Phenobarbital induction of detoxifying enzymes in resistant and susceptible houseflies

Houseflies, Musca domestica, L., were treated with the drugs phenobarbital and 3-methylcholanthrene to study the effects of these compounds as inducing agents of the microsomal oxidases, heptachlor epoxidase, and p-nitroanisole O-demethylase, and of DDT-dehydrochlorinase. Phenobarbital was active when applied by injection or as part of the diet but inactive when topically applied. The resulting increases in heptachlor epoxidase activity were as much as 25-fold that of the untreated controls. The net increase in enzyme activity after phenobarbital treatment was greater in an insecticide-susceptible strain, WHO-SRS strain, than in a carbamate-resistant strain. However, the phenobarbital induced increases in DDT-dehydrochlorinase were greater, about 2-fold, in the resistant strains than in the susceptible strain.The optimum dose for phenobarbital was 1% in the diet for a period of 3 days. None of the treatments with 3-MC, feeding, injection, exposure to residues, or topical, were effective in induction.     

Dehydrogenation: A previously unreported pathway of lindane metabolism in mammals

This study presents evidence for the dehydrogenation of lindane by a hepatic microsomal mixed-function oxidase system. Preliminary investigation established that the incubation of lindane with rat liver homogenates produces a chlorinated, nonpolar compound identified as hexachlorocyclohexene. Differential centrifugation resulted in the sedimentation of most of the dehydrogenase activity in the microsomal fraction. Optimum in vitro assay conditions were established and it was found that the dehydrogenase system required molecular oxygen and reduced pyridine nucleotide coenzyme for maximum activity. Inhibition by SKF 525-A and CO suggested that the enzyme was cytochrome P-450 dependent. Lack of inhibition by cyanide indicated that the cytochrome b₅ desaturase system was probably not involved. Pretreatment of rats with DDT, which stimulates lindane metabolism, also induced significantly higher dehydrogenase activity. Both the in vivo and in vitro metabolism of hexachlorocyclohexene produced previously identified lindane metabolites. The existence of a cytochrome P-450 dependent mixed-function oxidase which catalyzes the dehydrogenation of lindane has not previously been reported and may be of importance in the metabolism of other xenobiotics.     

Oxidative metabolism of tetrachlorocyclohexenes,pentachlorocyclohexenes, and hexachlorocyclohexenes with microsomes from rat liver and house fly abdomen

Microsomal mixed-function oxidase systems from rat liver and house fly abdomen effectively metabolized isomers of 3,4,5,6-tetrachlorocyclohexene, 1,3,4,5,6-pentachlorocyclohexene, and 1,2,3,4,5,6-hexachlorocyclohexene to tetrachlorocyclohexenol isomers, 2,4,5,-trichlorophenol, and 2,3,4,6-tetrachlorophenol, respectively. The $\text{(}\text{346}\text{5}\text{)-isomer}$ of pentachlorocyclohexene gave also an abundant amount of pentachlorocyclohexenol isomers. As the metabolites of ($\text{36}\text{45}$)-, ($\text{35}\text{46}$)-, and $\text{(}\text{34}\text{56}\text{)-hexachlorocyclohexene}$, some compounds such as 1,2,4-trichlorobenzene, 1,2,3,4-tetrachlorobenzene, and pentachlorobenzene were more abundantly formed, respectively, than 2,3,4,6-tetrachlorophenol. These oxidative metabolic reactions were shown to mainly proceed via “ene-like” hydroxylation accompanied by double bond migration. Inhibition by CO, piperonyl butoxide, and SKF 525-A suggested that the “ene-like” hydroxylating enzyme was cytochrome P-450 dependent. The formation of an isomer of pentachlorocyclohexenol from $\text{(}\text{36}\text{45}\text{)-hexachlorocyclohexene}$ was also observed, and this reaction was activated by SKF 525-A.     

Studies on the mechanisms of accumulation of phorate by the free-living nematode Panagrellus redivivus

Accumulation and metabolism of the organophosphate pesticide, phorate by the free-living nematode Panagrellus redivivus was studied under anaerobic conditions and in the presence of carbon monoxide or SKF 525A, and compared with results obtained under normal, aerobic conditions and using heat-killed nematodes. Both phorate hydrolysis and side-chain oxidation were inhibited under anaerobic conditions and with heat-killed nematodes, but only hydrolysis was affected in the presence of carbon monoxide. The much greater phorate accumulation in the nematodes under anaerobic conditions and with carbon monoxide could be explained by this metabolic inhibition. SKF 525A had no significant effect on phorate metabolism or accumulation. The possible mechanisms involved in phorate breakdown by P. redivivus are discussed.     

Induction of microsomal oxidases by host plants in the fall armyworm,Spodoptera frugiperda (J. E. Smith)

Larvae of the fall armyworm (Spodoptera frugiperda (J. E. Smith)) were maintained on a semidefined artificial diet until the end of the fifth instar and the newly molted sixth-instar larvae were then fed fresh leaves of various host plants for 2 days prior to microsomal oxidase assays. The order of the midgut aldrin epoxidase activity of larvae after feeding on these plants was: corn > Bermuda grass > cotton > cowpeas > cabbage > peanuts > sorghum > alfalfa > millet > soybean. The ratios of aldrin epoxidase, p-nitroanisole O-demethylase, and p-chloro-N-methylaniline N-demethylase activities for soybean- and corn-fed larvae were 4.5, 6.3, and 2.7-fold, respectively. The induction was affected by the age and developmental stage of the corn plant with mature leaves from old plants being more active. Higher epoxidase activities were also observed in mature larvae reared on corn plants since egg stage as compared with those on soybean plants. Monoterpenes such as α-pinene, β-pinene, limonene, menthol, and peppermint oil were found to induce the midgut epoxidase activity. Corn-fed larvae were more tolerant of the insecticides methomyl, acephate, methamidophos, diazinon, trichlorfon, monocrotophos, permethrin, and cypermethrin than soybean-fed larvae.     

Significance of phosphorylation in organophosphate-mediated β-glucuronidase release from the rat liver

The elevation of rat blood β-glucuronidase caused in vivo by O,O-dialkyl O-phenyl phosphates and phosphorothioates correlated well with the electron-withdrawing tendency (σ^?) of leaving group substituents indicating the importance of a phosphorylation mechanism in the enzyme release. Hydrophobic bonding of these compounds may facilitate the phosphorylation since hydrophobicity (π) of substituents also correlated with the enzyme release. SKF 525-A decreased the elevation of β-glucuronidase by parathion through the suppression of paraoxon production. Pretreatment of rats with phenobarbital or DDE resulted in lower and delayed enzyme release caused by parathion.     

Interactions of allelochemicals with detoxication enzymes of insecticide-susceptible and resistant fall armyworms

The induction of glutathione S-transferases and microsomal oxidases by host plants and allelochemicals was examined in sixth-instar larvae of insecticide-susceptible and resistant strains of the fall armyworm, Spodoptera frugiperda (J. E. Smith). Among 11 host plants studied, parsnip and parsley were the best inducers of glutathione S-transferase, resulting in increases of 39- and 19-fold, respectively, compared with the artificial diet. The inducer in parsnip leaves was identified by mass spectrometry, high-pressure liquid chromatography, gas chromatography, and thin-layer chromatography as xanthotoxin, a furanocoumarin. Xanthotoxin also showed a bimodal effect on the microsomal oxidase systems, increasing cytochrome P-450 content and heptachlor epoxidase activity but inhibiting aldrin epoxidase, biphenyl 4-hydroxylase, and p-chloro-N-methylaniline N-demethylase. Using indole 3-acetonitrile, indole 3-carbinol, and flavone as inducers, the inducing pattern of glutathione S-transferases was the same toward 3,4-dichloronitrobenzene, 1-chloro-2,4-dinitrobenzene, and methyl iodide. Microsomal oxidase and glutathione S-transferase were also inducible by host plants and allelochemicals in larvae of a carbaryl-resistant strain.     

Residues of organochlorine pesticides in broilers from feed fortified with known levels of these compounds

p, p'-DDT, HEOD, endrin, heptachlor epoxide, hexachlorobenzene and lindane were fed in combination to day-old broiler chickens at levels of 0.05, 0.15 and 0.30 part/million in the feed, for a period of about 7 weeks. Birds were killed at regular intervals and samples of abdominal fat and other tissues examined for residues of the chlorinated pesticides by gas-liquid chromatography. The lipid content of each sample was determined and the residue levels calculated for the whole tissue and also for the fat contained in the sample. At the end of the 7 weeks (when in practice broilers are slaughtered) residues were highest for heptachlor epoxide, followed by hexachlorobenzene, HEOD, DDT (total), endrin, and lindane. Heptachlor epoxide residue levels in fat were 13 times the levels in the feed, for lindane the ratio was 3.3. Fat residues of each pesticide reached a plateau level relatively quickly, and these levels were proportional to the fortification rates in the feed. HEOD and p, p'-DDT alone were fed to separate groups of birds. No differences in residue build-up of these compounds were found between these groups and the groups that had received these compounds in combination with the other pesticides. The total amounts of each pesticide in the tissues were reduced when the meat was fried, probably by loss of fat during preparation and frying. For p, p'-DDT and heptachlor epoxide also smaller residue were observed in the remaining fat after frying.     

Insect juvenile hormones: Induction of detoxifying enzymes in the housefly and detoxication by housefly enzymes

The cecropia juvenile hormone and three of its analogs were compared as inducers of microsomal epoxidase, O-demethylase, and DDT dehydrochlorinase in the housefly, Musca domestica L. The compounds were the cecropia juvenile hormone, methoprene, hydroprene, 6,7-epoxy-3,7-diethyl-1-[3,4-(methylenedioxy)phenoxy]-2-octene, and piperonyl butoxide, a well known insecticide synergist. The compounds were administered by feeding at levels up to 1% in the diet for 3 days to 1-day-old female adults. Enzymes were then prepared and assayed for their activity using heptachlor, p-nitroanisole, and DDT as substrates.There was approximately a twofold increase in the microsomal oxidases and a 50% increase in DDT dehydrochlorinase after the treatment with the cecropia juvenile hormone, while methoprene had some activity as an inducer of the epoxidase (30% increase) but no activity in the case of the O-demethylase or the dehydrochlorinase. Hydroprene had no effect on any of the enzyme systems, while 6,7-epoxy-3,7-diethyl-1-[3,4-(methylenedioxy)phenoxy]-2-octene was an inhibitor of the two microsomal oxidases. The latter compound and piperonyl butoxide were strong inducers of DDT dehydrochlorinase, causing approximately twofold increases in the activity of this enzyme.There was evidence that the microsomal preparations were able to metabolize and inactivate methoprene and hydroprene, the action being oxidative in the case of methoprene and both oxidative and hydrolytic in the case of hydroprene. The oxidative metabolism of the two juvenile hormone analogs by the microsomal preparations was inducible by the cecropia juvenile hormone and by phenobarbital and dieldrin.     

Host plant stimulation of detoxifying enzymes in a phytophagous insect

The midgut microsomal aldrin epoxidase of variegated cutworm larvae (Peridroma saucia, Hübner) fed bean or peppermint leaves was up to 10 and 45 times more active, respectively, than that of larvae fed a basic control diet. Large increases in oxidase activity and cytochrome P-450 levels also occurred in larvae fed mint plant constituents such as menthol menthone, α-pinene, and β-pinene. Mint-fed larvae were more tolerant of the insecticide, carbaryl, than bean-fed larvae.     

Uptake of [U-14C]Lindane (γ-hexachlorocyclohexane) following exposure of isolated hepatocytes for short intervals of time

The initial steps of the uptake of lindane by the liver were studied by using isolated rat hepatocytes. [U-¹⁴C]Lindane served as labeled indicator during the metabolite production. Results indicated a very fast uptake of the pesticide by the cells. During the first minute there were rapid exchanges between cells and medium; the rate of the uptake did not follow linear evolution. The metabolite production by the cells was time dependent. Up to eight metabolites were visualized during the first minute after the lindane addition. The time course of the uptake was concentration dependent. Profile evolution suggested that the cell metabolism was overloaded over 150 μM lindane. The depletion of hepatocytes in glutathion changed the partition of the radioactivity among the produced metabolites. At 37°C, the N₂ gassing decreased the quantities of cyclohexane rings in the cells. This effect was reversed by preliminary addition of SKF 525A to the incubation medium; SKF 525A had no effect under O₂ aeration. Isomers α and β of HCH, bromsulfophtalein interfered with the lindane transformation by the cells. Thus, isolated hepatocytes represented a sensitive intermediate preparation between perfused liver and homogenate experiments. They will be used for further comparison of the in vivo and in vitro metabolite formation corresponding to the elimination of lindane by the biliary pathway.     

A two generation chronic mixture toxicity study of Clophen A60 and diethyl phthalate after gestational and lactational exposure in female Wistar rats

Polychlorinated biphenyls and diethyl phthalate are both lipophilic in nature and are likely to be present in the same environmental compartment or bioaccumalate over a period of time, thus a mixture toxicity study was undertaken to evaluate the type of interaction between polychlorinated biphenyls (Clophen A60) and diethyl phthalate over two generations in female Wistar rats. Healthy male and female albino rats of Wistar strain weighing 75-100 g (6-7 weeks old) were randomly assigned to four groups of six each. Group I male and female rats were fed on normal diet and water ad libitum. Group II male and female rats were maintained on normal diet mixed with corn oil as oil control. GroupS III and IV male and female rats were given Clophen A60 and diethyl phthalate dissolved in corn oil mixed with the diet at 50 mg/kg of the diet individually to each group. Group V male and female rats received a mixture of diethyl phthalate and Clophen A60, each dissolved in corn oil mixed with the diet at 50 mg/kg of the diet. Hundred days after the treatment, females were mated with the males in each group for 10 days. Exposure to diethyl phthalate and Clophen A60 was continued throughout mating, gestation until termination at weaning, which was 150 days of total treatment period of the parental generation female rats. Treatment for F1 generation male and female pups (6 males & 6 females) with Clophen A60 and diethyl phthalate individually and in mixture was continued at doses reduced to 25 mg/kg of the diet after they reached 75-100 g in weight. The treatment was carried out similar to the parental generation for a period of 150 days. Liver and serum aspartate aminotransferase, liver cholesterol and glycogen were significantly increased in the F1 generation Clophen A60 + diethyl phthalate treated group, whereas serum cholesterol, liver glutathione and glutathione reductase showed a significant decrease in the F1 generation Clophen A60 + diethyl phthalate treated group as compared to the parental generation mixture and individually treated groups as well as the individually treated F1 generation groups. A significant increase was observed in the liver and serum aspartate aminotransferase activity of Clophen A60 and serum aspartate aminotransferase levels of diethyl phthalate treated F1 generation rats as compared to the parental generation Clophen A60 and diethyl phthalate individually and mixture treated rats. Liver glutathione levels were significantly decreased in the F1 generation Clophen A60 and diethyl phthalate individually treated rats which was similar to the parental generation individually treated rats as compared to the controls. Liver glutathione reductase level was also significantly declined in the diethyl phthalate treated F1 individual group as compared to diethyl phthalate individually treated parental generation rats. Histology of the liver showed fatty degeneration in the mixture treated F1 generation rats as compared to Clophen A60 and diethyl phthalate individually treated F1 rats and parental generation Clophen A60 and diethyl phthalate individually and mixture treated rats. Thus, in spite of dose reduction and continuous exposure over two generation’s to a mixture of diethyl phthalate and Clophen A60 exposed through gestation, lactation and diet leads to a synergistic toxic effect in the F1 generation.     

Bimodal effect of methylenedioxyphenyl compounds on detoxifying enzymes in the housefly

Thirteen methylenedioxyphenyl (MDP) compounds, including commercial insecticide synergists and juvenile hormone analogs, were compared in their effect on detoxifying enzymes in the housefly (Musca domestica). Flies were fed a diet containing 1% of the compounds for 3 days. Enzymes were then assayed in vitro for their activity using aldrin and DDT as substrates. Piperonyl butoxide (PB), sesamex, propyl isome, sulfoxide, safrole, isosafrole, 6,7-epoxy-3,7-diethyl-1-[3-4(methylenedioxy) phenoxy]-2-octene (MDP-JH I) and 6,7-epoxy-3-methyl-7-ethyl-1-[3,4-(methylenedioxy) phenoxy]-2-octene (MDP-JH II) all caused a bimodal effect, inhibiting microsomal epoxidase and inducing DDT-dehydrochlorinase in the resistant Isolan-B strain. Two of these, PB and MDP-JH I, gave similar results with the susceptible strain, stw;w⁵ and two resistant strains, Fc-B and Orlando-DDT. However, o-safrole, piperonylic acid, piperonal, 3,4-methylenedioxybenzyl acetate and methyl-(3,4-methylenedioxy) benzoate had little or no effect on the enzyme systems studied. The standard susceptible strain (WHO-SRS) responded to these compounds very differently. Among those tested, piperonyl butoxide, sesamex, safrole, and isosafrole were inducers of microsomal epoxidase, a 4-fold increase occurring after treatment with sesamex. Only MDP-JH II showed a marked inhibition of the epoxidase. These treatments did not effect DDT-dehydrochlorinase activity in this strain.The enhancement of DDT-dehydrochlorinase activity by the MDP compounds is associated with an increased rate of DDT dehydrochlorination in vivo. The stimulatory effect could be blocked by treatment with actinomycin D or cycloheximide.     

Reproductive toxicity in pseudopregnant and pregnant rats following postimplantational exposure: Effects of the herbicide dinoseb

In Day 10 decidualized pseudopregnant rats fed dinoseb in the diet from Days 6 through 9 of pseudopregnancy, uterine wet weight was reduced in the groups fed 100 to 750 ppm (the highest dose tested). Uterine protein and glycogen concentrations in these animals were reduced in a dose-related manner. In Day 16 pregnant rats, orally fed dinoseb from Days 6 through 15 of pregnancy, placental protein and glycogen concentrations were diminished at 200 ppm and greater. Pregnant rats fed dinoseb (Days 6–16) showed reductions in their embryonic survival rates at 200 ppm and greater. The fetal survival rates at birth, from pregnant rats which were similarly administered dinoseb, were reduced at 150 ppm and greater. Fetal weight of fetuses from dams given dinoseb (200 ppm) was diminished. These results from decidualized pseudopregnant and pregnant rats indicate a toxic role for dinoseb in the intrauterine environment. This toxicity is apparently responsible for a decline in fertility in pregnant rats. Moreover, 200 ppm dinoseb seems to be reproductively critical.     

Induction of detoxification enzymes by triazine herbicides in the fall armyworm, Spodoptera frugiperda (J.E. Smith)

The inductive effect of six triazine herbicides on a variety of detoxification enzymes was investigated in fall armyworm (Spodoptera frugiperda) larvae maintained on an artificial diet. Dietary atrazine induced nine microsomal oxidase activities ranging from 1.3- to 21.6-fold, 12 glutathione S-transferase activities ranging from 1.3- to 4.2-fold, four hydrolase activities ranging from 1.3- to 2.9-fold, and two reductase activities ranging from 1.5- to 5.1-fold, depending on the enzyme assayed and tissue source (midgut vs. fat body) used. Simazine, cyanazine, ametryn, tebutryn, and terbuthylazine also induced these detoxification enzymes. The induction of microsomal oxidase (aldrin epoxidase) ranged from 1.2- to 11-fold, glutathione S-transferase (CDNB) ranged from 1.3- to 4-fold, and general esterase ranged from 1.4- to 4.1-fold, depending on the tissue source examined. In general, fat bodies were more inducible than midguts with respect to these detoxification enzymes, especially the microsomal oxidases. The induction by atrazine was associated with decreased toxicity of carbaryl, permethrin and indoxacarb, but increased toxicity of methyl parathion, phorate, and trichlorfon.     

Sequential changes in lactate,isocitrate, and malate dehydrogenases in mice exposed to technical grade hexachlorocyclohexane (BHC) and their possible relationship to liver tumors

Hexachlorocyclohexane (BHC) was fed at a 500-ppm-dose level in diet to pure inbred Swiss mice for 2, 4, 6, and 8 months. Later BHC was discontinued for 4 months and subsequently the animals were refed BHC for 1 month. The protein, lactate dehydrogenase (E.C.1.1.1.27), isocitrate dehydrogenase (E.C.1.1.1.42), and malate dehydrogenase (E.C.1.1.1.37) were studied from liver. The liver weight as percentage of body weight was also determined. The results showed an increase in the liver weight/body weight ratio and a decrease in protein and all three enzymes after BHC feeding for the different time intervals. The discontinuation of BHC in the diet resulted in the reversion of values of the above parameters toward the normal and after refeeding BHC for 1 month, the decreased values, as seen after initial BHC feeding, were again observed. This indicates that the changes in the values of lactate dehydrogenase, isocitrate dehydrogenase, and malate dehydrogenase are associated with hexachlorocyclohexane feeding in the diet. The significance of these changes is discussed.     

Effect of endosulfan in female rats growing on low- and high-protein cereal diet

Endosulfan (α,β-1,2,3,4,7,7-hexachlorobicyclo(2,2,1)-heptene-(2)-bis-hydroxymethylene(5,6)sulfite) fed daily 0.5 and 100 ppm in the diet proved significantly toxic to female rats (initial weight 40–50 g) growing on a low-protein (5%) diet as compared to those growing on a high-protein (24%) diet. The toxic symptoms which developed exclusively in low-protein-fed rats included growth retardation, low blood counts, low RNA and protein levels in liver, and high glutathione levels in liver and blood. However, no changes in the activities of liver RNase, blood glucose-6-phosphate dehydrogenase, and glutathione reductase were observed. Liver glucose-6-phosphatase activity increased significantly. Endosulfan induced an increased accumulation of perirenal adipose in rats raised both on low- and high-protein diets. Endosulfan elicited cumulative toxic manifestations as evident from the relative degree of toxicity caused by 0.5 ppm and 18 weeks feeding. α-Endosulfan, β-endosulfan, and endosulfan sulfate were recovered from adipose tissue, the recovery being higher in adipose tissue of rats raised on a low-protein diet.     

The relationship between the pharmacokinetics of intravenous cismethrin and bioresmethrin and their mammalian toxicity

The distribution and excretion of [¹⁴C]alcohol-labeled cismethrin and bioresmethrin was determined after intravenous administration to rats. Initially the label distribution of both isomers was similar, but differences occurred at later times mainly due to the retention of 5-benzyl-3-furylcarboxylic acid, a metabolite of bioresmethrin, in high concentration in the blood. Retention of this metabolite accounted for the slower excretion of bioresmethrin label compared to cismethrin. After administration of either isomer, parent pyrethroid was rapidly cleared from the blood and liver, and both isomers rapidly entered the central nervous system reaching peak concentrations within 2–5 min. Brain cismethrin concentrations exceeding 3.5 nmol/g were associated only with animals showing tremors. These levels of cismethrin are maintained for up to 30 min but bioresmethrin was depleted more rapidly possibly due to brain metabolism. It is concluded that the low toxicity of bioresmethrin is possibly due to the inability of this isomer to interact with the site of action in the central nervous system and not, as previously suggested, primarily because of more rapid metabolism in the liver.     

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.     

Determination of residues of promecarb and its metabolite, 3-methyl-5-isopropylphenol,in cattle

A procedure is described for the colorimetric determination of promecarb as 3-methyl-5-isopropylphenol in tissues using 2,6-dichlorpbenzoquinone-4-chlorimine as the chromogen. Cattle were exposed to either 1, 3, 5 or 8 spray applications of a 0·2% promecarb emulsion at 3-day intervals or were fed promecarb at 2 ppm or 20 ppm daily in the diet for 20 days. All animals were slaughtered 30 h after final exposure. Of the animals in the spraying trial, residues in omental fat ranged from 0·9 ppm to 1·5 ppm promecarb and 0·4 ppm to 1·9 ppm 3-methyl-5-isopropylphenol. Residues in perirenal fat ranged from 0·8 ppm to 1·9 ppm promecarb and 0·9 ppm to 1·6 ppm 3-methyl-5-isopropylphenol. Residues in diaphragm muscle did not exceed 0·06 ppm. In the feeding trial, the maximum residues induced were 2·2 ppm promecarb and 1·4 ppm 3-methyl-5-isopropylphenol in omental fat of an animal fed for 20 days on a diet containing 20 ppm promecarb.