Parathion and methyl parathion toxicity and metabolism in piperonyl butoxide and diethyl maleate pretreated mice

Pretreatment of male mice with piperonyl butoxide, 400 mg/kg 1 h before challenge with insecticides, resulted in a 40-fold antagonism of the acute i.p. toxicity of methyl parathion but potentiated the toxicity of parathion two-fold. Piperonyl butoxide had no effect on the toxicity of the oxygen analogs of these insecticides, methyl paraoxon and paraoxon. Diethyl maleate (1 ml/kg) depleted liver glutathione by 80% after one hour, potentiated the toxicity of both methyl parathion and methyl paraoxon, and partially counteracted the protective effect of piperonyl butoxide on methyl parathion toxicity. Piperonyl butoxide delayed the onset of brain cholinesterase inhibition by parathion. Studies of the metabolism of the insecticides by liver homogenates in vitro demonstrated that piperonyl butoxide inhibited both the oxidative formation of the oxygen analogs (activation) and oxidative cleavage to p-nitrophenol and dialkylphosphorothioic acid (detoxification). While parathion metabolism was mostly oxidative, methyl parathion metabolism appeared to be predominantly via glutathione-dependent enzymes. Studies of in vitro distribution of the insecticides demonstrated that piperonyl butoxide pretreatment resulted in elevated tissue concentrations of parathion and methyl parathion; however, the rate constant for elimination from plasma for both insecticides was unaffected by piperonyl butoxide. The overall rate of metabolism of methyl parathion in vivo was approximately twice that of parathion. These results suggest that during piperonyl butoxide inhibition of oxidative activation and cleavage, methyl parathion detoxification continues through uninhibited glutathione-dependent pathways of metabolism. The net result is a reduction in the acute toxicity of methyl parathion. Lack of an effective alternate pathway of detoxification may explain the delayed but greater toxicity of parathion in piperonyl butoxide pretreated mice.     

Oxidative stress in liver and brain of the hatchling chicken (Gallus domesticus) following in ovo injection with TCDD

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was injected into chicken eggs prior to incubation to study possible mechanisms of toxicity and teratogenicity. One of the suggested mechanisms of teratogenicity is oxidative stress. Eggs were injected simultaneously with TCDD and cotreatment compounds in an attempt to prevent oxidative stress or to block cytochrome P450 activity. Indicators of oxidative stress were assessed in livers and brains of hatchling chicks. In ovo, exposure to TCDD caused significant effects on indicators of oxidative stress in liver, but not in the brain of the hatchling chicks. TCDD did not significantly affect superoxide production. In liver, TCDD treatment caused a decrease in glutathione content and glutathione peroxidase activity and an increase in the ratio of oxidized to reduced glutathione. TCDD increased the susceptibility to lipid peroxidation and oxidative DNA damage in liver. Administration of the antioxidants vitamin E and vitamin A provided partial protection against TCDD-induced oxidative stress in liver. The lack of effect of TCDD in chicken brain could be due to the low cytochrome P4501A activity in this tissue and little accumulation of TCDD in brain compared to liver. Phenytoin, a known inducer of oxidative stress, caused a decrease in glutathione content and an increase in susceptibility to lipid peroxidation in both liver and brain and increased oxidative DNA damage in brain. Responsiveness varied among individual animals, but measures of the oxidative stress were correlated.     

Effect of piperonyl butoxide on organic anion and cation transport in rabbit kidneys

Piperonyl butoxide has been shown to reduce accumulation of cephaloridine in rabbit renal cortex; however, the mechanism responsible for this effect remains unclear. Cephaloridine is a zwitterion and its accumulation in renal cortex has been suggested to be regulated by both organic anion and cation transport systems. Thus, it was of interest to determine the effect of piperonyl butoxide on renal transport of p-aminohippurate (PAH, an organic anion) and tetraethylammonium (TEA, an organic cation). Although pretreatment with piperonyl butoxide markedly inhibited renal cortical uptake of cephaloridine, the same treatment had less inhibitory effect on either PAH or TEA uptake. Efflux of PAH from preloaded renal cortical slices was enhanced by pretreatment with piperonyl butoxide; however, TEA efflux was unaffected. Thus, piperonyl butoxide appears to have effects on renal membrane functions which result in differential effects on PAH, TEA, and cephaloridine transport.     

截形叶螨抗哒螨灵品系和敏感品系体内解毒酶活性的变化

【目的】通过对截形叶螨 Tetranychus truncatus Ehara抗哒螨灵品系体内解毒酶活性分析和增效剂与哒螨灵混用的增效作用测定,明确截形叶螨对哒螨灵的抗性动态及抗性机理,获得抗性治理的途径。【方法】采用室内生测法培育截形叶螨抗哒螨灵品系,微量滴度酶标板测定抗性和敏感品系体内解毒酶比活性、米氏常数（K_m）及最大反应速度（V_max）,再用增效醚（PBO）、顺丁烯二酸二乙酯（DEM）和磷酸三甲苯酯（TPP）进行增效作用测定。【结果】室内筛选的截形叶螨对哒螨灵产生了抗性,筛选至49代,抗性倍数达到955.25;PBO,TPP和DEM对哒螨灵药剂有不同程度的增效作用,相对增效系数分别为95.97%, 85.14%和97.37%;抗哒螨灵品系体内的羧酸酯酶（CarE）、谷胱甘肽转移酶（GSTs）、多功能氧化酶（MFO）活性较敏感品系显著性提高（P＜0.05）,酸性磷酸酯酶（ACP）和碱性磷酸酯酶（ALP）的活性与敏感品系差异不大(P>0.05);抗性品系中的CarE,GSTs和MFO 3种解毒酶的米氏常数（K_m）下降,最大反应速度（V_max）高于敏感品系。【结论】截形叶螨对哒螨灵产生抗性可能与其体内CarE,GSTs和MFO 3种解毒酶与底物的亲和力提高和代谢能力增强有关;3种增效剂(PBO, TPP和 DEM)与哒螨灵混用能提高对截形叶螨的毒杀效果。     

Induction of metallothionein in the livers of female Sprague-Dawley rats treated with 2,3,7 ,8-tetrachlorodibenzo-p-dioxin

Metallothioneins (MTs) are cysteine-rich metal-binding proteins that exert cytoprotective effects against metal toxicity and external stimuli including ionizing or ultraviolet B irradiation. Since 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is known to cause an exaggerated oxidative stress response in animals and in different organs, we have studied possible involvement of MT in the oxidative responses induced by TCDD. Female Sprague-Dawley (SD) rats (6-week old) were administered a single oral dose of TCDD that varied from 1.0 to 4.0 microg/kg body weight. The serum and tissues were collected 7 days after dosing. Indicators of oxidative damage were assessed. Significant increases in serum 8-hydroxydeoxyguanosine (8-OHdG) levels were observed in the rats dosed with 2.0 and 4.0 microg TCDD/kg bw. Only 4.0 microg TCDD/kg bw produced a decrease in reduced glutathione concentration in the liver. Immunohistochemical staining revealed a TCDD-induced increase in heme oxygenase-1 (HO-1) expression in the hepatic macrophages (Kupffer cells). Under these conditions, MT protein as well as the mRNAs of MT-I and MT-II, were dose-dependently induced in the liver by TCDD doses from 1.0 microg/kg bw. TCDD-induced MT was found to localize in the parenchymal cells of the liver. Serum concentrations of cytokines (TNF-alpha, IL-1beta and IL-6) were not affected by TCDD. The hepatic concentrations of Cu, Zn and Fe were all increased significantly by TCDD administration. Our results suggest that MT levels are increased in the liver upon exposure to TCDD, perhaps by TCDD-generated reactive oxygen species, and that it may play a protective role in TCDD-induced oxidative stress responses as an antioxidant.     

Effect of a single in ovo injection of 2,3,7,8-tetrachlorodibenzo-p-dioxin on protein expression in liver and ovary of the one-day-old chick analyzed by fluorescent two-dimensional difference gel electrophoresis and mass spectrometry

The polyhalogenated aromatic hydrocarbon 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an ubiquitously distributed environmental pollutant which can induce a broad spectrum of toxic responses in animals, including birds. In this study, we investigated the impact of 0 or 20 ng TCDD injections into the yolk of chicken eggs before start of development, on liver and ovarian protein expression in hatchlings using fluorescent two-dimensional difference gel electrophoresis (2-D-DIGE) under a pH range of 4-7, combined with MS. Despite considerable interindividual variability, exposure to TCDD prior to the start of embryonic development resulted in significant changes in expression of a small set of proteins. Expression of fibrinogen gamma chain precursor in the liver and 60 kDa heat shock protein in the ovary were significantly higher as a result of the very early exposure to TCDD. NADH ubiquinone oxidoreductase (42 kDa subunit) and regucalcin expression was decreased by early TCDD treatment in the liver and ovary, respectively. These proteins could not be directly linked with drug metabolism per se but are involved in blood clotting, oxidative stress, electron transport, and calcium regulation. It remains to be elucidated how these changes in the hatchling might be linked to the observed long-term consequences during posthatch life of the chicken.     

Multiple resistance mechanisms to abamectin in the two-spotted spider mite

The two-spotted spider mite, Tetranychus urticae, is one of the most destructive pests of various orchard trees and garden plants. Biochemical mechanisms of abamectin resistance in two T. urticae strains (PTF, 239-fold resistance; AbaR, ca. 4753-fold resistance) were investigated. The involvement of both esterase (Est) and mixed function oxidase (MFO) in abamectin resistance was suggested by synergistic bioassays, in which median lethal time (LT₅₀) values were significantly reduced by pretreatment with triphenyl phosphate (TPP) and piperonyl butoxide, respectively. Detoxification enzyme assays confirmed that Est and MFO were related to abamectin resistance as metabolic factors. Moreover, some Est bands on a native isoelectric focusing gel were specifically inhibited by TPP, implying their association with resistance. Pretreatment with verapamil in synergistic bioassays did not reduce the LT₅₀ to abamectin, suggesting that the ABC transporter is not likely involved in resistance. However, enhanced MFO and Est activities in the AbaR strain were not enough to account for the extremely high level of abamectin resistance, which suggests the involvement of additional resistance mechanisms, such as target site insensitivity.     

Frequency of kdr gene in house fly field populations: correlation of pyrethroid resistance and kdr frequency

The frequency of the L1014 F kdr mutation was determined in 14 field populations of house flies, Musca domestica L., with resistance factors at LD50 for pyrethrin/piperonyl butoxide and bioresmethrin/piperonyl butoxide from 4 to 29 and 2 to 98, respectively. A polymerase chain reaction test for identifying kdr homo- or heterozygote house flies was used to determine the frequency of kdr. The L1014 F allele was found in all populations tested. The frequency of kdr in the field populations was high and varied from 0.46 to 0.99. Eleven of the populations were in Hardy-Weinberg equilibrium, whereas two strains had higher number of heterozygotes than expected, indicating a possible heterozygote advantage. The frequency of kdr was strongly correlated with the reduced mortality observed in the bioassays with pyrethrum and bioresmethrin synergized by piperonyl butoxide. This indicates that kdr is a major mechanism for pyrethroid resistance in these field populations. Five field populations had resistance factors >25 and >10 for bioresmethrin/piperonyl butoxide and pyrethrin/piperonyl butoxide, respectively. The frequencies of kdr in these five populations varied from 0.89 to 0.99. The frequencies of kdr in the field populations showing no or a low level of resistance had frequencies of kdr from 0.46 to 0.75, which indicates that the L1014 F kdr allele is a fully recessive genetic trait in house flies. We have shown that the molecular diagnostic PASA method to determine the resistance phenotypes and the frequency of kdr is a powerful tool, which could be used to get information to make recommendations about pest and resistance management.     

Toxicity of Piperonyl Butoxide — Carbaryl Synergism on the Snail Lymnaea acuminata

Effects of piperonyl butoxide and carbaryl synergism were studied on the metabolism of the snail Lymnaea acuminata. Snails were exposed to 40 % and 80 % of the 48 h LC₅₀ of carbaryl or carbaryl + piperonyl butoxide mixture (1:5). The amount of carbaryl present in the LC₅₀ mixture was only 0.23 % of the LC₅₀ of carbaryl alone. The treatments caused a dose-dependent decrease in glycogen and protein levels and acetylcholinesterase (AChE) and alkaline phosphatase activity; simultaneously, there was an increase in levels of lactic acid, reducing sugars and amino acid and the activity of acid phosphatase. Significant differences in AChE and phosphatase activity were also observed between the effects of equivalent concentrations of carbaryl and carbaryl-synergist.     

褐飞虱抗甲胺磷品系的交互抗性和抗性生化机制

用甲胺磷筛选的褐飞虱Nilaparvata lugens品系（R）,对甲胺磷的抗性达到43.74倍,对马拉硫磷、二嗪磷、异丙威、仲丁威及醚菊酯都表现出一定的交互抗性,而对氰戊菊酯和吡虫啉的交互抗性不显著。为了研究褐飞虱对甲胺磷抗性和对其它药剂交互抗性产生的机制,进行了活体增效试验和离体生化实验。用2 μg/头的增效剂预处理试虫的活体增效实验结果显示,在甲胺磷筛选品系（R）中, TPP(triphenyl phosphate, 磷酸三苯酯)对甲胺磷的增效倍数达到4.54,TPP对马拉硫磷、二嗪磷、仲丁威、异丙威都表现出一定的增效作用,增效比分别为2.76、2.07、2.17和1.64;PBO(piperonyl butoxide,胡椒基丁醚)对甲胺磷、马拉硫磷和醚菊酯有一定的增效作用;DEM(diethyl meteate, 顺丁烯二酸二乙酯)的增效作用不明显。研究离体情况下增效剂对三种解毒酶活性的影响发现,TPP对R品系酯酶活力抑制作用很强（抑制率69.04%）,PBO对多功能氧化酶（MFO）具有一定的抑制作用（抑制率29.30%）,而TPP和PBO在F品系和S品系中对酯酶和MFO的抑制作用都较小;DEM在三个品系中对谷胱甘肽-S-转移酶的抑制作用都很小。由此可见,酯酶在褐飞虱对甲胺磷的抗性中起最主要作用,在马拉硫磷、二嗪磷、异丙威和仲丁威的交互抗性中起很重要作用;MFO可能在甲胺磷抗性和醚菊酯、马拉硫磷的交互抗性中起一定作用。     

670 nanometer light treatment attenuates dioxin toxicity in the developing chick embryo

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is an acutely toxic anthropogenic chemical. Treatment with a red to near-infrared (630-1000 nm) light-emitting diode (LED) attenuates the toxicant-induced oxidative stress and energy deficit in neuronal cell culture. For this study, fertile chicken (Gallus gallus) eggs were injected once at the start of incubation with sunflower oil vehicle or 200 pg TCDD/g egg (200 parts per trillion), an environmentally relevant dose. Daily LED treatment after TCDD exposure reduced embryonic mortality by 47%. LED treatment of TCDD-exposed eggs also decreased the hepatic oxidized-to-reduced glutathione ratio by 88%. Activities of other hepatic indicators of oxidative stress, such as glutathione reductase and catalase, were increased after LED treatment of TCDD-exposed eggs. Our study demonstrates that 670 nm phototherapy can mitigate the oxidative stress and energy deficit resulting from developmental exposure to TCDD while reducing TCDD-induced embryo mortality. Moreover, LED treatment restores hepatic enzyme activities to control levels in TCDD-exposed embryos. The effective attenuation of TCDD-induced embryo toxicity by LED treatment could extend to mitigating the effects of other teratogens that induce oxidative and energy stress.     

Uroporphyrin accumulation produced by halogenated biphenyls in chick-embryo hepatocytes. Reversal of the accumulation by piperonyl butoxide.

Cultures of chick-embryo hepatocytes were used to study the mechanism by which 3,4,3',4'-tetrachlorobiphenyl and 2,4,5,3',4'-pentabromobiphenyl cause accumulation of uroporphyrin. In a previous paper, an isoenzyme of cytochrome P-450 induced by 3-methylcholanthrene had been implicated in this process [Sinclair, Bement, Bonkovsky & Sinclair (1984) Biochem. J. 222, 737-748]. Cells treated with 3,4,3',4'-tetrachlorobiphenyl and 5-aminolaevulinate accumulated uroporphyrin and heptacarboxyporphyrin, whereas similarly treated cells accumulated protoporphyrin immediately after piperonyl butoxide was added. Piperonyl butoxide also restored haem synthesis as detected by incorporation of radioactive 5-aminolaevulinate into haem, and decrease in drug-induced 5-aminolaevulinate synthase activity. The restoration of synthesis of protoporphyrin and haem by piperonyl butoxide was not affected by addition of cycloheximide, indicating recovery was probably not due to protein synthesis de novo. Piperonyl butoxide also reversed uroporphyrin accumulation caused by 3,4,5,3',4',5'-hexachlorobiphenyl, mixtures of other halogenated biphenyls, lindane, parathion, nifedipine and verapamil. The effect of piperonyl butoxide was probably not due to inhibition of metabolism of these compounds, since the hexachlorobiphenyl was scarcely metabolized. Other methylenedioxyphenyl compounds, as well as ellipticine and acetylaminofluorene, also reversed the uroporphyrin accumulation caused by 3,4,3',4'-tetrachlorobiphenyl. SKF-525A (2-dimethylaminoethyl-2,2-diphenyl valerate) did not reverse the uroporphyrin accumulation caused by the halogenated biphenyls, but did reverse that caused by phenobarbital and propylisopropylacetamide. We conclude that the mechanism of the uroporphyrin accumulation cannot be due to covalent binding of activated metabolites of halogenated compounds to uroporphyrinogen decarboxylase.     

Thymoquinone is protective against 2,3,7,8-tetrachlorodibenzo-p-dioxin induced hepatotoxicity

We investigated changes in rat liver tissues following administration of thymoquinone (TQ) against 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced hepatotoxicity. Fifty rats were assigned randomly to five groups of 10 as follows: control, corn oil, TCDD, TQ and TCDD + TQ. Biochemical and histopathological analyses were conducted on liver tissue. We found that 30 day TCDD administration caused histopathological changes in liver including thickening of Glisson’s capsule, intracytoplasmic vacuolization in hepatocytes, sinusoidal dilation, vascular and sinusoidal congestion and inflammatory cell infiltration. TCDD administration increased malondialdehyde (MDA), total oxidant status (TOS), alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALP) levels in rat liver tissue and reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and total antioxidant status (TAS) levels compared to all other groups. In the TQ treated group, GSH, SOD, CAT and TAS levels increased compared to all other groups. MDA, TOS, ALT, AST, ALP levels decreased compared to all other groups. Our histological findings were consistent with the biochemical findings. The oxidative and histologic effects of TCDD were eliminated by TQ treatment. TCDD administration caused oxidative stress in rat liver and TQ administered with TCDD prevented TCDD induced hepatotoxicity. TQ could be considered an alternative anti-TCDD toxicity agent.     

Interaction of estrogen and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) with hepatic fatty acid synthesis and metabolism of male chickens (Gallus domesticus)

This study tested the hypothesis that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) antagonizes estrogen-induced hepatic lipid synthesis and metabolism in birds. Twenty immature male chickens (Gallus domesticus) were divided evenly into four groups: (1) vehicle control; (2) estrogen alone (1.0 mg/kg estradiol cypionate injected on three consecutive days); (3) TCDD alone (50 μg/kg injected on the fourth day); and (4) a combination of the estrogen and TCDD treatments. On day 14, liver samples were collected for quantitative fatty acid analysis by capillary gas chromatography. Birds treated with estrogen alone had increased total triacylglyceride concentrations with specific increases in the Δ9 desaturase products 16:1n7, 18:1n7, 18:1n9, and 20:1n9. In addition, estrogen treatment specifically increased 22:6n3 concentrations in both triacylglycerides and phospholipids. However, these increases in Δ9 desaturase products or 22:6n3 did not occur for birds treated with estrogen in combination with TCDD. TCDD and estrogen plus TCDD treatments increased phospholipid concentrations of the diet-derived polyunsaturated fatty acids 18:2n6, 18:3n6, 20:3n6, 18:3n3, and 20:5n3, although only the estrogen plus TCDD group had significantly increased total phospholipids. In cholesterol esters, all three treatments decreased concentrations of total fatty acids, saturated fatty acids, and Δ9 desaturase products compared to the control group.     

Modulation of restraint stress induced oxidative changes in rats by antioxidant vitamins

In the present study we examined immobilization stress-induced antioxidant defense changes in rat plasma and also observed the antioxidant effects of pre and post vitamins A, E and C administration (15 mg/Kg of body weight) individually and in combination (vit E + C) on these alterations.Following immobilization stress the circulating activities of superoxide dismutase, catalase and glutathione-S-transferase were decreased, while the level of thiobarbituric acid reactive substances (TBARS) was increased as compared to non-stressed control rats.Post treatment with individual vitamins A, E and C (after exposure to stress) resulted in a less marked alteration of plasma TBARS levels and activities of SOD, GST and catalase as compared to pre vitamin stress or stress alone treatments. Both pre and post vitamin treatments were effective in preventing stress induced derangement of free radical metabolism with a relative dominance by latter. The combined treatment with vitamin E and C did not show any additive antioxidant effect on restraint stress induced altered free radical metabolism, rather a predominant effect similar to vitamin E alone was observed. The prevention of oxidative stress generated in response to restraint stress by the vitamins can be summarized as: vitamin (E + C) i.e. vit E > vit C > vit A, thus combined vitamin (E + C) treatment though showed maximum preventive effect, but was similar to vitamin E treatment alone, in terms of the circulating activities of SOD, GST, catalase and TBARS levels.     

TCDD-mediated oxidative stress in male rat pups following perinatal exposure

2,3,7,8-tetrachlorododibenzo-p-dioxin (TCDD) is a highly persistent trace environmental contaminant and is one of the most potent toxicants known. Exposure to TCDD has been shown to cause oxidative stress in a variety of animal models. In this study, pregnant Long Evans rats were dosed with 1 microg TCDD/kg on gestational day (GD) 15 so as to investigate oxidative stress in the liver of male pups following gestational exposure to TCDD. Lipid peroxidation (TBARS), production of reactive oxygen species (ROS), and total glutathione (GSH) were assayed to identify changes in oxidative stress parameters in the pup liver at GD 21 and postnatal days (PND) 4, 25, 32, 49, and 63. Mean ROS levels in pups were elevated at all time points tested with a significant elevation at PND 4 and PND 25. However, pup hepatic lipid peroxidation was unchanged throughout the time course. In addition, hepatic total GSH levels were not significantly changed although the means for the TCDD-treated groups were less than those of the controls at all time points except PND 49. The results indicate that although the levels of ROS are increased following gestational/lactational exposure, this increase does not translate to direct oxidative damage or significant changes to endogenous antioxidant defense mechanisms. Further investigation into the effect of gestational/lactational exposure in pups should include additional endpoints for further characterization of the time course of the response, the effect upon extrahepatic tissues, and investigation of differences between male and female offspring.     

Piperonyl Butoxide Increases Oxidative Toxicity of Fenthion in the Brain of Oreochromis niloticus

The present study was designed to understand the effects of piperonyl butoxide (PBO), modulator of cytochrome P450 (CYP 450), on the neurotoxicity of organophosphate pesticide fenthion in the brain of Oreochromis niloticus used as a model organism. Fish were exposed to one‐fourth of the LC₅₀ value of fenthion (0.567 mg/L) and 0.5 mg/L PBO concentration for 24 h, 96 h, and 15 days. Glutathione (GSH)‐related antioxidant system, lipid peroxidation, stress proteins, and acetylcholinesterase (AchE) activity were investigated. Our results showed that PBO induced the neurotoxic effect of fenthion with increasing oxidative stress in long‐term exposure. GSH‐related antioxidant system might take a role in protecting the brain from these oxidative effects. PBO possibly inhibited the biotransformation of fenthion by inhibiting CYP 450; thereby preventing the brain from AChE inhibition in short‐term exposure. Changes in parameters indicated that PBO caused biphasic response by affecting CYP 450 in the brain of O. niloticus.     

Evidence for cytochrome P-450 mediated metabolism in the bronchiolar damage by naphthalene

Intraperitoneal administration of the volatile hydrocarbon, naphthalene, resulted in severe bronchiolar epithelial cell necrosis in mice, while hepatic or renal necrosis was not observed. Pulmonary damage and mortality by naphthalene were increased by prior treatment with diethyl maleate and decreased by prior treatment with piperonyl butoxide (1600 mg/kg). SKF 525A pretreatment had no effect on naphthalene-induced pulmonary damage. Administration of [¹⁴C]naphthalene resulted in the covalent binding of radiolabel to tissue macromolecules. Highest levels of binding occurred in lung, liver and kidney. Levels of covalent binding reached a maximum 2–4 h after treatment and corresponded to rapid glutathione depletion in lung and liver. Covalent binding was dose-dependent and showed a threshold between 200 and 400 mg/kg which coincided with almost total depletion of tissue glutathione levels. Covalent binding of reactive metabolites was increased 3–4-fold by prior treatment with diethyl maleate, and was decreased 3–4-fold by pretreatment with piperonyl butoxide. These studies support the view that naphthalene-induced pulmonary damage is mediated by the cytochrome P-450-dependent metabolism of naphthalene and that glutathione plays an important role in the detoxification of the lung damaging metabolite(s).     

Multiple resistance and biochemical mechanisms of pyridaben resistance in Tetranychus urticae (Acari: Tetranychidae)

A field colony of Tetranychus urticae (Koch) (Acari: Tetranychidae) resistant to pyridaben was selected with pyridaben successively for 20 generations to produce the PR-20 strain. Resistance and multiple resistance levels of the PR-20 strain to 15 acaricides were determined using a spray bioassay. The PR-20 strain was extremely resistant to pyridaben (resistance ratio [RR] = 240]. The strain exhibited extremely strong resistance to fenpyroximate (RR=373) and acrinathrin (RR=329) and strong resistance to benzoximate (RR=84). An RR = 10-40 was observed with abamectin, fenazaquin, fenbutatin oxide, fenpropathrin, and tebufenpyrad. The PR-20 strain showed low levels of resistance (RR <10) to azocyclotin, bromopropylate, chlorfenapyr, dicofol, milbemectin, and propargite. Synergist experiments with different metabolic inhibitors revealed that piperonyl butoxide (PBO), a mixed function oxidase (MFO) inhibitor, had the greatest effect on pyridaben resistance. PBO significantly caused pyridaben resistance in the PR-20 strain to drop to the full susceptibility level of the susceptible (S) strain. However, there was no significant difference in MFO activities measured using a model substrate between the S and PR-20 strains. These results suggest that use of certain acaricides with little multiple resistance or PBO will be useful for the management of pyridaben resistance in the field.     

Analysis of putative inhibitors of anthelmintic resistance mechanisms in cattle gastrointestinal nematodes

Effects of the cytochrome P450 inhibitor piperonyl butoxide and the P-glycoprotein inhibitor verapamil on the efficacy of ivermectin and thiabendazole were studied in vitro in susceptible and resistant isolates of the cattle parasitic nematodes Cooperia oncophora and Ostertagia ostertagi. The effects of combined use of drug and piperonyl butoxide/verapamil, respectively, were investigated in the Egg Hatch Assay, the Larval Development Assay and the Larval Migration Inhibition Assay. The effects of piperonyl butoxide and verapamil as inhibitors of thiabendazole and ivermectin responses were particularly marked for larval development, where both inhibitors were able to completely eliminate all differences between susceptible and resistant isolates. Even the lowest concentrations of anthelmintics used in combination with inhibitors caused complete inhibition of development. Differences and/or similarities among responses in different isolates were only obtained in the two other assays: in the Egg Hatch Assay piperonyl butoxide caused a shift in concentration–response curves obtained with thiabendazole to the left for all isolates tested, changing relative differences between isolates. In contrast, an effect of verapamil in the Egg Hatch Assay was only apparent for benzimidazole-resistant isolates. In the Larval Migration Inhibition Assay only ivermectin was tested and piperonyl butoxide shifted the concentration–response curves for all isolates to the left, again eliminating differences in EC₅₀ values between susceptible and resistant isolates. This was not the case using verapamil as an inhibitor, where curves for both susceptible and benzimidazole-resistant isolates shifted to the left in Ostertagia isolates. In Cooperia the picture was more complex with ivermectin-resistant isolates showing a larger shift than the susceptible isolate. Single nucleotide polymorphisms in the β-tubulin isotype 1 gene were investigated. Significantly increased frequencies of resistance-associated alleles were observed for the codons 167 and 200 in one benzimidazole-resistant isolate but not in an isolate selected for benzimidazole resistance at an early stage of selection.