Impact of phorate on malate dehydrogenases, lactate dehydrogenase and proteins of epigeic, anecic and endogeic earthworms

The effects of an organophosphate pesticide phorate on cytoplasmic malate dehydrogenase (cMDH), mitochondrial malate dehydrogenase (mMDH), lactate dehydrogenase (LDH), supernatant and mitochondrial proteins of an epigeic (Perionyx sansibaricus), anecic (Lampito mauritii) and endogeic (Metaphire posthuma) earthworms were studied. The treatment of different concentrations (20, 40, 80 and 160 ppm) of phorate for 16 days gradually decreased the specific activities of cMDH, mMDH and LDH as well as cytoplasmic and mitochondrial protein contents. This showed the inhibitory effect of phorate on metabolic enzymes and proteins in tropical earthworms. The inhibition was dose- and time-dependent. The inhibitory response in mitochondrial enzyme (mMDH) and protein was somewhat earlier and more as compared to the inhibitory effect of phorate on cytoplasmic enzymes (cMDH, LDH) and protein. This indicates a greater interference of phorate in cellular respiration of earthworms. The phorate related decreases in enzyme and protein profiles were about 60% and 58% in P. sansibaricus, 54% and 49% in L. mauritii and 47% and 42% in M. posthuma, respectively. It reflects phorate-induced substantial decline in protein synthesis and aerobic and anaerobic capacity of earthworms. The maximum effect of phorate was on epigeic earthworm followed by anecic and endogeic species. The present findings suggest the differential sensitivity of different earthworm species in enzymatic and protein responses to phorate and the sensitivity was associated with the ecophysiological categories of earthworms.     

Ecophysiological category based toxicological responses in metabolism of earthworms: Impact of a pyrethroidal insecticide

Effects of alphamethrin (0.04 ppm, 0.08 ppm, 0.16 ppm, 0.32 ppm) on some metabolic dehydrogenases and proteins for 1, 2, 3, 4, 8 and 16 days using three ecologically different earthworm species (Perionyx sansibaricus, Lampito mauritii and Metaphire posthuma) were studied. The first significant effect was on 2nd/3rd day and maximum response was achieved on 16th day of exposure of alphamethrin at all concentrations. Similarly, maximum effect was obtained at 0.32 ppm alphamethrin at different exposure periods. It showed a dose- and duration-dependent inhibitory effects of a pyrethroid on enzymes and proteins of earthworms. There were approximately 47%, 43% and 41% declines in specific activities of cytoplasmic malate dehydrogenase (cMDH) of P. sansibaricus, L. mauritii and M. posthuma, respectively, in response to 0.32 ppm exposure of alphamethrin for 16 days. In case of similar treatment, specific activities of mitochondrial malate dehydrogenase (mMDH) showed 57%, 51% and 45% reductions in epigeic (P. sansibaricus), anecic (L. mauritii) and endogeic (M. posthuma) earthworms, respectively. Exposure of alphamethrin (0.32 ppm) also decreased the specific activities of lactate dehydrogenase (LDH) by 47% (P. sansibaricus), 35% (L. mauritii) and 39% (M. posthuma) in different types of earthworms. The reductions in specific activities of a Krebs cycle enzyme (mMDH) were greater than that of a glycolytic enzyme (LDH). The protein contents declined significantly in the earthworms exposed to alphamethrin. The decreases of 41%, 36% and 30% were obtained in cytoplasmic proteins of P. sansibaricus, L. mauritii and M. posthuma, respectively. However, the mitochondrial proteins showed reductions of 45% in P. sansibaricus, 46% in L. mauritii and 38% in M. posthuma in response to alphamethrin (0.32 ppm) intoxication for 16 days. The decrease in the protein content reflected an inhibitory effect of alphamethrin on protein turnover. The most pronounced effect of alphamethrin was on metabolic enzymes and proteins of P. sansibaricus and least effect was found on M. posthuma. This clearly showed that surface dwelling species of earthworms (epigeic) are more vulnerable to toxic chemical than deep burrowing species (endogeic). Thus a possible ecophysiological link exists between the toxicological responses and ecological categories at metabolic level in tropical earthworms.     

Concentration related responses of chlorpyriphos in antioxidant, anaerobic and protein synthesizing machinery of the freshwater fish, Heteropneustes fossilis

Impacts of chlorpyriphos (2, 4, 6 ppm) on the profiles of antioxidant (catalase) and anaerobic (lactate dehydrogenase) enzymes and other macromolecular contents (DNA, RNA, protein) of various tissues of the freshwater fish, Heteropneustes fossilis were studied. Chlorpyriphos significantly decreased the specific activity of catalase (CAT) and lactate dehydrogenase (LDH) in the brain, liver, gill and skeletal muscle of the fish. The reduction in specific activity might be due to the binding of chlorpyriphos or its metabolites with the enzyme molecules or affecting the synthesis and/or degradation of the enzymes. Like enzymes, the DNA, RNA and protein contents decreased in the brain, liver, gill and skeletal muscle of the fish as a function of increase in chlorpyriphos concentrations (2-6 ppm). The chlorpyriphos-induced reduction in these biochemical constituents might be because of alteration in their turnover (synthesis/degradation) in different tissues. The maximum effects on CAT, LDH, DNA, RNA and protein were obtained in response to 6 ppm chlorpyriphos. The present findings suggest chlorpyriphos concentration related impairment in antioxidative, anaerobic and protein synthesizing capacity of the fish. Therefore, the use of higher concentrations of chlorpyriphos should be avoided to protect the health of economically important freshwater food fish.     

Methylparathion- and carbofuran-induced mitochondrial dysfunction and oxidative stress in Helicoverpa armigera (Noctuidae: Lepidoptera)

The cotton bollworm, Helicoverpa armigera is a polyphagous pest of several crops in Asia, Africa, and the Mediterranean Europe. Organophosphate and carbamate insecticides are used on a large-scale to control Helicoverpa. Therefore, we studied the effect of methylparathion and carbofuran, an organophosphate and carbamate insecticide, respectively, on oxidative phosphorylation and oxidative stress in H. armigera larvae to gain an understanding of the different target sites of these insecticides. It was observed that state III and state IV respiration, respiratory control index (RCI), and P/O ratios were inhibited in a dose-dependent manner by methylparathion and carbofuran under in vitro and in vivo conditions. Methylparathion and carbofuran inhibited complex II by ∼45% and 30%, respectively. Lipid peroxidation, H₂O₂ content, and lactate dehydrogenase (LDH) activity increased and glutathione reductase (GR) activity decreased in a time- and dose-dependent manner in insecticide-fed larvae. However, catalase activity was not affected in insecticide-fed larvae. Larval growth decreased by ∼64% and 67% in larvae fed on diets with 100 μM of methylparathion and carbofuran. The results suggested that both the insecticides impede the mitochondrial respiratory functions and induced lipid peroxidation, H₂O₂, and LDH leak, leading to oxidative stress in cells, which contribute to deleterious effects of these insecticides on the growth of H. armigera larvae, along with their neurotoxic effects.     

Carbofuran-induced biochemical changes in Clarias batrachus

The effect of carbofuran, an organo-carbamate pesticide, upon the level of protein as well as the activity of lactate dehydrogenase (EC 1.1.1.28, LDH) was studied by exposing the teleost fish, Clarias batrachus, to different subacute con-centrations (0·01 and 0·02 mg litre^-1) for 96 h and 15 days. The results showed a drastic decrease in the protein content in different body organs of the fish. The pesticide also caused a significant decrease in the level of activity of LDH in different body tissues of the fish, the effect being more pronounced in the gills, muscle, brain and liver than in kidney and heart. The decrease in protein content and the activity of LDH in fish tissues was more marked at the higher concentration of the pesticide for the longer duration of treatment. The results suggested that carbofuran has an effect at very low concentration (compared to its LC₅₀ value) possibly at the level of protein metabolism, and also inhibits the activity of LDH, the terminal glycolytic enzyme. © 1998 SCI     

Dose-dependent sub-chronic toxicity of diethyl phthalate in female Swiss mice

The experiment was conducted to study the after effects of administering DEP at different doses to female Swiss mice for a period of 90 days. Group I mice were fed on normal diet and water ad libitum. Group II mice were maintained on normal diet mixed with corn oil at 8.25 mg/kg of the diet/day as oil control. Group III, IV and V mice were given diethyl phthalate dissolved in corn oil mixed with the diet at 10, 25 and 50 mg/kg of the diet/day, which is approximately equal to 1.25, 3.125 and 6.25 mg/kg body weight/day. A significant dose dependent increase was observed in serum acid phosphatase (ACP) whereas, serum and liver triglycerides levels showed a significant increase only in the high-dose treated group. Significant dose-dependent increase in serum aspartate and alanine aminotransferase (AST and ALT) and liver glycogen was observed. Serum lactate dehydrogenase (LDH) was significantly increased only in 25 and 50 ppm DEP-treated mice. Liver cholesterol was significantly increased in all the treated groups. Liver histology by light microscopy showed intracellular vacuolations in all the treated groups which was much more evident in the 25 and 50 ppm DEP-treated mice while hepatocellular degeneration and hypertrophy of the hepatocytes was evident in 50 ppm DEP-treated mice. Proliferation of mitochondria and peroxisomes was evident in the electron micrographs of the 10 ppm DEP-treated mice while 25 and 50 ppm DEP-treated mice showed increase in lipid droplets and severe mitochondrial proliferation.     

Cloning of the acetylcholinesterase 1 gene and identification of point mutations putatively associated with carbofuran resistance in Nilaparvata lugens

Molecular mechanisms of carbofuran resistance in the brown planthopper, Nilaparvata lugens Stål, were investigated. A carbofuran-resistant strain (CAS) showed approximately 45.5- and 15.1-fold resistance compared with a susceptible strain (SUS) and a non-selected field strain (FM), respectively. Activities of the esterase and mixed-function oxidase were approximately 2.8- and 1.6-fold higher, respectively, in the CAS strain than in the SUS strain, suggesting that these enzymes play a minor role in carbofuran resistance. Interestingly, the insensitivity of acetylcholinesterase (AChE) to carbofuran was approximately 5.5- and 3.7-fold higher in the CAS strain compared to the SUS and FM strains, respectively, indicating that AChE insensitivity is associated with carbofuran resistance. Western blot analysis identified two kinds of AChEs, of which the type-1 AChE (encoded from Nlace1, which is paralogous to the Drosophila AChE gene) was determined to be the major catalytic AChE in N. lugens. The open reading frame of Nlace1 is composed of 1989 bp (approximately 74 kD) and revealed 52.5% and 24.3% amino acid sequence identities to those of Nephotettix cincticeps and Drosophila melanogaster, respectively. Screening of point mutations identified four amino acid substitutions (G119A, F/Y330S, F331H and H332L) in the CAS strain that likely contribute to AChE insensitivity. The frequencies of these mutations were well correlated with resistance levels, confirming that they are associated with reduced sensitivity to carbofuran in N. lugens. These point mutations can be useful as genetic markers for monitoring resistance levels in field populations of N. lugens.     

The effect of single, binary, and tertiary combination of few plant derived molluscicides alone or in combination with synergist on different enzymes in the nervous tissues of the freshwater snail Lymnaea (Radix) acuminata (Lamark)

The effect of single, binary, and tertiary combination of few plant derived molluscicides alone or in combination with synergist on different enzymes (acetylcholinesterase—AChE, lactic dehydrogenase—LDH, and acid/alkaline phosphatase—ACP/ALP in the nervous tissue of the freshwater snail Lymnaea acuminata were studied. Sublethal in vivo 24 h exposure to 40 and 80% of LC₅₀ of Azadirachta indica oil (AI), oleoresin of Zingiber officinale (OL), Cedrus deodara oil (CD), Allium sativum (AS), and Polianthes tuberosa (PT) bulb powder singly, their binary combination of AI + OL, AS + CD, AS + PT, CD + OL, CD + PT, OL + PT, and tertiary combination of these binary combinations with the synergist piperonyl butoxide (PB) or MGK-264 significantly altered the activity of these enzymes. Tertiary combination with PB or MGK was very effective. Combination of CD + PT + MGK was more effective against AChE whereas, the combination of CD + OL + PB, CD + AS + PB, and CD + PT + PB were more effective against LDH, ACP, and ALP, respectively.     

Food consumption, utilization, and detoxification enzyme activity of the rice leaffolder larvae after treatment with Dysoxylum triterpenes

The toxicity and physiological (enzyme and nutritional indices) effect of Dysoxylum triterpenes 3β,24,25-trihydroxycycloartane and beddomei lactone were evaluated on the rice leaffolder Cnaphalocrocis medinalis (Guenée). The LC₅₀ [6.66 ppm (SD = 0.31), 5.79 ppm (SD = 0.33) for 3β,24,25-DHCL and BL, respectively] and LC ₉₀ [14.63 ppm (SD = 0.36), 13.49 ppm (SD = 0.27) for 3β,24,25-DHCL and BL, respectively] were identified by probit analysis. Fourth instars were exposed to various concentrations (1.5, 3, 6, and 12 ppm) of Dysoxylum triterpenes. Results showed that treated larvae exhibited reduced food consumption and enzyme activity. Food consumption, digestion, relative consumption rate, efficiency of conversion of ingested food, efficiency of conversion of digested food, and relative growth rate values declined significantly but the approximate digestibility of treated larvae was significantly higher as a result of treatment (in particular 6 and 12 ppm). Likewise, the gut enzymes acid phosphatases, alkaline phosphatases, and adenosine triphosphatases were significantly inhibited by the Dysoxylum triterpenes. The high biological activity of these triterpenes from Dysoxylum sp. could be used as an active principle during the preparation of botanical insecticides for insect pest like rice leaffolder.     

Comparative molluscicidal action of extract of Ginko biloba sarcotesta, arecoline and niclosamide on snail hosts of Schistosoma japonicum

In search for new local plant molluscicides for the control of the vectors of schistosomiasis, we compared the molluscicidal action of the extract of Ginkgo biloba sarcotesta by benzinum (EGSB) to that of arecoline (ARE) and niclosamide (NIC) against Oncomelania hupensis snails. NIC showed the highest toxicity on snails with 24 h LC₅₀ vales of 0.12 mg/L and LC₉₀ of 0.98 mg/L, while the LC₅₀ and LC₉₀ of EGSB were much lower than that of ARE. Sublethal in vivo 24 h exposure to 40% and 80% LC₅₀ of NIC, EGSB and ARE altered the activities of different enzymes in different body tissues of snails. EGSB could significantly inhibit Choline esterase (ChE), Alanine aminotransferase (ALT), Alkaline phosphatase (ALP) and Malic dehydrogenase (MDH) activities both in the cephalopodium and liver. ARE could significantly cause a reduction in ChE, ALP activities in the cephalopodium and ChE, ALT, ALP, Succinodehydrogenase (SDH), MDH activities in the liver. NIC significantly altered activities of ChE, ALT, ALP, SDH, and MDH in the cephalopodium and ChE, ALT, ALP, SDH activities in the liver. All molluscicides could not affect Lactate dehydrogenase (LDH) activity in the cephalopodium and the liver. Maximum inhibition of ALT and MDH activities was found in the cephalopodium and liver of snails treated with 80% of 24 h LC₅₀ of EGSB. However, NIC and ARE caused maximum reduction in ALP and SDH activities, respectively. The results indicated that molluscicidal action of EGSB was different to that of ARE and NIC in some extent.     

The impact of carbofuran on acetylcholinesterase activity in Anisakis simplex larvae from Baltic herring

The aim of this study was to investigate the impact of carbofuran, a carbamate pesticide on the enzymatic activity of acetylcholinesterase (AChE) in the larvae of the herring nematode parasite Anisakis simplex. A. simplex larvae collected from herring were exposed to carbofuran in vivo at concentrations of 50, 100, 500 and 1000 μg/l, for 24, 48 and 72 h, at a temperature of 4 °C. Generalized Linear Models (GLM) were applied to analyze the relationship between AChE activity and carbofuran concentration, the time of exposure and the biological parameters of the host. The results indicate that A. simplex larvae have a high threshold of sensitivity to carbofuran. The average enzymatic activity was higher in parasites obtained from male hosts, when compared with female hosts. These data suggest that host sex-dependent biological processes may also influence AChE enzymatic activity in parasites.     

Biological and biochemical responses of Biomphalaria alexandrina to some extracts of the plants Solanum siniacum and Artemisia judaica L.

The molluscicidal activity of cold water, boiled water, methanol, ethanol, acetone and chloroform extracts of Solanum siniacum and Artemisia judaica L. plants against Biomphalaria alexandrina snails was carried out. The tests revealed plant’s ethanol extract was more toxic to the snails than the other tested extracts. Therefore, it was tested against snails’ fecundity (M_x), reproduction rate (R₀) and their infection with Schistosoma mansoni miracidia. In addition, biochemical parameters and the activities of some enzymes in tissues of snails treated with the two tested plants were determined. As well, glucose concentration in snails’ hemolymph was evaluated. Exposure of B. alexandrina snails to plant’s ethanol extract led to a significant reduction in their survival and snails’ fecundity, reproduction rate. In addition, it caused a considerable reduction in the infectivity of S. mansoni miracidia to the snails. Also, it caused a reduction in number of cercariae per snail during the patent period and in the period of cercarial shedding. The results revealed that the glucose concentration in hemolymph and Lactate level in soft tissues of treated snails were increased (P < 0.001) while glycogen, total protein, the lipid content and the pyruvate level in snail’s tissues decreased (P < 0.001). The activities of lactic dehydrogenase (LDH), pyruvatekinase (PK) and cytochrome oxidase (CY) enzymes in homogenate of snail’s tissues were reduced (P < 0.001) in response to treatment with the two tested plants while protease (PR) activity increased (P < 0.001). It is concluded that the application of LC₂₅ of ethanol extract of S. siniacum and A. judaica L. may be helpful in snail control as it interferes with the snails’ biology and physiology.     

Influence of Atrazine and Roundup pesticides on biochemical and molecular aspects of Biomphalaria alexandrina snails

The excessive use of pesticides in agriculture has sparkled the interest of scientists in investigating the harmful effects of these compounds. The present study evaluates the pesticides Atrazine and Roundup (glyphosate) on biochemical and molecular aspects of Biomphalaria alexandrina snails. The results showed that LC₁₀ of these two pesticides caused considerable reduction in survival rates and egg production of treated snails. Additionally, Atrazine proved to be more toxic to B. alexandrina snails than Roundup. In treated snails, glucose concentration (GL) in the hemolymph as well as lactate (LT) and free amino acid (FAA) in soft tissues of treated snails increased while glycogen (GN), pyruvate (PV), total protein (TP), nucleic acids (DNA and RNA) levels in snail’s tissues decreased. The activities of glycogen phosphorylase (GP), superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR), succinic dehydrogenase (SDH), acetylcholinesterase (AChE), lactic dehydrogenase (LDH) and phosphatases (ACP and ALP) enzymes in homogenate of snail’s tissues were reduced in response to the treatment with the two pesticides while lipid peroxide (LP) and transaminases (GOT and GPT) activity increased (P < 0.001). The changes in the number, position and intensity of DNA bands induced by pesticides may be attributed to the fact that pesticide can induce genotoxicity through DNA damage. It was concluded that the pollution of the aquatic environment by Atrazine and Roundup pesticides, would adversely affect the metabolism of the B. alexandrina snails, and have adverse effects on its reproduction.     

Endocrine disruption and metabolic changes following exposure of Cyprinus carpio to diethyl phthalate

Diethyl phthalate (DEP) enter into aquatic environment from industries manufacturing cosmetics, plastic and many commercial products and can pose potential fish and human health hazard. This experiment evaluated effects of DEP in adult male (89 g) common carp (Cyprinus carpio) by exposing them to fractions of LC₅₀ (1/500-1/2.5) doses with every change of water for 28 days. Vitellogenin induction metabolic enzymes, somatic indices and bioaccumulation were studied on 7th, 14th, 21st and 28th day. The 96th hour LC₅₀ of DEP in fingerlings was found to be 48 mg/L. Compared to control, except increase (P < 0.01) in alkaline phosphatase activity (EC 3.1.3.1) and liver size, there was decrease (P < 0.01) in activity of acid phosphatase (EC 3.1.3.2), aspartate aminotransferase (EC 2.6.1.1), alanine aminotransferase (EC 2.6.1.2) and testiculosomatic index following exposure to 1, 5 and 20 ppm DEP. Significant (P < 0.01) dose dependant vitellogenin induction was observed with exposure of fish to 0.1, 1 and 5 ppm DEP. The bioaccumulation of DEP in testis, liver, brain, gills and more importantly in muscle tissues of fish increased significantly (P < 0.01) with increase of dose from 1 to 5 ppm. Significant interaction (P < 0.01) of dose and duration of exposure indicated that exposure period of a week to two was sufficient to bring about changes in quantifiable parameters studied. Fish exposed to 20 ppm DEP became lethargic and discolored during onset of the 4th week. This is the first report describing metabolic changes and vitellogenin induction following exposure of C. carpio to DEP dose that is as low as 1/500th fraction of LC₅₀.     

Effects of a fungicide, an insecticide, and a biopesticide on Tolypothrix scytonemoides

Lower concentrations of Bavistin supported good growth of Tolypothrix scytonemoides with maximum protein and pigment contents. The rates of photosynthetic oxygen evolution declined in cells grown in the pesticide-amended medium but rates of respiratory oxygen consumption were enhanced in cells grown in lower concentrations of Monocrotophos and Nimbicidin. Activities of nitrogenase and glutamine synthetase were affected in all the pesticide treatments but nitrogenase activity was enhanced in the presence of Bavistin. Release of ammonia and carbohydrates was enhanced in cells grown in pesticide-amended medium except for cells grown in medium containing Bavistin where the release of carbohydrates was reduced. New polypeptides of ∼280, 152, and 25 kDa (in 250 ppm Bavistin), ∼58 and 28 kDa (in 0.3 and 0.2-0.4 ppm Monocrotophos, respectively) and ∼31, 28, and 26 kDa (in 0.5 and 1.0 ppm Nimbicidin) were detected in the treated cells.     

Determination of toxicity of trichloroacetic acid in rats: 50 days drinking water study

This study aims to investigate the effects of the trichloroacetic acid (TCA) on serum marker enzymes [aspartate aminotransferase (AST), alanin aminotransferase (ALT), creatine phosphokinase (CPK), acid phosphatase (ACP), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH)], antioxidant defense systems [Reduced glutathione (GSH), glutathione reductase (GR), superoxide dismutase (SOD), glutathione-S-transferase (GST) and catalase (CAT)] and lipid peroxidation content (Malondialdehyde, MDA) in various tissues of rats. TCA (2000 ppm) as drinking water was administered orally to rats (Sprague-Dawley albino) ad libitum for 50 days continuously. TCA treatments caused different effects on the serum marker enzymes, antioxidant defense systems and the MDA content in experimented rats compared to controls. Results showed that TCA caused a significant increase in serum AST, ALT, CPK and ACP activity. The lipid peroxidation end product MDA slightly increased in the erythrocytes, liver and kidney of rats treated with TCA, whereas did not change in the brain. In addition, antioxidant enzyme activity such as CAT and SOD significantly increased in the brain, liver and kidney tissues of TCA induced group whereas the ancillary enzyme GR and the drug metabolizing enzyme GST activity did not significantly change in the all tissues. The observations presented led us to conclude that the administration of subchronic TCA promotes lipid peroxidation content, elevates tissue damage serum marker enzymes and fluctuates in the antioxidative systems in rats. Also the rats resisted to oxidative stress via antioxidant mechanism but the antioxidant mechanism could not prevent the increases in lipid peroxidation in rat’s tissues. These data, along with the determined changes suggest that TCA produced substantial systemic organ toxicity in the erythrocyte, liver, brain and kidney during the period of a 50-day subchronic exposure.     

Influence of subacute and subchronic treatment of abcisic acid and gibberellic acid on serum marker enzymes and erythrocyte and tissue antioxidant defense systems and lipid peroxidation in rats

This study describes the subacute and subchronic effects of two plant growth regulators (PGRs) [abcisic acid (ABA) and gibberellic acid (GA₃)] on serum marker enzymes [aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine phosphokinase (CPK) and lactate dehydrogenase (LDH), γ-glutamil transpeptidase (GGT)], antioxidant defense systems [reduced glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPx), superoxide dismutase (SOD), glutathione-S-transferase (GST) and catalase (CAT)] and lipid peroxidation level (Malondialdehyde = MDA) in various tissues of rats. Rats (Sprague-Dawley albino) were exposed to 75 ppm (parts per million) of ABA and GA₃. Seventy-five parts per million of PGRs as drinking water was administered orally ad libitum for 25 and 50 days continuously. The PGRs treatments caused different effect on the serum marker enzymes, antioxidant defense systems and the content of MDA in comparison to those of control rats. Results show that ABA caused a significant decrease in serum LDH and CPK activity with both periods. Also, GA₃ significantly decreased serum AST, CPK, and LDH activity with subacute and decreased serum ALT, CPK, LDH, and GGT treated with subchronic periods. The lipid peroxidation end product MDA significantly increased in the erythrocyte, liver, brain, and muscle of rats treated with both the period of GA₃ without significantly change in the erythrocyte and muscle of rats treated with the subacute period of ABA. The GSH levels were significantly depleted in the erythrocyte and brain of rats treated with both the period of GA₃ without any change in the erythrocyte, liver, brain, and muscle of rats treated with both the period of ABA. Also GSH levels in the muscle significantly depleted with the subchronic period of GA₃. Antioxidant enzyme activities such as SOD significantly decreased in the erythrocyte, liver and brain tissues but increased in the muscle tissue of rats treated with both the periods of GA₃. Meanwhile, SOD significantly decreased in liver and brain, and increased in muscle of rats treated with both the period of ABA. While CAT significantly decreased in the all tissues of rats treated with both the period of GA₃, decreased in the liver and muscle of rats treated with both the periods of ABA too. On the other hand, the ancillary enzyme GPx and GR activity in the erythrocytes, liver, brain and muscle were either significantly depleted or not changed with two periods of PGRs. The drug metabolizing enzyme GST activity significantly decreased in the brain of rats treated with subacute period of PGRs but increased in the erythrocytes of rats treated with subacute period of GA₃. As a conclusion, ABA and GA₃ had significantly increased the activity of hepatic damage enzymes. Also the rats resisted to oxidative stress via antioxidant mechanism. However, the antioxidant mechanism could not prevent the increases in lipid peroxidation in rat’s tissues. These data, along with changes, suggest that PGRs produced substantial systemic organ toxicity in the erythrocyte, liver, brain, and muscle during the period of a 25-day subacute and 50-day subchronic exposure.     

Determination of genotoxicity of Fenaminosulf by Allium and Comet tests

Genotoxic effects of Fenaminosulf, fungicide and micro-biocide, were examined by using mitotic index (MI), mitotic phase, and Comet assay on the root meristem cells of Allium cepa. In the Allium root growth inhibition test, EC₅₀ value was firstly determined as 25 ppm and, 12.5 (0.5 × EC₅₀), 25 (EC₅₀) and 50 (2 × EC₅₀) ppm concentrations of Fenaminosulf were introduced to onion tuber roots. Distilled water was used as a negative control. All obtained data were subjected to statistical analyses by using SPSS 15.0 for Windows software. For comparison purposes, Duncan multiple range tests by using one-way analysis of variance (ANOVA) were employed and p < 0.05 was accepted as significant value. While MI (except in 24 h at 12.5 and 50 ppm) and prophase index increased, metaphase, anaphase and telophase indexes decreased in all concentrations compared to control at each exposure time. A significant increase in DNA damage was also observed at the concentration of 25 ppm in 24 h, 25 and 50 ppm in 96 h by Comet assay.     

Combined cytogenetic and ultrastructural effects of substituted urea herbicides and synthetic pyrethroid insecticide on the root meristem cells of Allium cepa

Combined cytogenetic and ultrastructural effects of substituted urea herbicides-isoproturon (ISO) or diuron (DIU) and a synthetic pyrethroid insecticide-deltamethrin (DEL) were examined in the root meristem cells of Allium cepa. For cytogenetic analysis root meristem cells were exposed to the mixtures of ISO (25 or 50 ppm) or DIU (20 or 40 ppm) and DEL (0.25 or 0.5 ppm) for 6 h and analyzed at 24 or 48 h post-exposure whereas for ultrastructural studies roots were exposed for 6 or 24 h to similar concentrations of combinations and examined. Both the combinations, ISO + DEL or DIU + DEL, were found to induce chromosomal breaks and variety of mitotic aberrations at 24 and 48 h post-exposure. The combinations containing higher concentration of DEL (0.5 ppm) induced statistically significant (p < 0.001) frequencies of aberrations than that of the combinations containing low concentration of DEL (0.25 ppm). Chromosome aberrations in all the treatment groups were less frequent than that of mitotic aberrations. Electron microscopic study revealed drastic alterations in the membranous organelles like concentric arrangement of endoplasmic reticulum, crescented or circular structure of Golgi complex dictyosomes and swollen mitochondria. Further, the combination of DIU + DEL appeared to be more toxic than that of ISO + DEL. Present findings suggest that the coexistence of ISO or DIU and DEL in plants synergies the toxicity inducing drastic ultrastructural alterations which are different from its independent effects reported earlier.     

Protein metabolism in Spodoptera litura (F.) is influenced by the botanical insecticide azadirachtin

Azadirachtin, as a botanical insecticide, affects a wide variety of biological processes, including reduction of feeding, suspension of molting, death of larvae and pupae, and sterility of emerged adults in a dose-dependent manner. However, the mode of action of this toxin remains obscure. By using proteomic techniques, we analyzed changes in protein metabolism of Spodoptera litura (F.) induced by azadirachtin. Following feeding 4th instar larvae of Spodoptera litura (F.) with an artificial diet containing 1 ppm azadirachtin until pupation, 48 h old pupae were collected and protein samples prepared. Total soluble protein content was measured and the results showed that azadirachtin significantly influenced protein level. Moreover, the proteins were separated by 2-DE (two-dimensional polyacrylamide gel electrophoresis) and 10 proteins were significantly affected by azadirachtin treatment when compared to an untreated control. Six of these proteins were identified with peptide mass fingerprinting using MALDI-TOF-MS after in-gel trypsin digestion. These proteins are involved in various cellular functions. One identified protein may function as an ecdysone receptor, which regulates insect development, and reproduction. It is suggested that the botanical insecticide azadirachtin affects protein expression and the azadirachtin-related proteins would be essential for a better understanding of the mechanisms by which neem toxins exert their effects on insects.