Pyridostigmine bromide, chlorpyrifos, and DEET combined Gulf War exposure insult depresses mitochondrial function in neuroblastoma cells.

Gulf War Illness (GWI) is defined by the Centers for Disease Control and Prevention (CDC) as a multi-symptom illness having at least one symptom from two of three factors, which include: fatigue, mood-cognition problems, and musculoskeletal disorders. The cluster of long-term symptoms is unique to military personnel from coalition countries including United States, Australia, and the United Kingdom that served in Operation Desert Storm from 1990 to 1991. Reporting of these symptoms is much lower among soldiers deployed in other parts of the world like Bosnia during the same time period. The exact cause of GWI is unknown, but combined exposure to N,N-diethyl-m-toluamide (DEET), organophosphates like chlorpyrifos (CPF), and pyridostigmine bromide (PB), has been hypothesized as a potential mechanism. Mitochondrial dysfunction is known to occur in most neurodegenerative diseases that share symptoms with GWI and has therefore been implicated in GWI. Although exposure to these and other toxicants continues to be investigated as potential causes of GWI, their combined impact on mitochondrial physiology remains unknown. In this study, the effects of combined GWI toxicant exposure on mitochondrial function were determined in a commonly used and readily available immortalized cell line (N2a), whose higher rate of oxygen consumption resembles that of highly metabolic neurons in vivo. We report that combined exposure containing pesticide CPF 71 µM, insect repellants DEET 78 µM, and antitoxins PB 19 µM, causes profound mitochondrial dysfunction after a 4-h incubation resulting in decreased mitochondrial respiratory states in the absence of proapoptotic signaling, proton leak, or significant increase in reactive oxygen species production.

Gulf war illness-related chemicals increase CD11b/c+ monocyte infiltration into the liver and aggravate hepatic cholestasis in a rodent model.

Gulf War Illness (GWI) is a chronic multisymptom disorder affecting veterans of the 1990-91 Gulf war. GWI was linked with exposure to chemicals including the nerve gas prophylactic drug pyridostigmine-bromide (PB) and pesticides (DEET, permethrin). Veterans with GWI exhibit prolonged, low-level systemic inflammation, though whether this impacts the liver is unknown. While no evidence exists that GWI-related chemicals are hepatotoxic, the prolonged inflammation may alter the liver's response to insults such as cholestatic injury. We assessed the effects of GWI-related chemicals on macrophage infiltration and its subsequent influence on hepatic cholestasis. Sprague Dawley rats were treated daily with PB, DEET and permethrin followed by 15 minutes of restraint stress for 28 days. Ten weeks afterward, GWI rats or naïve age-matched controls underwent bile duct ligation (BDL) or sham surgeries. Exposure to GWI-related chemicals alone increased IL-6, and CD11b+F4/80- macrophages in the liver, with no effect on biliary mass or hepatic fibrosis. However, pre-exposure to GWI-related chemicals enhanced biliary hyperplasia and fibrogenesis caused by BDL, compared to naïve rats undergoing the same surgery. These data suggest that GWI patients could be predisposed to developing worse liver pathology due to sustained low-level inflammation of the liver when compared to patients without GWI.

Responses of the aquatic midge Chironomus riparius to DEET exposure.

N,N-diethyl-3-methylbenzamide (DEET) is the active ingredient of many commercial insect repellents. Despite being detected worldwide in effluents, surface water and groundwater, there is still limited information on DEET's toxicity toward non-target aquatic invertebrates. Thus, our main objective was to assess the effects of DEET in the life cycle of Chironomus riparius and assess its biochemical effects. Laboratory assays showed that DEET reduced developmental rates (reduced larval growth, delayed emergence) of C. riparius larvae and also caused a decrease in the size of adult midges. Concerning the biochemical responses, a short exposure to DEET caused no effects in lipid peroxidation, despite the significant inhibition of catalase and glutathione-S-transferase activities and of total glutathione contents. Moreover, inhibition of acetylcholinesterase activity was also observed showing neurotoxic effects. Environmental risk assessment of insect repellents is needed. Our results showed moderate toxicity of DEET toward C. riparius, however, due to their mode of action, indirect ecological effects of DEET and of other insect repellents cannot be excluded and should be evaluated.

Corticosterone primes the neuroinflammatory response to DFP in mice: potential animal model of Gulf War Illness.

Gulf War Illness (GWI) is a multi-symptom disorder with features characteristic of persistent sickness behavior. Among conditions encountered in the Gulf War (GW) theater were physiological stressors (e.g., heat/cold/physical activity/sleep deprivation), prophylactic treatment with the reversible AChE inhibitor, pyridostigmine bromide (PB), the insect repellent, N,N-diethyl-meta-toluamide (DEET), and potentially the nerve agent, sarin. Prior exposure to the anti-inflammatory glucocorticoid, corticosterone (CORT), at levels associated with high physiological stress, can paradoxically prime the CNS to produce a robust proinflammatory response to neurotoxicants and systemic inflammation; such neuroinflammatory effects can be associated with sickness behavior. Here, we examined whether CORT primed the CNS to mount neuroinflammatory responses to GW exposures as a potential model of GWI. Male C57BL/6 mice were treated with chronic (14 days) PB/ DEET, subchronic (7-14 days) CORT, and acute exposure (day 15) to diisopropyl fluorophosphate (DFP), a sarin surrogate and irreversible AChE inhibitor. DFP alone caused marked brain-wide neuroinflammation assessed by qPCR of tumor necrosis factor-α, IL6, chemokine (C-C motif) ligand 2, IL-1ß, leukemia inhibitory factor, and oncostatin M. Pre-treatment with high physiological levels of CORT greatly augmented (up to 300-fold) the neuroinflammatory responses to DFP. Anti-inflammatory pre-treatment with minocycline suppressed many proinflammatory responses to CORT+DFP. Our findings are suggestive of a possible critical, yet unrecognized interaction between the stressor/environment of the GW theater and agent exposure(s) unique to this war. Such exposures may in fact prime the CNS to amplify future neuroinflammatory responses to pathogens, injury, or toxicity. Such occurrences could potentially result in the prolonged episodes of sickness behavior observed in GWI. Gulf War (GW) veterans were exposed to stressors, prophylactic medicines and, potentially, nerve agents in theater. Subsequent development of GW Illness, a persistent multi-symptom disorder with features characteristic of sickness behavior, may be caused by priming of the CNS resulting in exaggerated neuroinflammatory responses to pathogens/insults. Nerve agent, diisopropyl fluorophosphate (DFP), produced a neuroinflammatory response that was exacerbated by pre-treatment with levels of corticosterone simulating heightened stressor conditions. While prophylactic treatments reduced DFP-induced neuroinflammation, this effect was negated when those treatments were combined with corticosterone.

Effects of triclocarban, N,N-diethyl-meta-toluamide, and a mixture of pharmaceuticals and personal care products on fathead minnows (Pimephales promelas).

Pharmaceuticals and personal care products (PPCPs) have been detected widely in aquatic ecosystems, but little is known about their mechanisms of toxicity. We exposed adult fathead minnows (Pimephales promelas) for 48 h to triclocarban (1.4 µg/L), N,N-diethyl-meta-toluamide (DEET; 0.6 µg/L), or a mixture of PPCPs consisting of atenolol (1.5 µg/L), caffeine (0.25 µg/L), diphenhydramine (0.1 µg/L), gemfibrozil (1.5 µg/L), ibuprofen (0.4 µg/L), naproxen (1.6 µg/L), triclosan (2.3 µg/L), progesterone (0.2 µg/L), triclocarban (1.4 µg/L), and DEET (0.6 µg/L). Quantitative real-time polymerase chain reaction revealed an upregulation in vitellogenin (vtg) in livers of females and males exposed to triclocarban. Also, an upregulation of hepatic lipoprotein lipase (lpl) and a downregulation of androgen receptor (ar) and steroidogenic acute regulatory protein (star) were observed in testes. The group treated with DEET only showed a significant decrease in ar in females. In contrast, the PPCP mixture downregulated vtg in females and males and expression of estrogen receptor alpha (erα), star, and thyroid hormone receptor alpha 1 (thra1) in testes. The authors' results show that the molecular estrogenic effects of triclocarban are eliminated (males) or reversed (females) when dosed in conjunction with several other PPCP, once again demonstrating that results from single exposures could be vastly different from those observed with mixtures. Environ Toxicol Chem 2014;33:910-919. © 2013 SETAC.

Pesticide and insect repellent mixture (permethrin and DEET) induces epigenetic transgenerational inheritance of disease and sperm epimutations.

Environmental compounds are known to promote epigenetic transgenerational inheritance of disease. The current study was designed to determine if a "pesticide mixture" (pesticide permethrin and insect repellent N,N-diethyl-meta-toluamide, DEET) promotes epigenetic transgenerational inheritance of disease and associated DNA methylation epimutations in sperm. Gestating F0 generation female rats were exposed during fetal gonadal sex determination and the incidence of disease evaluated in F1 and F3 generations. There were significant increases in the incidence of total diseases in animals from pesticide lineage F1 and F3 generation animals. Pubertal abnormalities, testis disease, and ovarian disease (primordial follicle loss and polycystic ovarian disease) were increased in F3 generation animals. Analysis of the pesticide lineage F3 generation sperm epigenome identified 363 differential DNA methylation regions (DMR) termed epimutations. Observations demonstrate that a pesticide mixture (permethrin and DEET) can promote epigenetic transgenerational inheritance of adult onset disease and potential sperm epigenetic biomarkers for ancestral environmental exposures.

The insect repellent DEET (N,N-diethyl-3-methylbenzamide) increases the synthesis of glutathione S-transferase in cultured mosquito cells.

DEET (N,N-diethyl-3-methylbenzamide) is the active ingredient used in many commonly used insect repellents, but its mode of action remains poorly understood. Efforts to identify properties that could lead to the development of more effective active ingredients have distinguished among DEET's repellent, deterrent, and insecticidal activities. We used an Aedes albopictus mosquito cell line to evaluate DEET's toxicological properties in the absence of sensory input mediated by the olfactory system. When cells were treated with DEET and labeled with [(35)S]methionine/cysteine, a single 25-kDa protein was induced, relative to other proteins, on SDS-polyacrylamide gels. The 25-kDa band from DEET-treated cells was enriched in peptides corresponding to glutathione S-transferase D10 and/or theta in the Aedes aegypti genome. Consistent with the increased expression of the labeled protein, DEET-treated cells had increased glutathione S-transferase activity, and the radiolabeled band bound to Sepharose 4B containing reduced glutathione. By analyzing partial tryptic digests, we established that DEET induces the homolog of A. aegypti glutathione S-transferase, class theta, corresponding to protein XP_001658009.1 in the NCBI database. This specific effect of DEET at the subcellular level suggests that DEET induces physiological responses that extend beyond recognition by the peripheral olfactory system.

Enzyme induction and cytotoxicity in human hepatocytes by chlorpyrifos and N,N-diethyl-m-toluamide (DEET).

Xenobiotics, including drugs and environmental chemicals, can influence cytochrome P450 (CYP) levels by altering the transcription of CYP genes. To minimize potential drug-pesticide and pesticide-pesticide interactions it is important to evaluate the potential of pesticides to induce CYP isoforms and to cause cytotoxicity in humans. The present study was designed to examine chlorpyrifos and DEET mediated induction of CYP isoforms and also to characterize their potential cytotoxic effects on primary human hepatocytes. DEET significantly induced CYP3A4, CYP2B6, CYP2A6 and CYP1A2 mRNA expression while chlorpyrifos induced CYP1A1, CYP1A2 and CYP3A4 mRNA, and to a lesser extent, CYP1B1 and CYP2B6 mRNA in primary human hepatocytes. Chlorpyrifos and DEET also mediated the expression of CYP isoforms, particularly CYP3A4, CYP2B6 and CYP1A1, as shown by CYP3A4-specific protein expression, testosterone metabolism and CYP1Al-specific activity assays. DEET is a mild, while chlorpyrifos is a relatively potent, inducer of adenylate kinase and caspase-3/7, an indicator of apoptosis, while inducing 15-20% and 25-30% cell death, respectively. Therefore, DEET and chlorpyrifos mediated induction of CYP mRNA and functional CYP isoforms together with their cytotoxic potential in human hepatocytes suggests that exposure to chlorpyrifos and/or DEET should be considered in human health impact analysis.

Widespread detection of N,N-diethyl-m-toluamide in U.S. streams: comparison with concentrations of pesticides, personal care products, and other organic wastewater compounds.

One of the most frequently detected organic chemicals in a nationwide study concerning the effects of wastewater on stream water quality conducted in the year 2000 was the widely used insect repellant N,N-diethyl-m-toluamide (DEET). It was detected at levels of 0.02 microg/L or greater in 73% of the stream sites sampled, with the selection of sampling sites being biased toward streams thought to be subject to wastewater contamination (i.e., downstream from intense urbanization and livestock production). Although DEET frequently was detected at all sites, the median concentration was low (0.05 microg/L). The highest concentrations of DEET were found in streams from the urban areas (maximum concentration, 1.1 microg/L). The results of the present study suggest that the movement of DEET to streams through wastewater-treatment systems is an important mechanism that might lead to the exposure of aquatic organisms to this chemical.

Insect repellents, phenoxyherbicide exposure, and non-Hodgkin's lymphoma.

OBJECTIVE: We sought to test a hypothetical explanation of contradictory results in studies of phenoxyherbicides and NHL, that the exposure of rubber gloves recommended for use by farmers when mixing or applying pesticides simultaneously to 2,4-D (2,4-dichlorophenoxyacetic acid), DEET (N,N-diethyl-m-toluamide), and ultraviolet rays increased their permeability to 2,4-D. METHODS: We conducted a case (NHL n = 513)/control (n = 1506) study among men using age; province of residence; exposure to insect repellents containing DEET, phenoxy-herbicides, or dicamba; and gloves when handling pesticides. RESULTS: Using conditional logistic regression, the stratum with reported exposure to mecoprop, to DEET and the use of rubber gloves had higher odds ratios (3.86; 95% confidence interval = 1.57-9.49) compared with strata with other combinations. CONCLUSIONS: In conclusion, the etiologic complexity of NHL was demonstrated.

Testicular germ-cell apoptosis in stressed rats following combined exposure to pyridostigmine bromide, N,N-diethyl m-toluamide (DEET), and permethrin.

This study reports and characterizes the testicular apoptosis following daily exposure of male Sprague-Dawley rats to subchronic combined doses of pyridostigmine bromide (PB, 1.3 mg/kg/d in water, oral), a drug used for treatment of myasthenia gravis and prophylactic treatment against nerve agents during the Persian Gulf War; the insect repellent N,N-diethyl m-toluamide (DEET, 40 mg/kg/d in ethanol, dermal); and the insecticide permethrin (0.13 mg/kg in ethanol, dermal), with and without stress for 28 d. Combined exposure to these chemicals was implicated in the development of illnesses including genitourinary disorders among many veterans of the Persian Gulf War. Previous studies from this laboratory have shown that exposure to combination of these chemicals produced greater toxicity compared to single components. Exposure to stress alone did not cause any significant histopathological alterations in the testes. Administration of combination of these chemicals induced apoptosis in rat testicular germ cells, Sertoli cells, and Leydig cells, as well as in the endothelial lining of the blood vessels. Testicular damage was significantly augmented when the animals were further exposed to a combination of chemicals and stress. Histopathological examination of testicular tissue sections showed that apoptosis was confined to the basal germ cells and spermatocytes, indicating suppression of spermatogenesis. Increased apoptosis of testicular cells coincided, in timing and localization, with increased expression of the apoptosis-promoting proteins Bax and p53. Furthermore, significant increase of 3-nitrotyrosine immunostaining in the testis revealed oxidative and/or nitrosation induction of cell death. In conclusion, combined exposure to real-life doses of test compounds caused germ-cell apoptosis that was significantly enhanced by stress.

Pyridostigmine bromide modulates topical irritant-induced cytokine release from human epidermal keratinocytes and isolated perfused porcine skin.

Gulf War personnel were given pyridostigmine bromide (PB) as a prophylactic treatment against organophosphate nerve agent exposure, and were exposed to the insecticide permethrin and the insect repellent N,N-diethyl-m-toluamide (DEET). The purpose of this study was to assess the effects of PB to modulate release of inflammatory biomarkers after topical chemical exposure to chemical mixtures containing permethrin and DEET applied in ethanol or water vehicles. Treatments were topically applied to isolated perfused porcine skin flaps (IPPSFs). Concentrations of interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha) and prostaglandin E(2) (PGE(2)) were assayed in perfusate to probe for potential inflammatory effects after complex mixture application. IPPSFs (n=4/treatment) were topically dosed with mixtures of permethrin, DEET, and permethrin/DEET, in ethanol. Each treatment was repeated with perfusate spiked with 50 ng/ml of PB. Perfusate was also spiked with 30 ng/ml diisopropylfluorophosphate to simulate low level organophosphate nerve agent exposure. Timed IPPSF venous effluent samples (0.5,1,2,4, and 8 h) were assayed by ELISA for IL-8 and TNF-alpha and by EIA for PGE(2). Overall, PB infusion caused a decrease or IL-8 and PGE(2) release. Effects on TNF-alpha were vehicle dependent. To probe the potential mechanism of this PB effect, human epidermal keratinocyte HEK cell cultures were exposed to permethrin DEET permethrin/DEET, with and without PB in DMSO. IL-8 was assayed at 1, 2, 4, 8, 12 and 24 h. PB suppressed IL-8 in permethrin and ethanol treatment from 4 to 24 h confirming the IPPSF results. In conclusion, these studies suggest that systemic exposure to PB suppressed IL-8 release at multiple time points in two skin model systems. This interaction merits further study.

Genotoxicity studies on permethrin, DEET and diazinon in primary human nasal mucosal cells.

Possible genotoxic effects exerted by three widely used pesticides, permethrin, N,N-diethyl-m-toluamide (DEET) and diazinon, in primary human nasal mucosal cells were investigated. Primary nasal mucosa cells were prepared from tissue biopsies taken from 21 patients who underwent nasal surgery. Cells were exposed to 0.5-1.0 mM concentrations of permethrin, DEET and diazinon for 60 min. Genotoxic effects were detected by the alkaline microgel electrophoresis assay ("comet assay"). Within the concentration range, no significant cytotoxic effects were observed, but all three tested pesticides showed a significant genotoxic response that was concentration dependent. More pronounced genotoxic effects were observed in mucosal cells from the middle turbinate than in the inferior turbinate. The results provide some evidence for the potential carcinogenicity of these agents to human nasal mucosal cells. This should be further investigated.

Effect of selected pesticides and their ozonation by-products on gap junctional intercellular communication using rat liver epithelial cell lines.

The non-genotoxic effects of two commonly used pesticides, 1,1-bis (p-chlorophenyl)-2,2,2-trichloroethane (DDT) and malathion, and one widely used commercial insect repellent N,N-diethy-m-toluamide (DEET) on gap junction intercellular communication (GJIC) were determined using a rat liver epithelial cell line. Malathion and DDT reversibly inhibited GJIC in a treatment time- and dose-dependent manner at non-cytotoxic doses, whereas, DEET did not inhibit GJIC. Malathion was very reactive with ozone, while DEET and DDT did not react to any appreciable extent with ozone. The mixtures of ozonation products from malathion and DEET did not inhibit GJIC. The mixtures of ozonation by-products formed from DDT inhibited GJIC, but to a lesser extent than did DDT, itself. These results suggest that ozone can effectively remove malathion from solution without forming GJIC-toxic products, but is less effective in eliminating DEET and DDT from solution.

Protection against the co-operative toxicity of nitric oxide and oxygen free radicals by overexpression of antioxidant enzymes in bioengineered insulin-producing RINm5F cells.

AIMS/HYPOTHESIS: The importance of different antioxidative enzymes for the defence of insulin-producing cells against the toxicity of nitric oxide (NO) was characterised in bioengineered RINm5F cells. METHODS: RINm5F insulin-producing cells stably overexpressing glutathione peroxidase (GPX), catalase (CAT) or Cu/Zn superoxide dismutase (SOD) were exposed to S-nitroso-N-acetyl-D,L-penicillamine (SNAP), sodium nitroprusside (SNP) and 3 morpholinosydnonimine (SIN-1), which generate both NO and reactive oxygen species, and to the polyamine/ NO, complex DETA/NO which generates NO alone. The viability of the cells was tested by the MTT assay. RESULTS: Overexpression of antioxidant enzymes provided significant protection against the toxicity of SNAP, SNP and SIN-1, with an individual specificity related to their chemical characteristics, but was without effect upon the toxicity of DETA/NO. Cells overexpressing GPX were well protected against SNP and SNAP, while CAT was most effective against SIN-1. SOD overexpression provided less protection against the toxicity of SNAP and SNP than overexpression of GPX but was more effective in protecting against SIN-1. Co-incubation of cells with NO donors and hydrogen peroxide or hypoxanthine and xanthine oxidase showed an overadditive synergism of toxicity. CONCLUSION/INTERPRETATION: The results emphasise the importance of a synergism between NO and reactive oxygen species for pancreatic beta-cell death. Such a synergism has also been observed after exposure of beta cells to cytokines. The component of the toxicity that is mediated by oxygen radicals can be suppressed effectively through overexpression of CAT, GPX or SOD or both.

Evaluation of the chronic toxicity and oncogenicity of N,N-diethyl-m-toluamide (DEET).

Chronic toxicity and/or oncogenicity studies were conducted in rats, mice, and dogs with the insect repellent DEET. DEET was mixed in the diet and administered to CD rats for two years at concentrations that corresponded to dosage levels of 10, 30 or 100 mg/kg/day for males and 30, 100, or 400 mg/kg/day for females; to CD-1 mice for 18 months at dosage levels of 250, 500, or 1000 mg/kg/day; and to dogs for one year, via gelatin capsules, at dosage levels of 30, 100, or 400 mg/kg/day. In the rodent studies, each group consisted of 60 animals of each sex, and two concurrent independent control groups, each containing 60 animals/sex were included in each study. Each group in the dog study consisted of four male and four female dogs and one control group was included in the study. Treatment-related effects were observed at the highest dose level in all three studies. For rats, the effects included decreases in body weight and food consumption and an increase in serum cholesterol in females only. In mice, the effects observed were decreases in body weight and food consumption in both sexes. The effects observed in dogs included increased incidences of emesis and ptyalism, and levels of transient reduction in hemoglobin and hematocrit, increased alkaline phosphatase (males only), decreased cholesterol, and increased potassium. One male dog in the high-dose group also exhibited ataxia, tremors, abnormal head movements, and/or convulsions on several occasions during the study. The highest no-observed-effect levels (NO-ELs) for rats, mice and dogs were determined to be 100, 500, and 100 mg/kg/day, respectively. No specific target organ toxicity or oncogenicity was observed in any of the studies.

Review of the biodistribution and toxicity of the insect repellent N,N-diethyl-m-toluamide (DEET).

A review of the biodistribution and toxicity of the insect repellent N,N-diethyl-m-toluamide (DEET) is presented. Workers using repellent containing this compound may be exposed to greater than 442 g in 6 mo. In human studies, variable penetration into the skin of from 9 to 56% of a topically applied dose and absorption into the circulatory system of approximately 17% have been reported. Excretion of DEET by humans was initially rapid but not as complete as in animal models. Only about one-half of the absorbed DEET was excreted by humans over 5 d. Depot storage of DEET in the skin was also documented. Skin irritant effects, including scarring bullous dermatitis in humans, were reported. One animal study that reported embryotoxicity could not be confirmed by other investigators. The limited testing for mutagenicity and carcinogenicity provided negative results. Neurotoxic effects were observed in workers exposed to 4 g or more per week. Six young girls developed encephalopathies after exposure to unspecified amounts of DEET ranging from small to massive doses. Three of these girls later died. The cause of their death has not been resolved. Because of the lack of information, further research into the absorption, carcinogenicity, and neurotoxic effects is needed.