Increased glutathione-S-transferase activity in antioxidant-deficient rats

Rats fed a diet deficient in vitamin E and selenium show an increased activity of glutathione-S-transferase (EC 2.5.1.18) in all tissues tested, with the possible exception of the retina. Glutathione-S-transferases are detoxifying enzymes that are induced by a variety of electrophilic drugs or toxins. Therefore, the induction of glutathione-S-transferase in vitamin E- and selenium-deficient rats indicates that substrates for the enzyme probably increase in vivo with dietary antioxidant deficiency. These substrates are likely to be lipid peroxides and/or other lipid peroxidation products.     

Antihyperglycemic, hypolipidemic and antioxidant activities of ethanolic extract of Commiphora mukul gum resin in fructose-fed male Wistar rats

High fructose feeding (66?% of fructose) induces type-2 diabetes in rats, which is associated with the insulin resistance, hyperinsulinemia, hypertriglyceridemia and oxidative stress. The present study was undertaken to evaluate the effect of ethanol extract of Commiphora mukul gum resin (CMEE) on blood glucose, plasma insulin, lipid profiles, reduced glutathione, lipid peroxidation, protein oxidation and enzymatic antioxidants like superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase, glutathione-S-transferase in fructose-induced type-2 diabetic rats. A significant gain in body weight, hyperglycemia, hyperinsulinemia, increased lipid profiles, lipid peroxidation, protein oxidation and decreased reduced glutathione, activities of enzymatic antioxidants and insulin sensitivity (increased homeostasis assessment assay) were observed in high-fructose-induced diabetic rats. The administration of CMEE (200?mg/kg/day) daily for 60?days in high-fructose-induced diabetic rats reversed the above parameters significantly. CMEE has the ability to improve insulin sensitivity and delay the development of insulin resistance, aggravate antioxidant status in diabetic rats and may be used as an adjuvant therapy for patients with insulin resistance.     

Oxidative stress in digestive gland and gill of the brown mussel (Perna perna) exposed to air and re-submersed

Mussels Perna perna were exposed to air for 24 h showing a clear increase in the levels of lipid peroxidation and oxidative DNA damage, measured as 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo). The levels of lipid peroxidation increased both in the digestive gland and gills, while oxidative DNA damage increased only in the gills. After the 24 h of air exposure, mussels were re-submersed for a period of 3 h, leading values to return to a pre-aerial exposure levels. Control animals were kept immersed during the whole period. Several antioxidant and complementary enzymes (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6PDH), glutathione S-transferase (GST) and the levels of total glutathione (Total GSH) were assayed in a second set of experiments where one group of mussels were exposed to air for 18 h and other to 1 h re-submersion after 18 h aerial exposure. Only a 52% increase in the glutathione S-transferase activity was observed in the digestive gland, which remained elevated to about 40% after 1 h re-submersion, showing that defense systems can be modulated even during oxygen deprivation in P. perna. The DNA and lipid oxidative damage observed after aerial exposure indicates that mussels face an oxidative challenge, and are able to counteract such an “insult” as values of lipid peroxidation and DNA damage returned to control values after 3 h re-submersion.     

Tellurium-induced dose-dependent impairment of antioxidant status: differential effects in cerebrum,cerebellum, and brainstem of mice

The effect of various doses of sodium tellurite (0.4, 0.8, and 2.0 mg/kg body weight, orally) on the activity of antioxidant enzymes (glutathione peroxidase, glutathione reductase, glutathione-S-transferase, and catalase) and content of glutathione and thiobarbituric acid reactive substances (TBARSs) in the cerebrum, cerebellum, and brainstem of male albino mice was studied after 15 d of treatment. All of the doses of tellurium (0.4, 0.8, and 2.0 mg/kg body weight, orally) have depleted the activity of antioxidant enzymes and the content of glutathione dose dependently in the cerebrum, cerebellum, and brainstem and it was significant with the dose of 2.0 mg/kg. On the other hand, the 2.0-mg/kg dose of tellurium has significantly elevated the content of TBARSs in the cerebrum and cerebellum. The 0.8-mg/kg dose of tellurium has significantly depleted the activities of glutathione peroxidase in the cerebrum and brainstem, glutathione-S-transferase in the cerebrum and cerebellum, catalase in the brainstem, and the content of glutathione in the cerebrum and cerebellum. In contrast, this dose has significantly elevated the content of TBARSs in the cerebrum and cerebellum. However, the depletion in the activity of glutathione reductase with various doses of sodium tellurite was not significant in any brain part of mice. The result suggests that sodium tellurite differentially affects the antioxidant status within various parts of the mice brain.     

Protective effect of naringenin on hepatic and renal dysfunction and oxidative stress in arsenic intoxicated rats

Arsenic has a long history as a potent human poison, chronic exposure over a period of time may result in the manifestation of toxicity in practically all systems of the body. In the present investigation the efficacy of naringenin (NRG), a naturally occurring citrus flavanone against arsenic-induced hepatotoxic and nephrotoxic manifestations have been studied in rats. Arsenic trioxide was administered orally at the dose of 2 mg/kg/day with or without combination of NRG (20 or 50 mg/kg/day) for 28 days. At the end of the experimental period the hepatic and renal dysfunction was evaluated by histological examination, serum biomarkers and markers of oxidative stress; lipid peroxidation (LPO), reduced glutathione (GSH) and antioxidant enzymes. Arsenic intoxication increased serum bilirubin, urea, uric acid and creatinine levels, additionally enhanced the activities of hepatic marker enzymes aspartate transaminase, alanine transaminase and alkaline phosphatase. Also, the hepatic and renal tissues showed a marked elevation in LPO levels with a decrease in GSH content and the activities of antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase and glutathione-S-transferase on arsenic treatment. Simultaneous treatment with NRG restored the activities of serum biomarkers and antioxidant enzymes in the tissues in a dose-dependent manner. Furthermore, the histopathological studies confirmed the protective effect of NRG co-treatment by reducing the pathological changes due to arsenic intoxication in both liver and kidney. Thus, our present study demonstrates that NRG has a potential to protect arsenic-induced oxidative hepatic and renal dysfunction.     

Oxidative stress and antioxidant defense responses by goldfish tissues to acute change of temperature from 3 to 23 °C

The effects of a rapid transfer from a low (3 °C) to a warm (23 °C) temperature on oxidative stress markers and antioxidant defenses were studied in the brain, liver and kidney of the goldfish, Carassius auratus. Cold-acclimated fish were acutely moved to 23 °C and sampled after 1, 6, 12, 24, 48 or 120 h of warm temperature exposure. Lipid peroxide levels increased quickly during the first few hours at 23 °C, but thiobarbituric acid-reactive substances changed little. Protein carbonyl content was reduced by 20–40% in the liver over the entire experimental course, but increased transiently in the kidney. The content of high-molecular mass thiols decreased by two-thirds in the brain and was affected slightly in other organs. By contrast, total low-molecular mass thiols (e.g. glutathione and others) increased transiently. Activities of the primary antioxidant enzymes—superoxide dismutase and catalase—were generally unaffected in goldfish organs, whereas glutathione-dependent enzymes were elevated in the brain and kidney after 24–48 h at 23 °C. Glutathione peroxidase increased by 1.5–2.3-fold and glutathione-S-transferase by 1.7-fold. Hence, a short-term exposure to warm temperature disturbed several oxidative stress markers, but only slightly affected the activities of antioxidant enzymes. However, comparison of the current data for cold-acclimated winter fish with the same parameters in summer fish suggests that longer exposure to high ambient temperature requires the enhancement of activities of glutathione-dependent enzymes for maintaining the steady-state levels lipid peroxidation and protein oxidation in goldfish tissues.     

Oxidative brain and cerebellum injury in diabetes and prostate cancer model: Protective effect of metformin

The body can host the spread of prostate cancer cells. Metastases from prostate cancer are more frequently seen in the brain, liver, lungs, and lymph nodes. A well-known antidiabetic drug, metformin, is also known to have antitumor effects. Our study focuses on the evaluation of potential metformin protective effects on brain and cerebellum damage in streptozotocin (STZ)-induced diabetic and Dunning prostate cancer models. In this investigation, six groups of male Copenhagen rats were created: control, diabetic (D), cancer (C), diabetic + cancer (DC), cancer + metformin, and diabetic + cancer + metformin. The brain and cerebellum tissues of the rats were taken after sacrifice. Oxidative stress markers including reduced glutathione level, lipid peroxidation, glutathione reductase, glutathione peroxidase, glutathione-S-transferase, catalase, superoxide dismutase activities, reactive oxygen species, total oxidant and total antioxidant status, lactate dehydrogenase, xanthine oxidase, acetylcholinesterase activities, protein carbonyl contents, nitric oxide and OH-proline levels, sodium potassium ATPase, carbonic anhydrase, and glucose-6-phosphate dehydrogenase activities; glycoprotein levels including hexose, hexosamine, fucose, and sialic acid levels; and histone deacetylase activity as a cancer marker were determined. Oxidative stress markers were impaired and glycoprotein levels and histone deacetylase activity were increased in the D, C, and DC groups. Metformin therapy reversed these effects. Metformin was found to protect the brain and cerebellum of STZ-induced diabetic rats with Dunning prostate cancer from harm caused by MAT-Lylu metastatic cells.     

Age Dependent Damage and Glutathione Metabolism in Ozone Fumigated Barley: A Leaf Section Approach

First, second, and third leaves of barley (Hordeum vulgare L.cv. Atem) seedlings fumigated with 200 nl 1^–1 ozone for5 d were divided into equal length quarter sections before analysisof glutathione metabolism. Since the first leaf is the oldestand the third leaf is the youngest and since monocotyledonousleaves grow from the base, sections were of a widely differingage. Lipid peroxidation rose moderately in all sections in all leaves.However, severe damage, detected visibly as chlorosis and analyticallyas loss of soluble protein, occurred only in the oldest tissue. The specific activity of glutathione reductase was slightlyelevated in old and middle-aged tissue whilst the specific activityof glutathione-S-transferase rose markedly in old, middle-aged,and young tissue following fumigation. This may reflect theimportance of glutathione-S-transferase in the protection ofplant cells from oxidative stress products formed in membranes. Fumigation with ozone also caused a loss of total glutathionein the tip sections of all leaves. Explanations for these results, including age dependent proteinloss, are discussed. Key words: Barley, ozone, glutathione, developmental age, glutathione-S-transferase     

Proline and betaine provide protection to antioxidant and methylglyoxal detoxification systems during cold stress in <Emphasis Type="Italic">Camellia sinensis</Emphasis> (L.) O. Kuntze

The aim of this study was to monitor the influence of proline and betaine exposure on antioxidant and methylglyoxal (MG) detoxification system during cold stress in Camellia sinensis (L.) O. Kuntze. Cold stress enhanced MG and lipid peroxidation levels in tea bud (youngest topmost leaf). This increase was resisted upon the exposure of tea bud to proline and betaine. Exposure of tea bud with proline and betaine also help in maintaining thiol/disulfide ratio during cold stress. Proline exposure enhanced glutathione-S-transferase and glutathione reductase (GR) activity, while betaine exposure increased only GR activity during cold stress. Furthermore, effect of proline/betaine was studied on glyoxalase pathway enzymes that are involved in MG detoxification and comprise of two enzymes glyoxalase I and glyoxalase II. Both proline and betaine showed protective effect on glyoxalase I and activating effect on glyoxalase II during cold stress in tea bud. This investigation, therefore, suggest that proline and betaine might provide protection to cold stress in tea by regulating MG and lipid peroxidation formation as well as by activating or protecting some of antioxidant and glyoxalase pathway enzymes.     

Cytotoxic effects of 4-octylphenol on fish hepatocytes

The present study was conducted to determine cytotoxic effects of 4-octylphenol (4-OP) on primary cultured hepatocytes of pearl mullet (Alburnus tarichi). Lactate dehydrogenase (LDH) release, malondialdehyde (MDA) level, antioxidant enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione-S-transferase (GST)] and glutathione (GSH) content were measured after 24-h exposure to 4-OP. 4-OP caused dose- and time-dependent increases in LDH release. Significant induction of MDA level and decrease in GSH content were found. SOD and GPx activities were decreased while GST activity was increased. These findings suggest that 4-OP leads to cytotoxicity by depressing antioxidant defenses in fish hepatocytes.     

Comparative and combined effect of chlorogenic acid and tetrahydrocurcumin on antioxidant disparities in chemical induced experimental diabetes

The present study evaluates the combined effect of tetrahydrocurcumin and chlorogenic acid on oxidative stress in streptozotocin–nicotinamide-induced diabetic rats. Rats were rendered diabetic by a single intraperitoneal injection (i.p) of streptozotocin (45 mg/kg BW), 15 min after an i.p injection of nicotinamide (110 mg/kg BW). The levels of fasting plasma glucose and insulin were estimated. As an index of oxidative stress, the levels of enzymic antioxidants and lipid peroxidation products were analyzed in liver and kidney. Diabetic rats showed an increase in the levels of fasting plasma glucose, lipid peroxidative products such as thiobarbituric acid reactive substances and lipid hydroperoxides and a decrease in plasma insulin, and enzymic antioxidants viz., superoxide dismutase, catalase, glutathione peroxidase and glutathione-S-transferase. Combined administration of tetrahydrocurcumin (80 mg/kg BW) and chlorogenic acid (5 mg/kg BW) to diabetic rats for 45 days, reversed the biochemical changes to near normal. The above findings were supported by histological observations of the liver and kidney. Together the present study clearly reflects that combined dosage of tetrahydrocurcumin and chlorogenic acid augments enzymic antioxidants with a concomitant decrease in lipid peroxidation and protects against streptozotocin–nicotinamide-induced type 2 diabetes in experimental rats.     

Effects of isoquinoline alkaloid berberine on lipid peroxidation,antioxidant defense system,and liver damage induced by lead acetate in rats

Objectives: Liver is considered a target organ affected by lead toxicity. Oxidative stress is among the mechanisms involved in liver damage. Here we investigated the effects of the natural alkaloid berberine on oxidative stress and hepatotoxicity induced by lead in rats.

Methods: Animals received an aqueous solution of lead acetate (500?mg Pb/l in the drinking water) and/or daily oral gavage of berberine (50?mg/kg) for 8 weeks. Rats were then weighed and used for the biochemical, molecular, and histological evaluations.

Results: Lead-induced oxidative stress, shown by increasing lipid peroxidation along with a concomitant decrease in hepatic levels of thiol groups, total antioxidant capacity, the activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione-S-transferase, and reduced versus oxidized glutathione ratio. Berberine corrected all the disturbances in oxidative stress markers induced by lead administration. Berberine also prevented the elevated levels of enzymes (alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase) and the decrease in body weight and albumin. The protective effects of berberine were comparable with silymarin. Furthermore, berberine attenuated liver damage, shown by decreased necrosis and inflammatory cell infiltration.

Discussion: Berberine represents a potential therapeutic option against lead-induced hepatotoxicity through inhibiting lipid peroxidation and enhancing antioxidant defenses.

Conclusion: Berberine exerted protective effects on lead-induced oxidative stress and hepatotoxicity in rats.     

Effect of D,L-buthionine-S,R-sulfoximine on the ratio of glutathione forms and the growth of Tatar buckwheat calli

We studied the intracellular content of reduced (GSH) and oxidized (GSSG) glutathione, glutathione reductase activity, glutathione-S-transferase, and ascorbate peroxidase in morphogenic and nonmorphogenic Tatar buckwheat calli during the culture cycle as well as under the treatment with D,L-buthionine-S,R-sulfoximine (BSO), an inhibitor of γ-glutamylcysteine synthase, the first enzyme of glutathione biosynthesis. We found that, during passaging, cultures only slightly differed in total glutathione content; however, the content of GSH was higher in the morphogenic culture, whereas the content of GSSG was higher in the nonmorphogenic culture. In the morphogenic callus, the glutathione-S-transferase activity was 10–20 times higher and the glutathione reductase activity was 2–2.5 times lower than in the nonmorphogenic callus. Under the treatment with BSO, the decrease in the GSH content in the morphogenic callus was temporary (on day 6–8 of passage), whereas that in the nonmorphogenic callus decreased within a day and remained lower than in the control throughout the entire passage. In the morphogenic callus, BSO did not affect the content of GSSG, whereas it caused GSSG accumulation in the nonmorphogenic callus. These differences are probably due to the fact that, in the BSO-containing medium, glutathione reductase is activated in the morphogenic callus and, conversely, inhibited in the nonmorphogenic callus. Although BSO caused a decrease in the total glutathione content only in the nonmorphogenic culture, the cytostatic effect of BSO was more pronounced in the morphogenic callus. In addition, BSO also had a negative effect on the differentiation of proembryonic cell complexes in the morphogenic callus. The role of the glutathione redox status in maintaining the embryogenic activity of cultured plant cells is discussed.     

Potential chemoprevention of N-nitrosodiethylamine-induced hepatocarcinogenesis by polyphenolics from Acacia nilotica bark

Chemopreventive potential of Acacia nilotica bark extract (ANBE) against single intraperitoneal injection of N-nitrosodiethylamine (NDEA, 200 mg/kg) followed by weekly subcutaneous injections of carbon tetrachloride (CCl₄, 3 ml/kg) for 6 weeks induced hepatocellular carcinoma (HCC) in rats was studied. At 45 day after administration of NDEA, 100 and 200 mg/kg of ANBE were administered orally once daily for 10 weeks. The levels of liver injury and liver cancer markers such as alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), γ-glutamyl transferase (γ-GT), total bilirubin level (TBL), α-feto protein (AFP) and carcinoembryonic antigen (CEA) were substantially increased following NDEA treatment. However, ANBE treatment reduced liver injury and restored liver cancer markers. ANBE also significantly prevented hepatic malondialdehyde (MDA) formation and reduced glutathione (GSH) in NDEA-treated rats which was dose dependent. Additionally, ANBE also increased the activities of antioxidant enzymes viz., catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) in the liver of NDEA-administered rats. Eventually, ANBE also significantly improved body weight and prevented increase of relative liver weight due to NDEA treatment. Histological observations of liver tissues too correlated with the biochemical observations. HPLC analysis of ANBE showed the presence of gallic, protocatechuic, caffeic and ellagic acids, and also quercetin in ANBE. The results strongly support that A. nilotica bark prevents lipid peroxidation (LPO) and promote the enzymatic and non-enzymatic antioxidant defense system during NDEA-induced hepatocarcinogenesis which might be due to activities like scavenging of oxy radicals by the phytomolecules in ANBE.     

Daily variations of the antioxidant defense system of the lithodid crab Lithodes santolla

Several physiological processes can induce daily variations in aerobic metabolism. Lithodes santolla is a decapod crustacean of special concern since it is a sub-Antarctic species of commercial interest. The aim of this work was to study in L. santolla the daily variations in levels of enzymatic and non-enzymatic antioxidants, lipid peroxidation and protein oxidation, and haemolymphatic pH. Males of L. santolla of commercial size were randomly dissected every 4 h during a period of 24 h. Enzymatic activities of superoxide dismutase (SOD), catalase (CAT), glutathione-S-transferase and glutathione peroxidase were determined in samples of gills, muscle, hepatopancreas and haemolymph. Ascorbic acid, total glutathione, lipid peroxidation and protein oxidation were also determined in all tissues. Gills showed the highest enzymatic activity and hepatopancreas the highest concentration of non-enzymatic antioxidants. Maximum antioxidant activity was during the dark phase in gills and during the photophase in the haemolymph. Muscle showed significant daily variations, with peaks during the photophase and scotophase. Overall, an antioxidant protective mechanism is present in all tissues, where SOD and CAT represent the first line of defense. The defense mechanism in L. santolla seems to be more active during the dark phase, with slight differences among the analyzed tissues, indicating a higher metabolic rate.     

Podophyllum hexandrum aqueous extract as a potential free radical scavenger

Abstract

The present study was undertaken to evaluate the effect of the aqueous extract of Podophyllum hexandrum against free radical-mediated damage and also explore its anticancer activity. The extract exhibited significant activity in scavenging 1, 1-diphenyl-2-picryl-hydrazyl radicals, ^?OH radical-mediated DNA damage, and lipid peroxide production in rat liver microsomes. The extract was also tested for its reducing abilities. The activity of liver marker enzymes and antioxidant defense enzymes in rat liver homogenate was assessed in control and carbon tetrachloride (CCl₄)-treated animals. It was observed that CCl₄-induced changes viz., increases in the activities of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase, a decrease in reduced glutathione as well as decreases in the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, and glutathione-S-transferase. All these parameters showed reversal when pretreated with aqueous extract of P. hexandrum. Podophylotoxin and etoposide are the two known anticancer agents derived from P. hexandrum and interestingly the aqueous extract of P. hexandrum showed a typical DNA ladder formation in HL-60 cells confirming its role as an inducer of apoptosis. The results obtained suggest that the plant extract exhibits inhibition of and free radical production and lipid peroxidation, increase in antioxidant enzyme activities, revealing its antioxidant properties, and is also able to show potent anticancer activity as depicted by its ability to cause fragmentation of DNA.     

Melatonin reduces oxidative stress induced by ochratoxin A in rat liver and kidney

Melatonin (MEL) displays antioxidant and free radical scavenger properties. In the present study, the effect of MEL on the oxidative stress induced by ochratoxin A (OTA) administration in rats was investigated. Four groups of 15 rats each were used: controls, MEL-treated rats (5 mg/kg body mass), OTA-treated rats (250 μg/kg) and MEL+OTA-treated rats. After 4 weeks of treatment, the levels of malondialdehyde (MDA), a lipid peroxidation product (LPO) were measured in serum and homogenates of liver and kidney. Also, the levels of glutathione (GSH), and activities of glutathione reductase (GR), glutathione peroxidase (GSPx), superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST) in liver and kidney were determined. In OTA-treated rats, the levels of LPO in serum and in both liver and kidney were significantly increased compared to levels in controls. Concomitantly, the levels of GSH and enzyme activities of SOD, CAT, GSPx and GR in both liver and kidney were significantly decreased in comparison with controls. In rats received MEL+OTA, the changes in the levels of LPO in serum and in liver and kidney were not statistically significant compared to controls. Concomitantly, the levels of GSPx, GR and GST activities in both liver and kidney tissues were significantly increased in comparison with controls. Similar increases in GSPx, GR and GST activities were also observed in MEL-treated rats when compared with controls. In conclusion, the oxidative stress may be a major mechanism for the toxicity of OTA. MEL has a protective effect against OTA toxicity through an inhibition of the oxidative damage and stimulation of GST activities. Thus, clinical application of melatonin as therapy should be considered in cases of ochratoxicosis.     

Prevention of N-nitrosodiethylamine-induced hepatocarcinogenesis by S-allylcysteine

Chemopreventive effect of S-allylcysteine (constituent of garlic) on N-nitrosodiethylamine (NDEA)-induced hepatocarcinogenesis was evaluated in Wistar rats. Significantly decreased lipid peroxidation products (thiobarbituric acid reactive substances-TBARS and lipid hydroperoxides) with increased level of reduced glutathione, increased activities of glutathione S-transferase, and glutathione peroxidase were observed in liver of NDEA-treated rats when compared with control rats. The activities of superoxide dismutase and catalase were significantly decreased in tumor tissue when compared with control. Administration of S-allylcysteine (SAC) showed the inhibition of tumor incidence, modulated the lipid peroxidation, and increased the reduced glutathione, glutathione-dependent enzymes, superoxide dismutase, and catalase in NDEA-induced carcinogenesis. From our results, we speculate that S-allylcysteine mediates its chemopreventive effects by modulating lipid peroxidation, GST stimulation, and by increasing the antioxidants. Hence SAC prevents cells from loss of oxidative capacity in NDEA-induced hepatocarcinogenesis.     

Biochemical and proteomic effects in Procambarus clarkii after chlorpyrifos or carbaryl exposure under sublethal conditions

In vivo effects of two sublethal doses of chlorpyrifos and carbaryl were studied in Procambarus clarkii after 2 and 7 days of exposure, and after pesticide removal. Chlorpyrifos inhibited carboxylesterase activity in a concentration-dependent manner, but acetylcholinesterase was less sensitive. Compared with chlorpyrifos, carbaryl had a less marked effect on esterase activity. The effects of selected pesticides on biotransformation or oxidative stress biomarkers were contradictory. Chlorpyrifos lowered ethoxyresorufin-O-deethylase (EROD), catalase and oxidized glutathione (GSSG) levels but raised glutathione-S-transferase activity, while carbaryl raised EROD, catalase and glutathione-S-transferase, but lowered glutathione peroxidase and reduced glutathione (GSH) levels. The effects on protein expression patterns depending on pesticide type and the tissue used for analysis were studied in parallel by 2-DE. In gill and nervous tissue about 2000 spots (pI 4–7) were resolved, with quite different expression patterns. Chlorpyrifos altered 72 proteins, mostly in nervous tissue, and carbaryl 35, distributed evenly between organs. Several specific spots were selected as specific protein expression signatures for chlorpyrifos or carbaryl exposure in gills and nervous tissue, respectively.