Joseph H. Gans

Effects of short-term and long-term theobromine administration to male dogs☆

Abstract Thirty-three dogs were each given a single oral dose of theobromine which ranged from 15 to 1000 mg/kg. Three dogs died, one each given theobromine 300, 500, and 1000 mg/kg. Ten dogs were continued on theobromine (75 to 150 mg/kg/day) for periods of 21 or 28 days; seven died and the others were killed with pentobarbital sodium. None had thymic or testicular atrophy, as has been reported in rats, but a degenerative, fibrotic cardiomyopathy limited to the right atrial appendage occurred in 6 of these 10 dogs. Theobromine was given to two groups of dogs for 1 year in doses of 25 and 50 mg/kg/day, respectively. Other dogs were given theobromine 25 or 50 mg/kg/day for 4 months and the dose was then increased to 100 or 150 mg/kg/day for 8 months. Three dogs (one each at 50, 100, and 150 mg/kg/day) died during the course of the year. The right atrial appendage of one, the dog at 100 mg/kg/day, was obliterated by fibrosis, but no heart lesions were found in the other two dogs. At the end of 1 year all surviving dogs including controls were killed and subjected to complete necropsies. As in the short-term study, the only gross and microscopic change associated with theobromine was a fibrotic lesion in the right atrium of the heart, in three of five dogs given theobromine 150 mg/kg/day and in two of the four dogs given 100 mg/kg/day. Plasma theobromine concentrations were determined during the last 2 months of the year. While right atrial cardiomyopathy occurred in dogs given theobromine 100 or 150 mg/kg/day, a clear relationship between dose, plasma concentration, frequency, and severity of the lesion could not be determined.

Short-term effects of graded levels of theobromine in laboratory rodents

Abstract Theobromine was added to the diet of mature and immature male and female rats in concentrations of 0.0, 0.2, 0.4, 0.6, 0.8, or 1.0% in diets containing 22 or 10% casein and these diets were fed for 28 days. The prominent effects of increasing concentrations of dietary theobromine were anorexia (except in female rats given the 22% casein diet), decreases in body weight in mature rats, growth retardation in immature rats and atrophy of the thymus glands in rats of both sexes and testicular atrophy in male rats. Atrophy of the thymus gland and of the testes became prominent at the 0.6% die any theobromine level. There was a progressive decrease in thymic cortical lymphocytes with increasing intake of theobromine, and at the 1% level the shrunken thymus gland was composed only of stromal cells and scattered medullary lymphocytes. Testicular changes included seminiferous tubular cell degeneration and necrosis and multinucleate cell formation with degeneration and necrosis at the higher theobromine levels. A low protein diet enhanced the severity of theobromine effects on body weight and growth and on thymic and testicular weight changes and histopathology. The daily dose of theobromine which produced retrogressive changes in weight patterns and in the morphology of the thymus in both sexes and of the testes in males was approximately 250–300 mg/kg/day in mature rats and approximately 500 mg/kg/day in immature rats. Hamsters and mice were much more resistant to theobromine than were rats. A decrease in growth and in thymic weights occurred only at the highest dose levels of theobromine and testicular and thymic changes were completely absent in hamsters. Testicular changes in mice were seen only at dietary theobromine concentrations which produced considerable mortality.

Norepinephrine induced cardiac hypertrophy in dogs

Abstract The daily administration of norepinephrine in oil to dogs, 0.8 to 1.0 mg/kg, over periods of 63 to 82 days results in significant increases in heart weight. Comparable treatment with epinephrine in oil does not produce any significant changes in heart weight. Heart tissue from dogs given norepinephrine in oil for 3 days incorporates significantly greater amounts of 14C from 14C-amino acids into protein than does control tissue. Histologically, myocardial sections from dogs treated for 3 days with norepinephrine in oil show a striking increase in the number of interstitial cells and well defined fatty changes when compared with tissue from control dogs. Myocardial sections from dogs treated with norepinephrine in oil for 63 to 82 days did not reveal either increased interstitial hypercellularity or marked evidence of fatty change.

Effects of continuous administration of dietary theobromine on rat testicular weight and morphology.

Previous studies have shown that high dietary theobromine levels fed to rats results in testicular atrophy with changes in testicular cytology. An experiment was performed to determine whether these changes in rat testes are reversible. Proven breeder male rats were fed Purina lab chow containing 0, 0.2, 0.6, or 0.8% theobromine for 49 days. At that time, unilateral orchiectomy was performed on all rats. Testicular weights in the rats fed 0.6 and 0.8% theobromine-containing diets were significantly less than those from control rats and from rats fed 0.2% dietary theobromine (p < 0.01). The seminiferous tubules from rats fed the 0.6 or 0.8% theobromine diets showed extensive severe cellular degeneration and necrosis and contained many multinucleated cells. The interstitial tissue was edematous and there was evidence of apparent proliferation of arterioles. The rats were allowed to recover and were fed the diet without theobromine for an additional 49 days. Testicular weights of the rats given 0.6 and 0.8% dietary theobromine in the first 49-day period remained significantly below those of control rats and of rats fed 0.2% dietary theobromine (p < 0.01). Histologically, 70 to 90% of the seminiferous tubules from the rats given the two highest dietary levels of theobromine appeared almost deplete of well-formed spermatozoa. These results indicate that the theobromine-induced testicular injury appears to be irreversible over time in the affected seminiferous tubules.

Dietary influences on theobromine-induced toxicity in rats

Abstract Theobromine-induced toxicity was compared at 8, 16, 21, and/or 28 days in rats given either a semisynthetic diet or a pulverized commercial diet in each of which 0.6 or 0.8% theobromine was incorporated. Rats given the pulverized commercial diet containing theobromine consumed more food and therefore, more theobromine over a 28-day period, and gained more weight than did rats given theobromine incorporated into the semisynthetic diet. Serum theobromine concentrations were significantly higher in rats given 0.8% theobromine in the semisynthetic diet than in rats given 0.8% theobromine in the pulverized commercial diet. Spermatogenic cell degeneration and necrosis in the testes of rats fed 0.8% theobromine in the semisynthetic diet for 16 days seemed to be limited to seminiferous tubular cross sections containing stages X to XIV of spermatogenesis, but after 28 days all tubular cross sections showed extensive spermatogenic cell destruction. No significant pathological changes were observed in the testes of rats fed 0.8% theobromine in the pulverized commercial diet for 21 days; spermatogenic cell degeneration and necrosis and multinucleate cell formation were seen after 28 days in seminiferous tubular cross sections containing the early stages of spermatogenesis, but sections containing stages X to XIV were spared or were much less involved. Atrophy of the thymus gland occurred earlier than testicular damage on each respective dietary regimen containing theobromine and was more severe in rats given theobromine in the semisynthetic diet.

Liver nuclear DNA synthesis in mice following carbon tetrachloride administration or partial hepatectomy

Abstract Long-term, continuous (twice per week) administration of CCL4 to male mice resulted in a high incidence of liver nodules which appeared to be resistant to the necrotizing effects of CC14 but showed no features of malignant neoplasia. Liver nuclear DNA synthesis was compared in mice given CC14 and in mice subjected to partial hepatectomy (PH). Mice were given by gavage corn oil or CC14 in corn oil for periods of 2 to 25 weeks and several mice were subjected to PH after 12 and 25 weeks of corn oil treatment. Mice were given [3H]TdR during liver regeneration and newly synthesized liver nuclear DNA was isolated and separated by BND-cellulose chromatography. Greater than 85% of the labeled DNA from PH mice eluted from BND-cellulose columns as double-stranded (ds) DNA with single-stranded (ss) regions or ends and less than 15% as ds DNA. When mice were treated with CC14 for 8 weeks or longer a significantly greater portion of liver nuclear DNA eluted as ds DNA. Administration of HU and 5-FU with [3H]TdR decreased [3H]TdR incorporation into DNA to low levels incompatible with unscheduled DNA synthesis. Single doses of CC14 given to mice treated with corn oil for 2 to 12 weeks provided newly synthesized DNA which was primarily (>80%) ds DNA with ss regions or ends, but after 25 weeks of corn oil administration, a single dose of CC14 resulted in newly synthesized DNA with a greater proportion of ds DNA. The high labeling of ds DNA in mice treated with CC14 may have resulted from an alternate pathway of DNA synthesis catalyzed by the enzymes or enzyme complexes associated with semiconservative DNA synthesis or from proliferation of nonparenchymal cells with a rapid turn-over rate.

Hypercholesterolemia of streptozotocin-induced diabetes mellitus in dogs

Abstract Diabetes mellitus was produced in dogs by the intravenous administration of streptozotocin 50 to 55 mg/kg, dissolved in citrate buffer, pH 4.5. The vehicle alone was administered to control dogs. Eight diabetic dogs and 7 control dogs were maintained for periods in excess of 2 months. Diabetic dogs were kept in a relatively stable condition by the administration of isophane insulin 0.4 U/kg/ day and by feeding a high fat dietary supplement. Plasma cholesterol concentrations in diabetic dogs increased significantly within 2 weeks after the administration of streptozotocin and remained at these elevated levels for periods in excess of 2 months. Electrophoresis of plasma lipoproteins indicated that the increase in plasma cholesterol concentrations resulted from an increase in both the high density and low density lipoproteins. The prominent post-mortem lesion in all diabetic dogs was marked degeneration or virtual absence of islet tissue from sections of pancreas observed under light microscopy.

Metabolic effects of clofibrate and of cholestyramine administration to dogs

Abstract Dogs were given clofibrate, 50 mg/kg of body weight/day for 6 days or 50 mg/kg of body weight/day for 2 days, followed by 40 mg/kg of body weight every 30–36 hr over an additional 9-day period. Cholestyramine, 0.7 g/kg of body weight/day, was administered to an additional group of dogs over an 11-day period. Plasma cholesterol concentrations in dogs treated with clofibrate for either 6 or 11 days decreased 24 per cent and decreased 16 per cent in dogs given cholestyramine. 14 C incorporation into bicarbonate from 3- 14 C-pyruvate, from [U- 14 C]-L-alanine and from [1- 14 C]glucose was significantly increased by liver slices from clofibrate-treated dogs when compared with tissue from control animals, but cloflbrate did not result in any changes in [ 14 C]cholesterol or [ 14 C]fatty acid formation from either [3- 14 C]pyruvate or from [U- 14 C]alanine. [ 14 C]Cholesterol formation from 6-[ 14 C]glucose was virtually abolished in liver slices taken from dogs treated with clofibrate for 11 days. Liver slices from dogs treated with cholestyramine showed a 3.5- to 4.0-fold increase in 14 C incorporation into cholesterol from both [3- 14 C]pyruvate and [6- 14 C] glucose, and a significant increase in 14 C incorporation from [1- 14 C]glucose into bicarbonate. Clofibrate administration resulted in decreased liver glycogen concentrations and decreased 14 C incorporation into glycogen from [ 14 C]glucose, but 14 C incorporation into glycogen from [U- 14 C]L-alanine was unaffected. If the hypocholesterolemic effect of clofibrate in dogs results from decreased cholesterol synthesis, changes in glucose metabolism may form the metabolic basis rather than alterations in pyruvate (or acetyl CoA) metabolism.

Diethylnitrosamine-lnduced Changes in Mouse Liver Morphology and Function

Summary Long-term administration of DEN to adult Swiss mice for periods of up to 24 weeks resulted in intense bile duct proliferation, formation of hepatic nodules, some of which appeared to be hyperplastic, and foci of lymphocytic infiltration. No hep-atocellular carcinomas or well-defined hepa-tomas were found in these DEN-treated mice. The only tumors observed were one hemangioma, and in 2 sections, tissue suggestive of cholangiocarcinoma. The significant increases in hexobarbital sleeping times and the striking resistance to the hepa-totoxic effects of CC14 in DEN-treated mice indicated that long-term exposure to DEN resulted in a progressive inhibition of liver DMES activity. Phenobarbital administration concurrent with DEN prevented the decreases in hexobarbital sleeping times but did not alter the morphologic changes in the liver produced by DEN, at least at the light microscopic level.

The effect of carbon tetrachloride administration on cholesterol metabolism in mice.

Abstract There was a profound depression of liver cholesterol synthesis 17–19 h after the intruperitoneal administration of carbon tetrachloride-peanut oil mixture to mice which was accompanied by an equally drastic impairment of hexobarbital metabolism as evidenced by a marked prolongation in hexobarbital sleeping times. 48 h after the single dose of carbon tetrachloride liver cholesterol synthesis was at control levels. Liver cholesterol synthesis in mice given repeated injections of carbon tetrachloride over a 13- or 20-day period also was at control levels despite prominent changes in hepatocellular morphology and liver lipid composition and a significant prolongation of hexobarbital sleeping times. Prominent effects of carbon tetrachloride on liver fatty acid synthesis also were observed; 17–19 h after the administration of a single dose of carbon tetrachloride, liver fatty acid synthesis was severely decreased, 48 h after the injection of the single dose of carbon tetrachloride liver fatty acid synthesis was at control levels while in mice treated chronically with carbon tetrachloride liver fatty acid synthesis was significantly increased. The distribution of [14C]cholesterol in vivo following the administration of [2-14C]acetate was significantly different in mice chronically treated with carbon tetrachloride when compared to mice acutely poisoned with carbon tetrachloride. Kidney cholesterol specific radioactivities in mice treated chronically with carbon tetrachloride were significantly greater than those of control mice while the reverse occurred in acutely treated mice. The labelling pattern of cholesterol extracted from the small intestine, in conjunction with that of kidney cholesterol support the conclusion that carbon tetrachloride administration markedly alters the distribution of [14C]cholesterol following an in vivo pulse of [2-14C]acetate.