Comet assay in gill cells of Prochilodus lineatus exposed in vivo to cypermethrin

Agricultural chemicals can induce genetic alterations on aquatic organisms that have been associated with effects on growth, reproduction and population dynamics. The evaluation of DNA damage in fish using the comet assay (CA) frequently involves the utilization of erythrocytes. However, epithelial gill cells (EGC) can be more sensitive, as they are constantly dividing and in direct contact with potentially stressing compounds from the aquatic environment. The aim of the present study was to evaluate (1) the sensitivity and suitability of epithelial gill cells of Prochilodus lineatus in response to different genotoxic agents through the application of the CA, (2) the induction of DNA damage in this cell population after in vivo exposure to cypermethrin. Baseline value of the CA damage index (DI) for EGC of juvenile P. lineatus was 144.68 ± 5.69. Damage increased in a dose-dependent manner after in vitro exposure of EGC to methyl methanesulfonate (MMS) and H₂O₂, two known genotoxic agents. In vivo exposure of fish to cypermethrin induced a significant increase in DNA DI of EGC at 0.150 μg/l (DI: 239.62 ± 6.21) and 0.300 μg/l (270.63 ± 2.09) compared to control (150.25 ± 4.38) but no effect was observed at 0.075 μg/l (168.50 ± 10.77). This study shows that EGC of this species are sensitive for the application of the CA, demonstrating DNA damage in response to alkylation (MMS), oxidative damage (H₂O₂), and to the insecticide cypermethryn. These data, together with our previous study on DNA damage induction on erythrocytes of this species, provides useful information for future work involving biomonitoring in regions where P. lineatus is naturally exposed to pesticides and other genotoxic agents.     

Monocrotophos augments the early alterations in lipid profile and organ toxicity associated with experimental diabetes in rats

The present study was undertaken to investigate the potential of monocrotophos (MCP), a widely used organophosphorus insecticide, to induce hyperglycemia and its interactive ability to accentuate the early diabetic outcomes and associated complications in experimentally rendered diabetic rats. Rats were rendered diabetic with an acute dose (60 mg/kg b.w, i.p) of streptozotocin. MCP was orally administered at a sublethal dose (1/20 LD_50, 0.9 mg /kg b.w./d, 5 days) to both normal and diabetic rats. While MCP per se moderately increased (by 25%) the blood glucose levels in normal rats, it significantly aggravated the hyperglycemic outcome in diabetic rats (56% above diabetic rats). Further, experimentally-induced diabetes was associated with only a marginal increase in total and HDL-cholesterol levels, while serum triglyceride levels were significantly enhanced. Although MCP per se did not affect these parameters, it caused a marked increase in triglyceride levels in serum of diabetic rats (54% above diabetic control). Furthermore, MCP-induced higher activity levels of serum transaminases viz., ALT and AST (51% and 71% higher as compared to diabetic control) suggestive of enhanced hepatic damage. Collectively, our findings provide evidence that MCP on repeated exposure has the propensity to augment the secondary complications associated with diabetes in a rat model.     

May antioxidants status depletion by Tetradifon induce secondary genotoxicity in female Wistar rats via oxidative stress?

Tetradifon is a potent organochlorine acaricide with an estrogen-like structure. The wide spread use of this chemical is likely to pollute the environment. Only few studies have been reported for the evaluation of its short- and long-term toxic effects including genotoxicity and carcinogenicity and there have been conflicting results in in vitro and in vivo test systems. In this work, we have evaluated Tetradifon for its ability to induce genotoxic damages in female Wistar rats. A single cumulative dose of 2430 mg/kg BW was administrated orally for 12 female rats of 190 g BW during 12 weeks. Twelve additional rats, no treated, have served as control. Animals were sacrificed after 6 and 12 weeks of treatment. Genetic toxicity studies were conducted in rats bone marrow, by chromosomal aberrations (CAs) and sister-chromatid exchanges (SCEs) assays. The oxidative stress status of treated animals has been also evaluated by assessment of lipid peroxidation by measuring thiobarbituric acid reactive substances (TBARS). Some serum parameters: vitamins (A and E), triglyceride (TG) and total antioxidants status (TAS) were determined. Our results showed a significant increase in tissue TBARS concentrations in the two treated groups suggesting that Tetradifon induce an oxidative stress. Elsewhere, rats treated with Tetradifon exhibited a statistical decrease in serum level of vitamin E and a significant depletion of serum total antioxidant status. Whereas, in comparison to control rats, treated animal cells did not show a significant increase in either the frequency of SCEs or CAs. These results indicate that Tetradifon did not present direct genotoxic effect in female Wistar rats. But we suggest that its inducting of an oxidative stress may lead to indirect mutagenecity that should be evaluated by other series of in vivo genotoxicity assays as micronucleus test or comet assay.     

Modulation of ethion-induced hepatotoxicity and oxidative stress by vitamin E supplementation in male Wistar rats

Organophosphorus insecticides (OPIs) may induce oxidative stress leading to generation of free radicals and alteration in antioxidant system of animals. Many studies reported that enzymatic and non-enzymatic antioxidant may play protective role against OPIs induced toxicity in human and rats. The aim of present study was to investigate the possible protective role of vitamin E on ethion-induced hepatotoxicity in rats using qualitative, quantitative and biochemical approaches. Adult male albino rats of Wistar strain were randomly divided into four groups; each group consists of six animals. Animals were treated for a period of 28 days. Group I (control group received corn oil); Group II [ethion treated (2.7 mg/kg bw/day)]; Group III (vitamin E treated (50 mg/kg of bw/day)]; Group IV (ethion + vitamin E treated). Animals were sacrificed after 7, 14, 21 and 28 days by decapitation and liver tissue was used for the measurement of proteins, lipid peroxidation (LPO), reduced glutathione (GSH) content and activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) glutathione reductase (GR) and glutathione-S-transferase (GST). Erythrocytes were analyzed for acetyl cholinesterase activity. The result of this study shows that in vivo administration of ethion caused a significant induction of oxidative damage in liver tissue as evidenced by increased level of LPO and decreased GSH content. Ethion toxicity also led to a significant increase in the activities of SOD, CAT, GPx and GST in liver tissue. In addition, decrease in GR activity was observed in ethion administered rats compared to control. Histopathological findings revealed that exposure to ethion caused damage in liver tissue. However, simultaneous supplementation with vitamin E restored these parameters partially. In conclusion, the results of the current study revealed that ethion-induced toxicity caused lipid peroxidation, alterations in the antioxidant enzymes and histopathological changes in liver. Supplementation of vitamin E exhibited protective effect by inhibiting ethion-induced toxicity in liver and erythrocytes.     

Oxidative parameters of Rhamdia quelen in response to commercial herbicide containing clomazone and recovery pattern

In this study, fish Rhamdia quelen, were exposed to different concentrations of herbicide clomazone: 0.0 (control), 0.45 and 0.91 mg L⁻¹. After exposure for 8 days to herbicide, fish were transferred to clean water for a recovery period (8 days). Oxidative stress indicators such as thiobarbituric acid reactive substances (TBARS) levels and protein carbonyl content, as well as antioxidant defenses, such as catalase (CAT), superoxide dismutase (SOD), glutathione S-transferase (GST), ascorbic acid and non-protein thiols levels were studied, using the liver, brain and muscle tissues. Herbicide exposure increased TBARS in muscle and in liver at higher concentration. In liver protein carbonylation increased and catalase activity did not change in fish exposed to herbicide. SOD enhanced in liver at concentration of 0.91 mg L⁻¹. GST, ascorbic acid and non-protein thiols levels increase at both concentrations. At the end of the recovery period the most of the parameters recovered whereas GST and ascorbic acid remain elevated. The present study demonstrates the occurrence of disorders in antioxidant parameters and importance in the assessment of the potential risk of herbicides as clomazone on fish species.     

Subacute oral toxicity of chlorpyriphos and protective effect of green tea extract

This paper reports the effect of green tea administration following subacute toxicity caused by exposure to organophosphorus pesticide chlorpyriphos in liver of rats. Four groups containing five male Sprague-Dawley rats each were selected. Group I served as control. Group II rats were permitted free access to solubilised crude extract of green tea (1.5%w/v in water) as the sole drinking fluid. Group III rats were given a single daily oral dose of chlorpyriphos (30 mg/kg bodyweight in corn oil). Group IV rats received oral dose of pesticide and green tea extract simultaneously. All rats were sacrificed after 15 days. Significant damage to liver was observed via increased serum levels of transaminases and alkaline phosphatase. Lipid peroxidation showed a 5-fold increase in pesticide exposed rats compared to control. In contrast, levels of antioxidant GSH, glutathione-dependent enzymes like glutathione peroxidase (GPx), glutathione S-transferase (GST) and free radical scavengers like catalase (CAT) and superoxide dismutase (SOD) were significantly lower than those of the control group reinforcing oxidative damage. The use of green tea extract appeared to be beneficial to rats, although not to a great extent in significantly reducing and reversing the damage sustained by pesticide exposure and favors recovery.     

Mutagenic, genotoxic and enzyme inhibitory effects of carbosulfan in rainbow trout Oncorhynchus mykiss

Rainbow trout (Oncorhynchus mykiss; 116.88 ± 21.69 g) were exposed to sublethal concentrations (25 μg/L) of carbosulfan for 60 days to test if the long term exposure of fish to carbosulfan affects red blood cells acetylcholinesterase (AChE), δ-aminolevulinic acid dehydratase (ALA-D) and paraoxonase (PON) enzyme activity and induces genotoxic and/or mutagenic effects. The exposure resulted in inhibition of AChE and ALA-D activity of rainbow trout when compared to control fish. The activity of PON was not affected by carbosulfan. Interestingly, carbosulfan was found to induce DNA damage in red blood cells (comet assay) and proved to be mutagenic as revealed by the Ames test. Results indicate that blood AChE and ALA-D of rainbow trout may be a sensitive biomarker for assessment of carbosulfan contaminated water bodies. Furthermore, because the Ames test and comet assay were proven successful to detect the genotoxicity of carbosulfan, we proposed that nonlethal techniques such as blood collection from caudal vein of fish should be used to determine potential toxic effects of other pesticides to surrounding environment.     

Oxidative stress and locomotor behaviour response as biomarkers for assessing recovery status of mosquito fish, Gambusia affinis after lethal effect of an organophosphate pesticide, monocrotophos

Recovery study was performed at regular intervals to establish the time course of 50% and 100% recovery in neurotransmitter enzyme (acetylcholinesterase, AChE, EC 3.1.1.7) and locomotor behaviour response of mosquito fish, Gambusia affinis exposed to lethal concentration (20.49 mg L⁻¹) of an organophosphorous pesticide, monocrotophos (MCP) for 96 h. In vitro AChE activity studies indicated that MCP could cause 50% inhibition (I₅₀) at 10.2 × 10⁻⁵ M. A positive correlation was observed between brain AChE activity and swimming speed during the recovery study. Also, the recovery response of the antioxidant enzymes superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6) and glutathione reductase (GR, EC 1.6.4.2) as well as lipid peroxidation (LPO) as biomarkers of oxidative stress were assessed in viscera of G. affinis. The results showed that the MCP besides its inhibitory effect on target enzyme AChE activity and induction in antioxidant enzyme activities as a characteristic of oxidative stress, which can be used as biomarkers in the pesticide contaminated aquatic streams.     

Nephrotoxicity in rats induced by organophosphate insecticide methidathion and ameliorating effects of vitamins E and C

The effects of organophosphate insecticide methidathion (MD) on kidney tissue and the ameliorating effects of a combination of vitamins E and C against subchronic MD toxicity were evaluated in rats. Experimental groups were: control group (group I), 5 mg/kg body weight MD-treated group (group II), and 5 mg/kg body weight MD plus vitamin E plus vitamin C treated group (group III). The groups II and III were treated orally with MD on five days a week for four weeks. Vitamins E and C were injected at doses of 50 mg/kg body weight, i.m. and 20 mg/kg body weight, i.p., respectively, 30 min after the treatment of MD in the group III. Rats were anaesthesized and venous blood samples were collected by direct right ventricle heart puncture, in addition, the right kidney was removed for histopathological examinations and malondialdehyde (MDA) analyses after four weeks. The serum activity of cholinesterase (ChE) and the kidney level of malondialdehyde, and kidney histopathology were studied in rats. MD caused decreased ChE activity (group I: 2114 ± 63 U/L, group II: 1455 ± 100 U/L) and increased MDA level (group I: 147 ± 20.2 nmol/mg protein, group II: 236 ± 25.6 nmol/mg protein), and kidney damage in rats. Furthermore, a combination of vitamins E and C restored partially (ChE activity: 1670 ± 111 U/L, MDA level: 159 19.4 nmol/mg protein) this changes in MD-treated rats.     

Block of electron transport by surangin B in bovine heart mitochondria

Exposure of mitochondria isolated from bovine heart to the insecticidal coumarin surangin B results in inhibition of complex II (IC₅₀ = 0.2 μM), III (IC₅₀ = 14.8 μM), and IV (IC₅₀ = 3.1 μM), but in contrast, the NADH:ubiquinone reductase (complex I) was completely insensitive to this compound at 100 μM. Kinetic analysis of surangin B’s interaction with complex II was then investigated using sub-mitochondrial particles. With succinate as the substrate, surangin B, like carboxin, acted with non-competitive kinetics and clearly contrasted in its action with malonate, a competitive inhibitor of complex II. Likewise, surangin B acted as a non-competitive inhibitor of decylubiquinone-dependent interception of electrons at complex II. Difference spectra of reduced complex III equilibrated with surangin B were found to closely parallel those of antimycin A, but were different in nature to those of the Q_o site inhibitors myxothiazol and famoxadone. Investigation of surangin B-dependent functional perturbation of complex III used the synthetic electron acceptor 2-nitrosofluorene, which intercepts electrons specifically from the Q_i site. These experiments demonstrated that like antimycin A, surangin B acts as a selective blocker of electron diversion to 2-nitrosofluorene through Q_i within complex III. We conclude that surangin B blocks electron transport at several points in bovine heart mitochondria, however, complex I is spared. The potent inhibitory action of surangin B on complex II involves binding to a site which is distinct from both the succinate binding site and the domain responsible for interacting with ubiquinone. Surangin B apparently blocks complex III by interacting with the Q_i (antimycin A-binding) pocket.     

Determination of genotoxic effects of chlorfenvinphos and fenbuconazole in Allium cepa root cells by mitotic activity, chromosome aberration, DNA content, and comet assay

Genotoxic effects of Chlorfenvinphos and fenbuconazole were examined by using mitotic index, mitotic phase, chromosomal abnormalities, 2C DNA content and Comet assay on the root meristem cells of Allium cepa. The roots were treated with 10, 20, 40, 60, 80, and 100 ppm concentrations for 24 and 48 h. The results indicated that Chlorfenvinphos and fenbuconazole significantly decreased the mitotic index in all treatments when compared with their controls. The percentages of mitotic phases have changed. Chlorfenvinphos and fenbuconazole significantly increased the abnormal cell frequency at all concentrations and treatment periods when compared with their controls. Different abnormal mitotic figures were observed in all mitotic phases. Among these abnormalities were stickiness, anaphase bridges, c-mitosis, laggards, and micronucleus. These pesticides remarkably depressed the 2C DNA content in the root meristems of A. cepa. The genotoxicity of chlorfenvinphos and fenbuconazole in A. cepa root cells was analyzed using comet assay, which allows the detection of single strand breaks. In all concentrations, chlorfenvinphos and fenbuconazole induced a significant increase in DNA damage. Additionally, it was also researched to determine if there is a relation between the amount of DNA and the DNA damage and a regression analyses was conducted. When the data that was accumulated via comet analysis from A. cepa root tip cells that are treated with type chlorfenvinphos and fenbuconazole, was compared to the data that was acquired as the result of the measurement of 2C DNA amount, a relation with negative correlation was found, (respectively, r = −0.80 and r = −0.82). This relation factor is statistically important and strong (p < 0.05).     

Protective roles of vitamin E (α-tocopherol), selenium and vitamin E plus selenium in organophosphate toxicity in vivo: A comparative study

In the present study, we investigated the possible protective role of vitamin E, selenium (Se) and vitamin E plus Se in fenthion-induced organophosphates (OP) toxicity in rats. Serum concentrations of ascorbic acid, retinol, β-carotene, ceruloplasmin, nitrite and nitrate as well as levels of malondialdehyde (MDA) and reduced glutathion (GSH) in whole blood and in some tissues such as brain, heart, jejunum, kidney, liver, lung, muscle and pancreas were measured in sham, control, vitamin E, Se and vitamin E + Se groups. Compare to the sham group, the MDA (p < 0.001) and GSH (p < 0.01) levels in whole blood and some in tissues were significantly higher in the control animals. Ceruloplasmin levels of the control (p < 0.05), vitamin E (p < 0.05) and vitamin E + Se (p < 0.01), groups were higher than the sham group. Ascorbic acid, retinol, β-carotene as well as nitrite and nitrate levels in the control group were significantly lower than sham, vitamin E, Se and vitamin E + Se groups. We concluded that fenthion toxicity-induced lipid peroxidation and generation of free radicals in whole blood and tissues. Additionally, the antioxidants we tested did show a significant protective effect against OP-induced tissue and blood injury at the biochemical level.     

Neurotoxic effects of subacute exposure of dichlorvos and methyl parathion at sublethal dosages in rats

The present study was designed to understand the effects of sublethal dosages of dichlorvos (DIC) and methyl parathion (MP) on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in various tissues of rats exposed to 5 and 10 ppm of DIC and MP in drinking water ad libitum for 28 days continuously. According to the results, AChE activity was significantly decreased in all the tissues of rats treated with both dosages of DIC and MP except for in the lungs treated with both dosages of DIC. With regard to the BChE, MP caused a significant decrease in the liver, heart and lungs with 5 ppm dosage whereas it did not change the BChE activity in the other tissues with two dosages. Also, DIC caused a significant decrease in BChE activity in the heart tissue treated with both dosages and in the brain of rats treated with 5 ppm. The observations presented led us to conclude that the administrations of MP and DIC at sublethal concentrations inhibited AChE and BChE activities in the rats. These results suggest that inhibition of AChE may be a better biomarker for the assessment of neurotoxic effects in the living.     

Chronic chlorpyrifos-induced oxidative changes in the testes and pituitary gland of Wistar rats: Ameliorative effects of vitamin C

Chlorpyrifos (CPF), a chlorinated organophosphate insecticide that is widely used in agriculture and public health, has been implicated in male reproductive toxicity. Apart from acetylcholinesterase inhibition, CPF has been shown to induce changes characteristic of oxidative stress. Therefore, the aim of the present study was to evaluate the effects of vitamin C on oxidative changes in the testes and pituitary gland of rats chronically exposed to CPF. Twenty adult male Wistar rats were divided into four groups of five animals each: Group I (S/oil) received soya oil (2 ml/kg); Group II (VC) was administered with vitamin C (100 mg/kg); Group III (CPF) was given CPF (10.6 mg/kg; ∼1/8th LD₅₀); Group IV (VC + CPF) was pretreated with vitamin C (100 mg/kg) and then given CPF (10.6 mg/kg), 30 min later. The regimens were administered orally by gavage once daily for 15 weeks. Thereafter, the rats were sacrificed and the testes and pituitary glands were evaluated for the concentration of malonaldehyde (MDA) and activities of superoxide dismutase (SOD) and catalase (CAT). The result shows that CPF increased MDA concentration and reduced activities of SOD and CAT, which were ameliorated by pretreatment with vitamin C.     

Protective effects of caffeic acid phenethyl ester on rotenone-induced myocardial oxidative injury

Rotenone, an insecticide, causes toxicity through inhibition of mitochondrial electron transport chain at complex I and oxidative injury to the tissues. The aim of the present study was to determine in vivo effects of rotenone on myocardium and cardio-protective effects of caffeic acid phenethyl ester (CAPE), an antioxidant agent, against rotenone toxicity in rats. The rats were divided into three groups: untreated control, rotenone (2.5 mg/kg/day for 60 days, i.p.) and rotenone + CAPE groups. CAPE was administrated i.p. 10 μmol/kg/day for 62 days started two days before first dose rotenone injection. The malondialdehyde, nitric oxide levels and xanthine oxidase activity of rotenone group was significantly higher than control and rotenone + CAPE groups (p < 0.05). However, catalase activity in the rotenone group was decreased in comparison with the other groups (p < 0.05). The superoxide dismutase activity of rotenone group was insignificantly decreased compared to the others. In conclusion, rotenone caused lipid peroxidation in myocardial tissue and CAPE treatment prevented this rotenone-induced lipid peroxidation in rats. CAPE might be a cardio-protective agent against myocardial toxicities.     

Dimethoate induced biochemical perturbations in rat pancreas and its attenuation by cashew nut skin extract

The significant antiradical activity of cashew skin extract was previously described. In this investigation, the extent of protection offered by cashew nut skin extract (CSE) against the damage induced in rat pancreas by sub chronic doses dimethoate (DM), an organophosphorous pesticide was studied. Rats were supplemented with CSE at 20 mg/kg b.w./d after a daily dose of DM at 40 mg/kg/d b.w. for 2 months. Weekly random blood glucose, oral glucose tolerance test (OGTT); pancreatic damage markers like amylase and lipase; oxidative damage markers such as reactive oxygen species generated, extent of lipid peroxidation, host antioxidant defenses like reduced glutathione (GSH); GSH-dependent enzyme activities viz., glutathione peroxidase (GPx), glutathione S-transferase (GST), glutathione reductase (GR); free radical scavenger enzymes viz., catalase and superoxide dismutase (SOD); xenobiotic metabolizing enzymes like DT-diaphorase and NADPH-diaphorase were measured in the four different groups namely (1) control, (2) DM treated, (3) CSE supplemented, (4) CSE supplements following DM treatment. Random blood glucose levels increased significantly on exposure to DM compared to that in control rats (119 ± 5 mg/dl vs. 92 ± 4 mg/dl), while the blood glucose levels in CSE supplemented rats were comparable to that of controls. DM treated rats exhibited impaired glucose tolerance at the end of two months as indicated by OGTT, while DM treated rats with CSE supplements showed normal glucose tolerance. Pancreatic specific marker enzymes like amylase and lipase in serum were restored to normalcy in rats supplemented with CSE following treatment with DM which otherwise was increased in the DM treated rats. Distinctly lower levels of GSH, increased levels of ROS, higher extent of lipid peroxidation, along with alterations in antioxidant enzymes and increase in xenobiotic metabolizing enzymes were evident in pancreas of DM treated rats. However, CSE supplement ameliorated the biochemical alterations in the pancreatic milieu in DM treated rats. Treatment with CSE significantly protected rat pancreas from injury, thus ameliorating and restoring tissue antioxidant status and also conferring normal glucose tolerance. The active components present in cashew skin extract can perhaps be effective in reducing the extent of pancreatic injury and in overcoming tissue damage caused by exposure to dimethoate.     

Effects of diazinon at different doses on rat liver and pancreas tissues

Diazinon is one of the most widely used organophosphates in agriculture. Toxic effects of diazinon are due to the inhibition of acetylcholinesterase, an enzyme needed for proper nervous system function. This study was designed to investigate the effects of diazinon at different doses on pancreas and liver tissues and in which dose level diazinon shows its effects. Sixty male Wistar albino rats were included in this study. Animals were initially divided into control and diazinon given groups. There were 10 animals in the control group and 50 animals in diazinon administered group. The latter was divided into five equal subgroups: 25, 50, 100, 200 and 300 mg/kg of diazinon administered groups. Control group was given only saline. All animals in 300 mg/kg diazinon group died. After 24 h, rats were sacrificed under ether anesthesia. Tissue and blood samples were taken for biochemical and histopathological analysis. Sample tissues were examined under light microscope. In biochemical analysis, AST, ALT, LDH, amylase and lipase enzyme activities were measured. One-way ANOVA test was used to compare the groups. In 200 mg/kg diazinon given group, it has been observed some histopathological changes in pancreas and liver tissues. Cholinesterase activities were significantly decreased and alkaline phosphatase levels were increased in all diazinon given groups, when compared with the controls. There was statistically significant difference between the control and diazinon given groups by means of serum amylase, lipase, ALT and AST activities (p < 0.05). LDH activities were significantly increased in 100 and 200 mg diazinon given groups, when compared with the controls (p < 0.05). Histopathological changes were observed only in 200 mg diazinon given group. This evidence suggest that diazinon effect is dose dependent and this is possibly 10-15% of the LD₅₀ dose (200 mg/kg), which cause acute pancreatitis and histopathological changes in liver.     

A two generation chronic mixture toxicity study of Clophen A60 and diethyl phthalate after gestational and lactational exposure in female Wistar rats

Polychlorinated biphenyls and diethyl phthalate are both lipophilic in nature and are likely to be present in the same environmental compartment or bioaccumalate over a period of time, thus a mixture toxicity study was undertaken to evaluate the type of interaction between polychlorinated biphenyls (Clophen A60) and diethyl phthalate over two generations in female Wistar rats. Healthy male and female albino rats of Wistar strain weighing 75-100 g (6-7 weeks old) were randomly assigned to four groups of six each. Group I male and female rats were fed on normal diet and water ad libitum. Group II male and female rats were maintained on normal diet mixed with corn oil as oil control. GroupS III and IV male and female rats were given Clophen A60 and diethyl phthalate dissolved in corn oil mixed with the diet at 50 mg/kg of the diet individually to each group. Group V male and female rats received a mixture of diethyl phthalate and Clophen A60, each dissolved in corn oil mixed with the diet at 50 mg/kg of the diet. Hundred days after the treatment, females were mated with the males in each group for 10 days. Exposure to diethyl phthalate and Clophen A60 was continued throughout mating, gestation until termination at weaning, which was 150 days of total treatment period of the parental generation female rats. Treatment for F1 generation male and female pups (6 males & 6 females) with Clophen A60 and diethyl phthalate individually and in mixture was continued at doses reduced to 25 mg/kg of the diet after they reached 75-100 g in weight. The treatment was carried out similar to the parental generation for a period of 150 days. Liver and serum aspartate aminotransferase, liver cholesterol and glycogen were significantly increased in the F1 generation Clophen A60 + diethyl phthalate treated group, whereas serum cholesterol, liver glutathione and glutathione reductase showed a significant decrease in the F1 generation Clophen A60 + diethyl phthalate treated group as compared to the parental generation mixture and individually treated groups as well as the individually treated F1 generation groups. A significant increase was observed in the liver and serum aspartate aminotransferase activity of Clophen A60 and serum aspartate aminotransferase levels of diethyl phthalate treated F1 generation rats as compared to the parental generation Clophen A60 and diethyl phthalate individually and mixture treated rats. Liver glutathione levels were significantly decreased in the F1 generation Clophen A60 and diethyl phthalate individually treated rats which was similar to the parental generation individually treated rats as compared to the controls. Liver glutathione reductase level was also significantly declined in the diethyl phthalate treated F1 individual group as compared to diethyl phthalate individually treated parental generation rats. Histology of the liver showed fatty degeneration in the mixture treated F1 generation rats as compared to Clophen A60 and diethyl phthalate individually treated F1 rats and parental generation Clophen A60 and diethyl phthalate individually and mixture treated rats. Thus, in spite of dose reduction and continuous exposure over two generation’s to a mixture of diethyl phthalate and Clophen A60 exposed through gestation, lactation and diet leads to a synergistic toxic effect in the F1 generation.     

Avermectin exposure induces apoptosis in King pigeon brain neurons

The effect of avermectin was studied on King pigeon brain nerve cells by cytotoxicity [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, MTT] and apoptosis [acridine orange/ethidium bromide (AO/EB) assay, transmission electron microscope (TEM) evaluation, measurement of mitochondrial membrane potential (Δψm), phosphatidylserine (PS) exposure, caspases activities, DNA fragmentation, reactive oxygen species (ROS) and caspase-3 mRNA expression] within the 2.5–10 μg L⁻¹ concentration-range. The results revealed that within the concentrations of 2.5–10 μg L⁻¹, avermectin showed obvious cytotoxicity and induced apoptosis in a dose-dependent manner to neurons of King pigeon in vitro. Cell viability were 99.93 ± 8.52%, 82.02 ± 4.99% and 78.23 ± 5.67% after 24 h of treatment with avermectin at the concentrations of 0, 2.5 and 5 μg L⁻¹, which decreased to 56.36 ± 2.17% of 10 μg L⁻¹. Treated cells showed typical apoptosis morphological changes including cytoplasmic vacuolation, chromatin condensation, unclear nuclear membrane and decreased/swollen mitochondria. Typical biochemical hallmarks of apoptosis including Δψm loss, PS exposure, activations of caspase-3, caspase-8 and caspase-9, DNA fragmentation were observed too. Moreover, the levels of ROS in the avermectin treatment groups increased significantly compared to control group. Furthermore, the caspase-3 mRNA levels increased significantly following AVM treatment. In conclusion, our experimental results show that avermectin has cytotoxicity to brain neurons of King pigeon in vitro and the mechanism of neurotoxicity induced by avermectin is closely related to apoptosis.