Effect of length of exposure to malathion on xenobiotic biotransformation in male rat liver

193

Toxicology Letters, 38 (1987) 193-199 Elsevier

TXL 01839

EFFECT

OF LENGTH

OF EXPOSURE

BIOTRANSFORMATION (Cytochrome S-transferase;

G.F.

REIDY,

IN MALE

P-450; monooxygenases; induction; inhibition)

H.A.

ROSE” and N.H.

TO MALATHION

ON XENOBIOTIC

RAT LIVER epoxide

hydrolase;

glutathione

STACEY

Occupational Health Division, National Occupational Health and Safety Commission, and “Department of Plant Pathology and Agricultural Entomology, University of Sydney, Sydney (Australia) (Received

2 March

(Revision

received

(Accepted

1987) 18 May 1987)

20 May

1987)

SUMMARY The effect

of exposure

was studied

to malathion

on several

in male Sprague-Dawley

200 mg/kg

malathion

glutathione

S-transferase

was decreased

after

showed

an increase

activity

1 week of exposure to 40 mg/kg

hepatic

biotransformation

to high doses of malathion activities.

Inductive

respective

of whether

results

effects

hydrolase

activity

and by both dosage

biotransformation

cytochrome

(1 week,

200 mg/kg) Aldrin

regimens

of epoxide

hydrolase

P-450 monooxygenase

no changes

that only continuous and glutathione activity

and

epoxidation

after 2 weeks. After

i.p. 3 times per week, however,

The results demonstrate

in an induction

was short-

xenobiotic

2 weeks 40 and 200 mg/kg).

administered

were noted.

on hepatic

exposure

in epoxide

to 200 mg/kg

malathion

of hepatic

of rats dosed i.p. daily for 1 or 2 weeks with 40 or

(1 week, 200 mg/kg;

9 weeks exposure xenobiotic

parameters

rats. Groups

in

exposure

S-transferase

were not observed

ir-

or medium-term.

INTRODUCTION

Malathion (S-l ,2-di(ethoxycarbonyl)ethyl O, 0-dimethyl phosphorodithioate) widely used non-systemic insecticide and acaricide of low mammalian toxicity

Address

for correspondence:

of Sydney,

Sydney,

Abbreviations: DCNB,

Dr. N.H.

AE,

aldrin

EH, epoxide

037%4274/87/$

Occupational

Health

Division,

Building

A27, University

NSW 2006, Australia. epoxidase;

1,2-dichloro-4-nitrobenzene;

deethylase;

Stacey,

is a [l].

03.50

0

hydrolase;

AH, ECOD,

aniline

GST, glutathione

1987 Elsevier

hydroxylase;

ethoxycoumarin

Science

S-transferase;

Publishers

AMD,

aminopyrine

0-deethylase;

EROD,

N-demethylase; ethoxyresorufin

P-450,

cytochrome

B.V. (Biomedical

Division)

P-450.

O-

194

In addition

to a range

of agricultural

and horticultural

uses it is used to control

animal ectoparasites and human head and body lice [2]. Several studies have shown that in vivo exposure to malathion results in the induction of hepatic microsomal cytochrome P-450 monooxygenases (P-450 monooxygenases) [3-61. In particular, the results of a study on factory workers exposed to malathion suggested that long-term exposure (over 2 years) to low levels of malathion caused induction of P-450 monooxygenases [3]. However, other studies have shown malathion to cause inhibition [7, 81 or to have no effect on P-450 monooxygenase activity after in vivo exposure [9]. Previous studies, however, are difficult to compare because of differences in the animals used, dosage levels and the type and length of malathion exposure. No single study has investigated both short- and medium-term malathion exposure to determine the importance of this variable. We therefore investigated the effect of exposure time to clarify the nature of the inductive/inhibitive effects of malathion. Intraperitoneal (i.p.) administration of malathion was preferred because of the surety of dose and the avoidance of confounding factors such as stability and bioavailability. Short-term (7- and 1Cday) exposures were employed for comparative purposes with other studies while the 63-day dosage regimen was considered an appropriate model for medium-term exposure. MATERIALS

AND

METHODS

Technical malathion (96%) was a generous gift from Cyanamid (Australia). Aldrin and dieldrin were purchased from Shell (Australia); ethoxycoumarin, 7-hydroxycoumarin, resorufin and 1,2-dichloro-4-nitrobenzene (DCNB) from Aldrich Chemical Co., Milwaukee, WI; aminopyrine from Sigma Chemical Co., St. Louis, MO; ethoxyresorufin from Pierce Chemical Company, Rockford, IL and [14C]styrene oxide from Amersham (Sydney). Enzymes and co-factors chased from Sigma Chemical Co. and Boehringer Mannheim (Sydney). chemicals used were of the highest grade commercially available.

were purAll other

Animals Male (body weight 220-400 g) Sprague-Dawley rats from the University of Sydney Animal House were allowed food and water ad libitum during these studies. Malathion (40 and 200 mg/kg) dissolved in corn oil (1 ml/kg) was administered i.p. either daily for 1 or 2 weeks or 3 times per week for 2 or 9 weeks. Microsomes and assays Microsomes and microsomal supernatants were prepared as previously described [l 11. Cytochrome P-450 content was determined according to Omura and Sato 1101 as previously described [l 11. Ethoxycoumarin and ethoxyresorufin-O-deethylation were determined by spectrofluorometric quantification of the respective phenolic products following the procedure of Prough et al. [12]. N-Demethylation of

195

aminopyrine

was determined

and

epoxidation

aldrin

as described were

measured

by Maze1 [ 131. Aniline-p-hydroxylation as

described

[ll].

Conjugation

of

1,2-dichloro-4-nitrobenzene (DCNB) by cytosolic glutathione S-transferases was measured as described by Kulkarni et al. [14] with minor modifications [I 11. Microsomal epoxide hydrolase activity was determined using [ 14C]styrene oxide following the method of Guengerich [ 151 with minor modifications [I 11. Protein content was determined by the modified Lowry method of Chaykin [16].

Statistics All results were evaluated by an analysis of variance and compared significant difference. Significance level was set at P
using the least-

RESULTS

The effects of malathion pre-treatment on hepatic microsomal epoxide hydrolase and cytosolic glutathione S-transferase activities are shown in Fig. 1. Epoxide hydrolase activity was significantly elevated after 1 week of exposure to 200 mg/kg of malathion. However, after 2 weeks exposure no significant increase was detected.

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Fig.

1. Effects

of daily

administration

of malathion

Glutothione S-transferore

on hepatic

microsomal

cytosolic glutathione S-transferase activity in male rats. Data are presented 3ignificantly different from control; bsignificantly different from 40 mg/kg

epoxide as means group.

hydrolase,

and

+ SE (n = 5).

196

1.0

UIn Control 0 4Omg/kg I ZOOmg/kg

1 Week 1

P-450

Fig. 2. Effects

AE

of daily administration

rats. Data are presented ferent

from 40 mg/kg

TABLE

as means

t

AH

of malathion

on hepatic

SE (n = 5). “Significantly

ECOD

cytochrome

different

P-450

parameters

from control;

in male

bsignificantly

dif-

group.

I

EFFECTS MALE

OF EXPOSURE

RAT LIVER

TO

MALATHION

ON XENOBIOTIC

BIOTRANSFORMATION

__~_,

~_______~

~

IN

~~~. ~

Treatment 2 Weeks Parameter

9 Weeks

Control

Malathion”

Control

Malathion”

P-450b

0.75

+ 0.02’

0.76

k 0.04d

0.72

AEf

2.98

+ 0.07

2.47

+ 0.08

3.78 zb 0.30

AMD’

3.16

+ 0.17

2.12

* 0.41

ERODg

19.0 & 2.0

22.0

+ 1.0

n.d.

3.71

c 0.04’ + 0.19

0.83

f

3.36

+ 0.14

0.03e

4.10

t

0.39

n.d.

EH’

21.87

* 2.78

22.27

+ 1.25

31.30

+ 2.84

32.90

+ 1.82

GST’

199.3

+ 18.2

210.5

+ 15.75

191.4

I

166.8

& 12.40

n.d.,

not

resorufin

determined; O-deethylase;

’ Rats dosed b Cytochrome

AE,

aldrin

EH, epoxide

epoxidase;

3 times per week with 40 mg/kg P-450 content

AMD,

hydrolase;

in nmol/mg

aminopyrine

GST, glutathione

8.40

N-demethylase;

EROD,

ethoxy-

S-transferase.

malathion.

protein.

‘Mean

5 SE; n=3.

’ Mean k SE; n=5. ’ Activity in nmol product/min/mg

protein.

’ Mean

& SE; n=4.

g Activity

protein.

in pmol product/min/mg

197

Glutathione group.

S-transferase

activity

was also increased

After 2 weeks dosing both treated

in the

groups showed elevation

l-week

200 mg/kg

of transferase

ac-

tivity. The effects of malathion on various parameters of hepatic microsomal cytochrome P-450are shown in Fig. 2. Aldrin epoxidase activity was significantly lowered after 1 week of exposure to 200 mg/kg of malathion. After 2 weeks exposure both treated groups showed a decrease in aldrin epoxidation. Cytochrome P-450 content was not altered though a trend to reduced levels with-treatment was observed. Both aniline hydroxylase and ethoxycoumarin-0-deethylase activities were unaffected by malathion treatment. Table I reveals the effects of dosing rats 3 times per week with 40 mg/kg malathion for 2 or 9 weeks on various hepatic xenobiotic biotransformation systems. As can be seen none of the activities investigated was affected after 2 or 9 weeks of dosing. This is in contrast to the results obtained in the daily 14-day dosing schedule (at 40 mg/kg) in which an increase in glutathione S-transferase activity and a decrease in aldrin epoxidation were noted. DISCUSSION

Much work has been done concerning the in vivo effects of malathion on hepatic P-450 monooxygenation in various animal groups. Little work, however, has been done on other xenobiotic biotransformation systems such as epoxide hydrolase and glutathione S-transferases. Short-term exposure studies in mammals have shown malathion to induce, or to have no effect on, monooxygenase activity. Stevens et al. [6] found that mice dosed orally with malathion had decreased hexobarbital sleeping times after 3 and 5 days of treatment. After 10 days exposure to lower doses no significant difference in sleeping time was noted. Madhuker and Matsumura [4] administered malathion i.p. to rats at 250 mg/kg daily for 7 days. In agreement with our l-week results (Fig. 2) they detected no change in P-450 levels or aniline hydroxylation. However, they also found several P-450 monooxygenase activities induced, in contrast to our results. Madhuker and Matsumura’s results, however, must be interpreted cautiously as no statistics were offered as support for induction by malathion. Instead they rather arbitrarily defined induction as any increase in excess of 120% of control. Using female rats Lechner and Abdel-Rahman [9] found that oral administration of 25 mg/kg of malathion for 7 days had no effect on aminopyrine demethylase or aniline hydroxylase activity. Medium- to long-term exposure studies have shown malathion to cause both inhibition and induction of P-450 activity. In humans Uppal et al. [3] showed that factory workers occupationally exposed to malathion had a reduced antipyrine half life when compared to matched controls. In contrast to this study other investigators have shown malathion to have an inhibitory effect on P-450 monooxygenations. Uppal et al. [8] found that repeated i.p.

198

administration

of 40 mg/kg

of malathion

resulted in increased pentobarbital paper the authors also noted that

for 15 days or 20 mg/kg

for 60 days

sleeping time in mice. However, in the same the well-documented monooxygenase inducer

DDT increased rather than decreased pentobarbital sleeping time. Varshneya et al. [7] found that dietary administration of malathion to cockerels over 90 days resulted in dose-dependent decreases in P-450 monooxygenase activity. Thus, the data concerning the effects of malathion on hepatic cytochrome P-450 monooxygenation are somewhat contradictory. Our data suggest that malathion administered to rats daily for 1 or 2 weeks acts as a selective inhibitor of cytochrome P-450 monooxygenation. Further, in contrast to the human study of Uppal et al. [3] our results suggest that exposure to low doses of malathion over longer periods of time has little effect on P-450 activity. An explanation for this observation may involve the different exposure routes of the studies. It should be noted that in the human study [3] workers were presumably exposed not only to malathion but also to precursors and by-products some of which may be potent P-450 inducers. The decrease in aldrin epoxidase activity noted in the 7- to 14-day dosing experiment (Fig. 2) is likely due to the destruction of specific P-450 isozymes by metabolic liberation of atomic sulfur from malathion as outlined by Neal et al. [17]. Indeed, the specific decrease in aldrin epoxidase activity while aniline hydroxylation and ethoxycoumarin-O-deethylation remained unaltered suggests that malathion may be metabolised by P-450 isozymes UT-A (P-450h) and PCN-E, both of which have been suggested to catalyse aldrin epoxidation in untreated male rats [18]. We found hepatic glutathione S-transferase activity was induced by daily malathion treatment for 1 or 2 weeks. An earlier report [14] using mice dosed with malathion for 3 days failed to detect induction of DCNB conjugation. It appears that induction of glutathione S-transferase activity requires high levels of malathion exposure. Given the high doses used and the relatively minor effects noted, the toxicological significance of malathion exposure on hepatic xenobiotic biotransformation systems appears minimal. In summary, the data presented here show that daily short-term repeated dosing of rats with malathion at 40 mg/kg or 200 mg/kg induces glutathione S-transferase activity, decreases aldrin epoxidase activity and causes a variable increase in epoxide hydrolation. Medium-term exposure to malathion at 40 mg/kg, 3 days per week had no effect on xenobiotic biotransformation. Our study therefore indicates that malathion is not a P-450 monooxygenase inducer in male rats under short- to medium-term exposure conditions.

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