM glutathione (oxidized). To initiate the reaction, 50 ml of enzyme source was added. The oxidation of NADPH was measured at 340 nm. For calculations, an extinction coefficient of 6.3 M − 1 cm − 1 was used. Results are expressed as nmol NADPH oxidized/min per mg protein. Glucose 6-phosphate dehydrogenase (G6PD) activity was determined by the method described by Bergmeyer et al. [18]. Reduction of NADP to NADPH was followed at 340 nm using a molar extinction coefficient of 6.3 M − 1 cm − 1. Results are expressed as nmol of NADP reduced/min per mg protein. Glutathione S-transferase activity measurement was based on the spectrophotometeric determination of 1-chloro-2,4-dinitrobenzene (CDNB)-conjugate formed in a reduced glutathione (GSH) coupled reaction [19]. The reaction was initiated by adding 10 mM CDNB (in acetone) to a reaction mixture containing 1 mM GSH and 20 ml of PMS in 0.1 M phosphate buffer. A molar extinction coefficient of 9.6 M − 1 cm − 1 at 340 nm was used to calculate the specific activity. Malondialdehyde was measured as an index of oxidative tissue damage. It was measured as thiobarbituric acid reacting substances in the total lung homogenate [20]. Since the absorbence of the treated/control samples was less than 0.025 at time 0, the samples were incubated for 30 min in a water bath at 37°C to obtain measurable absorption values. The samples were then precipitated in a 1:1 (v/v) ratio with 20% trichloroacetic acid (w/v). The mixture was centrifuged at 3000 rpm for 10 min, and then the supernatant was heated with 0.67% thiobarbituric acid (w/v) at 100°C for 20 min in the presence of 100 mM butylhydroxyanisol to avoid further cleavage of hydroperoxides. After cooling, the absorbence of the supernatant was measured at 432 nm. Protein content was determined by the method of Lowry et al. [21] using bovine serum albumin as reference standard. The data are expressed as mean 9 S.E. of six animals. Values obtained in each experimental group of animals were individually compared with the respective normal control group using Student’s t-test.
S. Ali et al. / Chemico-Biological Interactions 125 (2000) 79–91
83
2.5. Sur6i6al studies Effect of catalase inhibitor hydroxylamine hydrochloride (HA-HCl) on the survival in PQ treated Swiss mice was studied. Mice were administered with HA-HCl (70 mg/kg body weight, i.p.) 30 min before injecting the LD10 dose of PQ. PQ alone, or HA-HCl alone treated group of mice served as positive controls. Each group consisted of ten mice. Survival in each group was monitored every 12 h for 6 days.
3. Results
3.1. Pulmonary XOD acti6ity in PQ treated Swiss mice and Wistar rats XOD contributes to tissue damage by generating oxygen free radicals as byproduct while catalyzing its reaction. As shown in Fig. 1, an increase in the activity of XOD is seen in rats sacrificed 36 h after PQ administration. When compared with the untreated group, the increase was 26% more in the PQ treated group of rats. However, in mice killed at the same time point no change in XOD activity is observed.
3.2. Pulmonary catalase and NAD(P)H-quinone reductase acti6ity in PQ treated animals Catalase and NAD(P)H:quinone reductase are the two defense system enzymes, which were studied in the present investigation. A marked increase in the activity of catalase with a peak at 12 h after PQ administration is observed in Swiss mice (Fig.
Fig. 1. Effect of paraquat on pulmonary XOD activity in mice and rats sacrificed 36 h after a single injection of the toxicant to each animal. Dose and the treatment protocol have been described in Section 2. Each value represents mean 9S.E. of six animals. *PB 0.05, when compared with the respective saline treated control group of animals. A significant increase in the activity of XOD suggests that the enzyme contributes to tissue damage only in Wistar rats.
84
S. Ali et al. / Chemico-Biological Interactions 125 (2000) 79–91
Fig. 2. Effect of paraquat on pulmonary catalase activity in Swiss mice and Wistar rats sacrificed at different time points after a single injection of the toxicant to each animal. Dose and the treatment protocol have been described in Section 2. Each value represents mean 9S.E. of six animals. *P B0.05, when compared with the respective saline treated control group of animals. Catalase is induced in paraquat treated Swiss mice only whereas the enzyme activity in rats remains unaffected.
2), while NAD(P)H:quinone reductase is induced in Wistar strain of rats with a peak value seen 72 h following the administration of PQ (Fig. 3). Pulmonary catalase activity in mice and rats was also studied at an earlier time point than 12 h. The results were consistent with the changes seen at 12 h time point; rats did not show any change in enzyme activity, while the activity in mice induced. The results are, however, not shown in order to keep consistency with the other enzymes, which were measured at 12-h time point onward.
Fig. 3. Effect of paraquat on pulmonary NAD(P)H:quinone reductase activity in Swiss mice and Wistar rats sacrificed at different time points after a single injection of the toxicant to each animal. Dose and the treatment protocol have been as described in Section 2. Each value represents mean 9S.E. (n =6). *P B 0.05, when compared with the respective saline treated control group of animals. The enzyme is induced in rats only and reaches a peak 3 days after the treatment. Mice show no change in enzyme activity.
S. Ali et al. / Chemico-Biological Interactions 125 (2000) 79–91
85
Fig. 4. Effect of paraquat on the activity of pulmonary glutathione reductase in Swiss mice and Wistar rats sacrificed at different time points after a single injection of the toxicant to each animal. Dose and the treatment protocol have been described in Section 2. Each value represents mean 9S.E. (n =6). *P B 0.05, when compared with the respective saline treated control group of animals. The enzyme activity is inhibited in rats whereas in mice the specific activity remains unaffected.
3.3. Acti6ities of GR, G6PD and glutathione S-transferase (GST) in PQ treated mice and rats The results of the effect of PQ on the activity of glutathione redox cycle enzyme GR in Swiss mice and Wistar rats are shown in Fig. 4. GR is involved in the regeneration of reduced glutathione from oxidized glutathione at the expense of NADPH. In Swiss mice, the activity of pulmonary GR remains unaffected. However, a decrease in GR activity with time is shown in Wistar rats. Since G6PD recycles NADP to NADPH, it was thought that the activity of G6PD would be perturbed only in Wistar rats. We observed a time-dependent increase in the activity of glucose-6 phosphate dehydrogenase in PQ treated Wistar rats reaching a maximum at 72 h (Fig. 5). As expected G6PD remained unperturbed in Swiss mice. The activity of a phase II drug metabolizing enzyme glutathione S-transferase (GST) was also determined in the two species of animals treated with PQ (Fig. 6). GST helps in detoxification by conjugate formation. In rats, GST activity level increased to its maximum by the 36th hour and then remains unchanged at this peak value in the 72-h group also. In mice, the activity could only be affected significantly (decrease) at 72 h time point.
3.4. Lipid peroxidation in PQ treated Swiss mice and Wistar rats The lipid peroxidation profile in PQ treated rats and mice is shown in Fig. 7. A significant increase is observed 72 h after the treatment. Before this time point, lipid peroxidation value does not differ from the control value. Interestingly, the results can be related to the activity of XOD, which show maximum induction following
86
S. Ali et al. / Chemico-Biological Interactions 125 (2000) 79–91
Fig. 5. Effect of paraquat on the activity of pulmonary G6PD in paraquat treated Swiss mice and Wistar rats. Animals were sacrificed at different time points after a single injection of the toxicant to each animal. Dose and the treatment protocol have been described in Section 2. Each value represents mean9 S.E. (n= 6). *PB 0.05, when compared with the respective saline treated control group of animals. Enzyme activity is induced in Wistar rats, whereas the G6PD measured in the tissue of Swiss mice shows no change when compared with the respective normal values.
36 h after PQ intoxication. This can be explained assuming that the oxidants generated as byproduct in XOD-catalyzed reaction damage the unsaturated lipids present in the cell. PQ treated mice, however, did not exhibit increase in lipid peroxidation (Table 1). Rather a decreasing trend in the lipid peroxidation is observed with time in mice. This may be attributed to an early increase in the activity of catalase, which seems to scavenge the hydrogen peroxide in PQ treated Swiss mice; thus making the hydrogen peroxide unavailable to induce tissue damage in mice.
Fig. 6. Changes in the activity of pulmonary GST in paraquat treated Swiss mice and Wistar rats. Animals were sacrificed at different time points after a single injection of the toxicant to each animal. Dose and the treatment protocol were the same as described in Section 2. Each value represents mean9 S.E. (n= 6). *PB 0.05, when compared with the respective saline treated control group of animals.
S. Ali et al. / Chemico-Biological Interactions 125 (2000) 79–91
87
Fig. 7. Comparison of changes in pulmonary lipid peroxidation and the activity of XOD in paraquat treated Wistar rats. Estimations were performed 12, 36, or 72 h after a single injection of the toxicant. Dose and treatment protocol has been described in Section 2. For comparison, values were calculated as % change with respect to the saline treated control group. Lipid peroxidation increases 72 h after paraquat treatment, i.e. only after the XOD has contributed to the toxicity of paraquat. Note the XOD peak is seen 36 h after paraquat treatment.
3.5. Effect on sur6i6al of catalase inhibitor in PQ treated Swiss mice Administration of HA-HCl, an in situ inhibitor of catalase, 30 min before the administration of PQ significantly affect survival in Swiss mice. When compared with the PQ alone treated group, a 40% decrease in survival was observed in HA-HCl pretreated group of mice (Fig. 8). This clearly suggests that induction of catalase constitute an important protective response against PQ-induced oxidative damage in mice. The experiment with the inhibitor was not performed with rats because the activity level of catalase remained unaffected in PQ treated Wistar rats. Table 1 Lipid peroxidation in the lungs of paraquat treated Wistar rats and Swiss albino strain of micea The nmol of malondialdehyde/mg protein Group
Swiss mice
Wistar rats
Control 12 h 36 h 72 h
13.39 1.27 12.89 1.23 10.99 0.89 09.39 2.3*
10.2 9 0.96 08.4 9 0.85 09.99 0.57 14.1 90.45*
a Dose and the treatment protocol have been described in Section 2. Animals were sacrificed at 12, 36, or 72 h after paraquat treatment and the whole tissue homogenate was used for the determination of lipid peroxidation. * PB0.05, when compared to the respective normal control group of animals. A decreasing trend in lipid peroxidation values in mice may be attributed to an early increase in the activity of catalase, which seem to scavenge the hydrogen peroxide moieties produced in paraquat treated mice. The removal of the toxic hydrogen peroxide by catalase at an earlier time point makes these species unavailable to induce tissue damage in Swiss mice.
88
S. Ali et al. / Chemico-Biological Interactions 125 (2000) 79–91
Fig. 8. Effect on survival of HA-HCl pretreatment on the manifestation of paraquat toxicity in Swiss mice. Dose and treatment protocol has been described in Section 2. Results are expressed as change in survival with time. Hydroxylamine hydrochloride is a known in vivo inhibitor of catalase. Its pretreatment is shown to result in a decrease in survival in paraquat treated group of mice. The dose of paraquat injected in mice caused 10% mortality. The group of mice treated with the inhibitor alone, however, shows no mortality. Wistar rats were not treated in the same way because the activity level of catalase remained unaffected in rats treated with paraquat.
4. Discussion Species variations in the LD50 doses of PQ treated animals have been reported [22]. However, little work has been done on the comparison of the biochemical responses elicited in different species of animals. In many species including plants, PQ redox cycling leading to the concomitant generation of reactive oxygen species (ROS) has been held responsible for mediating tissue damage [10,23]. In animal tissues, a variety of mechanisms including one electron reduction of PQ to PQ radical by microsomal NADPH-dependent cytochrome c reductase catalyzed reaction may be responsible for the generation of ROS. Abstraction of electron from PQ radical by molecular oxygen further leads to the formation of superoxide anion radical and other oxidants which subsequently attack various cell constituents including unsaturated lipids and DNA [10,24]. ESR spectrophotometeric data provide direct evidence for the formation of PQ radical as a tissue damaging species [26]. Recently, Cheng et al. [25] have used transgenic mice in order to establish the role of peroxide decomposing enzyme glutathione peroxidase against paraquat lethality. Importance of PQ reduction via xanthine-xanthine oxidase system as well as by NADPH-dependent reduction has also been emphasized in rat [13]. The present paper reports that XOD contributes to the PQ toxicity in Wistar rats only; PQ does not induce XOD in Swiss mice (Fig. 1). It is further reported here that different protective biochemical responses are induced in the two animal species treated with PQ. In Swiss mice, a several fold increase in the activity catalase is seen merely 12 h after PQ administration (Fig. 2). In Wistar rats, a time dependent increase in rat pulmonary NAD(P)H-specific quinone reductase (Fig. 3)
S. Ali et al. / Chemico-Biological Interactions 125 (2000) 79–91
89
is observed with the progression of time, reaching a peak at 72 h following PQ administration. No change in the activity of catalase is observed in rats. This finding suggests that H2O2 is probably the predominant oxidant moiety generated in Swiss mice as a result of PQ toxicity. An early induction of catalase in mice may explain why mice can tolerate relatively high doses of paraquat, which are otherwise lethal to rats. The observation also provides a reasonable explanation, as to why in mice, induction of any other protective response is not required. Whereas in rats, protective response (in the form of NAD(P)H-specific quinone reductase) is induced after the XOD has contributed to the toxicity. There are reports describing changes in the antioxidant status of the tissues of animals exposed to the redox cycling compounds. In one such study, an in situ inducer of reduced glutathione, L-2-oxothiazolidine 4-carboxylate has recently been reported to protect the tissue against PQ-mediated pulmonary damage [27]. We observe different responses in the activities of the enzymes related to glutathione redox cycle in PQ treated Wistar rats and Swiss mice. Change in the activities of pulmonary glutathione reductase and of G6PD is seen in PQ treated Wistar strain of rats; activity level of these enzymes in Swiss mice remain unaffected (Figs. 4 and 5). Activity of GR, which generates reduced glutathione at the expense of NADPH, decreases with time showing a maximum inhibition at 72 h after PQ administration. However, a time dependent increase in the activity of G6PD, which recycles NADP to NADPH in order to restore the reduced status of the cell via different mechanisms including acting as a substrate for GR, is observed in Wistar rats (Fig. 5). However, Swiss mice showed no significant change in either GR or G6PD activity. Further, GST, a phase II drug metabolizing enzyme which helps in detoxification by conjugate formation, showed a marked time-dependent increase in activity in paraquat treated rats (Fig. 6). On comparison of these responses of the rat tissue with that of mice, it is clear that the two animal species are responding to PQ in different ways. It should be noted that in Swiss strain of mice, neither of the antioxidant enzymes studied in this study is induced except catalase. However, in rats, induction in the activities of quinone reductase, glutathione reductase, G6PD, and GST is observed with time. This indicates towards the activation of different sets of biochemical responses against PQ mediated pulmonary damage in the two animal species. A several-fold increase in the activity of catalase during the early hours of PQ intoxication appears to be an important factor in providing protection against PQ toxicity in Swiss strain of mice. In Wistar rats the protective response (i.e. the induction of quinone reductase) appears late at a stage where XOD has already contributed to the tissue damage as can be seen in Fig. 1. Lipid peroxidation peak is seen only after the XOD has attained its peak value (Fig. 7). These findings and aforementioned changes in enzyme activities suggest that in PQ treated Wistar strain of rats, XOD-derived ROS may be responsible for enhanced lipid peroxidation and that a different protective response against PQ is elicited in rats. However, in Swiss mice, an early induction of catalase prevents the onset of damage at an early stage by scavenging the hydrogen peroxide before they can mediate the tissue damage. A decrease in the survival is reported in Swiss mice
90
S. Ali et al. / Chemico-Biological Interactions 125 (2000) 79–91
treated with the catalase inhibitor HA-HCl 30 min prior to PQ administration (Fig. 8). Hydroxylamine hydrochloride is a known in vivo inhibitor of catalase. Decrease in survival in PQ treated Swiss mice by this inhibitor clearly provides evidence supporting the protective role of catalase in Swiss mice. In conclusion, the results presented in the study show that different pulmonary biochemical responses are induced in paraquat treated Wistar strain of rats and Swiss strain of mice. Further, it is suggested that constitutive pulmonary catalase activity levels may dictate the potency for tissue injury by paraquat only in Swiss mice.
Acknowledgements Professor M. Athar, Department of Toxicology (Jamia Hamdard) is thanked for extending some of the necessary facilities to carry out the present research work. Shaukat Naqvi of Central Animal House Facility, Jamia Hamdard is acknowledged for her timely support in providing the animals. CSIR is acknowledged for providing grant in the form of fellowship.
References [1] C. Bullivant, Accidental poisoning by paraquat. Report of two cases in man, Br. Med. J. 1 (1966) 1272–1273. [2] R.D. Fairshter, A.F. Wilson, Paraquat poisoning manifestation and therapy, Am. J. Med. 59 (1975) 751–753. [3] T.J. Halley, Review of the toxicology of paraquat, J. Clin. Toxicol. 14 (1976) 1 – 46. [4] P. Smith, D. Heath, Paraquat, CRC Crit. Rev. Toxicol. 4 (1976) 411 – 445. [5] M.S. Rose, H.C. Crabtree, K. Fletcher, I. Wyatt, Biochemical effects of diquat and paraquat. Disturbance of control of corticosteroid synthesis in rat adrenal, and subsequent effects on the control of liver glycogen utilization, Biochem. J. (BIJONA) 138 (1974) 437 – 443. [6] H.J. Forman, T.K. Aldrich, M.A. Posner, A.B. Fisher, Differential paraquat uptake and redox kinetics of rat granular pneumocytes and alveolar macrophages, J. Pharmacol. Exp. Ther. 221 (1982) 428–433. [7] A.A. Akhavein, D.I. Linscott, The dipyridilium herbicides: paraquat and diquat, Residue Rev. 22 (1968) 97–145. [8] M. Tomita, Comparison of one electron reduction activity against bipyridilium herbicides, paraquat and diquat in microsomal and mitochondrial fractions of liver, lung and kidney in vitro, Biochem. Pharmacol. 42 (1991) 303–309. [9] J.S. Bus, S.D. Aust, J.E. Gibson, Superoxide- and singlet oxygen-catalyzed lipid peroxidation as a possible mechanism for paraquat (methyl viologen) toxicity, Biochem. Biophys. Res. Commun. 58 (1974) 749–755. [10] J.S. Bus, S.D. Aust, J.E. Gibson, Lipid peroxidation: a possible mechanism for paraquat toxicity, Res. Commun. Chem. Pathol. Pharmacol. 11 (1975) 31 – 38. [11] J.F. Turrens, C. Giulivi, C. Pinus, C. Lavagno, A. Boveris, Spontaneous lung chemiluminescence upon paraquat administration, Free Radic. Biol. Med. 5 (1988) 319 – 323. [12] H.K. Fisher, J.A. Clements, D.F. Tierney, R.R. Wright, Pulmonary effects of paraquat in the first day after injection, Am. J. Physiol. 228 (1975) 1217 – 1223. [13] Y. Kitazawa, M. Matsubara, N. Takayama, T. Tanaka, The role of xanthine oxidase in paraquat intoxication, Arch. Biochem. Biophys. 288 (1991) 220 – 224.
S. Ali et al. / Chemico-Biological Interactions 125 (2000) 79–91
91
[14] F. Stirpe, E. Della Corte, The regulation of rat liver xanthine oxidase. Involvement of thiol groups in the conversion of enzyme activity from dehydrogenase (Type D) into oxidase (Type O) and purification of the enzyme, Biochem. J. 126 (1972) 739 – 745. [15] A.M. Benson, M.J. Hunkeler, P. Talalay, Increase of NAD(P)H:quinone reductase by dietary antioxidants: possible role in protection against carcinogenesis and toxicity, Proc. Natl. Acad. Sci. USA 77 (1980) 5216–5220. [16] H. Luck, Catalase, in: H.U. Bergmeyer (Ed.), Methods in Enzymatic Analysis, Academic Press, New York, 1963, pp. 885–888. [17] I. Carlberg, B. Mannervik, Purification and characterization of the flavoenzyme glutathione reductase from rat liver, J. Biol. Chem. 250 (1975) 5475 – 5480. [18] H.U. Bergmeyer, K. Gawehn, M. Grassi, Glucose 6-phosphate dehydrogenase, in: H.U. Bergmeyer (Ed.), Methods der Enzymatischen Analyse, Verlag Chemie, Weinheim, 1970, pp. 417 – 418. [19] W.H. Habig, M.J. Pabst, W.B. Jakoby, Glutathione S-transferase. The first enzymatic step in mercapturic acid formation, J. Biol. Chem. 249 (1974) 7130 – 7139. [20] F. Bernheim, M.L.C. Bernheim, K.M. Wilburn, The reaction between thiobarbituric acid and the oxidation products of certain lipids, J. Biol. Chem. 174 (1948) 257 – 264. [21] O.H. Lowry, N.J. Rosenbrough, A.L. Farr, R.J. Randall, Protein measurement with Folin phenol reagent, J. Biol. Chem. 193 (1951) 265 – 275. [22] D.G. Clark, T.F. McElligott, E.W. Hurst, The toxicity of paraquat, Br. J. Ind. Med. 23 (1966) 126–132. [23] A.D. Dodge, N. Harns, B.C. Baldwin, The mode of action of paraquat, Biochem. J. 118 (1970) 43–44. [24] S. Ali, S.K. Jain, M. Abdulla, M. Athar, Paraquat induced DNA damage by reactive oxygen species, Biochem. Mol. Biol. Int. 39 (1996) 63 – 67. [25] W.-H. Cheng, Y.-S. Ho, B.A. Valentine, A.R. Deborah, G.F. Combs Jr, X.G. Lei, Cellular peroxidase is the mediator of body selenium to protect against paraquat lethality in transgenic mice, J. Nutr. 128 (1998) 1070–1076. [26] T. Sata, E. Kubota, H.P. Misra, M. Mojarad, H. Pakbaz, S.I. Said, Paraquat-induced lung injury: prevention by N-tert-butyl-a-phenylnitrone, a free radical spin trapping agent, Am. J. Physiol. 262 (1992) L147–L152. [27] S. Ali, M. Abdulla, M. Athar, L-2-Oxothiazolidine 4-carboxylate, an in situ inducer of glutathione protects against paraquat-mediated pulmonary damage in rat, Med. Sci. Res. 24 (1996) 699 – 701.
.