Organophosphate (OP) agents are continuously utilized in large amount throughout the globe for crop protection and public health, thereby creating a potential concern on human health. OP agent as an anticholinesterase also acts on the endocannabinoid (EC)-hydrolases, i.e., fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), to reveal unexpected adverse effects including ADHD-like behaviors in adolescent male rats. The present investigation examines a hypothesis that OP compound inhibiting the EC-hydrolase(s) dysregulates the EC-signaling system, triggering apoptosis in neuronal cells. Ethyl octylphosphonofluoridate (EOPF), as an OP probe, preferably acts on FAAH over MAGL in intact NG108-15 cells. Anandamide (AEA), an endogenous FAAH substrate, is cytotoxic in a concentration-dependent manner, although 2-arachidonoylglycerol, an endogenous MAGL substrate, gives no effect in the concentrations examined here. EOPF pretreatment markedly enhances AEA-induced cytotoxicity. Interestingly, the cannabinoid receptor blocker AM251 diminishes AEA-induced cell death, whereas AM251 does not prevent the cell death in the presence of EOPF. The consistent results are displayed in apoptosis markers evaluation (caspases and mitochondrial membrane potential). Accordingly, FAAH inhibition by EOPF suppresses AEA-metabolism, and accumulated excess AEA overstimulates both the cannabinoid receptor- and mitochondria-mediated apoptotic pathways.
High urinary levels of dialkylphosphates (DAPs), which are common structures of organophosphate pesticides (OPs), have been associated with several adverse health outcomes in human biomonitoring studies. Previous studies have indicated that dietary OP exposure and ingestion of environmentally degraded DAP, which is inactive with acetylcholinesterase, can lead to an increase in urinary DAP levels in the general population. However, the specific food sources contributing to the intake of OPs and DAPs have not been identified. In this study, we analyzed the levels of OPs and preformed DAPs in various food items. DAP levels were markedly high in certain fruits, such as persimmon, apple juice, kiwi, and mandarin. In contrast, only moderate levels of OPs were detected in these foods. Furthermore, the levels of OPs and DAPs were positively associated with vegetables, whereas no such association was observed in fruits. Increased consumption of certain fruits presumably leads to a marked increase in urinary DAP levels in individuals despite limited exposure to OPs, resulting in reduced reliability of urinary DAPs as a marker of OP exposure. Therefore, the possible effects of dietary habits and the resulting intake of preformed DAPs should be considered when interpreting biomonitoring data of urinary DAPs. Additionally, DAP levels in most organic foods were much lower than those in conventional foods, suggesting that the reduction in urinary DAPs by organic diet intervention may be mainly attributed to the reduced intake of preformed DAPs rather than reduced exposure to OPs. Therefore, urinary DAP levels may not be suitable indicators for evaluating ingested OP exposure.
Title: Non-linear model analysis of the relationship between cholinesterase activity in rats exposed to 2, 2-dichlorovinyl dimethylphosphate (dichlorvos) and its metabolite concentrations in urine Sato H, Ito Y, Hanai C, Nishimura M, Ueyama J, Kamijima M Ref: Toxicology, :152679, 2021 : PubMed
Urinary dialkylphosphates (DAPs) are measured to assess exposure to organophosphorus pesticides (OPs), but they are common metabolites of OPs and not specific indices for individual agents. Biomonitoring (BM) of urinary DAPs has been widely adopted as an assessment of individual exposure in general environments, however, guidance values for DAPs based on health effects have yet to be established. The present study aimed to clarify the relationship between the amount of urinary dimethylphosphate (DMP), a metabolite of dichlorvos (DDVP), and the inhibition of cholinesterase (ChE) activity in rats exposed to DDVP. The relationship was analyzed using a nonlinear model analysis, and the excretion level of urinary DMP equivalent to ChE 20% inhibition (EL20) and the lower limit of the 95% confidence interval of EL20 (ELL20) were estimated. EL20 and ELL20 (mg/24 h urine) of brain, erythrocyte, and plasma ChE activities after 10-day administration of DDVP were 0.21 and 0.15, 0.11 and 0.06, and 0.23 and 0.09, respectively. Extrapolating ELL20 of the brain ChE to humans, the range of 24 h urinary DMP concentration according to the 20% inhibition of cholinesterase activity was estimated to be 20.5-30.8 mg/l. In conclusion, the amount of urinary DMP as ELL20 for DDVP exposure was identified and could probably be used as a novel index for the assessment of risk from OP exposure. Further studies are needed to clarify the ELL20 s derived from OPs other than DDVP, for informing efforts to establish guidance values of urinary OP metabolites that should prevent neurotoxicity.
OBJECTIVES: This study was conducted to assess inter-species and inter-individual differences in the metabolism of di(2-ethylhexyl)phthalate (DEHP) in humans and mice. METHODS: The activities of four DEHP-metabolizing enzymes [lipase, UDP-glucuronocyltransferase (UGT), alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH)] were measured in the livers of 38 human subjects of various ages and in eight 129/Sv male mice. RESULTS: Microsomal lipase activity was significantly lower in humans than in mice. The V max/K m value in humans was one-seventh of that in mice, microsomal UGT activity in humans was a sixth of that in mice, and cytosolic ALDH activity for 2-ethylhexanal in humans was one-half of that in mice. In contrast, ADH activity for 2-ethylhexanol was twofold higher in humans than in mice. The total amount of DEHP urinary metabolites and the concentration of mono(2-ethylhexyl)phthalate (MEHP) were much higher in intact mice than in the U.S. general population based on data reported elsewhere, regardless of the similar estimated DEHP intake between these mice and the human reference population. However, mono(2-ethyl-5-oxo-hexyl)phthalate (5oxo-MEHP) and mono(2-ethyl-5-carboxypentyl)phthalate (5cx-MEPP) levels were higher in the latter than in the former. Of note, inter-subject variability in the activities of all enzymes measured was 10-26-fold. CONCLUSION: The inter-individual variation in the metabolism of DEHP in humans may be greater than the difference between mice and humans (inter-species variation), and both may affects the risk assessment of DEHP.
Title: Organophosphate agents induce plasma hypertriglyceridemia in mouse via single or dual inhibition of the endocannabinoid hydrolyzing enzyme(s) Suzuki H, Ito Y, Noro Y, Koketsu M, Kamijima M, Tomizawa M Ref: Toxicol Lett, 225:153, 2014 : PubMed
Diverse serine hydrolases including endocannabinoid metabolizing enzymes fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) have been suggested as secondary targets for organophosphate (OP) agents to exert adverse toxic effects such as lipid homeostasis disruption. The goal of this investigation is to verify that a major OP insecticide fenitrothion (FNT) induces plasma hypertriglyceridemia through the inhibition of FAAH and/or MAGL in comparison with that elicited by isopropyl dodecylfluorophosphonate (IDFP), a potent FAAH/MAGL inhibitor. Fasted mice were treated intraperitoneally with FNT or IDFP and were subsequently sacrificed for evaluations of plasma triglyceride (TG) levels and liver FAAH/MAGL activities. Plasma TG levels were significantly enhanced by the FNT or IDFP treatment (1.7- or 4.8-fold, respectively) compared with that of vehicle control. The IDFP exposure reduced the liver FAAH and MAGL activities, whereas the FNT exposure led to the preferential FAAH inhibition. The brain acetylcholinesterase was almost unaffected by the FNT or IDFP treatment, thus leading to no neurotoxic sign. Intriguingly, the TG elevations were averted by concomitant administration with the cannabinoid receptor antagonist AM251. The present findings suggest that OP agents induce plasma hypertriglyceridemia in mouse through single or dual inhibition of FAAH or/and MAGL, apparently leading to overstimulation of cannabinoid signal regulating energy metabolism.
Title: New analytical method for sensitive quantification of urinary 3-methyl-4-nitrophenol to assess fenitrothion exposure in general population and occupational sprayers Okamura A, Saito I, Ueyama J, Ito Y, Nakajima T, Kamijima M Ref: Toxicol Lett, 210:220, 2012 : PubMed
The measurement of blood cholinesterase (ChE) activities is adopted worldwide for biological monitoring of exposure to organophosphorus insecticides (OPs). Recent development of analytical chemistry has made sensitive quantification possible of non-specific OP metabolites, dialkylphosphates, in urine as a biomarker of low-level OP exposure. In this study, we established a method for quantification of urinary 3-methyl-4-nitrophenol (MNP), a specific metabolite of fenitrothion (FNT), and a parathion metabolite p-nitrophenol (PNP), using gas chromatography-mass spectrometry. The limits of detection of MNP and PNP were 0.3 and 0.5mug/L, respectively. The method enabled the quantification of both free and conjugated metabolites. This method was actually applied to monitor human urine in summer and winter in FNT sprayers (N=29 and 9, respectively) and control workers (N=17 and 29, respectively). Geometric mean total MNP concentrations (mug/gcreatinine) in the FNT sprayers (28.8 in summer and 8.6 in winter) were significantly higher than those of the controls (3.1 in summer and 2.3 in winter) in both seasons. Among the sprayers, total MNP concentrations in summer were significantly higher than in winter. In contrast, no significant difference in total PNP concentrations was observed between FNT sprayers (geometric mean 3.4 in summer and 3.0 in winter) and controls (3.6 in summer and 2.1 in winter). No seasonal difference was observed in each group. In conclusion, the present new method is sensitive enough for biological monitoring of FNT and parathion metabolites even in a non-spraying population.
The potential toxicity resulting from combinatorial effects of organophosphorus and pyrethroid insecticides are not completely known. We evaluated male reproductive toxicity in mice co-exposed to diazinon and cis-permethrin. Nine-week-old male Sv/129 mice were exposed to diazinon (10 mumol/kg/day) or cis-permethrin (90 mumol/kg/day) alone or in combination (100 mumol/kg/day), or vehicle (corn oil), for 6 weeks. Diazinon and the diazinon-permethrin mixture inhibited plasma and liver carboxylesterase activities. In the mixture group, urinary excretion of cis-permethrin metabolite 3-phenoxybenzoic acid decreased along with increased plasma and testicular concentrations of cis-permethrin, while excretion of diazinon metabolites, diethylphosphate and diethylthiophosphate, did not change, versus mice exposed to each chemical alone, which suggested that inhibition of carboxylesterase decreased the metabolic capacity to cis-permethrin. Though the co-exposure decreased testosterone biosynthesis, increased degenerate germ cells in seminiferous tubule and sperm morphological abnormalities versus controls more clearly than exposure to cis-permethrin alone, the expected potentiation of toxicity was not evident.
Acetylcholinesterase and butyrylcholinesterase (BChE) activities in blood are widely used as the biomarkers for organophosphorus insecticide (OP) exposure. In the present study, we conducted a cross-sectional study to evaluate plasma beta-glucuronidase (BG), a sensitive biomarker candidate for OP exposure, BChE activities and urinary dialkyl phosphates (DAPs), OP metabolites. We assessed the relationship between these biomarker levels in the following groups: 32 controls (control), 21 pest control operators and their co-workers who had not sprayed OPs within 3 days prior to sample collection (PCO1), and 21 pest control operators who sprayed OPs within those 3 days (PCO2). Logarithmically transformed age-adjusted means of DAPs were 3.88, 5.62 and 6.45 nmol/g creatinine for control, PCO1 and PCO2, respectively (P<0.001 for difference, P<0.001 for trend). Logarithmically transformed age-adjusted means of BG were 1.40, 1.52 and 1.85 micromol/L/h for control, PCO1 and PCO2, respectively. BG activity, but not BChE, was increased according to their OP exposure level (P=0.038 for difference, P=0.026 for trend). It was concluded that plasma BG activity is more sensitive biomarker as well as urinary OP metabolites than BChE for low-level exposure in humans.
OBJECTIVES: Recent reports have shown significant associations between organopshophorus pesticide (OP) exposure and decreased sperm motility in workers and laboratory animals. However, the notion that OPs possess spermatotoxicity has yet to be established. The aim of this study was to clarify the effects of OP exposure on detailed sperm toxicity markers, i.e., motility, morphology and sperm adenine nucleotide contents, and the histopathology of the testis and epididymis. METHODS: Ten-week-old Wistar rats were divided into 4 groups (n=10) and orally administered corn oil, dichlorvos (DDVP; 5, 10 mg/kg) or diazinon (DZN; 3 mg/kg) 6 days a week for 9 wk. Sperm motility and morphology markers were analyzed with a computer-assisted sperm analysis (CASA) system. RESULTS: In addition to a significant decrease in acetylcholinesterase (AChE) activities and a significant increase in urinary OP metabolites, DDVP and DZN significantly reduced sperm motility, but they did not influence sperm adenine nucleotide contents. The OPs also significantly increased the percentage of broken sperm, and DDVP significantly increased the percentage of cytoplasmic droplets. Importantly, both OPs significantly increased cytoplasmic vacuolation and nuclear shrinkage in the epithelial cells of the ductus epididymis, whereas the testes did not show significant histopathological changes. CONCLUSIONS: The broken sperm and cytoplasmic droplets as well as reduced sperm motility were the relevant spermatotoxicity makers of DDVP and DZN. To our knowledge, this is the first report to suggest that the above-mentioned OP-induced spermatotoxicity is related to histopathological impairment of the caput epididymis.
We have reported that the toxicity of the organophosphorus pesticide diazinon (DZN) and its metabolites is increased in streptozotocin-induced diabetic rats (type 1 diabetic rats). In the present study, we have investigated the effect of DZN on glucose tolerance in genetic type 2 diabetic rats, Goto-Kakizaki (GK) rats. Oral glucose tolerance test (OGTT) (2g/(5 ml kg)) was assessed before, and 1 and 2 weeks after intraperitoneal injection of DZN (6.5 mg/kg) in Wistar and GK rats. DZN significantly increased the levels of glucose in plasma at designated blood sampling points in GK rats. The activity of hepatic drug-metabolizing enzymes and expression of hepatic cytochrome P450 (CYP) 1A2, CYP3A2 and CYP2D1, which oxidize DZN to DZN-oxon, a potent ChE inhibitor, were measured before DZN injection. There were no significant differences in the activity and expression of CYPs between both rat groups, indicating that the ability of metabolic activation might be almost the same in Wistar and GK rats. DZN dramatically decreased the activity of cholinesterase (ChE) in plasma by approximately 40% in both Wistar and GK rats. However, no significant differences in the activity of ChE in plasma were observed between Wistar and GK rats for 5 days after DZN injection. No massive necrotic and apoptotic areas, leukocyte infiltration and immunoreactive insulin-positive cells (beta-cells) were observed in pancreas 2 weeks after DZN injection. Moreover, DZN might not affect plasma insulin levels in Wistar and GK rats. These results suggest that DZN deteriorates the glucose tolerance in GK rats. It is unlikely that this phenomenon is due to differences in ChE activity and/or DZN-oxon production levels between Wistar and GK rats.
Permethrin, the most popular insecticide among the synthetic pyrethroids, has been used worldwide to control a wide range of insects in agriculture, forestry, public health, and homes. Humans may have suffered potential exposure to this compound. The commercial formulation of permethrin contains trans and cis isomers. Here, at the same dosage, we made a comparison of the reproductive effects between these two isomers. Male adult ICR mice were orally administered trans- or cis-permethrin daily for 6 weeks at a dose of 0 or 70 mg/(kg day). In the cis-permethrin exposure group, the caudal epididymal sperm count and sperm motility were significantly reduced, and testosterone levels in testes and plasma also fell. Moreover, cis-permethrin induced abnormal seminiferous tubules in testes and suppressed testicular mRNA expression levels of peripheral benzodiazepine receptor, steroidogenic acute regulatory protein, and the cytochrome P450 side-chain cleavage enzyme. Although such adverse effects were not observed in the trans-permethrin exposure group, testicular and urinary metabolite 3-phenoxybenzoic acid levels in trans-permethrin-exposed mice were about three- and sevenfold higher than those in cis-permethrin-exposed mice, respectively. Furthermore, in vitro, hepatic microsomal hydrolase activity for trans-permethrin was nearly 62-fold higher than that for cis-permethrin. Taken together, the difference in metabolic activity between cis- and trans-permethrin might contribute to the difference in the reproductive toxicity between both isomers.
The effect of diazinon (DZN) on the activities of cholinesterase (ChE) in plasma and acetylcholinesterase (AChE) in erythrocyte and brain was investigated in normal and streptozotocin-induced diabetic rats. Hepatic drug-metabolizing enzyme activity was also estimated by measuring the systemic clearance of antipyrine, and the expression of hepatic cytochrome P450 (CYP) 3A2 and CYP1A2, which is closely related to the metabolism from DZN to DZN-oxon, a strong inhibitor of both ChE and AChE. No significant differences in the activities of ChE in plasma and AChE in erythrocyte were observed between normal and diabetic rats. Treatment with DZN significantly decreased these activities in diabetic rats more than in normal rats 6h after injection (6.5 mg/kg). Treatment with DZN significantly decreased the activity of AChE in brain of diabetic rats than normal rats 3h after injection (65 mg/kg), although no significant difference in the activity was found between normal and diabetic rats. The urinary recovery of diethylphosphate (DEP), a metabolite of DZN-oxon, was significantly increased in diabetic rats, but that of diethylthiophosphate (DETP), a metabolite of DZN, was unchanged. Significant increases in the systemic clearance of antipyrine and protein levels of hepatic CYP1A2, not CYP3A2, were observed in diabetic rats. These results suggest the possibility that a metabolite of DZN, DZN-oxon, causes higher toxicity in diabetic rats due to the enhancement of hepatic CYP1A2-mediated metabolism of DZN.
Assessments of the reproductive toxicity of organophosphorus insecticides are important public health issues. This study aimed at defining the testicular toxicity of dichlorvos (DDVP) since this toxicity was suspected by our previous survey on pesticide sprayers and in some earlier publications during the 1970s. Ten-week-old Wistar rats were divided into four groups (n=8 or 9) and were injected subcutaneously with DDVP (0, 1, 2 or 4 mg/kg) 6 days a week for 9 weeks. After that period, erythrocyte cholinesterase (ChE) activities decreased dose-dependently, showing 44-55% inhibition among the treated groups. No significant difference was observed in the reproductive organ weights in any treated groups compared with the control group. Sperm motility decreased slightly but significantly in the 1 and 4 mg/kg groups, and significant regressions were observed between sperm motility and both blood ChE activity and urinary concentration of dimethyl phosphate (DMP), a urine metabolite of DDVP. However, sperm counts and sperm morphology in the cauda epididymidis, plasma testosterone concentrations, and histopathology in the testes in all the treated groups were not significantly different from those of the control group. Since only the sperm motility deteriorated by DDVP exposure at doses inducing marked inhibition of cholinesterase activities in the rats, it was suggested that the risk of testicular dysfunction posed to occupationally exposed humans would be small in terms of the effect of DDVP exposure alone. This conclusion was also supported by an estimate of the decrease in human sperm motility based on the urinary DMP concentrations observed in actual occupational settings.
This study aims at clarifying the semen indices of insecticide sprayers who are exposed mainly to organophosphorus and pyrethroid insecticides. Eighteen male sprayers out of 54 working for 9 companies in central Japan and 18 age-matched students or medical doctors as unexposed controls participated in detailed reproductive check-ups conducted in summer and the following winter. The sprayers were exposed to insecticides more in summer, the busiest season, than winter, the off-season (p<0.05). Erythrocyte true cholinesterase activities in the sprayers were lower than in the controls in summer (p<0.05), and decreased in significant association with the increase in exposure frequency. Testicular volumes in the sprayers tended to be smaller than in the controls (p=0.06). The serum testosterone concentration in winter in the sprayers was higher than in the controls (p<0.05), though luteinizing hormone and follicle stimulating hormone concentrations were not significantly different. The sperm counts and vitality were comparable between the groups, but detailed sperm motility analysis in summer revealed that the percentages of slow progressive and nonprogressive motile sperm were twice as high in the sprayers (p<0.05), and that of rapid progressive sperm tended to be lower (p=0.06). Such differences were not observed in winter. Differential sperm morphology counts showed that interaction of group and abstinence effects were significant in sperm with normal morphology and with head deformity only in the summer check-up. Despite possible inherent differences between the groups, the above season-dependent differences suggested that the observed lower semen quality in the sprayers was associated with pesticide spraying work.
Title: Localization of [3H]octylphosphonyl-labeled neuropathy target esterase by chicken nervous tissue autoradiography Kamijima M, Casida JE Ref: Neuroscience Letters, 273:101, 1999 : PubMed
Neuropathy target esterase (NTE) undergoes phosphorylation and aging as the initial steps in organophosphorus (OP)-induced delayed neuropathy (OPIDN). Localization of NTE is an important step in characterizing the mechanism of OPIDN. Earlier histochemical immunoreactivity or esterase assays localized NTE in areas of the brain and spinal cord rich in neuronal cell bodies and in the dorsal root ganglion. We use a more direct and quantitative autoradiographic approach of forming phosphorylated and aged [3H]octylphosphonyl-NTE on treatment with the highly potent [octyl-3H]octyl-4H-1,3,2-benzodioxaphosphorin 2-oxide to determine NTE as the labeling site resistant to the non-neuropathic paraoxon and sensitive to the neuropathic mipafox. NTE is observed in the cerebral cortical layer, some layers of the optic tectum, the gray matter of the spinal cord and the sensory neurons of the dorsal root ganglion to a higher extent than in adjacent areas.
