The role of octopamine receptor agonists in the synergistic toxicity of certain insect growth regulators (IGRs) in controlling Dengue vector Aedes aegypti (Diptera: Culicidae) mosquito.

The synergistic action of octopamine receptor agonists (OR agonists) on many insecticide classes (e.g., organophosphorus, pyrethroids, and neonicotinoids) on Aedes aegypti L. has been reported recently. An investigation of OR agonist's effect on insect growth regulators (IGRs) was undertaken to provide a better understanding of the mechanism of action. Based on the IGR bioassay, pyriproxyfen was the most potent IGR insecticide tested (EC50=0.0019ng/ml). However, the lethal toxicity results indicate that diafenthiuron was the most potent insecticide (LC50=56ng/cm(2)) on A. aegypti adults after 24h of exposure. The same trend was true after 48 and 72h of exposure. Further, the synergistic effects of OR agonists plus amitraz (AMZ) or chlordimeform (CDM) was significant on adults. Among the tested synergists, AMZ increased the potency of the selected IGRs on adults the greatest. As results, OR agonists were largely synergistic with the selected IGRs. OR agonists enhanced the lethal toxicity of IGRs, which is a valuable new tool in the field of A. aegypti control. However, further field experiments need to be done to understand the unique potential role of OR agonists and their synergistic action on IGRs.

Genotoxicity of 4-chloro-o-toluidine in Salmonella typhimurium, human lymphocytes and V79 cells.

In the absence of a metabolizing system (S9 mix) 4-chloro-o-toluidine (4-COT) was found to be ineffective in a combination of assays for gene mutations in Salmonella typhimurium, for chromosome aberrations and sister chromatide exchanges in human lymphocytes, and for the induction of spindle disturbances in V79 Chinese hamster cells. In the presence of S9, 4-COT was also ineffective in producing structural or numerical changes in mammalian cells, but the yields of 4-COT induced revertants in S. typhimurium strains TA 100 and TA 98 were about 2-fold higher than those in controls.

Mutagenicity testing of nine herbicides and pesticides currently used in agriculture.

Nine herbicides and pesticides were tested for their mutagenicity using the Drosophila sex-linked recessive lethal mutation assay. These are Ambush, Treflan, Blazer, Roundup, 2,4-D Amine, Crossbow, Galecron, Pramitol, and Pondmaster. All of these are in wide use at present. Unlike adult feeding and injection assays, the larvae were allowed to grow in medium with the test chemical, thereby providing long and chronic exposure to the sensitive and dividing diploid cells, i.e., mitotically active spermatogonia and sensitive spermatocytes. All chemicals induced significant numbers of mutations in at least one of the cell types tested. Some of these compounds were found to be negative in earlier studies. An explanation for the difference in results is provided. It is probable that different germ cell stages and treatment regimens are suitable for different types of chemicals. larval treatment may still be valuable and can complement adult treatment in environmental mutagen testing.

Caveats regarding the use of the laboratory rat as a model for acute toxicological studies: modulation of the toxic response via physiological and behavioral mechanisms.

The rodent, specifically the laboratory rat, is the primary experimental animal used in toxicology testing. Despite its popularity, recent studies from our laboratory and others raise a number of questions concerning the rat's appropriateness as an animal model for toxicological studies. While there may be additional areas in which the rat and other small rodents fail to adequately mimic the human response to xenobiotic agents, this article will focus on the area of temperature regulation. Thus, this article will review the thermoregulatory response of the laboratory rat following acute exposure to toxic agents and examine the impact of this response on the extrapolation of toxicological data from experimental animals to humans. In general, the rat responds to acute intoxication by lowering its core temperature via both physiological and behavioral mechanisms, thereby attenuating the induced toxicity. Similar responses have not been reported in humans.

Joint action between binary mixtures of chlordimeform and insecticides.

Chlordimeform (CDF) was tested for its ability to act jointly with an organophosphorus insecticide (parathion) and a series of carbamate insecticides. Three arthropod species were used as test subjects: the two-spotted spider mite, Tetranychus urticae; the German cockroach, Blatella germanica; and the flour beetle, Tenebrio castaneum. However, CDF antagonized the toxicity of parathion toward B. germanica while it acted in a greater than additive fashion toward T. castaneum. No combination of CDF and insecticide tested acted jointly toward T. urticae. Species-specific differences in sensitivity, absorption, metabolism, and mode of delivery account for the varying results. Metabolic studies of pairs of compounds using two radiolabeled carbamates showed that CDF altered the metabolic detoxification of both carbaryl and aldicarb. The results suggest that CDF may inhibit MFOs as its mode of action.

The formamidine pesticides chlordimeform and amitraz decrease hepatic glutathione in mice through an interaction with alpha 2-adrenoceptors.

Recent studies have provided evidence that formamidine pesticides, such as chlordimeform (CDM; N'-4-chloro-o-tolyl-N,N-dimethylformamidine) or amitraz (AMZ; N'-2-4-(dimethylphenyl)-N-[((2,4-dimethylphenyl)imino)methyl]-N- methanimidamide) exert some of their toxic effects by an interaction with alpha 2-adrenoceptors. Since epinephrine and clonidine have been shown to decrease hepatic glutathione (GSH) by activating alpha 2-adrenoceptors, and alpha 2-antagonists partially antagonize GSH depletion and hepatotoxicity caused by bromobenzene and cocaine, we have investigated whether the formamidines would affect hepatic GSH levels in mice. Both CDM and AMZ decreased hepatic nonprotein sulfydryls (NPSH) to a maximum of about 40%, in a dose-dependent manner. The effect of AMZ was longer lasting than that of CDM. For both compounds, decrease of hepatic NPSH was antagonized by the alpha 2-antagonist yohimbine but not by the alpha 1-antagonist prazosin or the beta-antagonist propanolol. The alpha 2-agonist clonidine also caused a dose-dependent decrease of hepatic NPSH (to a maximum of 40%), which was prevented only by yohimbine. The effects of AMZ, CDM, and clonidine were not additive, suggesting that all compounds act on a common site and/or with a common mechanism. Adrenalectomy or destruction of peripheral sympathetic nerves with 6-hydroxydopamine did not alter the ability of CDM and AMZ to decrease hepatic NPSH. These results indicate that formamidine pesticides can affect the levels of hepatic GSH, possibly through a direct interaction with hepatic alpha 2-adrenoceptors.

Effects of chlordimeform and its metabolite 4-chloro-o-toluidine on rat splenic T, B and tumoricidal effector cells.

The pesticide chlordimeform (CDF) and its metabolite 4-chloro-o-toluidine (4CT) are documented animal carcinogens. Various immunological and toxicological parameters were examined following CDF or 4CT exposure in Sprague-Dawley rats: spleen wt./body wt. ratio; spleen cells/mg; splenocyte viability; T and B cell mitogenesis; natural killer (NK) and natural cytotoxic (NC) cell activity. In this study CDF produced a dose-dependent inhibition of NK activity (E:T ratio 100:1). Significant decreases in NK activity also occurred at all CDF doses, while the spleen wt./body wt. ratio was reduced only by the highest CDF dose. The compound 4CT produced no significant effects on NK or NC activity. No changes were observed in spleen cells/mg, splenocyte viability, or in T and B cell proliferation mediated by concanavalin A (Con-A) and lipopolysaccharide (LPS), respectively, with either CDF or 4CT treatment. These results have demonstrated that CDF exposure has a selective effect on splenic functionally distinct tumoricidal effector cell populations, and that this effect is evident at 1 mg/kg, a dose not inconsistent with the maximum exposure levels in workers.

Formamidine pesticides and alpha 2-adrenoceptors: studies with amitraz and chlordimeform in rats and development of a radioreceptor binding assay.

The interaction of the formamidine pesticides chlordimeform (CDM) and amitraz (AMZ) with rat brain alpha2-adrenoceptors was investigated. Both compounds inhibited the binding of 3H-clonidine and 3H-yohimbine in vitro with IC50 values of 62-68 microM (CDM) and 95-110 nM (AMZ). In vivo administration of AMZ and CDM caused a dose-dependent inhibition of 3H-clonidine binding in rat forebrain. The inhibition was short-lasting (24 hr) following CDM administration, while after AMZ recovery of 3H-clonidine binding occurred only after 72 hr. Good correlations were found between inhibition of brain 3H-clonidine binding by the formamidines and "plasma equivalents" of these compounds and/or their biologically active metabolites, as measured by a new radioreceptor assay. These results suggest that 1) formamidines can interact in vivo with brain alpha 2-adrenoceptors when administered at doses previously shown to cause toxic effects on the central nervous system: and 2) this effect is reversible, both in vivo and in vitro, and appears to be linked to the presence of the formamidines and/or their active metabolites at the receptor sites.

Modulating effect of body temperature on the toxic response produced by the pesticide chlordimeform in rats.

Previous studies from this laboratory have demonstrated significant deficits in cardiovascular function in rats exposed to the pesticide chlordimeform (CDM) when body core temperature (TCO) was maintained at 37 degrees C. To investigate the role of TCO on CDM toxicity, similar experiments were conducted over a range of TCO values. Adult rats (n = 30) were anesthetized with sodium pentobarbital (35 mg/kg) and randomly assigned to one of six equal groups. Groups were paired and TCO was maintained in the rats in each of the respective group pairs at one of three levels (37, 35, or 33 degrees C). Rats in one group at each temperature level (groups T37, T35, and T33) were injected intraperitoneally with 60 mg/kg of CDM. Animals in the corresponding temperature-matched groups (groups C37, C35, and C33, respectively) received volume-matched injections of normal saline vehicle and served as time-paired controls. The electrocardiogram and heart rate (HR) were monitored throughout the experimental procedure. There was a significant decrease in HR in all CDM-treated groups when compared to the control group animals. The magnitude of the observed cardiac effect was attenuated in the T35 group when compared to that of the other treated groups. Similarly, lethality rates (number of deaths/total) for the T37, T35, and T33 groups were 2/5, 0/5, and 3/5, respectively; there were no deaths among the control-group animals. From these and previous data from this laboratory, we conclude there may be a beneficial effect of moderate hypothermia in rats exposed to toxic agents while more severe hypothermia appears to offer no advantage and may actually exacerbate the toxic effect.

Comparison of chlordimeform and carbaryl using a functional observational battery.

The effects of the formamidine pesticide chlordimeform (CDM), and the carbamate carbaryl (CAR) were compared using a functional observational battery (FOB). The FOB, a series of observations and measurements that can be rapidly administered to toxicant-treated rats, includes home-cage and open-field observations, neuromuscular and sensorimotor tests, and physiological measures. Evaluations were made according to U.S. Environmental Protection Agency testing guidelines so as to determine dose-, time-, and sex-related toxicant effects. Long-Evans hooded rats of both sexes were tested initially and then dosed ip with either vehicle, CDM (1, 25, 56 mg/kg) or CAR (3, 10, 30 mg/kg), and tested at various times after dosing (for CDM 1, 5, 24 hr; for CAR 0.5, 3, 24, 48 hr). Both compounds affected general activity (home-cage and open-field), equilibrium, CNS excitability, and sensory responsiveness. Whereas similar decreases were obtained on rearing, gait, and arousal, there were important qualitative differences in the effects of CAR and CDM on reactions to handling and the reflex tests in that CDM increased excitability and enhanced responses to several stimuli but CAR either had no effect or decreased these measures. Only CDM produced an increase in muscle tone as measured by grip strength, and only CAR produced cholinergic autonomic signs of intoxication. Body weight and temperature were decreased by both compounds. Thus, the profiles of effect produced by these two pesticides could be clearly differentiated using the FOB.

Alpha 2-adrenoceptors as a target for formamidine pesticides: in vitro and in vivo studies in mice.

While the toxicity in insects of formamidines such as chlordimeform (CDM), its demethylated metabolite DCDM, and amitraz (AMZ) appears to involve activation of an octopamine-sensitive adenylate cyclase, their mechanism of action in mammals remains elusive. There is increasing evidence, however, that alpha 2-adrenoceptors might mediate certain effects of CDM, DCDM, and AMZ. In the present study, we investigated whether formamidines can interact directly with adrenoceptors in mouse forebrain both in vitro and after in vivo administration. Formamidines were potent inhibitors of the binding of [3H]clonidine to alpha 2-adrenoceptors with IC50's of 13 microM, 29 nM, and 130 nM for CDM, DCDM, and AMZ, respectively. Binding of [3H]yohimbine was inhibited with similar potencies. All compounds also inhibited with equal (CDM) or lower potency the binding of [3H]spiperone to dopamine D2 receptors and were weak inhibitors or inactive toward alpha 1- and beta-adrenoceptors, cholinergic muscarinic, GABAA, opiate mu, benzodiazepine, and histamine 1 receptors. Administration of formamidines to mice caused a dose-dependent decrease of [3H]clonidine binding. [3H]Clonidine binding returned to control values within 5 hr following administration of CDM and DCDM, but was still significantly decreased up to 48 hr after AMZ. Among different brain regions, [3H]clonidine binding was decreased to a larger extent in cerebral cortex, hippocampus, and midbrain. In vitro and ex vivo kinetic binding studies indicated that the effect of formamidines on alpha 2-adrenoceptors was due to a decrease in affinity and not to an alteration of the density of [3H]clonidine binding sites. The results of these biochemical studies support the hypothesis that alpha 2-adrenoceptors represent an important target for formamidine neurotoxicity in mammals.

An alpha 2-adrenergic mode of action of chlordimeform on rat visual function.

The hypothesis that chlordimeform increased the amplitude of components N1P1 and P1N3 in rat pattern-reversal visual evoked potentials through actions on alpha 2-adrenergic receptors was tested with two sets of experiments. First, rats received single injections of either vehicle, an alpha 2-adrenergic antagonist yohimbine (0.1, 0.5, or 2.0 mg/kg), or an alpha 2-adrenergic agonist clonidine (0.05, 0.1, or 0.5 mg/kg). Yohimbine alone had no effect on pattern-reversal evoked potential amplitude. Clonidine treatment produced a dosage related increase in amplitude of both components similar to that produced by chlordimeform (W.K. Boyes and R.S. Dyer, 1984, Brain Res. Bull., 10, 817-823). Second, rats were given double injections of either vehicle or yohimbine (0.05, 0.5, 2.0, or 5.0 mg/kg) followed by either vehicle, clonidine (0.1 or 0.2 mg/kg) or chlordimeform (10, 20, or 40 mg/kg). Yohimbine pretreatment attenuated the effects of subsequent treatment with either clonidine or chlordimeform. These results support the hypothesis that chlordimeform alters rat pattern-reversal evoked potentials through actions as a central nervous system alpha 2-adrenergic agonist.

Investigations of amitraz neurotoxicity in rats. I. Effects on operant performance.

Amitraz (AMZ) is a formamidine pesticide which is often compared to chlordimeform (CDM). The effects of AMZ (6.25-75 mg/kg) and CDM (5-20 mg/kg) on the schedule-controlled performance of rats were examined using a multiple fixed-ratio (FR) 10 fixed-interval (FI) 300-sec schedule of milk reinforcement. Following dose-effect determinations, rats received three daily doses of AMZ (50 mg/kg). Low to intermediate doses of AMZ (6.25-25 mg/kg), administered 20 min presession, significantly decreased FI responding but not FR responding. In contrast, CDM appeared to decrease responding similarly under both components. Both compounds disrupted the temporal pattern of responding within the interval; AMZ had only a moderate influence at all effective doses but CDM produced a marked dose-dependent disruption of temporal response patterning. The effects of the high doses of AMZ (50-75 mg/kg) were more pronounced 24 hr after dosing, whereas the rats had recovered from lower doses at this time. Performance was progressively disrupted and the rats' health rapidly deteriorated during the course of three daily injections of AMZ (50 mg/kg). Operant performance recovered more quickly than did the general health of the rats. Thus AMZ produced effects of multiple-schedule performance that were distinct from the effects of CDM. Moreover, the signs of intoxication and the effects on schedule-controlled behavior following a high dose (50 mg/kg) were augmented and persistent with short-term repeated administration.

Investigations of amitraz neurotoxicity in rats. II. Effects on visual evoked potentials.

As a part of a series of studies investigating the possible neurotoxicity of amitraz (AMZ), a formamidine pesticide, visual evoked potentials were recorded from Long-Evans rats following acute and short-term repeated exposures to AMZ. The first of three experiments examined the relationship between a single ip injection of AMZ (0, 50, and 100 mg/kg) and the latency and peak-to-peak amplitude of pattern-reversal (PREP) and flash-evoked potentials (FEP). The effects of another formamidine, chlordimeform (CDM; 40 mg/kg), were also studied for comparison purposes. Two hours after treatment, AMZ exposure produced large, dose-related increases in PREP amplitudes. Exposure to CDM produced similar changes. Neither compound changed FEP amplitudes. Body temperatures were reduced and evoked potential peak latencies were increased by both compounds. The latency increases were probably a secondary consequence of hypothermia. In the second experiment, PREPs were recorded before and 2, 24, 48, and 72 hr after treatment with AMZ (100 mg/kg). The time course of changes was biphasic in nature, with increases in amplitudes (N1P1, P1N2, and N2P3) 2 hr after treatment followed by subsequent depression in amplitude (P2N3) at 48 hr. Recovery occurred by 72 hr after treatment. The third experiment examined the effects of three daily treatments with either vehicle or 50 or 100 mg/kg AMZ. Body weights and body temperatures showed dose-related reductions which progressed with each additional treatment and recovered partially by 6 days after cessation of treatment. The PREPs of AMZ-treated rats agains showed biphasic changes, with N1P1 and P1N2 amplitudes significantly increased on each day of treatment and 1-2 days following the third treatment. Amplitude P2N3 showed an initial increase on the first 2 days of treatment, followed by subsequent, progressive amplitude reductions. In summary, AMZ produced two phases of change in visual evoked potentials. The first phase was characterized by large increases in PREP amplitudes without increasing FEP amplitudes in the same rats. The second phase was characterized by suppression of PREP P2N3 amplitude. Short-term repeated exposure produced signs of accumulating intoxication including progressive loss of body weight, lowered body temperature, and prolonged duration of evoked potential changes.

Interaction of the pesticide chlordimeform with adrenergic receptors in mouse brain: an in vitro study.

Chlordimeform (N'(4-chloro-o-tolyl)-N, N-dimethylformamidine; CDM) is a formamidine insecticide acaricide whose major active metabolite is its N-monomethyl analog, desmethylchlordimeform, (DCDM). While their pesticidal action in invertebrates appears to be related to activation of octopamine receptors, their mechanism of action in mammals has not been established. Because of similarities between octopamine and adrenergic receptors and suggestions of CDM and DCDM action on adrenoceptors, the in vitro interactions of CDM and DCDM with adrenoceptors were studied. In mouse brain membrane preparations CDM inhibited the binding of [3H]-clonidine to alpha 2- adrenoceptors and of [3H]-WB4101 to alpha 1-adrenoceptors with IC50 values of 18.2 and 87 microM, respectively. DCDM was a much more potent inhibitor, with IC50 values toward alpha 2-, and alpha 1-adrenoceptors of 44 nM and 1 microM, respectively. Both compounds were only weak inhibitors of the binding of [3H]-dihydroalprenolol to beta-adrenoceptors and of [3H]-quinuclidinyl benzilate to muscarinic receptors and were inactive toward benzodiazepines and gamma aminobutyric acid (GABAA) receptors. Inhibition of [3H]-clonidine binding by both compounds was competitive, as indicated by a decreased receptor affinity without changes in receptor density. Interaction of CDM and DCDM with [3H]-WB4101 binding, on the other hand, was more complex, and not of the competitive type. These results show that CDM and its metabolite DCDM can interact directly in vitro with alpha-adrenergic receptors, suggesting that these receptors could mediate some of the effects of CDM and DCDM in vivo.

Acute and sub-acute effects of deltamethrin and chlordimeform on schedule-controlled responding in the mouse.

The effects of deltamethrin (0.03-3 mg/kg) and chlordimeform (0.3-56 mg/kg) were examined on schedule-controlled responding in the mouse. The response (interruption of a photocell beam) was maintained under a fixed-interval (FI) 60-sec schedule of milk delivery. Acute doses of deltamethrin larger than 0.1 mg/kg decreased responding in a dose-related manner. The ED50 was approximately 1 mg/kg; 3 mg/kg abolished responding. Repeated administration of 3 mg/kg (once daily for 10 days) did not result in a change in the rate-decreasing effects of that dose; however, daily (pre-injection) control response rates decreased about 50%. When dosing was discontinued, rates of responding failed to completely return to pre-drug control levels. Acute doses of chlordimeform larger than 10 mg/kg decreased responding in all mice, but in mice not previously receiving drugs lower doses had a greater rate-decreasing effect. The ED50 was between 10-30 mg/kg; 56 mg/kg abolished responding. Repeated administration of 3 and 10 mg/kg had little effect. Repeated administration of 30 and 56 mg/kg abolished daily (pre-injection) control performances completely after 5-6 days. Responding gradually returned to control levels after dosing was discontinued. The behavioral toxicity of both chlordimeform and deltamethrin was augmented by repeated administration.

Effects of chlordimeform on cardiovascular functional parameters: Part 2. Acute and delayed effects following intravenous administration in the postweanling rat.

The effects of intravenous (iv) administration of chlordimeform (CDM) were investigated in 22- to 30-d-old pentobarbital-anesthetized Sprague-Dawley rats. Animals (n = 25) were given sequential iv injections of 5, 10, 30, 60, and 120 mg CDM/kg, or normal saline vehicle followed by a single injection of 60 mg CDM/kg. Heart rate (HR), arterial blood pressure (BP), and electrocardiogram (ECG) were monitored for all animals, while body temperature was maintained at control levels (37 degrees C). CDM produced profound acute effects on all cardiovascular parameters monitored and persistent delayed effects on HR and BP. These effects were similar to but less severe than those observed in a previous study using geriatric animals. Younger animals also appeared to be more resistant to the lethal actions of CDM.

Effects of chlordimeform on cardiovascular functional parameters: Part 3. Comparison of different routes of administration in the postweanling rat.

The effects of intraperitoneal (ip) administration of chlordimeform (CDM) were investigated in 22- to 24-d-old pentobarbital-anesthetized Sprague-Dawley rats. Animals (n = 32) received a single ip injection of CDM at either 10, 30, or 60 mg/kg, or of normal saline vehicle. Heart rate (HR) and electrocardiogram (ECG) were monitored for all animals, while body temperature was maintained at control levels (37 degrees C). HR decreased 21-29% in the treated animals, and there were no differences observed across dose groups or over time. ECG effects were confined to rate-related changes only. The decrease in HR produced by CDM in these animals was comparable to the delayed HR effect seen in previous studies from this laboratory, despite major differences in dosing regimens and routes of administration.

Induction of hepatic drug-metabolising enzymes following treatment of rats and mice with chlordimeform.

We investigated the effects of the formamidine insecticide chlordimeform upon the activities of various hepatic drug metabolising enzymes in rats and mice. Chlordimeform treatment induced several enzyme activities. However, the extent of induction depended upon the activity studied, the sex of the animal and the species selected. Microsomal cytochrome P-450 content was elevated in both male and female rats and mice. Ethoxycoumarin O-deethylase activity was induced in male and female rats but not in mice, whilst ethylmorphine N-demethylase activity was elevated in mice, but not in rats. Benzo(a)pyrene hydroxylase activity was increased in female rats and mice, but not in males. UDP-glucuronyl transferase, glutathione S-transferase and microsomal epoxide hydrolase were induced in a dose-dependent manner in male rats, and female rats and mice, but not in male mice.