Effects of the herbicide LASSO MTX (alachlor 42% W/V) on biometric parameters and liver biomarkers in the common carp (Cyprinus carpio)

The aim of the study was to evaluate the effect of subchronic exposure to the herbicide LASSO MTX (alachlor 42% W/V) on biometric parameters and important liver biomarkers in the common carp (Cyprinus carpio). One year old fish were exposed for 28 days to LASSO MTX added to the tank water at concentrations of 240 and 2400 μg L⁻¹. The exposure did not affect fish biometric parameters. Glutathione-S-tranferase (GST) activity in liver (hepatopancreas) remained unchanged in exposed fish when compared to controls. However, significant induction of total cytochrome P 450 (CYP 450), ethoxyresorufin-O-deethylase (EROD) activity and elevated glutathione (GSH) in liver of exposed fish were detected.     

Cloning and expression of an atrazine inducible cytochrome P450, CYP4G33, from Chironomus tentans (Diptera: Chironomidae)

Previous studies performed in our laboratory have measured the effect of atrazine exposure on cytochrome P450-dependent monooxygenase activity and have found increased activity in midge larvae (Chironomus tentans) as a result of atrazine exposure (1-10 ppm). Here we report the cloning and expression of a specific C. tentans CYP4 gene that is responsive to atrazine induction with an open reading frame of 1678 bp which encodes a putative protein of 559 amino acid residues. Alignments of deduced amino acid sequences with other insect P450 genes and phylogenetic analysis indicated a high degree of similarity to other insect CYP4 genes. Northern blotting analysis employing a fragment of 1200 bp from the CYP4 gene as a probe indicated that the CYP4 gene was expressed in all developmental stages, but was expressed at highest levels in late instar larvae. Additionally, over-expression of CYP4 in C. tentans exposed to atrazine (10 mg/l) confirms the ability of atrazine to induce specific P450 genes and provides insight into potential consequences of atrazine exposure in aquatic organisms.     

Nonthiol ACE inhibitors, enalapril and lisinopril are unable to protect mitochondrial toxicity due to paraquat

Angiotensin-converting enzyme inhibitors (ACEi) were shown to ameliorate endothelial dysfunction in various human diseases and some of these inhibitors have been reported to enhance antioxidant defenses. The objective of the present study was to shown the abilities of enalapril and lisinopril as two nonthiol ACEi on mitochondrial toxicity due to paraquat. In this study, mitochondrial isolation from rat liver was divided into six groups. Group 1 was considered as control, group 2 received paraquat (5 mM), group 3 received enalapril (0.25 mM), group 4 received lisinopril (0.01 mM), group 5 received paraquat (5 mM) + enalapril (0.25 mM), and group 6 received paraquat(5 mM) + lisinopril (0.01 mM). Viability, lipid peroxidation, catalase activity, GSH (reduced glutathione) and GSSG (oxidized glutathione) concentrations were also determined. Simultaneous treatment of mitochondria with enalapril (0.25 mM) + paraquat (5 mM) and lisinopril (0.0.01 mM) + paraquat (5 mM) did not significantly ameliorate the mitochondrial toxicity induced by paraquat (5 mM) alone (p > 0.05). However, the nonthiol ACEi, enalapril showed to partially improve target of lipid peroxidation due to paraquat. In conclusion, nonthiol ACEi treatment did not improve the increased oxidative stress and the decreased antioxidant mechanisms.     

Nile tilapia (Oreochromis niloticus), liver morphology, CYP1A activity and thyroid hormones after Endosulfan dietary exposure

Endosulfan is a worldwide used insecticide suspected to be highly toxic to aquatic organisms, including fish. Most of the available studies have focused in water exposures, although this pollutant can be transferred through food chain. Therefore, in the present study, the effects of Endosulfan on tilapia (Oreochromis niloticus), when administered through the diet. Fish were fed 21 days with diets containing 1 and 0.5 μg g⁻¹ of Endosulfan, after which qualitative histological liver analysis showed that Endosulfan induced hepatocyte destruction, vessel endothelium rupture and increased melanomacrophages aggregates. To test lower environmentally relevant doses of Endosulfan could induce hepatic damage, as well as other negative effects, such as altered phase I metabolism and plasma thyroid hormone levels. Hence, tilapia were orally exposed to 0.1 and 0.001 μg g⁻¹ for 35 days. Low environmentally realistic doses of Endosulfan were still able to induce liver histopathological damage such as increased hepatocyte vacuolization and increased eosinophil granular cell aggregates. Liver cytochrome P450 1A activity, evaluated through ethoxyresorufin-o-deethylase (EROD), was enhanced in tilapia exposed to 0.001 μg g⁻¹, whereas the highest dose had no measurable effects in this enzyme activity. Fish exposed to 0.1 μg g⁻¹ of Endosulfan had depressed T4 plasma levels. Overall, the results of the present study further demonstrate the toxic effects of Endosulfan in tilapia when administered in the diet at environmentally relevant concentrations, which indicates that in the field food chain transfer may also be an importance source of this pollutant.     

Synergistic and antagonistic effects of atrazine on the toxicity of organophosphorodithioate and organophosphorothioate insecticides to Chironomus tentans (Diptera: Chironomidae)

The acute toxicities of two organophosphorodithioate (dimethoate and disulfoton) and two organophosphorothioate (omethoate and demeton-S-methyl) insecticides were evaluated individually and in binary combination with the herbicide atrazine using fourth-instar larvae of the aquatic midge, Chironomus tentans. Atrazine alone up to 1000 μg/L did not show significant toxicity to the midges in a 48-h bioassay. However, atrazine concentrations as low as 1 μg/L in combination with dimethoate at EC₂₅ (concentration to affect 25% of tested midges), 100 μg/L in combination with disulfoton (EC₂₅), and 10 μg/L in combination with demeton-S-methyl (EC₂₅) significantly enhanced the toxicity of each organophosphate insecticide. In contrast, atrazine concentrations of 10 μg/L and above in combination with omethoate (EC₂₅) significantly decreased the toxicity of the insecticide. Biochemical analysis indicated that increased toxicity of dimethoate, disulfoton, and demeton-S-methyl in binary combination with atrazine correlated to the increased inhibition of acetylcholinesterase. Furthermore, cytochrome P450-dependent O-deethylation activity in the midges exposed to atrazine at 1000 μg/L was 1.5-fold higher than that in the control midges. Thus, atrazine appeared to induce cytochrome P450 monooxygenases in the midges. Elevated cytochrome P450 monooxygenase activity may increase the toxicities of dimethoate, disulfoton, and demeton-S-methyl by enhancing the oxidative activation of dimethoate into omethoate, and disulfoton and demeton-S-methyl into their sulfoxide analogs with increased anticholinesterase activity. In contrast, atrazine reduced the toxicity of omethoate possibly by enhancing the oxidative metabolic detoxification since omethoate does not require oxidative activation.     

Cytochrome P450 monooxygenase-mediated permethrin resistance confers limited and larval specific cross-resistance in the southern house mosquito, Culex pipiens quinquefasciatus

The cytochrome P450-dependent monooxygenases (P450s) are an important enzymatic system that metabolizes xenobiotics (e.g., pesticides), as well as endogenous compounds (e.g., hormones). P450-mediated metabolism can result in detoxification of insecticides such as pyrethroids, or can be involved in the bioactivation and detoxification of insecticides such as organophosphates. We isolated (from the JPAL strain) a permethrin resistant strain (ISOP450) of Culex pipiens quinquefasciatus, having 1300-fold permethrin resistance using standard backcrossing procedures. ISOP450 is highly related to the susceptible lab strain (SLAB) and the high resistance to permethrin is due solely to P450-mediated detoxification. This is the first time in mosquitoes that P450 monooxygenase involvement in pyrethroid resistance has been isolated and studied without the confounding effects of kdr. Resistance in ISOP450 is incompletely dominant (D = +0.3), autosomally linked, and monofactorally inherited. It is expressed in the larvae, but not in adults. Cross-resistance to pyrethroids lacking a 3-phenoxybenzyl moiety (tetramethrin, fenfluthrin, bioallethrin, and bifenthrin) ranged from 1.5- to 12-fold. ISOP450 had only limited (6.6- and 11-fold) cross-resistance to 3-phenoxybenzyl pyrethroids with an α-cyano group (cypermethrin and deltamethrin, respectively). Examination of cross-resistance patterns to organophosphate insecticides in ISOP450 showed an 8-fold resistance to fenitrothion, while low, but significant, levels of negative cross-resistance were found for malathion (RR = 0.84), temephos (RR = 0.73), and methyl-parathion (RR = 0.55). The importance and uniqueness of this P450 mechanism in insecticide resistance is discussed.     

Effects of dimethoate (O,O-dimethyl S-methyl carbamoyl methyl phosphorodithioate) and Ethanol in antioxidant status of liver and kidney of experimental mice

Organophosphorus insecticides and ethanol individually cause free radical production induced by oxidative stress and alter the antioxidants and scavengers of free radicals. The present study indicates the effect caused by dimethoate in combination with ethanol on antioxidant status in mice. Daily, dimethoate at a dose of 18 mg/kg body weight and ethanol at 1 g/kg body weight were orally administered concurrently in a subacute study for 14 days. After the experimental period, the liver and kidney homogenates were analysed for various antioxidant enzymes. The results compared with dimethoate alone treated control indicated an increase in hepatic cytochrome P450 and lipid peroxidation. Decrease in superoxide dismutase, glutathione peroxidase, glutathione reductase, glutathione-S-transferase, and glutathione in liver was observed. In kidney, decrease in CAT, SOD, GR, GST, and GSH was observed. Acetyl cholinesterase activity of RBC was increased. No significant change was observed in catalase in liver and glutathione peroxidase in kidney. The results of the study allow us to hypothesize that dimethoate along with ethanol disturbs the antioxidant status.     

Effects of carbaryl and azinphos methyl on juvenile rainbow trout (Oncorhynchus mykiss) detoxifying enzymes

In this study, the effects of sublethal exposures to the anticholinesterase insecticides azinphos methyl (AzMe) and carbaryl on the detoxifying responses of juvenile rainbow trout Oncorhynchus mykiss were investigated. Juvenile specimen were exposed to sublethal concentrations of AzMe (2.5 and 5 μg/L) and carbaryl (1 and 3 mg/L) for 24, 48 and 96 h. Carboxylesterase (CbE), catalase (CAT) and glutathione S-transferase (GST) activities as well as reduced glutathione (GSH) and cytochrome P450-1A (CYP1A) levels were monitored in liver and/or kidney. In all exposed groups liver CbE was significantly inhibited. Liver and kidney GSH level was reduced after sublethal exposure to both compounds. Carbaryl induced CAT activity during the first 48 h of exposure, followed by a significant decrease, whereas AzMe continuously decreased CAT activity. GST activity and CYP1A were transiently induced at 24 h by carbaryl exposure (3 mg/L) but sublethal exposure to AzMe did not affect GST activity or CYP1A. Our results show that the O. mykiss detoxifying system are a target for carbaryl and AzMe action, probably affecting redox balance. Although the responses showed similar trends in both organs, they were more important in liver than in kidney. The early inhibitory effect in CAT activity and GSH content produced by AzMe may be associated with a high degree of oxidative stress. Early induction of CYP1A, GST and CAT by carbaryl followed by enzyme inhibition suggests a milder or delayed oxidative stress, revealing differences between both pesticides metabolization. CbE inhibition is a good biomarker for AzMe and carbaryl exposure.     

Prochloraz,a potent inducer of the microsomal cytochrome P-450 system

Prochloraz (N-propyl-N-[2-(2,4,6-trichlorophenoxy)ethyl]-imidazole-1-carboxamide), a recently developed agricultural fungicide, is a potent inducer of microsomal enzymes. Rats fed 7 days with a prochloraz-contaminated diet (2500 ppm) showed an increase in hepatic cytochrome P-450, cytochrome b₅, and microsomal protein level; aniline hydroxylase, 7-ethoxycoumarin dealkylase, 7-ethoxyresorufin dealkylase, NADPH-cytochrome c reductase, and epoxide hydrolase were significantly induced. At a lower dose (100 ppm), only an increase in cytochrome P-450 and 7-ethoxyresorufin dealkylase was noticed. As shown with aniline hydroxylase and 7-ethoxycoumarin dealkylase, prochloraz is also a potent inhibitor of drug-metabolizing enzymes. The interaction of prochloraz with hepatic microsomal fraction from rat liver was also studied. Prochloraz binds to oxidized cytochrome P-450 to produce a type II spectral change; the compound also binds to reduced cytochrome P-450. The binding of some ligands (7-ethoxycoumarin, n-octylamine, aniline, and imidazole) to oxidized cytochrome P-450 was determined after induction by prochloraz. Japanese quails (Coturnix coturnix) fed 7 days with a prochloraz-contaminated diet (2000 ppm) showed a dramatic increase in liver weight (2.5-fold) and both hepatic and duodenal cytochrome P-450 (9- and 12-fold, respectively).     

Characterization of the cytochrome b gene fragment of Puccinia species responsible for the binding site of QoI fungicides

The fragment of the cytochrome b (cyt b) gene responsible for the binding site of QoI fungicides was sequenced for different Puccinia species by using DNA and RNA as template for PCR and RT-PCR, respectively. Degenerated primers for the cyt b gene amplified in P. recondita f.sp. tritici a 450 bp fragment, which was cloned and sequenced. At cDNA level, several Thermal Asymmetric InterLaced (TAIL)-PCR cycles were needed to produce a 996 bp long fragment, which corresponded to almost the whole cyt b gene (about 1160-1180 bp, without introns). This fragment was sequenced and specific primers were designed. Amplification with cyt b specific primers using genomic DNA as template revealed the presence of an intron of about 1500 bp length after the codon for glycine at amino acid position 143. By using the same primer pair, the cyt b gene fragment was amplified and sequenced both at cDNA and genomic DNA level also for other rust species, including P. graminis f.sp. tritici (length: 506 bp), P. striiformis f.sp. tritici (755 bp), P. coronata f.sp. avenae (644 bp), P. hordei (660 bp), P. recondita f.sp. secalis (687 bp), P. sorghi (709 bp), and P. horiana (478 bp). At the same position as for P. recondita f.sp. tritici, an intron of about 1500-1600 bp length was detected also in all other Puccinia species. High homologies were observed among all Puccinia species for both the exonic and intronic fragments of the cyt b gene. Specific primers for the cyt b gene of all eight Puccinia species were developed, which easily amplified the fragment of the gene including all possible mutations known to confer resistance to QoIs in several plant pathogens. However, in all tested isolates of the Puccinia species included in this study, the sequence of cyt b gene fragment did not contain any point mutations.     

Human metabolism of atrazine

Atrazine (ATZ) metabolism by human liver microsomes (HLM), cytochrome P450 (CYP) isoforms, and human liver (HL) S9 fractions, was investigated using HPLC/PDA and LC/MS/MS. CYP-dependent metabolites from pooled HLM are desethylatrazine (DEA), desisopropylatrazine (DIA), 1-hydroxyisopropylatrazine (HIATZ), and 2-hydroxyethyl atrazine (HEATZ). DEA and DIA were major metabolites in pooled HLM. CYP1A2 and 2C19, respectively, were major isoforms for DEA and DIA production. CYP3A4, while less active, is generally at high concentrations, produces both DEA and DIA and is significant. The percent total normalized rates (%TNR) for CYP1A2 and 3A4 in pooled HLM were 63% and 24% for DEA, and 35% and 56% for DIA production. Single donor HLM samples, showed correlations for CYP1A2 (r = 0.92) and 3A4 (r = 0.81) for DEA and DIA production, while variations in production of DEA and DIA were 8.5- and 6.0-fold, respectively. Pooled S9 fractions also mediate glutathione conjugation of atrazine, DEA and DIA.     

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.     

The basis for the safening of clomazone by phorate insecticide in cotton and inhibitors of cytochrome P450s

Clomazone may be safely used in cotton to manage weeds when applied following treatments of the organophosphate insecticides phorate or disulfoton. The loss of chlorophyll and carotenoids with 6 days of 100 nM clomazone treatment of cotton seedlings was partially prevented with phorate in hydroponic solution in a rate-dependent manner. In a study to examine the timing of safening from a one-day clomazone (100 nM) treatment, maximum safening was achieved when phorate (50 μM) was applied the same day as clomazone. Phorate decreased metabolism of ¹⁴C-clomazone to polar metabolites in excised cotton shoots and shoots of intact cotton plants. Microsomal studies of corn shoots showed an NADPH-dependent/cytochrome P450 reaction was inhibited by phorate. Additional studies with corn microsomes, corn seedlings and cotton seedlings supported the basis of clomazone safening is the inhibition of toxic clomazone metabolism by P450 inhibitors.     

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.     

Induction of cytochrome P450 monooxygenase activity in the two-spotted spider mite Tetranychus urticae and its influence on acaricide toxicity

Detoxification by cytochrome P450 monooxygenases is an important mechanism involved in pesticide resistance in insects and mites. The activity of these enzymes can be induced by a variety of chemicals. The aim of this study was to evaluate the effect of six P450 inducers (phenobarbital, barbital, 3-methylcholanthrene, geraniol, isosafrole, pentamethylbenzene), known to have an inducing activity in insects and mammals, on the O-deethylation activity in the two-spotted spider mite Tetranychus urticae. Treatment with barbital, phenobarbital and geraniol resulted in a dose-dependent increase in activity. Neither 3-methylcholanthrene, isosafrole nor pentamethylbenzene were effective inducers. Time course studies showed that induction by geraniol and barbital started rapidly within a period of 1-4 h after initiation of the treatment, while maximal activity was reached within 4 and 48 h, respectively. In addition, it was shown that induction with xenobiotic compounds can alter the monooxygenase-mediated acaricide tolerance in a susceptible strain of T. urticae. Although barbital induced higher levels of P450 activity, geraniol proved to be a better compound to decrease toxicity of the tested acaricides.     

Expression of a wheat cytochrome P450 monooxygenase cDNA in yeast catalyzes the metabolism of sulfonylurea herbicides

A wheat cytochrome P450 cDNA (CYP71C6v1) was cloned by RT-PCR and heterologously expressed in yeast. The microsomal fractions derived from this strain could catalyze the metabolism of some sulfonylurea herbicides such as chlorsulfuron, triasulfuron, metsulfuron-metyl, bensulfuron-metyl, and tribenuron-metyl, but not sulfonylurea herbicides such as thifensulfuron and pyrazosulfuron. Kinetic parameters K_m for chlorsulfuron and triasulfuron were 57 (±15) μM and 38 (±16) μM in vitro, respectively. Analysis of the metabolites demonstrated that the CYP71C6v1 functioned as a 5-phenyl ring hydroxylase when chlorsulfuron and triasulfuron were the substrates.     

Hepatoprotective activity of angiotensin-converting enzyme (ACE) inhibitors, captopril and enalapril, against paraquat toxicity

Paraquat is a highly toxic herbicide that is used in most countries without restriction. The cytotoxic action of paraquat is mediated by reactive radicals that are products of its metabolic reduction in cells. It has already been hypothesized that some angiotensin-converting enzyme inhibitors (e.g., captopril and enalapril) could show antioxidant and radical scavenging activity through their structural thiol groups, increasing antioxidant enzymes production or nitric oxide synthesis. In this study the hepatoprotective effect of captopril and enalapril against paraquat induced oxidative stress cytotoxicity was evaluated in isolated rat hepatocyte. Subtoxic concentrations of captopril (0.2 mM) and enalapril (0.2 mM) significantly (p < 0.05) protected the hepatocytes against paraquat (2 mM) induced oxidative stress cytotoxicity markers including: cell lysis, reactive oxygen species (ROS) generation, lipid peroxidation, glutathione depletion, mitochondrial membrane potential decrease, lysosomal membrane oxidative damage and cellular proteolysis. Moreover, we showed that non-thiol enalapril acts as well as thiol containing captopril at inhibiting oxidative stress cytotoxicity markers. Finally, our results support the hypothesis that it is the increase in nitric oxide synthesis and not the presence of the thiol group that accounts for the antioxidant activity of ACE inhibitors.     

Effect of fraxinellone on growth and digestive physiology of Asian corn borer, Ostrinia furnacalis Guenee

A limonoid, fraxinellone significantly reduced the relative growth rate, food consumption rate as well as the efficiency of conversion of ingested food of the Asian corn borer, Ostrinia furnacalis when incorporated into artificial diets at concentration of 10 ppm and above. After being fed with diets that contained fraxinellone for 24 h, the larval midguts of O. furnacalis possess significantly lower activities of α-amylase and non-specific proteases and higher activities of cytochrome P450s. In vitro, the compound did not inhibit the activities of α-amylase and non-specific proteases extracted from the larval midguts. The modes of action of fraxinellone against the insect were discussed.     

Behavioral effects of acute exposure to the insecticide fipronil

The effects of fipronil (Frontline® Top Spot) were investigated in 40 days old rats utilizing open field (OF), hole-board (HB) and elevated plus-maze (EPM) apparatus. Rats (N = 15) received topical application of fipronil (70, 140 and 280 mg/kg) in the neck region and behavior was tested 3 h after administration. Animals treated with corn oil (vehicle) were used as controls. In the OF test animals treated with fipronil at 140 mg/kg showed increased rearing, whereas animals exposed to 280 mg/kg showed increased freezing, grooming, and rearing. In the HB test fipronil at 280 mg/kg increased head-dip and head-dipping behaviors. In the EPM test the only observed effect was increased number of entries in both open and closed EPM arms in animals treated with 280 mg/kg. In conclusion, dermal exposure to fipronil causes effects related to emotionality, fear, and exploratory activity; results add strength to the growing concern that pirazole insecticides can be neurotoxic to humans.     

Activation of paraquat in the earthworm Allolobophora chlorotica is mediated by NAD(P)H-cytochrome c reductase activities

This study reports that earthworms, Allolobophora chlorotica, are capable of biotransforming paraquat, a toxic herbicide, resulting in the formation of reactive oxygen species (ROS). We found that in earthworms the reduction of paraquat is mediated by NADPH- and NADH-cytochrome c reductase activities. The formation of superoxide anion (O₂⁻) from the incubation of paraquat with the earthworm extracts was demonstrated by using both Cypridina luciferin analog (CLA) chemiluminescence and the SOD-inhibitable cytochrome c reduction reaction. In addition, in vivo exposure of earthworms to paraquat in solution (24 and 48 h) was performed to investigate whether or not the herbicide affects the levels of the NAD(P)H-cytochrome c reductase activities. Although in vitro NADPH-cytochrome c reductase reduces paraquat more easily than the NADH-dependent activity, after the in vivo exposure an increase of NADH-cytochrome c reductase activity(s) by 12% compared to control values was observed, whereas NADPH-cytochrome c reductase activity was not affected. Xanthine oxidase (XO) is an enzyme implicated in paraquat toxicity, however, no XO was detected in earthworm extracts nor hypoxanthine was a source of electrons for the herbicide reduction. For comparative reasons menadione, a redox cycling quinone, was also incubated with the earthworm extracts. It was found that the incubation of menadione with earthworm extracts formed about two times more (O₂⁻) than with paraquat. It is concluded that the exposure of paraquat to earthworms could elicit radical formation and consequently toxic effects via oxidative stress-mediated mechanisms. The reduction of paraquat by the reductases leads to the formation of paraquat radical, which reacts with molecular oxygen, accounting for the formation of superoxide anion. Further studies are required to conclude that the observed increase of NADH-cytochrome c reductase activity(s) should be used as a biomarker for paraquat exposure in earthworms.