Comparative effects of diethylhexyl phthalate or monoethylhexyl phthalate on male mouse and rat reproductive organs

TOXICOLOGY AND APPLIED PHARMACOLOGY 62, 12 1- 125 ( 1982)

Comparative Effects of Diethylhexyl Phthalate or Monoethylhexyl Phthalate on Male Mouse and Rat Reproductive Organs’ KAREN A. CURTO AND JOHN A. THOMAS* Department of Pharmacology and Toxicology, West Virginia University Medical Center, Morgantown, West Virginia 26506

Received May 28, 1981: accepted September 1, 1981 Comparative Effects of Diethylhexyl Phthalate or Monoethylhexyl Phthalate on Male Mouse and Rat Reproductive Organs. CURTO, K. A., AND THOMAS, J. A. (1982). Toxicol. Appl. Pharmacol. 62, 121-125. Sexually mature mice injected (ip or SC) with doses ranging from 1 to 100 mg/kg monoethylhexyl phthalate (MEHP) or 50 to 100 mg/kg diethylhexyl phthalate failed to exhibit any significant alterations in testicular weight or zinc levels. Further, no changes in mouse seminal vesicle or anterior prostate weight and zinc levels occurred. Rats injected with MEHP (50 mg/kg) showed a 37% decrease in prostatic zinc; DEHP (100 mg/ kg) caused a 33% decrease in prostatic zinc and a 31% decrease in gonadal zinc. These studies indicated that the reproductive system of the male rat is more sensitive to phthalate-induced toxicities than the mouse. Finally, phthalate acid ester-induced gonadal zinc depletion was evident despite the fact that the MEHP or DEHP was administered ip or SC,thus apparently eliminating altered intestinal absorption as the cause for the species differences.

Recent interest in the toxic effects of diethylhexyl phthalate (DEHP) and its principal metabolite monoethylhexyl phthalate (MEHP) centers on the action of phthalate acid esters (PAE) on the male gonad (Oishi and Hiraga, 1980a,b,c,d). Previously, Shaffer et al. (1945) observed testicular degeneration in rats receiving DEHP. Studies by Gray et al. (1977) examined the short-term toxicity of DEHP in rats and reported significant decreases in testicular weights of animals receiving 2% dietary DEHP for 6, or 17 weeks. In the same study, testicular weights from rats fed a 0.2% DEHP diet were not reduced, but histologically there was evidence of decreased spermatogenesis. Foster et al. ( 1980) reported that rats fed different n-alkyl phthalates showed varying degrees of gonadal damage depending upon

the particular phthalate. Of a number of PAEs studied, dietary DEHP or MEHP produced severe testicular atrophy in rats (Oishi and Hiraga, 1980a,b). The testicular concentrations of zinc were reduced by DEHP while gonadal testosterone levels were increased (Oishi and Hiraga, 1980a). Zinc concentrations in testes obtained from rats previously treated with MEHP were likewise reduced, but gonadal testosterone levels remained unchanged by this monoester (Oishi and Hiraga, 1980b). Dietary DEHP or MEHP also caused a reduction in testicular zinc in mice (Oishi and Hiraga, 1980c,d). The present study examined changes in testes and sex accessory weight as well as gonadal zinc and compared these effects in the rat and in the mouse injected with varying doses of DEHP or MEHP. Because DEHP is hydrolyzed in the gastrointestinal tract (Albro and Thomas, 1973), the present study used ip and SCroutes of administration.

I This work was supported by NIH Training Grant GM07039 and biomedical research grants. Z To whom all correspondence should be addressed. 121

0041-008X/82/010121-05$02.00/0 Copyright Co 1982 by Academic Press,Inc. All rights of reproduction

in any form rcscrwd.

122

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AND

METHODS Male Swiss-Webster mice weighing between 35 and 40 g from Hilltop Laboratory Animals (Scottsdale, Pa.) and male Sprague-Dawley rats weighing between 200 and 300g from Harlan Sprague-Dawley (Madison, Wise.) were used. Six animals were housed per cage. Rats were housed in hanging metal cages while mice were placed in polystyrene cages with hardwood chips as bedding. Food (Purina Rodent Laboratory Chow 5001) and water were available ad libitum. A 12-hr light cycle was maintained (0700 to 1900 hr) in the animal quarters. DEHP was purchased from ICN Pharmaceuticals, Inc. (Plainview, N.Y.). MEHP was supplied by Travenol Laboratories (Deerfield, Ill.). Experiments with DEHP employed a peanut oil vehicle while those experiments with MEHP employed a 0.9% NaCl vehicle. Control groups of animals received the appropriate vehicle (e.g., 0.9% NaCl or peanut oil). Mice received one of the following dose regimens: (a) daily SC injections of 1, 5, or 10 mg/kg MEHP for 5 days; (b) daily SC injections of 5, 10, or 20 mg/kg MEHP for 10 days; (c) daily ip injections of 50 or 100 mg/kg MEHP or DEHP for 5 days; or (d) alternate daily ip injections of 50 or 100 mg/kg MEHP or DEHP for 20 days (10 injections). Rats received the dose regimen as described in (d) above. None of these dose regimens caused any significant change between initial and final body weights when compared to vehicle-treated controls. Regardless of duration or dose regimens, all animals were killed by cervical dislocation 24 hr following the last injection. The testes, lobes of prostate, and seminal vesicles were dissected and weighed on a torsion balance. Sex accessory tissues analyzed for zinc were digested in concentrated nitric acid at room temperature overnight. The digests were diluted with 1 ml water and centrifuged at 1000 X g for 10 min. Zinc levels in the supernatant fractions were determined with a PerkinElmer 305B atomic absorption spectrophotometer operating with an acetylene flame and a zinc lamp source of 214.8 nm. Zinc values (ng/mg of tissue) were calculated on a wet weight basis. All results were analyzed for statistical significance by Dunnett’s test (Wirier, 1971).

RESULTS The effects of varying doses of MEHP (daily X 5 or 10, SC) upon mouse reproductive organs are presented in Table 1. Dose increments of 1, 5, or 10 mg failed to affect either prostate gland or seminal vesicle weights. Further, these doses exerted no ef-

THOMAS

feet upon sex accessory zinc levels (Table 1A). Since dose regimens of 1, 5, or 10 mg daily for 5 days exerted no alterations in testicular zinc, the duration of injection was doubled to 10 days (Table 1B). Extending the duration of treatment also failed to alter gonadal zinc levels. A comparison of the 10 mg/kg daily X 5 group (Table 1A) with the 5 mg/kg daily X 10 group (Table 1B), or treatment regimens where the two groups received the same total dose (viz., 50 mg/ kg), revealed no significant differences despite the longer duration of exposure to MEHP. Even with considerably higher doses of the PAEs, gonadal zinc remained unaffected by daily injections of either MEHP or DEHP (Table 2). However, an ip dose of 100 mg/ kg MEHP lead to 50% mortality (Table 2A). DEHP was not as toxic and no mortality was recorded in either the 50 or 100 mg/kg groups (Table 2B). In an effort to confirm the previously reported gonadal toxicity caused by high dietary levels of either MEHP or DEHP, experiments were conducted employing a longer duration of exposure (Table 3). Despite this more extended treatment period as well as a higher total dose, neither the sex accessory organs (viz., prostate gland or seminal vesicle) nor the testes of the mouse were affected by MEHP or DEHP (Table 3A). The effects of the PAEs on the rat (Table 3B), unlike the mouse, revealed significant reductions in both gonadal and prostate gland zinc. The prostate gland was particularly sensitive to the actions of either MEHP or DEHP, and organ concentrations of zinc were significantly reduced (p 5 0.05). Similarly, DEHP treatment resulted in a significant loss of testicular zinc in the rat (p I 0.05) (Table 3B). DISCUSSION Unlike several previous reports of phthalate-induced changes in the male reproduc-

PHTHALATES

EFFECTS

OF MEHP

Anterior

RODENT

TABLE

1

ON MOUSE

TESTES

prostate

Weight (mg)

Group

AND

Seminal

Zinc

Weight bid

Ww) A. 1, 5, or 10 mg/kg

Control 1 (6) 5 (6) 10 (7)

( 12)

28.6 32.8 34.0 34.4

+ 2 * I

3.1b 2.8 5.3 4.8

29.3 32.8 25.5 26.4

AND

2 8.4 zk 10.8 f 4.6 f 4.9

B. 5, 10, or 20 mg/kg

+ + 2 + MEHP

Control ( 12) 5 (6) 10 (‘3) 20 (12)

SEX ACCESSORY

daily 8.9 10.9 4.4 12.3

Testes Weight (4

X 5 days, 25.5 26.0 24.2 24.8

daily

ORGANS

vesicle Zinc (ng/md

MEHP

64.2 83.5 78.8 78.7

123

TESTES

f k + f

SC

3.5 3.6 2.3 5.5

X 10 days,

Zinc Wmg)

220.6 241.7 219.0 252.6

f f f f

36.5 21.4 16.5 20.6

20.0 21.4 20.6 25.7

k f + +

1.6 10.6 6.4 6.0

220.6 221.0 255.8 231.5

+ + f +

36.5 26.1 12.9 23.2

20.0 20.4 19.5 21.2

k k f +

1.6 0.9 1.3 1.6

SC

L?Number of animals/group. *X+ SEM.

tive system (Oishi and Hiraga, 1980a,b,c,d), the current findings indicated that lower dose regimens show few adverse effects on male mouse gonads or sex accessory organs. With regard to dose, and based upon earlier

TABLE

2

EFFECTS OF MEHP OR DEHP (50 DAILY X 5, ip) ON MOUSE TESTICULAR ZINC

or 100 mg/kg WEIGHTS

AND

CONCENTRATIONS

Weight b-4

w/kg

Zinc (ng/md

A. MEHP Control (6) 50 (6) 100 (3)’

251.5 216.8 219.3

r f f

22.7’ 22.4 16.6

11.7 + 1.6 19.3 f 0.9 16.0 -+ 2.5

B. DEHP Control 50 (6) 100 (6)

(6)

257.5 238.0 223.9

y Number of animals/group. ‘5f SEM. ’ Of the six mice injected, imen.

Ifr 28.2 + 18.5 k 18.1

three

18.6 f 20.2 f 19.8 f

1.5 1.8 0.9

died at this dose reg-

studies (Jaeger and Rubin, 1972) DEHP was found to leach from polyvinyl chloride plastic bags into either human or dog blood at a rate of 0.25 mg/ 100 ml/day for 2 1 days. For example, a whole-body transfusion of 20-day stored blood based on this rate of DEHP leaching would result in an iv administration of approximately 300 mg (or about 4 mg/kg infused over a period of a few hours). Such an example assumes that DEHP would be metabolized principally to MEHP (Albro et al., 1981). Therefore, in the present study (Tables 2 and 3), the amount of DEHP injected would approximate those of a whole-body exchange transfusion. Accordingly, even doses of MEHP much higher than this calculated amount of 4 mg/kg employed in a whole-body exchange transfusion failed to adversely affect the parameters measured in male mouse reproductive organs. In studies by Oishi and Hiraga (1980a,b, c,d), rats or mice were fed either MEHP or DEHP in amounts considerably higher (2% in diet) than those employed in the present studies. The probable daily ingestion of MEHP in the Oishi and Hiraga studies was

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AND

THOMAS

TABLE EFFECTS

OF EITHER

DEHP

Anterior

OR MEHP

MOUSE

OR RAT

Seminal

vesicle

prostate

Weight bd

Group

ON MALE

Zinc

3

Weight bd

(w/w9

REPRODUCTIVE

ORGAN

RELSPONS~ Testes

Zinc (w/md

Weight (mid

Zinc Ww)

A. Mice Control MEHP

(6) 50

(6) DEHP

29.5 f

3.5’

20.5

f

1.4

58.8

k

4.9

30.5 *

9.1

20.7

+

3.0

61.3

+

32.2 f

9.5

18.2 +

2.0

72.7

+

20.9

k

1.9

243.5

+

42.4

17.6 +

1.7

12.7

19.6 +

1.8

235.7

-+

28.2

17.7 f

1.4

13.7

17.8 k

0.6

261.0

f

40.6

16.9 +

1.6

100

(6)

B. Rats Control MEHP

(6) 50

(6) DEHP (5)

113.0? 92.8 f

19.1

24.3

f

4.2

236.8

t

19.4

19.1 t

4.2

3657.0

+

308.0

24.2 _t 5.4

12.2

15.3 rf: 1.8’

222.5

f

29.8

15.7 +

2.8

3421.0

k

160.0

26.5

+

8.6

5.7

16.0 _t 4.7’

242.6

f

24.8

17.2 k

1.9

3410.8

i:

402.0

16.7 k

4.2’

100

Note. Animals

109.4

+

were injected a Number of animals/group. *Ek SEM. ’ Significantly different from

(ip)

every

appropriate

other

day

control

for a period

of 20 days.

(p 5 0.05).

approximately 4000 mg/kg assuming complete absorption over a 24-hr feeding period. This large dose of 4 g/kg may explain the gonadal toxicity observed in rats and mice. Dietary zinc deficiency reportedly depresses growth and development of rat testes, inhibits sex accessory organ weight, and produces severe atrophy of germinal epithelium (Millar et al., 1958; 1960). Experimental zinc deficiency in man causes oligospermia and can adversely affect testicular function. Such gonadal toxicities can be reversed when adequate amounts of zinc are replaced in the diet (Abbasi et al., 1980). Likewise, certain phthalates can cause a reduction in reproductive organ zinc. Rat testicular zinc levels are lowered by dietary DEHP (Oishi and Hiraga, 1980a) and by dietary MEHP (Oishi and Hiraga, 1980b). In their studies (Oishi and Hiraga, 1980a,b), it is possible that these toxic effects of the phthalate acted by interfering with gastrointestinal absorption of zinc. A number of dietary constituents,

including calcium and phytate, interfere with zinc absorption (Halstead et al., 1974). The present study avoided the oral route of administration in an effort to exclude phthalate-induced reduction in the gastrointestinal absorption of zinc. Although the mechanism of zinc depletion in the present study is unresolved, it may be explained by the formation of a zinc-phthalate complex similar to that described by Foster et al, (1980) where 65Zn chromatographed with the major urinary metabolite of dibutyl phthalate following treatment to rats. Despite phthalate-induced zinc reductions in the rat gonad, the mouse testes and prostate were more resistant to such depletion in this divalent ion. Somewhat similar results have been reported (Oishi and Hiraga, 1980d) where the authors suggested that mice may be protected more from exposure to PAE because of their higher basal zinc levels than rats. However, in the present study, a comparison of basal levels of zinc

PHTHALATES

AND RODENT

in mouse and rat testes revealed essentially the same level. Zinc is normally present in relatively high concentrations in sex accessory organs, particularly in the prostate where it selectively concentrates (Gunn et al., 1955). The present study is unique in reporting phthalateinduced zinc depletion in the rat prostate. Studies are planned to compare the effects of this depletion with instances of known zinc reductions in the prostate such as in prostatitis (Marmar et al., 1975). Saito et al. (1967) postulated that the prostate in the dog may provide a reservoir of zinc for the sperm. Zinc content in canine epididymal spermatozoa was much less than in seminal plasma and when epididymal sperm were bathed in ZnCl,, their motility was enhanced to a level similar to that of ejaculated sperm (Saito et al., 1967). Phthalate-induced zinc reduction of the prostate might, therefore, interfere with sperm motility and hence could impair reproductive capability. ACKNOWLEDGMENTS The authors acknowledge the scientific comments of senior scientists at Travenol Laboratories, and Dr. Leo Martis for the monoethylhexyl phthalate.

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