Anticonvulsant-resistant seizures following pyridostigmine bromide (PB) and N,N-diethyl-m-toluamide (DEET).

An acute toxic interaction has been described, in which sublethal doses of pyridostigmine bromide (PB) and the insect repellent N,N-diethyl-m-toluamide (DEET), when administered concomitantly, resulted in seizures and lethality. To investigate the possible relationships between seizures and lethality and the role of the cholinergic system in this interaction, PB (5 mg/kg), DEET (200 mg/kg) or PB (3 mg/kg) + DEET (200 mg/kg) were administered i.p. to male ICR mice, alone or following i.p. pretreatment, with one of several anticonvulsant agents: diazepam, 10 mg/kg; fosphenytoin, 40 mg/kg; phenobarbital, 45 mg/kg; or dextrophan, 25 mg/kg), or the anticholinergic agents, atropine (5 mg/kg), atropine methyl nitrate (2.7 mg/kg), or mecamylamine (2.5 mg/kg). The anticonvulsants selected for this study act through different mechanisms to reduce seizures. None of the anticonvulsants was able to reduce the incidence of seizures following treatment with PB, DEET or PB + DEET. Only diazepam delayed the onset of seizures. Fosphenytoin or diazepam significantly prolonged the time to lethality following PB, but only fosphenytoin reduced the incidence of PB-induced lethality. Diazepam or phenobarbital significantly prolonged the time to lethality following PB + DEET. Both atropine and atropine methyl nitrate protected against PB and PB + DEET-induced lethality and PB-induced seizures. Neither agent blocked seizures resulting from DEET or PB + DEET. Mecamylamine reduced seizures and lethality in PB-treated mice, but not in mice treated with DEET or PB + DEET. The results indicate that seizure activity is not a causative factor in the toxic interaction between PB and DEET. Furthermore, PB, DEET and PB + DEET induce seizures that are resistant to standard anticonvulsants, and each appears to operate through different mechanisms to produce seizures. Peripheral muscarinic receptors may play a specific role in lethality caused by PB + DEET.     

Some cardiovascular effects of the insect repellent N,N-diethyl-m-toluamide (DEET)

Initial toxicological safety evaluations of the insect repellent N,N-diethyl-m-toluamide (DEET) indicated a potential hypotensive effect. The current study was initiated in order to pursue this aspect of DEET toxicity and to elucidate potential mechanisms for this response. Sublethal intraperitoneal injections of DEET in anesthetized rats were found to decrease mean blood pressure and heart rate in a dose-related fashion. Doses ranged from 56 to 225 mg/kg. Dogs treated with 225 mg/kg of DEET exhibited a similar hypotension and bradycardia. Cardiac output was also significantly reduced but stroke volume and total peripheral resistance were not altered. Lead II ECG changes included small increases in P-R and Q-T intervals. In a series of pharmacological studies in rats, DEET was found to decrease the hypotension and bradycardia associated with acetylcholine injection; epinephrine, norepinephrine, and histamine responses were not altered. Atropine pretreatment reduced but did not eliminate the hypotensive effects of DEET.     

Percutaneous absorption of topical N,N-diethyl-m-toluamide (DEET): effects of exposure variables and coadministered toxicants

Exposure to N,N-diethyl-m-toluamide (DEET) commonly occurs in the general population and has been implicated as a contributory factor to the Gulf War Illness. The focus of the present studies was to determine the effect of coexposure factors, potentially encountered in a military environment, that could modulate transdermal flux of topically applied DEET. Factors investigated were vehicle, dose, coexposure to permethrin, low-level sulfur mustard, occlusion, and simultaneous systemic exposure to pyridostigmine bromide and the nerve agent stimulant diisopropylfluorophosphate (DFP). Studies were conducted using the isolated perfused porcine skin flap (IPPSF), with a few mechanistically oriented studies conducted using in vitro porcine skin and silastic membrane diffusion cells. DEET was quantitated using high-performance liquid chromatography. The vehicle-control transdermal DEET flux in the IPPSF was approximately 2 micrograms/cm2/h for both 7.5 and 75% DEET concentrations, a value similar to that reported in humans. DEET absorption was enhanced by coinfusion of pyridostigmine bromide and DFP, by the presence of sulfur mustard, or by dosing under complete occlusion. The greatest increase in baseline flux was fivefold. In vitro diffusion cell studies indicated that silastic membranes had two orders of magnitude greater permeability than porcine skin, and showed vehicle effects on flux that were not detected in the IPPSF. These results suggest that coexposure to a number of chemicals that potentially could be encountered in a military environment may modulate the percutaneous absorption of topically applied DEET beyond that seen for normal vehicles at typically applied concentrations.     

Insect repellent [correction of repellant] interactions: sunscreens enhance DEET (N,N-diethyl-m-toluamide) absorption.

Toxicology studies are typically performed on single compounds, which we hypothesized would miss adverse synergies from chemical mixtures. This hypothesis was tested using an insect repellant and sunscreens because both groups include known permeation enhancers, with prior pediatric reports of toxicity from highly concentrated DEET (N,N-diethyl-m-toluamide). Using real-time mass spectroscopy in a hairless mouse skin model, we confirmed substantial penetration of a 20% DEET standard. Despite a lower (10%) DEET content, a commercially marketed sunscreen formulation had a 6-fold more rapid detection (5 versus 30 min) and 3.4-fold greater penetration at steady state. We also tested the efficacy of DEET microemulsion products and confirmed that one successfully slowed the onset of absorption, but not the steady-state permeation. Risks from mixtures of potential toxins are worthy of routine testing, which can be accomplished by simple assays, and are of utmost importance for pediatric applications.     

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.     

Microsomal metabolism of N,N-diethyl-m-toluamide (DEET,DET): the extended network of metabolites

1. The aim was to set out to establish the complete network of metabolites arising from the phenobarbital-treated rat liver microsomal oxidation of N,N-diethyl-m-toluamide (DEET). The products formed from DEET and all its subsequent metabolites were identified by HPLC retention times, UV spectroscopy, mass spectrometry and by comparison with authentic standards. 2. DEET (1a) produces three major metabolites, N-ethyl-m-toluamide (1b), N,N-diethyl-m-(hydroxymethyl)benzamide (2a) and N-ethyl-m-(hydroxymethyl)benzamide (2b), and, at low substrate concentrations or extended reaction times, two minor metabolites, toluamide (1c) and N,N-diethyl-m-formylbenzamide (3a). 1b and 2a are primary metabolites and their formation follows Michaelis-Menten-type kinetics. At low DEET concentrations, ring methyl group oxidation is favoured; at saturation concentrations, methyl group oxidation and N-deethylation proceed at similar rates. The rate of formation of 2b decreases with increasing DEET concentration; 2b is therefore a secondary metabolite of DEET and DEET acts as a competitive inhibitor of the metabolism of 1b and 2a. 3. Except for the primary amides, where N-dealkylation is impossible, metabolism of all subsequent compounds, 1b,c, 2a-c, 3a-c and 4a,b, involves an N-deethylation (NEt₂ → NHEt or NHEt → NH₂) competitive with a ring substituent oxidation (CH₃ → CH₂OH, CH₂OH → CHO or CHO → CO₂H). Surprisingly, the aldehydes 3a-c are also reduced to the corresponding alcohols 2a-c (CHO → CH₂OH); CO inhibits the oxidative metabolism of 3a-c, but reduction to 2a-c continues uninhibited. 4. The outcomes of this work are that (1) previously unreported aldehydes 3b and 3c form part of the DEET network of metabolites, (2) the reduction of the aldehydes 3a-c has the potential to inhibit the formation of the more highly oxidized DEET metabolites, (3) amide hydrolysis was not observed for any substrate and (4) no evidence was obtained for N-(1-hydroxyethyl)amide intermediates.     

In-vitro permeation of the insect repellent N,N-diethyl-m-toluamide (DEET) and the sunscreen oxybenzone

The permeation behaviours of the insect repellent N,N-diethyl-m-toluamide (DEET) and the sunscreen oxybenzone were assessed in a series of in-vitro diffusion studies, using piglet skin and poly (dimethylsiloxane) (PDMS) membrane. The transmembrane permeability of DEET and oxybenzone across piglet skin and PDMS membrane was dependent on dissolving vehicles and test concentrations. An enhanced permeation increase across piglet skin was found for DEET and oxybenzone when both compounds were present in the same medium (DEET: 289% in propylene glycol, 243% in ethanol and 112% in poly(ethylene glycol) (PEG-400); oxybenzone: 139% in PEG-400, 120% in propylene glycol and 112% in ethanol). Permeation enhancement was also observed in PDMS membrane (DEET: 207% in ethanol, 124% in PEG-400 and 107% in propylene glycol; oxybenzone: 254% in PEG-400, 154% in ethanol and 105% in propylene glycol). PDMS membrane was found to be a suitable candidate for in-vitro diffusion evaluations. This study shows that the permeations of the insect repellent DEET and the sunscreen oxybenzone were synergistically enhanced when they were applied simultaneously.     

Microsomal metabolism of N,N-diethyl-m-toluamide (DEET, DET): the extended network of metabolites.

1. The aim was to set out to establish the complete network of metabolites arising from the phenobarbital-treated rat liver microsomal oxidation of N,N-diethyl-m-toluamide (DEET). The products formed from DEET and all its subsequent metabolites were identified by HPLC retention times, UV spectroscopy, mass spectrometry and by comparison with authentic standards. 2. DEET (1a) produces three major metabolites, N-ethyl-m-toluamide (1b), N,N-diethyl-m-(hydroxymethyl)benzamide (2a) and N-ethyl-m-(hydroxymethyl)benzamide (2b), and, at low substrate concentrations or extended reaction times, two minor metabolites, toluamide (1c) and N,N-diethyl-m-formylbenzamide (3a). 1b and 2a are primary metabolites and their formation follows Michaelis-Menten-type kinetics. At low DEET concentrations, ring methyl group oxidation is favoured; at saturation concentrations, methyl group oxidation and N-deethylation proceed at similar rates. The rate of formation of 2b decreases with increasing DEET concentration; 2b is therefore a secondary metabolite of DEET and DEET acts as a competitive inhibitor of the metabolism of 1b and 2a. 3. Except for the primary amides, where N-dealkylation is impossible, metabolism of all subsequent compounds, 1b,c, 2a-c, 3a-c and 4a,b, involves an N-deethylation (NEt2 --> NHEt or NHEt --> NH2) competitive with a ring substituent oxidation (CH3 --> CH2OH, CH2OH --> CHO or CHO --> CO2H). Surprisingly, the aldehydes 3a-c are also reduced to the corresponding alcohols 2a-c (CHO --> CH2OH); CO inhibits the oxidative metabolism of 3a-c, but reduction to 2a-c continues uninhibited. 4. The outcomes of this work are that (1) previously unreported aldehydes 3b and 3c form part of the DEET network of metabolites, (2) the reduction of the aldehydes 3a-c has the potential to inhibit the formation of the more highly oxidized DEET metabolites, (3) amide hydrolysis was not observed for any substrate and (4) no evidence was obtained for N-(1-hydroxyethyl)amide intermediates.     

Acute manic psychosis following the dermal application of N,N-diethyl-m-toluamide (DEET) in an adult

Extensive animal testing and 30 years of human experience have established the general safety of DEET when applied episodically to skin or bedclothes. Local and systemic toxic and allergic reactions to DEET have been observed in man. Three weeks prior to admission, for the purpose of self-medication, a 30 year old man began daily applications of the insect repellant, DEET followed by a 1-2 hour period in a light-bulb heated box. Sedation and incoherence were noted for short periods following each application session. Aggressiveness and psychotic ideation led to hospital admission where he displayed psychomotor hyperactivity, rapid and pressured speech, tangentiality, flight of ideas, and grandiose delusions. Treatment was begun with haloperidol. Clinical improvement was complete within 6 days, atypical for classic endogenous mania. Drug and metabolites were identified in the urine more than 2 weeks after the last drug application.     

Dermal absorption of the insect repellent DEET (N,N-diethyl-m-toluamide) in rats and monkeys: effect of anatomical site and multiple exposure

The dermal absorption of 14C-ring-labeled DEET (N,N-diethyl-m-toluamide) applied in acetone to the skin of Sprague-Dawley rats and rhesus monkeys for 24 h was determined. Absorption in rats dosed middorsally was 36 +/- 8% with a urinary excretion half-life (t1/2) of 20 h. Both the extent and rate of absorption in monkeys were highly dependent on anatomic site, with 14 +/- 5% (t1/2 = 4 h) penetrating the forearm, 33 +/- 11% (t1/2 = 6 h) the forehead, 27 +/- 3% (t1/2 = 7 h) the dorsal forepaw, and 68 +/- 9% (t1/2 = 8 h) the ventral forepaw. Since DEET is commonly applied frequently by the same individual, the effect of multiple exposure was investigated. No significant difference (p greater than or equal to .3) was obtained either between the total percentage absorbed dermally with single (36 +/- 8%; t1/2 = 20 h) as compared with three (31 +/- 5%; t1/2 = 16 h) DEET applications at 2-h intervals to rats, or between single (14 +/- 5%; t1/2 = 4 h) as compared with three (12 +/- 1%; t1/2 = 4 h) applications at 0.5-h intervals to monkey forearm. A DEET metabolite detected in urine 4 h following topical exposure in humans was extractable following either acid (HCl) hydrolysis or urine treatment with beta-glucuronidase and was identified as ethyltoluamide (parent ion 163; base ion 119) following HPLC purification and characterization by GC/MS.     

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

Ecotoxicology - 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...     

Cardiorespiratory effects following acute exposure to pyridostigmine bromide and/or N,N-diethyl-m-toluamide (DEET) in rats

The acute lethal interaction that occurs in rodents when high doses of a peripherally restricted cholinesterase inhibitor, pyridostigmine bromide (PB), and the insect repellent N, N-diethyl-m-toluamide (DEET) are combined was first described during studies of chemical mixtures that were targeted as potential causative agents of Gulf War illnesses. This study was intended to provide insight into possible mechanisms of that lethal interaction. Following a single intraperitoneal injection of PB (2 mg/kg) and/or DEET (300 or 500 mg/kg), respiratory activity was measured in conscious freely moving rats using whole-body plethysmography. Cardiovascular function was also monitored simultaneously through an arterial catheter. PB (2 mg/kg) given alone stimulated respiration and increased blood pressure. Arterial pH levels were decreased, whereas pO(2) and pCO(2) remained at control levels. Administration of DEET (300 mg/kg) alone increased tidal volume and decreased blood pressure. Blood gases and pH levels were unaltered. A higher dose of DEET (500 mg/kg) also decreased respiratory and heart rate. Coadministration of PB (2 mg/kg) and DEET (300 mg/kg) increased tidal volume, decreased arterial pH, and elevated pCO(2). Heart rate and blood pressure declined progressively after drug coadministration. Pretreatment with atropine methyl nitrate (AMN), a peripherally selective competitive antagonist at nicotinic and muscarinic receptor sites, reduced the individual effects of PB or DEET, and significantly increased survival after coexposure to these agents. Although changes in respiratory function may have contributed to the lethal interaction, it was concluded that the primary cause of death was circulatory failure.     

Combined exposure to DEET (N,N-diethyl-m-toluamide) and permethrin-induced release of rat brain mitochondrial cytochrome c.

The release of cytochrome c from the mitochondrial intermembrane space can induce apoptosis. The levels of mitochondrial cytochrome c in rat brain following a single dermal dose of 400 mg/kg of DEET, and of 1.3 mg/kg of permethrin, alone or in combination were determined. Rats were sacrificed at a time interval of 0.5, 1, 2, 4, 8, 16, 24, 48, or 72 h after dosing. Brain mitochondria were isolated and the levels of cytochrome c were measured using reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. Average percentage recovery of cytochrome c spiked with control rat brain mitochondria was 83.2 +/- 8.9%. Limits of detection and quantitation were 1 and 5 ng, respectively. The results showed that a single dermal dose of a combination of DEET and permethrin significantly increased the release of brain mitochondrial cytochrome c starting 24 h after treatment. DEET and permethrin alone did not affect the release of cytochrome c. The results indicate that combined exposure to DEET and permethrin might induce the apoptotic processes in rat brain as seen by the release of cytochrome c.     

Absorption and evaporation of N,N-diethyl-m-toluamide from human skin in vitro

The penetration of DEET through split-thickness cadaver skin was measured in non-occluded Franz cells placed either in a fume hood or on a laboratory workbench. DEET, dissolved in a small volume of ethanol and spiked with (14)C radiolabel was applied to skin at doses from 0.02 to 11000 microg/cm(2). DEET penetration was greater for cells placed on the workbench, and the percentage of radioactivity penetrated after 72 h increased gradually with dose, for doses up to 680 microg/cm(2). At higher doses, it declined. Percent penetration ranged from 11.5 +/- 3.2% for a dose of 0.021 microg/cm(2) in the fume hood to 71.9 +/- 5.5% for a dose of 260 microg/cm(2) on the workbench. Results were interpreted in terms of a diffusion/evaporation model having three parameters-a solubility value for the chemical in the upper stratum corneum, M(sat); a mass transfer coefficient for evaporation, k(evap); and a characteristic time for diffusion, h(2)/D. The parameters obtained from fitting the model to the data (normalized to the fume hood environment) were M(sat) = 18 microg/cm(2) and k(evap) = 2.6 x 10(-5) cm/h. The value of h(2)/D decreased from 16 h at a DEET dose of 25 microg/cm(2) to 10 h at 1480 microg/cm(2), consistent with an increase in skin permeability of about 1.5-fold over this dose range. This effect was confirmed by means of an additional study in which skin samples pretreated with increasing amounts of unlabeled DEET were washed and redosed with (14)C-benzyl alcohol. A small (1.7-fold), but significant, increase in benzyl alcohol penetration with increasing amount of DEET was obtained. Thus, DEET enhanced its own skin permeation rate as well as that of another compound, but the effect was modest and not likely to be a major concern for compounds coadministered with DEET.     

Metabolism of N,N-diethyl-m-toluamide (DEET) by liver microsomes from male and female rats. Simultaneous quantitation of DEET and its metabolites by high performance liquid chromatography

A simple and sensitive HPLC procedure was developed to separate and quantitate N,N-diethyl-m-toluamide (DEET) and its in vitro metabolites using a gradient elution, a reversed phase C18 column, and an internal standard. This procedure was applied to examine the metabolism of this insect repellent in liver microsomes from normal (untreated) male and female Wistar rats. At a pH of 8.6 and at a substrate concentration of 200 microM, the microsomal preparations from males degraded DEET faster than did those from females. The half-life of DEET was 10 min and 15 min, respectively. The benzylic methyl hydroxylated metabolite and the N-deethylated metabolite were determined over 2 hr in fortified microsomal suspensions. Rate constants for appearance of the metabolites showed significantly higher values for males than for females. These observations suggests that a sex difference may be present in the metabolism of DEET.     

Simultaneous determination of malathion, permethrin, DEET (N,N-diethyl-m-toluamide), and their metabolites in rat plasma and urine using high performance liquid chromatography

A method was developed for the separation and quantification of the insecticide malathion (O,O-dimethyl-S-(1,2-carbethoxyethyl) phosphorodithioate), its metabolite malaoxon (O,O-dimethyl-S-(1,2-carbethoxyethyl) phosphorothioate), the insecticide permethrin (3-(2,2-dichloro-ethenyl)-2,2-dimethylcyclopropanecarboxylic acid(3-phenoxyphenyl)methylester), two of its metabolites m-phenoxybenzyl alcohol and m-phenoxybenzoic acid, the insect repellent N,N-diethyl-m-toluamide (DEET), and its metabolites m-toluamide and m-toluic acid in rat plasma and urine. The method used high performance liquid chromatography (HPLC) with reversed phase C(18) column, and UV detection at 210 nm. The compounds were separated using gradient of 45--99% acetonitrile in water (pH 3.5) at a flow rate ranging between 0.5 and 2 ml/min in a period of 15 min. The retention times ranged from 7.4 to 12.3 min. The limits of detection ranged between 20 and 100 ng/ml, while limits of quantitation were 50-150 ng/ml. Average percentage recovery of five spiked plasma samples were 80.1+/-4.2, 75.2+/-4.6, 84.5+/-4.0, 84.3+/-3.4, 82.8+/-3.9, 83.9+/-5.5, 82.2+/-6.0, 83.1+/-4.3, and from urine 78.8+/-3.9, 76.4+/-4.9, 82.3+/-4.5, 82.5+/-3.9, 81.4+/-4.0, 83.9+/-4.3, 81.5+/-5.0, and 84.5+/-3.8 for, malathion, malaoxon, DEET, m-toluamide, m-toluic acid, permethrin, m-phenoxybenzyl alcohol, and m-phenoxybenzoic acid, respectively. The method was reproducible and linear over range between 100 and 1000 ng/ml. This method was applied to analyze the above chemicals and metabolites following combined dermal administration in rats.     

Neurotoxicity Evaluation of N,N-Diethyl-m-toluamide (DEET) in Rats

The neurotoxic potential of N,N-diethyl-m-toluamide (DEET) wasevaluated following acute oral administration or following multigenerationplus chronic dietary administration to the rat. For the acutestudy, rats were administered undiluted DEET at dose levelsof 50, 200, or 500 mg/kg by gavage. A dose level of 500 mg/kgwas considered to be the highest practical dose that could beevaluated in this study based upon observations of overt toxicityat 500 mg/kg and mortality at 1000 mg/ kg in a dose range-findingstudy. The two measures of neurotoxicity evaluated in the acutestudy were functional observational battery (FOB) and motoractivity measurements. An apparent treatment-related effectin thermal response time (increased) was noted for both sexes1 hr after dosing at the 500 mg/kg dose level. A questionableeffect on rearing activity (decreased) also was noted at thesame dose level. For the multigeneration plus chronic dietaryadministration study, rats were administered DEET at dietaryconcentrations of 0, 500, 2000, or 5000 ppm continuously overtwo generations and then chronically for 9 months. A dietaryconcentration of 5000 ppm meets the criteria for a maximum tolerateddose (MTD) based on traditional chronic toxicology assessments.Evaluations included FOB, motor activity, discriminative acquisitionand reversal in an Mmaze, acoustic startle habituation, passiveavoidance acquisition and retention, and microscopic examinationof central and peripheral nervous tissue. The only effect thatwas considered to be possibly treatment-related was a slightincrease in exploratory locomotor activity at the 5000 ppm doselevel. Based on the results of these studies, the nervous systemdoes not appear to be a selective target when DEET is administeredto rats either as a single oral dose at high dose levels orchronically at the MTD.     

DEET (N,N-diethyl-m-toluamide) alone and in combination with permethrin increased urinary excretion of 6beta-hydroxycortisol in rats, a marker of hepatic CYP3A induction.

In this study, the ratio of 6beta-hydroxycortisol (6beta-OHF) to free cortisol (F) was determined in urine following a single dermal dose of 400 mg/kg of DEET (N,N-diethyl-m-toluamide), and 1.3 mg/kg of permethrin, alone and in combination, in rats. Urine samples were collected at 2, 4, 8, 16, 24, 48, and 72 h after application. Recoveries of 6beta-OHF and cortisol (F) from control urine samples were between 75 and 85%, with limits of detection at 30 and 10 ng/ml for cortisol and 6beta-OHF, respectively. A single dermal dose of DEET alone and in combination with permethrin significantly increased urinary excretion of 6beta-hydroxycortisol 24 h after dosing. Permethrin did not significantly alter the urinary excretion of 6beta-hydroxycortisol. These results indicate that DEET, alone and in combination with permethrin, increased urinary excretion of 6beta-OHF in rats following a single dermal dose application.     

Induction of urinary excretion of 3-nitrotyrosine, a marker of oxidative stress, following administration of pyridostigmine bromide, DEET (N,N-diethyl-m-toluamide) and permethrin, alone and in combination in rats

In this study, we determined levels of 3-nitrotyrosine in rat urine following administration of a single oral dose of 13 mg/kg pyridostigmine bromide (PB) (3-dimethylaminocarbonyloxy-N-methylpyridinum bromide), a single dermal dose of 400 mg/kg N,N-diethyl-m-toluamide (DEET) and a single dermal dose of 1.3 mg/kg permethrin, alone and in combination. Urine samples were collected from five treated and five control rats at 4, 8, 16, 24, 48, and 72 h following dosing. Solid-phase extraction coupled with high-performance liquid chromatography with ultraviolet detection at 274 nm was used for the determination of tyrosine and 3-nitrotyrosine. A single oral dose of PB and a single dermal dose of DEET or their combination significantly (P<0.05) increased levels of 3-nitrotyrosine starting 24 h after dosing compared with control urine samples. The maximum increase of 3-nitroytyrosine was detected 48 h after combined administration of PB and DEET. The ratio of 3-nitrotyrosine to tyrosine in urine excreted 48 h after dosing was 0.19+/-0.04, 0.20+/-0.05, 0.28+/-0.03, 0.32+/-0.04, 0.19+/-0.05, 0.42+/-0.04, 0.27+/-0.03, 0.36+/-0.04, and 0.48+/-0.04 following administration of water, ethanol, PB, DEET, permethrin, PB+DEET, PB+permethrin, DEET+permethrin, and PB+DEET+permethrin, respectively. The results indicate that an oral dose of PB and a dermal administration of DEET, alone and in combination, could generate free radical species, and thus increase levels of 3-nitrotyrosine in rat urine. Induction of 3-nitrotyrosine, a marker of oxidative stress, following exposure to these compounds could be significant in understanding the proposed enhanced toxicity following combined exposure to these compounds.