Enhancing effects of luteinizing hormone-releasing hormone on testicular damage induced by di-(2-ethylhexyl)phthalate in rats

271

Enhancing effects of luteinizing hormone-releasing hormone on testicular damage induced by di-(2-ethylhexyl)phthalate in rats

Shinshi Oishi

K~J‘ nor&: Testicular

Di-(2-ethylhexyl)phthalate;

Luteinizing

hormone-releasing

hormone;

Co-administration:

atrophy

SUMMARY The administration mone-releasing

of

hormone

1.5g/kg of di-(2-ethylhexyl)phthalate (LRH)

to male Crj:Wistar

static gland weights.

Co-administration

dent with decreases

in zinc and sulfhydryl

testicular

specific lactate

administration triglycerides

of DEHP

dehydrogenase alone.

and phosphoIipids)

of DEHP

and LRH,

concentration

isozyme.

50 or IO @g/kg of luteinizing

however.

induced

were similar

and hypolipidemia

sometimes

testicular

in the testis and reduction

These changes

Liver enlargement occurred

(DEHP),

hor-

1 week did not affect their testicular and pro-

rats for

after co-administration

atrophy

coinci-

of the activity

of

to the results of high-dose

(reduction

of serum cholesterol,

of DEHP

and LRH.

1NTRODIJCTION

Phthalic acid esters are used as plasticizers for a wide range of synthetic polymers. The health hazards of these compounds have been reviewed recently [I]. Recent interest in their toxic effects has focussed on their carcinogenic~ty and action on the male gonads (Conference on Phthalic Acid Esters, Guildford, 1984). Di-(2-ethylhexyl)phthalate (DEHP) is the phthalic acid ester most extensively used in polyvinyl chloride manufacture and is known to be widely distributed in the environment [2]. DEW has a low acute toxicity in experimental animals [3, 41, but it

Address

for correspondence:

Laboratory

of Public Health,

0378-4274/89]$3,50

Shinshi Oishi, Department 3-24-1, Hyakunincho,

of Toxicology.

Shinjuku-ku,

@ 1989 Elsevier Science Publishers

Tokyo

B.V. (Biomedical

Tokyo

Metropolitan

169, Japan.

Division)

Research

has been reported to have antifertility, mutagenic and carcinogenic effects in rodents at high dose levels [4, 51 and its administration to rats has been shown to cause testicular atrophy lar atrophy,

[6 91. Since the monoester it has been suggested

the gonadotoxic

metabolite

of DEHP

that this metabolite

also produces

of DEHP

testicu-

is responsible

for

effects [lo, Ill.

Testicular atrophy induced by DEHP also induced an alteration of testosterone levels in serum and testis [Xl. Furthermore, testicular venous plasma testosterone concentrations were significantly lower in DEHP-treated rats than in controls both before and after stimulation of human chorionic gonadotrophin (hCG), although hCG increased testosterone levels in serum and testes [ 121. The purpose of this study is to determine the effects of luteinizing hormone-releasing hormone on the testicular damage incurred by DEHP. MATERIALS

AND

METHODS

Chemids DEHP was purchased from Tokyo Kasei Kogyo Ltd., Tokyo. Its chemical purity was found to be greater than 99% by gas-liquid chromatography. Luteinizing hormone-releasing hormone (LRH) was purchased from Protein Institute, Protein Research Foundation, Osaka, and 2-keto-n-valeric acid was obtained from Tokyo Kasei Kogyo Ltd.. Tokyo. Nitric acid and perchloric acid were used for atomic absorption analysis grade produced by Wako Pure Chemicals Industries Ltd., Osaka. Other reagents were purchased from Wako Pure Chemicals Industries Ltd., Osaka. Animuls and trecltments Male Crj:Wistar rats were obtained

from Charles

River Japan

Inc., Kanagawa.

The animals were housed, 3 or 2 per cage, in suspended wire-floor cages equipped with an automatic water supply system and were given constant access to standard laboratory diet (CE-2, Clea Japan Inc., Tokyo). Environmental conditions were as follows: temperature, 23325 ‘C; relative humidity, 50&60%; 12-h light-dark cycle. Rats at 35 days of age, weighing 100&l 10 g (mean k SD, 105 f 4.05) were divided into 6 groups, each containing 5 rats. The experimental schedule is summarized in Table I. DEHP was administered without a vehicle. LRH was dissolved in saline solution to prepare the dosing solution in total volumes of 2.0 ml/kg body weight. Each chemical was administered daily for 7 days, and the rats were killed by decapitation 24 h after the last administration. Blood was collected from the wound, and the fresh weights of testis, ventral prostate and liver were recorded.

For determination of zinc concentration in the testis, the tissue was wet digested in 1 ml each of nitric acid and perchloric acid, and the concentration was then measured with a Perkin-Elmer Model 4000 atomic absorption spectrometer [8].

273

TABLE

I

EXPERIMENTAL

GROUPS

Group

AND ADMINISTRATION No. of rats

ROUTES

Administration

AND MATERIALS

materials

p.0.

i.p.

Control

5

saline (2 ml/kg)

saline (2 ml/kg)

LRHSO

5

saline (2 ml/kg)

LRH (50 fig/kg)

LRH IO

5

saline (2 ml/kg)

DEHP

5

DEHP (1.5 g/kg)

LRH (10 pg/kg) saline (2 ml/kg)

5

DEHP(l.5g/kg)

LRH (50 pg/kg)

5

DEHP (1.5 g/kg)

LRH (10 pg/kg)

DEHP+

LRHSO

DEHP+LRHlO _

A portion of the testis was homogenized in IO volumes of ice-cold 0.05 M phosphate buffer (pH 7.3) and aliquots were used to assay total sulkydryl content by the method of Sedlak and Lindsay [ 131. The crude homogenate was centrifuged at 10 000 x g for 30 min at 4°C and the supernatant was used to assay lactate dehydrogenase isozyme X (LDH-X) by the method of Meistrich et al. [14]. The protein concentration of samples was determined by the method of Bradford [I51 using crystalline bovine serum albumin as standard. The concentrations of triglycerides, total and free cholesterol, and phospholipids in serum were determined using diagnostic kits purchased from Wako Pure Chemicals Industries, Ltd., Osaka. Statistics

The results were evaluated by Bartlett’s test and if they were not significant, the data were analyzed by ANOVA and compared by Duncan’s multirange test. If the results were significant by Bartlett’s test, the treatment means were compared by the distribution free multirange test [ 161. All P-values less than 0.05 were considered statistically significant. RESULTS

Fig. 1 demonstrates the changes in the final body weight and relative organ weights of rats administered DEHP and/or LRH. Doses of DEHP or LRH alone did not affect body weight gain and organ weights except for the liver weight of the DEHP group. However, the combined administration of DEHP and LRH significantly lowered the weights of the testes and prostate compared to those of the control, DEHP or corresponding treatment groups. Zinc and SH concentrations, and LDH-X activity of testes are shown in Fig. 2. The zinc concentration in the testes was not affected by DEHP administration, but that in the group treated with the combined doses of DEHP and higher dose of LRH

120 5 3 100 g U % M

80

q DEHP+LRH50

60

N DEHl+I.RHlO

40

0

Fig

Body

weight

Testis

Prostate

Liver

I. Body and rcla~~vc oryn weights of rats administered

& SD for 5 rats. (0) Signilicanlly corrcspondlng

treatment

group

different

from control

DFII IP and or LRI

value,

I Value\ are the moan

P< 0.05. (/I) Significantly

v;ilues (cx., LRHSO to DEHP+LRtlO),

P
diffcrcnt

(c) Sigmlicantly

from dlll’cr-

was significantly lower than in the control or DEHP-treatment groups. The serum zinc concentration was not altered in any of the treatment groups. The activities of LDH-X, testicular specific LDH isozyme. were severely decreased only in groups coadministered DEHP and LRH. The total SH content in the testis was not affected by the administration of DEHP or LRH alone, but it was significantly reduced by DEHP and LRH administration. The concentrations of cholesterol, triglycerides and phospholipids in serum are shown in Fig. 3. No significant changes in serum free cholesterol were observed in

[iIIDEHP+LRHBO RI DEHP+LRHlO

SH

Zn

Fig. 2. SH and rinc concentrations Values are the mean cantly

different

i

LDH-X

and LDH-X

act&y

SD for 5 rats. (a) Significantly

Serum

Zn

of rat testis administered different

from control

DEHP

and/or

value, PcO.05.

from corresponding treatment group values (ex., LRHSO to DEHPf (c) Significantly different from DEHP value, PiO.05.

LRHSO),

LRH.

(h) SignifiP x 0.05.

275

_

E LRHlO

W DEHP ItUDEHP+LRHBO N DEHP+LRHlO

0

Phoapholipid

Triglyceride

Fig. 3. Serum lipid concentration

Free cholesterol

of rats administered

DEHP

for 5 rats. ((I) Significantly

different

sponding

values (ex., LRHSO to DEHP+

treatment

group

from control

Total cholesterol and/or

value, P~0.05.

LRH.

Values are the mean & SD

(h) Significantly

different

from corre-

LRHSO), Pi 0.05. (c) Significantly different

from DEHP value. P~0.05.

any of the groups. The total cholesterol concentration of the DEHP-treated group had a decreasing tendency but this was not significant. The DEHP + LRH group had a significantly lower total cholesterol concentration than control, but not significantly so compared to the group given DEHP alone. Phospholipid concentrations in serum were decreased only in the DEHP+ LRH-treated groups as compared to control. DISCUSSION

Testicular

atrophy

induced

by DEHP coincided

with a decrease in the zinc concen-

tration in testis and in the testosterone level in serum [S]. It is well established that zinc and testosterone are essential for testicular development and spermatogenesis, although the effects of the interaction of zinc and testosterone in testis are not known [ 17, 181. In our previous paper, co-administration against or enhance testicular damage resulting [191. Reduction

of LDH-X

activities.correlated

of zinc and DEHP did not protect from treatment with DEHP in rats with the testicular

injury

caused

by

DEHP [20]. The SH content has been known to be influenced by alterations in mineral metabolism such as zinc [21, 221 and its content in the testis coincided with the loss of testicular zinc concentration and testicular injury [23]. The results reported here, zinc concentrations, LDH-X activities and SH content were not influenced by the administration of DEHP alone, but co-administration of DEHP and LRH decreased those concentrations and activities. These results are in agreement with the alterations of testicular atrophy induced by high-dose administration of DEHP alone [g,

231.

376

The present

results suggest that testicular

ed to hormonal control LRH may have occurred Gray and Butterworth ministration

atrophy

induced

by

DEHP may be rclat-

in the testis and that the synergistic effects of DEHP and via negative feedback mechanisms of sex-related hormones. [24] reported

of testosterone

that in 4-week-old

or FSH with DEHP

rats the simultaneous

ad-

did not affect the development

of

testicular atrophy. but did prevent that of the accessory sex glands. The discrepancy between the results of Gray and Butterworth [24] and the present experiments may have occurred because ofdifferences in the dose levels of DEHP and hormones. Gray and Butterworth [34] used a dose level of 2.8 g/kg and at this level the extreme testicular atrophy was induced by DEHP alone. The synergistic effect of DEHP and tcstosterone or FSH on testicular weight may therefore be masked by the el‘fects of the high dose of DEHP. DEHP and a principal metabolite. 2-ethyhexanol, reduced serum lipid concentration [25]. In the present study. the cholesterol concentration in serum was significantly decreased by DEHP+ LRH as compared to controls, and was not significantly different from that of DEHP alone. The results suggest that the enhancement of DEHP toxicity by LRH may be limited to the gonadal organs, since non-gonadal effects of DEHP. such as serum cholesterol concentration, occurred independently of administration of hormones. These results resemble those of the report of Agarwal et al. [23] in that the enhancement of DEHP toxicity by dietary zinc deficiency is limited to the gonadal tissues. REFERENCES I Thomas. phthalic 2 Peakal.

J.A.

and

Thomas.

M.J.

(1983)

Biological

acid esters. CRC Crit. Rev. Towicol. D.B. (1975) Phthalate

3 Callcy. D., Autian.

esters: occurencc

J. and Guess,

efrects

of di(2.ethylhcxyl)phthalate

and

other

13. 2X3 317. and biological

W.L. (1966) Toxicology

effects. Reslduc

Rev. 54, I II.

of a series of phthalate

esters. J. Pharm.

Sci. 55. I58 162. 4 Singh. A.R.. Lawrcncc.

W.H. and Autian.

J. (1972) Tcratogcnicity

ofphthalatc

cstcrs in rats. J. Pharm

Sci. 61. 51 55. 5 Kluwc. W.M.. Haseman, ethylhexyl)phthalate 6 Gray.

T.J.B.,

J.K.. Douglas.

J.R. and Huff. J.E. (19X2) The carcinogenicity

in Fischer 344 rats and B6C3F,

Buttcrworth.

K.R.. Gaut.

study of di(2-cthylhexyl)phthalate 7 Oishi. S. and Hiraga.

mice. J. Toxicol.

I.F.. Grasso.

P. and Gangolli,

in rats. I:ood Cosmet.

K. (1977) Toxicity

Environ.

Toxicol.

of dietary

Health

di(?-

IO. 797 Xl 5.

S.D. (1977) Short-term

toxIcit)

15. 389 399.

of di(2-ethylhexyl)phthalate

in rats. Tokyo

Eiken Nempo

26.

I3 I 13X (in Japancsc). X Oichi. S. and Hiraga.

K.

(1980).Testicular

ronc and rinc concentrations.

Toxicol.

9 Oishi, S. (1985) Reversibility

atrophy

induced

Appl. Pharmacol.

of testicular

atrophy

by phthalic

acid esters: effects on tcstoatc-

53. 35 41.

induced

by di(2-ethylhexyl)phthalate

in rats. Envi-

ron. Rcs. 36, 160 169. 0 Foster.

P.M.D..

and changes

Thomas.

L.V.. Cook.

in fine excretion

M.W. and Gangolli.

produced

S.D. (1980) Study of the testicular

by some ,z-alkyl phthalates

in the rat. Toxicol.

etfccts

Appl. Pharma-

col. 54. 392 ~398. I &hi,

S. and Hiraga.

and testosterone

K. (1980) Testicular

conccntrdtionh.

Toxicology

atrophy

induced

15, 197 202.

by phthalic

acid monocstcrs:

cffccts of zinc

277

12 Oishi, S. and Hiraga. Environ.

Contam.

I3 Sedlak,

K. (1979) Effects of phthalic

Toxicol.

J. and Lindsey,R.H.

groups

(1968) Estimation

in tissue with Ellman’s

14 Meistrich,

M.L., Trostle,

of lactate dehydrogenase

acid esters on gonadal

function

in male rats. Bull.

21, 65 67. reagent.

P.K.,

of total,

Anal. Biochem.

Frapart.

protein

M. and Erickson,

isozyme X in pachytene

bound

and non protein

sulfhydryl

25, 1922205. R.P. (1977) Biosynthesis

spermatocytes

and spermatids

and localization

of mouse testis. Dev.

Biol. 60.428441. I5 Bradford.

M.M.

of protein I6 Shane.

(1976) A rapid

utilizing the principle

and sensitive of protein-dye

A.R. and Weil, C.S. (1982) Statistics

Methods

of Toxicology.

17 Millar, M.J., Elcoate. on the reproductive I8 Desjardins.

C. (1985) Morphological,

Toxicol.

K. (1983) Testicular Appl. Pharmacol.

cific enzyme activities

don.

atrophy

atrophy

70,43

induced

J.H. (1970) Nutrition.

S.A. and Gould,

Biophysiol.

and

36, 557 569.

and biochemical

aspects of male reproduction.

induced

In:

pp. I31 146.

by di(2-ethylhexyl)phthalate:

by di(2-ethylhexyl)phthalate:

changes

in rat testis. Arch. Toxicol.

In: A.D. Johnson,

T.C. (1970) Cadmium

(Ed.). Principles

effect of zinc

48.

Press, New YorkkLondon.

and N.L. Vandemark

Hayes

quantities

C.A. (1958) The effect of dietary zinc deficiency

Raven Press, New York,

and zinc concentrations

Testis, Vol. 3. Academic son, W.R. Gomes

physiological

Toxicology.

In: A.W.

of microgram

pp. 273 320.

P.V., Fischer, M.I. and Mawson,

20 Oishi. S. (1986) Testicular

22 Gunn.

for toxicologist.

system of male rats. Can. J. Biochem.

I9 Oishi. S. and Hiraga.

21 Leathman,

for the quantitation

Anal. Biol. 72, 2488254.

Raven Press, New York,

R.L. Dixon (Ed.), Reproductive supplement.

method binding.

W.R. Comes

in histology,

cellspe-

59,290&295.

and N.L. Vandemark

(Eds.), The

pp. 169 204.

and other mineral elements

in the testis. In: A.D. John-

(Eds.). The Testis, Vol. 3. Academic

Press, New York

Lon-

pp. 37748 I.

23 Agarwal.

D.K..

Eustis, S., Lamb

ethylhexyl)phthalate

induced

IV, J.C. and Kluwe, W.M. (1986) Influence

testicular

atrophy

and zinc depletion

of dietary

in adult rats. Toxicol.

zinc on di(2Appl. Phar-

macol. 84. I2 24. 24 Gray, Toxicol. 25 Moody.

T.J.B. and Butterworth.

K.R. (1980) Testicular

atrophy

produced

by phthalate

esters. Arch.

Suppl. 4. 452 455. D.E. and Reddy, J.K. (1982) Serum triglyceride

diets containing

plasticizers

and analogues

and cholesterol

of ester 2-ethylhexanol.

contents

Toxicol.

in male rats receiving

Lett. IO, 379- 3X3.