Effects of four bisphenolic antioxidants on lipid contents of rat liver

Letters, 8 (1981) 77-86

Toxicology

Elsevier/North-Holland

77

Biomedical

Press

EFFECTS OF FOUR BISPHENOLIC OF RAT LIVER

OSAMU

TAKAHASHI

and KOGO

ANTIOXIDANTS

ON LIPID CONTENTS

HIRAGA

Department of Toxicology, Tokyo Metropolitan Research Laboratory of Public Health, Tokyo 160

(Japan) (Received

August

(Accepted

December

22nd.

1980)

23rd,

1980)

SUMMARY

Hepatic phenol), phenol)

lipids were studied

in Sprague-Dawley

or 4,4’-methylenebis(2,6-di-terf-butylphenol)

idenebis(3-methyl-6-tert-butylphenol) esteryl-ester

concentrations,

and non-esterified mainly lipids.

male rats given 2,2’-methylenebis(4-ethyl-6-tert-butyl-

2,2’-methylenebis(4-methyl-6-tert-butylphenol),

fatty

from defective

induced

fatty liver and increased

7.1-, 5.8- and 6.1-fold, acids were markedly

fat excretion

4.4’.butylidenebis(3-methyl-6-tert-butylat a level of 1.135 mmol% respectively.

decreased.

It would

triglyceride,

Plasma

diglyceride

triglycerides,

appear

from the liver. The other bisphenols

for 1 week. 4,4’-Butyl-

that the fatty

produced

and chol-

total cholesterol liver resulted

a little change

in liver

INTRODUCTION

It has been reported that the antioxidants, butylated hydroxytoluene, 6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline or N,N’-diphenyl-p-phenylene diamine, protect choline-deficient and ethanol-, carbon tetrachlorideand erotic acid-induced fatty livers [l-12]. These compounds do not induce fatty liver by themselves. A fatty liver is produced when 2,4,6-tri-tert-butylphenol is incorporated in the purified diet of ddY mice (0. Takahashi, unpublished data). The present study was undertaken to determine the fatty composition of liver of rats given the four bisphenols. 0378-4274/81/OOOC-0000/$02.50

0 ElsevierINorth-Holland

Biomedical

Press

78

MATERIALS

AND METHODS

Male Sprague-Dawley laboratory

ration

rats (CLEA

(CLEA

CE-2)

ethyl-6-tert-butylphenol),

Japan

either

Inc.) approx.

alone,

173 g, were fed powdered

or containing

2,2’-methylenebis(C 4,4’ -

2,2’-methylenebis(4-methyl-6-tert-butylphenol),

butylidenebis(3-methyl-6-tert-butylphenol) or 4,4’-methylenebis(2,6-di-terfbutylphenol) at a level of 1.135 mmol% for 1 week. All compounds were purchased from Tokyo Kasei Kogyo. Other procedures were as previously described [13]. Lipid analysis was as described by Ogura et al. [14] and Takahashi [15]. Hepatic lipids were extracted by the method of Folch et al. [ 161. Total lipids were determined gravimetrically. Each lipid class was separated on silica gel 60F254 precoated glass plates (E. Merck, Darmstadt) with n-hexane-ether-acetic acid (80:20: 1). Total phospholipids were calculated by multiplying the Pi content by 25, the Pi content being determined by Nakamura’s modification of Allen’s method [17]. Cholesterol and cholesteryl esters were determined by the method of Zak [18], triglycerides and diglycerides by that of Fletcher [19] and non-esterified fatty acids by that of Duncombe [20] and plasma triglycerides, total cholesterol and non-esterified fatty acids by those of Fletcher [19], Henly [21] and Duncombe [20], respectively. Livers were fixed in 10% buffered formalin, sectioned with cryostat and stained with Sudan

black B.

RESULTS

The effects

on body-weight

body and liver weights

TABLE BODY

change

and total

and food intake

lipid content

are shown

are shown

in Table

in Fig.

1. Final

I. Microscopic

I WEIGHT,

BISPHENOLS

LIVER

WEIGHT

FOR 1 WEEK

AND

TOTAL

LIVER

LIPIDS

OF RATS

GIVEN

DIETARY

(N = 5) Final body weight

(g)

Liver weight Absolute

Total

(g)

Relative

lipid

(470) (mg/g liver)

Control

233.4 + 8.8

15.97 t 0.88

6.83 k 0.12

33.04 + 1.16

2,2’-Methylenebis(4-ethyl-6-

218.4 i 9.4

15.35 f 0.66

7.04 k 0.15

31.30 + 0.84

ferr-butylphenol 2,2’-MethyIenebis(4.methyl-6-

201.8 f 4.@

16.20 rk 0.61

8.03 ? 0.26b

34.42 + 0.54

tert-butylphenol) 4,4’-Butylidenebis(3-methyl-

213.8 + 5.1

16.86 ? 0.50

7.88 of-0.12’

73.04 + 2.76~

6-tert-butylphenol) 4,4’-Methylenebis(2.6.di-terr-

225.4 f 3.6

15.19 t 0.29

6.74 k 0.08

36.96 zk 1.12”

butylphenol) Values

represent

mean k S.E.

Significantly

values: aP < 0.05; bf < 0.01; CP < 0.001.

different

(Student’s

f-test)

from

the appropriate

control

79

photographs

of liver tissue

stained

by Sudan

Butylidenebis(3-methyl-6-tert-butylphenol) fold

and

black

increased

4,4’-methylenebis(2,6-di-tert-butylphenol)

B are shown the hepatic increased

in Fig. 2. 4,4’-

lipid content it

slightly.

2.2The

composition of liver lipids is shown in Table II. 4,4’ -Butylidenebis(3-methyl-6-tertbutylphenol) significantly increased triglycerides, diglycerides and cholesteryl esters, 7.1-, 5.8- and 6.1-fold, respectively. The proportions of these lipid classes to total lipids were also significantly elevated by this compound. Fat-deposition occurred in the middle to central zone of the hepatic lobule. 2,2’-Methylenebis(4-ethyl-6-tertbutylphenol) significantly decreased triglycerides and increased free cholesterol. 2,2’-Methylenebis(4-methyl-6-tert-butylphenol) also significantly decreased triglycerides and slightly increased diglycerides. 4,4’ -Methylenebis(2,6-di-tertbutylphenol) increased free cholesterol and diglycerides. Plasma triglycerides, total cholesterol and non-esterified fatty acids were markedly decreased in rats given 4,4’-butylidenebis(3-methyl-6-tert-butylphenol). 22’ -Methylenebis(4-methyl-6-tert-butylphenol) decreased plasma triglycerides and increased plasma total cholesterol (Table III).

130

3 ;120 01 T J 10 -0 B .?lOO z T [L 90

0

I

Fig. 1. Effects Control;

(C),

butylphenol); butylphenol).

2 3 Feeding of dietary

4 period

5 6 (days)

four bisphenolic

7

antioxidants

on body-weight

2,2’-methylenebis(4-ethyl-6-tert-butylphenol); (D),

4,4’-butylidenebis(3-methyl-6-tert-butylphenol);

(E),

changes

and food intakes.

2,2’-methylenebis(4-methyl-6-tert(A), 4,4’-methylenebis(2,6-di-tert-

(B),

80 TABLE

II

COMPOSITION

OF HEPATIC

LIPIDS

OF RATS GIVEN

Cholesteryl mg/g

DIETARY

esters

liver

BISPHENOLS

FOR 1 WEEK (N = 5)

Triglycerides

% total lipid

mg/g

liver

% total lipid

Control

0.47 + 0.06

1.43 f 0.16

2.00 f 0.12

6.02 + 0.17

2,2’-Methylenebis(4-ethyl-6tert-butylphenol)

0.48 + 0.05

1.52 t 0.16

1.16 _+0.12’

3.71 k 0.33c

2,2’-Methylenebis(4-methyl-6tert-butylphenol)

0.40 + 0.03

1.16 k 0.09

1.33* 0.22a

3.87 f 0.64a

4,4’-Butylidenebis(3-methyl6-tert-butylphenol)

2.87 f 0.18~

3.93 t 0.18c

14.25 f 1.49

19.41 t 1.51~

4,4’-Methylenebis(2,6-di-lerrbutylphenol)

0.79 + O.loa

2.15 k 0.27

2.95 k 0.61

7.85 f 1.44

Non-esterified mg/g

fatty acids

liver

Diglycerides

Cholesterol

% total lipid

mg/g

liver

% total lipid

mg/g

liver

Phospholipids % total lipid

mg/g

liver

% total lipid 58.14k2.70

20.21 + 0.40

64.64 k 0.691

0.82kO.15

0.29 k 0.01

0.93 + 0.06

0.17~0.02 4.00t0.58 1.99 + 0.12b 6.38 k 0.40h 0.14 i 0.02

0.17 f 0.03

0.48 k 0.07

1.83 _t 0.11” 5.32 i 0.32

0.27 + 0.02” 0.78 t 0.04” 21.55 t 0.36”

62.62 2 0.51

0.41 f 0.03”

0.57 k 0.05

2.10 k 0.05’ 2.89 f 0.08

0.98 zk 0.07~ 1.38 f 0.08’ 25.36 + 0.53~

34.85 + 1.05~

0.27 t 0.04

0.72 -+ 0.09

2.07 + 0.08b 5.61 i 0.16” 0.32 + 0.04b 0.86 k 0.10.’ 21.48 + 0.52h

Values

represent

values:

aP c 0.05; bp < 0.01;

TABLE

1.30+0.15

mean k S.E.

Significantly

different

(Student’s

0.51 kO.07 0.46 k 0.06

19.09kO.34

0.26 k 0.04

t-test)

from

58.2,

the appropriate

+

, JO

control

‘P < 0.001.

111

TRIGLYCERIDES, PLASMA

TOTAL

OF RATS GIVEN

CHOLESTEROL DIETARY

AND

BISPHENOLS

NON-ESTERIFIED

FATTY

AClDS

IN

FOR 1 WEEK (N = 5)

Triglycerides

Total

(mgjdl)

(mg/dl)

cholesterol

Non-esterified fatty acids (mg/dl)

12

47 i 2

20 i- 2

2,2’-Methylenebis(4.ethyl-6-lert-

Control

117*

83 -t 11

49 * 2

19 + 3”

butylphenol) 2,2’-Methylenebis(4-methyl-6-rerr-

79 k 6”

64 i 2’

21 +2c

butylphenol) 4,4’-Butylidenebis(3.methyl-h-terf-

28 k 3~

22 f 3c

131 i 16

48 -t 2

butylphenol) 4,4’-Methylenebis(2.6~di-lerl-

8 f O”.d 32,9~

butvlphenol) Values represent

mean k S.E. Significantly

different

aP < 0.05; “P < 0.01; CP < 0.001; dN = 4; CN = 3.

(Student’s

I-test) from appropriate

control

values:

81

a2

83

DISCUSSION

2,2’-Methylenebis(4-ethyl-6-tert-butylphenol) increases fetal resorption rates in the rat [22]. 2,2’ -Methylenebis(Cmethyl-6-tert-butylphenol) induces functional changes in the nervous system and liver, and morphological changes in the testes [23], decreases the plasma prothrombin [13], and delays the appearance and development at malignancy of tumors and prolongs the survival of the experimental

Fig. 2. Photomicrograph Fatty

deposition

butylphenol); [err-butylphenol);

of the liver tissue

is shown

as black

stained

droplets.

with Sudan

(A),

Control;

(C), 2.2’.methylenebis(4.methyl-6-rerf-butylphenol); (E), 4,4’-methylenebis(2,6-di-tert-butylphenol).

black (B),

B; Original

magnification

2,2’-methylenebis(4-ethyl-6.Bert-

(D), 4,4’-butylidenebis(3-methyl-6-

x 40.

84

animals [24]. 4,4’-Butylidenebis(3-methyl-6-tert-butylphenol) and antidiabetic effects [25] and the hepatic fatty infiltration

has anticholesteremic of rats given 0.005%

of this compound in the diet for 90 days was described by Lefaux [26]. About 20% of a dose of 4,4’-methylenebis(2,6-di-tert-butylphenol), can be absorbed from the gastrointestinal tract and approx. 13%14% can be metabolized. The major metabolites are 3,5-di-tert-4-hydroxybenzoic quinone methide [27]. This bisphenol hypocholesteremic properties [29].

acid is

and

less

its glucuronides,

toxic

[28]

and

or the has

oral

2,2’-Methylenebis(4-ethyl-6-tert-butylphenol) and 2,2’-methylenebis(4-methyl-6tert-butylphenol) were found to decrease liver triglycerides but to increase liver cholesterol and phospholipids slightly (Table II). Butylated hydroxytoluene decreases liver triglycerides [31, 32, 151 or is without effect [33]. 2,2’-Methylenebis(4-methyl-6-tert-butylphenol) increases plasma cholesterol (Table III) as well as butylated hydroxytoluene [33-361 or ethoxyquin (0. Takahashi, unpublished data). 4,4’-Butylidenebis(3-methyl-6-tert-butylphenol) can induce fatty liver, fat deposition being associated mainly with increase of triglycerides and to a lesser extent, cholesteryl ester (Table II). Triglycerides, cholesterol, and non-esterified long-chain fatty acids are decreased by this bisphenol, suggesting that the fat excretion from the liver may be defective as in the case of carbon tetrachloride, ethionine, phosphorus, puromycin, choline-deficient diet and erotic acid-induced fatty livers, in which fat deposition is associated with depression of triglyceride excretion from liver to circulating blood [37]. Centrilobular fat deposition in the livers of ddY-mice given 2,4,6-tri-tertbutylphenol and slight fat deposition in Sprague-Dawley rats given 1,3,5-trimethyl2,4,6-tris(3,5-di-tert-butyl-4-hydroxybenzyl)benzene diet had previously been observed (0. Takahashi, unpublished data). On the other hand ethanol-induced fatty liver is prevented by N,N’-diphenyl-p-phenylenediamine, butylated hydroxytoluene, butylated hydroxyanisole or a-tocopheryl acetate [l-5], carbon liver by N, N’-diphenyl-p-phenylenediamine or tetrachloride-induced fatty ethoxyquin [5-g], erotic acid-induced fatty liver by N,N’-diphenyl-pphenylenediamine or butylated hydroxytoluene and dietary or choline-deficiencyinduced fatty liver by butylated hydroxyanisole or butylated hydroxytoluene 111, 121. 4,4’-Methylenebis(2,6-di-tert-butylphenol) slightly increases total liver lipids and this may be due to the increase

in cholesteryl

esters and cholesterol.

ACKNOWLEDGEMENTS

The authors are grateful to Mr. Yoshimitsu Hisatoshi Mikuriya for their histopathological

Sakamoto, Ms. Yukie Tada and Mr. observations.

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