1979

I’OXICOLOGY AND APPLIED PHARMACOLOGY 49, 127-149 (1979)

Short-Term Effects of Graded Levels of Theobromine in Laboratory Rodents

STANLEY M. TARKA, JR.,’ BARRY L. ZOUMAS,~ AND JOSEPH H. GANS*

Research Laboratokes, Hershey Foods Corporation, Hershey, Pennsylvania 17033, and Department of Pharmacology, College of Medicine, University of Vermont,

Burlington, Vermont 05405

Received December 22, 1977; accepted December 4, 1978

Short-Term Effects of Graded Levels of Theobromine in Laboratory Rodents. TARKA,

S. M., JR., ZOUMAS, B. L., AND GANS, J. H. (1979). Toxicol. Appi. Pharmacoi. 49, 127-149. 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 czmcentrations of dietary theobromine were anorexia (except in female rats given the 22 % casein diet), de-creases in body weight in mature rats, growth retardation in immature ra’s and atrophy of the thymus glands in rats of both sexes and testicular atrophy in ma e Fats. Atrophy of the thymus gland and of the testes became prominent at the 0.6% die a y 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.

As part of his food supply, man ingests three alkaloids with well-documented pharma- cologic activity-caffeine, theophylline, and theobromine. Caffeine occurs naturally in coffee, tea, and cocoa and as an additive to cola beverages. Theophylline is present in tea while theobromine is obtained from cocoa and its chief derivative, chocolate. Theo-

1 Research Laboratories, Hershey Foods Corpora- tion.

2 Department of Pharmacology, University of Vermont.

bromine, therefore, is a constituent of foods and condiments which are conswmed by humans from childhood through old age.

Cocoa and its derivatives, as normally consumed, produce no adverse or toxic consequences in humans. Undesired effects have been reported only after the ingestion of large amounts of cocoa by humans. For example, the daily intake of 50-100 g of cocoa (1 .I to 2.2 g of theobromine) by human subjects has been associated with sweating, trembling, and severe headaches (Czok,

127 0041-008x/79/070127-23502.00/0 Copyright 0 1979 by Academic Press, Inc.

All rights of reproduction in any form reserved. Printed in Great Britain

128 TARKA, ZOUMAS, AND GANS

1974). Cocoa also contains caffeine although the concentration of caffeine in cocoa is one- eighth that of theobromine (Kreiser and Martin, 1978). Therefore, the adverse effects which have been reported following the consumption of large quantities of cocoa probably represent the effects of its theobromine content.

Experimental studies have indicated that a broad spectrum of toxicity occurs when caffeine or theobromine are fed in high concentrations to laboratory rodents on a daily basis for periods in excess of 14 days. Feeding high levels of caffeine to rats resulted in decreased growth rates, anorexia, and decreased thymus weights with death, when it occurred, resulting from automutilation, convulsions, and apparent respiratory failure (Peters and Boyd, 1967). High levels of dietary caffeine and theobromine have also been associated with a high incidence of reproductive disorders in rats (Fujii and Nishimura, 1972; Fujii and Nishimura, 1973) as well as adverse effects on rat testicular morphology and function (Friedman et al., 1975). By contrast, the feeding of relatively low levels of dietary theobromine was accompanied by a modest growth-stimulating effect in rats (Zoumas and Tarka, 1976).

The concentrations of caffeine and theobromine which produce serious toxic effects in laboratory rats are far in excess of what even the most extravagant human consumption would attain. Nonetheless, these reports indicated to us a need for more knowledge of the nutritional pharmacological and toxicological effects of cocoa and its constituents. We have selected theobromine as the focus of this research effort because of its quantitative predominance in cocoa. This report summarize the effects of adding graded increases of theobromine to the diet of laboratory rats, hamsters, and mice.

METHODS

The first series of experiments was performed to determine the effects of graded increments of dietary theobromine in mature rats. All rats were of the

Sprague-Dawley strain obtained from A.R.S. Sprague-Dawley, Madison, Wisconsin. Rats were received in the research animal facilities in the Hershey Foods Corporation Research Laboratories, males when 6 weeks old and females when 8 weeks old. The rats were given constant access to water and food (Purina Rat Chow) for an additional 6 weeks and then were fed the standard diet in agar form pre- pared according to the method of Shank and New- berne (1976) for 1 week. The nutritional compo- sition of this diet was: A.N.R.C. (Animal Nutrition Research Council) Reference Casein, 22 %; Vitamin mix A.O.A.C. (Association of Official Analytical Chemists), 1%; Mineral mix U.S.P. XVII, 5 %; corn oil (Mazola), 10%; cellulose, 1 % and Cerelose (glu- cose monohydrate), 61 ‘A. One group of male rats was given a modified diet composed of A.N.R.C. Reference Casein, lo%, corn oil, 8 %, Cerelose, 75 %, the other ingredients being of the same concentration as indicated above.

Each group of rats-male rats given the 22% casein diet, female rats given the 22 % casein diet and male rats given the 10 % casein diet-was divided into six subgroups, each containing 10 rats. Each subgroup was given one of six diets containing one of the following concentrations of theobromine: 0.0 % (control), 0.2, 0.4, 0.6, 0.8, or 1% for a period of 28 days. The rats were weighed at the start of the feeding trials and weight gain (or loss) and food intake were monitored twice a week. At the conclusion of the 28-day feeding period each rat was weighed and killed by decapitation with a guillotine and ex- sanguinated. The following organs were removed and weighed: brain, thymus, liver, kidneys, testes of male rats, and reproductive tract of female rats. Segments of these organs and of lung, heart, adrenal glands, pancreas, stomach, duodenum, jejunum, terminal ileum, and colon were fixed in 10% neutral buffered formaldehyde solution for histological examination. Sections were stained with hematoxylin-eosin.

In a second series of experiments theobromine was fed to immature rats in order to determine its effects on an early phase of growth and maturation. Rats of both sexes were obtained from A.R.S. Sprague- Dawley when 20 days old and weighing 35 to 40 g. The rats were maintained on the purified diets previously described for 1 week, each of which contained either 22% casein or 10% casein. The rats of each sex were separated into groups of 10 and were fed diets containing various theobromine concentrations according to the protocol outlined above.

A final series of experiments was performed with hamsters and mice. Outbred male, golden Syrian hamsters were obtained from A.R.S. Sprague- Dawley, Madison, Wisconsin, when the hamsters were 23 days of age. The hamsters were housed in individual stainless steel cages and were allowed

TOXICITY OF THEOBROMINE 129

RESULTS constant access to water and to the purified diet in agar form containing 22% casein. One week later the hamsters were randomized into groups of 10 hamsters per group and each group was given one of the purified diets containing the levels of theobromine as indicated for the rat studies. The experimental period with hamsters, however, was 35 days and food intake and body weight were monitored at weekly intervals. The rest of the protocol was identical to that described for rats.

Mature male rats fed the 22% casein diet without added theobromine (controls) gained 14.8% of their original body weight over the 4-week feeding trial (Fig. 1A). Theobromine added to the diet decreased weight gain and

Noninbred male and female Swiss albino mice were obtained from A.R.S. Sprague-Dawley, Madison, Wisconsin, when the mice were 25 to 27 days of age. The mice were fed the standardized purified diet containing 22% casein for 1 week and then randomized into groups of 5 or 10 male or female mice per group. Each group was given the purified diet containing theobromine in concentrations of 0.0 (control), 0.2, 0.4,0.6, 0.8, 1.2 and 1.6%. The experimental period was 28 days and the protocol, therefore, was identical to that described for rats.

. CONTROL $ 1 A 0.2% u! 4

n 0.4%

0 0.6%

As the investigation proceeded it became clear that feeding high levels of theobromine to rats produced not only marked changes in the morphology of certain organs but also marked decreases in food in- intake. Therefore, the changes in organ weights and structure could well have been a reflection of decreased food intake rather than any specific effects of theobromine. The following series of experiments were performed in order to obtain some distinction between the effects of decreased food intake and direct effects of theobromine in these experiments.

2001 4 A Weeks of Feeding Trials

Male mature rats and male immature rats were maintained on purified diets containing either 22 or 10% casein as previously described for a period of 1 week prior to experimentation. The rats were then divided into a number of subgroups of 20 rats. Ten rats in each subgroup were given diets containing theobromine concentrations of 0.0 (control), 0.6, and 0.8 % for a period of 28 days. The food consumption was monitored every day and changes in weight were recorded daily. The other 10 animals of each subgroup were then given a restricted diet containing only the quantity which represented the food consumption of the theobromine fed rats. Thus a careful attempt was made to produce a pair-fed control for each group of rats given dietary theobromine. The pituitary and thymus glands and testes were removed at autopsy from each rat in this group of experiments, fixed in neutral buffered 10% formaldehyde solution, and histologic preparations stained with hematoxylin- eosin and pituitaries also with PAS.

l WN-rFaL

-I A 0.2% :

m 0,4% 0 0.6% A 0.8% 0 1.0%

I I I 1 f I 2 3 4

B Weeks of FeedingTrials

Statistical methods. t Tests were performed on all data according to the method of Snedecor and Cochran (1971).

FIG. 1. Changes in body weights (A) and cumulative food consumption (B) of mature male rats given varying levels of theobromine in the diet over a 28- day period. The diet in these experiments contained 22 % casein. Bars indicate + SE.


TABLE 1

AVERAGE THEOBROMINE INTAKE BY ADULT RATS DURING LAST 3 WEEKS

0~ A ~-WEEK EXPERIMENT

Sex

Theobromine intake from dietary theobromine Dietary protein

levels (%) ~~g/Wday)

(%) 0.2 0.4 0.6 0.8 1.0

Male 22 94 215 310 422 492 Female 22 110 236 355 489 596 Male 10 144 291 382 425 562

L? Each group consisted of 10 animals.

male rats given the 1 y0 theobromine-containing diet lost 15 % of their original body weight during the 4-week period (Fig. 1A). Cumulative food intake was increased in male rats given the 0.2 and 0.4% diets as the result of a marked increase during Week 4 of the experiment (Fig. 1B). Small (19 %) decreases in food intake were recorded in the rats given the 0.6 and 0.8% theobromine- containing diets and a larger (30.4%) decrease in food consumption occurred in rats given the 1% diet (Fig. 1B).

Theobromine intake (Table I), expressed as mg/kg/day, in adult male rats given the 22% casein diet was calculated from the data shown in Fig. 2. The dose of theobromine was taken as the average for the last 3 weeks of the feeding trial. The linearity of theobromine intake in the latter periods of this group of experiments permits an accurate estimation of the dose over the major portion of the experimental period.

Mature female rats given the 22% casein diet without added theobromine (controls)

1.0% 0.6%

06%

0,4X

0.2%

FIG. 2. Theobromine intake in the groups of mature male rats described in Fig. 1.

lost weight during Week 1 of the feeding trial and over the next 3 weeks regained their initial body weights (Fig. 3A). Similar changes were seen in rats given the 0.2, 0.4, and 0.6 % theobromine-containing diets. Fe- male rats given the highest dietary theobromine levels did not regain their initial body weight, and those given 1 % dietary theobromine lost 10% of their initial body weight over the 4-week period (Fig. 3A). Food consumption by adult female rats given each level of dietary theobromine was greater than the food consumption by the control rats (Fig. 3B).

Average theobromine intake (Table 1) by adult female rats was comparable to that of adult male rats for the dietary levels of 0.2 to 0.8 %. At the 1% dietary level, the dose of theobromine was greater in female rats.

Mature male rats given the 10 % casein diet without added theobromine (controls) gained an average of 9.4 % of their initial body weight over the 4-week period (Fig. 4A). Progressive decreases in body weight occurred in adult male rats given the 10% casein diet with dietary theobromine levels of 0.6 to 1 .O %. At the end of the experimental period, rats given the 1% theobromine-containing diets had lost 32.8 % of their initial body weight.

Food consumption in mature male rats given the 10% casein diet without added theobromine (controls) was 30 % greater than that in rats given the 22% casein diet (Fig. 4B, compare with Fig. 1B). Marked, progressive decreases in food consumption


i

l c%imia A 0.2% n 0.4% 0 0.6% A 0.6%

350 0 1.0%

;w!!@** I 2 3 4 A Weeks of FeedingTrials

600- l CONTROL A 0.2% . 0,4% 0 0.6%

1 4

B Weeks af Feeding Triils

FIG. 3. Changes in body weights (A) and cumulative food consumption (B) of mature female rats given varying levels of theobromine in the diet over a 2%day period. The diet in these experiments contained 22 % casein. Bars indicate + SE.

occurred when adult male rats were given the 10 % casein diet with 0.6 to 1 .O % theobromine (Fig. 4B). The average dose of theobromine at each comparable dietary concentration of theobromine tended to be greater than the

*CONTROL A 0.2%

A Weeks of FeedingTrials

600

t

l cOtlT60L A 0.2% n 0.4% 0 0.6%

I 4

B Weeks of Feeding Trials

FIG. 4. Changes in body weights (A) and cumulative food consumption (B) of mature male rats given varying levels of theobromine in the diet over a 2% day period. The diet in these experiments contained 10% casein. Bars indicate I!I SE.

dose of theobromine to rats fed the 22 % casein diet (Table 1).

Changes in organ weights are summarized in Tables 2, 3, and 4. In all three groups of rats, males and females given the 22 %


FIG. 5. Histologic appearance of thymus tissue. (A) Mature male rat fed the 22% casein diet. The lobules are packed with cells and separated by thin interlobular septa. Cortex and medulla are distinct. (B) Thymus gland from a mature male rat fed the 22 y0 casein diet containing 0.6 y0 theobromine. There is extensive destruction of cortical lymphocytes and the depletion of cortical lymphocytes leaves a less distinct demarcation between cortex and medulla. (C) Atrophied thymus tissues from a mature male rat fed the 22% casein diet containing 1.0% theobromine. Widespread lymphocyte destruction has led to almost complete loss of cortical and medullary zonation. The interlobular septa are thickened with collagen. (hematoxylin-eosin; 40 x ).

TOXICITY OF THBOBROMINE 133

TABLE 2

THYMUS, KIDNEY, AND TESTICULAR WEIGHTS IN ADULT MALE RATS GIVEN VARYING LEVELS OF THEOBROMINE IN A 22 % CASEIN DIET

Organ weight at dietary theobromine levels (%) (% body wt)

Organ 0.0 0.2 0.4 0.6 0.8 1.0

Thymus 0.117+0.006 0.106~0.011 0.088_+0.006” 0.081_+0.009” 0.053+0.004” 0.039f0.003”

Kidneys 0.334+0.004 0.336kO.003 0.343 kO.005 0.368 +0.007 0.378 50.005 0.447 f 0.022” Testes 0.456+0.007 0.458kO.012 0.433+0.006 0.451~0.008 0.393+0.008” 0,279_+0.011”

‘p < 0.01 when compared with controls, t test.

TABLE 3

ORGAN WEIGHTS IN ADULT FEMALE RATS GIVEN VARYING LEVELS OF THEOBROMINE IN A 22% CASEIN DIET


Organ 0.0 0.2 0.4 0.6 0.8 1.0

Thymus 0.120_+ 0.010 0.063 + 0.003“ 0.057 + 0.006” 0.057 f 0.006” 0.043 + 0.003” 0.033 t 0.003” Kidneys 0.364+0.008 0.334+0.005 0.362_+0.005 0.386?0.006 0.407 f 0.009” 0.422 + 0.013” Reproductive

system 0.473 + 0.042 0.709+0.138 0.686 f 0.08 1 0.938+0.105 0.803 f 0.096 0.619+0.105

*p<O.Ol when compared to controls, r test.

casein diet and males given the 10% casein male rats at higher theobromine concentra- diet, there was a progressive decrease in tions with earlier and greater decreases ob- thymus weights with increasing concentra- served in male rats given the 10 “/, protein diet tions of dietary theobromine. Significant de- (Tables 2 and 4). Since thymus and testicular creases in testicular weights occurred in the weights are expressed as percentage of body


FIG. 6. Testicular sections. (A) Mature rats fed the 22% casein diet. (B) Section from a rat fed the diet containing 0.6% theobromine. The normal cellular relationship within the seminiferous tubules are distorted and there is degeneration particularly of the spermatocytes. In most seminiferous tubules the spermatozoa1 complement appeared normal. (C) Seminiferous tubule from a rat fed the diet containing 1% theobromine. Cellular degeneration and necrosis with prominent multinucleate cells. There is virtually complete aspermia and absence’ of spermatids. (Hematoxylin-eosin; 400x.)


weight, the actual decreases in the weights of these glands were profound. By contrast, the increase in kidney weights as percentage body weight seen in all three groups at the higher theobromine levels represented either no change or a modest increase in actual organ weight.

The progressive decreases in thymus and testicular weights with increasing doses of dietary theobromine concentrations in male rats given the 22% casein diet were accompanied by striking changes in tissue morphology as viewed by light microscopy (Fig. 5). There was a decreased lymphocyte density and blurring of the demarcation between cortex and medulla in the thymus glands from rats given diets containing 0.6%

theobromine (Fig. 5B). At the 0.8 % dietary theobromine concentration, the cortex of the thymus was almost devoid of lymphocytes and zonal demarcations were absent. Thymic remnants from rats given diets containing 1.0% theobromine were composed only of stromal elements and scattered medullary lymphocytes (Fig. 5C).

Testicular cytology (Fig. 6) was normal in rats fed diets containing up to and including 0.4% theobromine. An abrupt change was obvious in testes taken from rats given 0.6% dietary theobromine and was characterized by seminiferous tubular cell degeneration, by distortion of the normal cellular relationships and the presence of multinucleate cells (Fig. 6B). Testicular tissue from rats given the

TABLE 4

THYMUS, KIDNEY, AND TESTICULAR WEIGHTS IN ADULT MALE RATS GIVEN VARYING LEVELS OF

THEOBROMINE IN A 10% CASEIN DIET


Organ 0.0 0.2 0.4 0.6 0.8 I.0

Thymus 0.144*0.01 0.086f.O.008” 0.066~0.005” 0.038+0.002 0.028+0.004 b

Kidneys 0.304+0.005 0.315f0.003 0.323+0.004 0.402kO.022 0.475kO.02 0.530+ 0.038 Testes 0.481 f 0.006 0.486 + 0.063 0.477kO.014 0.34OkO.023 0.228AO.03 0.186f0.004

‘p < 0.01 when compared to controls, t test. b Thymic remnants too small to weigh accurately.


0.8 % dietary theobromine showed extensive widespread tubular cell degeneration and tubular cell degeneration and necrosis, oligo- necrosis, and the presence of many multi- spermia, and many multinucleate cells, some nucleate cells, some of which were in various of which resembled giant cells. In the testes stages of degeneration and some obviously of rats given 1% theobromine-containing necrotic (Fig. 6C). diets, there was obvious atrophy, aspermia, Although the weights of the thymus glands

FIG. 7. Thymus tissue. (A) Mature female rat fed the 22% casein diet containing 0.6% theobromine. (B) Thymic remnant from a mature female rat fed the 22% casein diet containing 1.0% theobromine. (Hematoxylinveosin; 40 x .)


were significantly reduced in female rats given levels of theobromine. At 0.6 and 0.8 %, there dietary theobromine at the 0.2 and 0.4% was a marked decrease in lymphocyte den- concentrations, significant histopathologic sity and in the depth of the thymic cortex but changes were only observed in the thymus zonal demarcations were distinct (Fig. 7A). glands from female rats fed 0.6% and higher The thymus in adult female rats given the

FIG. 8. Testicular sections-from mature rats given the 10% casein diet. (A) Seminiferous tubule from a rat fed diet with 0.6% theobromine showing multinucleate cells. (B) Section of testis from a rat fed the diet containing 1.0% theobromine with extensive tubular cell and multinucleate cell destruction. (Hematoxylineosin; 400 x .)


l CoNlRoL A (x2% m 0.4% o 0.6% A 0.6% 0 LOX P

I I I I

B we3ks;f Fi3edi&a:

FIG. 9. Body weight changes (A) and cumulative food consumption (B) of immature male rats given varying levels of theobromine in the diet over a 2% day period. The diet in these experiments contained 22 % casein. Bars indicate + SE.

1.0 % theobromine-containing diets was severely atrophied, almost depleted of lymphocytes and zonal demarcations were either faint or more often completely absent (Fig. 7B).

Morphologic lesions in mature male rats

given the 10% casein diet occurred in the same glands-thymus and testes-with a comparable but not identical sequence as in the mature male rats given the 22% casein diet. The testes of adult male rats given the 10% casein diet with 0.6 % theobromine contained many multinucleate cells, some of which resembled giant cells, against a back- ground of seminiferous tubular cell degeneration and necrosis (Fig. 8A). At the higher levels of dietary theobromine, both tubular cell destruction and multinucleate cell for- formation and degeneration were exaggerated and were accompanied by oligospermia or aspermia (Fig. 8B).

Male immature rats given the 22% casein diet gained 224% of their original weight in the 4-week period and the addition of theobromine to the diet resulted in dosage related decreases in growth weight (Fig. 9A). Parallel decreases were observed in cumulative food intake beginning with the diet containing 0.4 % theobromine (Fig. 9B).

Changes in organ weights of male immature rats are summarized in Table 5. The prominent changes in weight and in histologic morphology were in the thymus glands and testes (Fig. 10) and were essentially similar to those reported for mature male rats.

Female immature rats gained 123 % of their initial body weight over the 4-week period and food consumption was less than that of immature male rats (Figs. 11A and B). Theobromine intake (on the basis of.mg/kg/ day) was quantitatively similar to that of the immature male rats (Table 6) and increases in theobromine intake were accompanied by dose-related decreases in weight gain and food consumption.

A significant decrease in the weight of the thymus glands was observed in female immature rats beginning with the group fed the 0.6 % theobromine containing diet (Table 7). Histologic changes in the thymus glands of immature female rats given varying levels of theobromine in the 22% casein diet were identical to those previously described in mature rats and in immature male rats.


TABLE 5

THYMUS, KIDNEY, AND TESTICULAR WEIGHTS IN IMMATURE MALE RATS GIVEN VARYING LEVELS OF THEOBROMINE IN A 22 yO CASEIN DIET


Organ 0.0 0.2 0.4 0.6 0.8 1.0

Thymus Kidneys Testes

0.362 + 0.011 0.291+0.008” 0.24OrtO.009” 0.197+0.122” 0.148+0.008’ 0.128+0.012’ 0.420+0.004 0.400~0.004 0.397+0.004 O/448+0.004 0.507+0.016” 0.531 +o.olo” 0.590+0.014 0.559+0.009 0.524+0.015 O-362+0.026” 0.310~0.0090 0.279 +0.018”

‘p < 0.01 when compared to controls, f test.

TABLE 6

AVERAGE THEOBROMINE INTAKE BY IMMATURE RATS DURING LAST 3 WEEKS OF A b-WEEK EXPERIMENT

Dietary Theobromine intake from dietary theobromine

protein levels (%) (mg/kg/day)

Sex (%I 0.2 0.4 0.6 0.8 1.0

Male 22 200 389 623 872 1049 Female 22 199 405 654 909 1054 Male 10 237 501 731 705 912’ Female 10 787 869 973

o Each group consisted of 10 animals. b Weeks 2 and 3 only.

TABLE 7

ORGAN WEIGHTS IN IMMATURE FEMALE RATS GIVEN VARYING LEVELS OF

THEOBROMINE IN A 22 % CASEIN DIET


Organ 0.0 0.2 0.4 0.6 0.8 1.0

Thymus 0.252+0.018 0.221+ 0.008 0.175~0.007” 0.130+0.011” 0.112+0.009” 0.168~0.012” Kidneys 0.463+0.006 0.503&0.014 0.522+0.016 0.518+0.014 0.502rtO.012 0.618f0.020” Reproductive

system 0.658+0.070 0.647+0.064 0.560f0.046 0.580+0.057 0.408 + 0.056” 0.343 f 0.024”

@p < 0.01 when compared to controls, t test.

The few deaths which occurred during the course of these experiments with male and female immature rats given theobromine in the 22% casein diet were random and were not correlated with a specific dietary level of theobromine.

Immature male rats given the 10% casein diet showed an increase of 137 % of initial

body weight over the 4-week period (Fig. 12A)---a marked decrease from the weight gains recorded in immature male rats given

the 22% casein diet (Fig. 9A). A significant decrease in weight gain (when compared to the controls given the 10% casein diet) was observed when dietary theobromine levels

were 0.6% and actual decreases in body


weight below the starting level occurred in rats given the 0.8 and 1% theobromine containing diets.

The combination of the 10% casein diet and the higher levels of dietary theobromine decreased survival. In immature male rats given 0.8 % dietary theobromine, 6 of 10 rats survived 4 weeks. Only 4 of 10 rats given 1 % dietary theobromine survived 3 weeks and

none of the rats in this group survived the 4-week period.

Average cumulative food consumption paralleled the changes in body weights (Fig. 12B). Theobromine intake was approximately similar to that observed in immature male rats given the varying levels of theobromine in the 22% casein diet (Table 6). Because of the high rates of mortality, organ


FIG. 10. Testicular sections from immature rats fed the 22% casein diet. (A) Control. (B) Semini- ferous tubule from an immature rat given the diet containing 0.6% theobromine. There are few spermatids or spermatozoa, but several multinucleate cells are present. There has been extensive loss of germinal cells. (C) Seminiferous tubule from an immature rat given the diet containing 1.0% theobromine. In addition to extensive destruction of germinal cells, there has been necrosis of the multinucleate cells. (Hematoxylin-eosin; 400 x .)

weights are not shown for this series of experiments. Decreases in thymic and testicular size, however, were obvious beginning with 0.6 % theobromine. Histopathologic changes were comparable to those described for mature male rats and testicular changes were particularly striking in these immature rats given the 10% casein diet.

Immature female rats given the 10 % casein diet doubled their body weight in 4 weeks (Fig. 13A)-a weight gain comparable to the control immature female rats given the 22% casein diet; however, average food consumption in these control immature female rats given the 10 % casein diet was 56.7 % greater than that in immature female rats given the 22% casein diet (Fig. 13B, compare with Fig. 10B). Theobromine intake was somewhat greater in female than in male immature rats (Table 6). Five of the immature female rats given the 0.8 % theobromine-containing diet and five of the immature female rats

given the 1% diet survived the 4-week experimental period.

Changes in the weights of the thymus glands of female immature rats given the 10 % casein diet were comparable to those reported for male immature rats (Table 8). Similarly, histologic changes in the thymus glands were essentially the same.

Gross and histologic examinations of other tissues included heart, lungs, gut, liver, pancreas, spleen, and ovaries. No significant pathologic changes were evident in any of these tissues.

The pair-feeding experiments produced the following results: restricting dietary intake produced a decrease in growth rates which were not, however, as severe as those produced by theobromine in a comparable diet (Fig. 14). Most impressive was the failure of quantitative dietary restriction of controls to exert any effect on thymus or testicular weights (Tables 9 to 12). Additionally, the


AO.Z%

.Q4% 0 0.6%

4 A w&s of i&&gT&la

l cohlRoL A 0.2%

n OAX 0 0.6%

I I

B Weeks if Feed& Tds4

FIG. 11. Body weight changes (A) and cumulative food consumption (B) of immature female rats given varying levels of theobromine in the diet over a 2% day period. The diet in these experiments contained 22 % casein. Bars indicate + SE.

histologic morphology of the thymus and testes from these rats was identical to those of the controls given continuous access to food. By contrast, the effects of the theobromine-containing diets on weight changes, and thymus and testicular weights and morphology were virtually identical to those already described (Tables 9 to 12). Finally, no castration cells were observed in the adenohy- pophyses of the theobromine treated animals.

I

beaks d FiecfngliidIi 4

. CONTROL A 0.2%

n 0.4% 0 0.6% A 0.6%

B ‘Weeks if Fee& -Iii& 4

FIG. 12. Body weight changes (A) and cumulative food consumption (B) of immature male rats given varying levels of theobromine in the diet over a 28- day period. The diet in these experiments contained 10% casein. Bars indicate +_SE.

Hamsters fed the control diets gained 166% of their initial body weight over the 35-day experimental period (Fig. 15). In- creasing dietary theobromine concentrations produced parallel decreases in weight gain, Hamsters fed the 1% theobromine-containing diet maintained 81 y0 of their original body weight. A significant decrease in food consumption occurred in hamsters fed 0.8 and 1 y0 theobromine (Table 13). Theo- bromine intake increased progressively with increasing concentrations of theobromine in the diet (Table 14). A decrease in thymus


l CONTRX

ISO- o 0.6% A 0.6% 0 LOX

1

A I Weeks of lhdngTrio%

4’

500.

. CONTEOL 0 0.6% AaeX 0 1.0%

I B Weeks of Feeding Hals

FIG. 13. Body weight changes (A) and cumulative food consumption (B) of immature female rats given varying levels of theobromine in the diet over a 28- day period. The diet in these experiments contained 10% casein. Bars indicate f SE.

weight in hamsters fed the 1% theobromine containing diet was the only significant change in organ weights which was observed (Table 15). No abnormal histologic changes were seen in any of the hamster tissues examined.

Male mice fed the control diet gained 57 % of their initial body weight over the 28-day period and only the 1.2 % theobromine containing diet produced a significant decrease in weight gain (Fig. 15). Of the 10 male mice in the group fed the diet containing 1.2 % theobromine, five survived the 28-day period. None of the mice given the 1.6% theobromine-containing diet survived 3 weeks, and 7 of 10 animals in this group were dead within the first week. Cumulative food consumption by male mice was significantly decreased when the diets contained 0.8 and 1.2 % theobromine (Table 13). Theobromine intake (as mg/kg) by male mice was 25-35x greater than that of hamsters at comparable dietary theobromine levels (Table 14).

Histologic changes were seen in the thymus glands and in the testes of mice fed the 1.2% theobromine-containing diet. Thymic changes were similar to those seen in rats given higher dietary concentrations of theobromine. Testi- cular changes in mice given 1.2% theobromine were similar to the changes seen in the testes from rats given the 0.6% theobromine in a 22% casein diet. Degeneration and necrosis of seminiferous tubular cells occurred throughout the tissue and multinucleate cells also were seen. In some tubules

TABLE 8

ORGAN WEIGHTS IN IMMATURE FEMALE RATS GIVEN VARYING LEVELS OF THEOBROMINE

IN A lOok PROTEIN DIET

Organ weights at dietary theobromine levels (%) (% body wt)

Organ 0.0 0.6 0.8 1.0

Thymus 0.309 + 0.011 0.095 + 0.006” b b

Kidneys 0.399 fO.01 0.461 kO.026 0.584 +_0.036” 0.659+_0.053” Reproductive system 0.232 + 0.043 0.132+0.015 0.203 f 0.046 0.222 k 0.027

“p < 0.01 when compared to controls, t test. * Thymic remnants too small to be weighed accurately.

144

450.

E* 0

TARKA, ZOUMAS, AND GANS

0 0.6% 0 wIR.FED (0 0.6% A 0.6%

250 c

L 2 3 4

A d&of FeedingTtiils

45O- . COMRCL 0 0.6% CI PAlR.FED to 0.6% A 0.6% A PAIR-FED to 0.6%

xy)- l CONTROL 0 0.6% 0 F’AIR-FED bO.6% A 0.6%

E A PAIR-FED to 0.6%

0, m-

2

50

i

0 0.6% 0 MRTED b 0.6% A (x0% A WR-FE0 tot&e%

FIG. 14. Body weight changes in pair feeding experiments. (A) Mature male rats given a 22% casein diet. (B) Immature male rats given a 22 % casein diet. (C) Mature male rats given a 10% casein diet. (D) Immature male rats given a 10% casein diet. Bars indicate f SE.

TABLE 9

THYMLJS, LIVER, AND TESTICULAR WEIGHTS IN IMMATURE MALE RATS FED 0,0.6 AND 0.8 % THEOBROMINE IN A 10 % CASEIN DIET

Organ weight (% body wt)

Pair-fed

Organ Control 0.6% Control” 0.8 % Control”

Thymus 0.30 rk 0.05 0.07 +0.02b 0.29 kO.06 0.05 +0.01b 0.26k 0.06 Liver 4.04kO.36 4.7O&O.W 3.44kO.28 4.76+0.41b 3.37+ 1.02 Testes 1.28k0.12 0.57 * 0.09b 1.49kO.18 0.46+_0.12b 1.57kO.07

’ Controls were pair-fed. bp < 0.01 when compared to controls, t test.


TABLE 10

THYMUS, LIVER, AND TESTICULAR WEIGHTS IN IMMATURE RATS PAIR-FED 0, 0.6, AND 0.8 % THEOBROMINE IN A 22 % CASEIN DIET


Pair-fed

Organ Control 0.6% Controie 0.8% ControP

Thymus

Liver

Testes

0.29 -+ 0.04 0.16f0.03b 0.31 k 0.05 0.08 + O.OY 0.28 k 0.07

3.89kO.92 3.85kO.37 3.61 kO.29 3.62k0.39b 3.00f0.22

0.98_+0.11 0.74 * 0.20b 1.10_+0.09 0.55f0.13b 1.34f0.21

g Controls are pair fed. bp < 0.01 when compared to controls, t test.

TABLE 11

THYMUS, LIVER, AND TESTICULAR WEIGHTS IN MATURE RATS PAIR-FED 0, 0.6, AND 0.8 % THEOBROMINE IN A 10 % CASEIN DIET


Pair-fed

Organ Control 0.6% Control” 0.8% Control”

Thymus

Liver

Testes

0.14kO.02 0.05 + 0.02b 0.12+0.02 0.04 f 0.02b 0.12f0.03

3.33kO.14 3.29kO.36 2.99kO.34 3.12kO.60 2.64_+0.20

0.82+ 0.08 0.57+0.14b 0.94+ 0.09 0.43+0.1Y 1.00~0.07

D Controls were pair-fed. bp < 0.01 when compared to controls, t test.

TABLE 12

THYMUS, LIVER, AND TESTICULAR WEIGHTS IN MATURE RATS PAIR-FED 0,0.6, AND 0.8 % THEOBROMINE IN A 22 % CASEIN DIET


Pair-fed

Organ Control 0.6%

Thymus 0.11 kO.02 0.06 i- 0.02”

Liver 3.37 + 0.26 3.18kO.48

Testes 0.78 + 0.04 0.78 L- 0.08

a Controls were pair-fed. bp < 0.01 when compared to controls, t test.

Control”

0.14+0.02

3.01 f0.37

0.84 rt 0.06

0.8 % Control@

0.07 + 0.04b 0.13+0.04

3.39 + 0.22 3.1OkO.59

0.59 * 0.07b 0.91 kO.14


tw- .uJNlliu 0 0.6%

1 2 3 4 5

Weeks of F&ding Trial

FIG. 15. Body weight changes of male hamsters and of male mice given varying concentrations of theobromine in the diet for 35 or 28 days respectively. Bars indicate + SE.

there were many immature spermatids and few spermatozoa while other tubules contained what appeared to be a normal complement of spermatozoa.

Theobromine produced the same effects on growth in female as in male mice; significant reductions in weight gain occurred in female mice given the 0.8% diet. Female mice given the 1.2% diet for 28 days weighed 19.0 f 2 g, an average of 4 g less than the initial body weight at the start of the experimental period. Of the 10 female mice given the 1.2 % theobrominecontaining diet, four survived the 28-day period. None of the mice given 1.6 % theobromine survived 3 weeks of the experimental period. Cumulative food consumption was significantly decreased when the theobromine concentration was 0.4% or greater (Table 13). Theobromine intake by female mice was similar to that of the males (Table 14).

DISCUSSION The outstanding changes in rats associated

with increasing dietary concentrations of theobromine were growth retardation and thymic atrophy in both male and female rats, and in male rats testicular atrophy with bizarre changes in testicular cytology. We would like to consider each of the three responses recognizing, however, their likely interdependence.

Weight loss in mature female rats given theobromine at all dietary levels in a 22% casein diet clearly could be associated with theobromine intake since these female rats consumed more food over the Cweek period than did the control rats. Weight loss and growth retardation in mature male rats and in immature rats of both sexes may have resulted from both theobromine intake and from decreased food consumption. This conclusion is supported by the results of the pair-feeding study. Quantitative restriction of food intake itself resulted in decreases in body weight and in growth retardation in immature rats. However, a comparable decrease in food consumption with theobromine administration resulted in a greater decrease in body weight.

Decreasing the protein concentration of the diet to 10% casein produced a different pattern of food consumption, body weight and growth changes, and in the response to theobromine. Since carbohydrate replaced protein and this change was accompanied by a large increase in the basal level of food intake, the total available energy to rats given the 10% casein diet apparently was con- siderably increased. Yet this diet did not sustain the same weight increases in male rats as did the 22% casein diet. In rats of both sexes of mature and immature rats the 10% casein diet markedly enhanced the depressant effects of dietary theobromine on weight and growth changes and food consumption. Thus high concentrations of dietary theobromine produce complex changes in the relationships between energy metabolism and body weight maintenance or growth.


TABLE 13

CUMULATIVE Foot INTAKE BY HAMSTERS AND BY MICE GNEN VAR~NG LEVELS OF DIETARY THEOBROMINE

Cumulative food intake by hamsters and mice fed theobromine levels (%) (g/animal)

Species Sex 0.0 0.2 0.4 0.6 0.8 1.0 1.2

Hamsters” Male 256 239 246 231 208 188 b

Mice’ Male 151 150 141 142 120 I, 104* Mice’ Female 141 130 119 120 llld ‘b 94*

U Thirty-five-day period. * Animals did not receive this dietary level. c Twenty-eight-day period. dp-cO.O1, t test.

TABLE 14

AVERAGE THEOBROMINE INTAKE BY HAMSTERS AND MICE DURING THE L~sr WEEKS OF THE EXPERIMENTAL PERIOD

Theobromine intake from dietary theobromine levels (%) (mg/kg/day)

Species Sex 0.2 0.4 0.6 0.8 1.0 1.2

Hamsters” Male 182 406 638 848 1027 b

Mice’ Male 301 634 928 1138 b 1843 Mice’ Female 297 600 862 1411 b 1886

a Determined from average of last 4 weeks of a 35-day experimental period. * Animals did not receive this dietary level. c Determined from average of last 3 weeks of 28day experimental period.

TABLE 15

ORGAN WEIGHTS IN HAMSTERS GIVEN VARYING LEVERS OF THEOBROMINE IN A 22% CASEIN DIET

Organ weight at dietary theobromine levels (“/,) (% body wt)

Organ 0.0 0.2 0.4 0.6 0.8 1.0

Thymus 0.09 * 0.02 O.lOf0.04 0.08 k 0.03 0.10+0.06 0.05 f 0.02’ 0.05 f 0.03” Liver 5.0 +0.4 5.0 +9.6 5.0 50.5 5.0 f0.5 5.0 +0.7 6.0 +0.7* Testes 1.6 f0.5 1.4 +0.5 1.4 +0.5 1.3 +0.5 1.7 +0.7 1.1 +0.4

“p-zO.01, t test. bp<0.05, t test.

Decreases in thymus weights accompanied in adrenal weights (Peters and Boyd, 1967). the daily administration of high levels of Since thymic atrophy is one of the most caffeine to rats (Peters and Boyd, 1967). Our characteristic responses to adrenal steroids in studies showing the same response to theo- the rat (Weaver, 1955; Dougherty et al., bromine suggest that thymic atrophy pro- 1964; Lee and Dom, 1964; Munck et al., bably is a characteristic effect of the methyl- 1972), hypersecretion of adrenal steroids may xanthines in rats. Caffeine-induced thymic have produced the decreases in thymus atrophy occurred concurrently with increases weights in rats given either caffeine or theo-


bromine. However, thymic atrophy in the rat has been reported in response to chemicals such as the hepatotoxin thioacetamide (Barker and Smuckler, 1973), organoplatinum compounds (Ward and Fauvie, 1976), and organotin compounds (Sienen and Willems, 1976). Moreover, the cytotoxic effects of thioacetamide on thymic lymphocytes were produced in adrenalectomized rats (Barker and Smuckler, 1973). Thus, adrenal steroid secretion is not essential to chemically induced thymic atrophy. Whatever the physio- logic and molecular mechanisms may be, the pair-feeding experiments again indicate clearly that thymic atrophy is an effect of high concentrations of theobromine and not the result of decreased food intake.

Testicular lesions comparable to those seen in theobromine-treated rats have been observed in vitamin E-deficient rats (Mason, 1933), in rats exposed to X-irradiation (Metcalf er al., 1954; Metcalf and Inda, 1954; Berliner et al., 1964), and following the parenteral administration of serotonin (Lui and Kinson, 1973), epinephrine (Chatter- jee and Paul, 1968) the monoamine oxidase inhibitor, pargyline (Urry and Dougherty, 1975), and phthalate esters (Gray er al., 1977). The diversity of agents which evoke the same lesion makes difficult any speculation of the molecular processes involved. It is also pos- sible that decreases in food intake, particularly in developing young rats may lead to poor testicular development or testicular atrophy. The pair-feeding studies in this investigation demonstrate clearly that dietary restriction did not result in testicular atrophy or significant changes in testicular cytology. The testicular lesions of theobromine-treated rats, therefore, were the result of the effects of the methylxanthine itself. The absence of castration cells from the adenohypophysis indicates that theobromine-induced testicular atrophy may reflect either (1) persistence of interstitial cell secretion or (2) effects of theobromine at several levels of the hypothalamo- pituitary-gonadal system.

Extrapolation of data obtained in experi-

mental animals to humans is difficult under the best of circumstances. In this instance it is further complicated by the enormous doses of theobromine required to produce toxic side effects. For example, the 0.6% theobromine- containing diet provided the mature male rat with a theobromine intake of some 310 mg/ kg/day. For a 65-kg human this would mean a daily intake of approximately 18 g of theobromine, which could be attained by the daily ingestion of one hundred seventy-one 1.05-0~ milk chocolate bars. However, the comparative pharmacokinetics of theobromine in humans when compared to the rat may further decrease this factor. The plasma half-life of theobromine in the human is about 6 hr (Resman et al., 1977; Drouillard et al., 1978) while in the rat the half-life of theobromine in plasma may be of the order of 3 hr (Welch et al., 1977). If this were the sole determinant of the species differences in theobromine toxicity, then a 65-kg human would be required to eat eighty-five to eighty-six 1.05-0~ milk chocolate bars per day in order to attain a toxic level of theobromine.

The striking resistance of hamsters and to a lesser extent of mice to the toxic effects of theobromine add a further complication to any extrapolation of these experimental data to humans. The results of these experiments with hamsters and mice emphasize the importance of interspecies differences in resistance and susceptibility. Toxicity studies of methylxanthines in other animal species, moreover, may indicate that organs other than the thymus and testes are targets and that additional differences in pharmacokinetics present further uncertainties in making interspecies comparisons.

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CHAITERJEE, A., AND PAUL, B. S. (1968). Testicular atrophy in rats foItowing epinephrine administration. Endokrinologie 52, 406.

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DOUGHERTY, T. F., BERLINER, M. L., SCHNEEBLI, G. L., AND BERLINER, D. L. (1964). Hormonal control of lymphatic structure and function. Ann. N. Y. Acad. Sci. 113, 823-843.

DROUILLARD, D. D., VESELL, E. S., AND DVORCHICK, B. N. (1978). Studies on theobromine disposition in normal subjects. Clin. Pharmacol. Ther. 23, 296- 302.

FRIEDMAN, L., WEINBERGER, M. A., AND PETERS, E. L. (1975). Testicular atrophy and aspermato- genesis in rats fed caffeine or theobromine in the presence or absence of sodium nitrite. Fed. Proc. Fed. Amer. Sot. Exp. Biol. 32, 228. (Abstract)

FUJII, T., AND NISHIMURA, H. (1972). Adverse effects of prolonged administration of caffeine on the rat fetus. ToxicoI. Appl. Pharmacol. 22, 449-457.

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animals exposed to single doses of X radiation. In Biological E$ects of External Radiation (H. A. Blair (ed.)), Ch. 2. McGraw-Hill, New York.

METCALF, R. G., and INDA, F. A. (1954). Pathology in animals subjected to repeated daily exposure to X rays. In Biological Effects of External Radiation (H. A. Blair, ed.), Ch. 12. McGraw-Hill, New York.

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URRY, R. L., AND DOUGHERTY, K. A. (1975). In- hibition of rat spermatogenesis’ and seminiferous tubular growth after short-term and long-term administration of a monoamine oxidase inhibitor. FertiI. Steril. 26, 232-239.

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1979

Documents

initial body weight

original body weight

cumulative food consumption

hershey foods corporation

decreased food intake

mature male rats

adult male rats

feeding high levels