Effects of di-2-ethylhexyl phthalate on lipid composition of serum and testis in rats

61

Toxicology Letters, 23 (1984) 67-72

Elsevier TOXLett.

1289

EFFECTS OF DE2-ETHYLHEXYL PHTHALATE TION OF SERUM AND TESTIS IN RATS (Di-2-ethylhexyl phthalate; testicular atrophy; zinc)

fatty acid compositions;

ON LIPID COMPOSI-

lipid compositions;

rats;

SHINSHI OISHI Department Hyakunincho,

of

Toxicology,

Shinjuku-ku,

Tokyo

Metropolitan

Research

Laboratory

of Public

Helath,

3-24-l

Tokyo 160 (Japan)

(Received Mareh 17th, 1984) (Revision received April 19th, 1984) (Accepted May 17th, 1984)

SUMMARY Dietary administration of di-2-ethylhexyl phthalate (DEHP; 28/o) to male rats caused testicular atrophy. Serum non-esterified fatty acid increased while cholesteroi, triglyceride and phospho~ipid decreased in treated rats. Testicular concentration of cholesterol and non-esterified fatty acid increased in DEHP-treated rats. Fatty acid composition of phospholipid and triglyceride were also changed. These changes were similar to those in rats fed a zinc(&)-deficient diet.

INTRODUCTION

DEHP is widely used as a plasticizer. Although the acute toxicity of DEHP is very low [I], we reported previously that DEHP and its monoester metabolic intermediate caused testicular atrophy in rats accompanied by decreased Zn concentration in the testis [2, 31. There are many similarities between Zn and fatty acid deficiency symptoms [4, 5]. For example, both deficiencies are accompanied by growth retardation, parakeratosis, lengthened gestation period, parturition accompanied by excessive

Abbreviations: DEHP, di-2-etbylhexy1 phthalase; 14:O; myristrate; f&O, palmitate; l&l, paimito~~ate; 18:0, stearate; 18:1, oleate; 18:2, linoteate; 18:3, linolenate; 20:3, eicosatrienate; 20:4, arachidonate; 225, docosapentaenate. 0378-4274/84/$ 03.00 0 Elsevier Science Publishers B.V.

68

bleeding, and sterility of males. These similarities suggest that there might be physiological interactions between Zn and fatty acids. The purpose of this study was to determine whether DEHP alters the lipid composition in the testis and serum as Zn concentration decrease in those organs. MATERIALS

AND

METHODS

Animals and diet

Young male Jcl:Wistar rats were purchased from Clea Japan Ltd., Tokyo. The rats were acclimatized for 1 week prior to the experiment. They were fed a synthetic diet (Formula A, Oriental Yeast Co., Ltd., Tokyo) containing 2% DEHP. After 1 week of treatment, the rats were killed by decapitation. Samples of blood were collected and the fresh weights of the testes and liver were recorded. Serum and testes were stored at -30°C until analysis. Zn analysis

Serum and testicular Zn concentrations methods [2].

were measured by previously reported

Lipid analysis

Serum lipid composition was measured by previously reported methods [6]. The testicular lipids were extracted by the method of Folch et al. [7]. Total lipid was measured gravimetrically and lipid phosphorus, cholesterol and cholesteryl ester, triglyceride, and non-esterified fatty acid were determined by the methods of Allen [S], Zak [9], Van Handel [lo] and Ducombe [ll], respectively, after fractionation of lipid by thin-layer chromatography with n-hexane:ether:acetic acid (80:20: 1). Fatty acid composition was determined by gas-liquid chromatography after transmethylation with 5% m-trifluoromethylphenyl trimethylammonium hydroxide-methanol [12]. Gas-liquid chromatography was performed on a Shimadzu GC-9A gas chromatograph equipped with flame-ionization detector. A glass column (2 m x 3 mm) was packed with silanized Chromosorb W containing 15% poly(diethylene glycol succinate). Each fatty acid methyl ester was identified by the comparison of retention time with authentic material. Data analyses were performed with Shimadzu Chromatopack CR-1B integrator. Statistical analysis

Statistical analysis was performed of significance.

by Student’s t-test taking P
69 RESULTS

Body and testicular weights were significantly lower than those of control rats, whereas liver weights of DEHP-treated rats were higher than those of controls (Table I). Zn concentration of serum and testis decreased in DEHP-treated rats (Table II). In the serum, non-esterified fatty acid increased and choIestero1, triglyceride and phospholipid decreased in DEHP-treated rats (Table III). In the testes, the concentration of cholesterol and non-esterified fatty acid increased. Fatty acid composition of phospholipid of the serum and testis are shown in Table XV. 16:0 and 18:l and fatty acids were increased and 18:O fatty acid was decreased TABLE I BODY AND ORGAN WEIGHTS OF RATS FED DIET CONTAINING

Zoy, DEHP”

Treatment

Body weight

Liver weight

Testicular weight

Control DEHP

188 f 2.65(g) 14Ok 2.09b

5.29 ztz0.068 8.19~k0.18~

1.07+0.029 0.88 It 0.0546

‘Values are the means k SE for 5 animals and organ weights are expressed as percentages of body weight. bSignificantly different from control, P
Control DEHP

IN TESTIS AND SERUM OF RATS FED DIET CONTAINING Serum (&ml)

Testis @g/g)

1.25 + 0.05 1 1.07 + 0.038’

16.3 kO.39 14.2~0@’

2%

aValues are the mean + SE for 5 animals. bSignificantIy different from control, PcO.05. TABLE III LIPID COMPOSITION OF TESTIS AND SERUM OF RATS FED DIET CONTAINING 2% DEHP= Serum Control Total Choiesterol Cholesteryl ester Non-esterified fatty acid Triglyceride Phosphoiipid

105 79.8 3.82 312 310

Testis DEHP

Control

DEHP

79.6b 56.5 6.03b lllb 196b

18.3 1.54 0.058 1.28 1.07 10.8

23.2b 1.88b 0.16b 2.31b 1.33 13.5

aValues are the means for 5 animals and expressed as mg/dI (serum) or m&g wet tissue. bSignificantIy different from control, P
TABLE

IV

FATTY

ACID

COMPOSITIONS

DIET CONTAINING

OF PHOSPHOLIPIDS

OF SERUM

Serum

16:0

Testis

Control

DEHP

Control

DEHP

32.4b

46.2

40.3b

16:l

_c 26.0

0.31

25.2

llsb

11 .o 4.51

3.38b

0.17

0.42b

16.9

16.2

11.1 for 5 animals from control,

the limits of detection

and expressed P< 0.05.

TABLE

V

FATTY

ACID

COMPOSITIONS

DIET CONTAINING

as percentages

of the serum and testes are shown in Table 18:2 fatty acids was decreased and that of

OF TRIGLYCERIDE

OF SERUM

AND TESTIS

Control

DEHP

0.55

0.24

1.89

0.91b

2.51

2.61

3.73

6.03

38.5b

48.3 _c

34.6b

2.58

50.8

18:0 18:l

23.4

DEHP

2.29

22.6

16:l

18:3 20:3 20:4

60.0 3.18 8.07

18.0

14.3b 1.39b

5.64 0.57

0.30b 1.12

0.90 3.05

3.80

9.35

22:5 aValues are the means bSignificantly different

OF RATS

Testis

23.4

18:2

acids.

2% DEHPa

Control

16:0

7.84b fatty

18:0, 18:l and 18:3 fatty acids in the testis of 16:0, 18:2 and 22:5 fatty acids were

Serum

14:o

of total

or not present.

in serum of DEHP-treated rats, whereas were increased while the concentration reduced. Fatty acid composition of triglyceride V. In serum, the percentage of 16:l and

‘Under

0.70

17.5

13.4

22:5 “Values are the means bSignificantly different ‘Under

18.1b

0.74

2.49

20:4

10sb

23.5

18:3 20:3

0.22

6.65

16.7b

5.26

18:2

OF RATS FED

26.1

18:0 18:l

AND TESTIS

2% DEHP”

for 5 animals from control,

the limits of detection

and expressed P
or not presented.

as percentages

of total

5.33 fatty acid.

FED

71

18:l fatty acid was increased

in DEHP-treated

rats. In the testis, the percentage

18:1, 18:2 and 18:3 fatty acids were decreased

in treated

of

rats.

DISCUSSION

Since altered lipid metabolism in the testis is frequently associated with testicular atrophy [13], lipid changes in composition were investigated in DEHP-induced testicular injury. Rats receiving 2% DEHP for one week had testicular atrophy as indicated by a significant decrease in the weight of the testes. Several lipids in the testis and serum were changed. The feeding of DEHP to rats has resulted in diverse biochemical effects, such as inhibition of cholesterol synthesis in the liver, testis and adrenal gland, decreased plasma cholesterol levels and decreased synthesis of hepatic phospholipid and triglycerine [14]. In the present experiment, serum total cholesterol was decreased, whereas the concentration of testicular cholesterol was increased. In rats maintained on a Zn-deficient diet, total lipid in the testis was increased; phospholipid had a downward trend; total cholesterol was increased [ 151. Zn deficiency sharply decreased 22:5 fatty acid and to some extent, other polyunsaturated fatty acids in the testis microsome [16]. The present results resemble the changes in lipid composition in the testis and serum of rats maintained on a Zn deficient diet. However, testicular atrophy induced by DEHP was not prevented by Zn administration [17]. Castration significantly decreased 20:4 fatty acid in plasma but increased 20:3 fatty acid, which was negligible in normal rats or rats fed Zn-deficient diet [ 161. In the present study, 20:3 fatty acid in serum phospholipid fraction was increased in DEHP-treated rats. The percentage of 20:4 fatty acid in serum phospholipid was not changed but that of the cholesterol ester fraction was reduced markedly (from 49.3% to 31.4%). In hypophysectomized rats, testicular without supplemental hormones, cholesterol, especially cholesteryl ester, increased and 22:5 fatty acid markedly increased [18]. In the present experiment, cholesteryl ester in the testis was increased but 22:5 fatty acid in both phospholipid and triglyceride fractions was decreased. In conclusion, lipid alteration after DEHP administration was similar to the lipid changes in rats fed Zn-deficient diets and/or castration. Therefore, testicular atrophy induced by DEHP may relate to Zn concentrations in the testis and/or hormonal abnormalities.

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