Tsutomu Nakatsugawa

Microsomal Oxidation and Insecticide Metabolism

Once in the animal body, organic insecticides are subject to metabolism by a variety of enzymes. Depending on their chemical structure, the compounds may be hydrolyzed, oxidized, conjugated with endogenous metabolites, or otherwise modified. Of particular importance are reactions mediated by the oxidative enzymes known as microsomal oxidases, so called because of their localization in the microsomal fraction of cell homogenates. Since these enzymes as a class have an extremely broad spectrum of substrates and catalyze a wide variety of biotransformations, they play a central role in the metabolism of insecticides. In fact, most of the biodegradability of current insecticides is dependent on their successful biotransformation by microsomal oxidases of various species. These enzymes are also intimately associated with the phenomena of synergism, enzyme induction, and insecticide resistance.

Aldrin epoxidation in the earthworm, Lumbricus terrestris L.

Abstract Aldrin epoxidase of the earthworm, Lumbricus terrestris L., has been shown to occur mainly in the intestine and seemed to increase in activity with the development of the animal. The microsomal fraction was identified by electron microscopy as the locus of the epoxidase. Although sesamex was not inhibitory, inhibition of expoxidase by carbon monoxide suggested the involvement of cytochrome P-450. However, the carbon monoxide difference spectrum of the microsomes was dominated by a material which was spectroscopically identical to earthworm hemoglobin.

Hepatic disposition of parathion: Uptake by isolated hepatocytes and chromatographic translobular migration

Abstract Isolated rat hepatocytes suspended in Waymouths medium absorbed parathion rapidly and reversibly until the intracellular parathion concentration reached more than 300 times the ambient concentration. The distribution quotient q, defined as the ratio of intra- and extracellular concentrations at equilibrium, decreased when horse serum was added to the medium. The high hepatocyte affinity and rapid uptake of parathion suggested that this compound might be almost completely absorbed by periportal hepatocytes in the perfused liver and migrate downstream chromatographically through the lobule. Parathion infusion experiments verified this prediction and showed that the migration rate is dependent on the q value. The chromatographic feature may be useful as a basis for nonhistological investigation of intralobular hepatocyte heterogeneity. The lobule may function as a reverse-phase chromatograph also for many other unionized xenobiotics. Implications of the findings in the hepatic disposition of xenobiotics in vivo are discussed.

The toxicological significance of paraoxon deethylation

Abstract The in vivo formation of deethylation and hydrolytic products of paraoxon degradation after parathion or paraoxon administration was nearly equal in control male rats, and the relative abundance of metabolites was not appreciably altered by pretreatment of rats with enzymeinducing agents. However, pretreatment with inducers dramatically increased the oxidative paraoxon O -deethylase of male rat liver while having little effect on hydrolytic enzymes. Prior to induction, the hepatic O -deethylase activity was greatly inferior to the various hydrolytic enzymes, but nearly equal levels of both enzyme systems were found after induction. These results indicate that a large portion of the hepatic hydrolases detected in vitro is not active in vivo . It also appears that the majority of the induced hepatic deethylase was not involved in vivo at the dosage levels employed. The in vivo metabolism of monoethyl paraoxon was also demonstrated. The predominant metabolite of ethyl-[1- 14 C]monoethyl paraoxon is 14 CO 2 , while phenyl-[1- 14 C]monoethyl paraoxon yielded 4-nitro[1- 14 C]phenol. Paraoxon deethylation was also shown to be an important detoxication mechanism in female rats and male mice and must be considered in interpreting the toxicological properties of parathion and paraoxon.

inactivation in vitro of microsomal oxidases during parathion metabolism

Abstract The inactivation in vitro of microsomal oxidases during parathion metabolism was examined in relation to the binding to the microsomal membrane of sulfur resulting from desulfuration. Modulation of sulfur binding with sulfhydryl compounds revealed that the majority of bound sulfur is unrelated to inactivation. It was concluded that inactivation was related to either the binding of a very small fraction of detached sulfur to a high-affinity component of the oxidase system or a mechanism other than the binding of a reactive sulfur.

Perfusion analysis of periportal and centrilobular metabolism of paraoxon in the rat liver

Abstract Paraoxon infused into the rat liver during perfusion in situ with Waymouths medium underwent chromatographic translobular migration with an apparent hepatic transit time of 3 min. Intralobular heterogeneity of paraoxon metabolism was examined by analyzing metabolites produced under conditions minimizing the chromatographic translobular migration of paraoxon. Periportal and centrilobular activities were estimated following forward and retrograde infusion of paraoxon, respectively. Centrilobular hepatocytes exhibited nearly twice the metabolic rate of the periportal cells. Pretreatment of the rat with DDE resulted in about a threefold increase in the ratio of oxidative deethylation to hydrolytic dearylation in the centrilobular region. The differentials observed by these analyses were less pronounced than expected from enzyme analyses in vitro , possibly reflecting secondary metabolism or intracellular heterogeneity of metabolic activities.

Rat liver paraoxonase (paraoxon arylesterase)

Abstract Paraoxonase in the liver of male Sprague-Dawley rats was studied by using [ phenyl -1- 14 C]paraoxon. Examination of the enzyme activity in subcellular fractions of liver homogenates indicated that hepatic paraoxonase is essentially a microsomal enzyme with a pH optimum of 7.5 to 7.8. Effects of calcium ions and EDTA on the enzyme suggested that active paraoxonase is a protein-calcium complex possibly with a range of affinity to calcium ion. Activity in homogenates declined with a half-life of 6 to 9 hr when stored at 0°C, apparently reflecting dissociation of calcium ions. Experiments with homogenates of perfused liver provided evidence that even without the contribution of calcium from blood, paraoxonase is almost fully active at the moment of homogenization. Possible reasons for the much reduced activity of paraoxonase in in vivo metabolism are discussed.

Sulfur oxyacid production as a consequence of parathion desulfuration

Abstract Parathion desulfuration in vitro by the rat liver microsomal oxidase system gives rise to reactive sulfur atoms which either covalently bind to microsomal macromolecules, most likely as hydrodisulfides (-SSH) or appear as water-soluble metabolites. The latter were tentatively identified as sulfate, thiosulfate, and a minor amount of sulfite. Production of sulfate was enhanced by glutathione. Moreover, glutathione and other thiols removed covalently bound sulfur from the microsomal membranes and partially restored activity to enzymes known to be inactivated during parathion metabolism. A semilogarithmic plot of sulfur removal with time indicated at least two populations of covalently bound sulfur differing in the stability of the hydrodisulfide bond or accessibility to thiol compounds. The significance of transient hydrodisulfide formation with regard to the production of sulfur oxyacids is discussed. Both macromolecular and low molecular weight endogenous sulfhy dryl-containing biochemicals may play a role in preventing sulfur binding in vivo since only low level sulfur binding in liver homogenates was observed after the in vivo administration of parathion.

Liver damage induced in rats by malathion impurities.

Administration of a single oral dose of the malathion impurity, O,O,S-trimethyl phosphorothioate (OOS-Me) or O,S,S-trimethyl phosphorodithioate (OSS-Me), to the rat resulted in hemostatic disorders, e.g. prolongation of blood clotting, prothrombin and thrombin time. Deficiency of coagulation Factors II, V and VII was also observed. OOS-Me and OSS-Me also caused dose-dependent increases of beta-glucuronidase in the blood with a maximum of 15- and 31-fold observed following treatment with 60 mg/kg OOS-Me and 40 mg/kg OSS-Me, respectively. Analysis of serum beta-glucuronidase by isoelectrofocusing electrophoresis showed that the liver endoplasmic reticulum was the source of this enzyme released into the blood. Co-treatment of OOS-Me with 5% O,O,O-trimethyl phosphorothioate (OOO-Me), a potent antagonist of OOS-Me-induced delayed toxicity, prevented hemostatic disorders but had no effect in reducing beta-glucuronidase levels. However, pretreatment of rats with piperonyl butoxide reduced the amount of beta-glucuronidase released into the blood. Of other O,O,S-trialkyl phosphorothioates examined, the O,O-diethyl S-alkyl phosphorothioates showed the highest activity in increasing beta-glucuronidase levels.

CHROMATOGRAPHIC TRANSLOBULAR MIGRATION OF XENOBIOTICS

Abstract The structure of the liver lobule is well adapted for efficient exchange of materials between hepatocytes and blood. Only recently, however, has it been realized during a rat liver perfusion study that the exchange of certain chemicals can be so fast as to cause their complete absorption by the periportal hepatocytes. Because of the high affinity to hepatocytes and the reversibility of uptake, the chemical thus absorbed migrates chromatographically toward the central venule. A recirculating autologous blood perfusion system was developed to provide evidence that such translobular behavior also occurs in vivo with various chemicals including parathion, naphthalene, 4-nitroanisole and dichlorobenzenes. Autoradiography of liver sections following an i. p. injection of 0.8 mg/Kg ethyl-1-H3 parathion showed the total absorption and metabolism of the dose by the periportal half of the liver lobule. These results indicate that chemicals exhibiting chromatographic behavior may be degraded in a single passage through the liver (first-pass effect) and may have a threshold oral dose for systemic toxicity. For these chemicals, the numerical product of the hepatic transit time and the enzyme activity rather than the enzyme activity alone should better predict metabolic consequences in the liver.