spermatogenic disturbance induced by di-(2-ethylhexyl) phthalate is significantly prevented by...

Spermatogenic disturbance induced by di-(2-ethylhexyl)

phthalate is signi®cantly prevented by treatment with

antioxidant vitamins in the rat

MASARU ISHIHARA,* MASAHIRO ITOH, KENSAKU MIYAMOTO, SHIGERU SUNA,à YOSHIKI TAKEUCHI, IKUMASA TAKENAKA* and

FUMIHIKO JITSUNARIà

Departments of *Urology, Anatomy, and àHygiene and Public Health, Kagawa Medical

University, Kagawa, Japan

SummaryPhthalate esters, now regarded as endocrine disruptors, are widely used in the plastics

industry. In particular, di-(2-ethylhexyl) phthalate (DEHP) is produced in large

quantities, and is used in blood storage bags, catheters and haemodialysis instruments.

Previous studies have demonstrated that treatment of rats with DEHP induces testicular

atrophy with liver enlargement, although the precise nature and mechanism of the action

of DEHP on these organs remains unclear. In the present study, we produced an

experimental model of DEHP-induced spermatogenic disturbance in rats by feeding

them a DEHP-containing diet. Liver enlargement occurred in rats fed either a 1 or 2%

DEHP-containing diet. However, testicular atrophy accompanied by aspermatogenesis

was induced by feeding with the 2% but not with the 1% DEHP-containing diet. This

suggests that the critical DEHP dose for gonadotoxicity is higher than that for

hepatotoxicity. Using the 2% DEHP-dose, the effect of simultaneous administration of

antioxidant vitamins (� vitamins C and E) was next examined. It was found that the

vitamin supplementation signi®cantly prevented the testicular injury. The results suggest

that antioxidant vitamins can protect the testes from DEHP-toxicity.

Keywords: DNA ¯ow cytometry, phthalate, spermatogenesis, testicular toxicity,

vitamin C, vitamin E

IntroductionPhthalic acid esters are widely used as plasticizers in several

plastic formulations. Di-(2-ethylhexyl) phthalate (DEHP),

one of the most commonly used plasticizers, has been shown

to leach out from the ®nished plastics into the air, water and

ground, and thereby to enter various foods (Thomas et al.,

1978; Albro, 1987). DEHP also directly enters the human

system from blood storage bags, catheters and haemodialysis

instruments. In fact, a high DEHP residue concentration has

been found in the blood and tissues of patients after numerous

blood transfusions (Hillman et al., 1975; Sjoberg et al., 1985;

Faouzi et al., 1999; Manojkumar et al., 1999).

Recent interest in DEHP has been focused on its toxicity

to spermatogenesis (Oishi & Hiraga, 1983; Oishi, 1984).

Indeed, many studies have demonstrated that treatment of

laboratory animals with DEHP induces testicular atrophy

with liver enlargement (Oishi, 1989a, 1989b). Although the

mechanism underlying the testicular damage caused by

DEHP remains unclear, it was recently found that the

administration of vitamin B12 is effective in preventing

DEHP-induced spermatogenic disturbance in rats (Oishi,

1994). Like vitamin B12, vitamins A, C and E are also

essential for normal spermatogenesis (Mason, 1933, 1940;

Correspondence: Dr Masahiro Itoh, Department of Anatomy,

Kagawa Medical University, Miki-cho, Kita-gun, Kagawa

761±0793, Japan.

international journal of andrology, 23:85±94 (2000)

Ó 2000 Blackwell Science Ltd.

Coward et al., 1966; Mason & Mauer 1975; Chinoy et al.,

1986; Sapra et al., 1987; Bensoussan et al., 1998). This raises

the possibility that the administration of these vitamins

would also have some bene®cial effect on spermatogenesis in

DEHP-treated rats. It is well known that both vitamins C

and E are antioxidative ( � oxido-reductive) agents which

act synergistically as potent scavengers of free radicals and

terminators of free-radical chain reactions (Tappel, 1962;

Summer®eld & Tappel, 1984; McCay, 1985; Niki et al.,

1985; Niki, 1987; Burton & Ingold, 1989). Therefore, the

simultaneous administration of the two vitamins may have

some in¯uence on DEHP-induced spermatogenic lesions

through their cooperative antioxidant function. The aim of

the present study was to determine the histological appear-

ance of the seminiferous epithelium of rats fed a DEHP-

containing diet with or without supplementation of the two

antioxidant vitamins.

Materials and methods

AnimalsSprague-Dawley (SD) male rats (aged 4 weeks) were

purchased from Charles River (Kanagawa, Japan) and kept in

the Laboratory Animal Center of Kagawa Medical Univer-

sity for 1 week before use. They were maintained at

22±24 °C and 50±60% relative humidity with a 12 h light-

dark cycle. The approval of the Kagawa Medical University

Animal Committee was obtained for this study.

ChemicalsDEHP was purchased from Tokyo Chemical Industries

(Tokyo, Japan). The chemical purity of DEHP was found to

be >98% by gas-liquid chromatography. CE-2 diets (Clea,

Tokyo, Japan) containing 1 and 2% DEHP were prepared by

Oriental Yeast Company (Chiba, Japan). Vitamin powders

(Chocola EC) comprising ascorbic acid (� vitamin C) and

water-soluble d-a-tocopherol (� vitamin E), respectively,

were provided by Eizai (Tokyo, Japan). The vitamin

C:vitamin E ratio in the powders was 2:1.

Experimental designIn the ®rst experiments, the animals were housed in three

groups (Groups I±III) of 15 animals each, and treated daily

for 2, 4 or 6 weeks in the following manner. Group I:

control rats fed the DEHP-free diet, Group II: Experimental

rats fed the diet containing 1% DEHP, Group III:

Experimental rats fed the diet containing 2% DEHP.

In the second experiment, the effects of vitamins C and E

on DEHP-treated rats were examined. The vitamin powders

were dissolved in drinking water at doses of 3.0 and 1.5 mg/mL,

respectively. Four groups (Groups A±D) composed of ®ve

animals each and were treated daily for 2 weeks, as follows.

Group A: negative control rats fed the DEHP-free diet and

vitamin-free water. Group B: Experimental rats fed the

DEHP-free diet and vitamin-supplemented water. Group C:

Experimental rats fed the 2% DEHP-containing diet and

vitamin-free water. Group D: Experimental rats fed the 2%

DEHP-containing diet and vitamin-supplemented water.

In both the ®rst and second experiments, all rats were

5 weeks of age at the time of the ®rst dosing, and the diet

and tap water were freely available. The average bodyweight

of rats at the start of the experiments was ~130 g. The

average volumes of diet taken and drinking water per rat

were approximately 50 g and 30 mL per day, respectively,

and there were no signi®cant differences in these volumes

between the DEHP-free and DEHP-administered groups, or

between the vitamin-free and vitamin-supplemented groups.

After completion of the treatments, the animals were deeply

anaesthetized with diethyl ether and their body weights were

recorded. Thereafter, the animals were killed and the testes,

kidneys and the liver of each animal were then removed and

weighed. Organ weight/body weight ´ 100 is shown as the

relative organ weight.

HistologyAfter recording the organ weights, the testes were

immediately ®xed in Bouin's solution for 2 days. Then,

the organs were washed, dehydrated in an ethanol series and

embedded in plastic (Technovit 7100; Kuizer, Germany).

The organs were sectioned at 5 lm with a microtome

(HN340E; Microme, Germany), after which the sections

were stained with Gill's haematoxylin III and 2% eosin Y.

The degree of spermatogenic disturbance was determined

according to Johnsen's scoring system, ranging from score1

(�no cells in the seminiferous tubules) to score10 (�com-

plete spermatogenesis)(Johnsen, 1970). At least 500 round or

oval sections of seminiferous tubules were examined and the

mean score was calculated for each animal.

Flow cytometryIn the second experiment, the left testis of each rat was

minced and suspended in a solution comprising RPMI1640

tissue culture medium (Biowhittaker, Maryland, USA)

containing 0.1% collagenase type IV (Sigma, Missouri,

USA) for 1 h at 37 °C. The cellular elements were then

centrifuged at 400 g for 10 min and ®xed in 70% cold

ethanol for 1 h at 4 °C. After removing the ethanol and

washing the cells with phosphate-buffered saline (pH 7.2),

the resulting pellet was resuspended in 1 mL 0.1% ribonuc-

lease A (Sigma) at 37 °C for 30 min with frequent vortex

mixing. The cells were then washed again with phosphate-

buffered saline by centrifugation under the same conditions.

The resulting pellet was stained with 1 mL of 5 lg/mL

propiduim iodide (Sigma) and then ®ltered through a nylon

mesh. Flow-cytometric analysis was performed using a

Facscan (Becton Dickinson, New Jersey, USA), ®tted with

a 488-nm argon ion laser line at 250 mW to induce the

¯uorescence of propidium iodide. Approximately 2±3 ´ 105

cells were examined for each specimen and DNA histograms

86 M. Ishihara et al.

Ó 2000 Blackwell Science Ltd, International Journal of Andrology, 23, 85±94

were generated by counting at least 1 ´ 105 nuclei per

sample. The low signal events (cell debris) were subtracted

from the histograms. The populations of cells containing

quantitatively similar amounts of DNA are represented by

discrete peaks on the histograms i.e. each of the peaks

represents a cell subpopulation with a speci®c DNA content

or ploidy. The ®rst peak for the normal control sample

represents haploid cells (1n), the second peak diploid cells

(2n), and the third peak tetraploid cells (4n). The areas under

these peaks were integrated by computer analysis to yield

quantitative information on the relative proportion of each

cell type with respect to the total cell population. Sper-

matids and spermatozoa contain haploid DNA. G0/G1

spermatogonia, Sertoli cells, interstitial cells and secondary

spermatocytes produce the diploid peak, whereas G2/M

spermatogonia and primary spermatocytes are responsible for

the tetraploid peak.

Statistical analysisValues are presented as means � standard deviation (SD).

The signi®cance of the differences between groups was

determined by ANOVA, with the aid of Statview-J-4.5

(Abacus Concepts, CA).

Results

Effect of DEHP-containing diets on the testesIn the 1% DEHP-treated rats (Group II), liver enlarge-

ment was noted at 2 and 4 weeks (p < 0.05), but not at

6 weeks (Fig. 1). However, there were no signi®cant

changes in the weights of the body, testes and kidneys

compared with controls (Group I), except that bodyweight

at 6 weeks was slightly lower than in Group I (p < 0.05)

(Fig. 1). In the 2% DEHP-treated rats (Group III), a

signi®cant decrease in bodyweight and marked testicular

atrophy were detected compared with either Group I or II

(p < 0.01) (Fig. 1). The relative weight of testes in Group III

was also dramatically decreased (p < 0.01 compared with

either Group I or II). However, the degree of liver

enlargement in Group III was not signi®cantly different

from that in Group II. Kidney weight in Group III

compared with Group I showed a slight reduction at 2 and

4 weeks (p < 0.05), however, the relative weights exhibited

no signi®cant change (Fig. 1).

Histopathological examination revealed that testes from

Group I showed complete spermatogenesis whereas severe

aspermatogenesis was induced in Group III. Some semin-

iferous tubules in the Group II testes exhibited spermato-

genic disturbance, however, the lesions were not signi®cant

compared with Group I (Fig. 2). In Group III, marked

atrophy of the seminiferous tubules, and disappearance of

spermatids and spermatocytes were observed (Fig. 3). Sertoli

cells with no or few spermatogonia remained in most

seminiferous tubules. However, destruction of the tubular

architecture or an apparent loss of interstitial cells was not

seen. Therefore, it was found that the testicular damage was

inducible in rats by feeding a DEHP-containing diet without

injection of the chemical. Furthermore, the results showed

that 1% DEHP in the diet was enough to cause liver

enlargement, whereas a critical point for gonadotoxicity

existed with diets of between 1 and 2% DEHP.

Effects of vitamins C and E on 2% DEHP-inducedtesticular damageAs seen in the ®rst experiments, experimental rats fed the

2% DEHP-containing diet for 2 weeks (Group C) exhibited

a signi®cant decrease in bodyweight and dramatical atrophy

of the testes compared with controls (Group A)

(p < 0.0001). In addition, a signi®cant increase in relative

liver weight and no signi®cant change in relative kidney

weight were also con®rmed in Group C (Fig. 4). However,

rats fed the 2% DEHP-containing diet with supplementation

of vitamins C and E (Group D) showed signi®cant

prevention of these changes (p < 0.05 compared with

Group C). The relative testis weights in Groups A and D

were not signi®cantly different from each other, although

the absolute organ weight in Group D did not reach that in

Group A (p < 0.01) (Fig. 4). Rats fed the DEHP-free diets

and vitamin-supplemented water (Group B) did not exhibit

signi®cant changes in body and organ weights except that

slight liver enlargement was unexpectedly induced (p < 0.05

compared with Group A) (Fig. 4).

Histopathological examination revealed that spermato-

genic status in Group D did not return to that in Group A

(p < 0.01) but was signi®cantly improved compared with

Group C (p < 0.0001) (Fig. 5). Severe aspermatogenesis, a

typical histopathological feature in Group C, was never seen

in Group D (Fig. 6). Group B testes exhibited complete

spermatogenesis similar to that in Group A (Fig. 6). Flow

cytometric analysis revealed apparent reductions of haploid

and tetraploid cells, and an increase of diploid cells in Group

C (p < 0.005 compared with Group A), showing that the

remaining cells were mainly G0/G1 spermatogonia, Sertoli

cells and interstitial cells (Fig. 7). However, the germ cell

populations in Group D were comparable with those in

Group A (Fig. 7), indicating that many haploid cells

(spermatozoa and spermatids) and tetraploid cells (G2/M

spermatogonia and primary spermatocytes) were continu-

ously differentiated from diploid cells (G0/G1 spermato-

gonia) in Group D. Therefore, these results showed that

supplementation of vitamins C and E could protect the testes

from DEHP-gonadotoxicity.

DiscussionIn the present study, disruption of spermatogenesis was

induced in rats by feeding them a diet containing 2% DEHP.

The amount of DEHP producing the aspermatogenic lesions

was extremely large as the rats ingested approximately 1 g

DEHP/day. It is unlikely that such a large dose of DEHP


Prevention of phthalate-induced aspermatogenesis 87

enters the human system. However, the spermatogenic

lesions induced by the 2% DEHP-diet could be signi®cantly

improved by supplementation of antioxidant vitamins C and

E. This shows that these vitamins can protect the testes from

the gonadotoxicity of DEHP. This is the ®rst demonstration

of prevention of DEHP-induced aspermatogenesis by

antioxidant vitamins.

DEHP is now regarded as an endocrine disrupter (Reiter

et al., 1998; Mylchreest et al., 1999). However, there has

been no direct evidence of an oestrogen-like action of the

chemical (Nakai et al., 1999). Although the basic metabolic

disturbance which elicits aspermatogenesis remains obscure,

there have been some attempts at prevention of DEHP-

induced testicular damage. Oishi (1989a) reported that

Figure 1. Body and organ weights in Group I (rats fed DEHP-free diet, light circle), Group II (rats fed 1% DEHP-containing diet, dark triangle) and Group III (ratsfed 2% DEHP-containing diet, dark square). Relative weight of organ � organ weight/bodyweight ´ 100. The means � SD for ®ve individual rats per group ateach point are plotted. P-values were derived by ANOVA.



testosterone levels in the testes and serum decreased in

DEHP-treated rats. However, contrary to expectations,

coadministration of testosterone did not prevent but

enhanced the testicular atrophy in rats. Coadministration of

gonadotrophin hormone-releasing hormone also failed to

afford protection against DEHP-induced testicular atrophy

in rats (Oishi, 1989b). Selective depletion of zinc from the

testes has also been detected in DEHP-treated rats (Oishi,

1984). It is well established that dietary zinc de®ciency

induces disturbance of spermatogenesis (Millar et al., 1958).

However, the testicular atrophy induced by DEHP was not

prevented by coadministration of zinc in rats (Oishi &

Hiraga, 1983). Like zinc de®ciency, vitamin A de®ciency is

also known to result in impaired spermatogenesis (Mason,

1933; Coward et al., 1966). However, Oishi (1984) reported

the unexpected ®nding that vitamin A concentrations in the

serum and testes increased in DEHP-treated rats. This

suggests that the testicular damage induced by DEHP was

not due to a dietary or intrinsic de®ciency of vitamin A.

Prevention of DEHP-induced aspermatogenesis was ®rst

achieved by coadministration of vitamin B12 in rats (Oishi,

1994). It is known that chronic de®ciency of vitamin

B12 induces defective synthesis of DNA in any cells that

undergo chromosomal replication and division, resulting in

Figure 2. Spermatogenic status of Group I(rats fed DEHP-free diet, light circle), Group II(rats fed 1% DEHP-containing diet, dark triangle)and Group III (rats fed 2% DEHP-containing diet,dark square). The means � SD for ®ve individualrats per group at each point are plotted. P-valueswere derived by ANOVA.

Figure 3. Representative seminiferous tubule cross-sections from (a) Group I (rats fed DEHP-free diet) (b) Group II (rats fed 1% DEHP-containing diet) and(c) Group III (rats fed 2% DEHP-containing diet). Severe aspermatogenic lesions were noted in Group III (magni®cation ´150).



disturbance of spermatogenesis in rats (Kawada et al., 1990,

1993). Therefore, the phenomenon of protection of

spermatogenesis by vitamin B12 in DEHP-treated rats

suggests the presence of a de®ciency of the vitamin.

Like vitamins A and B12, antioxidant vitamins

(� vitamins C and E) are also essential for normal sperma-

togenesis judging from evidence that animals de®cient in the

vitamins exhibit spermatogenic disorders (Mason, 1933,

1940; Mason & Mauer, 1975; Chinoy et al., 1986; Sapra

et al., 1987; Bensoussan et al., 1998). In the present study, we

demonstrated that the simultaneous administration of vita-

mins C and E signi®cantly improved the spermatogenic

Figure 4. Body and organ weights in Group A (rats fed DEHP-free diet and vitamin-free water), Group B (rats fed DEHP-free diet and vitamin-supplementedwater), Group C (rats fed 2% DEHP-containing diet and vitamin-free water) and Group D (rats fed 2% DEHP-containing diet and vitamin-supplemented water).Relative weight of organ � organ weight/bodyweight ´ 100. The means � SD for ®ve individual rats per group are plotted. P-values were derived by ANOVA.



status of DEHP-treated rats. Although further study is

needed to determine which vitamin is more important for

the prevention of DEHP-induced aspermatogenesis, both

vitamins have often been simultaneously used in clinical and

experimental medicine, because the two vitamins synergis-

tically exert a potent antioxidant action which affords

protection against excessive peroxidative reactions (Leung

et al., 1981; Reddy et al., 1982). Water-soluble vitamin C is

localized in the cytosol, while lipid-soluble vitamin E is

localized almost exclusively in cell membranes (Tappel et al.,

1973; Giasuddin & Diplock, 1981). The former scavenges

oxygen radicals in the aqueous phase, and the latter scavenges

oxygen radicals in the membrane (Hill & Burk, 1984; Kagan,

1989). Ginter et al. (1982) reported that the simultaneous use

of vitamins C and E synergistically increased the activities

of liver detoxi®cation enzymes dependent on cytochrome

Figure 5. Spermatogenic status of Group A(rats fed DEHP-free diet and vitamin-free water),Group B (rats fed DEHP-free diet and vitamin-supplemented water), Group C (rats fed 2%DEHP-containing diet and vitamin-free water)and Group D (rats fed 2% DEHP-containing dietand vitamin-supplemented water). The Means� SD for ®ve individual rats per group areplotted. P-values were derived by ANOVA.

Figure 6. Representative seminiferous tubule cross-sections from (a) Group B (rats fed DEHP-free diet and vitamin-supplemented water) (b) Group C (rats fed2% DEHP-containing diet and vitamin-free water) and (c) Group D (rats fed 2% DEHP-containing diet and vitamin-supplemented water) (c). Active spermatogenesiswas noted in Group D (magni®cation ´150).



P-450 in guinea pigs. Chorvatovicova et al. (1991) showed

that simultaneous treatment with vitamins C and E had a

synergistic preventive effect on lipoperoxide production in

experiments on the toxicity and mutagenicity of K2Cr2O7.

Niki et al. (1985) demonstrated that supplementation of

vitamin C signi®cantly increased vitamin E levels in the rat

liver. Further study demonstrated that vitamin C regenerates

vitamin E by reducing vitamin E radicals formed when

vitamin E scavenges oxygen radicals (Niki, 1987). In

previous studies on irradiation- or cadmium-induced

gonadotoxicity, it became evident that spermatogenesis is

very sensitive to oxidative stress (Koizumi & Li, 1992; Narra

et al., 1993, 1994). Therefore, in our experiments, it may be

that free radicals were excessively produced in the testis due

to the DEHP-treatment, with resultant de®ciency of the

antioxidant vitamins in this organ.

ConclusionsThe preventive effects of vitamins C and E on DEHP-

induced testicular damage have been demonstrated for the

®rst time in the present study. This suggests that the

cooperative redox systems comprising supplemented vita-

mins C and E play a role in maintenance of spermatogenesis.

In a further study, we will determine the concentrations of

vitamins C and E, and free radicals in the testicular tissue of

DEHP-treated rats. Besides vitamins C and E, a signi®cant

number of complementary antioxidant systems prevent

oxidative damage in the body under normal conditions.

These include superoxide dismutase, glutathione, peroxid-

ase, uric acid, cystein and catalase (Tappel et al., 1973;

Docampo et al., 1981; Harman, 1981; Reddy et al., 1982;

Hill & Burk, 1984; McCay, 1985). Therefore, it might be

that the amounts of these antioxidants in the testes also

decrease after DEHP-treatment, but that their supplemen-

tation is capable of protecting the testes from gonadotoxicity.

To our knowledge, there has been no study of histological

changes to the damaged seminiferous epithelium after

cessation of DEHP administration. Thus, in future experi-

ments we will determine whether or not the severely

damaged spermatogenesis of DEHP-treated rats recover, with

or without later supplementation with vitamins C and E.

AcknowledgementsWe thank Mrs. M. Miyawaki, Mr W. Nagata and Mrs. E.

Turumi for their secretarial and technical assistance. This

work was supported by a Grant-in-Aid for General Scienti®c

Research (11470108) from the Ministry of Education,

Science, Sports and Culture, Japan.

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Received 29 September 1999; accepted 27 November 1999

1. VII International Congress of Andrology, hosted by the American Society of Andrology

(also the 26th Annual Meeting of the ASA)

Montreal, Quebec, Canada

Friday 15 June 2001 ± Tuesday 19 June 2001

2. 27th Annual Meeting of the American Society of Andrology

Sheraton Seattle Hotel and Towers, Seattle, Washington, USA

Thursday 26 April ± Saturday 29 April

(Postgraduate course: 29 April 2002. Laboratory workshop: 24 April 2002

For more information on all meetings or to request a 2000 abstract submission form please contact:

American Society of Andrology

74 New Montgomery, Suite 230

San Francisco, CA 94105

USA

phone: (415) 764 4823

fax: (415) 764 4915

email: [email protected]

homepage: www.cvm.uiuc.edu/HomePages/rhess/asa/front



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