Reproductive Toxicology, Vol. 6, pp. 347-353, 1992

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REPRODUCTIVE

TOXICITY OF THEOBROMINE EXTRACT IN MALE RATS

AND COCOA

Y I N G W A N G , * D O N A L D P. W A L L E R , * A M I Y A P. S I N H A HIKIM,'~

a n d LONNIE n . RUSSELL t *Program for Collaborative Research in the Pharmaceutical Sciences, University of Illinois at Chicago; "~Department of Physiology, Southern Illinois University, Carbondale, Illinois Abstract - - The toxicities of theobromine and cocoa extract on the reproductive tract of male rats were compared in the present study. A cocoa powder extract containing 117 mg theobromine/g extract was prepared using 85% boiling methanol. Sprague-Dawley rats were weighed and dosed daily for 31 days with vehicle, 250 mg/kg theobromine, 2.14 g/kg cocoa extract (117 mg theobromine/g extract), or 0.43 g/kg cocoa extract by oral garage. The animals were sacrificed on day 32. One testis and epididymis were removed and weighed. The epididymis was saved for the determination of epididymal sperm reserves. The remaining testis was fixed by whole body glutaraldehyde perfusion and processed for morphologic examination. A decrease in body weight gain and epididymal weights were observed in theobromine and high-dose cocoa-extract-treated groups. Theobromine and high-dose cocoa extract caused vacuolation within the Sertoli cell, abnormally shaped spermatids, and failed release of late spermatids in treated animals. Most of the vacuolations were found in the earlier and middle stage seminiferous tubules (stages I to VIII). However, the frequency of some parameters of testis alterations were significantly lower in the high-dose cocoa-extract-treated group compared to the theobromine-treated group. These data demonstrate the ability of a cocoa extract containing theobromine to alter testis structure in a similar pattern but with reduced intensity compared to that observed after oral exposure to pure theobromine. Key Words: theobromine; cocoa; Sertoli cell; vacuolations;multinucleate cells; testis morphology.

INTRODUCTION

reproductive tract of a cocoa extract that contains theobromine and other phytochemicals normally consumed with cocoa. It would be necessary to determine ifa cocoa extract containing a known amount of theobromine would cause an equivalent effect on the male reproductive tract compared to the theobromine alone. A variety of substances, such as carbohydrates, proteins, tannins, and other methylxanthines found in a cocoa extract may act synergistically with theobromine or inhibit theobromine's pharmacologic effects. These alterations of activity may occur through a variety of pharmacodynamic and pharmacokinetic mechanisms. Previous studies have demonstrated the ability of a substance to alter theobromine effects when mixed together with theobromine in the diet. A low protein diet enhances the severity oftheobromine toxicity (3), whereas, a high fiber diet may inhibit theobromine absorption or increase its metabolism and thus decreases theobromine toxicity in rats (6,7). In the present study, theobromine was quantitated in a cocoa extract. Animals were then dosed with pure theobromine or a cocoa extract containing an equivalent amount oftheobromine, and reproductive tract alterations of treated animals were evaluated.

Methylxanthines are found in a wide variety of foods and drinks, including coffee, cocoa products, and cola soft drinks. They are constituents of foods and condiments consumed by humans from childhood through old age. Theobromine is the primary methylxanthine in cocoa. Commercial cocoa powder contains about 2% theobromine, 0.2% caffeine, and trace amounts of theophylline (1). Previous experiments have investigated the effects of the pure methylxanthines on the male reproductive tract (2-6). Theobromine causes extensive damage to the rat testis when added to the feed (0.6% to 1%) for 4 to 75 weeks. The damage includes degeneration and necrosis of seminiferous tubular epithelium, multinucleate cell formation; interstitial tissue edema, and proliferation of arterioles. Body weight loss is also observed following the dosing of methylxanthines (2-5). However, none of the studies have investigated the effects on the male Address correspondence to Donald P. Waller, Ph.D., Department of Pharmacodynamics, M/C 865, University of Illinois at Chicago, Chicago, IL 60612. Received 25 February 1991; Revision received 9 January 1992; Accepted 19 January 1992. 347

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Reproductive Toxicology

MATERIALS AND M E T H O D S Preparation of cocoa extracts Cocoa powder (3.2 kg) distributed by DLDI, Inc. (Batavia, IL) was extracted by refluxing the cocoa powder in boiling 85% methanol (12 L, bp 66°C) for 15 min. The extract was filtered through Whatman # 1 filter paper and the residue washed with 4 L of boiling 85% methanol. The cocoa marc was re-extracted with 16 L of boiling 85% methanol following the same procedure. Methanol was then removed using a rotary evaporator and the residue dried by lyophilization. The final extract was weighed (566.5 g) and stored with Drierite in a refrigerator (4°C). Determination of theobromine concentration in cocoa extract by HPLC Theobromine concentration was determined according to the liquid chromatographic method described by Blauch and colleagues (8) using a Beckman liquid chromoatograph (Model I lOA with a 100A solvent delivery system and a Model 421 controller) with a Shimadzu SPD-6AV variable wavelength detector (sensitivity of 0.04 and a wavelength of 254 nm), and a Waters u-Bondapak C18 column (4 × 250 mm). The mobile phase was acetonitrile/water (8:92) with a flow rate of 2.0 mL/min. Theobromine was purchased from Fisher Scientific Co. (Itasca, IL). The internal standard j3-hydroxyethyltheophylline

Volume 6, Number 4, 1992

(¢3-HETP) was obtained from Sigma Chemical Company (St. Louis, MO). Crude cocoa extract (3.0 g) was refluxed in boiling water (115 mL) for 30 min. Internal standard (10 mL of 1 mg/mL/3-HETP) was then added and the mixture refluxed for another 30 min. The mixture was filtered through Whatman # 1 filter paper and refiltered through Nylong-66 filters (pore size 0.2 mm, Rainin Instrument Co. Inc., Woburn, MA). The concentration oftheobromine in the filtrate was then determined by HPLC. The retention time oftheobromine and/3-HETP in cocoa extract under our HPLC conditions were 5.5 and 11.3 min, respectively (Figure 1). The peak area ratio of theobromine to ~-HETP versus theobromine concentration was plotted as a standard curve (Figure 2). The concentration of theobromine in the cocoa extract was 117 mg/g. The recovery rate of the extraction was > 99%. Animal procedures Male Sprague-Dawley rats, 230 to 250 g, were randomly divided into 4 groups of 4 animals each. The animals were orally gavaged for 31 days using a stainless steel dosing needle. The groups were dosed with vehicle (5% gum acacia), 250 mg/kg oftheobromine, 2.14 g/kg of cocoa extract (equal to 250 mg/kg theobromine), or 0.43 g/kg of cocoa extract (equal to 50 mg/kg theobromine). On day 32, heparin (3.12 U/g body weight) was injected intraperitoneally and animals were anesthetized with 80 mg/kg body weight of pentobarbital. One testis and epididymis were removed, weighed, and saved for counting of epididymal sperm. Saline was then perfused through the heart of animals to remove most of the blood, followed by 5% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.4) to fix the tissues (9). The fixed testis was removed, diced into 1.5

"7"

I

. . . .

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Fig. 1. HPLC chromatogram of theobromine in cocoa extract with internal standard. Column: u-Bondapak C 18 column; Mobile phase: Acetonitrile:Water 8:92; Flow rate: 2.0 mL/min; Absorption: 254 nm; Internal standard: /3-hydroxyethyltheophylline (/3-HETP). 1: Theobromine, 2: ~HETP.

1.0

0.5

Q_

0.0 0.00

I

0.02

0.04,

Theobromine concentration

0.06

0.08

(mg/ml)

Fig. 2. HPLC standard curve for theobromine quantitation.

Theobromine and cocoa extract in male rats

cubes with dimensions of 1 mm, and fixed in the solution described above for an additional hour. Tissue cubes were washed in the buffer and postfixed in an osmium ferrocyanide mixture for 1 h (10). Subsequently, tissues were dehydrated in ascending concentrations of ethanol, infiltrated with propylene oxide, and embedded in epoxy (Araldite 212 Polyscienses, Inc., Warrington, PA). Five tissue cubes from each animal were processed. Sections (1 #m) were cut on an ultramicrotome, stained with Mallory's methylene blue and examined under a light microscope. A minimum of 50 tubules from at least three different plugs were evaluated from each animal for morphologic characteristics. Statistics ANOVA with Sheffr's test for significance were used to determine the P values for the reproductive tract parameters. Fisher's two-tailed test was used to examine the significance of testis morphologic alterations.

RESULTS The theobromine and high-dose cocoa extract groups exhibited a decreased body weight gain from day 16 of treatment (Figure 3). The rat epididymal weights were also decreased in these two groups (Table 1). A decrease in cauda epidydymal weight compared to controls was observed only in the highdose cocoa extract group. No changes in testis weight or sperm concentration were observed in any of the treated groups. Animals treated with theobromine had widespread vacuolation of the seminiferous epithelium (Figure 4B) and displayed frequent multinucleate cells (Figure 4C). Of the evaluated tubules, 18% had at least one vacuolation, and 7% had multinucleate cells. The frequencies ofvacuolation and multinucle-



WAN6 ET AL.

349

0 Control • Theobromlne (250ml/kli) tx Cocoa extract (0.43R/ki) • Cocoa extract (2,14s/kli)

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280

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Testis wt (g) Testis wt/BW (X 10-3) Epidid vet (g) Epidid wt/BW (X l0 -3) Cauda epid wt (g) Sperm count ( X 106) Sperm count/cauda epid wt ( × 106/g)

1.76 4.20 0.56 1.33 0.27 236.3 889.5

+ 0.20 + 0.57 + 0.04 _+ 0.13 + 0.02 + 88.6 _+ 375.7

*P < 0.05. CE50 = cocoa extract containing 50 mg/kg theobromine. CE250 = cocoa extract containing 250 mg/kg theobromine. TB250 = theobromine 250 mg/kg.

I

3"2

Days of T r e a t m e n t

Fig. 3. Body weight o f rats treated daily with t h e o b r o m i n e a n d c o c o a extracts. T h e d a t a was expressed as m e a n _+ S D (n = 4).

ate cell formation in theobromine-treated animals were significantly higher than in the control and cocoa extract groups. Most of the vacuolations (80%) were present in stage I to VIII seminiferous tubules. Close examination of vacuoles suggested that they were associated with the Sertoli cell and were probably intracellular, as has been described by Russell (11). Failed release of late spermatids and presence of abnormally shaped spermatids were observed in stages VIII to X tubules in this group (Figure 4C). The number of seminiferous tubules with failed release of late spermatids was significantly higher than in controis and the cocoa-extract-treated rats (P < 0.001). Abnormally shaped elongate spermatids showed irregular shapes with hooks, triangular, or squared sides. These were not observed in any control slides (Figure 4A). A similar pattern oftesticular alterations was observed in the high-dose cocoa-extract-treated group, but with a greatly reduced frequency. Vacuolation in

T a b l e 1. R e p r o d u c t i v e t r a c t p a r a m e t e r s o f rats t r e a t e d w i t h 250 m g / k g o f t h e o b r o m i n e o r 2.14 g/kg o f c o c o a extract for 31 days Control (n = 4)

I

CE50 (n) = 4) 1.69 4.34 0.5t 1.32 0.26 208.8 813.6

+ 0.17 +_ 0.35 + 0.06 _ 0.11 _+ 0.03 + 32.8 _+ 101.1

CE250 (n) = 4 1.68 4.67 0.44 1.22 0.19 150.0 827.1

+ + + + + + +

0.13 0.63 0.03* 0.15 0.03* 25.2 252.5

TB250 (n = 4) 1.64 4.50 0.46 1.26 0.22 131.3 605.1

+ 0.17 _+ 0.49 + 0.02* + 0.05 + 0.01 + 28.9 ___ 121.2

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Reproductive Toxicology

Volume 6, Number 4, 1992

Fig. 4. Alterations of testis morphology of rats treated with 250 mg/kg of theobromine or 2.14 g/kg of cocoa extract for 31 days. A Control testis (X 86). B Theobromine-treated testis (X 86); wide-spread vacuolation (V) in seminiferous tubular epithelium. C Theobromine treated testis (X 138); multinucleate cell formation (MNC), failed release of late spermatid (arrow), and abnormally shaped spermatids (arrow head). D High dose cocoa extract treated testis ( X 86); vacuolation (V) of seminiferous tubular epithelium was observed. E High dose cocoa extract treated testis (X 138); abnormally shaped spermatids (arrow head). F High-dose cocoa-extract-treated testis ( X 86); failed release of late spermatids (arrow).

T h e o b r o m i n e and cocoa extract in male rats • WANG ET AL. T a b l e 2. T e s t i c u l a r d a m a g e o f r a t s t r e a t e d w i t h 2 5 0 m g / k g o f t h e o b r o m i n e e x t r a c t f o r 31 d a y s

351 or 2.14 g/kg of cocoa

Control (n = 4)

CE50 (n = 4)

CE250 (n = 4)

TB250 (n = 4)

235 0 0 0 0 0 0 0

326 0 0 0 0 0 0 9a

291 11 b 0.04 9b 7 0 0 14a

253 113 ¢ 0.45 46 ¢ 37 23 ¢ 18¢ 20 b

N u m b e r ST counted Total n u m b e r o f vacuolations Number of vacuo/ST N u m b e r o f ST with vacuolation N u m b e r o f stage I to VIII ST with vacuo Total n u m b e r o f M N C N u m b e r o f ST with M N C N u m b e r o f ST with failed release

ST = seminiferous tubule. M n C = multinuclate cells. Vacuo = vacuolation. ap < 0.05 c o m p a r e d to the control group. bp < 0.05 compared to the control a n d low cocoa extract group. cp < 0.001 compared to the control a n d all-cocoa-extract group. O t h e r abbreviations as in Table 1.

the seminiferous epithelium (Figure 4D) was observed in this group, but only 3% of the evaluated seminiferous tubules had vacuolations with P less than 0.001 compared to 18% in the theobrominetreated group (Table 2). Similarly, the abnormally shaped spermatids (Figure 4E) were also observed at a lower frequency. No significant difference in the number of seminiferous tubules with failed release of late spermatids was observed between the high- (Figure 4F) and low-dose cocoa extract group (Table 2). No multinucleate cells were found in the high-dose cocoa extract group. The low-dose cocoa-extract-treated animals did not exhibit the extensive morphologic alterations observed in the other two treated groups. Only failed release of late spermatids was observed. The frequency of failed release of late spermatids in the evaluated seminiferous tubules was significantly higher than in the controls (Table 2). DISCUSSION An aqueous methanol extraction was utilized to provide a suspension with a reasonable volume for dosing. Aqueous methanol extracts most hydrophobic and hydrophilic biologically active compounds, whereas the relatively biologically inactive polysaccharides remain in the extract residues. Removal of these polysaccharides from the cocoa powder was necessary to reduce the viscosity of the cocoa extract solution, permit easy filtration, and provide a dosing suspension with a reasonable volume. Boiling methanol (bp 66"C) also closely approximates the temperature usually involved when consumers use cocoa in products such as hot chocolate. Also, the higher temperature increases the solubility of theobromine in

the aqueous methanol solution and results in a higher recovery rate. The final concentration of theobromine in the cocoa extracts was 117 mg/g extract, which is about a 6-fold increase in concentration compared to the original powder (1). In the present experiment, one group of animals was orally treated with 250 mg/kg of theobromine. This is approximately equivalent to the 0.5% dose of theobromine in the diet used by Weinberger and colleagues (2). Both the theobromine-treated group and the high-dose cocoa-extract-treated group had significantly lower body weights when compared to controis by the 16th day of dosing (Figure 3). There was no significant difference in the body weight of lowdose cocoa-extract-treated group compared to the control. Reduced weight gain is normal for theobromine-treated rats and usually occurs in a dose-dependent manner (2-5). The decrease in weight gain of the theobromine-treated rats was less than that observed in previous experiments of theobromine treatments in the diet. This was probably due to the difference in exposure time of the rats to the theobromine. Previous experiments included theobromine in the diet, resulting in the doses of theobromine being administered during the primary feeding time of the rat. In the current experiment the dosing was performed in the morning after the primary feeding period. The highest blood levels of theobromine would thus be attained in our experiment during a time when the rat is not engaged in feeding activities. In spite of this, the decreased weight gain was still observed in both the theobromine-treated and the high-dose cocoaextract-treated animals. Alterations in reproductive tissues were not observed for testis weight, caudal epididymal sperm count, or epididymal weight per body weight. The ab-

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Reproductive Toxicology

solute weights of the epididymides, however, were reduced in both the theobromine-treated group and the high-dose cocoa-extract-treated group. Utilization of reproductive tissue weight parameters to determine alterations in the reproductive tract is problematic. Frequently, high doses of test compounds are administered, which result in reduced body weights, and it becomes difficult to accurately determine early and minor alterations in the reproductive tissues based upon tissue weight only. The cauda epididymal weight was significantly different for only the highdose cocoa-extract-treated group. This decrease, however, is not an accurate predictor of early damage to the testes that may have led to a decrease in spermatozoal reserves. The dosing period was not long enough to allow a manifestation of alterations in the early stages of spermatogenesis. Sperm count in the cauda epididymis did not change, but a trend for decreased numbers in both the theobromine and highdose cocoa-extract-treated groups was present. Larger numbers of animals in the test groups may provide more accurate data and result in significant values. The possibility of damage in later stages ofspermatogenesis that results in decreased numbers of epididymal spermatozoa should be more closely evaluated. Morphologic examination of the testicular tissues in the test groups of this experiment supports previous results, which demonstrated damage to the male reproductive system by theobromine. Weinberger and colleagues reported that feeding male rats for 14 to 75 weeks with 0.5% theobromine mixed with animal diet caused severe testicular atrophy with aspermatogenesis or oligospermatogenesis (2). Tarka and colleagues reported that male rats fed for 28 days with a diet containing at least 0.6% theobromine had extensive and severe testicular damage, including seminiferous tubular cell degeneration, necrosis, and multinucleate cell formation (3) as well as interstitial tissue edema and arteriole proliferation (4). Similar results were reported by Gans and colleagues (5,6). In our experiment, 250 mg/kg of theobromine administered in a single daily oral dose by gavage, caused a wide-spread vacuolation in the seminiferous tubule epithelium, multinucleate cell formation, failed release of late spermatids, and abnormally shaped spermatids. The staging of the seminiferous tubules identified the presence of vacuoles primarily in earlier and middle stages (Stage I to VIII). In addition to the above effects, failed release of late spermatids and abnormally shaped spermatids observed in the group treated with the cocoa extracts were of the same pattern as testicular alterations observed previously (2-6) and in this experiment following treatment with pure theobromine. This is rea-

Volume 6, Number 4, 1992

sonably good evidence that the testicular injury caused by the cocoa extract is probably due primarily to the presence oftheobromine in the extract. The intensity of the testicular alterations caused by treatment with the cocoa extract, however, was greatly reduced compared to the effects caused by the administration of a similar amount of pure theobromine. Multinucleate cells were only observed in the animals treated with pure theobromine. Vacuolation, abnormally shaped spermatids, and failed release of late spermatids were observed in the high-dose cocoaextract-treated group containing 250 mg/kg of theobromine with a much lower frequency than the group treated with 250 mg/kg of pure theobromine. The decrease in testicular effects of theobromine when administered as a component of cocoa could be caused by a variety of mechanisms. Components in cocoa extracts may alter pharmacokinetic parameters of theobromine. There is a wide variety of compounds in the cocoa extract that may alter the bioavailability of the theobromine by direct chemical action such as binding or even the alteration of intestinal tract physiology. There may also be compounds that directly compete with theobromine for interactions with specific sites of testicular damage. Previous investigations demonstrated the ability of different diets to alter testicular damage caused by theobromine (6,7). The mechanisms by which theobromine effects are reduced through interactions with cocoa components should be further investigated. Failed release of late spermatids was observed in the cocoa extract and pure theobromine-treated groups. Similar failed release of late spermatids was observed by Ettlin and colleagues following very short exposure of rats to theobromine (12). Failed release of late spermatids may be one of the earliest signs of theobromine and cocoa extract toxicity. Release of late spermatids (spermiation) is highly associated with the normal function of Sertoli cells (11,13). In addition, most of the vacuolations in our studies appeared within the Sertoli cells. These data support Ettlin's conclusion that impairment of Sertoli cell function induced by theobromine may account for the early as well as later toxicity observed in rat testes (12). The mechanisms oftheobromine toxicity and reduced damage following the administration of cocoa extracts containing an equivalent amount oftheobromine needs further investigation. The ability of a cocoa extract to cause testicular damage, but at a greatly reduced level compared to the damage caused by the suspected compound, theobromine, provides evidence that the quantitative extrapolation of toxic effects of pure compounds cannot be directly applied

Theobromine and cocoa extract in male rats • WANG ET AL.

to the concentrations of the toxins contained within natural sources. This is of importance when attempting to determine the potential toxic effects of any natural product ingested either as food, as food supplements, or for medicinal purposes. REFERENCES 1. ShivelyCA, TarkaSM. Methylxanthine composition and consumption patterns of cocoa and chocolate products. In: Spiller GA, ed. The methylxanthine beverages and foods: Chemistry, consumption, and health effects. New York: Alan R. Liss; 1984:149-78. 2. Weinberger MA, Friedman L, Farber TM, et al. Testicular atrophy and impaired spermatogenesis in rats fed high levels of the methylxanthines caffeine, theobromine, or theophylline. J Environ Pathot Toxicol. 1978;1:669-88. 3. Tarka SM, Zoumas BL, Gans JH. Short-term effects of graded levels of theobromine in laboratory rodents. Toxicol Appl Pharmacol 1979;49:127-49. 4. Tarka SM, Zoumas BL, Gans JH. Effects of continuous administration of dietary theobromine on rat testicular weight and morphology. Toxicol Appl Pharmacol. 1981;58:76-82. 5. Gans JH. Comparative toxicities of dietary caffeine and theobromine in the rat. Food Chem Toxicol. 1984;22:365-9.

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6. GansJH. Dietary intluences on theobromine-inducedtoxicity in rats. Toxicol Appl Pharmacol. 1982;63:312-20. 7. Shively CA, White DM, Tarka SM. Diet-induced alterations in theobromine disposition and toxicity in the rat. Toxicol Appl Pharmacol. 1986;84:593-8. 8. Blauch JL, Takra SM. HPLC determination of caffeine and theobromine in coffee, tea, and instant hot cocoa mixes. J Food Sci. 1983;48:745-50. 9. Sprando RL. Appendix A: Perfusion of the rat testis through the heart using heparin. In: Russell LD, Ettlin RA, Sinha Hikim AP, Clegg ED, eds. Histological and histopathological evaluation of the testis. Clearwater, FL: Cache River Press; 1990:277-80. 10. Russell LD, Burguet S. Ultrastructure of Leydig cell as revealed by secondary tissue treatment with a ferrocyanide-osmium mixture. Tissue Cell. 1977;9:751-6. 11. Russell LD. Histopathology ofthe testes. In: Russell LD, Ettlin RA, Sinha Hikim AP, Clegg ED, eds. Histological and histopathological evaluation of the testis. Clearwater, FL: Cache River Press; 1990:210-66. 12. Ettlin RA, Armstrong JM, Buser S, Hennes U. Retardation of spermiation following short-term treatment of rats with theobromine. Arch Toxicol. Suppl. 1986;9:441-6. 13. Russell LD. Spermiation--the sperm release process: ultrastructure observations and unresolved problems. In: Blerkom JP, Motta PM, eds. Ultrastructure of reproduction. Hingham, MA: Martinus Nijhoff; 1984:46-66.

Reproductive toxicity of theobromine and cocoa extract in male rats.

The toxicities of theobromine and cocoa extract on the reproductive tract of male rats were compared in the present study. A cocoa powder extract cont...
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