Liver lipid peroxidation-related parameters after short-term administration of hexachlorocyclohexane isomers to rats

Toxicology

Letters,

56 (1991) 137-144

@ 1991 Elsevier Science Publishers ADONIS

037842749100058P

TOXLET

02541

137

B.V. (Biomedical

Division)

03784274/91/%

3.50

Liver lipid peroxidation-related parameters after short-term administration of hexachlorocyclohexane isomers to rats

Silvia B.M. Barrosl, Kiyoko Simizu2 and Virginia B.C. Junqueira’ ‘Faculdade de Cihcias

FarmacPuticas and ZDepartamento de Bioquimica. Institute de Quimica,

Universidade de Srio Paulo. Sa’o Paul0 (Brazil) (Received

23 July 1989)

(Accepted

30 October

1990)

Key words: Lipid peroxidation;

a-Hexachlorocyclohexane;

y-Hexachlorocyclohexane;

Short-term

intoxication

SUMMARY Rats treated

with diets containing

showed increased acid reactants

by liver homogenates

somes. In these animals between

20 ppm of a- or y-hexachlorocyclohexane

levels of liver cytochrome

SOD activity

15 days of treatment.

superoxide

and microsomes dismutase

and microsomal However,

P-450 followed by increased

superoxide

(CAT) activities.

or 30 days of treatment mechanism

of toxicity

radical

Among

(02-.)

dehydrogenase,

production

of a xenobiotic,

It is suggested

this parameter

for 15 or 30 days

of both thiobarbituric

production

by liver micro-

In consequence,

peroxidase,

showed

the ratio

showed a slight increase

for this ratio to decrease.

glutathione

them, only CAT activity

with the u-isomer.

anion

was also increased.

after 30 days, there was a tendency

eters studied were liver glucose-6-phosphate and catalase

and superoxide

(SOD) activity

(HCH)

production

glutathione

reductase

a 26% and 38% increase

that when lipid peroxidation can be used to determine

after

Other paramafter

is involved

15

in the

the no-observed-effect

level.

INTRODUCTION

Hexachlorocylohexane

Address for correspondence: Faculdade

de Ciencias

Abbreviations:

(HCH)

is an organochlorine

Dr. Silvia B.M. Barros,

Farmactuticas,

Universidade

DDT =dichlorodiphenyltrichloroethane;

Departamento

CAT =catalase;

GPx = glutathione

peroxidase;

ane;

no-observed-effect

level; MDA = malondialdehyde;

superoxide

dismutase;

TBAR = thiobarbituric

de Analises

that has been used

Clinicas

e Toxicologicas,

de Sao Paulo, P.O. Box 30.786, Slo Paulo,

dehydrogenase; NOEL =

insecticide

GR = glutathione acid reactants.

reductase;

Brazil.

G-6-PD = glucose-6-phosphate HCH = hexachlorocyclohex-

02m’ = superoxide

radical;

SOD =

138

world-wide during the last 40 years. The technical isomers but only one of them, namely the y-isomer, this reason

and mainly

active isomers,

because

product is a mixture of stereo has insecticidal properties. For

of the high environmental

the use of the technical

product

was forbidden

persistence

of the non-

in many countries.

One of the most studied properties of chlorinated insecticides is the capacity to induce the hepatic cytochrome P-450 system which is responsible for the biotransformation of xenobiotics [I]. This property has been used to determine the no-effect level of many chlorinated insecticides [2,3]. Enzyme induction has always been considered a mechanism of cellular adaptation [4]. However, the increased activity of the cytochrome P-450 system can sometimes lead to the formation of free radicals that can be hazardous to the liver cell. One of these radicals is 0~~. (superoxide anion) produced by the oxidative activity of cytochrome P-450 [5,6]. Moreover, free radicals can also be produced by the reductive activity of cytochrome P-450 such as during carbon tetrachloride (CC&) [7,8], dichloro-diphenyl-trichloro-ethane (DDT) (9) and y-HCH biotransformation [lo]. These free radicals can bind irreversibly to biological molecules such as proteins, lipids or nucleic acids. As a result, many alternations, e.g. enzyme inactivation, mutation and lipid peroxidation, can occur [I 11. The latter has been responsible for many pathological conditions such as liver lesions following CC14 and ethanol intoxication [ 121. HCH, like other chlorinated insecticides, is also a liver enzyme inducer [2,13]. Most of the literature concerning HCH enzyme-inducing properties is related to the yisomer. However, Schorter et al. [14] demonstrated that both the c1- and y-isomers have similar behavior with respect to this property and that the p-isomer is less potent. Increased levels of liver cytochrome P-450 are measured after treatment of rats with diets containing 900 ppm of technical HCH for 60 or 90 days. This increase is followed by increased lipid peroxidation rates of liver homogenates [ 151. In acute y-HCH intoxication, Junqueira et al. [ 161 demonstrated that after 24 h of intoxication there is an increase in the liver cytochrome P-450 content. This is followed by an increase in both endoplasmic reticulum Oz-. production and lipid peroxidation levels. Although the property of liver enzyme induction the response of liver lipid peroxidation parameters

is shared by all the HCH isomers, to subchronic treatment with the

isolated isomers of HCH is still unknown. The aim of this work was to study the effect of subchronic and y-HCH isomers on liver lipid peroxidation parameters. MATERIALS

treatment

with the CI-

AND METHODS

Ninety-day-old male Wistar rats, weighing about 200 g, were individually housed in wire cages and received ad libitum a commercial diet (Produtor 49, Anderson Clayton, Brazil) with and without 20 ppm of c(- and y-HCH. After 15 or 30 days of

139

treatment,

the animals

were killed by cervical concussion

cold 0.9% NaCl. The livers were then removed,

and the livers perfused

weighed and homogenized

with

with 3 vol-

umes of 140 mM KC1 and 10 mM potassium phosphate buffer (pH 7.0). This homogenate was centrifuged at 900 x g in a RC2B Sorvall for 20 min at 4°C. Part of the supernatant previously obtained was employed as homogenate for thiobarbituric acid reactant (TBAR) rate determination as described elsewhere [16]. Microsomes were prepared from this homogenate by conventional procedures [ 171 and the 105 000 x g supernatant was considered as the cytosolic fraction. The microsomal pellet was resuspended as previously described [ 161 and used for the determination of cytochrome P-450 content [ 171, NADPH-cytochrome P-450 reductase activity [ 181 and for superoxide radical production rate [ 191. The activities of glucose-6-phosphate dehydrogenase (G6PD) [20], glutathione reductase (GR) [21], glutathione peroxidase (GPx) [21] and catalase (CAT) [22] were determined in the cytosolic fraction and expressed as units/mg of protein considering the molar extinction coefficients originally described. Superoxide dismutase (SOD) activity was determined according to Beauchamp and Fridovich [23] in the same fraction. The protein content of each subcellular fraction was determined as described by Layne [24]. For histological observations, liver fragments were excised just before liver perfusion with 0.9% NaCl. Histopathological studies were performed on liver slices stained with hematoxylin-eosin and Sudan III when necessary [25]. Student’s r-test [26] for unpaired results was used for significance in the studies carried out. RESULTS

After TABLE

15 days of treatment

both a- and y-HCH

induced

an increase

respectively

I

CYTOCHROME

P-450

LIVER

MICROSOMES

7-HCH

FOR

CONTENT FROM

AND

CYTOCHROME

CONTROL

RATS

AND

P-450 RATS

REDUCTASE

TREATED

ACTIVITY

WITH

I5 OR 30 DAYS Cytochrome

Treatment

(nmol/mg

Cytochrome

P-450 protein

P-450 reductase

(Ujmg protein

+ SEM)

k SEM)

15 days

30 days

I5 days

30 days

Control

0.858~0.026(13)

0.870+0.091

(13)

0.083 f 0.003 (7)

0.079 +0.004

wHCH

1.031 kO.025 (8)*

1.188+0.011

(6)*

0.084+0.003

(7)

0.108 kO.007 (7)*

y-HCH

1.154f0.016(16)*

1.469+0.014

(13)*

0.090f0.006

(6)

0.077 kO.004 (6)

SEM = standard *Statistical

OF

20 ppm a- OR

error

significance

of the mean.

Numbers

at PC 0.01 from controls

in brackets by Student’s

represent f-test.

the number

of animals

(7)

per group.

140

TABLE

II

RATE OF TBAR PRODUCTION TROL

BY LIVER

Treatment

HOMOGENATES

AND MICROSOMES

WITH 20 ppm a- AND y-HCH

RATS AND RATS TREATED

FROM

CON-

FOR 15 OR 30 DAYS

TBAR (nmol MDA/120

min/mg

protein It SEM)

Homogenate

Microsomes

15 days

30 days

I5 days

30 days

4.028+0.117(22)

l.037~0.0~(13)

I .062&0.057

(12) (7)’

~Control

3.808_tO.198

a-HCH

6.108~0.~6(7)*

5.760 kO.237 (6)”

1.673 + 0.098 (8)*

2.064+0.075

y-HCH

5.076~0.374(13)*

5.396kO.139

1.492~0.~3

1.821 kO.077 (12)*

SEM=standard *Statistical

error

significance

(21)

of the mean.

Numbers

(1 I)*

in brackets

at PcO.05 from controls

represent

by Student’s

(14)’ the number

of animals

per group.

f-test.

of 20% and 34% in the cytochrome P-450 content of liver microsomes. However, NADPH-cytochrome P-450 reductase activity was not altered (Table I). On the other hand, after 30 days, the increased levels of liver cytochrome P-450 (37% and 69% for 01-and y-HCH, respectively) were followed, only for the g-isomer, by a 37% increase in the activity of NADPH-cytochrome P-450 reductase (Table I). These increased levels of cytochrome P-450 were accompanied by an increased production of TBAR substances by liver homogenates and microsomes (Table II). An increased rate of superoxide anion production by liver microsomes was also observed (Table III). However. superoxide dismutase activity was increased in all treat-

TABLE

III

MICROSOMAL DISMUTASE

SUPEROXIDE ACTIVITY

20 ppm OF a- OR y-HCH

ANION

OF LIVER FOR

PRODUCTION FROM

AND CYTOPLASMATIC

CONTROL

RATS

AND

RATS

SUPEROXIDE TREATED

WITH

15 OR 30 DAYS ~-__I

Microsomal 02

Treatment

production

(nmol adren~hrome/~n/g

SOD activity

SOD/C& --.

(U/g liver f SEM)

liver + SEM)

I5days

30 days

Control

364.88&0.65(8)

366.07&0.74(8)

a-HCH

530.06&0.69(8)*

588.69&0.92(8)*

y-HCH ~__..

598.05,0.71(8)*

737.65*0.99(8)*

SEM=standard *Statistical

error

significance

of the mean.

Numbers

at P~O.001 fron controls

15days

30 days

15

30

days

ddys

1295.00& 1.45(8) 2390.71 f4.90(8)*

1289.49+ 1.38(7) 1888.33i2.20(8)*

3.56 4.51

3.21

2315.62_f3.97(7)* -_ .._-.

2146.62&3.13(8)*

3.87

2.91

in brackets

represent

by Student’s

r-test.

the number

of animals

3.52

per group.

141

TABLE

IV

ACTIVITY

OF GLUCOSE-6-PHOSPHATE

DUCTASE

(GR),

FROM

GLUTATHIONE

CONTROL

DEHYDROGENASE

PEROXIDASE

RATS AND RATS TREATED

(GPx)

AND

(G6PD),

GLUTATHIONE

CATALASE

WITH 20 ppm G(-OR y-HCH

(CAT)

OF

RELIVER

FOR 15 OR 30 DAYS

G6PD

GR

GPx

CAT

(Ujmg protein + SEM)

(Ujmg protein f SEM)

(Ujmg protein + SEM)

(Ujmg protein k SEM)

Control

0.018~0.001

(7)

0.110+0.006

(8)

1.260 f 0.053 (7)

271.75 & 16.92 (8)

a-HCH

0.017+0.001

(7)

0.127+0.008

(7)

1.371+0.074

(7)

342.53 & 14.99 (8)*

y-HCH

0.016+0.002

(7)

0.112,0.005

(6)

1.169&0.038

(7)

301.09+

Control

0.016+0.001

(8)

0.093 kO.003 (8)

1.156*0.037(8)

282.72 + 13.88 (8)

a-HCH

0.017f0.004

(8)

0.107~0.002

(8)*

1.143+0.020(8)

389.45 + 26.65 (7)*

y-HCH

0.016~0.001

(7)

0.090~0.004

(8)

1.113+0.044(8)

303.72*

1s ahys

14.02 (8)

30 day.9

SEM = standard *Statistical

error of the mean. Numbers

significance

19.01 (7)

in brackets represent the number of animals per group.

at P< 0.01 from controls by Student’s f-test.

ed animals. In this way, the ratio between superoxide dismutase activity and microsomal 02-. production showed a slight increase between 15-day treated and untreated animals. Moreover, after 30 days this ratio showed a tendency to decrease (Table III). Among the other parameters studied, G6PD, GPx and GR activities were not altered after 15 or 30 days of treatment with either isomer (Table IV). However, catalase activity showed an increase of 26% or 38% after 1.5 or 30 days of treatment, respectively, but only for the a-isomer (Table IV). Microscopic observation of liver sections showed no significant alterations in any of the treated

animals.

DISCUSSION

HCH isomers

have been shown

to produce

different

acute and chronic

responses

in living organisms. Liver necrosis and steatosis have been described among the hazards following acute and chronic lindane intoxication [ 16,27,28]. Free radicals have been implicated in the liver injury consequent to xenobiotic exposure [29]. These radicals can be produced by liver biotransformation reactions, when relatively harmless substances are transformed into highly reactive free radicals [7,12,30]. Other xenobiotics act by increasing oxygen-derived free radical production within liver cells [31]. In both cases these free radicals are able to enhance lipid peroxidation of hepatocyte membranes with consequent liver injury [ 111. Recently, Baker et al. [lo] proposed reductive liver microsomal dehalogenation of lindane with the formation of a pentachlorocyclohexane radical. On the other hand,

142

a causal

relationship

cytochrome demonstrated

between

P-450 induction, in acute lindane

increased

superoxide

anion

production,

and increased liver lipid peroxidation intoxication in rats [ 161.

secondary

to

rate has been

In this experiment we demonstrated that t(- and y-HCH induced an increase in liver P-450 levels with no changes in cytochrome P-450 reductase activity. This effect was more pronounced after y-isomer treatment. However, although both isomers induced an increase in liver lipid peroxidation rates, it was more pronounced in the M-HCHtreated animals. As previously demonstrated, increased cytochrome P-450 levels can generate superoxide anion [16]. In fact, the liver microsomal production of superoxide anion was increased in both U- and y-HCH-treated animals. However, this increase was followed by enhanced activity of SOD. In consequence, the relation between SOD activity and superoxide anion production in HCH-treated animals did not differ from that in control animals. In fact, an increase in SOD activity is expected when cells are submitted to oxidative stress mainly during short- or long-term experiments. Thus high levels of O?-. generation cannot be directly responsible for the increased levels of TBAR produced, as in acute lindane treatment. On the other hand, the proposed carbon-centered radical formation suggested by Baker et al. [lo] must be considered as a possible explanation for the increased levels of TBAR observed. Histological observations of liver showed no differences between HCH-treated and untreated animals. However, the increased levels of lipid peroxidation, demonstrated in both Z- and y-HCH-treated animals, could lead to an alteration in homeostatic levels with long-term pathological consequences. Den Tonkelaar and Van Esch [3] and Pelissier and Albrecht [2], based on the induction of microsomal liver enzymes in short-term toxicity experiments, proposed a no-observed-effect level (NOEL) for lindane of 20 ppm. In a short-term experiment rats exposed for 30 days to diets containing IO ppm of r- or y-HCH showed no alterations in liver microsome P-450 content or lipid peroxidation levels (unpublished results). In this experiment we demonstrated that the NOEL for both I’- and a-HCH is below 20 ppm. As a correlation exists between liver cytochrome P-450 levels and lipid peroxidation production rates we suggest that the liver lipid peroxidation rate could also be used to establish the NOEL when this mechanism is involved in the toxic action of the xenobiotic. ACKNOWLEDGEMENTS

This work was supported by grant 83-0034-5 from FAPESP (Fundaclo de Amparo a Pesquisa do Estado de Sao Paulo, Brazil) and CNP (Conselho de Desenvolvimento Cientifico e Tecnologico, Brazil).

143

REFERENCES

1 Guengerich,

F.P. and Liebler,

CRC Crit. Rev. Toxicol.

2 Pelissier, M.A. and Albrecht, oxygenases

microsomales

3 Den Tonkelaar, on induction 4 World

Health

C., Prost.

Chemical 7 Dianzani,

Society, Washington,

binding.

Chem.-Biol.

chlorine

experiments.

pesticides

Toxicology

for evaluating

based

2, 371-80.

the toxicity

of chemicals.

Press, London,

Interact.

production

P-450: its role in oxygen activation

Concepts.

Symposium

from

for drug

Series No. 44. American

In: T.F. Slater (Ed.), Biochemical

Mecha-

p. 669.

H. (1982) Metabolic

activation

of carbon

or 3-methylcholanthrene

tetrachloride:

in-

and its effects on covalent

40, 193-208.

(I 984) Metabolism-dependent

DDD, to microsomal

protein

binding

of lindane in rat liver microsomes

L.A. (1985) Citotoxidad

de xenobioticos:

of the chlorinated

and lipids. Biochem.

R.M. and Van Dyke R.A. (1985) The formation

metabolism

by liver microsomes

1.

H.J. and Remmer,

DDT and its metabolite

anion

J. (1977) Cytochrome

9 Baker, M.T. and Van Dyke. R.A.

by the reductive

les mono-

27, 355-358.

P-450 with phenobarbital

10 Baker, M.T. Nelson,

levels of organ0

toxicity

U. (1978) Lipid peroxidation.

Academic

of cytochrome

I1 Videla,

Pharmacol.

DC, p.

M.V. and Ugazio,

H. Haussmann,

induisant

14,297-301.

and methods

In: D.M. Jerina (Ed.), Drug Metabolism

duction

to toxic metabolites.

du regime en hndane

Toxicol.

J. (1978) Superoxide

rat. Biochem.

nism of Liver Injury. 8 Frank,

minimale

in short-term

(1987) Principles

R.W. and Werringloer,

metabolism.

of chemicals

Crit. 6.

D. and Hakim,

treated

6 Estabrook.

liver enzymes

Organization

phenobarbital

activation

E.M. and Van Esch, G.J. (1974) No-effect

Part I. Environ. 5 Auclair,

T.R. (1976) Teneur

chez le rat. Food Cosmet.

of microsomal

Health

D.C. (1985) Enzymatic

14. 259-307.

Pharmacol.

of cholorobenzene

Arch. Biochem.

un problema

insecticide

33,2555260.

metabolico.

and benzene

Biophys.

236, 506-514.

Biol. Educ. Bioquim.

4,

3540. 12 Comporti.

M., Biology

of disease.

Lipid peroxidation

and cellular

damage

in toxic liver injury.

Lab.

Invest. 53,599623. 13 Parzefall,

W.. Munster,

hexachlorocyclohexane activities

in rat liver. Biochem.

14 Schorter,

C., Parzefall,

fects of Z-, p- and growth

J. and Schulte-Hermann, and its metabolite

16 Junqueira.

V.B.C.,

17 Lu. A.Y.H.,

Simizu,

Junk,

hydroxylation

K., Videla,

R. Doseeresponse

preneoplastic

studies

on the ef-

foci. monooxygenases,

and

C.H. and Kamin.

anion production.

study of the

antioxidant

enzyme

41, (1969) 1933204.

M. (1969) Resolution

system of liver microsomes

and renal lipid peroxida-

S.B.M. (1986) Dose-dependent

on rat liver superoxide

Toxicology

and Coon,

S.B.M. (1985) Hepatic

An. Fam. Quim. 25,20-24.

L.A. and Barros,

administration

K.W.

and mono-oxygenase

Res. 47,8&88.

and lipid peroxidation.

18 Williams,

on putative

with hexachlorocyclohexane.

effects of acute hndane activities

H. and Schulte-Hermann,

V.B.C., Simizu, K.. Boveris, A. and Barros,

tion in rats treated

study on the effects of X-

on growth

29, 216992178.

y-hexachlorocyclohexane

in rat liver. Cancer

15 Junqueira,

Pharmacol.

W., Schorter,

R. (1980) A comparative

/%pentachlorocyclohexane

of the cytochrome

into three components.

H. (1962) Microsomal

P-450-containing

w-

J. Biol. Chem. 244, 3714 3721.

triphosphopyridine

nucleotide-cytochrome

c reduc-

tase of liver. J. Biol. Chem. 237, 5877595. 19 Boveris,

A., Fraga,

superoxide

production

C.G.,

Varsavski,

A.I. and Koch,

in the liver of chronically

O.R. (1983) Increased

ethanol

treated

rats. Arch.

chemiluminescence

and

Biochem.

227,

Biophys.

534-541. 20 Clock,

G.E. and McLean,

dehydrogenase

P. (1953) Further

and 6-phosphogluconate

21 Sies, H., Koch, O.R., Martino, in chronically

ethanol

treated

studies on the properties

dehydrogenase

and assay of glucose-6-phosphate

of rat liver. Biochem.

E. and Boveris, A. (1979) Increased rats. FEBS Lett. 103. 2877290.

J. 55,40&408.

biliary glutathione

disulfide

release

144

22 Beutler, E. (1975) Red Cell Metabolism, 2nd ed. Grune and Stratton, New York. pp, 89990. 23 Beauchamp, C. and Fridovich, I. (1971) Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44,276287. 24 Layne, E. (1957) S~trophotometric and turbidimet~c methods for measuring proteins. Methods Enzymol. 3,447454. 25 Pearse, A.G.E. (1968) Histochemistry, 2 vols., 3rd cd. Churchill, London. 26 Snedecor. G.W. (1956) Statistical Methods Applied to Experiments on Agriculture and Biology, 5th ed. Iowa State University Press, Ames, Iowa, 534 pp. 27 Barros, S.B.M. and Saliba, A.M. (1978) Toxicity of the hexachlorocychohexane in rats. Toxicology 10,271-279. 28 Fitzhugh, O.G., Nelson, A.A. and Frawley, J.P. (1950) The chronic toxicities of technical benzene hexachloride and its a-, /?- and y-isomers. J. Pharmacol. Exp. Ther. 100, 59-66. 29 Wilson, R.L. (1978) Free radicals and tissue damage: mechanistic evidence from radiation studies. In: T.F. Slater (Ed.), Biochemical Mechanism of Liver Injury. Academic Press, London, pp. 123-224. 30 Benedetti, A.. Ferrali, M., Chieli, E. and Comporti, M. (1974) A study on the relationships between carbon tetrachloride-induced lipid peroxidation and liver damage in rats pretreated with vitamin E. Chem.-Biol. Interact. 9, 117--134. 31 Bus, J.S., Aust, S.D. and Gibson, J.E. (1976) Paraquat toxicity: proposed mechanism of action involving lipid peroxidation. Environ. Health Perspect. 16, 1399146.