- Email: [email protected]
The effects of diazinon on pancreatic damage and ameliorating role of vitamin E and vitamin C
PESTICIDE Biochemistry & Physiology
Pesticide Biochemistry and Physiology 81 (2005) 123–128 www.elsevier.com/locate/ypest
The effects of diazinon on pancreatic damage and ameliorating role of vitamin E and vitamin C Osman Gokalpa,*, Bora Buyukvanlıb, Ekrem Ciceka, Mehmet Kaya Ozera, Ahmet Koyub, Irfan Altuntasc, Halis Koylub a
Department of Pharmacology, School of Medicine, Suleyman Demirel University, 32260 Isparta, Turkey Department of Physiology, School of Medicine, Suleyman Demirel University, 32260 Isparta, Turkey Department of Biochemistry and Clinical Biochemistry, School of Medicine, Suleyman Demirel University, 32260 Isparta, Turkey b
c
Received 27 September 2004; accepted 1 November 2004
Abstract The aim of this study was to investigate effects of an organophosphate insecticide, diazinon (DI), on pancreas, and possible ameliorating role of vitamins E and C. We examined both in vivo and in vitro effects of DI on serum activities of alkaline phosphatase (ALP), c-glutamyltransferase (GGT), amylase, and lipase enzymes. We also evaluated possible ameliorating effects of vitamins E and C combination against DI toxicity and blood levels of thiobarbituric acid reactive substances (TBARS) only in vivo. In vivo experimental groups were: control group, DI-treated group, and DI + vitamins E plus C-treated group. In vitro study groups were: control group and DI-treated group. The biochemical analyses were determined in in vitro experiments at both hour 0 and 24 while in in vivo experiments were determined only at hour 24. Lipase activity and TBARS level were found increased by DI in in vivo experiments while lipase activity was found decreased in in vitro experiments. Amylase and ALP activities were found decreased by DI in both in vivo and in vitro experiments. Also, the combination of vitamins E and C was found to partially improve these disorders. These results suggest that DI treatment causes pancreas damage via increasing oxidative stress in rats, and a combination vitamins E and C can reduce this lipoperoxidative effect. 2004 Elsevier Inc. All rights reserved. Keywords: Organophosphate; Pancreatitis; Diazinon; Vitamin E; Vitamin C
1. Introduction
*
Corresponding author. Fax: +90 246 237 11 65. E-mail address: [email protected] (O. Gokalp).
Various complications can be seen in organophosphate insecticide (OPI) intoxication cases [1– 4]. OPI compounds are so various, however, only parathion, malathion, diphonate, coumaphos,
0048-3575/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2004.11.001
124
O. Gokalp et al. / Pesticide Biochemistry and Physiology 81 (2005) 123–128
diazinon (DI), mevinphos, and dimethoate have been reported as acute pancreatitis causes in the literature (approximately 12% rate) [1,2]. It has also been reported that when dimethoate was absorbed by skin, it could cause pancreatitis [1]. Although pancreatitis incidence is high in OPI intoxications, there are few articles regarding this issue in the literature, most of which are related to case reports on pathophysiological aspect. It has been reported that DI is one of the most pronounced OPIs responsible for OPI-induced pancreatitis [1,5–11]. It has been thought that OPIs could cause acute pancreatitis via increased pressure in pancreas and its canals due to the excessive cholinergic stimulation [1]. In previous studies, we have found that OPIs have caused lipid peroxidation in kidney, liver, and erythrocytes, increment in lactate dehydrogenase leaking to extracellular fluid [12–16]. It has also been indicated that the reactive oxygen types and/or free radicals produced by DI have given rise to these toxic effects [12]. Also we found that vitamins E and C combination therapy could decrease lipid peroxidation caused by various OPIs [13,15,17]. Furthermore, in the other two studies, we found that both fenthion and methidathion altered activities of the pancreatic enzymes, and these changes were partially prevented by vitamins E and C [18,19]. The precise molecular mechanisms of OPI-induced acute pancreatitis are still undefined. They may involve obstruction of pancreatic ducts, and/or enhancement of reactive oxygen species [5,20]. Therefore, we aimed to investigate DI mediated acute pancreatitis by means of serum amylase, lipase activities, pancreatic duct obstruction by means of serum alkaline phosphatase (ALP), c-glutamyltransferase (GGT) activities, increment oxidative stress by thiobarbituric acid reactive substances (TBARS) level and, to determine possible protective effects of vitamins E and C combination therapy against DI toxicity.
2. Materials and methods 2.1. In vivo study: animals and treatment Twenty Wistar albino rats weighing between 200 and 290 g were divided into three experimental
groups: control group (n = 8), DI-treated group (DI, n = 6) and DI + vitamin E + vitamin C-treated group (DI + Vit, n = 6). DI and DI + Vit groups were treated orally with a single dose of 335 mg/kg body weight of DI (0.25 LD50) (Basudin; Syngenta, Turkey) [21] in corn oil at 0 h. Only corn oil was given in the same way to the control goup. Vitamin E as a-tocopherol acetate (Evigen; Aksu Farma, Istanbul, Turkey) and vitamin C as sodium-L -ascorbate (Redoxon; Roche, Basel, Switzerland) were injected at doses of 150 mg/kg body weight i.m. [22] and 200 mg/kg body weight i.p. [22], respectively, 30 min after the treatment of DI in DI + Vit group. Equal amounts of physiologic saline instead of vitamins were given to the rats of control and DI groups. After all the rats received the above treatments they were fed ad libitum until midnight. The animals were starved overnight for 12 h before the blood was collected. Rats were anaesthetized with ether and venous blood samples were collected by direct heart puncture at 24 h. Blood samples were centrifuged and serum was discarded. We hereby declare that the experiments reported here comply with the current laws and regulations of the Turkish Republic on the care and handling of experimental animals. 2.2. In vitro study Effects of various concentrations of DI on the activities of amylase, lipase, GGT, ALP, and cholinesterase (ChE) in sera were studied. A 10-ml venous blood sample was obtained from each of seven volunteers (four male, three female). Blood samples were centrifuged and serum discarded. The activities of serum enzymes were determined in each sample and these served as 0 h. Each sample was divided into five portions and each one served as experimental groups, as follows: control group and DI-treated groups. DI was dissolved in water at concentrations ranging from 0.0, 0.0033, 0.033, 0.33, 3.3, and 33 mM [22]. Each DI concentration was incubated with previously prepared sera at +4 C for 24 h. In control group, physiologic saline was used instead of DI. The activities of serum enzymes were determined in each sample at 24 h.
O. Gokalp et al. / Pesticide Biochemistry and Physiology 81 (2005) 123–128
2.3. Biochemical parameters An autoanalyser, Abbott Aeroset (IL, USA), was used to determine the serum activities of ChE, amylase, lipase, ALP, and GGT. TBARS was determined by the double heating method of Draper and Hadley [23]. The principle of the method was spectrophotometric measurement of the colour produced during the reaction to thiobarbituric acid (TBA) with malondialdehyde (MDA). For this purpose, 2.5 ml of 100 g/L trichloroacetic acid (TCA) solution was added to 0.5 ml serum in a centrifuge tube and placed in a boiling water bath for 15 min. After cooling in tap water, the mixture was centrifuged at 1000g for 10 min, and 2 ml of the supernatant was added to 1 ml of 6.7 g/L TBA solution in a test tube and placed in a boiling water bath for 15 min. The solution was then cooled in tap water and its absorbance was measured using a Shimadzu UV1601 spectrophotometer (Japan) at 532 nm. The concentration of TBARS was calculated by the absorbance coefficient of MDA–TBA complex 1.56 · 105 cm 1 M 1 and expressed in nmol/ml. 2.4. Statistical evaluation For statistical analyses of in vivo results, normality was first investigated, and it was shown that some values of parameters did not fit to the normal distribution. Therefore, as stated by Dawson– Saunders and Trapp considering the small number of cases, non-parametric Kruskal–Wallis test and Mann–Whitney U test were used to compare the groups [24]. The p values smaller than 0.05 (p < 0.05) were accepted as statistically meaningful.
125
For statistical analyses of in vitro results, first the degree of normality was investigated, and it was found that the values of the parameters fitted a normal distribution. Therefore, ANOVA test was performed to demonstrate the significance of experimental treatments.
3. Results The in vivo results are shown in Table 1. The activity of lipase and level of TBARS increased significantly in DI group compared with control group (p < 0.05). There was no statistically significant difference in lipase activity between DI + Vit and control groups. The activity of amylase decreased in DI group compared with control group. The activity of amylase remained unchanged in DI + Vit group compared with DI group. There was no statistically significant difference in GGT activity between DI and control groups. The activity of ALP decreased significantly in DI and DI + Vit groups compared with control group (p < 0.05) but the activity of ALP increased significantly in the DI + Vit group compared with control group (p < 0.05). The results of in vitro experiment are shown in Table 2. In vitro experiment showed that all enzyme activities remained unchanged in control groups compared with value at 0 h. The ChE and lipase activities decreased dose dependently (p < 0.05). The activities of ALP, GGT, and especially amylase variably changed at low doses, but their activities significantly decreased at high doses in the DI groups compared with 0 h values (p < 0.05).
Table 1 Activities of serum enzymes and levels of thiobarbituric acid reactive substances (TBARS) in the control, diazinon (DI), and DI plus vitamins E and C (DI + Vit) treatment groups (in vivo)
Control DI DI + Vit
n
TBARS (nmol/mL)
Amylase (U/L)
Lipase (U/L)
GGT(U/L)
ALP(U/L)
8 6 6
4.9 ± 0.8 7.0 ± 0.5* 6.2 ± 0.5**
1335.67 ± 148.0 384.2 ± 63.0* 444.5 ± 45.6
13.1 ± 2.0 25.0 ± 6.9* 16.2 ± 2.1**
1.7 ± 1.2 2.1 ± 1.0 2.3 ± 3.3
307.12 ± 39.6 178.5 ± 29.9* 225.33 ± 33.6**
Values are expressed as means ± SD for groups. (ChE cholinesterase, ALP alkaline phosphatase, GGT c-glutamyltransferase). * p < 0.05, as control group is compared with the DI group. ** p < 0.05, as DI group is compared with the DI + Vit group.
126
O. Gokalp et al. / Pesticide Biochemistry and Physiology 81 (2005) 123–128
Table 2 Activities of enzymes following incubation of sera for various times with diazinon in the different concentration range at time 0 h (initial values) and 24 h (end values), in control and diazinon treatment groups (in vitro) 0h
Amylase (U/L) Lipaze (U/L) GGT (U/L) ALP (U/L) ChE (U/L)
95 ± 5.59 18 ± 1.2 20 ± 1.3 90 ± 6.91 15,238 ± 1113
Control
97 ± 5.33 19 ± 1.0 22 ± 1.0 87 ± 4.08 14,990 ± 1593
Diazinon concentrations (mM) 0.0033
0.033
0.33
3.3
33
95.0 ± 7.11 18 ± 2.2 20 ± 2.1 89 ± 5.93 8899 ± 752*
96 ± 3.57 17 ± 1.7 22 ± 1.9 92 ± 8.32 6476 ± 1105*
94 ± 4.77 16 ± 1.1 19 ± 2.1 90 ± 6.19 2533.8 ± 302*
100 ± 5.03 16 ± 0.8 18 ± 0.8 88 ± 6.5 1563 ± 79*
86 ± 6.3* 16 ± 0.8* 18 ± 1.4* 80 ± 6.6* 617 ± 55*
Note. There was no statistically significant difference in control group compared with 0 h. Values are expressed as means ± SD for seven samples each group. (ChE, cholinesterase; ALP, alkaline phosphatase; and GGT, c-glutamyltransferase in each group.) * p < 0.05 (DI-treated groups compared with the control group).
4. Discussion The present study focused on the issues: (1) Can DI also induce pancreatitis at a single dose? (2) If it induces pancreatitis, does oxidative stress have an important role in this pancreatitis? (3) If oxidative stress has an important role in DI-induced pancreatitis, can pharmacological concentrations of vitamin E and vitamin C decrease pancreatitis caused by DI? For clarifing above issues, we have, therefore, examined both in vivo and in vitro effects of DI on serum activities of lipase, amylase, ChE, ALP, GGT, and TBARS level concerning pancreatic damage and discussed them, respectively. We have observed that DI significantly increased the serum lipase activity. This finding indicates that DI causes acute pancreatitis since the increase in lipase activity by twofold is specific for the diagnosis for pancreatitis. In the previous studies, methidathion increased the serum lipase activity [18], whereas it decreased in rats treated with fenthion [19]. The other parameter to evaluate pancreatitis is the increased amylase activity. However, the amylase activity decreased in the present study. Dressel et al. [5] reported that the amylase activity was increased after DI administration at 75 mg/kg in dogs. These contradictory results in the amylase activity could be due to species used and the administration dose of DI. For example, the dogs were reported to be more susceptible to DI-induced pancreatitis [25,26]. Also in our previous studies, methidathion and fenthion
have slightly increased the amylase activity [18,19]. In the present study, we found significant reductions in amylase activity when high doses of DI used in vitro. According to the present data, it can be suggested that high dose of DI significantly inhibits the amylase activity. In most cases, pancreatitis is related to high doses of OPI intoxication. All changes in levels of lipase and amylase were significantly restored by incorporation of vitamins E and C. Other investigators have reported similar findings. For example, in a study carried out by Nonaka et al. [27], an antioxidant (CV 3611) produced from the derived products of ascorbic acid increased the amylase activity. Probably, these effects are due to the direct outcomes of vitamins E and C combination and OPIs. Our other questions were that if it induce pancreatitis, does oxidative stress have an important role in this pancreatitis model and if oxidative stress has an important role in DI-induced pancreatitis, can pharmacological concentrations of vitamin E and vitamin C decrease pancreatitis resulting from DI? The sing of oxidative stress is the increased level of TBARS. In the present study, the level of TBARS was increased. Treatment of rats with vitamin E and vitamin C decreased the level of TBARS. Gultekin et al. [17] have shown that pretreatment of rats with melatonin or a combination of vitamins E and C with the repeated doses—one per day—for six days consecutively prior to the administration of chlorpyrifos-ethyl reduced lipid peroxidation. The results of previous studies showed that methidathion,
O. Gokalp et al. / Pesticide Biochemistry and Physiology 81 (2005) 123–128
fenthion, and DI caused a significant increase in lipid peroxidation, in addition, the treatment with a combination of vitamins E and C 30 min after the administration of methidathion and fenthion led to a significant decrease in lipid peroxidation [12–17]. The results of present study confirm previous findings that oxidative stress has important role in the DI-induced pancreatitis in rats [18– 20,28]. In these studies, it has been observed that pancreatic duct obstruction increased oxidative stress, which in turn can cause acute pancreatitis by impairing the enzymatic and non-enzymatic antioxidant system. It has also been reported that acute pancreatitis can be prevented by antioxidant therapies. The corrosive effect of OPIs in gastrointestinal system can be another possible pathophysiologic mechanism [5,11]. The corrosive effect can inhibit pancreatic and biliary secretion by leading to inflammation and edema in Oddi sphincter during acute period. However, this mechanism of mode of action could not explain the pathophysiology of pancreatitis that is specifically induced by OPIs since DI can induce pancreatitis when it is absorbed by skin [1]. It has been suggested that a possible cause of pancreatitis in DI intoxication was butyrylcholinesterase inhibition, due to the high level of butyrylcholinesterase activity in pancreas [5,7,8]. Dressel et al. [5] have reported that DI could cause acute pancreatitis by increasing pressure in pancreas and its canals due to the excessive cholinergic stimulation in dogs. However, in the study of Dressel et al., pancreatic duct cannulation was seen to prevent the increased lipase activity, but not prevent histopathological damage of pancreas. In this study, DI did not change GGT activity but decreased ALP activity; in addition, the treatment with a combination of vitamins E and C partially restored the decreased ALP activity. These findings suggest that DI-induced pancreatitis may not occur via obstruction of pancreatic duct with excessive cholinergic stimulation in rat. ALP activity decrement is unexpectable. This was confirmed by our two studies where methidathion and fenthion did not change GGT and ALP activities in vitro, and similarly GGT and ALP activity increased in vivo. This was partially prevented by
127
the supplementation of vitamins E and C [13– 15]. On the other hand, it is well known that various insecticides have different effects on digestive enzymes [29]. As a result, DI with 0.25 LD50 or higher doses can cause acute pancreatitis in rat. Probably, this damage is due to the oxidative stress mechanism. Single-dose treatment with a combination of vitamins E and C 30 min after the administration of DI can reduce the toxicity. Adjuvant antioxidants may be of significant use in the treatment of OPIinduced acute pancreatitis.
References [1] C.T. Hsiao, C.C. Yang, J.F. Deng, M.J. Bullard, S.J. Liaw, Acute pancreatitis following organophosphate intoxication, J. Toxicol. Clin. Toxicol. 34 (1996) 343–347. [2] I. Sahin, K. Onbasi, H. Sahin, C. Karakaya, Y. Ustun, T. Noyan, The prevalence of pancreatitis in organophosphate poisonings, Hum. Exp. Toxicol. 21 (2002) 175–177. [3] E. Panieri, T.E. Krige, P.C. Bornman, D.M. Linton, Severe necrotizing pancreatitis caused by organophosphate poisoning, J. Clin. Gastroenterol. 25 (1997) 463–465. [4] J.C. Martin Rubi, F. Yelamos Rodriguez, F. Laynez Bretones, J. Cordoba Escamez, F. Diez Garcia, A. Lardelli Claret, J.L. BlancoCorodona et al., Poisoning caused by organophosphate insecticides, Study of 506 cases, Rev. Clin. Esp. 196 (1996) 145–149. [5] T.D. Dressel, R.L. Goodale Jr., B. Zweber, J.W. Borner, The effect of atropine and duct decompression on the evolution of Diazinon-induced acute canine pancreatitis, Ann. Surg. 195 (1982) 424–434. [6] H.S. Lee, Acute pancreatitis and organophosphate poisoning: a case report and review, Singapore Med. J. 30 (1989) 599–601. [7] Y. Oguchi, T.W. Frick, T.L. Satterberg, T.D. Dressel, J.W. Borner, R.L. Goodale, Effect of the organophosphate iso-OMPA on amylase release by pancreatic lobules of dog, guinea pig, and cat, Pancreas 2 (1987) 664–668. [8] T.W. Frick, S. Dalo, J.F. OÕLeary, W. Runge, J.W. Borner, H. Baraniewski, T. Dressel, J.G. Shearen, R.L. Goodale, Effects of insecticide, diazinon, on pancreas of dog, cat and guinea pig, J. Environ. Pathol. Toxicol. Oncol. 7 (1987) 1–11. [9] M.M. Harputluoglu, B. Kantarceken, M. Karincaoglu, M. Aladag, R. Yildiz, M. Ates, B. Yildirim, F. Hilmioglu, Acute pancreatitis: an obscure complication of organophosphate intoxication, Hum. Exp. Toxicol. 22 (2003) 341–343. [10] A.J. Bone, D.J. Gwillam, Animal Models of InsulinDependent Diabetes Mellitus, 1998, Chapter 16, pp. 16.1– 16.16.
128
O. Gokalp et al. / Pesticide Biochemistry and Physiology 81 (2005) 123–128
[11] A. Sood, V. Midha, N. Sood, Gastric outlet obstruction as a late complication of ingestion of diazinon, Indian J. Gastroenterol. 22 (2003) 106–107. [12] I. Altuntas, I. Kilinc, H. Orhan, R. Demirel, H. Koylu, N. Delibas, The effects of diazinon on lipid peroxidation and antioxidant enzymes in erythrocytes in vitro, Hum. Exp. Toxicol. 23 (2004) 9–13. [13] I. Altuntas, N. Delibas, M. Demirci, I. Kilinc, N. Tamer, The effects of methidathion on lipid peroxidation and some liver enzymes: role of vitamins E and C, Arch. Toxicol. 76 (2002) 470–473. [14] I. Altuntas, N. Delibas, D.K. Doguc, S. Ozmen, F. Gultekin, Role of reactive oxygen species in organophosphate insecticide phosalone toxicity in erythrocytes in vitro, Toxicol. In Vitro 17 (2003) 153–157. [15] I. Altuntas, N. Delibas, The effects of Fenthion on lipid peroxidation and some liver enzymes: role of vitamins E and C, Turk. J. Med. Sci. 32 (2002) 293–297. [16] I. Altuntas, N. Delibas, R. Sutcu, The effects of organophosphate insecticide methidathion on lipid peroxidation and anti-oxidant enzymes in rat erythrocytes: role of vitamins E and C, Hum. Exp. Toxicol. 21 (2002) 681–685. [17] F. Gu¨ltekin, N. Delibasß, S. Yasßar, I_. Kilnc¸, In vivo changes in antioxidant systems and protective role of melatonin and a combination of vitamin C and vitamin E on oxidative damage in erythrocytes induced by chlorpyrifos-ethyl in rats, Arch. Toxicol. 75 (2001) 88–96. [18] H. Mollaog˘lu, H.R. Yılmaz, O. Go¨kalp, I_. Altuntasß, MethidathionÕun pankreas u¨zerine etkileri: Vitamin E ve CÕnin rolu¨, Van Tip Dergisi 10 (2003) 98–100. [19] O. Gokalp, H. Mollaog˘lu, H.R. Yılmaz, I_. Altuntasß, FenthionÕun pankreas u¨zerine etkileri: Vitamin E ve CÕnin ¨ niversitesi Tip Faku¨ltesi Dergisi rolu¨, Su¨leyman Demirel U 10 (2003) 21–23. [20] S. Sevillano, A.M. de la Mano, M.A. Manso, A. Orfao, I.N. De Dios, N-Acetylcysteine prevents intra-acinar oxygen free radical production in pancreatic duct obstruc-
[21]
[22]
[23]
[24] [25]
[26]
[27]
[28]
[29]
tion-induced acute pancreatitis, Biochem. Biophys. Acta 20 (2003) 177–184. Office of Prevention, Pesticides and Toxic Substances, United States Environmental Protection Agency, Washington, DC, Diazinon-RED Chapter Toxicology, 1999, pp. 1–26. D. Appenroth, S. Fro¨g, L. Kersten, F.K. Splinter, Protective effects of vitamin E and C on Cisplatin nephrotoxicity in developing rats, Arch. Toxicol. 71 (1997) 677–683. H.H. Draper, M. Hadley, Malondialdehyde determination as index of lipid peroxidation, Methods Enzymol. 186 (1990) 421–431. B. Dawson-Sanders, R.G. Trapp, Basic and Clinical Statistics, Appleton and Lange, Norwalk, CT. L.G. Costa, T.B. Cole, C.E. Furlong, Polymorphisms of PON1 (paraoxonase) and their significance in clinical toxicology of organophosphates, J. Toxicol. Clin. Toxicol. 41 (2003) 37–45. M.M. Rodriguez, J. Bisset, M. Ruiz, A. Soca, Crossresistance to pyrethroid and organophosphorus insecticides induced by selection with temephos in Aedes aegypti (Diptera: Culicidae) from Cuba, J. Med. Entomol. 39 (2002) 882–888. A. Nonaka, T. Manabe, T. Tobe, Effect of a new synthetic ascorbic acid derivative as a free radical scavenger on the development of acute pancreatitis in mice, Gut 32 (1991) 528–532. J.A. Gomez, X. Molero, E. Vaquero, A. Alonso, A. Salas, J.R. Malagelada, Vitamin E attenuates biochemical and morphological features associated with the development of chronic pancreatitis, Am. J. Physiol. Gastrointest. Liver Physiol. 287 (2004) G162–G169. B.Q. Gary, J.E. Casida, Sensitivity of blood-clotting factors and digestive enzymes to inhibition by organophosphores pesticides, J. Biochem. Mol. Toxicol. 14 (2000) 51–56.
Pesticide Biochemistry and Physiology 81 (2005) 123–128 www.elsevier.com/locate/ypest
The effects of diazinon on pancreatic damage and ameliorating role of vitamin E and vitamin C Osman Gokalpa,*, Bora Buyukvanlıb, Ekrem Ciceka, Mehmet Kaya Ozera, Ahmet Koyub, Irfan Altuntasc, Halis Koylub a
Department of Pharmacology, School of Medicine, Suleyman Demirel University, 32260 Isparta, Turkey Department of Physiology, School of Medicine, Suleyman Demirel University, 32260 Isparta, Turkey Department of Biochemistry and Clinical Biochemistry, School of Medicine, Suleyman Demirel University, 32260 Isparta, Turkey b
c
Received 27 September 2004; accepted 1 November 2004
Abstract The aim of this study was to investigate effects of an organophosphate insecticide, diazinon (DI), on pancreas, and possible ameliorating role of vitamins E and C. We examined both in vivo and in vitro effects of DI on serum activities of alkaline phosphatase (ALP), c-glutamyltransferase (GGT), amylase, and lipase enzymes. We also evaluated possible ameliorating effects of vitamins E and C combination against DI toxicity and blood levels of thiobarbituric acid reactive substances (TBARS) only in vivo. In vivo experimental groups were: control group, DI-treated group, and DI + vitamins E plus C-treated group. In vitro study groups were: control group and DI-treated group. The biochemical analyses were determined in in vitro experiments at both hour 0 and 24 while in in vivo experiments were determined only at hour 24. Lipase activity and TBARS level were found increased by DI in in vivo experiments while lipase activity was found decreased in in vitro experiments. Amylase and ALP activities were found decreased by DI in both in vivo and in vitro experiments. Also, the combination of vitamins E and C was found to partially improve these disorders. These results suggest that DI treatment causes pancreas damage via increasing oxidative stress in rats, and a combination vitamins E and C can reduce this lipoperoxidative effect. 2004 Elsevier Inc. All rights reserved. Keywords: Organophosphate; Pancreatitis; Diazinon; Vitamin E; Vitamin C
1. Introduction
*
Corresponding author. Fax: +90 246 237 11 65. E-mail address: [email protected] (O. Gokalp).
Various complications can be seen in organophosphate insecticide (OPI) intoxication cases [1– 4]. OPI compounds are so various, however, only parathion, malathion, diphonate, coumaphos,
0048-3575/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2004.11.001
124
O. Gokalp et al. / Pesticide Biochemistry and Physiology 81 (2005) 123–128
diazinon (DI), mevinphos, and dimethoate have been reported as acute pancreatitis causes in the literature (approximately 12% rate) [1,2]. It has also been reported that when dimethoate was absorbed by skin, it could cause pancreatitis [1]. Although pancreatitis incidence is high in OPI intoxications, there are few articles regarding this issue in the literature, most of which are related to case reports on pathophysiological aspect. It has been reported that DI is one of the most pronounced OPIs responsible for OPI-induced pancreatitis [1,5–11]. It has been thought that OPIs could cause acute pancreatitis via increased pressure in pancreas and its canals due to the excessive cholinergic stimulation [1]. In previous studies, we have found that OPIs have caused lipid peroxidation in kidney, liver, and erythrocytes, increment in lactate dehydrogenase leaking to extracellular fluid [12–16]. It has also been indicated that the reactive oxygen types and/or free radicals produced by DI have given rise to these toxic effects [12]. Also we found that vitamins E and C combination therapy could decrease lipid peroxidation caused by various OPIs [13,15,17]. Furthermore, in the other two studies, we found that both fenthion and methidathion altered activities of the pancreatic enzymes, and these changes were partially prevented by vitamins E and C [18,19]. The precise molecular mechanisms of OPI-induced acute pancreatitis are still undefined. They may involve obstruction of pancreatic ducts, and/or enhancement of reactive oxygen species [5,20]. Therefore, we aimed to investigate DI mediated acute pancreatitis by means of serum amylase, lipase activities, pancreatic duct obstruction by means of serum alkaline phosphatase (ALP), c-glutamyltransferase (GGT) activities, increment oxidative stress by thiobarbituric acid reactive substances (TBARS) level and, to determine possible protective effects of vitamins E and C combination therapy against DI toxicity.
2. Materials and methods 2.1. In vivo study: animals and treatment Twenty Wistar albino rats weighing between 200 and 290 g were divided into three experimental
groups: control group (n = 8), DI-treated group (DI, n = 6) and DI + vitamin E + vitamin C-treated group (DI + Vit, n = 6). DI and DI + Vit groups were treated orally with a single dose of 335 mg/kg body weight of DI (0.25 LD50) (Basudin; Syngenta, Turkey) [21] in corn oil at 0 h. Only corn oil was given in the same way to the control goup. Vitamin E as a-tocopherol acetate (Evigen; Aksu Farma, Istanbul, Turkey) and vitamin C as sodium-L -ascorbate (Redoxon; Roche, Basel, Switzerland) were injected at doses of 150 mg/kg body weight i.m. [22] and 200 mg/kg body weight i.p. [22], respectively, 30 min after the treatment of DI in DI + Vit group. Equal amounts of physiologic saline instead of vitamins were given to the rats of control and DI groups. After all the rats received the above treatments they were fed ad libitum until midnight. The animals were starved overnight for 12 h before the blood was collected. Rats were anaesthetized with ether and venous blood samples were collected by direct heart puncture at 24 h. Blood samples were centrifuged and serum was discarded. We hereby declare that the experiments reported here comply with the current laws and regulations of the Turkish Republic on the care and handling of experimental animals. 2.2. In vitro study Effects of various concentrations of DI on the activities of amylase, lipase, GGT, ALP, and cholinesterase (ChE) in sera were studied. A 10-ml venous blood sample was obtained from each of seven volunteers (four male, three female). Blood samples were centrifuged and serum discarded. The activities of serum enzymes were determined in each sample and these served as 0 h. Each sample was divided into five portions and each one served as experimental groups, as follows: control group and DI-treated groups. DI was dissolved in water at concentrations ranging from 0.0, 0.0033, 0.033, 0.33, 3.3, and 33 mM [22]. Each DI concentration was incubated with previously prepared sera at +4 C for 24 h. In control group, physiologic saline was used instead of DI. The activities of serum enzymes were determined in each sample at 24 h.
O. Gokalp et al. / Pesticide Biochemistry and Physiology 81 (2005) 123–128
2.3. Biochemical parameters An autoanalyser, Abbott Aeroset (IL, USA), was used to determine the serum activities of ChE, amylase, lipase, ALP, and GGT. TBARS was determined by the double heating method of Draper and Hadley [23]. The principle of the method was spectrophotometric measurement of the colour produced during the reaction to thiobarbituric acid (TBA) with malondialdehyde (MDA). For this purpose, 2.5 ml of 100 g/L trichloroacetic acid (TCA) solution was added to 0.5 ml serum in a centrifuge tube and placed in a boiling water bath for 15 min. After cooling in tap water, the mixture was centrifuged at 1000g for 10 min, and 2 ml of the supernatant was added to 1 ml of 6.7 g/L TBA solution in a test tube and placed in a boiling water bath for 15 min. The solution was then cooled in tap water and its absorbance was measured using a Shimadzu UV1601 spectrophotometer (Japan) at 532 nm. The concentration of TBARS was calculated by the absorbance coefficient of MDA–TBA complex 1.56 · 105 cm 1 M 1 and expressed in nmol/ml. 2.4. Statistical evaluation For statistical analyses of in vivo results, normality was first investigated, and it was shown that some values of parameters did not fit to the normal distribution. Therefore, as stated by Dawson– Saunders and Trapp considering the small number of cases, non-parametric Kruskal–Wallis test and Mann–Whitney U test were used to compare the groups [24]. The p values smaller than 0.05 (p < 0.05) were accepted as statistically meaningful.
125
For statistical analyses of in vitro results, first the degree of normality was investigated, and it was found that the values of the parameters fitted a normal distribution. Therefore, ANOVA test was performed to demonstrate the significance of experimental treatments.
3. Results The in vivo results are shown in Table 1. The activity of lipase and level of TBARS increased significantly in DI group compared with control group (p < 0.05). There was no statistically significant difference in lipase activity between DI + Vit and control groups. The activity of amylase decreased in DI group compared with control group. The activity of amylase remained unchanged in DI + Vit group compared with DI group. There was no statistically significant difference in GGT activity between DI and control groups. The activity of ALP decreased significantly in DI and DI + Vit groups compared with control group (p < 0.05) but the activity of ALP increased significantly in the DI + Vit group compared with control group (p < 0.05). The results of in vitro experiment are shown in Table 2. In vitro experiment showed that all enzyme activities remained unchanged in control groups compared with value at 0 h. The ChE and lipase activities decreased dose dependently (p < 0.05). The activities of ALP, GGT, and especially amylase variably changed at low doses, but their activities significantly decreased at high doses in the DI groups compared with 0 h values (p < 0.05).
Table 1 Activities of serum enzymes and levels of thiobarbituric acid reactive substances (TBARS) in the control, diazinon (DI), and DI plus vitamins E and C (DI + Vit) treatment groups (in vivo)
Control DI DI + Vit
n
TBARS (nmol/mL)
Amylase (U/L)
Lipase (U/L)
GGT(U/L)
ALP(U/L)
8 6 6
4.9 ± 0.8 7.0 ± 0.5* 6.2 ± 0.5**
1335.67 ± 148.0 384.2 ± 63.0* 444.5 ± 45.6
13.1 ± 2.0 25.0 ± 6.9* 16.2 ± 2.1**
1.7 ± 1.2 2.1 ± 1.0 2.3 ± 3.3
307.12 ± 39.6 178.5 ± 29.9* 225.33 ± 33.6**
Values are expressed as means ± SD for groups. (ChE cholinesterase, ALP alkaline phosphatase, GGT c-glutamyltransferase). * p < 0.05, as control group is compared with the DI group. ** p < 0.05, as DI group is compared with the DI + Vit group.
126
O. Gokalp et al. / Pesticide Biochemistry and Physiology 81 (2005) 123–128
Table 2 Activities of enzymes following incubation of sera for various times with diazinon in the different concentration range at time 0 h (initial values) and 24 h (end values), in control and diazinon treatment groups (in vitro) 0h
Amylase (U/L) Lipaze (U/L) GGT (U/L) ALP (U/L) ChE (U/L)
95 ± 5.59 18 ± 1.2 20 ± 1.3 90 ± 6.91 15,238 ± 1113
Control
97 ± 5.33 19 ± 1.0 22 ± 1.0 87 ± 4.08 14,990 ± 1593
Diazinon concentrations (mM) 0.0033
0.033
0.33
3.3
33
95.0 ± 7.11 18 ± 2.2 20 ± 2.1 89 ± 5.93 8899 ± 752*
96 ± 3.57 17 ± 1.7 22 ± 1.9 92 ± 8.32 6476 ± 1105*
94 ± 4.77 16 ± 1.1 19 ± 2.1 90 ± 6.19 2533.8 ± 302*
100 ± 5.03 16 ± 0.8 18 ± 0.8 88 ± 6.5 1563 ± 79*
86 ± 6.3* 16 ± 0.8* 18 ± 1.4* 80 ± 6.6* 617 ± 55*
Note. There was no statistically significant difference in control group compared with 0 h. Values are expressed as means ± SD for seven samples each group. (ChE, cholinesterase; ALP, alkaline phosphatase; and GGT, c-glutamyltransferase in each group.) * p < 0.05 (DI-treated groups compared with the control group).
4. Discussion The present study focused on the issues: (1) Can DI also induce pancreatitis at a single dose? (2) If it induces pancreatitis, does oxidative stress have an important role in this pancreatitis? (3) If oxidative stress has an important role in DI-induced pancreatitis, can pharmacological concentrations of vitamin E and vitamin C decrease pancreatitis caused by DI? For clarifing above issues, we have, therefore, examined both in vivo and in vitro effects of DI on serum activities of lipase, amylase, ChE, ALP, GGT, and TBARS level concerning pancreatic damage and discussed them, respectively. We have observed that DI significantly increased the serum lipase activity. This finding indicates that DI causes acute pancreatitis since the increase in lipase activity by twofold is specific for the diagnosis for pancreatitis. In the previous studies, methidathion increased the serum lipase activity [18], whereas it decreased in rats treated with fenthion [19]. The other parameter to evaluate pancreatitis is the increased amylase activity. However, the amylase activity decreased in the present study. Dressel et al. [5] reported that the amylase activity was increased after DI administration at 75 mg/kg in dogs. These contradictory results in the amylase activity could be due to species used and the administration dose of DI. For example, the dogs were reported to be more susceptible to DI-induced pancreatitis [25,26]. Also in our previous studies, methidathion and fenthion
have slightly increased the amylase activity [18,19]. In the present study, we found significant reductions in amylase activity when high doses of DI used in vitro. According to the present data, it can be suggested that high dose of DI significantly inhibits the amylase activity. In most cases, pancreatitis is related to high doses of OPI intoxication. All changes in levels of lipase and amylase were significantly restored by incorporation of vitamins E and C. Other investigators have reported similar findings. For example, in a study carried out by Nonaka et al. [27], an antioxidant (CV 3611) produced from the derived products of ascorbic acid increased the amylase activity. Probably, these effects are due to the direct outcomes of vitamins E and C combination and OPIs. Our other questions were that if it induce pancreatitis, does oxidative stress have an important role in this pancreatitis model and if oxidative stress has an important role in DI-induced pancreatitis, can pharmacological concentrations of vitamin E and vitamin C decrease pancreatitis resulting from DI? The sing of oxidative stress is the increased level of TBARS. In the present study, the level of TBARS was increased. Treatment of rats with vitamin E and vitamin C decreased the level of TBARS. Gultekin et al. [17] have shown that pretreatment of rats with melatonin or a combination of vitamins E and C with the repeated doses—one per day—for six days consecutively prior to the administration of chlorpyrifos-ethyl reduced lipid peroxidation. The results of previous studies showed that methidathion,
O. Gokalp et al. / Pesticide Biochemistry and Physiology 81 (2005) 123–128
fenthion, and DI caused a significant increase in lipid peroxidation, in addition, the treatment with a combination of vitamins E and C 30 min after the administration of methidathion and fenthion led to a significant decrease in lipid peroxidation [12–17]. The results of present study confirm previous findings that oxidative stress has important role in the DI-induced pancreatitis in rats [18– 20,28]. In these studies, it has been observed that pancreatic duct obstruction increased oxidative stress, which in turn can cause acute pancreatitis by impairing the enzymatic and non-enzymatic antioxidant system. It has also been reported that acute pancreatitis can be prevented by antioxidant therapies. The corrosive effect of OPIs in gastrointestinal system can be another possible pathophysiologic mechanism [5,11]. The corrosive effect can inhibit pancreatic and biliary secretion by leading to inflammation and edema in Oddi sphincter during acute period. However, this mechanism of mode of action could not explain the pathophysiology of pancreatitis that is specifically induced by OPIs since DI can induce pancreatitis when it is absorbed by skin [1]. It has been suggested that a possible cause of pancreatitis in DI intoxication was butyrylcholinesterase inhibition, due to the high level of butyrylcholinesterase activity in pancreas [5,7,8]. Dressel et al. [5] have reported that DI could cause acute pancreatitis by increasing pressure in pancreas and its canals due to the excessive cholinergic stimulation in dogs. However, in the study of Dressel et al., pancreatic duct cannulation was seen to prevent the increased lipase activity, but not prevent histopathological damage of pancreas. In this study, DI did not change GGT activity but decreased ALP activity; in addition, the treatment with a combination of vitamins E and C partially restored the decreased ALP activity. These findings suggest that DI-induced pancreatitis may not occur via obstruction of pancreatic duct with excessive cholinergic stimulation in rat. ALP activity decrement is unexpectable. This was confirmed by our two studies where methidathion and fenthion did not change GGT and ALP activities in vitro, and similarly GGT and ALP activity increased in vivo. This was partially prevented by
127
the supplementation of vitamins E and C [13– 15]. On the other hand, it is well known that various insecticides have different effects on digestive enzymes [29]. As a result, DI with 0.25 LD50 or higher doses can cause acute pancreatitis in rat. Probably, this damage is due to the oxidative stress mechanism. Single-dose treatment with a combination of vitamins E and C 30 min after the administration of DI can reduce the toxicity. Adjuvant antioxidants may be of significant use in the treatment of OPIinduced acute pancreatitis.
References [1] C.T. Hsiao, C.C. Yang, J.F. Deng, M.J. Bullard, S.J. Liaw, Acute pancreatitis following organophosphate intoxication, J. Toxicol. Clin. Toxicol. 34 (1996) 343–347. [2] I. Sahin, K. Onbasi, H. Sahin, C. Karakaya, Y. Ustun, T. Noyan, The prevalence of pancreatitis in organophosphate poisonings, Hum. Exp. Toxicol. 21 (2002) 175–177. [3] E. Panieri, T.E. Krige, P.C. Bornman, D.M. Linton, Severe necrotizing pancreatitis caused by organophosphate poisoning, J. Clin. Gastroenterol. 25 (1997) 463–465. [4] J.C. Martin Rubi, F. Yelamos Rodriguez, F. Laynez Bretones, J. Cordoba Escamez, F. Diez Garcia, A. Lardelli Claret, J.L. BlancoCorodona et al., Poisoning caused by organophosphate insecticides, Study of 506 cases, Rev. Clin. Esp. 196 (1996) 145–149. [5] T.D. Dressel, R.L. Goodale Jr., B. Zweber, J.W. Borner, The effect of atropine and duct decompression on the evolution of Diazinon-induced acute canine pancreatitis, Ann. Surg. 195 (1982) 424–434. [6] H.S. Lee, Acute pancreatitis and organophosphate poisoning: a case report and review, Singapore Med. J. 30 (1989) 599–601. [7] Y. Oguchi, T.W. Frick, T.L. Satterberg, T.D. Dressel, J.W. Borner, R.L. Goodale, Effect of the organophosphate iso-OMPA on amylase release by pancreatic lobules of dog, guinea pig, and cat, Pancreas 2 (1987) 664–668. [8] T.W. Frick, S. Dalo, J.F. OÕLeary, W. Runge, J.W. Borner, H. Baraniewski, T. Dressel, J.G. Shearen, R.L. Goodale, Effects of insecticide, diazinon, on pancreas of dog, cat and guinea pig, J. Environ. Pathol. Toxicol. Oncol. 7 (1987) 1–11. [9] M.M. Harputluoglu, B. Kantarceken, M. Karincaoglu, M. Aladag, R. Yildiz, M. Ates, B. Yildirim, F. Hilmioglu, Acute pancreatitis: an obscure complication of organophosphate intoxication, Hum. Exp. Toxicol. 22 (2003) 341–343. [10] A.J. Bone, D.J. Gwillam, Animal Models of InsulinDependent Diabetes Mellitus, 1998, Chapter 16, pp. 16.1– 16.16.
128
O. Gokalp et al. / Pesticide Biochemistry and Physiology 81 (2005) 123–128
[11] A. Sood, V. Midha, N. Sood, Gastric outlet obstruction as a late complication of ingestion of diazinon, Indian J. Gastroenterol. 22 (2003) 106–107. [12] I. Altuntas, I. Kilinc, H. Orhan, R. Demirel, H. Koylu, N. Delibas, The effects of diazinon on lipid peroxidation and antioxidant enzymes in erythrocytes in vitro, Hum. Exp. Toxicol. 23 (2004) 9–13. [13] I. Altuntas, N. Delibas, M. Demirci, I. Kilinc, N. Tamer, The effects of methidathion on lipid peroxidation and some liver enzymes: role of vitamins E and C, Arch. Toxicol. 76 (2002) 470–473. [14] I. Altuntas, N. Delibas, D.K. Doguc, S. Ozmen, F. Gultekin, Role of reactive oxygen species in organophosphate insecticide phosalone toxicity in erythrocytes in vitro, Toxicol. In Vitro 17 (2003) 153–157. [15] I. Altuntas, N. Delibas, The effects of Fenthion on lipid peroxidation and some liver enzymes: role of vitamins E and C, Turk. J. Med. Sci. 32 (2002) 293–297. [16] I. Altuntas, N. Delibas, R. Sutcu, The effects of organophosphate insecticide methidathion on lipid peroxidation and anti-oxidant enzymes in rat erythrocytes: role of vitamins E and C, Hum. Exp. Toxicol. 21 (2002) 681–685. [17] F. Gu¨ltekin, N. Delibasß, S. Yasßar, I_. Kilnc¸, In vivo changes in antioxidant systems and protective role of melatonin and a combination of vitamin C and vitamin E on oxidative damage in erythrocytes induced by chlorpyrifos-ethyl in rats, Arch. Toxicol. 75 (2001) 88–96. [18] H. Mollaog˘lu, H.R. Yılmaz, O. Go¨kalp, I_. Altuntasß, MethidathionÕun pankreas u¨zerine etkileri: Vitamin E ve CÕnin rolu¨, Van Tip Dergisi 10 (2003) 98–100. [19] O. Gokalp, H. Mollaog˘lu, H.R. Yılmaz, I_. Altuntasß, FenthionÕun pankreas u¨zerine etkileri: Vitamin E ve CÕnin ¨ niversitesi Tip Faku¨ltesi Dergisi rolu¨, Su¨leyman Demirel U 10 (2003) 21–23. [20] S. Sevillano, A.M. de la Mano, M.A. Manso, A. Orfao, I.N. De Dios, N-Acetylcysteine prevents intra-acinar oxygen free radical production in pancreatic duct obstruc-
[21]
[22]
[23]
[24] [25]
[26]
[27]
[28]
[29]
tion-induced acute pancreatitis, Biochem. Biophys. Acta 20 (2003) 177–184. Office of Prevention, Pesticides and Toxic Substances, United States Environmental Protection Agency, Washington, DC, Diazinon-RED Chapter Toxicology, 1999, pp. 1–26. D. Appenroth, S. Fro¨g, L. Kersten, F.K. Splinter, Protective effects of vitamin E and C on Cisplatin nephrotoxicity in developing rats, Arch. Toxicol. 71 (1997) 677–683. H.H. Draper, M. Hadley, Malondialdehyde determination as index of lipid peroxidation, Methods Enzymol. 186 (1990) 421–431. B. Dawson-Sanders, R.G. Trapp, Basic and Clinical Statistics, Appleton and Lange, Norwalk, CT. L.G. Costa, T.B. Cole, C.E. Furlong, Polymorphisms of PON1 (paraoxonase) and their significance in clinical toxicology of organophosphates, J. Toxicol. Clin. Toxicol. 41 (2003) 37–45. M.M. Rodriguez, J. Bisset, M. Ruiz, A. Soca, Crossresistance to pyrethroid and organophosphorus insecticides induced by selection with temephos in Aedes aegypti (Diptera: Culicidae) from Cuba, J. Med. Entomol. 39 (2002) 882–888. A. Nonaka, T. Manabe, T. Tobe, Effect of a new synthetic ascorbic acid derivative as a free radical scavenger on the development of acute pancreatitis in mice, Gut 32 (1991) 528–532. J.A. Gomez, X. Molero, E. Vaquero, A. Alonso, A. Salas, J.R. Malagelada, Vitamin E attenuates biochemical and morphological features associated with the development of chronic pancreatitis, Am. J. Physiol. Gastrointest. Liver Physiol. 287 (2004) G162–G169. B.Q. Gary, J.E. Casida, Sensitivity of blood-clotting factors and digestive enzymes to inhibition by organophosphores pesticides, J. Biochem. Mol. Toxicol. 14 (2000) 51–56.