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.     

Impacts of hypersaline water on the biotransformation and toxicity of fenthion on rainbow trout (Oncorhynchus mykiss), striped bass (Morone saxatilis X Morone chrysops) and tilapia (Oreochromis mossambicus)

Hypersaline water derived from agricultural practices primarily in arid landscapes can impact fisheries in streams receiving run-off from fields. Previous studies have indicated significant elevation in the toxicity of thio-ether pesticides by hypersaline water in certain species of euryhaline fish due to enhanced formation of sulfoxide metabolites which may be more toxic than the parent compounds. The objectives of this study were to evaluate the effects of salinity on the toxicity of the thio-ether organophosphate pesticide, fenthion in three species of euryhaline fish: rainbow trout (Oncorhynchus mykiss), striped bass (Morone saxatilis X Morone chrysops), tilapia (Oreochromis mossambicus) and determine whether a relationship was observed between toxicity and enantioselective sulfoxidation. Stereochemical formation and total sulfoxide formation did not mirror acute toxicity in the three species exposed in fresh or hypersaline conditions. Mortality of striped bass and rainbow trout due to fenthion exposure was enhanced by hypersaline treatments and a trend towards increased toxicity was observed in tilapia. In liver microsomes of rainbow trout, inhibition of cytochrome P450 (CYP) caused a respective 7- and 3-fold increase in sulfoxide formation in liver microsomes fresh and saltwater fish, and a significant increase in the formation of the (+)R-sulfoxide. CYP inhibition also caused a significant elevation of the (+)R-sulfoxide in freshwater striped bass, but not in hypersaline-treated bass. The results indicated contribution by CYP in the sulfoxidation of fenthion as well as the formation of other metabolites in all three species. In summary, hypersaline conditions impacted fenthion toxicity under conditions that appeared to be independent of fenthion sulfoxide formation indicating a much more complex mechanism of action for compounds with phosphorothioate and thio-ether structural features potentially involving multiple oxidative pathways.     

An in vitro assay for the assessment of the effects of an organophosphate, paraoxon, and a triazine, atrazine, on the heart of the gilthead sea bream (Sparus aurata)

The effects of paraoxon and atrazine on the spontaneously beating auricle, isolated from the heart of Sparus aurata, were assessed. Paraoxon, 5 μM, eliminated the atria contraction within 28.4 ± 2.8 min, an effect which was fully reversed by 15 μM atropine, an antagonist of muscarinic cholinergic receptors. The IC₅₀ was estimated to be 3.2 ± 1.5 μΜ. Atrazine, 50 and 100 μM, induced a 22.5 ± 3.2 and 32.9 ± 2.3% increase in the force of auricle contraction, caused by excitation of sympathetic synaptic terminals releasing adrenaline. This effect was reversed by 50 μM propranolol, a blocker of β-adrenoreceptors. The results have shown that both sympathetic and parasympathetic nerve terminals are activated by atrazine. Also, the auricle contraction is mainly under sympathetic control, while the frequency is dominated by cholinergic system. Finally, the detailed parameters of the auricle contraction estimated during exposure to specific pesticides, force, frequency, time-response curves and electromechanical coupling can be further used to assess and compare the toxic effects of other compounds, anticholinesterases for example, on the heart of the fish.     

Mechanisms for multiple resistances in field populations of common cutworm, Spodoptera litura (Fabricius) in China

The mechanisms for multiple resistances had been studied with two field resistant strains and the selected susceptible and resistant strains of Spodoptera litura (Fabricius). Bioassay revealed that the two field strains were both with high resistance to pyrethroids (RR: 63-530), low to medium resistance to organophosphates and carbamates, AChE targeted insecticides (RR: 5.7-26), and no resistance to fipronil (RR: 2.0-2.2). Selection with deltamethrin in laboratory could obviously enhance the resistance of this pest to both pyrethroids and AChE targeted insecticides. Synergism test, enzyme analysis and target comparison proved that the pyrethroid resistance in this pest associated only with the enhanced activity of cytochrome P450 monooxygenase (MFO) and esterase. However the resistance to the AChE targeted insecticides depended on the target insensitivity and also the enhanced activity of MFO and esterase. Thus, the cross-resistance between pyrethroids and the AChE targeted insecticides was thought to be resulted from the enhanced activity of MFO and esterase.     

Deletion of Cyp6d4 does not alter toxicity of insecticides to Drosophila melanogaster

Cytochrome P450-dependent monooxygenases are important in the activation and detoxification of numerous insecticides. In this study, a Drosophila melanogaster Cyp6d4 null mutant was used to determine the role of this P450 in insecticide metabolism. This null mutant was generated by imprecise excision of a mobile P element located upstream to the P450 gene Cyp6d4. Comparative analysis between the non-functional mutant and relevant control strains shows that Cyp6d4 does not appear to be involved in the metabolism of chlorfenapyr, cypermethrin, diazinon, imidacloprid, malathion, oxamyl, parathion, or pyrethrum extract, even though these insecticides are known to be activated or detoxified by P450-monooxygenases. No obvious abnormalities in development were seen in the Cyp6d4 null mutant, indicating that Cyp6d4 is not critical for the metabolism of vital endogenous substrates.     

Cellular and biochemical effects induced by atrazine on blood of male Japanese quail (Coturnix japonica)

Atrazine a potent endocrine disruptor herbicide is broadly used to control rapidly growing unwanted weeds in various cereals crops which induce adverse effects both in mammalian and avian species. In present study 96 mature male Japanese quail (Coturnix japonica) were procured and randomly kept in eight groups (A to H) each having 12 birds. Atrazine was administered orally at 0, 10, 25, 50, 100, 250 and 500 mg/kg body weight to all experimental groups. The mitomycin C at 2 mg/kg body weight was given to the birds of group B which served as a positive control. From each group 4 birds were randomly selected and harvested at day 15, 30 and 45. A significant (P < 0.05) decrease in serum total protein, serum albumin and serum testosterone values were recorded at day 45 in all treated groups. A significant increase in serum ALT and AST concentration was also recorded. Moreover, morphological alterations in nucleus of erythrocytes were also observed including blebbed nuclei, notched nuclei, lobed nuclei, vacuolated cells, binucleated cell, and cell with pear shaped and micronucleus. Overall, our results show that atrazine at higher doses induces significant serum biochemical alterations and changes in nucleus of erythrocytes.     

The role of cytochrome P450 monooxygenase in the different responses to fenoxaprop-P-ethyl in annual bluegrass (Poa annua L.) and short awned foxtail (Alopecurus aequalis Sobol.)

Herbicide resistance or tolerance in weeds mediated by cytochrome P450 monooxygenase is a considerable problem. However, cytochrome P450 mediated resistance or tolerance in weeds was less studied. Thus, in this work, the role of the cytochrome P450 monooxygenase in the different responses of Poa annua and Alopecurus aequalis to fenoxaprop-P-ethyl was studied. We found that the effect of fenoxaprop-P-ethyl could be synergized by piperonyl butoxide (PBO) in P. annua, but not by malathion. After being treated with fenoxaprop-P-ethyl (containing mefenpyr-diethyl), the contents of cytochrome P450 and cytochrome b₅ in P. annua increased significantly compared to plants treated with mefenpyr-diethyl only or untreated plants. However, the increase was less in A. aequalis, which was susceptible to fenoxaprop-P-ethyl. The activities of ρ-nitroanisole O-demethylase (PNOD), ethoxyresorufin O-deethylase (EROD), ethoxycoumarin oxidase (ECOD) and NADPH-dependent cytochrome P450 reductase mediated by cytochrome P450 monooxygenase increased in P. annua after treatment with fenoxaprop-P-ethyl, especially the activities of ECOD and cytochrome P450 reductase. Besides this, cytochrome P450 monooxygenase activity toward fenoxaprop-P-ethyl in P. annua increased significantly compared to untreated or treated with mefenpyr-diethyl plants and treated or untreated A. aequalis. Cytochrome P450 monooxygenase may play an important role in the different responses to fenoxaprop-P-ethyl in P. annua and A. aequalis.     

In-vivo effects of 2,4-D and atrazine on cytochrome P-450 and insecticide toxicity in southern armyworm (Spodoptera eridania) larvae

After feeding 2,4-D or atrazine in a diet to southern armyworm (Spodoptera eridania Cram.) larvae for three days, the effect on total content and activities of cytochrome P450 and on insecticide toxicity were determined. Both 2,4-D and atrazine induced cytochrome P450-catalyzed aldrin epoxidation (AE) and methoxyresorufin O-demethylatin (MROD). The 2,4-D was a more potent inducer for total cytochrome P450 content, whereas atrazine disproportionately increased AE. Both compounds increased MROD significantly. The apparent kinetic characteristics of AE indicates that 2,4-D and atrazine induced similar P450 isozymes (K_m 8.78 and 7.80 μM, respectively), which may differ from the constitutive isozyme (K_m 3.14 μM). The 2,4-D-induced cytochrome P450 contributed to decreased carbaryl and permethrin toxicity, whereas the atrazine-induced cytochrome P450 caused decreased parathion and permethrin toxicity. The carbaryl toxicity correlated directly with 2,4-D-induced total P450 content and activities but not with atrazine-induced changes. The 2,4-D and atrazine also induced nonspecific esterase activity which may contribute to permethrin detoxification.     

Effect of celangulin V on detoxification enzymes in Mythimna separata and Agrotis ypsilon

Celangulin V (CA-V), a β-dihydroagrofuran sesquiterpene polyol ester, is extracted from the root bark of Chinese bittersweet, Celastrus augulatus Maxim. It exhibited selective toxicity against different insects. By CO-difference spectral and biochemical method, the effects of CA-V on two kinds of detoxification enzymes, cytochrome P450 (P450 and NADPH-cytochrome P450 reductase) and glutathione S-transferase, were investigated in oriental armyworm, Mythimna separata and black cutworm, Agrotis ypsilon. CA-V showed higher induction against P450 of M. separata than that of A. ypsilon. Treated by CA-V, the maximum absorption of M. separata increased 1.2 and 0.8 nm than the control, respectively. Meanwhile, compared with the control, the P450 content and NADPH-P450 reductase activity in treated M. separata larvae increased 1.46-, 2.26- and 1.26-, 2.56-fold, respectively. But in treated A. ypsilon larvae, they all increased a little more than those of control. So far as M. separata and A. ypsilon, whether there is exposure of CA-V or not, the P450 content and GST activity in A. ypsilon were obviously higher than those in M. separata. It suggested that the content or activity difference of these two kinds of detoxification enzymes may have important roles in the selective toxicity of CA-V in M. separata and A. ypsilon.     

Cytogenetic effects of commercially formulated atrazine on the somatic cells of Allium cepa and Vicia faba

In the present study cytogenetic effects of atrazine herbicide, were examined on the root meristem cells of Allium cepa and Vicia faba. Test concentrations were chosen by calculating EC₅₀ values of formulated atrazine against both the test systems which determined to be 30 mg l⁻¹ for A. cepa and 35 mg l⁻¹ for V. faba, respectively. For cytogenetic effects root meristem cells of A. cepa were exposed to 15, 30 or 60 mg l⁻¹ whereas V. faba to 17.5, 35 or 70 mg l⁻¹ for 4 or 24 h. Roots exposed for 4 or 24 h, after sampling, were left in water for 24 h recovery and sampled at 24 h post-exposure. A set of onion bulbs or seedlings of V. faba exposed to DMSO (0.3%) was run parallel for negative control. Treatment of atrazine significantly and dose-dependently inhibited the mitotic index (MI) and induced micronucleus formation (MN) chromosome aberrations (CA) and mitotic aberrations (MA) in both the test systems at 4 or 24 h. Root meristem cells examined at 24 h post-exposure also revealed significant (p < 0.001) frequencies of MN, CA or MA despite considerable decline. Chromosome breaks and fragments were found to be major CA whereas C-metaphase, chromosome bridges and laggards were prevalent MA. Results of our study, indicate that atrazine may produce genotoxic effects in plants. Further, both the plant bioassays found to be sensitive indicators for the genotoxicity assessment as the outcome of majority of in vivo/in vitro mammalian tests are comparable.     

Effect of atrazine and chlorpyrifos exposure on heat shock protein response in the brain of common carp (Cyprinus carpio L.)

Atrazine (ATR) and chlorpyrifos (CPF) are the most common pesticides found in freshwater ecosystems throughout the world. In the present study, the common carps were exposed to ATR, CPF and their mixture as describe in Table 1. Then we investigated the HSPs (HSP60, HSP70 and HSP90) expression in the brain of common carp by western blot and real-time PCR. Results indicated that HSP60 and HSP70 were significantly increased as compared to corresponding controls after 40 d exposure (P < 0.05). While the increased expression levels of HSP90 in exposure groups was only observed at 4.28 μg/L ATR group and 1.16 μg/L CPF group, and after 40 d recovery, the expression levels of HSP90 were higher than most of exposure groups (P < 0.05). It was suggested that the increased gene expression of HSPs was possible to protect injured cell mass caused by ATR, CPF and their mixture exposure. The present results provided new insights into the mechanisms used by fish to adapt to stressful environments.     

Induction effects of host plants on insecticide susceptibility and detoxification enzymes of Bemisia tabaci (Hemiptera: Aleyrodidae)

BACKGROUND: The polyphagous B‐biotype Bemisia tabaci (Gennadius) has developed a high resistance to commonly used insecticides in China. To illustrate the induced changes by host plant, bioassay and biochemical research on five different host populations were investigated. RESULTS: Except for bifenthrin, all tested insecticides showed lower toxicity to the B. tabaci poinsettia population compared with other host populations. Moreover, four insecticides, the exceptions being abamectin and fipronil, showed highest toxicity towards the tomato population. The LC₅₀ values of the poinsettia population, particularly towards acetamiprid, were 14.8‐, 10.3‐ and 7.29‐fold higher than those of tomato, cucumber and cabbage respectively. The CarE activities of B. tabaci cabbage and cucumber populations were all significantly higher than those of poinsettia, cotton and tomato populations. The ratio of the cabbage population was 1.97‐, 1.79‐ and 1.30‐fold higher than that of poinsettia, cotton and tomato respectively. The frequency profiles for this activity also have obvious differences. The GST and P450 activities of the cucumber population were the lowest in the five host populations. CONCLUSION: Long‐term induction of host plants for B‐biotype B. tabaci could influence their susceptibilities to several insecticides. Rational selection and usage of insecticides for particular hosts will be helpful for resistance management and control of this species. Copyright © 2010 Society of Chemical Industry     

Cross-resistance and biochemical mechanisms of abamectin resistance in the western flower thrips, Frankliniella occidentalis

Abamectin resistance was selected in the western flower thrips [Frankliniella occidentalis (Pergande)] under the laboratory conditions, and cross-resistance patterns and possible resistance mechanisms in the abamectin-resistant strain (ABA-R) were investigated. Compared with the susceptible strain (ABA-S), the ABA-R strain displayed 45.5-fold resistance to abamectin after 15 selection cycles during 18 generations. Rapid reversion of abamectin resistance was observed in the ABA-R strain in the absence of the insecticide selection pressure. Moderate and low levels of cross-resistance to chlorpyrifos (RR 11.4) and lambda-cyhalothrin (3.98) were observed in the ABA-R strain, but no significant cross-resistance was found to spinosad (2.00), acetamiprid (1.47) and chlorfenapyr (0.26). Our studies also showed that the esterase inhibitor S,S,S-tributyl phosphorotrithioate (DEF) and glutathione S-transferase inhibitor diethyl maleate (DEM) were not able to synergize the toxicity of abamectin, whereas the oxidase inhibitor piperonyl butoxide (PBO) conferred a significant synergism on abamectin in the ABA-R strain (SR 3.00). Biochemical analysis showed that cytochrome P450 monooxygenase activity of the ABA-R strain was 6.66-fold higher than that of the ABA-S strain. It appears that enhanced oxidative metabolism mediated by cytochrome P450 monooxygenases was a major mechanism for abamectin resistance in the western flower thrips.     

Identification of cytochrome P450 differentiated expression related to developmental stages in bromadiolone resistance in rats (Rattus norvegicus)

Adult, 20-week-old, rats from a Danish bromadiolone-resistant strain of rats (Rattus norvegicus) over-express the cytochrome P450 genes Cyp2e1, Cyp3a2 and Cyp3a3 upon bromadiolone exposure. Furthermore, adult female rats of this strain over-express the Cyp2c13 gene and suppress Cyp2c12, while males over-express the Cyp2a1 gene. The altered gene expression has been suggested to be involved in the bromadiolone resistance by facilitating enhanced anticoagulant metabolism. To investigate the gene expression of these cytochrome P450 genes in rats of different developmental stages we compared expression profiles from 8-, 12- and 20-week-old resistant rats of the Danish strain to profiles of anticoagulant-susceptible rats of same ages. The three age-groups were selected to represent a group of pre-pubertal, pubertal and adult rats. We found expression profiles of the pre-pubertal and pubertal resistant rats to concur with profiles of the adults suggesting that cytochrome P450 enzymes are involved in the Danish bromadiolone resistance regardless of developmental stage. We also investigated the relative importance of the six cytochrome P450s in the different development stages of the resistant rats. The P450-3a2 and -3a3 isoforms were proposed to be of higher importance in adult male resistance than in pre-pubertal resistance. In contrast, the P450-2c13 and -3a2 isoforms were proposed to be more important in sexual immature female resistance, while the P450-2e1 and -3a3 isoforms were suggested to play a more significant role in adult female resistance.     

Involvement of esterases in diazinon resistance and biphasic effects of piperonyl butoxide on diazinon toxicity to Haematobia irritans irritans (Diptera: Muscidae)

Resistance to insecticides remains a major problem for the successful control of the horn fly, Haematobia irritans irritans (L.), one of the most important pests of cattle in many countries including the United States. The organophosphate (OP) insecticide diazinon has been used to control pyrethroid-resistant populations of the horn fly. There are only a few reported cases of horn fly resistance to diazinon in the United States and Mexico. Piperonyl butoxide (PBO) has been used successfully as a synergist of pyrethroid insecticides to control horn flies. PBO-synergized diazinon products are also available for horn fly control in the United States, although PBO is known to inhibit the bio-activation of certain OP insecticides including diazinon. A study was conducted to evaluate the effect of PBO on diazinon toxicity to horn flies using a filter paper bioassay technique. These bioassays in both the susceptible and diazinon-resistant horn fly strains revealed a biphasic effect of PBO on diazinon toxicity to horn flies. PBO inhibited diazinon toxicity when the PBO concentration used was high (5%), and no effect was observed when PBO concentration was intermediate (2%). However, at low concentrations (1% and lower), PBO significantly synergized diazinon toxicity. We demonstrated that enhanced esterase activity was associated with survivability of horn flies exposed to diazinon alone. PBO has been shown to inhibit esterase activity in other insect species. However, results of biochemical assays with esterases from this study suggest that PBO did not have significant effect on the overall esterase activity in the horn fly. The observed synergistic effect of PBO at lower concentrations on diazinon toxicity to horn flies could not be explained by reduced esterase activity due to PBO inhibition. It is likely that PBO synergized diazinon toxicity at lower concentrations by facilitating penetration of diazinon through the cuticle and/or inhibiting the oxidative detoxification of diazinon, and reduced diazinon toxicity at high PBO concentration by inhibiting the bio-activation of diazinon.