Succinic Dehydrogenase and Cytochrome Oxidase in the Liver of Mice Infected with Trypanosoma cruzi

Abstract

In livers of mice infected with the Tulahuen strain of Trypanosoma cruzi the activity of the mitochondrial enzymes, succinic dehydrogenase and cytochrome oxidase, is significantly altered. Histochemical assays revealed that in infections older than 7 days, in which parasitemias were generally higher than 30 million trypanosomes per ml of blood and many tissue foci occurred, the periportal pattern of enzyme activity occurring in normal livers was lost. Instead, activity occurred at random, was less intense, and unstained areas occurred, indicating the progression of a necrotic process. Corresponding quantitative decreases in activity were 39% for succinic dehydrogenase and 29% for cytochrome oxidase. The alteration of enzyme activity was related to the breakdown in cellular structure and consequent loss of mitochondria, although the possibility that other factors, such as injury to the remaining mitochondria, and/or injury to the enzymes themselves, may have played a role was not excluded. The pathologic physiology of clinical and experimental Chagas' disease has been studied extensively, but studies have dealt primarily with abnormalities related to cardiac function (Magarinos-Torres, 1941; Marcuzzi, 1950; Laranja et al., 1956; Federici et al., 1964; Anselmi et al., 1965, 1966, 1967; Chapadeiro et al., 1966). Comparatively less emphasis has been given to studies on hepatic function, although as has been observed in experimental infections (Pizzi, 1953; Andrade and Lopes, 1963) Trypanosoma cruzi multiplies quite markedly in the liver, and a greater predilection may be shown for this organ than for heart muscle (e.g., the Tulahuen strain). It was thought of interest therefore to extend our previous physiological studies on experimental trypanosomiasis to a study of oxidative function, and to examine whether or not T. cruzi in the liver modifies the activity of such enzymes as succinic dehydrogenase and cytochrome oxidase. This seemed of further interest in view of reports indicating that in malaria (Plasmodium berghei, P. knowlesi) both respiration and oxidative phosphorylation of the liver mitochondria are impaired (Riley and Deegan, 1960; Riley and Maegraith, 1962; Maegraith et al., 1962). MATERIALS AND METHODS The Tulahuen strain of Trypanosoma cruzi was used in adult CFW male mice. It gave rise to many parasitic foci in the liver (Fig. 1). Parasitemia levels were determined by hemocytometer Received for publication 1 April 1969. counts of the blood in normal saline at pH 7. The leishmanial forms were stained with Giemsa (Lillie, 1954). Infections older than 7 days, in which parasitemias of 30 million trypanosomes per ml of blood or higher ocurred, were generally accompanied by a marked degree of tissue invasion and were classified as high infections. However, the level of parasitemia alone, regardless of age of infection, did not always indicate the degree of tissue invasion; in some specimens many foci occurred after 7 days even though the parasitemia levels were minimal. Infections younger than 7 days generally showed low parasitemias (2 to 4 million trypanosomes per ml of blood) and few tissue foci, and were classified as low infections. The animals were killed by dislocating the neck with an inverted beaker. For the histochemical assays blocks of liver were frozen in dry ice and sectioned as 7 tL in a cryostat maintained at -15 C. The method of Nachlas (1957) with some modification was used for the detection of succinic dehydrogenase, employing tetranitro-BT (2,2',5,5'tetra-p-nitrophenyl-3,3'( 3,3'dimethoxy-4,4-diphenylene )-ditetrazolium chloride with or without phenazine methosulfate. The sections were incubated at 37 C for 1 to 2 hr. When phenazine methosulfate was used a shorter period of incubation (10 to 15 min) was found to be sufficient, but the use of this reagent in the incubation medium was not necessary to visualize the activity of the enzyme. Prior to incubation the sections were fixed briefly (1 min) in absolute acetone at 2 C. This was followed by an acetone-distilled water rinse (50:50) for 1 min at the same temperature, and a distilled water rinse (2 changes, 1 min each) at room temperature. As observed by Novikoff et al. (1960), acetone treatment allowed better localiz tion of the reaction product (formazan), minimized cell destruction, and removed lipids in which the formazan tended to accumulate. Longer periods of incubation as used by Novikoff et al. (1960), and Hitzeman (1963), resulted in some loss of enzyme activity and were not neces-