Effects of chronic treatment with DI-(2-ethylhexyl) phthalate on rat liver microsomal activities

Toxicology Letters, 6 (1980) 51-58 o Elsevier/North-Holland Biomedical

51 Press

EFFECTS OF CHRONIC TREATMENT WITH DI-( 2ETHYLHEXYL) PHTHALATE ON RAT LIVER MICROSOMAL ACTIVITIES ETTORE ZUCCATO, ROBERTO CANTONI, FEDERICA BIDOLI, MILEMA RIZZARDINI, MARIO SALMONA, SILVIO CACCIA, KATIA GAMBAZZA, IVAN BARTOSEK AMALIA GUAITANI, FRANCA MARCUCCI and EMIL10 MUSSINI Istituto

di Ricerche

(Received (Accepted

February February

Farmacologiche

“Mario Negri”,

Via Eritrea, 62-20157

Milan (Italy)

14th, 1980) 18th, 1980)

SUMMARY

The effects of chronic di-( 2-ethylhexyl)phthalate (DEHP) on liver mi_ .crosomal activity were studied in rats. Daily doses of 50 and 500 mg/kg for 4 weeks did not affect 0-demethylation, aromatic hydroxylation, N-demethylation, C,-hydroxylation, styrene monooxygenase, glutarnic-oxalacetic and glutamic-pyruvic transaminases (GOT, GPT). Inhibition of glutathioneS-transferase A and C and induction of epoxide hydrase, glutathione-S’transferase B and nitroreductase activity were instead observed. Protein, cytochrome P-450 and reduced glutathione levels in liver did not appear to be affected by DEHP pretreatment.

INTRODUCTION

DEHP is the most commonly used plasticizer, and is found widely in our environment. Phthalate esters have been reported to have low toxicity [l--3] and a weak teratogenic effect [4] in laboratory animals. Guess et al. [ 51 have emphasized the subtle toxic effects of phthalate ester plasticizers. The present paper reports an investigation of the effects of chronic DEHP treatment on liver microsomal activities in rats. The microsomal activities investigated were iV- and 0-demethylation, hydroxylation, nitroreduction, and epoxide formation and hydration. This study was undertaken because it has been reported that phthalate esters affect hexobarbital sleeping time [6, 71 and zoxazolamine paralysis time [ 71.

Abbreviations:

Auc, area under

the curve; DEHP, di-( 2-ethylhexyl)phthalate.

52 MATERIALS

AND METHODS

Animals Charles River CD-COBS male rats, 150 f 10 g were used. They were housed 5 per cage in animal rooms (temperature 22”C, relative humidity 60%, lighting cycle, 12/12 h) and received a standard diet ad libitum (Altromin MT, Rieper, Italy) and water. Two groups of animals were treated for 4 weeks with 50 mg/kg and 500 mg/kg doses of DEHP dissolved in olive oil and administered by gavage. Treatment was for 5 days/wk, the animals being dosed at 3 p.m. each day. The total volume administered was 1 ml/kg b.w. of each compound. Control animals were given olive oil (1 ml/kg). Observations during the treatment Individual body weights of all rats were recorded daily and food consumption was measured once weekly (mean values of 5 animals). Parameters evaluated at the end of the treatment 20 h after the last dose the rats were killed and the liver, lungs, kidneys, heart and spleen rapidly removed. Microsomes were prepared by the method of Kato and Takayanagi [ 81. Concentrations of reduced glutathione were measured in whole liver homogenate by the method of Bemt and Bergmeyer [9], based on the enzymatic me~urement of glutathione with glyoxalose I in the presence of methylglyoxal. Glutathione-S-transferase activity was measured with 3 different substrates to take into account the different isoenzymes present in the supernatant of the 105 000 X g fraction [lo-131. Microsomal styrene monoxygen~e and epoxide hydrase were measured using styrene and styrene oxide as substrates 1141. O-Demethylation and aromatic hydroxylation were measured by the method of Kato and Gillette [ 3.51 using p-nitroanisol and aniline, respectively, as substrates and measuring the formation of o-NHa-phenol and p-NO,-phenol. ~-Demethylation and C,-hydroxylation were measured using diazepam (7-chloro-1,3-dihydro-1-methyl-5-phenyl2H-1,4-benzodiazepin-2-one) as substrate by the method of Marcucci et al. [16]. Diazepam is transformed by rat liver microsomal enzymes in 2 metabolites: ~-demethyldiazepam and methyloxazepam [ 161. Demethyldiazepam and methyloxazepam and the unmetabolized diazepam were measured after 10 min incubation. Cytochrome P-450 was measured by the method of Omura and Sato [ 171; proteins were measured by the method of Lowry et al. [18]. Aminopyrine disappearance from plasma of DEHP-treated animals was measured by the method of Brodie and Axelrod [ 191. Specifically DEHP-pretreated animals were treated 24 h after the last injection with a single i.v. dose of 15 mg/kg aminopyrine, DEHP-treated and control rats were killed after 15, 30, 60, 90 min and plasma samples were collected for analysis. Nitroreductase activity was measured by incubating liver 9000 X g fraction with nitrazepam as substrate.

53

The Gino-derive product (Aminazep~) was determined by the BrattonMarshall reaction [Xl-231 . GOT and GPT transaminases were measured in fresh non-haemolysed serum [ 241 (I The significance of the differences between untreat~ and the two treated groups was analysed by Dunnett’s test [27]. RESULTS AND DISCUSSION

Table I summarizes the protein content of the liver microsomal and cytosol fractions with results expressed in terms of g/fresh tissue. Cytochrome P-450 levels are shown in Table II. DEHP treatment did not appear to have any significant inducing effect, a finding in good agreement with the other results (Tables II and III) which indicated that aniline hydroxylase, p-nitroanisol demethylase, styrene monoxygenase, diazepam N-demethylase and diazepam hydroxylase activities were not si~ificantly modified in treated rats compared with control values. Acute treatment with 2.5 g/kg did not affect nitroreductase and diazepam in vitro metabolism. Table II presents the results also for epoxide hydrase. This enzyme, although particulate and belonging to the microsomal fraction, is not P-450 dependent and is sensitive to systemic treatments with foreign compounds [25, 261.

TABLE I PROTEIN CONTENT IN RAT LIVER FRACTIONS Supernatant (105 000 Control DEHPb (50 mg/kg) DEHPb (500 mg/kg)

(mglg FRESH TISSUE)a

X g)

42.56rt1.44 40.322 1.40 36.9Ot3.34

Microsomal suspension 23.39t0.25 22.0220.37 22.32+ 0.42

aEach value is the mean i S.E. of four determinations. bDEHP was given 5 times a week for 4 weeks. TABLE II EFFECT OF CHRONIC DEHP TREATMENT ON SOME RAT LIVER MICROSOMAL ACTIVITIES (mnol/min/mg PROT.)a AND ON MICROSOMAL CYTOCHROME P-450 CONTENT (nmol/mg: PROT.)a

Control DEHP (50 mg/kg) DEHP (500 m&kg)

Aniline hydroxylase

p-Nitroanisoio Q-demethylase

Styrene monoxygenase

185.40+12.32 192.45i16.07 161.56216.22

0.461+0.022

5.2Ok1.2 6.1720.3 6.95*0.5

0.499co.049

0.420*0.063

aEach value is the mean t S.E. of four determinations. bP < 0.05. cP < 0.01.

styrene

7,S-oxide hydrase 5.90*1.21 7.74+0.80b 10.02~1.30=

Cytochrome P-450

0.920t0.08 0.962+0.04 0.867t0.05

54

The levels of reduced glutathione in liver are reported in Table IV. This compound is an important biochemical parameter that may help in identification of the formation of nucleophilic agents in the body. It is known that foreign compounds frequently form unstable intermediates that covalently bind to DNA or proteins, and a decrease in liver glutathione levels after chronic treatment with a foreign compound frequently indicates the formation of such intermediates. In our experimental conditions, the decrease of reduced glutathione was not significant. Table V reports the activity of

TABLE III KN VITRO METABOLISM OF DIAZEPA~ BY LIVER MIC~OSO~ES ~EHP-TRRATED RATS (~ol/m~/mg PROTEINS)= Diaxepam added I.~M

100 200 300

Demethyldiazepam DEHP Owh3

formed

FROM CONTROL

Methyloxazepam DEHP Ow/kgf

AND

formed

c

50

500

c

59

500

0.08+0.01 0.12~0.01 0.14~0.01

O.lO+-0.01 0.13+0.01 0.17*0.01

O.lOrO.O1 0.13r0.02 0.17+0.02

0.43t0.04 0.52t0.02 0.51r0.03

0.76-rO.03 0.87iO.04 0.93~0.06

1.03r0.02 1.09t0.04 1.21tO.05

aEach value is the mean -t S.E. of five determinations.

TARLE IV LEVELS OF REDUCED G~UTATHIGNE (pmol/g FRESH TISSU~)a Control DEHF (50 mg/kg) DEHF (500 mg/kg)

IN RAT LIVER CRUDE HOMOGENATE

4.19kO.37 3.63kO.23 3.51-r0.63

aEach value is the mean * S.E. of four determinations.

TABLE V GLUTATHIGNE-S-TRANSFERASE LIVER CYTOSOL

Control DEHP (50 mg/kg) DEHP (500 mg/kg)

ACTIVITY

(nmol/min/mg

PROTEIN)a IN RAT

3,4-Dichloro-lnitrobenzene

1-chloro-2,4dinitrobenzene

1,2-Dichioro-4nitrobenzene

61+-0.01 20*0.01c 19t0.1C

754kO.10 795t0.01 860t0.05b

46+ 0.02 48+ 0.01 33+0.01b

aEach value is the mean * SE. of four determinations. bP < 0.05. CP < 0.01.

2.8 t 0.3

28.1 f 0.4

aP < 0.05 vs. controls and vs. 50 mg/kg treated group.

3.1 + 0.3

2.1 3.4 1.9 3.7 3.1

27.1 29 27.1 29 28.1

Mean f S.E.

nmol/mg proteins

2.15 3.54 3.1 2.7 3.8

DEHP 50 mg/kg

Proteins mg/ml

nmol/mg proteins

IN 9000 x g FRACTION

Control

ACTIVITY

1 2 3 4 5

No.

NITROREDUCTASE

TABLE VI

27.7 f 0.6

29 27.1 29 26.1 27.1

Proteins mg/ml

Proteins mg/ml 25.2 26.1 27.1 27.1 28.1 26.7 ?; 0.5

4.3 4.1 5.4 3.1 3.5 4.1 f 0.4a

DEHP 500 mg/kg nmol/mg proteins

56

glutathione-~-t~nsferase with 3 substrates. The first, 3,4-dichloro-l-nitrobenzene, is specific for the A and C isoenzymes, and the other two for B isoenzyme (ligandin). It has been established that the ratio of glutathione transferase catalytic activities towards 1-chloro-2,4_dinitrobenzene and 1,2-dichloro-4-nitrobenzene indicates the fraction of conjugating activity in cytosol contributed by transferase B [ 121. The A and C transferases were significantly inhibited by both doses of DEHP, whereas ligandin was significantly induced only in animals treated with 500 mg/kg. Table VI sets out the nitroreductase activity in the 9000 X g fraction. This activity did not appear to be significantly modified by repeated doses of 50 mg/kg/day of DEHP, but was clearly enhanced by treatment with 500 mg/kg. The differences between the higher dose treated group, the controls and the lower dose group were s~tistically si~ificant (P < 0.05). Protein content in the 9000 X g fraction was unaffected. Serum transaminase levels, body weight and food consumption were taken as indicators of possible toxic effects and liver damage induced by repeated doses of DEHP. GOT and GPT transaminases were measured in rat serum at the end of the experiment (Table VII): there were no significant differences between untreated and DEHP treated groups. Body weight gain of rats treated with DEHP (50 and 500 mg/kg) was similar to that of controls over the whole period of treatment and the food consumption/rat/week was not affected by chronic administration of DEHP. No mortality was recorded during treatment and no gross lesions were seen in rats at autopsy. The ph~ma~okineti~ data of aminopyrine, calculated assuming a single comp~ment model, are reported in Tables VIII and IX. Half-lives (F/z) and AUC were similar in all experimental conditions.

TABLE

VII

TRANSAMINASES DOSES OF DEHP

No.

IN SERUM

GPT mu/ml

1 2 3 4 5 Mean * SE.

132 67.1 70 54.2 155 96r 20

TREATED

Transaminases

Control GOT

OF RATS

41.3 34 23 26 18.1 2%.5+4

DEHP

50 mg/kg

GOT

GPT

80 77.4 70 70 106

21 18.1 26 13 46.4

8127

25+6

CHRONICALLY

in serum

WITH TWO

DEHP 500 mg/kg GOT

GPT

85 108 52 67.1 75

28 26 15.5 21 21

77.4*$-i

22.352.2

57 TABLE VIII PLASMA LEVELS OF AMINOPYRINE Pretreatment (mg/kg x weeks) DEHP 50 x 4 DEHP 500 x 4

Treatment (mg/kg i.v.) Aminopyrine Aminopyrine Aminopyrine

IN RATS PRETREATED Aminopyrine 15 min

15 15 15

WITH DEHPa

concentrations (fig/ml) 30 min

13.06tO.24 10.97*0.30 13.77~0.24 9.15kO.35 12.98kO.23 8.79kO.14

60 min

90 min

6.23tO.80 5.99kO.74 5.81kO.25

3.20+0.02 3.12fO.14 3.09+0.11

aEach value is the mean + S.E. of four determinations.

TABLE IX HALF-LIVES (2%) AND AREAS UNDER THE CONCENTRATION OF AMINOPYRINE PRETREATED WITH DEHPa Pretreatment mg/kg x weeks

T% (min)

AUC (Crg/ml/min)

Control DEHP 50x 4 DEHP 500x 4

36 36 37

971 926 900

CURVES (AUC)

_.-

aTreatment as set out in Table VIII.

CONCLUSIONS

In our experimental conditions, chronic treatment with DEHP appears to change only one parameter related to the P-450 system, i.e. liver nitroreductase activity. Moreover this induction was seen only in animals given 500 mg/kg DEHP. Among the biochemical parameters taken into consideration and not directly related to the P-450 system, three major differences were observed: (a) induction of epoxide hydrase; (b) inhibition of glutathione-S-transferase A and C; and (c) induction of ligandin at 500 mg/kg. REFERENCES 1 C.B. Shaffer, C.P. Carpenter and H.F. Smyth Jr., Acute and subacute toxicity of di( 2-ethylhexyl)phthalate with note upon its metabolism, J. Ind. Hyg. Toxicol., 27 (1945) 130-135. 2 C.P. Carpenter, C.S. Weil and H.F. Smyth Jr., Chronic oral toxicity of bis(2-ethylhexyl)phthalate for rats, guinea pigs, and dogs, Arch. Ind. Hyg. Occup. Med., 8 (1953) 219-226. 3 R.S. Harris, H.C. Hodge, E.A. Maynard and H.J. Blanchet, Chronic oral toxicity of 2-ethylhexylphthalate in rats and dogs, Arch. Ind. Hlt., 13 (1956) 259-264. 4 A.R. Singh, W.H. Lawrence and J. Autian, Teratogenicity of phthalate esters in rats, J. Pharm. Sci., 61 (1972) 51-55.

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