Kenneth P. DuBois

Quantitative measurement of induction of hepatic microsomal enzymes by various dietary levels of DDT and toxaphene in rats

Abstract A study was conducted to develop procedures suitable for quantitative measurement of induction of hepatic microsomal enzyme activity by chemical agents fed in the diet of rats. The enzyme systems that catalyze the detoxification of EPN, the O-demethylation of p-nitroanisole, the N-demethylation of aminopyrine, and the reduction of p-nitrobenzoic acid were found suitable for this purpose. The activity of these enzymes can be measured quantitatively using whole-liver homogenates. Enzyme induction by DDT and toxaphene was studied by feeding various levels from 0.2 ppm to 50 ppm in the diet for periods up to 13 weeks. Induction of all the enzymes except reductase was produced by some dietary levels of both compounds. The lowest levels that produced a significant increase in any enzyme system were 1 ppm of DDT and 5 ppm of toxaphene. Maximal induction occurred within the first 3 weeks of the feeding period at all levels of each compound that caused enzyme induction; after this time the activity was maintained at a constant, elevated level until feeding of the pesticides was discontinued. The enzyme induction was more pronounced and occurred more rapidly in male rats than in females.

COMPARISON OF ACUTE TOXICITY OF ANTICHOLINESTERASE INSECTICIDES TO WEANLING AND ADULT MALE RATS.

Summary A comparison was made of the acute toxicity of a number of anticholinesterase insecticides in weanling and adult male rats. The results indicate that weanlings are about twice as susceptible as adults to parathion, methyl parathion, Systox, Di-Syston, Guthion, Malathion, Delnav, and Folex. A smaller increase in susceptibility of weanlings was noted with Ethion, Phosdrin, Dipterex and Sevin. Weanlings were about 5 times more susceptible to EPN and about 4 times more susceptible to Trithion than adult male rats. In the case of OMPA adults were about 5 times more susceptible than weanlings. The age differences in susceptibility are probably due to incomplete development of enzymes which catalyze the metabolism of anticholinesterase insecticides in the livers of young animals.

Quantitative Measurement of Inhibition of the Enzymatic Detoxification of Malathion by EPN (ethyl p-nitrophenyl thionobenzenephosphonate).

Summary A quantitative method for measuring the hydrolytic detoxification of malathion and its toxic metabolite, malaoxon, was developed. Application of the method to normal tissues revealed that the liver of several species contains the highest concentration of the enzyme but serum, kidney and lung also exhibited activity in some species. EPN was found to inhibit the enzyme system which detoxifies malathion in vitro and in vivo. This interference with the detoxification of malathion by EPN provides an explanation for the potentiation of toxicity which occurs when the two compounds are administered simultaneously and suggests the possibility that EPN may inhibit the detoxification of other esters which utilize the same detoxification pathway.

THE ACUTE MAMMALIAN TOXICITY OF RARE EARTH NITRATES AND OXIDES.

Acute toxicity studies were conducted on the rare earth nitrates and oxides. The approximate LD50 values for the rare earth nitrates given intraperitoneally to mice ranged from 225 mg/kg to 480 mg/kg, and for rats the values ranged from 210 mg/kg to 335 mg/kg. Rats were able to tolerate 1000 mg/kg of the rare earth oxides given orally or by the intraperitoneal route. When the salts of rare earths were administered orally to rats, the LD50 values ranged from 2750 mg/kg to 4200 mg/kg. With the exception of the transition elements for which the oral LD50 values were greater than 5000 mg/kg, the compounds generally exhibited an increase in toxicity with increasing atomic weight. Intravenous toxicity studies in rats showed that the light lanthanons are highly toxic to rats and an appreciable sex difference in susceptibility was observed. The nitrate salts of cerium, praseodymium, neodymium, and samarium were from 7 to 11 times more toxic to females than to male rats. In contrast, erbium nitrate, a member of the heavy lanthanons, did not show the marked sex difference in toxicity to rats.

Effect of Hexaethyl Tetraphosphate on Choline Esterase in vitro and in vivo.

Summary Hexaethyl tetraphosphate exerts a strong inhibitory effect on mammalian and insect cholinesterase in vitro and in vivo. This finding, in conjunction with its gross effects on animals suggests that its physiological effects may be at least in part due to its inhibition of this enzyme.

Comparative inhibition of aliesterases and cholinesterase in rats fed eighteen organophosphorus insecticides

Abstract Eighteen organophosphorus insecticides were fed to 30-day-old female rats for 1 wk at various dietary levels. Measurements were made of the inhibitory effects of the insecticides on aliesterases of serum and liver using diethylsuccinate and tributyrin as substrates. The cholinesterase activity of liver, serum, and brains of rats that received the same levels of the insecticides was also measured. Dose-related decreases in aliesterase activity were obtained with the appropriate dietary levels of each compound. The aliesterase activity of liver was more sensitive to inhibition than serum. The hydrolysis of tributyrin was inhibited at lower dietary levels than the hydrolysis of diethylsuccinate by liver, but hydrolysis of the two substrates by serum was affected to essentially the same extent. In most cases the dietary levels required to inhibit cholinesterase were considerably higher than the levels that inhibited aliesterases. When the aliesterase activity was depressed by feeding various levels of organophosphorus insecticides for 1 wk, the amount of potentiation of the toxicity of malathion was closely related to the amount of inhibition of liver aliesterases regardless of which compound was fed.

Potentiation of Toxicity of Malathion by Triorthotolyl Phosphate.

Summary Toxicity of malathion to male rats was markedly increased after treatment of animals with TOTP. At 24 hours after dermal application or intraperitoneal administration of 110 mg/kg of TOTP the LD50 of malathion was reduced from normal value of 1100 mg/kg to 12.5 and 8.2 mg/kg respectively. The increased susceptibility of rats to malathion persisted for as long as 14 days after treatment with TOTP. When TOTP was given 24 hours after malathion no potentiation was observed. TOTP inhibited enzymatic detoxification of malathion thus providing an explanation for its potentiating action. These findings indicate that the problem of potentiation of toxicity of organic phosphate insecticides extends beyond those agents commonly used for insecticidal purposes.

Studies on the enzyme inducing effect of polychlorinated biphenyls

Aroclor 1260 was fed to weanling male and female rats in the diet at levels of 1, 5, 25 and 50 ppm to determine its enzyme inducing capacity over various feeding periods. Dose-dependent increases in the activity of 3 hepatic microsomal enzymes were observed; maximal induction occurred in the first 3 weeks of the feeding period. Among the 3 enzyme systems studied, N-demethylase was the most sensitive to the inducing effect of Aroclor 1260. The enzyme induction was more pronounced in male rats than in females. An age difference in susceptibility of male rats to the enzyme inducing effect was also noted, young rats being more susceptible than adults. Data obtained by simultaneously feeding PCB and DDT to rats showed that these compounds have additive inducing effects on N-demethylase activity. However, PCB and DDT were not, when combined, additive on the O-demethylase and EPN-detoxification systems under the test conditions employed. Comparison of enzyme inducing effects of three Aroclor compounds, Aroclor 1221, 1254 and 1260, clearly indicates that both microsomal N-demethylase and EPN detoxification activity were increased in proportion to the chlorine content of the Aroclor compounds. Maximal induction of O-demethylase occurred at 54% chlorination.

INHIBITORY ACTION OF DITHIOCARBAMATES ON ENZYMES OF ANIMAL TISSUES

Summary A study of the effects of derivatives of dithiocarbamic acid was conducted in an attempt to explain their acute toxicity on a biochemical basis. Attention was focused on the actions of these compounds on the oxidative phase of intermediary carbohydrate metabolism. Dimethylammonium dimethyldithiocarbamate, diethylammonium diethyldithiocarbamate, sodium diethyldithiocarbamate, and bis(dimethyldithiocarbamyl) disulfide were found to be strong inhibitors of the tricarboxylic acid cycle of animal tissues in vitro as evidenced by inhibition of oxygen consumption, citrate synthesis, and pyruvate utilization. However, when maximum tolerated doses of dimethylammonium dimethyldithiocarbamate (500 mg/kg) and bis(dimethyldithiocarbamyl) disulfide (150 mg/kg) were given to rats, mice, and guinea pigs, the salt of dimethyldithiocarbamic acid exerted a pronounced inhibitory effect but the disulfide had no effect on the tricarboxylic acid cycle of liver and kidney. Further studies to locate the site of the inhibitory action of dimethylammonium dimethyldithiocarbamate demonstrated that this compound was a strong inhibitor of α-ketoglutarate oxidase, pyruvic dehydrogenase, and succinic dehydrogenase in vitro and in vivo . The disulfide was an effective inhibitor of these sulfhydryl enzymes in vitro but had no effect in vivo . The salts of dithiocarbamic acid also rapidly reduced cytochrome c and inhibited tyrosinase activity in vitro . The results of this investigation suggest that the acute mammalian toxicity of the dithiocarbamates is the result of their strong inhibitory action on enzymes that require sulfhydryl groups for activity.

Influence of Induction of Hepatic Microsomal Enzymes by Phenobarbital on Toxicity of Organic Phosphate Insecticides

Summary A comparison was made of the toxicity of 15 cholinergic organic phosphates to normal rats and mice and to animals pretreated with phenobarbital for 5 days. The purpose of the study was to determine whether the hepatic microsomal enzyme induction caused by phenobarbital changed the acute toxicity of the organic phosphates. The study demonstrated that phenobarbital treatment either decreased the toxicity or had no effect on the toxicity of any of the compounds in either species with one exception being an increased toxicity of OMPA to phenobarbitaltreated rats.