Effect of chlordane on hepatic mitochondrial respiration

Toxicology Letters, 48 (1989) 67-74

67

Elsevier

TXL 02183

Effect of chlordane on hepatic ~itoc~ondrial respiration

Masana Ogata, Fumio Izushi, Kohei Eto, Ritsue Sakai, Bunji Inoue and Nobuyuki Noguchi Department of Public Health, Okayama University Medical School, Okayama (Japan)

(Received 25 June 1988) (Revision received 30 November 1988) (Accepted I4 March 1989) Key words: Chlordane; Oxidative phosphorylation;

K+ efflux; Mitochondria

SUMMARY In order to clarify the cytotoxicity of chlordane, an industrial product used as an insecticide, its effect on oxidative phosphorylation in rat hepatic mitochondria was studied. The respiration rate, RCI and ADP/O ratios were inhibited by chlordane-related compounds; the degree of inhibition was in the descending order of trans-chlordane, cis-chlordane, heptachlor and heptachlorepoxide. Of the indices indicating various respiratory activities, state 3 respiration was the most sensitively inhibited by these compounds, suggesting that they inhibit energy transfer. However, electron transport was inhibited also by high concentrations of chlordane constituents. The inhibitory effect of the chlordane constituents on respiratory activity varied depending on the species of respiratory substrate, suggesting site-specificity of these compounds. The release of K+ ions paralleled the results of the respiratory activity study. Heptachlorepoxide, a metabolic product of heptachlor, had less effect on mitochondria than heptachlor.

INTRODUCTION

Technical chlordane, containing beans-chlordane, eis-chlordane, y-chlordene, heptachlor, trans-nonachlor and cis-nonachlor has been widely used in Japan for the protection of wooden structures from. termites, and its use had increased up to 1986 [I] when it was prohibited. Chlordane residues have been found in various foods Address for correspondence: Masana Ogata, Department Medical School, Shikata-cho, Okayama 700, Japan.

of Public Health, Okayama University of

0378-4274/89/$3,50 @ 1989 Elsevier Science Publishers B.V. (Biomedical Division)

68

[24]. However, technical chlordane is used currently in several countries of Southeast Asia. As shown in several reports, chlordane has been detected not only in the blood of pest control operators [5] but also in blood from non-occupationally exposed human subjects [6] and in mothers’ milk [7, 81. Studies on the fate of chlordane constituents indicate that they accumulate in the adipose tissue and liver of rats [9]. Thus, the toxicity of chlordane and the problem of environmental contamination with this chemical must be thoroughly investigated. Findings on the toxicity of chlordane in the rat include stimulation of the central nervous system, gastric ulcer, hepatomegaly [lo] and thyroid carcinoma [l 11.Moreover, cancer of the liver has been reported in mice treated with heptachlor and chlordane for 18 months [12]. The mechanisms by which the hepatic toxicity of chlordane is mediated are not clearly understood. From this point of view, the effects of chlordane on hepatic mitochondrial membrane were examined. This communication describes the effects of the major 3 constituents of technical-grade chlordane on the sites of oxidative phosphorylation in isolated rat liver mitochondria. MATERIALS AND METHODS

Preparation of rat liver mitochondria

Male Donryu rats, 8 weeks old, weighing approximately 200g were sacrificed by decapitation. The rats were kept in individual cages made of stainless steel (Okazaki Sangyo Co., Tokyo) with white wooden flakes (Oriental Yeast Co., Tokyo) and a 12 h day/night cycle at 22°C and 4&58 % relative humidity. Tap water and a standard pellet (Oriental Yeast Co., Tokyo) diet were given ad libitum during the experimental period. Liver mitochondria were isolated according to a modification [14] of the method of Hogeboom and Schneider [ 131. Mitochondrial protein was determined by the biuret reaction using bovine serum albumin as a standard. The biuret reaction of Gornall et al. [ 151was used. Measurement of respiratory activity of mitochondria

Mitochondrial oxygen uptake was measured with a galvanic oxygen electrode (Kyusui Kagaku kenkyusho Co., Tokyo) connected to an autorecorder. Mitochondria (4.665.48 mg of protein/ml) were incubated at 25°C with continuous stirring in a reaction medium (2.5 ml) containing 0.15 M KCl, 10 mM Tris-HCl buffer (pH 7.4), 5 mM MgCl2 and 10 mM phosphate buffer (pH 7.4). Five millimolar respiratory substrate [succinate, p-hydroxybutylate (B-OH) or ascorbate plus 0.1 mM iV,N,WJ’tetramethylphenylene-diamine (TMPD)], 0.25 mM ADP and 0.02 mM 2,4-dinitrophenol (DNP) were added in the presence or absence of chlordane. Respiratory control index (RCI: state 3/state 4) and ADP/O ratio were calculated according to the method of Hagihara [ 161.

69

Measurement Mitochondria

of K+ eflux (0.4 mg protein/ml)

were incubated

in the medium

containing

0.15

M choline chloride and 10 mM Tris-HCl buffer (pH 7.4) at 25°C. The measurement of K+ efflux was carried out by using a K + electrode (Orion Co.) connected to a pH meter. The signal of K+ concentration change was amplified and recorded with an autorecorder. Reagents Trans-chlordane, cis-chlordane and heptachlor as constituents of technical grade chlordane and heptachlorepoxide as metabolite were purchased from Wako Chemicals Co. (Tokyo) and dissolved in ethanol solution. The ethanol solution and the control were treated identically. The final concentration of ethanol was 0.36% in the reaction medium. This concentration of ethanol had no effect on mitochondrial respiratory activity. Na-ADP and Na-ATP were from Boehringer Mannheim Yamanouchi K.K. (F.R.G.), and oligomycin from Sigma Chemicals Co. (U.S.A.). Other chemicals used were commercial products of reagent grade. RESULTS

EfSect on respiratory activity of rat liver mitochondria Fig. 1 represents the change in respiratory activity of isolated mitochondria by trans-chlordane. As shown in Fig. IA, 50 PM (22-25 nmol/mg protein) trans-chlordane inhibited both state 3 and state 4 respiration. State 3 respiration was inhibited almost completely by 100 ,LLMtrans-chlordane; however, this inhibited respiration was released by DNP. DNP was added to all the reaction media at the time when the rate of oxygen uptake became stable. Thus, in oxygen uptake curves after addition of higher concentrations of chlordanes to the mitochondrial solution, the interval between the addition of ADP and the addition of DNP was shorter than that after the addition of lower concentrations of chlordanes. Concentrations of transchlordane higher than 0.5 mM completely inhibited oxygen mitochondrial consumption. Similar results were obtained when P-OH was used as the respiratory substrate (Fig. IB). On the other hand, when ascorbate + TMPD was used as an artificial electron-transfer system, state 4 respiration was maintained even in the presence of a high concentration of trans-chlordane (0.5 mM) (Fig. IC). This suggests specificity of trans-chlordane with respect to its active site: i.e., the sensitivity of electron transport to chlordane between cytochrome c and oxygen (site III) may be less than that of other sites (sites I and II). Fig. 2 shows the concentration dependence of the effects of the major chlordane constituents and heptachlorepoxide on state 3 and state 4 respiration, RCI and ADP/ 0 ratio with succinate as substrate. State 3 respiration was most sensitively inhibited; the concentration of chlordane required to produce 50% inhibition ranged from 40 to 60 PM.

70

5.48

4.59

mq

mg protein

protein

I l-

100)JMO2

2

n-l,”

71

+ DNP

001 Trons - chlordane

CIS - chlordane

(PM)

(JJM)

‘/.

‘1.

C

100

D 100

P

5 ”

b ,g

50

8 b a

OO1 Heptachlor

Fig. 2. Effect of chlordane

epox!de

compounds

4 and DNP-released

(PM

1

,

,

20

50

\state3 100

Heptachlor

and heptachlorepoxide (+ DNP) respiration],

(PM)

on mitochondrial

respiration

RCI and ADPjO

ratio.

[state 3, state

The RCI (state 3/state 4 respiration) in the presence of 50 ,LJMtram-chlordane, cischlordane, heptachlor, heptachlorepoxide and control without addition of the above reagents was calculated to be 1.8, 2.6, 3.1, 3.3 and 4.7, respectively. Thus the degree of inhibitory effect was in the descending order of tram-chlordane, cis-chlordane, heptachlor and heptachlorepoxide. Efect on K+ compartmentation of mitochondria

As shown in Fig. 3, little Kt release was seen during the incubation of untreated C

Fig.

I. Effect of rrans-chlordane

(Sue) as substrate; tochondria

on the respiratory

(B) 5 mM B-hydroxybutylate

with added

frans-chlordane.

activity

The oxygraph

preincubation

of rat liver mitochondria.

(B-OH); (C) 5 mM ascorbate with

on the extreme

(Astor)

right of(C)

I mM KCN for 30 s.

(A) 5 mM succinate + TMPD.

Mt’=mi-

shows that of Mt after

72

% lOO-

0 Heptachlor A Trans - chlordane 0 CIS -chlordane

10

20

30

OJM)

Concentration

Fig. 3. Potassium bols (0. chlordane

release from mitochondria

A, U) indicate

average

and cis-chlordane,

at various

values as percent

concentrations

of chlordane

of total intramitochondrial

Kf

compounds.

The sym-

of heptachlor,

trans-

respectively.

control mitochondria. By the addition of various concentrations of truns-chlordane, cis-chlordane and heptachlor to the medium, however, release of intramitochondrial K+ was induced in a concentration-dependent manner. Concentrations which induced 50% release of K+ during the initial 3 min incubation in 5 cases were 14.9 f 0.66 PM for trans-chlordane, 15.9 f 0.36 ,uM for cis-chlordane and 18.7 f 0.48 ,uM for heptachlor. A significant difference was obtained between trans-chlordane and heptachlor as tested by Scheffe’s method [ 171 after one-way classification in analysis of variance. This K+-releasing intensity of these substances paralleled the values of the respiratory control index. DISCUSSION

In the present

investigation,

low concentrations

(6 100 PM) of chlordane-related

compounds suppressed state 3 and state 4 respiration, resulting in a decrease in RCI and ADP/O ratio; the suppressed state 3 respiration was released by DNP. The results suggest that these compounds can be characterized chemically as energy transfer inhibitors. However, as illustrated by the effect of truns-chlordane (Fig. 1A and B), complete inhibition of oxygen consumption indicates that they are electron transport inhibitors at higher concentrations. Ogata et al. [ 181 classified various chemicals into 4 groups based on the combination of their effects on state 3 and state 4 respiration. According to this classification, chlordane was also included in the electron transport inhibitor group.

73

In the ascorbate -t- TMPD system, state 3 respiration was not affected even by high concentrations of trans-chlordane. This difference is probably due to the sitespecificity of chlordane, the sensitivity of electron transport to chlordane between cytochrome c and oxygen being less than that of the other two sites. The effect of chlordane compounds on the respiratory chain was similar to that of Zn2+ [19]. From the effect on mitochondrial K+ compartmentation, these chlordane-related compounds alter the permeability of the mitochondrial membrane, resulting in deterioration of mitochondrial oxidative phosphorylation. The effect of heptachlorepoxide, one metabolite of chlordane, was less than that of heptachlor on mitochondrial oxidative phosphorylation. Oxychlordane is a main metabolite of chlordane; however, it was not used in this experiment because it was difficult to obtain. It appears important to determine the effect of oxychlordane on mitochondrial respiration. Our present data may be of help in elucidating the toxicity and mechanism of chlordane. REFERENCES I Environment in Office of Health Studies (1983) Chemicals in the Environment. Environmental Health Department, Environment Agency (Eds.), Report Series No. 9, Tokyo, pp. 9-71. 2 NRC Canada (1974) Chlordane. Its Effects on Canadian Ecosystems and Its Chemistry. National Research Council, NRCC No. 14094, Ottawa, pp. 189. 3 US EPA (1976) Pesticidal Aspects of Chlordane and Heptachlor in Relation to Man and the Environment - A Further Review. 197221975, EPA/540, 4-76-005. US Environmental Protection Agency, Washington, DC, 3%258-339. 4 IPCS (1984) Chlordane. International Programme on Chemical Safety, Environmental Health Criteria 34, WHO, Geneva, pp. 82. 5 Noguchi, N. (1985) Epidemiological studies of chlordane for termite prevention. I. Chlordane residues of houses treated for termites and results of blood tests of pest control operators. Okayama Igakkai Zasshi 97, 315-326 (in Japanese). 6 Wariishi, M., Suzuki, Y. and Nishiyama, K. (1986) Chlordane residues in normal human blood. Bull. Environ. Contam. Toxicol. 36,635643. 7 Miyazaki, T., Akiyama, K., Kaneko, S., Horn, S. and Yamagishi, T. (1980) Chlordane residues in human milk. Bull. Environ. Contam. Toxicol. 25, 518-523. 8 Tojo, Y., Wariishi, M., Suzuki, Y. and Nishiyama, K. (1986) Quantitation of chlordane residues in mothers’ milk. Arch. Environ. Contam. Toxicol. 15, 3277332. 9 Noguchi, N. (1985) Epidemiological studies of chlordane for termite prevention. II. Metabolism of chlordane in exposed rats. Okayama Igakkai Zasshi 97,327-339 (in Japanese). 10 Boyd, E.M. and Taylor, F.I. (1969) The acute oral toxicology of chlordane in albino rats fed for 28 days from weaning on a protein deficient diet. Ind. Med. 38,434-441. 11 National Cancer Institute (1977) Bioassay of Chlordane for Possible Carcinogenicity. Technical Report Series, No. 8, pp. 777808. 12 Epstein, S.S. (1976) Carcinogenicity of heptachlor and chlordane. Sci. Total. Environ. 6, 103-154. 13 Hogeboom, G.H., Schneider, W.C. and Pallade, G.C. (1948) Cytochemical studies of mammalian tissues. I. Isolation of intact mit~hond~a from rat liver; some biochemical properties of mitochondria and submicroscopic particulate material. J. Biol. Chem. 172,619-635.

14 Utsumi,

K. (1963) Relation

mulation

between mitochondria

of P32 in mitochondrial

15 Gornall,

A.G., Bardawill,

reaction.

Pi fraction.

swelling induced

Acta Med. Okayama

by inorganic

phosphate

and accu-

17,258-271.

C.J. and David, M.M. (1949) Determination

of serum proteins

by the biuret

J. Biol. Chem. 177, 751-766.

16 Hagihara,

B. (1961) Technique

chim. Biophys. 17 Snedecor,

for the application

of polarography

to mitochondrial

respiration.

Bio-

Acta 46, 134142.

G.W. and Cochran,

W.G. (1980) Statistical

Methods,

7th edn. The Iowa State University

Press, Ames, IA, pp. 232-237. 18 Ogata,

M., Mori, T., Izushi,

of potentially Med. NMR 19 Miyaji,

toxic chemicals

F., Etoh,

K., Sakai,

R., Meguro,

T. and Inoue,

based on their effects on mitochondrial

B. (1983) Classification

respiration.

Phys. Chem. Phys.

15229-232.

Y. (1987) Effects of copper,

phosphorylation,

ATPase

and electron

Zasshi 99, 8633879 (in Japanese).

cadmium, transport

zinc and two mixtures of isolated

of these metals

rat liver mitochondria.

on oxidative

Okayama

Igakkai