Age-dependent variations in the response of several species of diptera to insecticidal chemicals

The toxicity of carbaryl to three species of fleshflies Sarcophaga bullata Parker, S. crassipalpis Macquart, and S. argyrostoma (Robineau-Desvoidy) varied considerably with age and sex. In contrast, the susceptibility of a blowfly, Phormia regina (Meigen) to carbaryl decreased with age and that of two muscid flies, Musca autumnalis DeGeer and Stomoxys calcitrans (L.), remained relatively constant. The synergistic activity of piperonyl butoxide varied inversely with the innate toxicity of carbaryl to each species suggesting that the observed age- and sex-dependent variations in carbaryl toxicity result mainly from differences in detoxifying capability. This was supported by in vitro measurements of oxidative microsomal metabolism. It was further established that differences in the rates of penetration and excretion of carbaryl and in the susceptibility of the head cholinesterase to carbaryl inhibition were of little importance in determining the susceptibility of the flies to this insecticide.     

In vitro metabolism of insecticides during midgut penetration

A study of the metabolism of ¹⁴C-labeled dieldrin, DDT, malathion, and carbaryl during penetration of the isolated midgut of two insects (Blaberus discoidalis and Manduca sexta) and a section of the intestine of a mammal (Mus musculus) is reported. There was appreciable metabolism of malathion during penetration, including differences in the activation reaction to malaoxon, between insects and mammals. Metabolism was relatively slow during penetration of carbaryl and the chlorinated hydrocarbon insecticides, and little difference in metabolic patterns was noted among the three species. The penetration studies were supported by experiments in which insecticides were incubated with intact and homogenized midgut preparations.     

Toxicity and metabolism of carbaryl in the European corn borer

Larvae from two strains of the European corn borer, Ostrinia nubilalis (Hübner), were compared for differences in their tolerance and metabolism of carbaryl (1-naphthyl N-methylcarbamate). The Geneva strain was about twice as susceptible to carbaryl, but both Valley and Geneva borers converted carbaryl to oxidative metabolites at similar rates in vivo and in vitro. Maximum carbaryl-metabolizing activity was present in last-instar larvae, particularly in the fat body and gut tissues. However, the specific activity of gut homogenates was highest in the Geneva strain and the specific activity of fat body was highest in the Valley strain. Other differences in the mixed-function oxidase systems of gut and fat body were also found. The major metabolite in vivo and in vitro was hydroxymethyl carbaryl.     

Comparative toxicity,absorption, and metabolism of chlorpyrifos and its dimethyl homologue in methyl parathion-resistant and -susceptible tobacco budworms

Chlorpyrifos (Dowco 179) and its dimethyl homologue, chlorpyrifosmethyl (Dowco 214), were used to study the influence of the O,O-dialkyl group of organophosphorus insecticides on toxicity, absorption, and metabolism among larvae of the tobacco budworm [Heliothis virescens (F.)] from strains that were resistant (R) and susceptible (S) to methyl parathion. In toxicity tests, chlorpyrifos and chlorpyrifosmethyl were more toxic than methyl parathion to 3rd-stage R larvae but less toxic to S larvae. Chlorpyrifosmethyl was more toxic (3–4 ×) than chlorpyrifos to both strains of larvae, and the results of absorption studies indicated that the toxicity differential of the homologues may be explained in part by the more rapid absorption of the dimethyl form. Studies of the in vivo metabolism of both Dowco compounds indicated that each was degraded mainly by the cleavage of the pyridylphosphate linkage. In vitro tests demonstrated that the NADPH-dependent microsomal oxidases were of primary importance in detoxification, while glutathione (GSH)-dependent mechanisms (aryl- and alkyltransferases) present in the soluble cell fractions were of lesser importance. O-dealkylation occurred only with chlorpyrifosmethyl. The R larvae demonstrated greater capability in detoxifying both compounds in the comparative in vivo and in vitro studies of metabolism, but the differences were more apparent during the 5th instar than during the 3rd instar.     

Affinities of parathion,DDT, dieldrin,and carbaryl for macromolecules in the blood of the rat and American cockroach and the competitive interaction of steroids

This investigation concerns the binding of DDT, dieldrin, parathion, and carbaryl to macromolecules of rat and roach blood. Plasma was filtered on Sephadex G-200, and the first protein peak (FPP) was used to investigate binding characteristics for the insecticides. Binding was demonstrated to be of a nonspecific hydrophobic nature, except for roach FPP and dieldrin in which, in addition to low-affinity binding, sites for high-affinity binding were also found. Competitive binding experiments were run with cholesterol, estrogen, DDT, and dieldrin. No significant competitive interactions were demonstrated.     

Effect of insecticides on the short-circuit current and resistance of isolated frog skin

The effects of aldrin, carbaryl, α- and γ-chlordane, dichlorodiphenyldichloroethane (DDD), dichlorodiphenyltrichloroethane (DDT), dieldrin, endrin, heptachlor, lindane, methoxychlor, and nonachlor on the short-circuit current and resistance of the isolated intact frog skin were studied. The short-circuit current is approximately equivalent to the rate of active transport of sodium, while the resistance indicates the summed ionic permeability of the skin. At a concentration of 2 × 10^?4M, only carbaryl, DDD, dieldrin, and lindane produced significant (P<0.05, paired t test) changes in the short-circuit current. Nonachlor produced a decrease (P=0.12) in the short-circuit current and also increased the resistance (P=0.07). DDD, dieldrin, and carbaryl caused significant increases in short-circuit current while at the same time the resistance was significantly decreased. Lindane increased both the short-circuit current and the resistance. It was concluded that the frog skin probably contains effective permeability barriers that prevent externally applied insecticides from reaching the site of active sodium transport. It appeared likely that most of the insecticides produced the observed effects on the frog skin by altering the sodium permeability of the outer barrier.     

Toxicity in the brain of organophosphate insecticides: Comparison of the toxicities of metabolites with parent compounds using an intracerebral injection method

The toxicity in the brain of several parathion, fenthion, and fensulfothion insecticides and their toxic metabolites was determined by a technique of directly injecting the compounds into the region of the third ventricle of conscious mice, an area rich in cholinesterase activity. The results were compared on a body weight basis to the toxicity of these compounds when given by ip and oral routes. The results show that there is a direct relationship between the relative inhibition of cholinesterase activity in the brain by the organophosphates (e.g., methyl paraoxon, Sumioxon, and some members of the fenthion series) and the toxicity of these compounds in the brain. Methyl paraoxon and Sumioxon were found to be very toxic in the brain, Sumioxon being three to four times less toxic than methyl paraoxon. This is of the same order of effect of these compounds in inhibiting cholinesterases. It is concluded that any selective effects of Sumithion compared with methyl parathion must be due to the greater rate of metabolism of Sumithion to less toxic metabolites as well as to the lower toxicity of the oxon metabolite and not due to the relative rates of penetration of the toxic oxygen metabolites as previously suggested [J. Miyamoto, Agr. Biol. Chem.28, 422 (1964)]. A gas-liquid chromatographic method was employed to assess the distribution in the brain following intracerebral injection of the parathion-type compounds. The results suggest that there may be intracerebral metabolism of thionophosphates in vivo.     

The biochemical basis of resistance to organophosphorus insecticides in the sheep blowfly, Lucilia cuprina

The metabolism in vivo and in vitro of [¹⁴C]parathion and [¹⁴C]paraoxon was studied in a susceptible (LS) and an organophosphorus-resistant (Q) strain of the sheep blowfly, Lucilia cuprina. Both strains detoxified the insecticides in vivo via a number of pathways, but the resistant strain produced more of the metabolites diethyl phosphate and diethyl phosphorothionate. No difference was found between strains in the rate of penetration of the compounds used. Also, in vitro studies showed no difference between strains in the sensitivity of head acetylcholinesterase to inhibition by paraoxon. Both the microsomal and the 100,000g supernatant fractions degraded paraoxon, but resistance in Q could be explained by the eightfold greater rate of diethyl phosphate production with or without added NADPH. Parathion was also degraded to diethyl phosphorothionate by an NADPH-requiring enzyme in microsomal preparations from both strains. However, Q produced significantly more diethyl phosphorothionate in vivo than LS. It was concluded that organophosphorus resistance in Q was due mainly to a microsomal phosphatase hydrolyzing phosphate but not phosphorothionate esters, probably enhanced by a microsomal oxidase detoxifying the latter.     

Why does DDT toxicity change after a blood meal in adult female Culex pipiens?

The toxicity of topically applied DDT to adult female anautogenous mosquitoes (Culex pipiens L.) showed dramatic variations in blood-fed insects. It decreased very rapidly about twofold to a minimum at 24 hr after a blood meal, then increased within 72 hr back to values typical of non-blood fed insects. A comparison of the metabolism of [¹⁴C]DDT in vivo revealed an increase in DDT dehydrochlorination to DDE at 72 hr after a blood meal, but this increase was not responsible for the variations in DDT toxicity at 24 hr. Changes in penetration rates were not observed and changes in the distribution of DDT could likewise not be related to the short period of decreased toxicity of DDT. Fenvalerate and trans-permethrin, two pyrethroid insecticides which are believed to have a mode of action similar to that of DDT, were also significantly less toxic 24 hr after a blood meal. By contrast, the cyclodiene insecticide aldrin and the carbamate insecticide propoxur were not less toxic 24 hr after a blood meal. The results suggest that after a blood meal an unidentified and transient change in C. pipiens specifically decreases DDT/pyrethroid toxicity. A hypothesis concerning this transient change is advanced. The results illustrate the difficulties in explaining physiological changes in insecticide toxicity.     

Effect of hepatic enzyme inducers on the in vivo and in vitro metabolism of dicrotophos,dimethoate, and phosphamidon in mice

Daily 75 mg/kg phenobarbital ip injections for 3 days or 25 ppm dieldrin in the diet of mice for 14 days caused an increase in liver cytochrome P-450 and blood B-esterase. Liver A-esterase was not significantly increased. Under in vitro conditions, phenobarbital and dieldrin induced the oxidative as well as hydrolytic metabolism of dicrotophos, dimethoate, and phosphamidon by liver homogenates or combined microsomes plus 105,000g supernatant fractions. The concentration of dimethoxon was increased more than fourfold by the pretreatments after incubation for 4 hr at 37.5°C with NADPH added. The organophosphorus insecticides used in this study were not metabolized as well by the liver microsomes alone or 105,000g supernatant alone, as by the combination of microsomes and 105,000g supernatant. Under in vivo conditions in mice, phenobarbital and dieldrin treatments increased the urinary recovery of metabolites in the initial 6 hr after [¹⁴C]carbonyl-dimethoate or [¹⁴C]N-ethyl-phosphamidon administration. Analysis of urine showed that the inducers caused a more than sixfold increase in dimethoxon recovered and twofold increase in water-soluble nontoxic metabolites within 6 hr after dimethoate administration. With phosphamidon both inducers increased the rate of metabolism, and the total recovery in aqueous and chloroform fractions was decreased. These results suggest that increased dimethoate toxicity after phenobarbital and dieldrin treatments in whole animals results from stimulation of the activation of dimethoate to dimethoxon, while the increase in hydrolytic products after both pretreatments results in decreased toxicity of the direct acetylcholinesterase inhibitors, dicrotophos and phosphamidon.     

Insensitive acetylcholinesterase,high glutathione-S-transferase,and hydrolytic activity as resistance factors in a tetrachlorvinphos-resistant strain of house fly

The gene producing insensitive acetylcholinesterase in a tetrachlorvinphos-resistant strain of house fly was introduced into the genome of a susceptible strain. The resistance to a number of organophosphorus insecticides, which was high in the original strain (234- and more than 800-fold for paraoxon and tetrachlorvinphos, respectively), was much lower in the resulting strain, ranging from 53-fold for paraoxon to 3.9-fold for tetrachlorvinphos. Synergists further decreased these factors. The reduction in sensitivity of the acetylcholinesterase in vitro was about 20-fold for both inhibitors. The Michaelis parameters for hydrolysis of acetylcholine and acetylthiocholine of the mutant and normal acetylcholinesterase showed only minor differences. An increased rate of metabolism of the insecticides was found in the original strain. In vitro, glutathione-dependent detoxication was from 9- to 120-fold that of a susceptible strain, depending on the insecticide and the conditions used. The enzyme de-alkylated parathion and methylparaoxon, but there was no de-arylation of parathion, although it is important in other strains. In vitro, paraoxon and methylparaoxon were also hydrolysed, and this is not so in the susceptible strain. The high resistance present in the original strain seems to be due to joint action of at least three mechanisms.     

Inhibition of permethrin-hydrolyzing esterases from Wiseana cervinata larvae

Inhibition of permethrin-hydrolyzing enzymes from larvae of the porina moth Wiseana cervinata has been examined in vivo and in vitro. Significant inhibition was shown by carbaryl and pirimiphos-methyl. 1-Dodecylimidazole substantially inhibited permethrin hydrolysis only in liver insects. The triphenylmethane dye tetrabromophenolphthalein was a moderate inhibitor only in vitro. TMDM (bis(N-dimethyl-4-aminophenyl)methane) had little effect on hydrolysis. These observations extend the range of species and substrates for which the triphenylmethane dyes and 1-dodecylimidazole are useful inhibitors of insecticide metabolism.     

Comparative ester hydrolysis of carbaryl and ethiofencarb in four mammalian species

The comparative ester hydrolysis and selective toxicity of carbamate insecticides were studied in four mammalian species. Hydrolysis rates of carbaryl and ethiofencarb (Croneton) were examined in the rat, mouse, guinea pig, and gerbil. Respiratory ¹⁴CO₂ resulting from the hydrolysis of orally administered [carbonyl-¹⁴C]carbamates (0.2 mg/kg) was taken as measure of in vivo hydrolytic capabilities. Ester hydrolysis was found to be greater for ethiofencarb than for carbaryl in all species tested, although the relative order of hydrolysis among species was the same with both compounds. After 24 hr, gerbils had hydrolyzed 91% of the ethiofencarb and 65% of the carbaryl. Guinea pigs hydrolyzed somewhat less of the compounds, 65 and 58%, but considerably more than rats and mice, about 40 and 25%. Comparing hydrolysis capabilities to acute toxicity data revealed that those species exhibiting the greatest hydrolysis were equally or more susceptible to carbamate poisoning than those having lesser hydrolytic capabilities. While ester hydrolysis destroys the anticholinesterase activity of carbamates, it is clear from these findings that factors other than hydrolysis are largely responsible for the variation in toxicity of the carbamates to different mammalian species.     

Excretion of unchanged organophosphorus compounds after their consumption by larvae of the Egyptian cotton leafworm,Spodoptera littoralis Boisd

The ingestion and excretion of sublethal doses of phosfolan, monocrotophos, parathion, and leptophos were studied in larvae of the Egyptian cotton leafworm, Spodoptera littoralis Boisd. Styropor (foamed polystyrene) lamellae treated with insecticide-sucrose mixtures were fed to the larvae and recovery of the undecomposed insecticides from the feces could be estimated by gas-liquid chromatography, without any cleanup of the sample. The insecticidal residues on Styropor were found to be stable for 5 days.As regards the percentage of insecticide recovered from the feces, two groups could be distinguished: (a) leptophos, practically complete; parathion, 59–67%; b) phosfolan, 10–23%; monocrotophos, 4–7%. A tentative hypothesis was advanced that both oral toxicity of the four compounds for S. littoralis larvae and their subsequent recovery in the feces were related to water solubility.     

Rapid in vitro screening assay for immunotoxic effects of organophosphorus and carbamate insecticides on the generation of cytotoxic T-lymphocyte responses

There has been an increasing need for rapid and easily interpreted techniques for the screening of possible immunotoxicants. Besides the obvious detrimental effects of exposure to immunosuppressive agents, the modulation of the immune system which results from exposure to these toxicants may be a sensitive index to the toxicologic effects of such agents. Other researchers have proposed assays to screen the effect of in vitro treatment with immunotoxicants on mitogenic and humoral immune responses. In this report, we have described an in vitro technique for screening the effect of immunotoxicants, in the presence and absence of a NADPH fortified liver postmitochondrial supernatant (S-9) from Arochlor 1254-treated rat, on another aspect of the mammalian immune system, the generation of a T-cell-mediated cytolytic (CTL) response. This enzyme system altered the effect of organophosphorus compounds on the generation of a CTL response. Malathion and fenitrothion were no longer suppressive following this pretreatment; however, ethyl and methyl parathion and fenthion were only partially detoxified. In contrast, the S-9 enzyme system did not alter the effect of carbamate pesticides, carbaryl and carbofuran, on the generation of CTL responses. This report describes the effects of these seven organophosphorus and carbamate pesticides on the generation of the CTL response. In addition, some of the in vivo data published on the immunomodulatory effects of these compounds were collated from the literature and a correlation between in vitro and in vivo studies was discussed.     

Penetration and metabolism of [14C]carbaryl in larvae of the gypsy moth,Lymantria dispar (L.)

The absorption and metabolism of topical doses of carbaryl by larvae of the gypsy moth, Lymantria dispar (L.), were determined using ¹⁴C-ring-labeled material. Carbaryl penetration followed four distinct phases of linear absorption, i.e., 0–5 min, 5–60 min, 1–3 hr, and 3–12 hr; the absorption rates for the four phases were 2.8, 0.57, 0.07, and 0.02% per minute, respectively. Ninety percent of the total metabolism of carbaryl occurred within the first 3 hr; over the next 9 hr, metabolism was exceedingly slow with a linear rate of ca. 0.8% per hour. Carbaryl was always the major radiocarbon in the larvae and the feces, amounting to ca. 16 and 9%, respectively, at 12 hr. At 12 hr the metabolite composition was 5-hydroxy carbaryl >N-hydroxy carbaryl > 1-naphthol > 4-hydroxy carbaryl > 5,6-dihydro-5,6-dihydroxy carbaryl. Small amounts of an additional product were detected but not identified. Much of the excretion of carbaryl and metabolites occurred without conjugation. The amounts of carbaryl metabolized by second, third, fourth, and fifth instars were directly correlated with the insecticide tolerances and mixed-function oxidase activity of the various larval stages. The synergistic action of 2,6-dichlorobenzyl-2-propynyl ether and piperonyl butoxide was also correlated with inhibition of the oxidative pathway.     

The mechanisms of carbaryl resistance in the fall armyworm,Spodoptera frugiperda (J. E. Smith)

The physiological mechanisms of resistance to carbaryl were investigated in a carbaryl-resistant strain of the fall armyworm, Spodoptera frugiperda (J. E. Smith). Piperonyl butoxide greatly reduced the resistance level from 90- to 6-fold, indicating that microsomal cytochrome P-450-dependent monooxygenases may play a major role in resistance. This finding is consistent with metabolic data in which the oxidative metabolism of carbaryl by midgut homogenates was five times more active in the resistant strain than in the susceptible strain. In addition, the resistant strain showed increased activities of microsomal hydroxylation and epoxidation compared to the susceptible strain. Cuticular penetration studies using [¹⁴C]carbaryl revealed that 55% of the applied radioactivity remained on the cuticle of resistant larvae while 32% remained on susceptible larvae 24 hr after topical treatment. The resistance appeared to be unrelated to target site insensitivity. It is concluded that the high level of resistance to carbaryl in this insect was mainly due to enhanced oxidative metabolism of the insecticide (via hydroxylation and epoxidation) with reduced cuticular penetration playing a very minor role, if any.     

Laboratory evaluation of fipronil,a phenylpyrazole insecticide,against adult Anopheles (Diptera: Culicidae) and investigation of its possible cross‐resistance with dieldrin in Anopheles stephensi

Adult mosquitoes from two strains of Anopheles gambiae and from three strains of Anopheles stephensi were exposed to 0.25% fipronil‐treated papers in WHO test kits or to 500 mg fipronil m⁻² impregnated mosquito netting in bioassay spheres. For comparison, tests were also carried out with the pyrethroid permethrin, using the same methods and doses, and on papers treated with 0.4 and 4% of the cyclodiene insecticide dieldrin. Compared with the same doses of permethrin, fipronil showed less and delayed activity. Two of the An stephensi strains were resistant to fipronil and dieldrin. To investigate whether this was due to a resistance mechanism in the An stephensi strains acting against both insecticides, the most fipronil‐ and dieldrin‐tolerant strain was further selected in two separate lines with one of the insecticides, followed by tests with the insecticide that the line had not been selected with. This indicated a concomitant rise of resistance to dieldrin in the fipronil‐selected line and vice versa. Repeated back‐crossing of the two lines with a susceptible strain and re‐selection with either dieldrin or fipronil gave evidence for the involvement of a single resistance mechanism to both insecticides. Permethrin resistance in both lines declined with selection for dieldrin or fipronil and confirms the absence of cross‐resistance between fipronil and pyrethroids. © 2001 Society of Chemical Industry     

Metabolism and absorption of methyl parathion by tobacco budworms resistant or susceptible to organophosphorus insecticides

Factors involved in insecticide resistance were evaluated by using ¹⁴C-labeled methyl parathion and aldrin to compare rates of absorption and metabolism by Heliothis virescens (F.) larvae that were resistant (R) and susceptible (S) to methyl parathion. Tests with third-stage R and S larvae suggested that the rate of insecticide absorption from the cuticular surface was not a major resistance factor. Further evidence for this assumption was demonstrated by the resistance of R larvae to injected and orally administered doses of methyl parathion. Smaller amounts of unmetabolized methyl parathion and aldrin were recovered from S larvae, an indication that differences in metabolism were probably related to the resistance.     

The mechanism of resistance to lindane and hexadeuterated lindane in the Third Yumenoshima strain of house fly

The factors which cause lindane resistance in the Third Yumenoshima strain, a strain of house flies highly resistant to insecticides, were studied using hexadeuterated lindane. Hexadeuterated lindane has the same physicochemical properties as lindane, but the former is much less biodegradable than the latter. The LD₅₀ ratio of lindane to hexadeuterated lindane in this strain, deuterium isotope effect on LD₅₀ values, was larger than that in S_NAIDM, a susceptible (nonresistant) strain. The penetration rates of labeled and nonlabeled lindane through the insect cuticle were about the same for both strains. Thus, penetration rate does not cause resistance. The metabolic degradation of lindane in the resistant strain in vivo occurred much faster than in the susceptible strain. This was also the case for lindane degradation processes in vitro such as microsomal oxidation and glutathione conjugation. In both strains, significant isotope effects were observed in the degradation rates in vitro of labeled and nonlabeled lindane. Therefore, principal biodegradation and detoxication pathways should include reactions which cleave the CH bonds. When the much less biodegradable d₆ counterpart of lindane was applied to both strains, the susceptible strain became much more highly intoxicated than the other within 20 to 30 min. This indicates that a combination of both greater degradability and probably lower sensitivity at the action site are the main factors underlying resistance in the Third Yumenoshima strain.