http://informahealthcare.com/dct ISSN: 0148-0545 (print), 1525-6014 (electronic) Drug Chem Toxicol, 2014; 37(4): 378–383 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/01480545.2013.866139
RESEARCH ARTICLE
Protective effect of crocin on diazinon induced vascular toxicity in subchronic exposure in rat aorta ex-vivo Bibi Marjan Razavi1, Hossein Hosseinzadeh2, Khalil Abnous3, and Mohsen Imenshahidi2 Drug and Chemical Toxicology Downloaded from informahealthcare.com by Mcgill University on 11/21/14 For personal use only.
1
Department of Pharmacodynamy and Toxicology, Targeted Drug Delivery Research Center, School of Pharmacy, 2Department of Pharmacodynamy and Toxicology, Pharmaceutical Research Center, School of Pharmacy, and 3Department of Medicinal Chemistry, Pharmaceutical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran Abstract
Keywords
Context: Diazinon (DZN) is a widely used organophosphate insecticide. Although mechanism of DZN cardiovascular toxicity is primarily mediated through inhibition of acetylcholinesterase, however, DZN causes remarkable atropine-insensitive hypotension in rats. It has been proved that oxidative stress is an important mechanism of DZN toxicity especially in chronic exposure. Crocin, an active ingredient of saffron, has been found to antagonize the hypotensive effects of DZN in rats, but do not reverse acetylcholinesterase inhibition. Objective: In this study the effects of DZN on contractile and relaxant responses in rat aorta as well as ex-vivo antioxidant actions of crocin have been investigated. Materials and methods: Rats were divided into 7 groups: corn oil (control), DZN (15 mg/kg/day, gavage), crocin (12.5, 25 and 50 mg/kg/day, i.p.) plus DZN, vitamin E (200 IU/kg, i.p., three days a week) plus DZN and crocin (50 mg/kg/day, i.p.) groups. Treatments were continued for 4 weeks. Contractile and relaxant responses were evaluated on the isolated aorta. Results: Our results showed that DZN not only decreased the contractile responses to KCl and Phenylephrine (PE) (p50.001), but also attenuated the relaxant response to acetylcholine (ACh) (p50.01). Crocin and vitamin E attenuated lipid peroxidation, improved the reduction of contractile responses by KCl and PE and restored the decrease in ACh relaxation in rat aorta. Conclusion: DZN induced vascular toxicity which may be due to oxidative stress and not to a cholinergic mechanism. Crocin improved toxic effects of DZN via reducing lipid peroxidation and restoring altered contractile and relaxant responses in rat aorta.
Crocin, Crocus sativus L., diazinon, isolated rat aorta, saffron
Introduction Organophosphate insecticides are used to control pests in agriculture, industries and domestic. In addition to the toxic effects of organophosphate on different tissues (Zhou et al., 2010), it was reported that they could have some effects on circulation system (Guvenic Tuna et al., 2011). Some studies reported that acute and chronic toxicity of OPs could lead to the degeneration of collagenous and elastic fibers of vascular wall (Akimov & Kolesnichenko, 1985; Antov et al., 1984; Yavuz et al., 2005). DZN is one of the widely used OPs in agriculture which can poison occupationally exposed workers and others. Cardiovascular and other toxic effects induced by DZN are primarily mediated through inhibition of acetylcholinesterase (AChE) (Ogutcu et al., 2006). In addition to the inhibition of acetylcholine esterase, DZN causes remarkable
Address for correspondence: Mohsen Imenshahidi, Department of Pharmacodynamy and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. Tel: 0098 511 882 3255. Fax: 0098 511 882 3251. E-mail: [email protected]
History Received 9 June 2013 Revised 11 September 2013 Accepted 1 October 2013 Published online 18 December 2013
atropine-insensitive hypotension in rats when administered intravenously in lethal doses and exerts vasorelaxant actions in endothelium-denuded rat aorta (Kojima et al., 1993). Moreover animal studies showed that DZN increases oxygen free radicals (Shadnia et al., 2005) and oxygen free radicals play a role in OPs toxicity including delayed neuropathy (Masoud & Sandhir, 2012; Ogutcu et al., 2006), a progressive neuropathic disorder that develops after many days of the cessation of cholinergic crisis. Furthermore some studies showed that cardiovascular toxicity induced by OPs could be related to the oxidative stress (Kumar & Jugdutt, 2003), and antioxidants can suppress toxicity (Guney et al., 2007; Kumar & Jugdutt, 2003). Crocus sativus L., commonly known as saffron, widely cultivated in Iran and other countries (Soeda et al., 2007). Crocin, a carotenoid isolated from this herb, is responsible for red color of saffron. Antioxidant (Hosseinzadeh et al., 2010) and antiapoptotic (Soeda et al., 2001) effects of crocin have been proved in modern pharmacological research (Xu et al., 2006, 2007). Moreover, the vascular protective effects of crocin have been also reported. Crocin may exert antiatherosclerotic effects by inhibition of the aortic endothelial
Crocin and diazinon induced vascular toxicity
DOI: 10.3109/01480545.2013.866139
cell apoptosis as a result of the increase the ratio of Bcl2/Bax expression. Furthermore, in our previous study we found that crocin, as an antioxidant, could antagonize the hypotensive effects of DZN in rats, but do not reverse AChE inhibition (Razavi et al., 2013). There is no study investigating the effects of subchronic DZN administration on the vascular system, therefore, the aims of this study were to examine the effects of DZN on KCl and PE-induced contractions as well as its effects on acetylcholine (ACh) or sodium nitroprosside (SNP) induced relaxations in rat aorta. Furthermore, ex-vivo antioxidant actions of crocin in rat aorta were also investigated.
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Methods Chemicals DZN (BazodinÕ , purity 96%) and Vitamin E were purchased from Syngenta, Singapore, and OSVE Pharmaceutical Co., Tehran, Iran. Stigmas of C. sativus L. from Novin Saffron (collected from Ghaen, Khorasan province, Northeast of Iran) was obtained and analyzed in accordance to the ISO/TS 36322. Crocin was extracted and purified as previously reported (Hadizadeh et al., 2010). PE, ACh and SNP were obtained from sigma-aldrich. All chemicals used in this study were analytical grade and purchased from Sigma-Aldrich (Germany). Animals and treatment Adult male Wistar rats (weight 200–250 g) were provided by animal center (School of Pharmacy, Mashhad University of Medical Sciences). They were kept on a 12 h light/dark cycle and at a temperature of 23 1 C with free access to food and water. These conditions were maintained constant throughout the experiments. All animal experiments were carried out in accordance to Mashhad University of Medical Sciences, Ethical Committee Acts. Rats were randomly divided into seven groups: (1) control group (corn oil); (2) DZN group (15 mg/kg); (3) DZN þ crocin 12.5 mg/kg group; (4) DZN þ crocin 25 mg/kg group; (5) DZN þ crocin 50 mg/kg group; (6) DZN þ vitamin E 200 IU/kg group and (7) Crocin 50 mg/kg group. All groups consisted of six rats. DZN was dissolved in corn oil and administrated via gavage once a day for 4 weeks. Control rats received corn oil in the same way. Crocin (once a day) and vitamin E (three days a week) were administrated via i.p. for 4 weeks. Determination of cholinesterase activity After 4 weeks of treatment, rats were killed and sera were collected. The plasma cholinesterase activity was analyzed using Ellman method (Ellman et al., 1961). This method is based on the hydrolysis of acetylthiocholine iodide. The reaction of the thoil compound with 5,50 -Dithiobis(2-nitrobenzoic acid) (DTNB) produces a color-forming compound with absorbance at 405 nm. The absorbances of different samples were recorded at 0.5 min intervals for 2 min. The cholinesterase activity was calculated as: Cholinesterase (mU/ml, at 25 C) ¼ change in absorbance in 30 sec. 23 400 (Rezg et al., 2008).
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Tissue preparation After 4 weeks treatment, rats were killed by decapitation. Then the thorax was opened and the thoracic aorta was quickly removed and cleaned from adherent connective tissues and cut into rings (4–5 mm in length). Special care was taken to ensure that the endothelial was not damaged during tissue preparation. Two stainless steel stirrups were passed through the lumen of each ring. One stirrup was connected to an isometric force transducer (POWERLAB, AD Instrument, Australia) to measure tension in the vessels. The rings were placed in a 25 ml organ chamber (Organ Bath, 4 channels, AD Instrument, Australia) containing Krebs solution gassed with 95% O2/5% CO2, and maintained at 37 C. The composition of Krebs solution was as follows: 118.0 mM NaCl, 4.7 mM KCl, 1.2 mM NaH2PO4, 1.2 mM MgSO4, 25.0 mM NaHCO3, 11.1 mM glucose, and 2.5 mM CaCl2 (pH ¼ 7.4) (Parsaee et al., 2006). Rings were placed under a resting tension of 2 g (in preliminary studies determined to be optimum), and allowed to equilibrate for 1 h. During this equilibrium period the physiological salt solution was replaced every 15 min. Concentration-response curve for KCl (20–80 mM) and PE (10–9–10–4 M) was obtained by cumulative addition of KCl or PE to the bath solution. The endothelium dependent relaxation responses were recorded in PE (10–6 M) preconstricted rings for ACh. Endothelium independent vasorelaxation to NO donor SNP (10–12–10–6 M) were also measured. The Labchart 7.3 (AD-Instruments) software was used for this study. Statistical analysis All results are expressed as mean SEM. ANOVA followed by Tukey–Kramer tests were performed to compare means. The software Pharm-PCS, was used to calculate of pD2 values. p Values less than 0.05 were considered as significant.
Results Effect of DZN and crocin on plasma cholinesterase activity A significant decrease was observed in plasma cholinesterase activity in the DZN treated group as compared with the control group (p50.001), but no significant difference was observed between DZN plus crocin groups (all three doses) or vitamin E and DZN treated groups (Table 1). Table 1. Effects of DZN and crocin (4 weeks) on plasma cholinesterase activity in rats.
Groups Control DZN 15 mg/kg Crocin 50 mg/kg DZN þ Vitamin E DZN þ Crocin 12.5 mg/kg DZN þ Crocin 25 mg/kg DZN þ Crocin 50 mg/kg
Cholinesterase activity (mU/ml) (mean SEM) 679.56 11.93 256.56 15.02*** 659.56 10.78 279.33 7.31*** 302.72 8.73*** 277.29 6.02*** 269.1 10.94***
Data are shown as mean SEM, ***p50.001 compared to the control group, one way ANOVA Tukey–Kramer test, n ¼ 6.
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Vasoconstrictor responses to KCl As shown in Figure 1, vasoconstrictor responses to cumulative concentrations of KCl (20–80 mM) in the aortic rings of rats were significantly decreased at concentration of 80 mM of KCl in DZN treated group as compared with the control and, the maximum responses in control and DZN groups were 1.11 0.06 and 0.62 0.070 g tension respectively (a 44.14% decrease, n ¼ 6, p50.001). Cotreatment with DZN and crocin (25 and 50 mg/kg) or vitamin E significantly increased vasoconstrictor responses to 80 mM of KCl as compared with DZN group (p50.05).
comparison with control (p50.05). Cotreatment with DZN and crocin (25 and 50 mM) or vitamin E significantly improved ACh-mediated relaxation in DZN groups (p50.05). Crocin alone did not significantly change the ACh-induced relaxation in the control group. Endothelium independent vasorelaxation to different concentrations of SNP (1012–107 M) remained unimpaired in DZN groups. Moreover maximum relaxations were not significantly different among studied groups (Figure 4). Control DZN (15mg/kg) Crocin 50 mg/kg DZN+Cro12.5 mg/kg DZN+Cro25 mg/kg DZN+Cro50 mg/kg DZN+vitE (200mg/kg)
Vasodilator responses to ACh and SNP
2.2
### ## 1.0
**
**
-5
-4
** *** *** -9
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-6
Log of PE concentration (M)
Figure 2. Concentration-response curve showing the effect of DZN and crocin on vasoconstrictor responses of rat aortic rings to PE. Values are expressed as mean SEM from 6 animals. **p50.01 versus control, #p50.05, ##p50.01 and ###p50.001 versus DZN.
Control DZN(15 mg/kg) Crocin50 mg/kg DZN+Cro12.5mg/kg DZN+Cro25mg/kg DZN+Cro50mg/kg DZN+vitE(200mg/kg)
***
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0 20 % relaxation
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DZN(15mg/kg) Crocin50mg/kg DZN+Cro12.5mg/kg DZN+Cro25mg/kg DZN+Cro50mg/kg DZN+Vit.E(200mg/kg)
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#
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Control
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As shown in Figure 3 and Table 2, the concentrationdependent relaxation responses to ACh in the aortic rings precontracted with PE (106 M) was significantly diminished in DZN groups. Maximum relaxation responses in control and DZN groups were 100.00 0.00 and 66.75 6.88 respectively (a 33.75% decrease, p50.01). A significant decrease in pD2 value (log EC50) was also observed in DZN group as
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1.8
Tension (g)
As shown in Figure 2, vasoconstrictor responses to cumulative concentrations of PE (109–104 M) in the aortic rings of rats were significantly decreased in DZN treated rats and the maximum responses in control and DZN groups at concentration of 106 M were 1.90 0.19 and 0.95 0.13 g tension respectively (a 50.00% decrease, n ¼ 6, p50.01). Cotreatment with DZN and crocin (25 and 50 mg/kg) or vitamin E significantly increased vasoconstrictor responses to PE as compared with DZN group (p50.05 and p50.001).
Tension (g)
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Vasoconstrictor responses to PE
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KCl Concentration (mM)
Figure 1. Concentration-response curve showing the effect of DZN and crocin on vasoconstrictor responses of rat aortic rings to KCl. Values are expressed as mean SEM from 6 animals. ***p50.001 versus control, #p50.05 and ##p50.01 versus DZN.
Figure 3. Concentration-response curve showing the effect of DZN and crocin on ACh-induced relaxation in PE-precontracted (106 M) rat aortic rings. Relaxation is expressed as the percentage of reduction in the PE-induced increase in tone. Values are expressed as mean SEM from 6 animals. **p50.01 versus control, #p50.05 versus DZN.
Crocin and diazinon induced vascular toxicity
DOI: 10.3109/01480545.2013.866139
Table 2. Effect of crocin and DZN on the Emax and pD2 values of ACh in isolated rat aorta. Groups
pD2 (log EC50)
Control DZN Crocin 50 mg/kg DZN þ cro 12.5 mg/kg DZN þ cro 25 mg/kg DZN þ cro 50 mg/kg DZN þ Vit E
Emax
(%)
100.00 0.00 66.75 6.88** 100.00 0.00 57.20 7.15 100.00 0.00## 100.00 0.00## 100.00 0.00##
7.64 (95%CI: 7.41–7.87) 5.37* (95%CI: 4.27–6.47) 7.05 (95%CI: 6.85–7.24) 4.42 (95%CI: 4.16–4.68) 7.42# (95%CI: 7.19–7.66) 7.59# (95%CI: 6.99–8.16) 6.81# (95%CI: 6.62–7.00)
Control DZN (15mg/kg) Crocin50mg/kg DZN+ Cro12.5 mg/kg DZN+ Cro25 mg/kg DZN+ Cro50mg/kg DZN+vitE(200mg/kg) 0 20 % relaxation
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Data are presented as mean SEM. pD2 ¼ (log EC50) Emax ¼ maximum Ach relaxation, and CI ¼ confidence intervals. *p50.05, **p50.01 versus control, ##p50.01 and #p50.05 versus DZN.
40 60 80 100 120 -12
-11
-10
-9
-8
-7
Log of SNP concentration (M)
Figure 4. Concentration-response curve showing SNP-induced relaxation in PE-precontracted (106 M) rat aortic rings. Relaxation is expressed as the percentage of reduction in the PE-induced increase in tone. Values are expressed as mean SEM from 6 animals.
Discussion This study showed that DZN (15 mg/kg) exhibits vascular toxicity in subchronic exposure (28 days) by changing vasoconstriction and vasorelaxation responses to PE and ACh in isolated rat aorta. 4 weeks treatment with crocin or vitamin E showed protective effects against DZN induced vascular toxicity by reducing toxic effects of DZN on isolated aortic rings. Furthermore, subchronic DZN administration caused a reduction in plasma cholinesterase activity in comparison with the control group. The concurrent administration of DZN and crocin or vitamin E did not affect the cholinesterase activity. So that it may be concluded that crocin does not have any effect on cholinesterase inhibition of DZN. In isolated rat aorta, subchronic DZN administration attenuated maximal contractile responses to KCl (80 mM) and PE (1 mM). It has been shown that i.v. administration of DZN and fenthion inhibit KCl and PE induced contraction in isolated rat aorta, through direct inhibitory effect on vessel smooth muscle cells which is a result of reduction of intracellular calcium concentration (Kojima et al., 1993).
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Also several studies reported that acute and chronic OPs toxicity could damage the vascular wall and led to degeneration of collagenous and elastic fibers (Akimov & Kolesnichenko, 1985; Antov et al., 1984; Yavuz et al., 2005). In our previous study, subchronic DZN administration increased MDA, a marker of lipid peroxidation, in rat aorta (Razavi et al., 2013), so it could be suggested that elevation of oxidative stress products led to the damages of smooth muscle cells of aorta. Similar to our results, it was reported that OPs like paraoxone and dichlorvos attenuated arterial smooth muscle contraction in rats (Zhou et al., 2010). It is well known that voltage dependent calcium channels of smooth muscle cells are affected by KCl. KCl depolarizes the cell membrane, increases the intracellular calcium and finally leads to contraction (Mashhoodi et al., 2004). So, the decreased effect of DZN on KCl induced contraction may be related to the inhibitory effect of DZN on voltage dependent calcium channels. Furthermore PE (an a1-receptor agonist) -induced contraction involves receptor-operated calcium and voltageoperated calcium channels, whereas KCl-induced contraction is mediated by voltage-operated calcium channels (Karaki et al., 1997; Shin et al., 2005). Thus, inhibitory effect of DZN on KCl- and PE-induced contraction seems to be associated with inhibition of voltage-operated calcium channels, leading to decreased intracellular calcium concentration. The maximum relaxation response to acetylcholine (ACh) as well as the pD2 (log EC50) value in the aortic rings precontracted with PE (106 M) was significantly attenuated in DZN group. Endothelium-independent relaxations induced by SNP were not affected by DZN. As DZN attenuated endothelium dependent relaxation induced by ACh and endothelium independent relaxation induced by SNP was not impaired by DZN, so it is concluded that endothelium might be damaged by DZN. According to the results of our previous study, which was shown that subchronic DZN exposure decreased systolic blood pressure (SBP) (Razavi et al., 2013), it might be postulated that toxic effects of DZN on aorta is not restricted to the endothelium. Because of a significant increase in the MDA level in the aortic tissue (Razavi et al., 2013), therefore it could be suggested that elevated products of oxidative stress during DZN administration, caused alterations in vascular system, so that tonicity and elasticity of vascular smooth muscle cells can be affected by DZN. Thus, subchronic exposure to DZN, caused disturbances in aorta smooth muscle cells and reduced SBP and contraction responses to KCl and PE. Another study revealed that chronic exposure to chlorpyriphos and dichlorvos decreased the strength of the aorta and it became less stiff, therefore might influence the response of the aorta to mechanical loading induced by blood pressure (Guvenic Tuna et al., 2011). Administration of crocin (25 and 50 mg/kg) with DZN improved the reduction of KCl and PE induced contraction as well as the impairment of ACh induced relaxation in isolated rat aorta, whereas crocin alone did not cause significant differences when compared with the control. These protective effects of crocin were similar to vitamin E. Vitamin E or a- tocopherol is one of the most biologically active
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antioxidants in the biological system. Through chain breaking action, it can protect cells or tissues against lipid peroxidation. Protective effect of vitamin E in vascular system has been previously shown. It was found that a-tocopherol can prevent hyperglycemia induced activation of PKC (protein kinase C) in vascular cells by lowering DAG (diacyleglycerole), possibly via its antioxidant effect (Kunisaki et al., 1994). Another study revealed that vitamin C improved endotheliumdependent vasodilatation in non-insulin-dependent diabetes mellitus patients through antioxidant effect (Ting et al., 1996). Moreover vitamin E can improve a decrease in systolic BP induced by DZN via inhibition of lipid peroxidation (Razavi et al., 2013). In this research cotreatment of crocin and DZN after 4 weeks restored DZN induced vascular toxic effects. Previous studies showed that antioxidant activity and modulation of antioxidant defense system are involved in protective effects of long term crocin exposures. For example cardioprotective effects of crocin (20 mg/kg/day, 21 days) against isoproterenol-induced toxicity via antioxidant effect, has been shown previously. Also, crocin restored the endogenous antioxidants and lipid peroxide productions (Goyal et al., 2010). It was reported that crocetin extracted from Gardenia jasminoides could improve endothelium dependent relaxation of aorta isolated from hypercholesterolemic rabbit, which could be partly due to the increased activity of eNOS, resulting increase of NO production and protection against atherosclerosis. It was also shown that crocetin inhibited down regulation of expression of eNOS as a result of oxidized LDL (low density lipoprotein) and increased NO production in BAECs (bovine aortic endothelial cells) (Tang et al., 2006). So, considering the similarity of crocin and crocetin, crocin may also increase NO production and improve DZN impaired endothelial dependent relaxation.
Conclusions Our results revealed that DZN induced vascular toxicity which may be due to oxidative stress and not to a cholinergic mechanism. Crocin like vitamin E improved the responses of the aorta to vasoconstrictor agents like KCl and PE as well as vasorelaxators like ACh in the presence of DZN. It can be concluded that crocin, as an antioxidant, has protective effect against DZN adverse effects in vascular system.
Acknowledgements The authors are thankful to the Vice Chancellor of Research, Mashhad University of Medical Sciences for financial support. The results described in this paper are part of a Ph.D. thesis.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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RESEARCH ARTICLE
Protective effect of crocin on diazinon induced vascular toxicity in subchronic exposure in rat aorta ex-vivo Bibi Marjan Razavi1, Hossein Hosseinzadeh2, Khalil Abnous3, and Mohsen Imenshahidi2 Drug and Chemical Toxicology Downloaded from informahealthcare.com by Mcgill University on 11/21/14 For personal use only.
1
Department of Pharmacodynamy and Toxicology, Targeted Drug Delivery Research Center, School of Pharmacy, 2Department of Pharmacodynamy and Toxicology, Pharmaceutical Research Center, School of Pharmacy, and 3Department of Medicinal Chemistry, Pharmaceutical Research Center, Mashhad University of Medical Sciences, Mashhad, Iran Abstract
Keywords
Context: Diazinon (DZN) is a widely used organophosphate insecticide. Although mechanism of DZN cardiovascular toxicity is primarily mediated through inhibition of acetylcholinesterase, however, DZN causes remarkable atropine-insensitive hypotension in rats. It has been proved that oxidative stress is an important mechanism of DZN toxicity especially in chronic exposure. Crocin, an active ingredient of saffron, has been found to antagonize the hypotensive effects of DZN in rats, but do not reverse acetylcholinesterase inhibition. Objective: In this study the effects of DZN on contractile and relaxant responses in rat aorta as well as ex-vivo antioxidant actions of crocin have been investigated. Materials and methods: Rats were divided into 7 groups: corn oil (control), DZN (15 mg/kg/day, gavage), crocin (12.5, 25 and 50 mg/kg/day, i.p.) plus DZN, vitamin E (200 IU/kg, i.p., three days a week) plus DZN and crocin (50 mg/kg/day, i.p.) groups. Treatments were continued for 4 weeks. Contractile and relaxant responses were evaluated on the isolated aorta. Results: Our results showed that DZN not only decreased the contractile responses to KCl and Phenylephrine (PE) (p50.001), but also attenuated the relaxant response to acetylcholine (ACh) (p50.01). Crocin and vitamin E attenuated lipid peroxidation, improved the reduction of contractile responses by KCl and PE and restored the decrease in ACh relaxation in rat aorta. Conclusion: DZN induced vascular toxicity which may be due to oxidative stress and not to a cholinergic mechanism. Crocin improved toxic effects of DZN via reducing lipid peroxidation and restoring altered contractile and relaxant responses in rat aorta.
Crocin, Crocus sativus L., diazinon, isolated rat aorta, saffron
Introduction Organophosphate insecticides are used to control pests in agriculture, industries and domestic. In addition to the toxic effects of organophosphate on different tissues (Zhou et al., 2010), it was reported that they could have some effects on circulation system (Guvenic Tuna et al., 2011). Some studies reported that acute and chronic toxicity of OPs could lead to the degeneration of collagenous and elastic fibers of vascular wall (Akimov & Kolesnichenko, 1985; Antov et al., 1984; Yavuz et al., 2005). DZN is one of the widely used OPs in agriculture which can poison occupationally exposed workers and others. Cardiovascular and other toxic effects induced by DZN are primarily mediated through inhibition of acetylcholinesterase (AChE) (Ogutcu et al., 2006). In addition to the inhibition of acetylcholine esterase, DZN causes remarkable
Address for correspondence: Mohsen Imenshahidi, Department of Pharmacodynamy and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. Tel: 0098 511 882 3255. Fax: 0098 511 882 3251. E-mail: [email protected]
History Received 9 June 2013 Revised 11 September 2013 Accepted 1 October 2013 Published online 18 December 2013
atropine-insensitive hypotension in rats when administered intravenously in lethal doses and exerts vasorelaxant actions in endothelium-denuded rat aorta (Kojima et al., 1993). Moreover animal studies showed that DZN increases oxygen free radicals (Shadnia et al., 2005) and oxygen free radicals play a role in OPs toxicity including delayed neuropathy (Masoud & Sandhir, 2012; Ogutcu et al., 2006), a progressive neuropathic disorder that develops after many days of the cessation of cholinergic crisis. Furthermore some studies showed that cardiovascular toxicity induced by OPs could be related to the oxidative stress (Kumar & Jugdutt, 2003), and antioxidants can suppress toxicity (Guney et al., 2007; Kumar & Jugdutt, 2003). Crocus sativus L., commonly known as saffron, widely cultivated in Iran and other countries (Soeda et al., 2007). Crocin, a carotenoid isolated from this herb, is responsible for red color of saffron. Antioxidant (Hosseinzadeh et al., 2010) and antiapoptotic (Soeda et al., 2001) effects of crocin have been proved in modern pharmacological research (Xu et al., 2006, 2007). Moreover, the vascular protective effects of crocin have been also reported. Crocin may exert antiatherosclerotic effects by inhibition of the aortic endothelial
Crocin and diazinon induced vascular toxicity
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cell apoptosis as a result of the increase the ratio of Bcl2/Bax expression. Furthermore, in our previous study we found that crocin, as an antioxidant, could antagonize the hypotensive effects of DZN in rats, but do not reverse AChE inhibition (Razavi et al., 2013). There is no study investigating the effects of subchronic DZN administration on the vascular system, therefore, the aims of this study were to examine the effects of DZN on KCl and PE-induced contractions as well as its effects on acetylcholine (ACh) or sodium nitroprosside (SNP) induced relaxations in rat aorta. Furthermore, ex-vivo antioxidant actions of crocin in rat aorta were also investigated.
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Methods Chemicals DZN (BazodinÕ , purity 96%) and Vitamin E were purchased from Syngenta, Singapore, and OSVE Pharmaceutical Co., Tehran, Iran. Stigmas of C. sativus L. from Novin Saffron (collected from Ghaen, Khorasan province, Northeast of Iran) was obtained and analyzed in accordance to the ISO/TS 36322. Crocin was extracted and purified as previously reported (Hadizadeh et al., 2010). PE, ACh and SNP were obtained from sigma-aldrich. All chemicals used in this study were analytical grade and purchased from Sigma-Aldrich (Germany). Animals and treatment Adult male Wistar rats (weight 200–250 g) were provided by animal center (School of Pharmacy, Mashhad University of Medical Sciences). They were kept on a 12 h light/dark cycle and at a temperature of 23 1 C with free access to food and water. These conditions were maintained constant throughout the experiments. All animal experiments were carried out in accordance to Mashhad University of Medical Sciences, Ethical Committee Acts. Rats were randomly divided into seven groups: (1) control group (corn oil); (2) DZN group (15 mg/kg); (3) DZN þ crocin 12.5 mg/kg group; (4) DZN þ crocin 25 mg/kg group; (5) DZN þ crocin 50 mg/kg group; (6) DZN þ vitamin E 200 IU/kg group and (7) Crocin 50 mg/kg group. All groups consisted of six rats. DZN was dissolved in corn oil and administrated via gavage once a day for 4 weeks. Control rats received corn oil in the same way. Crocin (once a day) and vitamin E (three days a week) were administrated via i.p. for 4 weeks. Determination of cholinesterase activity After 4 weeks of treatment, rats were killed and sera were collected. The plasma cholinesterase activity was analyzed using Ellman method (Ellman et al., 1961). This method is based on the hydrolysis of acetylthiocholine iodide. The reaction of the thoil compound with 5,50 -Dithiobis(2-nitrobenzoic acid) (DTNB) produces a color-forming compound with absorbance at 405 nm. The absorbances of different samples were recorded at 0.5 min intervals for 2 min. The cholinesterase activity was calculated as: Cholinesterase (mU/ml, at 25 C) ¼ change in absorbance in 30 sec. 23 400 (Rezg et al., 2008).
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Tissue preparation After 4 weeks treatment, rats were killed by decapitation. Then the thorax was opened and the thoracic aorta was quickly removed and cleaned from adherent connective tissues and cut into rings (4–5 mm in length). Special care was taken to ensure that the endothelial was not damaged during tissue preparation. Two stainless steel stirrups were passed through the lumen of each ring. One stirrup was connected to an isometric force transducer (POWERLAB, AD Instrument, Australia) to measure tension in the vessels. The rings were placed in a 25 ml organ chamber (Organ Bath, 4 channels, AD Instrument, Australia) containing Krebs solution gassed with 95% O2/5% CO2, and maintained at 37 C. The composition of Krebs solution was as follows: 118.0 mM NaCl, 4.7 mM KCl, 1.2 mM NaH2PO4, 1.2 mM MgSO4, 25.0 mM NaHCO3, 11.1 mM glucose, and 2.5 mM CaCl2 (pH ¼ 7.4) (Parsaee et al., 2006). Rings were placed under a resting tension of 2 g (in preliminary studies determined to be optimum), and allowed to equilibrate for 1 h. During this equilibrium period the physiological salt solution was replaced every 15 min. Concentration-response curve for KCl (20–80 mM) and PE (10–9–10–4 M) was obtained by cumulative addition of KCl or PE to the bath solution. The endothelium dependent relaxation responses were recorded in PE (10–6 M) preconstricted rings for ACh. Endothelium independent vasorelaxation to NO donor SNP (10–12–10–6 M) were also measured. The Labchart 7.3 (AD-Instruments) software was used for this study. Statistical analysis All results are expressed as mean SEM. ANOVA followed by Tukey–Kramer tests were performed to compare means. The software Pharm-PCS, was used to calculate of pD2 values. p Values less than 0.05 were considered as significant.
Results Effect of DZN and crocin on plasma cholinesterase activity A significant decrease was observed in plasma cholinesterase activity in the DZN treated group as compared with the control group (p50.001), but no significant difference was observed between DZN plus crocin groups (all three doses) or vitamin E and DZN treated groups (Table 1). Table 1. Effects of DZN and crocin (4 weeks) on plasma cholinesterase activity in rats.
Groups Control DZN 15 mg/kg Crocin 50 mg/kg DZN þ Vitamin E DZN þ Crocin 12.5 mg/kg DZN þ Crocin 25 mg/kg DZN þ Crocin 50 mg/kg
Cholinesterase activity (mU/ml) (mean SEM) 679.56 11.93 256.56 15.02*** 659.56 10.78 279.33 7.31*** 302.72 8.73*** 277.29 6.02*** 269.1 10.94***
Data are shown as mean SEM, ***p50.001 compared to the control group, one way ANOVA Tukey–Kramer test, n ¼ 6.
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Vasoconstrictor responses to KCl As shown in Figure 1, vasoconstrictor responses to cumulative concentrations of KCl (20–80 mM) in the aortic rings of rats were significantly decreased at concentration of 80 mM of KCl in DZN treated group as compared with the control and, the maximum responses in control and DZN groups were 1.11 0.06 and 0.62 0.070 g tension respectively (a 44.14% decrease, n ¼ 6, p50.001). Cotreatment with DZN and crocin (25 and 50 mg/kg) or vitamin E significantly increased vasoconstrictor responses to 80 mM of KCl as compared with DZN group (p50.05).
comparison with control (p50.05). Cotreatment with DZN and crocin (25 and 50 mM) or vitamin E significantly improved ACh-mediated relaxation in DZN groups (p50.05). Crocin alone did not significantly change the ACh-induced relaxation in the control group. Endothelium independent vasorelaxation to different concentrations of SNP (1012–107 M) remained unimpaired in DZN groups. Moreover maximum relaxations were not significantly different among studied groups (Figure 4). Control DZN (15mg/kg) Crocin 50 mg/kg DZN+Cro12.5 mg/kg DZN+Cro25 mg/kg DZN+Cro50 mg/kg DZN+vitE (200mg/kg)
Vasodilator responses to ACh and SNP
2.2
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** *** *** -9
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Log of PE concentration (M)
Figure 2. Concentration-response curve showing the effect of DZN and crocin on vasoconstrictor responses of rat aortic rings to PE. Values are expressed as mean SEM from 6 animals. **p50.01 versus control, #p50.05, ##p50.01 and ###p50.001 versus DZN.
Control DZN(15 mg/kg) Crocin50 mg/kg DZN+Cro12.5mg/kg DZN+Cro25mg/kg DZN+Cro50mg/kg DZN+vitE(200mg/kg)
***
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0 20 % relaxation
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DZN(15mg/kg) Crocin50mg/kg DZN+Cro12.5mg/kg DZN+Cro25mg/kg DZN+Cro50mg/kg DZN+Vit.E(200mg/kg)
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#
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Control
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As shown in Figure 3 and Table 2, the concentrationdependent relaxation responses to ACh in the aortic rings precontracted with PE (106 M) was significantly diminished in DZN groups. Maximum relaxation responses in control and DZN groups were 100.00 0.00 and 66.75 6.88 respectively (a 33.75% decrease, p50.01). A significant decrease in pD2 value (log EC50) was also observed in DZN group as
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Tension (g)
As shown in Figure 2, vasoconstrictor responses to cumulative concentrations of PE (109–104 M) in the aortic rings of rats were significantly decreased in DZN treated rats and the maximum responses in control and DZN groups at concentration of 106 M were 1.90 0.19 and 0.95 0.13 g tension respectively (a 50.00% decrease, n ¼ 6, p50.01). Cotreatment with DZN and crocin (25 and 50 mg/kg) or vitamin E significantly increased vasoconstrictor responses to PE as compared with DZN group (p50.05 and p50.001).
Tension (g)
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Figure 1. Concentration-response curve showing the effect of DZN and crocin on vasoconstrictor responses of rat aortic rings to KCl. Values are expressed as mean SEM from 6 animals. ***p50.001 versus control, #p50.05 and ##p50.01 versus DZN.
Figure 3. Concentration-response curve showing the effect of DZN and crocin on ACh-induced relaxation in PE-precontracted (106 M) rat aortic rings. Relaxation is expressed as the percentage of reduction in the PE-induced increase in tone. Values are expressed as mean SEM from 6 animals. **p50.01 versus control, #p50.05 versus DZN.
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DOI: 10.3109/01480545.2013.866139
Table 2. Effect of crocin and DZN on the Emax and pD2 values of ACh in isolated rat aorta. Groups
pD2 (log EC50)
Control DZN Crocin 50 mg/kg DZN þ cro 12.5 mg/kg DZN þ cro 25 mg/kg DZN þ cro 50 mg/kg DZN þ Vit E
Emax
(%)
100.00 0.00 66.75 6.88** 100.00 0.00 57.20 7.15 100.00 0.00## 100.00 0.00## 100.00 0.00##
7.64 (95%CI: 7.41–7.87) 5.37* (95%CI: 4.27–6.47) 7.05 (95%CI: 6.85–7.24) 4.42 (95%CI: 4.16–4.68) 7.42# (95%CI: 7.19–7.66) 7.59# (95%CI: 6.99–8.16) 6.81# (95%CI: 6.62–7.00)
Control DZN (15mg/kg) Crocin50mg/kg DZN+ Cro12.5 mg/kg DZN+ Cro25 mg/kg DZN+ Cro50mg/kg DZN+vitE(200mg/kg) 0 20 % relaxation
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Data are presented as mean SEM. pD2 ¼ (log EC50) Emax ¼ maximum Ach relaxation, and CI ¼ confidence intervals. *p50.05, **p50.01 versus control, ##p50.01 and #p50.05 versus DZN.
40 60 80 100 120 -12
-11
-10
-9
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-7
Log of SNP concentration (M)
Figure 4. Concentration-response curve showing SNP-induced relaxation in PE-precontracted (106 M) rat aortic rings. Relaxation is expressed as the percentage of reduction in the PE-induced increase in tone. Values are expressed as mean SEM from 6 animals.
Discussion This study showed that DZN (15 mg/kg) exhibits vascular toxicity in subchronic exposure (28 days) by changing vasoconstriction and vasorelaxation responses to PE and ACh in isolated rat aorta. 4 weeks treatment with crocin or vitamin E showed protective effects against DZN induced vascular toxicity by reducing toxic effects of DZN on isolated aortic rings. Furthermore, subchronic DZN administration caused a reduction in plasma cholinesterase activity in comparison with the control group. The concurrent administration of DZN and crocin or vitamin E did not affect the cholinesterase activity. So that it may be concluded that crocin does not have any effect on cholinesterase inhibition of DZN. In isolated rat aorta, subchronic DZN administration attenuated maximal contractile responses to KCl (80 mM) and PE (1 mM). It has been shown that i.v. administration of DZN and fenthion inhibit KCl and PE induced contraction in isolated rat aorta, through direct inhibitory effect on vessel smooth muscle cells which is a result of reduction of intracellular calcium concentration (Kojima et al., 1993).
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Also several studies reported that acute and chronic OPs toxicity could damage the vascular wall and led to degeneration of collagenous and elastic fibers (Akimov & Kolesnichenko, 1985; Antov et al., 1984; Yavuz et al., 2005). In our previous study, subchronic DZN administration increased MDA, a marker of lipid peroxidation, in rat aorta (Razavi et al., 2013), so it could be suggested that elevation of oxidative stress products led to the damages of smooth muscle cells of aorta. Similar to our results, it was reported that OPs like paraoxone and dichlorvos attenuated arterial smooth muscle contraction in rats (Zhou et al., 2010). It is well known that voltage dependent calcium channels of smooth muscle cells are affected by KCl. KCl depolarizes the cell membrane, increases the intracellular calcium and finally leads to contraction (Mashhoodi et al., 2004). So, the decreased effect of DZN on KCl induced contraction may be related to the inhibitory effect of DZN on voltage dependent calcium channels. Furthermore PE (an a1-receptor agonist) -induced contraction involves receptor-operated calcium and voltageoperated calcium channels, whereas KCl-induced contraction is mediated by voltage-operated calcium channels (Karaki et al., 1997; Shin et al., 2005). Thus, inhibitory effect of DZN on KCl- and PE-induced contraction seems to be associated with inhibition of voltage-operated calcium channels, leading to decreased intracellular calcium concentration. The maximum relaxation response to acetylcholine (ACh) as well as the pD2 (log EC50) value in the aortic rings precontracted with PE (106 M) was significantly attenuated in DZN group. Endothelium-independent relaxations induced by SNP were not affected by DZN. As DZN attenuated endothelium dependent relaxation induced by ACh and endothelium independent relaxation induced by SNP was not impaired by DZN, so it is concluded that endothelium might be damaged by DZN. According to the results of our previous study, which was shown that subchronic DZN exposure decreased systolic blood pressure (SBP) (Razavi et al., 2013), it might be postulated that toxic effects of DZN on aorta is not restricted to the endothelium. Because of a significant increase in the MDA level in the aortic tissue (Razavi et al., 2013), therefore it could be suggested that elevated products of oxidative stress during DZN administration, caused alterations in vascular system, so that tonicity and elasticity of vascular smooth muscle cells can be affected by DZN. Thus, subchronic exposure to DZN, caused disturbances in aorta smooth muscle cells and reduced SBP and contraction responses to KCl and PE. Another study revealed that chronic exposure to chlorpyriphos and dichlorvos decreased the strength of the aorta and it became less stiff, therefore might influence the response of the aorta to mechanical loading induced by blood pressure (Guvenic Tuna et al., 2011). Administration of crocin (25 and 50 mg/kg) with DZN improved the reduction of KCl and PE induced contraction as well as the impairment of ACh induced relaxation in isolated rat aorta, whereas crocin alone did not cause significant differences when compared with the control. These protective effects of crocin were similar to vitamin E. Vitamin E or a- tocopherol is one of the most biologically active
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antioxidants in the biological system. Through chain breaking action, it can protect cells or tissues against lipid peroxidation. Protective effect of vitamin E in vascular system has been previously shown. It was found that a-tocopherol can prevent hyperglycemia induced activation of PKC (protein kinase C) in vascular cells by lowering DAG (diacyleglycerole), possibly via its antioxidant effect (Kunisaki et al., 1994). Another study revealed that vitamin C improved endotheliumdependent vasodilatation in non-insulin-dependent diabetes mellitus patients through antioxidant effect (Ting et al., 1996). Moreover vitamin E can improve a decrease in systolic BP induced by DZN via inhibition of lipid peroxidation (Razavi et al., 2013). In this research cotreatment of crocin and DZN after 4 weeks restored DZN induced vascular toxic effects. Previous studies showed that antioxidant activity and modulation of antioxidant defense system are involved in protective effects of long term crocin exposures. For example cardioprotective effects of crocin (20 mg/kg/day, 21 days) against isoproterenol-induced toxicity via antioxidant effect, has been shown previously. Also, crocin restored the endogenous antioxidants and lipid peroxide productions (Goyal et al., 2010). It was reported that crocetin extracted from Gardenia jasminoides could improve endothelium dependent relaxation of aorta isolated from hypercholesterolemic rabbit, which could be partly due to the increased activity of eNOS, resulting increase of NO production and protection against atherosclerosis. It was also shown that crocetin inhibited down regulation of expression of eNOS as a result of oxidized LDL (low density lipoprotein) and increased NO production in BAECs (bovine aortic endothelial cells) (Tang et al., 2006). So, considering the similarity of crocin and crocetin, crocin may also increase NO production and improve DZN impaired endothelial dependent relaxation.
Conclusions Our results revealed that DZN induced vascular toxicity which may be due to oxidative stress and not to a cholinergic mechanism. Crocin like vitamin E improved the responses of the aorta to vasoconstrictor agents like KCl and PE as well as vasorelaxators like ACh in the presence of DZN. It can be concluded that crocin, as an antioxidant, has protective effect against DZN adverse effects in vascular system.
Acknowledgements The authors are thankful to the Vice Chancellor of Research, Mashhad University of Medical Sciences for financial support. The results described in this paper are part of a Ph.D. thesis.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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