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The effect of crocin and safranal, constituents of saffron, against subacute effect of diazinon on three biomarkers TNF-α, Direct 8-iso-prostaglandin F2α and S100β in rats
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Abstracts / Toxicology Letters 205S (2011) S180–S300
P2175 AOEL as a criterion of pesticides absorption, distribution and bioavailability S. Sergeiev ∗ , O. Kravchuk, I. Leposhkin, V. Lyshavski, N. Kolontaieva, V. Medvedev, E. Spynu Medved’s Institute of Ecohygiene and Toxicology, Kyiv, Ukraine The purpose of study was an establishment of AOELs on a basis of pesticides toxicity manifestation depending on anatomy and physiology of primary barrier of absorption, physicochemical characteristics of pesticides, penetration and ultimately their bioavailability. Pesticide operators are mainly exposed through skin contact and inhalation. It is apparent that studies for establishment of AOELs should reproduce the exposure route. Route-specific repeated dose toxicity studies correctly take into account the influence of physicochemical characteristics on pesticides penetration through primary barrier of absorption, bioavailability and the rate and the extent of pesticide toxicity manifestation. Such studies on laboratory animals allow to establish no effect doses (concentrations, converted into doses), mg/kg bw for the exposure route. The doses are registered by external dosimetry of pesticide on skin surface and in inspired air on the external border of respiratory system, the same as at measuring of dermal and inhalation operator exposure. In the presence of critical effect after oral exposure the lowest oral NOEL convert into equivalent dermal and inhalation doses using nonequivalence coefficients of toxicity effect, which reflects differences in expression of toxicity at oral, dermal and inhalation exposure. As a result, external values of dermal and inhalation operator exposure (mg/kg bw) are compared with dermal and inhalation AOELs, i.e. with dermal and inhalation NOELs (external value, mg/kg bw) for laboratory animals, divided by assessment factor. Thus, estimation reflects route-specific bioavailability of pesticide. Outline principle was priority in estimation of more than 300 pesticides authorized in Ukraine. doi:10.1016/j.toxlet.2011.05.799
P2176 Fatal pesticides-related self-poisonings in the southeastern region of Serbia—Alarming increase in suicides calls for restrictions ˇ skar Stojanˇcov 2 , G. Ilic´ 3 , B. ´ A. Simic´ 1,∗ , S. Milicevi c´ Misic´ 2 , S. Seˇ Milosavljevic 3 , M. Vujovic 3 , T.P. Flaten 1 Department of Chemistry, NTNU, Trondheim, Norway, 2 Department of Toxicology, Clinic of Endocrinology, University Clinical Centre, Niˇs, Serbia, 3 Institute of Forensic Medicine, Niˇs, Serbia
self-poisonings increased from 11 in 1996 to 32 in 2006 (10% and 25% of the total numbers in Serbia in these two years). Of these cases, 5 were related to OPs in 1996, and 16 in 2006. Thus, the number of OP-related fatal self-poisonings has increased by 320% in the Niˇs region in the ten-year period, and currently accounts for 50% of all the suicidal poisonings in this region. Conclusion: Measures should be taken to restrict the availability of pesticides. doi:10.1016/j.toxlet.2011.05.800
P2177 Inducibility of CYP1A by linuron in primary cultured human hepatocytes M. Sunouchi 1,∗ , A. Miyajima-Tabata 2 , R. Kikura-Hanajiri 3 , S. Kim 1 , T. Kubo 1 , S. Ishida 1 , M. Usami 1 , Y. Sekino 1 1
Division of Pharmacology, National Institute of Health Sciences, Tokyo, Japan, 2 Division of Medicinal Safety Science, National Institute of Health Sciences, Tokyo, Japan, 3 Division of Pharmacognosy and Phytochemistry, National Institute of Health Sciences, Tokyo, Japan Linuron is a substituted urea herbicide that is used in large quantities all over the world. In Japan, 76,000–111,000 kg of linuron was imported annually in recent three years (2006–2008), as reported by Japan Plant Protection Division (2009). So far, we have shown that linuron induced O-deethylation activities of ethoxycoumarin and ethoxyresorufin, in rat hepatocytes both in vitro and in vivo. In this study, we investigated the inducibility of CYP1A by linuron in human hepatocytes. The dose dependency of CYP1A induction by linuron was determined and compared with prototypical inducers, 3-methylcholanthrene (3-MC) and omeprazole (OPZ). Primary cultured human hepatocytes were exposed to linuron (1–125 M), 3-MC or OPZ for 48 h. Medium containing the inducer was renewed every 24 h. Induction of CYP1A was determined as increased ethoxyresorufin O-deethylation activity. The amounts of protein and mRNA of CYP were determined by Western blotting and RT-PCR, respectively. Cytotoxicity on hepatocytes was assessed by LDH leakage assay. Linuron induced CYP1A activity in a dose dependent manner at 5 M or more, with increased protein and mRNA of CYP1A1/2. Linuron showed strong inducibility of human CYP1A activity. The rank order of the inducibility was 3-MC > OPZ > linuron. The inducibility of CYP1A by linuron was stronger in humans than in rats. These results suggest that CYP1A induction by linuron affects the hepatic drug-metabolizing enzyme system and endogenous hormone regulation in humans.
1
Objectives: Pesticide self-poisoning is a major problem in developing countries, especially in rural areas, and an important reason is that hazardous pesticides are available without restrictions. Organophosphates (OPs) are the most commonly used pesticides for suicidal poisoning causing over 200,000 deaths in the world yearly. The authors present an alarming increase in fatal OP-related self-poisonings from 1996 to 2006 in the southeastern region of Serbia. Methods: Descriptive, retrospective epidemiological study. Results: National statistical data in Serbia show a 2.7% decrease in the total number of suicides from 1996 to 2006. In the same period, the number of fatal self-poisonings increased by 18%, from 108 to 128. Niˇs is the centre of the southeastern part of Serbia, which is a low-income region with a predominantly rural population. In the University Clinical Centre in Niˇs, the total number of cases of fatal
doi:10.1016/j.toxlet.2011.05.801
P2178 The effect of crocin and safranal, constituents of saffron, against subacute effect of diazinon on three biomarkers TNF-␣, Direct 8-iso-prostaglandin F2␣ and S100 in rats A. Timchehhariri 1,∗ , H. Hosseinzadeh 2 , S.A. Moalem 2 1
Pharmacy School, Mashhad University of Medical Sciences, Khorasan Razavi, Iran, 2 Mashhad University of Medical Sciences, Mashhad, Iran Diazinon after malathion is one of the most commonly used OPs in the world. Carotenoids by acting as biological antioxidants, protect cells and tissues from damaging effects of free radicals. In this study, the effect of crocin and safranal was studied
Abstracts / Toxicology Letters 205S (2011) S180–S300
against subacute toxicity of diazinon (DZN) on three biomarkers TNF-␣, ␣-iso prostan and S100 in rats. Vitamin E (200 IU/kg), safranal at doses 0.025, 0.05 and 0.1 ml/kg and crocin at doses 50, 100 and 200 mg/kg were injected intraperitoneally three times per week alone or with DZN (20 mg/kg/day, orally) for 4 weeks. Biomarkers were evaluated at the end of 4 weeks. The evaluation of biomarkers was done using ELISA. The levels of serum TNF-␣, Direct 8-iso-prostaglandin F2␣ and S100 increased significantly in diazinon treated group compared with the control group. Direct 8-iso-prostaglandin F2␣ level significantly decreased in diazinon + crocin (all doses) and diazinon + safranal (all doses) treated groups when compared to diazinon treated group. S100 level significantly decreased in diazinon + crocin 100 and 200 mg/kg and diazinon + safranal 0.1 ml/kg treated groups when compared to diazinon treated group. TNF-␣ level significantly decreased in diazinon + crocin 50 and 100 mg/kg treated group while no significant changes in diazinon + safranal (all doses) and diazinon + vitamin E when compared to diazinon treated group, and also this biomarker significantly increased in safranal 0.1 ml/kg treated group compared with the control group. Vitamin E, safranal (0.025 or 0.05 ml/kg) and all doses of crocin decreased this effect of diazinon. In all doses of crocin our study showed that diazinon changes these three biomarkers and even if it is administrated less than the LD50 dose level. Vitamin E, safranal in low doses and crocin in three doses reduced diazinon toxicity. doi:10.1016/j.toxlet.2011.05.802
P2179 Mechanism of nonlinear effects of plant growth regulators – N-oxide derivates O. Vasetska ∗ , O. Fedchenko, P. Zhminko, M. Prodanchuk Medved‘s Institute of Ecohygiene and Toxicology, Kyiv, Ukraine The prominent feature of some plant growth regulators (PGR) is amplification effects or change of the action directivity at low doses, that is, absence of classical linear dependence “dose-effect” and manifestation of the so-called “paradoxical effect”. The mechanism of nonlinear effects of the PGR (pyridine N-oxide derivatives) in the organism of animals can be explained by example of the Ivin (N-oxide 2,6-dimethylpyridine). Since the Ivin molecule may be presented in the hydrated/dehydrated state, then at high concentrations Ivin interacts with membranes in hydrated form. As a result of membrane modification, their destabilization occurs as well as the damage of the structural integrity of lipid matrix. This can lead to changes in cellular signal transduction, reduction of the protein-synthetic processes and decrease of reparative system function, therefore the toxic effect increases. At low concentrations, Ivin interacts with membrane lipids in dehydrated state. The dehydrated Ivin causes the lipid compaction and contributes to the membrane stabilization. Moreover, it can be observed changes in cellular signal transduction, increase of protein-synthetic processes in the organism, improvement of the repair system function, and in consequence of this an adaptation to acting factor takes place. Under the condition of balance of hydrated and dehydrated forms of Ivin molecule, there is the leveling of the Ivin effect and therefore the inversion occurs (no toxic effect). doi:10.1016/j.toxlet.2011.05.803
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Safety pharmacology P2180 Flavin-containing monooxygenase 3 gene polymorphisms in Turkish population B. Alpertunga ∗ , G. Ozhan, P. Topal Department of Pharmaceutical Toxicology, Istanbul University, Faculty of Pharmacy, Istanbul, Turkey Purpose: Flavin-containing monooxygenases (FMOs), one of the most important monooxygenase systems, are involved in xenobiotics metabolism and variability in drug response. FMO gene catalyzes the monooxygenation of numerous N-, Pand S-containing xenobiotics. Substrates of FMO include many drugs (cocaine, nicotine, imipramine, tamoxifen, cimetidine, etc.), chemicals (phorate, aldicarb, etc.) and endogenous substrates (methionine, trimethylamine, etc.). FMO3 is associated with the majority of FMO-mediated adult hepatic metabolism. In the adult human liver, FMO3 is the prominent form and other FMOs are not expressed or are expressed at very low levels. Methods: 13 of totally 40 genomic FMO3 variants are Single Nucleotide Polymorphisms (SNPs) which cause low function. Allele frequencies of three functional SNPs (E158K, V257M, and E308G) vary greatly among different ethnic populations with distinct genetic backgrounds. The present study aimed to determine frequencies of FMO3 polymorphisms and haplotypes in Turkish population. We genotyped in 120 healthy volunteers using polymerase chain reaction (PCR)restriction fragment length polymorphism (RFLP) methods. For statistical analysis, the computer software SPSS was used. Haplotype frequencies were estimated from genotype data by PHASE software based on Bayesian algorithm. The results were compared with those obtained from different populations. Results: The study on alleles of FMO3 gene could be useful in understanding the variant and haplotype distributions of FMO3 alleles in Turkish population because FMO3 alleles, which can be observed on various races and even on sub-populations, have not been researched in Turkey even though it has been researched on different populations. doi:10.1016/j.toxlet.2011.05.804
P2181 Anti-inflammatory effect of Thymus kotschyanus extract on rat’s hind paw edema induced by carrageenan A. Bakhtiarian 1,∗ , M. Khanavi 2 , F. Aarabi Moghaddam 1 1
Department of Pharmacology, Tehran University of Medical Sciences, Tehran, Iran, 2 Department of Pharmacognosy, Tehran University of Medical Sciences, Tehran, Iran Purpose: The advantages of herbal drugs have been known to human beings for many years and they have been used for the treatment of many diseases. Nowadays, inflammation is known to be one of the basic pathologic causes of diseases. In this study, the anti-inflammatory effects of Thymus kotschyanus were investigated. Methods: Forty two wistar rats were divided into three groups. In the first group, the extract of Thymus kotschyanus in doses of 50, 100, 150, and 200 mg/kg was injected intra peritoneal and after 1 h, they were treated with the hind paw edema test with carrageenan. In the second group, Indomethacin with the dose of 5 mg/kg was injected 1 h before carrageenan and in the third one, saline was injected before carrageenan, to the rat’s right hind paw.
Abstracts / Toxicology Letters 205S (2011) S180–S300
P2175 AOEL as a criterion of pesticides absorption, distribution and bioavailability S. Sergeiev ∗ , O. Kravchuk, I. Leposhkin, V. Lyshavski, N. Kolontaieva, V. Medvedev, E. Spynu Medved’s Institute of Ecohygiene and Toxicology, Kyiv, Ukraine The purpose of study was an establishment of AOELs on a basis of pesticides toxicity manifestation depending on anatomy and physiology of primary barrier of absorption, physicochemical characteristics of pesticides, penetration and ultimately their bioavailability. Pesticide operators are mainly exposed through skin contact and inhalation. It is apparent that studies for establishment of AOELs should reproduce the exposure route. Route-specific repeated dose toxicity studies correctly take into account the influence of physicochemical characteristics on pesticides penetration through primary barrier of absorption, bioavailability and the rate and the extent of pesticide toxicity manifestation. Such studies on laboratory animals allow to establish no effect doses (concentrations, converted into doses), mg/kg bw for the exposure route. The doses are registered by external dosimetry of pesticide on skin surface and in inspired air on the external border of respiratory system, the same as at measuring of dermal and inhalation operator exposure. In the presence of critical effect after oral exposure the lowest oral NOEL convert into equivalent dermal and inhalation doses using nonequivalence coefficients of toxicity effect, which reflects differences in expression of toxicity at oral, dermal and inhalation exposure. As a result, external values of dermal and inhalation operator exposure (mg/kg bw) are compared with dermal and inhalation AOELs, i.e. with dermal and inhalation NOELs (external value, mg/kg bw) for laboratory animals, divided by assessment factor. Thus, estimation reflects route-specific bioavailability of pesticide. Outline principle was priority in estimation of more than 300 pesticides authorized in Ukraine. doi:10.1016/j.toxlet.2011.05.799
P2176 Fatal pesticides-related self-poisonings in the southeastern region of Serbia—Alarming increase in suicides calls for restrictions ˇ skar Stojanˇcov 2 , G. Ilic´ 3 , B. ´ A. Simic´ 1,∗ , S. Milicevi c´ Misic´ 2 , S. Seˇ Milosavljevic 3 , M. Vujovic 3 , T.P. Flaten 1 Department of Chemistry, NTNU, Trondheim, Norway, 2 Department of Toxicology, Clinic of Endocrinology, University Clinical Centre, Niˇs, Serbia, 3 Institute of Forensic Medicine, Niˇs, Serbia
self-poisonings increased from 11 in 1996 to 32 in 2006 (10% and 25% of the total numbers in Serbia in these two years). Of these cases, 5 were related to OPs in 1996, and 16 in 2006. Thus, the number of OP-related fatal self-poisonings has increased by 320% in the Niˇs region in the ten-year period, and currently accounts for 50% of all the suicidal poisonings in this region. Conclusion: Measures should be taken to restrict the availability of pesticides. doi:10.1016/j.toxlet.2011.05.800
P2177 Inducibility of CYP1A by linuron in primary cultured human hepatocytes M. Sunouchi 1,∗ , A. Miyajima-Tabata 2 , R. Kikura-Hanajiri 3 , S. Kim 1 , T. Kubo 1 , S. Ishida 1 , M. Usami 1 , Y. Sekino 1 1
Division of Pharmacology, National Institute of Health Sciences, Tokyo, Japan, 2 Division of Medicinal Safety Science, National Institute of Health Sciences, Tokyo, Japan, 3 Division of Pharmacognosy and Phytochemistry, National Institute of Health Sciences, Tokyo, Japan Linuron is a substituted urea herbicide that is used in large quantities all over the world. In Japan, 76,000–111,000 kg of linuron was imported annually in recent three years (2006–2008), as reported by Japan Plant Protection Division (2009). So far, we have shown that linuron induced O-deethylation activities of ethoxycoumarin and ethoxyresorufin, in rat hepatocytes both in vitro and in vivo. In this study, we investigated the inducibility of CYP1A by linuron in human hepatocytes. The dose dependency of CYP1A induction by linuron was determined and compared with prototypical inducers, 3-methylcholanthrene (3-MC) and omeprazole (OPZ). Primary cultured human hepatocytes were exposed to linuron (1–125 M), 3-MC or OPZ for 48 h. Medium containing the inducer was renewed every 24 h. Induction of CYP1A was determined as increased ethoxyresorufin O-deethylation activity. The amounts of protein and mRNA of CYP were determined by Western blotting and RT-PCR, respectively. Cytotoxicity on hepatocytes was assessed by LDH leakage assay. Linuron induced CYP1A activity in a dose dependent manner at 5 M or more, with increased protein and mRNA of CYP1A1/2. Linuron showed strong inducibility of human CYP1A activity. The rank order of the inducibility was 3-MC > OPZ > linuron. The inducibility of CYP1A by linuron was stronger in humans than in rats. These results suggest that CYP1A induction by linuron affects the hepatic drug-metabolizing enzyme system and endogenous hormone regulation in humans.
1
Objectives: Pesticide self-poisoning is a major problem in developing countries, especially in rural areas, and an important reason is that hazardous pesticides are available without restrictions. Organophosphates (OPs) are the most commonly used pesticides for suicidal poisoning causing over 200,000 deaths in the world yearly. The authors present an alarming increase in fatal OP-related self-poisonings from 1996 to 2006 in the southeastern region of Serbia. Methods: Descriptive, retrospective epidemiological study. Results: National statistical data in Serbia show a 2.7% decrease in the total number of suicides from 1996 to 2006. In the same period, the number of fatal self-poisonings increased by 18%, from 108 to 128. Niˇs is the centre of the southeastern part of Serbia, which is a low-income region with a predominantly rural population. In the University Clinical Centre in Niˇs, the total number of cases of fatal
doi:10.1016/j.toxlet.2011.05.801
P2178 The effect of crocin and safranal, constituents of saffron, against subacute effect of diazinon on three biomarkers TNF-␣, Direct 8-iso-prostaglandin F2␣ and S100 in rats A. Timchehhariri 1,∗ , H. Hosseinzadeh 2 , S.A. Moalem 2 1
Pharmacy School, Mashhad University of Medical Sciences, Khorasan Razavi, Iran, 2 Mashhad University of Medical Sciences, Mashhad, Iran Diazinon after malathion is one of the most commonly used OPs in the world. Carotenoids by acting as biological antioxidants, protect cells and tissues from damaging effects of free radicals. In this study, the effect of crocin and safranal was studied
Abstracts / Toxicology Letters 205S (2011) S180–S300
against subacute toxicity of diazinon (DZN) on three biomarkers TNF-␣, ␣-iso prostan and S100 in rats. Vitamin E (200 IU/kg), safranal at doses 0.025, 0.05 and 0.1 ml/kg and crocin at doses 50, 100 and 200 mg/kg were injected intraperitoneally three times per week alone or with DZN (20 mg/kg/day, orally) for 4 weeks. Biomarkers were evaluated at the end of 4 weeks. The evaluation of biomarkers was done using ELISA. The levels of serum TNF-␣, Direct 8-iso-prostaglandin F2␣ and S100 increased significantly in diazinon treated group compared with the control group. Direct 8-iso-prostaglandin F2␣ level significantly decreased in diazinon + crocin (all doses) and diazinon + safranal (all doses) treated groups when compared to diazinon treated group. S100 level significantly decreased in diazinon + crocin 100 and 200 mg/kg and diazinon + safranal 0.1 ml/kg treated groups when compared to diazinon treated group. TNF-␣ level significantly decreased in diazinon + crocin 50 and 100 mg/kg treated group while no significant changes in diazinon + safranal (all doses) and diazinon + vitamin E when compared to diazinon treated group, and also this biomarker significantly increased in safranal 0.1 ml/kg treated group compared with the control group. Vitamin E, safranal (0.025 or 0.05 ml/kg) and all doses of crocin decreased this effect of diazinon. In all doses of crocin our study showed that diazinon changes these three biomarkers and even if it is administrated less than the LD50 dose level. Vitamin E, safranal in low doses and crocin in three doses reduced diazinon toxicity. doi:10.1016/j.toxlet.2011.05.802
P2179 Mechanism of nonlinear effects of plant growth regulators – N-oxide derivates O. Vasetska ∗ , O. Fedchenko, P. Zhminko, M. Prodanchuk Medved‘s Institute of Ecohygiene and Toxicology, Kyiv, Ukraine The prominent feature of some plant growth regulators (PGR) is amplification effects or change of the action directivity at low doses, that is, absence of classical linear dependence “dose-effect” and manifestation of the so-called “paradoxical effect”. The mechanism of nonlinear effects of the PGR (pyridine N-oxide derivatives) in the organism of animals can be explained by example of the Ivin (N-oxide 2,6-dimethylpyridine). Since the Ivin molecule may be presented in the hydrated/dehydrated state, then at high concentrations Ivin interacts with membranes in hydrated form. As a result of membrane modification, their destabilization occurs as well as the damage of the structural integrity of lipid matrix. This can lead to changes in cellular signal transduction, reduction of the protein-synthetic processes and decrease of reparative system function, therefore the toxic effect increases. At low concentrations, Ivin interacts with membrane lipids in dehydrated state. The dehydrated Ivin causes the lipid compaction and contributes to the membrane stabilization. Moreover, it can be observed changes in cellular signal transduction, increase of protein-synthetic processes in the organism, improvement of the repair system function, and in consequence of this an adaptation to acting factor takes place. Under the condition of balance of hydrated and dehydrated forms of Ivin molecule, there is the leveling of the Ivin effect and therefore the inversion occurs (no toxic effect). doi:10.1016/j.toxlet.2011.05.803
S235
Safety pharmacology P2180 Flavin-containing monooxygenase 3 gene polymorphisms in Turkish population B. Alpertunga ∗ , G. Ozhan, P. Topal Department of Pharmaceutical Toxicology, Istanbul University, Faculty of Pharmacy, Istanbul, Turkey Purpose: Flavin-containing monooxygenases (FMOs), one of the most important monooxygenase systems, are involved in xenobiotics metabolism and variability in drug response. FMO gene catalyzes the monooxygenation of numerous N-, Pand S-containing xenobiotics. Substrates of FMO include many drugs (cocaine, nicotine, imipramine, tamoxifen, cimetidine, etc.), chemicals (phorate, aldicarb, etc.) and endogenous substrates (methionine, trimethylamine, etc.). FMO3 is associated with the majority of FMO-mediated adult hepatic metabolism. In the adult human liver, FMO3 is the prominent form and other FMOs are not expressed or are expressed at very low levels. Methods: 13 of totally 40 genomic FMO3 variants are Single Nucleotide Polymorphisms (SNPs) which cause low function. Allele frequencies of three functional SNPs (E158K, V257M, and E308G) vary greatly among different ethnic populations with distinct genetic backgrounds. The present study aimed to determine frequencies of FMO3 polymorphisms and haplotypes in Turkish population. We genotyped in 120 healthy volunteers using polymerase chain reaction (PCR)restriction fragment length polymorphism (RFLP) methods. For statistical analysis, the computer software SPSS was used. Haplotype frequencies were estimated from genotype data by PHASE software based on Bayesian algorithm. The results were compared with those obtained from different populations. Results: The study on alleles of FMO3 gene could be useful in understanding the variant and haplotype distributions of FMO3 alleles in Turkish population because FMO3 alleles, which can be observed on various races and even on sub-populations, have not been researched in Turkey even though it has been researched on different populations. doi:10.1016/j.toxlet.2011.05.804
P2181 Anti-inflammatory effect of Thymus kotschyanus extract on rat’s hind paw edema induced by carrageenan A. Bakhtiarian 1,∗ , M. Khanavi 2 , F. Aarabi Moghaddam 1 1
Department of Pharmacology, Tehran University of Medical Sciences, Tehran, Iran, 2 Department of Pharmacognosy, Tehran University of Medical Sciences, Tehran, Iran Purpose: The advantages of herbal drugs have been known to human beings for many years and they have been used for the treatment of many diseases. Nowadays, inflammation is known to be one of the basic pathologic causes of diseases. In this study, the anti-inflammatory effects of Thymus kotschyanus were investigated. Methods: Forty two wistar rats were divided into three groups. In the first group, the extract of Thymus kotschyanus in doses of 50, 100, 150, and 200 mg/kg was injected intra peritoneal and after 1 h, they were treated with the hind paw edema test with carrageenan. In the second group, Indomethacin with the dose of 5 mg/kg was injected 1 h before carrageenan and in the third one, saline was injected before carrageenan, to the rat’s right hind paw.