Involvement of cytochrome P‐450 enzyme activity in the selectivity and safening action of pyrazosulfuron‐ethyl

To investigate the selectivity and safening action of the sulfonylurea herbicide pyrazosulfuron‐ethyl (PSE), pyrazosulfuron‐ethyl O‐demethylase (PSEOD) activity involving oxidative metabolism by cytochrome P‐450 was studied in rice (Oryza sativa L cv Nipponbare) and Cyperus serotinus Rottb. Cytochrome P‐450‐dependent activity was demonstrated by the use of the inducers 1,8‐naphthalic anhydride and ethanol, the herbicides PSE, bensulfuron‐methyl, dimepiperate and dymron, or the inhibitor piperonyl butoxide (PBO). Growth inhibition in C serotinus seedlings was more severe than that in rice seedlings. O‐Dealkylation activities of PSE were induced differently in rice and in C serotinus, with distinctly higher activity in rice seedlings. The induced PSEOD activities were slightly inhibited by PBO in rice seedlings, whereas they were strongly inhibited in C serotinus seedlings. Dimepiperate and dymron were effective safeners of rice against PSE treatment. Treatments with herbicide alone resulted in less induction of PSEOD activity compared with combined treatments of the herbicide and safener. PSEOD activity in rice seedlings induced with herbicide alone was strongly inhibited by PBO, whereas it was weakly inhibited in rice seedlings induced with combinations of PSE and two safeners. These results suggest that O‐demethylation by cytochrome P‐450 enzymes may be involved in the metabolism of PSE and may contribute to its selectivity and safening action. Furthermore, these results suggest the existence of a multiple form of cytochrome P‐450 in plants. © 2001 Society of Chemical Industry     

Responses to clomazone and 5-ketoclomazone by Echinochloa phyllopogon resistant to multiple herbicides in Californian rice fields

BACKGROUND: Late watergrass [Echinochloa phyllopogon (Stapf.) Koss.] is a major weed of Californian rice that has evolved P450-mediated metabolic resistance to multiple herbicides. Resistant (R) populations are also poorly controlled by the recently introduced herbicide clomazone. The authors assessed whether this cross-resistance was also P450 mediated, and whether R plants also had reduced sensitivity to photooxidation. Understanding mechanism(s) of resistance facilitates the design of herbicide management strategies to delay resistance evolution.RESULTS Ratios (R/S) of R to susceptible (S) GR(50) were near 2.0. [(14)C]Clomazone uptake was similar in R and S plants. Clomazone and its metabolite 5-ketoclomazone reduced chlorophyll and carotenoids in S more than in R plants. The P450 inhibitors disulfoton and 1-aminobenzo-triazole (ABT) safened clomazone in R and S plants. Disulfoton safened 5-ketoclomazone only in S plants, while ABT synergized 5-ketoclomazone mostly against S plants. Paraquat was more toxic in S than in R plants.CONCLUSION: Cross-resistance to clomazone explains failures to control R plants in rice fields, and safening by P450 inhibitors suggests that oxidative activation of clomazone is needed for toxicity to E. phyllopogon. Clomazone resistance requires mitigation of 5-ketoclomazone toxicity, but P450 detoxification may not significantly confer resistance, as P450 inhibitors poorly synergized 5-ketoclopmazone in R plants. Responses to paraquat suggest research on mechanisms to mitigate photooxidation in R and S plants is needed. Copyright (c) 2008 Society of Chemical Industry.     

Absorption, translocation, and metabolism of propoxycarbazone-sodium in ALS-inhibitor resistant Bromus tectorum biotypes

Experiments were conducted to investigate the absorption, translocation, and metabolism of propoxycarbazone-sodium in acetolactate synthase-inhibitor resistant (AR and MR) and susceptible (AS and MS) Bromus tectorum biotypes. Absorption and translocation of ^l4C-propoxycarbazone-sodium were similar in all biotypes. One major and three minor metabolites were identified using reverse-phase high performance liquid chromatography. In all biotypes, 80% of the propoxycarbazone-sodium was metabolized by 72 h after treatment (HAT). However, propoxycarbazone-sodium was metabolized more rapidly in the MR biotype than in the other biotypes. The half-life of propoxycarbazone-sodium in the MR biotype was 8.9 h, which was 30, 36, and 40% shorter than in the AS, AR, and MS biotypes, respectively. When ¹⁴C-propoxycarbazone-sodium was applied with 1-aminobenzotriazole, a known cytochrome P450 inhibitor, metabolism decreased 20% 12 HAT in the MR biotype. These results indicate that resistance of the MR biotype to propoxycarbazone-sodium is due to a relatively rapid rate of propoxycarbazone-sodium metabolism compared to other B. tectorum biotypes and that cytochrome P450s may be involved in the metabolism. The fact that these populations evolved so quickly and with different resistance mechanisms is a concern as more ALS inhibitors are introduced into the production systems.     

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.     

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.     

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.     

聚乙二醇8000对棉铃虫微粒体蛋白沉淀作用的研究

为建立棉铃虫Helicoverpa armigera微粒体P450的分离纯化方案,比较了不同浓度下聚乙二醇8000(PEG8000)对棉铃虫幼虫中肠和脂肪体微粒体蛋白的沉淀作用。结果表明,终浓度为8.0×104 mg/L的PEG8000可以使中肠和脂肪体微粒体蛋白的78%沉淀下来,其中包含的细胞色素P450分别占中肠和脂肪体P450总量的28%和34%。SDS-PAGE显示,中肠微粒体经8.0×104 mg/L的PEG8000沉淀后,被沉淀蛋白的分子质量主要集中在14.1 ~ 40 kD和66 ~97 kD范围内。     

Biotransformation of clomazone in rice (Oryza sativa) and early watergrass (Echinochloa oryzoides)

Rice (Oryza sativa), a relatively tolerant species, and early watergrass (Echinochloa oryzoides; EWG), a relatively susceptible species, were exposed to ¹⁴C-labeled clomazone to determine accumulation, biotransformation, and mass balance. On a total mass basis, rice absorbed more clomazone than EWG (p < 0.05), but on a nmol/g basis, there was no significant difference between the two species (p > 0.05). Rice contained more extractable ¹⁴C residues (7.7 ± 0.5 vs. 4.8 ± 0.5 nmol in rice vs. EWG, respectively; p < 0.5), but the concentration in EWG was significantly higher (4.2 ± 0.5 vs. 1.8 ± 0.1 nmol/g in EWG vs. rice, respectively; p < 0.01). More metabolized residue was measured in EWG compared to rice (84.1% vs. 67.9%; p < 0.01). Both species produced hydroxylated forms, β-d-glucoside conjugates, and several other unidentified polar metabolites, but EWG generally produced higher metabolite concentrations. The concentration of the suspected active metabolite, 5-ketoclomazone, was significantly higher in EWG vs. rice (21 ± 2 vs. 5.7 ± 0.5 pmol/g, respectively; p < 0.01). Differences in sensitivity to clomazone between rice and EWG appear to be due to differential metabolism, but in this case the more susceptible EWG qualitatively and quantitatively metabolized more clomazone than the more tolerant rice. This is consistent with the action of a metabolically activated herbicide. This metabolic difference could be exploited to develop herbicide safeners for use with clomazone.     

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.     

In vivo and in vitro metabolism of fipronil by larvae of the European corn borer Ostrinia nubilalis

In vivo and in vitro metabolism of [14C]fipronil was examined in a susceptible European corn borer (Ostrinia nubilalis, Hübner) laboratory strain. [14C]Fipronil penetrated the larval integument slowly, with 71.5% of the applied radioactivity recovered from surface rinses 24 h after topical application. Despite this slow penetration, radioactivity was detected in both the excrement and internal organo-soluble fractions. Radioactivity in the internal aqueous fraction and tissue pellet accounted for less than 0.8% of total radioactivity. The in vivo studies suggest that fipronil oxidation to its sulfone metabolite is the major route of metabolic conversion. In vitro studies were performed using subcellular microsomal fractions isolated from European corn borer larval midguts. Cytochrome P450-dependent monooxygenase activity (methoxyresorufin O-demethylase) was consistently observed in midgut preparations, and formation and detection of the sulfone metabolite in the same midgut preparations was also NADPH-dependent and inhibited by piperonyl butoxide. In vitro metabolism results indicate microsomal monooxygenases are responsible for the conversion of fipronil to its sulfone form in the European corn borer.     

Influences of sub-acute exposure to paraquat on cytochrome P450 3A2 expression in rat liver and lungs

This study was designed to investigate the possible effects of paraquat sub-acute poisoning on cytochrome P450 3A2 expression in the liver and lungs. Twenty adult male rats (150-200 g) were exposed either against saline normal as control group or various doses of paraquat (3.5, 7 and 10 mg/kg, s.c.) as test groups for 7 consecutive days. Paraquat-exposed animals showed loss of body weight and elevation in serum levels of alkaline phosphates and alanine aminotransferase in a dose-dependent fashion. Moreover, animals in the test groups demonstrated a significant (P < 0.05) increase of malondialdehyde content in both the liver and lung tissues. Ultimately, by using RT-PCR method it became clear that 7 days exposure to paraquat resulted in a total suppression of cytochrome P450 3A2 at the mRNA level in the lungs. By contrast, a considerable up regulation of the same gene occurred in the liver. This data suggest that paraquat not only affect the lungs as a main target tissue but also up regulates the predominant cytochrome P450 gene in the liver which may induce detoxification processes.     

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.     

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.     

Thiamethoxam is a neonicotinoid precursor converted to clothianidin in insects and plants

Neonicotinoid insecticides are compounds acting agonistically on insect nicotinic acetylcholine receptors (nAChR). They are especially active on hemipteran pest species such as aphids, whiteflies, and planthoppers, but also commercialized to control many coleopteran and some lepidopteran pest species. The most prominent member of this class of insecticides is imidacloprid. All neonicotinoid insecticides bind with high affinity (I₅₀-values around 1 nM) to [³H]imidacloprid binding sites on insect nAChRs. One notable ommission is the neonicotinoid thiamethoxam, showing binding affinities up to 10,000-fold less potent than the others, using housefly head membrane preparations. Electrophysiological whole cell voltage clamp studies using neurons isolated from Heliothis virescens ventral nerve cord showed no response to thiamethoxam when applied at concentrations of 0.3 mM, although the symptomology of poisoning in orally and topically treated noctuid larvae suggested strong neurotoxicity. Other neonicotinoids, such as clothianidin, exhibited high activity as agonists on isolated neurons at concentrations as low as 30 nM. There was no obvious correlation between biological efficacy of thiamethoxam against aphids and lepidopterans and receptor affinity in electrophysiological and binding assays. Pharmacokinetic studies using an LC-MS/MS approach to analyze haemolymph samples taken from lepidopteran larvae revealed that thiamethoxam orally applied to 5th instar Spodoptera frugiperda larvae was rapidly metabolized to clothianidin, an open-chain neonicotinoid. Clothianidin shows high affinity to nAChRs in both binding assays and whole cell voltage clamp studies. When applied to cotton plants, thiamethoxam was also quickly metabolized, with clothianidin being the predominant neonicotinoid in planta briefly after application, as indicated by LC-MS/MS analyses. Interestingly, the N-desmethylated derivative of thiamethoxam, N-desmethyl thiamethoxam, was not significantly produced in either lepidopteran larvae or in cotton plants, although it was often mentioned as a possible metabolite, being nearly as active as imidacloprid. In conclusion, our investigations show that thiamethoxam is likely to be a neonicotinoid precursor for clothianidin.     

Induction of defense response of Oryza sativa L. against Pyricularia grisea (Cooke) Sacc. by treating seeds with chitosan and hydrolyzed chitosan

Seeds of rice (Oryza sativa L.) were treated with chitosan and hydrolyzed chitosan at 100, 500 and 1000 mg L⁻¹. After 18 days of germination, spore suspension of Pyricularia grisea was applied. The enzyme activity of phenylalanine ammonia-lyase, β-1-3-glucanase, chitinase and chitosanase in leaves of rice seedlings was evaluated after 24, 72, 120 and 168 h of inoculation. Blast affected area (%) was evaluated 7 and 14 days after spraying spore suspension. Chitosan performance to elicit defense response induction was associated with the concentration and type of chitosan. The activity of most of the enzymes tested was induced in leaves of treated seeds with chitosan and hydrolyzed chitosan at 1000 and 500 mg L⁻¹, respectively. The highest enzyme activities were observed with hydrolyzed chitosan after 72 h however, compared to chitosan, the activity was not maintained during the entire post-inoculation period. The highest control (0 = no lesions) of P. grisea in rice seedlings was observed at 1000 mg L⁻¹ in both chitosan and hydrolyzed chitosan treated leaves. Symptoms of infection by P. grisea were evident after 14 days evaluation date, but according to the standard scale proposed by the International Rice Research Institute, these symptoms fell into the resistance category of blast diseases.     

Physiological and biochemical modes of action of the diphenylether aclonifen

Aclonifen belongs to the diphenylether (DPE) chemical family among which potent herbicides with a photodependent mode of action can be found. For years aclonifen has been used in several types of cultures. However its biochemical mode of action remains unclear although it was listed as a carotenoid synthesis inhibitor by the Herbicide Resistance Action Committee (HRAC). As a matter of fact, corn seedling leaves treated with 10⁻⁴ M aclonifen and maintained in the dark no longer contained carotenoids but showed an accumulation of a compound having all the characteristics of phytoene. That demonstrated aclonifen ability to inhibit the desaturases catalyzing the transformation of phytoene into carotenoids. Moreover, aclonifen (5 × 10⁻⁵ M) was responsible for a photodependent cell destruction (necrosis) of cucumber cotyledons typically due to protoporphyrin IX accumulation. The same phenomenon was demonstrated in aclonifen-treated etiolated corn seedlings (10⁻⁴ M) that showed an accumulation of protoporphyrin IX, reaching 62 ng/g of leaf fresh mass and reactive oxygen species production under light. On these two cases (cucumber cotyledons and etiolated corn seedlings), aclonifen was acting as a typical DPE, as demonstrated by the accumulation of protoporphyrin IX. As a whole, aclonifen was shown here, to act on two different biochemical pathways including carotenoid synthesis on the one hand, as well as protoporphyrinogen oxidase, in the chlorophyll synthesis pathway, on the other for the same range of concentrations.     

Relationship between safening activity of dymron and fenclorim to pretilachlor and glutathione S-transferase activity in rice

Dymron [1‐(α,α‐dimethybenzyl)‐3‐(p‐tolyl)urea] and fenclorim (4,6‐dichloro‐2‐phenylpyrimidine) were found to exhibit a safening activity on the growth of rice (Oryza sativa L.) seedlings against pretilachlor [2‐chloro‐2′,6′diethyl‐N‐(2‐propoxyethyl)acetanilide] injury. By pretilachlor treatment at 10^–6 and 10^–5 mol L^–1, the elongation of the third leaves of rice seedlings was reduced by approximately 20 and 40%, and that of the fourth leaves was reduced by approximately 40 and 80%, respectively. Upon the treatment of dymron at 3 × 10^–6 and 10^–5 mol L^–1 in combination with pretilachlor, the growth inhibition was half alleviated in the third leaves, and the length of the fourth leaves was almost recovered from 10^–6 mol L^‐1 pretilachlor injury, and was 20–25% recovered from 10^–5 mol L^–1 pretilachlor injury. Upon the treatment of fenclorim at 3 × 10^–6 and 10^–5 mol L^–1 in combination with pretilachlor, the growth inhibition of rice seedlings was almost alleviated in both the third and the fourth leaves. This result indicated that dymron and fenclorim showed almost the same safening effect on the fourth leaf growth against 10^–6 mol L^‐1 pretilachlor injury, although fenclorim showed higher effects at higher concentrations of pretilachlor. Glutathione S‐transferase (GST) activities in rice seedlings were investigated after being treated with a herbicide and safener. By pretilachlor treatment at 10^–6 and 10^–5 mol L^–1, the GST activity was approximately 32 and 72% increased in roots, respectively, and a little increased (7–13%) in shoots of two‐leaf‐stage rice seedlings. By dymron treatment at 3 × 10^–6?10^–5 mol L^–1, the GST activity was 2–30% increased in roots, but was not increased in shoots. By their combination treatment, the GST activity was almost the same or less than that by treatment with pretilachlor alone. In contrast, by fenclorim treatment alone, the GST activity was 43–52 and 33–45% increased in roots and shoots of rice seedlings, respectively. By the combination treatment of pretilachlor and fenclorim, the GST activity was increased 73–126% in shoots and 101–139% in roots, and was much more increased in both shoots and roots compared with treatment of pretilachlor or fenclorim alone. It was found that dymron showed less effect in increasing the GST activity than fenclorim. It is also suggested that dymron did not increase the GST activity in shoots but did increase it slightly in roots, and showed almost no effect on GST increase by pretilachlor in shoots, or rather reduced the increase in roots. From the above results, fenclorim and dymron may have different mechanisms of safening effects on the protection of rice seedlings against pretilachlor injury.     

Phase 1 and 2 Metabolism in Freshly Isolated Hepatocytes and Subcellular Fractions from Rat,Mouse, Chicken and Ox Livers

In toxicological studies hepatocytes offer an excellent alternative to whole-animal experiments, provided their metabolic competence has been established. We have compared Phase 1 and 2 metabolism in rat, mouse, chicken and ox liver microsomes and cytosol with freshly isolated hepatocytes. The relative amounts of total cytochrome P450 in microsomes and hepatocytes were equivalent. Rat liver had the highest P450 content while chicken liver had the lowest content (148·2(±75·7) and 20·6(±11·5) pmol mg^-1 hepatocellular protein, respectively). The metabolism of testosterone was assessed to determine selective cytochrome P450 isoenzyme activities. Only two metabolite products were common to all four species, namely 6β-hydroxytestosterone (6β-OHT) and androstenedione (ASD), which co-eluted with 6-dehydrotestosterone (6DHT). 16α-OHT was present in all incubations except for ox microsomes. The rate of metabolism of testosterone was generally lower in microsomes than hepatocytes, with the exception of the ox, but the pattern and quantity of metabolite formation was similar. The quantity of total products formed was 15- to 27-fold higher in rat and mouse livers than in chicken or ox. The major product formed in freshly isolated hepatocytes from mice and chickens was ASD/6DHT which accounted for 60% and 76% of the total metabolites, respectively. ASD/6DHT formation accounted for only 33% and 17% of the total metabolites formed by rat and ox hepatocytes, respectively. 2α-OHT production occurred in rat and mouse hepatocytes (14% of the total metabolites in rat and 7% in mouse hepatocytes) but was lacking in chicken or ox cells. The stability of P450 isoforms in culture was species-dependent. Rat and mouse hepatocyte cultures lost 54% and 31% of their initial P450 content after 72 h, while there was no loss in chicken hepatocytes over the same period. There was a good correlation between the relative glutathione S-transferase (GST) activities in cytosol and freshly isolated hepatocytes. Mouse liver exhibited highest GST activity (664·2(±203·5)) compared with rat, chicken or ox (320·4(±64·0), 341·5(±13·9) and 256·3(±109·9) nmol min^-1 mg^-1 cytosolic protein, respectively). © 1997 SCI.     

Co-uptake of atrazine and mercury by rice seedlings from water

The uptake of atrazine (ATR) by rice seedlings (Oryza sativa L.) from nutrient solution was investigated in the presence and absence of Hg²⁺ over a period of 96 h. Either ATR or Hg²⁺ was found phytotoxic to rice seedlings, as they inhibited the seedling growth. The seedlings showed about 50% biomass reduction when exposed to 1.0 mg/L Hg²⁺ alone in nutrient solution, and about 80% reduction when exposed to 12.0 mg/L ATR alone. Observed ATR and Hg²⁺ levels (in mg/kg) in seedlings are not related to biomass changes. When either ATR or Hg²⁺ was applied, the concentrations in seedlings increased largely in proportion to those in nutrient solution. The presence of Hg²⁺ at 0.1 and 1.0 mg/L in solution caused a small-to-moderate decline in ATR uptake by the seedlings, the effect being largely independent of the ATR concentration in nutrient solution (at 6.0 and 12.0 mg/L). The presence of ATR (at 6.0 and 12.0 mg/L) in the nutrient solution led to small-to-moderate irregular changes in the Hg²⁺ concentrations in rice seedlings. The overall results showed that there was no significant interdependence between the uptakes of ATR and Hg²⁺ by rice seedlings, which is in contrast to the enhanced ATR uptake noticed earlier with Pb²⁺ ion. Plant uptake of non-ionic organic compounds, such as ATR may be partly through water channels on the plasma membranes of plant cells.     

Fipronil metabolism,oxidative sulfone formation and toxicity among organophosphate‐ and carbamate‐resistant and susceptible western corn rootworm populations

Fipronil toxicity and metabolism were studied in two insecticide‐resistant, and one susceptible western corn rootworm (Diabrotica virgifera virgifera, LeConte) populations. Toxicity was evaluated by exposure to surface residues and by topical application. Surface residue bioassays indicated no differences in fipronil susceptibility among the three populations. Topical bioassays were used to study the relative toxicity of fipronil, fipronil + the mono‐oxygenase inhibitor piperonyl butoxide, and fipronil's oxidative sulfone metabolite in two populations (one resistant with elevated mono‐oxygenase activity). Fipronil and fipronil‐sulfone exhibited similar toxicity and application of piperonyl butoxide prior to fipronil resulted in marginal effects on toxicity. Metabolism of [¹⁴C]fipronil was evaluated in vivo and in vitro in the three rootworm populations. In vivo studies indicated the dominant pathway in all populations to be formation of the oxidative sulfone metabolite. Much lower quantities of polar metabolites were also identified. In vitro studies were performed using sub‐cellular protein fractions (microsomal and cytosolic), and glutathione‐agarose purified glutathione‐S‐transferase. Oxidative sulfone formation occurred almost exclusively in in vitro microsomal reactions and was increased in the resistant populations. Highly polar metabolites were formed exclusively in in vitro cytosolic reactions. In vitro reactions performed with purified, cytosolic glutathione‐S‐transferase (MW = 27 kDa) did not result in sulfone formation, although three additional polar metabolites not initially detectable in crude cytosolic reactions were detected. Metabolism results indicate both cytochromes P450 and glutathione‐S‐transferases are important to fipronil metabolism in the western corn rootworm and that toxic sulfone formation by P450 does not affect net toxicity. © 2000 Society of Chemical Industry