Theobromine toxicity on sertoli cells and comparison with cocoa extract in male rats

Toxicology Letters, IO (1994) H-164 0 1994 Elsevier Science Ireland, Ltd. All rights reserved

TOXLET

155 0378-4274/94/307.00

02972

Theobromine toxicity on Sertoli cells and comparison with cocoa extract in male rats

Ying Wang and Donald P. Waller Program for the Collaborative Research in the Pharmaceutical Sciences, University of Illinois at Chicago, Chicago, IL (USA) (Received (Accepted

5 January 1993) 7 March 1993)

Key words: Theobromine;

Cocoa

extract;

Sertoli cell; Testes

SUMMARY The target cell(s) of theobromine toxicity on rat testes and reproductive toxicity induced by pure theobromine and cocoa extract are evaluated in the present studies. Theobromine (500 mg/kg x 7 days) inhibited body weight gain in treated

rats. Decreased

cauda epididymal

sperm reserve (38%), seminiferous

tubule

fluid (STF) volume (33%), lactate concentration in STF (22%), inhibition of binding activity of androgen binding protein (ABP, 21%) and reduced ABP content in STF were also observed in theobromine-treated animals.

Cocoa extract

containing

an equivalent

amount

of theobromine

did not produce

significant

toxic-

ity in treated rats. Theobromine concentrations in serum and testes from pure theobromine-treated rats were 1.8- and 1.6-fold higher, respectively, than that in rats treated with cocoa extract. The results support Sertoli cells as the primary target cells of theobromine toxicity. The lower theobromine concentrations in serum and testes of cocoa extract-treated

rats could account

for the lower toxicity

in these animals.

INTRODUCTION

Theobromine is a primary methylxanthine in cocoa products which are consumed in large quantities by humans from childhood through old age. Commercial cocoa powder contains 2% theobromine, 0.2% caffeine and trace amounts of theophylline [l]. Large dosages and long-term dosing of pure theobromine produces severe morphological alterations in rat testes. Animals fed a diet containing 0.551% theobromine for 4 to 75 weeks were found to have reduced semen output and concentration, degeneration and necrosis of spermatogenic cells, multinucleate cell formation, inter-

Correspondence to: Donald P. Waller, Ph.D., Department Illinois at Chicago, Chicago, IL 60612, USA.

of Pharmacodynamics,

M/C 865, University

of

1.56

stitial tissue edema and proliferation of arterioles. Spermatogenic cells were considered the primary target cells of theobromine toxicity [2-51. Spermatogenesis is a stepwise process and spermatogenic cells are highly associated with each other and are dependent on the normal function of support cells f&7]; Large dosage and long-term period dosing of reproductive toxin will destroy the germ cells at all stages and/or support cells, and make it hard to identify the initial site of action. In 1986, Ettlin and colleagues treated rats with theobromine for a short period and obtained contrasting conclusions to previous work. The minor sloughing of germ cells and retarded release of late spermatids were observed in the rats treated with 500 mglkg of theobromine for 3 to 5 days. Based on these observations, they suggested that Sertoli cell toxicity induced by theobromine treatment may account for its early as well as late effects on the testes [8]. The evidence from this lab in rats treated with pure theobromine and cocoa extract suggested that Sertoli cells seemed to be the primary target cells of theobromine toxicity. Cocoa extract containing an equivalent amount of theobromine produced the same pattern of testicular injury as pure theobromine, but the intensity was greatly reduced [9]. Theobromine toxicities can be altered by mixing with different diets fed to animals. Tarka et al. reported that a low protein diet enhanced theobromine toxicity [3]. Rats fed with a pulverized commercial (high fiber) diet mixed with theobromine had a lower serum ~on~ntration of theobromine than the rats fed with a semisynthetic (lower fiber) diet containing the same quantity of theobromine. The author speculated that the semisynthetic diet might cause almost complete absorption of added theobromine. The pulverized commercial diet, however, contained more fiber and other materials which might limit the absorption of theobromine and thereby reduced its bioavailability [lo]. However, Shively and colleagues found that the lower theobromine concentration in serum of rats fed with commercial chow mixed with theobromine was due to the higher rate of theobromine metabolism and clearance in these animals compared to that in rats fed with a semisynthetic diet containing equivalent amounts of theobromine [l 11. A variety of substances, such as organic acids, proteins, tannins and other methylxanthines are found in cocoa [I]. These compounds may interact with theobromine through physical/chemical mechanisms such as co-solvent, binders and buffers. They may also act pharmacologically with synergistic and antagonistic actions. Therefore, the activities and/or toxicities of theobromine in a cocoa product may be altered compared to that induced by pure theobromine. To further elucidate the target cell(s) of theobromine toxicity and compare the toxicity of pure theobromine with cocoa extract, effects of theobromine and cocoa extract on biochemical functions of rat testes after short-term treatment were evaluated by examination of(i) reproductive tract parameters, (ii) testicular enzyme activity, (iii) seminiferous tubule fluid (STF) volume, lactate concentration, androgen binding protein (ABP) binding activity and content in STF, and (iv) theobromine concentrations in serum and testes of rats treated with pure theobromine or cocoa extract.

157

MATERIALS AND METHODS

materials Theobromine was purchased from Eastman Kodak Co..(Rochester, NY). Cocoa powder distributed by ALDI Inc. (Batavia, IL) was obtained from a local food store. Cocoa extract was prepared using 85% boiling methanol and the theobromine concentration in the cocoa extract was determined by HPLC as described by Wang et al. [9]. 5a-dihydro[1,2,4,5,6,7-3H]testosterone ([3H]DHT, specific activity 106.8 Ci/ mmol) was obtained from Amersham Co. (Arlington Heights, IL). Animals Male Sprague-Dawley rats, 250-270 g, were purchased from Sasco King Animal Lab (Oregon, WI) and randomly divided into control, theobromine- and cocoa extract-treated groups of six animals each. Animals were orally gavaged for 7 days with vehicle (5% gum acacia), 500 mg/kg theobromine or 4.28 g/kg cocoa extract (117 mg theobrominelg extract). The dosing volume was 1 ml/l00 g body weight. Three hours after the last dosing on day 8, animals were anesthetized with diethyl ether and the blood was withdrawn by cardiac puncture. Serum was prepared and saved for theobromine quantitation. Animals were then sacrificed and the epididymides were removed, weighed and saved for sperm counting. The testes were weighed and saved for determination of enzyme activities, lactate concentration, ABP binding activity and theobromine concentration. Assay of testicular enzyme activities Activities of testis sorbitol dehydrogenase (SDH), lactate dehydrogenase (LDH), ~-glycerophosphate dehydrogenase ((r-GPDH) and isocitrate dehydrogenase (ICDH) were determined according to the method of Shen and Lee [12]. Determination of lactate concentration, ABP binding activity and content in STF STF was prepared following the method described by Turner and colleagues [ 131. Protein concentration in STF was determined by the method of Lowry [14]. Lactate concentration in STF was determined by an enzyme method using an assay kit provided by Sigma Chemical Co. (St. Louis, MO}. Binding activity of ABP in STF was determined according to the method described by Ritzen [15]. Briefly, STF samples (800 lug protein) from control and treated rats were loaded on the top of 7% polyacrylamide disc gel (5 x 40 mm) containing 5 nM 13H’JDHT.The samples were run at 3 mA/tube and 4°C for 2 h. Following electrophoresis, the gels were sliced transversely into 2-mm thick slices and placed into counting vials containing 5 ml of scintillation fluid (RPI Co., Mont Prospect, IL) to measure the radioactivity. The maximum radioactive value from each sample was used to express ABP binding activity. ABP content in STF was examined using polyacrylamide slab gel electrophoresis using the method of Musto et al. [ 161.STF samples (300 ,ug) from control and treated rats were loaded on a SDS-free polyacrylamide gel containing 3.5% staking and 7% separation

158

gels. The samples were run under 75 V for 1 h, 50 V for 1 h and 25 V for 25 h. ABP band was identified based on its molecular mass (85 kDa) [16]. MW-SDS-6H kit (Sigma Chemical Co.) was used as protein standard.

Determination of theobromine concentrations in serum and testes Concentrations of theobromine in serum and testes were determined by HPLC. Serum (50 ~1) and testes (0.1 g) from control and treated rats were mixed with 200 ,ul and 150 ~1 of /?-hydroxyethyltheophylline (J-HETP, 0.4 mg/ml in ethanol), respectively. The mixture was extracted by 5 ml chloroform vortexing at full speed for 30 s and then centrifuged at 600 x g for 10 min. The supernatant (4 ml) was removed and dried by N, in a 37°C water bath. Mobile phase (100 ,~l; CH,CN/H,O, 8:92) was added to the dried residues and heated at 45°C for 30 min. Twenty microliters of the extract was injected into HPLC. HPLC assay was performed using a Beckman liquid chromatography (Model 110A and 1OOA solvent delivery system and a Model 421 controller) connected to Shimadzu SPD-6AV variable wavelength detector and a Waters y-Bondapak Cl8 column (4 x 250 mm). The mobile phase was acetonitrilei water (8:92) with flow rate of 2.0 ml/min. Theobromine (0.040.14 mg/ml) and internal standard (J?-HETP) were mixed with 50 ~1 of serum or 0.1 g of testes from untreated rats to prepared standard curve.

Statistical analysis The data were expressed as mean * standard deviation (SD). ANOVA with Scheffe’s multiple-comparison test was used to test for significance. A value of P less than 0.05 was considered as statistically significant. RESULTS

There was a slight, but statistically significant inhibition of body weight gain in theobromine-treated rats starting from day 2 of dosing until the day of sacrifice (Fig. 1). Theobromine treatment caused a 38% decrease of cauda epididymides sperm reserve in treated rats compared to the animals treated with vehicle (Table I). A significant alteration of testis biochemical functions was also observed in theobromine-treated rats. Theobromine produced a 33% decrease of STF volume, 22% decrease of lactate concentration and 21% decrease of ABP binding activity compared to the control (Table II). PAGE results showed that the density of ABP band from theobromine-treated rats was smaller and lighter than that from control and cocoa extract-treated animals, which indicated a lower ABP content in STF in theobrominetreated rats (Fig. 2). No significant changes in testicular enzyme activities were found in theobromine treated animals (Table III). Cocoa extract containing equivalent amounts of theobromine did not produce significant reproductive toxicity in treated rats. No significant differences in body weight gain and reproductive tract parameters were observed between control and cocoa extract-treated animals. STF volume, lactate concentration and ABP binding

159

450

250

I 0

1

2

3

Day

Fig. 1. Body weight of rats treated

4

5

6

7

8

of treatment

with 500 mg/kg of theobromine 7 days (n = 6). Control

(A) or 4.28 g/kg of cocoa extract

(0) for

(0). *P < 0.05.

activity in cocoa extract-treated rats were not significantly different compared to the control (Table II). Cocoa extract treatment increased testis protein concentration 21%, SDH and LDH activities 26% and 31%, respectively. However, no significant differences in a-GPDH and ICDH activities were observed between control and cocoa extract-treated animals (Table III). Theobromine concentrations in serum and testes from rats treated with pure theobromine were 1.8- and 1.6-fold higher, respectively, than that in rats treated with cocoa extract (Table IV). No theobromine could be detected in the serum and testes from control rats.

TABLE

I

EFFECT OF THEOBROMINE (500 mg/kg) AND COCOA EXTRACT 7 DAYS ON RAT REPRODUCTIVE TRACT PARAMETERS Control

(n = 6)

Theobromine

(4.28 g/kg) TREATMENT

(n = 6)

Cocoa

extract

(n = 6)

Testes wt.

3.44 f

0.08

2.97 f

0.55

3.18 f

0.19

(8) Epidid wt.

0.82 f

0.15

0.71 +

0.09

0.75 f

0.11

(9) Cauda

0.37 *

0.07

0.31 f

0.06

0.30 f

0.06

241.67 f

66.61

211.67 f

26.58

epidid wt.

(g) Sperm count (x 106) Sperm count/cauda (x 106/g) *P < 0.05 compared

epidid

to control.

682.56 f 157.06

150.0

f

26.08*

495.84 f 118.31*

724.15 f 196.3

FOR

160

C

TB

S

CE

ABP,

Fig. 2. Polyacrylamide

gel electrophoresis

of seminiferous

tubule

fluid of rats treated

theobromine or 4.28 g/kg of cocoa extract for 7 days. The electrophoresis Materials and Methods. C = control; TB = theobromine treatment; S = standard

with 500 mg/kg

of

was performed as described in the CE = cocoa extract treatment;

protein.

DISCUSSION

Rats treated with 500 mg!kg of theobromine for 7 days exhibited a significant decrease in body weight gain, which was consistent with our previous results [9] and

TABLE

II

EFFECT OF THEOBROMINE (500 mg/kg) AND COCOA EXTRACT (4.28 g/kg) TREATMENT 7 DAYS ON RAT BIOCHEMICAL PARAMETERS OF SEMINIFEROUS TUBULE FLUID Control Vol. STF (ml) Vol. STF/testes

Theobromine

(n = 6)

Cocoa

extract

0.27 f

0.05

0.18 f

0.06*

0.21 *

0.02

0.15 f

0.03

0.12 f

0.03

0.13 *

0.02

(ml/g) STF protein

66.66 f 15.98

72.81 f

8.02

70.02 2

7.49

(mg/ml) STF lactate

17.08 f

13.33 *

0.77*

15.15 *

0.99

(mg/dl) ABP binding

wt.

(n = 6)

activity

81.24 f 10.60

(cpm x 103) *P < 0.05 compared

3.52

to control

64.19 + 11.04*

81.76 * 11.43

(n = 6)

FOR

161

TABLE

III

EFFECT 7 DAYS

OF THEOBROMINE

(500 mg/kg) AND COCOA

ON RAT TESTICULAR Control

Testis protein

ENZYME

EXTRACT

(4.28 g/kg) TREATMENT

FOR

ACTIVITIES

(n = 6)

Theobromine

(n = 6)

Cocoa

extract

(n = 6)

27.09 f

1.53

28.88 +

2.01

32.80 rt

3.97*

63.40 +

9.12

70.99 +

6.09

79.78 +

4.87*

(mg/g tissue) SDH (,ffmoYmg/min) LDH ~mo~rn~rnin)

405.28 + 98.15

474.95 t 52.89

a-GPDH ~mo~rn~rnin)

38.32 f 12.00

34.60 + 11.93

33.73 i 13.94

ICDH ~mo~rn~rnin)

14.25 +

14.70 f

10.96 2

*P < 0.01 compared

5.23

531.84 + 39.09*

2.11

3.46

to control.

literature reports [224,10]. Theobromine directly inhibits appetite in treated animals to cause body weight loss. Results from pair-fed studies demonstrated that inhibition of body weight gain induced by theobromine was not the reason for the observed testicular damage [3,10]. Chapin et al. reported that commonly measured reproductive tract parameters of rats were not uniquely sensitive to the decrease of body weight [17]. It is likely, that the dietary restriction induced by theobromine treatment, which resulted in the decreased body weight gain in treated animals, is not the cause of testicular atrophy. Testicular damage induced by theobromine treatment is specifically related to theobromine, and not a secondary effect through the body weight loss

1101. Alterations of testicular enzyme activities are associated with changes in numbers of spermatogenic ceil types. Increased SDH and LDH activities were observed in the animals treated with procarbazine along with a decrease of a-GPDH and ICDH

TABLE

IV

THEOBROMINE THEOBROMINE

CONCENTRATIONS IN SERUM AND TESTES FROM RATS TREATED (500 mglkg) AND COCOA EXTRACT (4.28 g/kg) FOR 7 DAYS Theobromine

Theobromine (500 mg/kg) Cocoa

106.13 + 19.28

extract

(4.28 g/kg) Theobromine/cocoa *P < 0.0005 compared

in serum (,ug/ml)

60.03 + extract to theobromine

1I .49*

1.77 treatment

Theobromine 79.36 * 9.86 49.74 f 6.30* 1.60

in testes @g/g)

WITH

162

activities. It could be attributed to the loss of premeiotic spermatocytes, and a subsequent increase in the proportion of postmeotic cells in seminiferous tubules [18]. Testicular enzyme activities have, therefore, become a marker for certain type of spermatogenic cell injury [19]. No significant changes in testicular enzyme activities were observed in theobromine-treated rats. The results indicated that spermatogenic cells were not greatly affected by theobromine under the current dosing conditions. STF secreted by Sertoli cells is important for spermatogenesis and sperm transposition from testes to epididymides. There is no blood supply to the germ cells in seminiferous tubules. STF is the only medium to maintain the required environment (constant pH) and transport nutrients (lactate and hormones) required for the spermatogenesis. Mature spermatozoa in the testes are not motile. They are transported to epididymides by the flow of STF and seminiferous tubule contractions [6,20]. Reduced STF volume in theobromine-treated rats indicated theobromine toxicity on Sertoli cells. The lower STF volume may also account for the decreased sperm reserve in cauda epididymides of treated rats. Spermatogenic cells can not directly utilize glucose as a substrate for their energy metabolism. They must use lactate provided by Sertoli cells [21l23]. Lactate concentration in STF is another important marker for the Sertoli cell function. It has been used to monitor Sertoli cell function in vitamin A deficient rats [24]. Theobromine treatment significantly decreased lactate concentration in STF, indicating abnormal functions of Sertoli cells induced by theobromine treatment. Sertoli cells produce ABP, a protein to facilitate spermatogenesis in testes and sperm maturation in epididymides. The major function of ABP is to bind androgens and maintain the high concentration of androgen in the testes. Another function of APB is to transport androgen from the testes to the epididymides, where it plays an important role in the maturation of spermatozoa [25]. Theobromine significantly inhibited ABP binding activity in treated rats. Slab-PAGE results indicated that decreased ABP binding activity in theobromine-treated rats was due to the lower ABP content in their STF. Similar results were also obtained in rats treated with 500 mg/kg of theobromine for 3 days and sacrificed on day 10 after onset of dosing (data not showed). These data suggest that Sertoli cells are the primary target cells of theobromine toxicity in rat testes. No significant changes of total protein concentration in STF were found between control and theobromine-treated rats. Theobromine may selectively inhibit ABP production or secretion from the Sertoli cells. There was a trend in the decrease of testis and epididymal weight, sperm reserve, STF volume and lactate concentration in cocoa extract-treated rats. The changes, however, were not statistically significant, indicating that theobromine toxicity was greatly reduced when administrated to the animals in a cocoa extract form. These are consistent with our previous results in the morphological examinations [9]. An increased testis protein concentration, SDH and LDH activities were observed in cocoa extract-treated rats, but a-GPDH and ICDH activities were not affected. These data indicated that germ cells were not greatly affected by cocoa extract treatment. Un-

163

changed enzyme activity in theobromine-treated rats also give evidence that germ cells are not the primary target ceils of theobromine toxicity. The peak concentration of theobromine in rat blood appears to be around 3 h after a single dose (100 mg/kg) of theobromine treatment [26]. Based on this result, theobromine concentrations in the serum and testes of rats treated with theobromine or cocoa extract were determined 3 h after the last dosing. Theobromine concentrations in the serum and testes from theobromine-treated rats were significant higher than animals treated with cocoa extract. The lower serum and testis concentrations of theobromine in cocoa extract-treated animals could account for the lower toxicity observed in these animals. Gans reported that high fiber diet may reduce absorption of theobromine [lo]. High fiber diet could also increase the metabolism and clearance of theobromine [l 11.The cocoa extract used in the present study was prepared using 85% methanol [9]. Extraction of cocoa powder with 85% methanol can remove most of the water and alcohol insoluble polysaccharides such as cellulose. Moreover, all animals in the present experiment were fed with same commercial chow. Therefore, the lower theobromine concentrations in the serum and testes from cocoa extracttreated rats are not due to the action of fiber in the diet or cocoa extract. The different theobromine concentrations between cocoa extract and pure theobromine-treated animals is probably due to the actions of the components in the cocoa extract. Cocoa extract contains a variety of biologically active compounds such as organic acids, tannins and nitrogenous compounds. These compounds may stimulate theobromine metabolism and elimination by increasing the activities of liver cytochrome P-450 enzymes. In conclusion, the present results give evidence that Sertoli cells are the primary target cells of theobromine toxicity. Cocoa extract containing an equivalent amount of theobromine did not produce significant toxicity in treated rats. The lower theobromine concentrations in the serum and testes of cocoa extract-treated rats could account for the lower toxicity in these animals. REFERENCES 1 Shively, C.A. and Tarka, SM. (1984) Methylxanthine composition and chocolate products. In: G.A. Spiller (Ed.), The Methylxanthine Consumption, 2 Weinberger,

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