Recent advances in degradation of N,N-diethyl-3-toluamide (DEET)-an emerging environmental contaminant: a review.

N,N-Diethyl-3-toluamide (DEET) is a commonly used insect repellent, which acts as an organic chemical contaminant in water and considered as an emerging contaminant which has been observed worldwide. It gets discharged into the environment through sewage waste. The various methods have been used to degrade DEET, such as UV based, ozonation, photocatalytic degradation, and biodegradation (based on the metabolic activity of fungi and bacteria). However, less research has been done on the degradation of DEET by deploying nanoparticles. Therefore, biodegradation and nanotechnology-based methods can be the potential solution to remediate DEET from the environment. This review is an attempt to analyze the routes of entry of DEET into the atmosphere and its environmental health consequences and to explore physical, chemical, and biological methods of degradation. Furthermore, it focuses on the various methods used for the biodegradation of the DEET, including their environmental consequences. Future research is needed with the application of biological methods for the degradation of DEET. Metabolic pathway for biodegradation was explored for the new potent microbial strains by the application of physical, chemical, and microbial genomics; molecular biology; genetic engineering; and genome sequencing methods.

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.

Epigenome-wide association study for pesticide (Permethrin and DEET) induced DNA methylation epimutation biomarkers for specific transgenerational disease.

BACKGROUND: Permethrin and N,N-diethyl-meta-toluamide (DEET) are the pesticides and insect repellent most commonly used by humans. These pesticides have been shown to promote the epigenetic transgenerational inheritance of disease in rats. The current study was designed as an epigenome-wide association study (EWAS) to identify potential sperm DNA methylation epimutation biomarkers for specific transgenerational disease. METHODS: Outbred Sprague Dawley gestating female rats (F0) were transiently exposed during fetal gonadal sex determination to the pesticide combination including Permethrin and DEET. The F3 generation great-grand offspring within the pesticide lineage were aged to 1 year. The transgenerational adult male rat sperm were collected from individuals with single and multiple diseases and compared to non-diseased animals to identify differential DNA methylation regions (DMRs) as biomarkers for specific transgenerational disease. RESULTS: The exposure of gestating female rats to a permethrin and DEET pesticide combination promoted transgenerational testis disease, prostate disease, kidney disease, and the presence of multiple disease in the subsequent F3 generation great-grand offspring. The disease DMRs were found to be disease specific with negligible overlap between different diseases. The genomic features of CpG density, DMR length, and chromosomal locations of the disease specific DMRs were investigated. Interestingly, the majority of the disease specific sperm DMR associated genes have been previously found to be linked to relevant disease specific genes. CONCLUSIONS: Observations demonstrate the EWAS approach identified disease specific biomarkers that can be potentially used to assess transgenerational disease susceptibility and facilitate the clinical management of environmentally induced pathology.

Aquatic life criteria derivation and ecological risk assessment of DEET in China.

N,N-diethyl-meta-toluamide (DEET) is the most widely used active ingredient in commercial insect repellents. In addition to its adverse effects in insects, DEET can affect non-target organisms in surface water systems. Nevertheless, the aquatic life criteria of DEET are not available. This study conducted both acute and chronic toxicity tests on DEET in native Chinese aquatic species, and derived its criterion maximum concentration (CMC) and criterion continuous concentration (CCC). The determined CMC and CCC of DEET were 21.53 and 0.52 mg/L, respectively. The toxicity data indicated that DEET exposure posed a higher toxicity to some algae than other aquatic species. Compared with other insect repellents, DEET exposure posed a moderate toxicity to aquatic species. Therefore, the exposure concentration of DEET in Chinese surface water was collected to assess the potential ecological risk. The preliminary ecological risk assessment showed that DEET posed negligible risk to aquatic ecosystems in China. However, considering its toxic effects on the growth and reproduction to aquatic organisms, the ecological risk posed by DEET is worth further concern.

In vitro skin irritation assessment using EpiDerm™: applicability for updating toxicity information of oxybenzone and N,N-diethyl-m-toluamide.

A skin irritation test using in vitro reconstructed human epidermis (RhE) models was established for hazard identification of irritant chemicals in accordance with UN Globally Harmonized System of Classification and Labelling of Chemicals (GHS) category. In this study, EpiDerm™ was used to assess skin irritation by oxybenzone and N,N-diethyl-m-toluamide (DEET), which are widely used sunscreen and insect repellent components, respectively. EpiDerm™ was applied with oxybenzone and DEET, combined and sequentially with each single dose. Epidermal morphology and differentiation/proliferation were examined microscopically. Oxybenzone and sequential administration groups were determined as nonirritant with cell viability >50% and the morphology was comparable to the human epidermis. Contrastingly, the DEET and coadministration groups exhibited cell viability <50% and poor epidermal morphology. Interleukin (IL)-1α release from substance-treated EpiDerm™ increased inversely to cell viability, suggesting the pro-inflammatory reaction was initiated by DEET. CK-10, E-cadherin, Ki-67, laminin, and ceramide were identified as relevant markers to assess oxybenzone- or DEET-induced epidermal injury. In conclusion, these results may indicate to be aware of the possible skin irritation by indiscriminate use of oxybenzone and DEET without animal testing.

Stimulation of eryptosis by broad-spectrum insect repellent N,N-Diethyl-3-methylbenzamide (DEET).

N,N-Diethyl-3-methylbenzamide (DEET) is the most widely used insect repellent in the world. Adverse effects following DEET exposure are well documented. Moreover, DEET has been shown to possess cytotoxic and apoptotic properties in nucleated cells. Although red blood cells (RBCs) lack intracellular organelles, they nevertheless undergo programmed cell death termed eryptosis. Compromised RBC health contributes to the development of anemia; a condition affecting 25% of the global population. This study investigated the interaction between DEET and human RBCs, and explored accompanying biochemical and molecular alterations. RBCs at 5% hematocrit were incubated in presence and absence of 1-5 mM (0.02%-0.1%) of DEET for 6 h at 37 °C. Hemolysis was spectrophotometrically determined by hemoglobin release, while major eryptotic events were analyzed by flow cytometer. Phosphatidylserine (PS) exposure was detected with Annexin-V-FITC, cell volume by forward scatter (FSC) of light, intracellular calcium with Fluo-3/AM, and reactive oxygen species with 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). DEET caused slight hemolysis at 4 and 5 mM, and significantly increased Annexin-V-FITC and Fluo3 fluorescence, with reduced FSC at 5 mM. Removal of extracellular Ca2+ abolished DEET-induced Fluo3 fluorescence but had no effect on Annexin-V binding. Importantly, blockade of eryptotic signaling mediators p38 MAPK, caspases, protein kinase C, casein kinase 1, or necroptotic kinases receptor-interacting protein 1 and mixed lineage kinase domain-like protein, with small molecule inhibitors, did not ameliorate DEET-mediated PS externalization. In conclusion, DEET elicits suicidal erythrocyte death; an event characterized by loss of membrane asymmetry, cell shrinkage, and elevations in intracellular Ca2+ mainly through dysregulated Ca2+ influx.

High mortality in aquatic predators of mosquito larvae caused by exposure to insect repellent.

In the face of mosquito-borne disease outbreaks, effective mosquito control is a primary goal for public health. Insect repellents, containing active compounds such as DEET and picaridin, are a first defence against biting insects. Owing to widespread use and incomplete sewage treatment, these compounds are frequently detected in surface waters, but their effects on aquatic taxa such as mosquito larvae or their naturally occurring aquatic predators are poorly understood. We investigated the effects of environmentally realistic concentrations of commercial products containing DEET and picaridin on survivorship of mosquito larvae, and their potential indirect effects on survival of larval salamanders, a major predator of mosquito larvae. Larval mosquitos were not affected by exposure to repellents containing DEET or picaridin. We found no larval salamander mortality in control and DEET treatments, but mortality rates in picaridin treatments ranged from 45 to 65% after 25 days of exposure. Salamander larvae exposed to repellents containing picaridin began to display tail deformities and impaired development four days after the experiment began. Our findings suggest the possibility that environmentally realistic concentrations of picaridin-containing repellents in surface waters may increase the abundance of adult mosquitos owing to decreased predation pressure.

DEET potentiates the development and persistence of anticholinesterase dependent chronic pain signs in a rat model of Gulf War Illness pain.

Exposure to DEET (N,N-diethyl-meta-toluamide) may have influenced the pattern of symptoms observed in soldiers with GWI (Gulf War Illness; Haley and Kurt, 1997). We examined how the addition of DEET (400mg/kg; 50% topical) to an exposure protocol of permethrin (2.6mg/kg; topical), chlorpyrifos (CP; 120mg/kg), and pyridostigmine bromide (PB;13mg/kg) altered the emergence and pattern of pain signs in an animal model of GWI pain (Nutter et al., 2015). Rats underwent behavioral testing before, during and after a 4week exposure: 1) hindlimb pressure withdrawal threshold; 2) ambulation (movement distance and rate); and 3) resting duration. Additional studies were conducted to assess the influence of acute DEET (10-100µM) on muscle and vascular nociceptor Kv7, KDR, Nav1.8 and Nav1.9. We report that a 50% concentration of DEET enhanced the development and persistence of pain-signs. Rats exposed to all 4 compounds exhibited ambulation deficits that appeared 5-12weeks post-exposure and persisted through weeks 21-24. Rats exposed to only three agents (CP or PB excluded), did not fully develop ambulation deficits. When PB was excluded, rats also developed rest duration pain signs, in addition to ambulation deficits. There was no evidence that physiological doses of DEET acutely modified nociceptor Kv7, KDR, Nav1.8 or Nav1.9 activities. Nevertheless, DEET augmented protocols decreased the conductance of Kv7 expressed in vascular nociceptors harvested from chronically exposed rats. We concluded that DEET enhanced the development and persistence of pain behaviors, but the anticholinesterases CP and PB played a determinant role.

Impact of Environmental Chemicals on the Transcriptome of Primary Human Hepatocytes: Potential for Health Effects.

New paradigms for human health risk assessment of environmental chemicals emphasize the use of molecular methods and human-derived cell lines. In this study, we examined the effects of the insect repellent DEET (N,N-diethyl-m-toluamide) and the phenylpyrazole insecticide fipronil (fluocyanobenpyrazole) on transcript levels in primary human hepatocytes. These chemicals were tested individually and as a mixture. RNA-Seq showed that 100 µM DEET significantly increased transcript levels (α = 0.05) for 108 genes and lowered transcript levels for 64 genes and fipronil at 10 µM increased the levels of 2246 transcripts and decreased the levels for 1428 transcripts. Fipronil was 21-times more effective than DEET in eliciting changes, even though the treatment concentration was 10-fold lower for fipronil versus DEET. The mixture of DEET and fipronil produced a more than additive effect (levels increased for 3017 transcripts and decreased for 2087 transcripts). The transcripts affected for all chemical treatments were classified by GO analysis and mapped to chromosomes. The overall treatment responses, specific pathways, and individual transcripts affected were discussed at different levels of fold-change. Changes found in transcript levels in response to treatments will require further research to understand their importance in overall cellular, organ, and organismic function.

Behavioral and Toxicological Responses of Rhodnius prolixus (Hemiptera: Reduviidae) to the Insect Repellents DEET and IR3535.

,N-diethyl-3-methylbenzamide (DEET) is a broad-spectrum insect repellent used by millions of people since the 1950s. Ethyl 3-[acetyl(butyl)amino]propanoate (IR3535) is a repellent developed more recently that is still not used as extensively. This study compares the behavioral and toxicological effects of both substances in fifth-instar nymphs of the blood-sucking bug Rhodnius prolixus Stål (Hemiptera: Reduviidae), one of the main vectors of American trypanosomiasis (Chagas disease). Repellency was quantified using filter paper discs as experimental arenas. Half the discs were treated with solution of repellent in acetone, and the other half with acetone alone. The lowest observed effect level was identical for both substances, 74 µg/cm2. Nymph age (between 1-3 h and 99 d from last ecdysis) had no influence on repellency. Topical application of 750 µg of DEET per nymph produced a mortality rate between 0% (24 h after application) and 40% (7 d later). The same dose of IR3535 produced no mortality during the same period of time. Simultaneous treatment with piperonylbutoxide (a mixed function microsomal oxidase inhibitor) synergized the lethal effect of DEET. Only DEET increased locomotor activity in nymphs exposed to a treated surface. Nymph antennectomy abolished DEET repellency but not its effect on locomotor activity. The concentrations of both these compounds required to produce either behavioral or toxicological effects are too high to have any practical applications in the control of R. prolixus.

Acute toxicity assessment of N,N-diethyl-m-toluamide (DEET) on the oxygen flux of the dinoflagellate Gymnodinium instriatum.

Despite the ubiquitous occurrence of N,N-diethyl-m-toluamide (DEET) in aquatic systems, assessments evaluating the toxicity of DEET on phytoplankton species are summed to a single study on a unicellular green alga. In particular, the toxicological effects of DEET in dinoflagellates are unknown. In this study, we employed the mixotrophic dinoflagellate Gymnodinium instriatum as a study system to evaluate acute effects of DEET on the oxygen flux of laboratory cultures. This study reports an inhibitory reaction model of DEET described by the equation y = 4.99x(0.54), where y represents the percent inhibition of oxygen flux and x represents DEET concentration in mg L(−1) (r(2) = 0.98). Based on this model, the effective concentration of DEET needed to reduce O2 flux by 50% (EC50) for this species was found to be at 72.9 mg L(−1). The reported EC50 is more than five times lower than the EC50 reported previously for the unicellular green algae Chlorella protothecoides. This study raises the question of the potential toxicological effects of DEET in dinoflagellates, in particular those populations inhabiting systems characterized by low water circulation such as enclosed bays and lagoons.

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.

Toxicity assessment of binary mixtures of BP3 with 4-MBC (UV-filters), and BP3 with DEET (insect repellent) using the aquatic midge Chironomus riparius.

Personal care products have various organic ultraviolet filters (UV filters) in their composition to increase protection against ultraviolet radiation. Some of these products also contain insect repellents in their formulations. Consequently, these compounds reach freshwater ecosystems, exposing aquatic organisms to a cocktail of anthropogenic contaminants. In this study, the joint effects of two most frequently detected UV filters (Benzophenone - 3 (BP3) and Enzacamene (4-MBC)) and joint effects of BP3 combined with an insect repellent (N, N diethyl-3-methylbenzamide - DEET) were evaluated using life-history traits of the aquatic midge Chironomus riparius such as emergence rate, time to emergence and imagoes body weight. The results showed synergistic effects between BP3 and 4-MBC for C. riparius emergence rate. Regarding the effects of BP3 and DEET mixture, our analysis suggests synergism in the case of males but antagonism in the case of females' time to emergence. Our results imply that the effects of UV filters present in sediments within chemical mixtures are complex and that the evaluation of effects using different life-history traits can yield different patterns of responses. This study demonstrates the importance of assessing the combined effects of pollutants used/found concomitantly in aquatic systems for a more accurate risk assessment, as individual chemical testing can underestimate the toxicity of organic UV filters.

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.

Comparative effects of insect repellent N,N-diethylbenzamide, N,N-diethylphenylacetamide, and N,N-diethyl-3- methylbenzamide aerosols on the breathing pattern and respiratory variables in mice.

Comparative inhalation toxicity studies of aerosols of insect repellents N,N-diethylbenzamide (DEB), N,N-diethylphenylacetamide (DEPA), and N,N-diethyl-3-methylbenzamide (DEET) were carried out in mice. The respiratory pattern was monitored using a computer program that recognizes the modifications of the breathing pattern. Exposure to the aerosols caused a dose-dependent decrease in normal breath, with an increase in airway obstruction. All the three insect "sensilla irritants" showed no significant mammalian sensory irritation. The acute LC(50) value for a 4-h exposure of DEB, DEPA, and DEET aerosols in male mice was found to be >2.5 g/m(3), 1714 mg/m(3), and 1369 mg/m(3), respectively. Irreversible depression in respiratory frequency was observed after exposure to DEB aerosol at a concentration of 277 mg/m(3) and above, which did not revert back to normal level even after aerosol exposure was stopped. At a concentration of 156 mg/m(3) of DEB, no respiratory depression was observed. DEPA and DEET caused no depression in respiratory frequency up to a concentration 1292 and 950 mg/m(3), respectively. Hence the two insect repellents DEET and DEPA do not cause any harmful effect to the respiratory parameters in acute exposure, showing that they are more suitable chemicals to be used as insect repellents as compared to DEB.

Adverse events associated with the use of insect repellents containing N,N-diethyl-m-toluamide (DEET).

DEET is the major component of most topically applied insect repellents used in the US. The DEET Registry is a post-marketing surveillance system to provide systematic and detailed information about medical events temporally associated with DEET use. From 1995 to 2001, 296 moderate and major severity cases were included in the DEET Registry. Of these, 36 (14.5%) cases were deemed to be probably and 157 (65%) cases possibly related to DEET exposure. Insufficient data prevented determination of causality in the remaining 49 (20.2%) cases. Forty-one percent of the cases were in children 19 or younger. Forty-two percent of children experienced a seizure of moderate or major severity. The widely variable spectrum of other neurological symptoms reported in the Registry makes it unlikely they were due to one agent. People with an underlying neurological disorder were not disproportionately represented in the DEET Registry. Data showed no clear relationship between case severity and DEET concentration or concurrent use of common medicines. Recognizing the extensive use of DEET in the US and considering the information about the more serious adverse events described in the Registry, the risk of serious neurological events following the use of DEET repellents is quite low.

Evidence for inhibition of cholinesterases in insect and mammalian nervous systems by the insect repellent deet.

BACKGROUND: N,N-Diethyl-3-methylbenzamide (deet) remains the gold standard for insect repellents. About 200 million people use it every year and over 8 billion doses have been applied over the past 50 years. Despite the widespread and increased interest in the use of deet in public health programmes, controversies remain concerning both the identification of its target sites at the olfactory system and its mechanism of toxicity in insects, mammals and humans. Here, we investigated the molecular target site for deet and the consequences of its interactions with carbamate insecticides on the cholinergic system. RESULTS: By using toxicological, biochemical and electrophysiological techniques, we show that deet is not simply a behaviour-modifying chemical but that it also inhibits cholinesterase activity, in both insect and mammalian neuronal preparations. Deet is commonly used in combination with insecticides and we show that deet has the capacity to strengthen the toxicity of carbamates, a class of insecticides known to block acetylcholinesterase. CONCLUSION: These findings question the safety of deet, particularly in combination with other chemicals, and they highlight the importance of a multidisciplinary approach to the development of safer insect repellents for use in public health.

Tissue deposition of the insect repellent DEET and the sunscreen oxybenzone from repeated topical skin applications in rats.

Insect repellent N,N-diethyl-m-toluamide (DEET) and sunscreen oxybenzone are capable of enhancing skin permeation of each other when applied simultaneously. We carried out a cellular study in rat astrocytes and neurons to assess cell toxicity of DEET and oxybenzone and a 30-day study in Sprague-Dawley rats to characterize skin permeation and tissue disposition of the compounds. Cellular toxicity occurred at 1 µg/mL for neurons and 7-day treatment for astrocytes and neurons. DEET and oxybenzone permeated across the skin to accumulate in blood, liver, and brain after repeated topical applications. DEET disappeared from the application site faster than oxybenzone. Combined application enhanced the disposition of DEET in liver. No overt sign of behavioral toxicity was observed from several behavioral testing protocols. It was concluded that despite measurable disposition of the study compounds in vivo, there was no evidence of neurotoxicological deficits from repeated topical applications of DEET, oxybenzone, or both.

Effects of Incubation of Human Brain Microvascular Endothelial Cells and Astrocytes with Pyridostigmine Bromide, DEET, or Permethrin in the Absence or Presence of Metal Salts.

Gulf War Illness (GWI) is a chronic, multi-symptom illness suffered by over one-third of American military veterans who served in the Persian Gulf War between 1990 and 1991. No current single-exposure scenario accounts for all the symptoms observed in GWI, and instead may be due to a multi-exposure scenario. As a larger effort to understand how one category of multi-exposure scenarios of organic compounds such as nerve gas prophylactic pyridostigmine bromide, or insecticides/pesticides such as N,N-diethyl-m-toluamide (DEET) and permethrin, plus heavy metals found in inhaled dust particles (Al, Fe, Ni, Sr, DU, Co, Cu, Mn, and Zn) might play a role in neural aspects of GWI, we begin this initial study to examine the toxicity and oxidative damage markers of human brain endothelial cell and human astrocyte cell cultures in response to these compounds. A battery of cytotoxicity assessments, including the MTT assay, Neutral Red uptake, and direct microscopic observation, was used to determine a non-toxic dose of the test compounds. After testing a wide range of doses of each compound, we chose a sub-toxic dose of 10 µM for the three organic compounds and 1 µM for the nine metals of interest for co-exposure experiments on cell cultures and examined an array of oxidative stress-response markers including nitric oxide production, formation of protein carbonyls, production of thiobarbituric acid-reactive substances, and expression of proteins involved in oxidative stress and cell damage. Many markers were not significantly altered, but we report a significant increase in nitric oxide after exposure to any of the three compounds in conjunction with depleted uranium.

Metabolomic Investigations of the Temporal Effects of Exposure to Pharmaceuticals and Personal Care Products and Their Mixture in the Eastern Oyster (Crassostrea virginica).

The eastern oyster (Crassostrea virginica) supports a large aquaculture industry and is a keystone species along the Atlantic seaboard. Native oysters are routinely exposed to a complex mixture of contaminants that increasingly includes pharmaceuticals and personal care products (PPCPs). Unfortunately, the biological effects of chemical mixtures on oysters are poorly understood. Untargeted gas chromatography-mass spectrometry metabolomics was utilized to quantify the response of oysters exposed to fluoxetine, N,N-diethyl-meta-toluamide, 17α-ethynylestradiol, diphenhydramine, and their mixture. Oysters were exposed to 1 µg/L of each chemical or mixture for 10 d, followed by an 8-d depuration period. Adductor muscle (n = 14/treatment) was sampled at days 0, 1, 5, 10, and 18. Trajectory analysis illustrated that metabolic effects and class separation of the treatments varied at each time point and that, overall, the oysters were only able to partially recover from these exposures post-depuration. Altered metabolites were associated with cellular energetics (i.e., Krebs cycle intermediates), as well as amino acid metabolism and fatty acids. Exposure to these PPCPs also affected metabolic pathways associated with anaerobic metabolism, osmotic stress, and oxidative stress, in addition to the physiological effects of each chemical's postulated mechanism of action. Following depuration, fewer metabolites were altered, but none of the treatments returned them to their initial control values, indicating that metabolic disruptions were long-lasting. Interestingly, the mixture did not directly cluster with individual treatments in the scores plot from partial least squares discriminant analysis, and many of its affected metabolic pathways were not well predicted from the individual treatments. The present study highlights the utility of untargeted metabolomics in developing exposure biomarkers for compounds with different modes of action in bivalves. Environ Toxicol Chem 2020;39:419-436. © 2019 SETAC.