Functional and biochemical correlates of chlordecone exposure and its enhancement of CCl4 hepatotoxicity

Toxicology, 15 (1980) 91--103 © Elsevier/North-Holland Scientific Publishers Ltd.

FUNCTIONAL AND BIOCHEMICAL CORRELATES OF CHLORDECONE EXPOSURE AND ITS ENHANCEMENT OF CC14 HEPATOTOXICITY*

M.E. D A V I S and H.M. M E H E N D A L E

University of Mississippi Medical Center, Department of Pharmacology and Toxicology, 2500 North State Street, Jackson, MS 39216 (U.S.A.)

(Received August 20th, 1979) (Revision received December 14th, 1979} (Accepted February 10th, 1980)

SUMMARY

Animals pretreated with chlordecone exhibit a greatly increased hepatotoxic response to CC14 challenge. Possible mechanisms underlying this interaction were examined. A single p.o. administration of chlordecone (5 mg/kg) was followed by CC14 (200 ul/kg) administered i.p. 48 h later. Twenty-four hours later, animals treated with chlordecone + CC14 had decreased hepatic excretory function {20% of controls) and elevated plasma transaminase activities and bflirubin. Hepatic mixed function oxidase activity was assessed as pentobarbital sleeping time and was n o t affected by chlordecone pretreatment. Irreversible binding of label from 14CC14 to hepatic protein or lipid was n o t different in the chlordecone group compared to vehicle controls. Hepatic and renal glutathione concentrations were n o t affected by chlordecone alone (at 6 h, 1, 2, 3, 5 and 7 days) or by a combination of chlordecone (48 h) and CC14 (24 h). CC14-induced lipid peroxidation of liver tissue, measured in vitro or in vivo, was not increased by chlordecone treatment. Thus, while the mechanism for the enhanced toxicity remains to be elucidated, these results suggest that the interaction between chlordecone and CC14 is a subtle one, n o t causally involving increased covalent binding of the toxin, increased susceptibility of tissue lipids to peroxidative damage or decreased hepatic GSH.

*Preliminary results of these studies were reported at the 63rd Annual Meeting of the Federation of American Societies for Experimental Biology, at Dallas, Texas (Fed. Proc., 38 (1979) 683). These investigationswere supported by a P H S Grant ES-01369. M.E,D. is postdoctoral fellow supported by N I E H S Toxicology Training Grant, ES-

07045. Abbreviations: BUN, blood urea nitrogen; GSH, glutathione; HMFO, hepatic mixed function oxidase; MDA, n~londialdehyde; PMIMP, polar metabolites of imipramine; TCA, trichloracetic acid.

91

INTRODUCTION

Chlordecone (decachlorooctahydro-l,3,4-metheno-2H-cyclobuta(c,d)pentalen-2-one, Kepone®) is present in the environment as a result of direct contamination in the Hopewell, VA area [1] and from photolytic degradation of the pesticide mirex, used in the Southeastern U.S.A. in attempts to control fire ants [2]. Previous work has shown that exposure to chlordecone results in greater hepatotoxicity upon subsequent exposure to carbon tetrachloride or chloroform [3,4]. CC14 is an intrinsic hepatotoxin which rapidly produces steatosis and necrosis. While the mechanism underlying fat accumulation in liver is widely accepted to result from decreased synthesis of and coupling to the transport apolipoprotein, there is still considerable debate as to the mechanism(s) of cell death (see Zimmerman [5]). CCI4 is activated by the hepatic mixed function oxidase system (HMFO) to a toxic free radical species, either CC13 or CC130~ (see review by Slater [6] and [7,8]). The free radicals initiate lipid peroxidation or bind to cellular constituents; both of these events are considered to be responsible for cell death (see reviews by Gillette [9] and Recknagel and Glende [10]). Increased toxicity of halogenated hydrocarbon solvents, including CCI4, has been found after pretreatment with a variety of other chemicals, including ethanol [11--13], isopropanol [11,12], phenobarbital [14,15], polybrominated biphenyls [16], diethyl maleate [17] and 1,3-butanediol [18]. The enhancement of CCI4 hepatotoxicity has been attributed most often to increased activation of the solvent to the toxic free radical(s) [6,9,16], although depletion of hepatic glutathione (GSH) [17] and less well defined interactions involving ketone moieties [ 18] have also been proposed as underlying mechanisms. In the present report we have further characterized the interaction between chlordecone and CC14 and examined several possible mechanisms for this interaction. For this purpose, a low dose of chlordecone and short duration were used. While marked enhancement of CC14 hepatotoxicity was observed, this increased toxicity was not associated with increased irreversible binding of label from 14CC14 increased lipid peroxidation or depletion of hepatic glutathione. METHODS

Animals and treatments

Male, Sprague--Dawley derived rats (250--450 g) were purchased from Charles River Breeding Laboratories and maintained in central animal facilities away from any known inducers. Chlordecone (Allied Chemical Co., Morristown, NJ, 99% purity) was administered in vegetable or corn oil by garage, 1 ml/kg. CC14 was administered in vegetable or corn off solution by i.p. injection (1 ml/kg). Sodium phenobarbital was administered in saline ( 50 mg/kg, kp.) once daffy for 5 days.

92

Excretory function Rats were pretreated with chlordecone (5 mg/kg) or oil vehicle and 48 h later challenged with vehicle or CC14 (50--200 ul/kg for chlordecone pretreated and 200 ~l/kg for controls). Excretory function was determined 24 h after CC14 or vehicle challenge. Rats were anesthetized with sodium pentobarbital (50 mg/kg as Nembutal@). The bile duct and both ureters were each cannulated with PE-10 tubing. Saline was infused (0.5 ml/min) and 14C-labeled polar metabolites of imipramine ([14C]PMIMP, prepared as previously described [19]) were administered as a bolus through a cannula (PE-50 tubing) placed in a femoral vein. Blood samples were taken through a cannula in a femoral artery at the midpoint of each collection period. Bile and urine samples were collected, in calibrated tubes, for 4 15-min periods, after administration of [14C]PMIMP. Radioactivity in aliquots of bile, urine and plasma was determined by liquid scintillation spectrometry. Results were corrected for quench by external standard-sample channels ratio after calibration for various sample types.

Pentobarbital sleeping time The rats were pretreated with chlordecone (0, 7.6, 11.4 and 15.2 mg/kg) and 48 h later challenged with CC14 (200 ~l/kg) or vehicle. Twenty-four hours later sodium pentobarbital was administered in saline (40 mg/kg, i.p.) and the time required to regain fighting reflex was noted. The experiment was terminated at 420 min. At the time of waking or at termination of the experiment the rats were decapitated and blood collected in heparinized tubes for plasma transaminase determinations.

Lipid peroxidation For in vitro lipid peroxidation studies rats were pretreated with chlordecone (5 mg/kg) or vehicle 48 h prior to sacrifice. Liver tissue was homogenized in 0.154 M KC1 in 0.1 M phosphate buffer (pH 7.4) (20% w/v, using a Potter-Elvehjem type homogenizer) and centrifuged at 9000 g for 30 min. The supernatant was incubated with additional buffer and an NADPH generating system (2 ~mol NADP, 6 pmol glucose-6-phosphate, both from Sigma Chemical Co.); the total volume was 4 ml. The flasks were incubated at 37°C and the reaction begun by adding various volumes of CC14 to the sidearm of the flask [20]. After 15 min the reaction was stopped by adding 2 ml 10% trichloroacetic acid (TCA). Malondialdehyde (MDA) formation was determined by reaction with thiobarbituric acid and the chromogen extracted with n-butanol [6]. Malondialdehyde bis-(dimethyl acetal) (Aldrich Chemical Co.) was used as a standard. Vmax and Km were estimated by double reciprocal analysis, after subtraction of background (no CC14) MDA formation. Lipid peroxidation in vivo was measured as diene conjugation of tissue lipids. Rats were pretreated with chlordecone (5 mg/kg) 48 h prior and with sodium phenobarbital (50 mg/kg) for 5 days prior to challenge with

93

either a low dose (42.6 nl/kg) or hepatotoxic dose (200.4 pl/kg) of CC14. They were sacrificed 1 h after CC14; liver tissue was homogenized in 2 vols. of 0.32 M sucrose3 mM EDTA. Lipid was extracted from an aliquot (0.5~ml) of the homogenate into 9.5 ml of chloroform/methanol (2 : 1). The tissue precipitate was removed by filtration, the extract brought to 10 ml with chloroform/methanol and washed (to remove non-lipid material) with 2 ml of saline, followed by 2 washes, each 0.5 ml, with upper phase solvent (upper phase of mixture of 25 ml water and 95 ml chloroform] methanol). Methanol was added to clear the samples and they were scanned from 230 nm to 280 nm using a Cary 219 spectrophotometer. The lower phase solvent was used as the reference blank.

Covalent binding studies After pretreatment with chlordecone or phenobarbital as above, the rats were injected with either a tracer (42.6 nl/kg) or hepatotoxic tracer plus 200 pl/kg CC14; total dose 200.4 gl/kg 14CC14 (New England Nuclear Corp.). Sixty minutes later each animal was sacrificed by decapitation and blood collected in heparinized tubes. The liver was removed and rapidly weighed, homogenized (by use of a Polytron homogenizer) and aliquots added to scintillation fluid. Additional aliquots were assayed for binding to protein and lipid. Covalent binding to protein was taken as the label remaining associated with the tissue pellet after 3 TCA and 7 hot methanol/ ether (3 : 1, v/v) washes; the results were expressed as dpm/mg protein in the solubilized pellet divided by dpm/mg protein in the homogenate. Lipids were extracted, from 0.5 ml of the homogenate, as for the diene conjugation assay and covalently bound label was taken as the label remaining after the chloroform/methanol extract had been heated in an 80°C heating block for 3 h [21]. The results were expressed as dpm bound/total chloroform/methanol extract divided by dpm/0.5 ml homogenate. Miscellaneous biochemical parameters Tissue glutathione was estimated as non-protein sulfhydryl using bi~ (3-carboxy-4-nitrophenyl) disulfide as described by Mitchell et al. [22]. Lipid was measured by reduction of potassium dichromate [23] and protein by the biuret [24], Lowry [25] or Bradford [26] methods. Transaminase activity in plasma was assayed by the method of Reitman and Frankel [27] and bilirubin by the method of Jendmssik and Grof [28], using Sigma reagents. Blood urea nitrogen (BUN) was determined by the method of Natelson [ 29] using Pierce reagents. Statistics Data were analyzed by factorial analysis of variance appropriate for the experimental design (1 or 2 way or 2 way factorial with repeated measures on 1 factor) and the significance of differences between means determined using the Student-Newman-Keuls procedure [30]. The level of significance chosen was P < 0.05. 94

RESULTS

Excretion studies

Hepatic excretory function was measured to confirm the hepatotoxicity of the chlordecone-CCl4 treatment schedule used in the present study. Biliary excretion of [14C]PMIMP was not different in rats treated with either chlordecone or CC14 (Fig. 1) compared to untreated controls (51.4 + 5.7% of the dose administered excreted in 60 rain [31]). Chlordecone treated animals challenged with CC14 showed a marked decrease of biliary excretion. The effect was maximal at 50 pl/kg of CC14 and the results for 50, 100 and 200 pl/kg doses did not differ. Therefore, all the data were pooled (Fig. 1) and the average of these was decreased, to 1 2 % of the chlordecone control, confirming the hepatotoxic interaction. Bile flow rate in the chlordecone + CCI4 group was half that of the chlordecone group. The urinary excretion was increased, but this increase was inadequate to compensate for impaired biliary excretion. The plasma P M I M P concentration in the chlordecone + CC14 group was approximately twice that of the chlordecone or CCI4 controls. In contrast, 24 h after 1 rnl CCl4/kg biliary excretion of P M I M P was 75% of control [31]. Thus, while the chlordecone treatment alone had no effects on hepatic excretory function, the hepatotoxic response to CC14 was dramatically increased. The plasma and urine of the chlordecone + CC14 group was icteric and the bile pale, suggesting impaired biliary excretion of bilirubin. [] BILE [ ] URINE

6O

_L*

w 0

40

u,. 0 I--z w (o I.LI n

CD (7)

CCt4 (4)

CD+CCI 4

(6)

Fig. 1. Effect of chlordecone and CCI, on excretory function. Chlordecone (CD; 5 mg/kg) was administered 48 h prior to challenge with either CCI~ or oil vehicle. The C D group received vehicle challenge, the CCI 4 group received 200 ~I/kg and C D + CCI 4 received 50--200 ~I CCl,/kg 48 h after C D (since no differences were seen between 50 and 200 izl/kg doses the results were pooled). Excretory function was measured 24 h after CCI, or vehicle challenge. T h e results are expressed as percent of the dose of polar metabolites of imipramine excreted in 60 rain, by each route, and are shown as m e a n -+ S.E. of the number of animals indicated in parentheses. *Different from both C D and CCI 4, P < 0.05.

95

Chlordecone time course T h e e f f e c t s o f c h l o r d e c o n e t r e a t m e n t o n a n u m b e r o f liver a n d k i d n e y p a r a m e t e r s were d e t e r m i n e d 6 h, 1, 2, 5 a n d 7 d a y s a f t e r a single oral dose o f c h l o r d e c o n e (5 m g / k g ) . T h e f o l l o w i n g p a r a m e t e r s were m e a s u r e d : b o d y weight, liver o r k i d n e y w e i g h t t o b o d y w e i g h t ratios, p l a s m a t r a n s a m i n a s e activities ( G O T a n d G P T ) , p l a s m a b'dirubin c o n c e n t r a t i o n , B U N , h e p a t i c a n d renal g l u t a t h i o n e a n d p r o t e i n c o n t e n t , lipid c o n t e n t in h e p a t i c m i c r o s o m e s a n d d i e n e c o n j u g a t i o n o f lipids e x t r a c t e d f r o m h e p a t i c m i c r o s o m e s . N o d i f f e r e n c e s f r o m c o n t r o l were f o u n d in a n y o f t h e s e p a r a m e t e r s . C h l o r d e c o n e a n d CCI4 T h e e f f e c t s o f c h l o r d e c o n e , CC14 a n d c h l o r d e c o n e + CC14 o n h e p a t i c a n d renal g l u t a t h i o n e a n d p r o t e i n c o n t e n t s , p l a s m a bilirubin c o n c e n t r a t i o n a n d B U N were d e t e r m i n e d . N o e f f e c t s o n h e p a t i c o r renal G S H were f o u n d (Table I). I n t h e c h l o r d e c o n e + CC14 g r o u p h e p a t o t o x i c i t y was e v i d e n t as d e c r e a s e d p r o t e i n c o n t e n t a n d increased p l a s m a bilirubin c o n c e n t r a t i o n . While d e c r e a s e d renal p r o t e i n c o n t e n t suggests n e p h r o t o x i c i t y , B U N was n o t affected. P e n t o b a r b i t a l sleeping t i m e P e n t o b a r b i t a l sleeping time was d e t e r m i n e d as a n i n d e x o f H M F O activity. C h l o r d e c o n e a l o n e h a d n o e f f e c t (Fig. 2). Sleeping time was p r o l o n g e d in t h e animals t r e a t e d with C C L a l o n e a n d this e f f e c t was e n h a n c e d b y TABLE I EFFECTS OF CHLORDECONE A N D CCI 4 O N H E P A T I C A N D R E N A L G L U T A T H I O N E AND PROTEIN CONCENTRATION AND PLASMA BILIRUBIN AND BLOOD UREA NITROGEN a

Vehicle --CCI4 + CCI4

Chlordecone -CCI, + CCI4

Glutathione

Protein

Bilirubin

BUN

(~mol/g)

(mglg)

(mg/100 ml)

(mgll00 ml)

0.18 +- 0.05 0.17 -+ 0.06

22.9 -+ 3.1 21.2 -+ 3.6

173

0.22

3

+ 0.12

22.1 -+ 1.9 19.2 -+ 2.9

Hepatic

Renal

Hepatic

7.90 b -+ 0.31 8.79 -+ 0.37

2.67 -+ 0.12 2.45 -+ 0.33

253 +- 12 256 -+ 21

8.04 ± 0.23 7.62 -+ 0.77

2.70 *- 0.09 3.20 ± 0.41

254 -+ 10 200 c +- 3

Renal

177 3 175 -+ 5

+-

+

+-

159 c 5

2.28 c +- 0.33

aRats were treated with chlordecone (5 mg/kg) or vehicle, and CCI, (200 ~llkg) or vehicle administered 48 h later. The animals were sacrificed 24 h after CCI~ challenge. b M e a n -+ S . E . ( N = 7).

CDifferent from all other treatment groups, P <~ 0.05.

96

500 .~_

400

E a)

300

~-

200

_E t-

~

I00

(D

0

7.6

11.4

15.2

Dose of Chlordecone (mg/kg) Fig. 2. E f f e c t s o f c h l o r d e c o n e and CCI 4 o n p e n t o b a r b i t a l sleeping t i m e . M a l e rats w e r e t r e a t e d w i t h a single oral d o s e o f c h l o r d e c o n e i n d i c a t e d a n d 4 8 h l a t e r c h a l l e n g e d w i t h oil vehicle o r CC14 ( 2 0 0 u l / k g , i.p.). T w e n t y - f o u r h o u r s l a t e r s o d i u m p e n t o b a r b i t a l ( 4 0 m g / k g , i.p.) was a d m i n i s t e r e d ; t i m e t o r e c o v e r y o f r i g h t i n g reflex m e a s u r e d . T h e results are s h o w n as m e a n +- S.E. o f t h e n u m b e r o f a n i m a l s i n d i c a t e d in e a c h bar. * D i f f e r e n t f r o m CD + vehicle; * * d i f f e r e n t f r o m t h e vehicle + CCI 4, P < 0.5.

chlordecone pretreatment in a dose-dependent manner. In the 15.2 mg/kg group all the CC14 challenged rats slept for 420 rain, at which time the experiment was terminated. Plasma transaminase activities (Control GOT 91 -+ 6 SF units/ml, GPT 27 + 3) were unaffected b y chlordecone alone (i.e. 15.2 mg/kg; GOT 84 + 7, GPT 28 -+ 1 4 were slightly increased by CCI4 alone (GOT 121 + 11, GPT 48 + 5 ) a n d were greatly elevated, (GOT > 1080, GPT > 625) for all 3 chlordecone + CC14 groups.

Effect of phenobarbital pretreatment on CCl4 toxicity To m o n i t o r the efficacy o f the phenobarbital treatment to enhance CC14 toxicity, plasma bflirubin concentration and transaminase activities were measured 24 h after CC14 challenge (200 ul/kg) in animals pretreated for 5 days with sodium p h e n o b a r b i t a l Plasma bilirubin concentration was increased in the phenobarbital pretreated group (0.80 + 0.25 mg/100 ml} compared to CC14 controls (0.23 -+ 0.25). This increase was not as great as that seen with chlordecone pretreatment (Table I). Increases of plasma GOT activity (925 -+ 155 SF units/ml vs. 137 + 30 for CC14 control) and GPT activity (501 + 123 vs. 70 +- 19) were also found. The above treatm e n t p r o t o c o l was used as positive control for enhanced CC14 toxicity in the following studies dealing with lipid peroxidation and 14C (14CC14) binding to hepatic protein and lipids.

Lipid peroxidation Lipid peroxidation induced by CCI4 in vitro was determined in rats treated 48 h prior to sacrifice with chlordecone (5 mg/kg, p.o.) or oil vehicle. The results are shown {Fig. 3) as total malondialdehyde produced. Preliminary studies showed that w i t h o u t an NADPH generating system, MDA production at any CC14 volume was the same as that p r o d u c e d in the absence 97

of CC14. Neither V m a x n o r Km were significantly affected by chlordecone pretreatment. Lipid peroxidation in vivo was assessed in liver tissue from animals used in the 14CC14-covalent binding experiments. The shape of the UV-spectrophotometric scan was different with the low and high dose CC14 groups. The maximal absorbance for all the groups was at 235 nm, the minimum was 257 nm for the 42.6 nl/kg dose and 266 nm for the 200.4 ~l/kg dose CC14. Therefore the results were calculated as Emax-Emin. With the tracer (42.6 nl/kg) dose of ~4CC14 there was no difference between the control, phenobarbital and chlordecone groups {Fig. 4). The 200.4 pl/kg dose CC14 resulted in a small increase of lipid peroxidation in controls and a parallel increase in the chlordecone group. With the phenobarbital treated group the increase was larger. The amount of lipid extracted from the tissue was not different among any of the groups. ~4CCl4 binding to protein and lipid Binding of ~4CC14 to protein and lipid was determined as an index of activation of CC14 to reactive intermediates. With the tracer dose of 14CC14 binding to lipid was unaffected by any of the pretreatments, and binding

9 E

.08,

.300

06 /I::

tf) o

o, . 2 0 0 Q.

,T

o

E

E

Control

~ .100

Vmax .215 ± . 0 2 0 Km

a

2.7 +- .9

Chlordecone

uJ 0 4 <~

.250 -+.031

.02

6 II

1

0 5

I

,/yY"

1.7 -I- 2

/

I

lO 25 CCl 4 (pl in sidearm)

50

@

I

I

42.6

200.4

nl/kg

.vl/kg

Fig. 3. Effect o f chlordecone pretreatment on CCl~-induced lipid peroxidation in vitro. Male rats were treated with chlordecone (5 mg/kg) or oil vehicle and 48 h later sacrificed and liver tissue prepared for in vitro experiments. Lipid peroxidation was determined as M D A formation. Results are s h o w n as m e a n +- S.E. (N = 4) of total M D A formed; for V m a x and K m estimation the background M D A formation was subtracted. There were no significantdifferences between control ( ) and chlordecone (------) groups. Fig. 4. Effect o f pretreatments on CC14-induced lipid peroxidation in vivo. After pretreatment, rats (3 per group) were injected with the dose of CC14 indicated, sacrificed 60 rain later and diene conjugation o f lipids was measured. F o r control and chlordecone groups the difference between CCI4 doses was not significant. *Different from control 200.4 ~,l/kg and from phenobarbital ÷ 42.6 nl/kg, P < 0.05.

98

T A B L E II B I N D I N G O F 14C F R O M 14CC1, T O L I V E R T I S S U E Total homogenate

Bound

dpm/0.5

Protein

ml

dpm/mg

Lipid

protein

T r a c e r dose (42. 6 n l / k g ) Control Phenobarbital Chlordecone

2527 -+ 6 5 1 c 2636 + 713 3178 -+ 5 6 2

High dose ( 200.4 pl/kg) Control

476; 859 1002

Phenobarbital +

Chlordecone

84 +- 25 85 +- 25 107 +- 23

114

495 -+ 79

dpm/mg protein

%a

dpm/mg lipid

%b

32.8 -+ 10.5 18.3 + 5.8 43.3 -+ 7.7

39.1 +- 2.8 21.1 + 2.7 d 41.2 -+ 1.6

127 45 153 • 57 129 + 25

12 +- 1 16 + 1 13 -+ 2

18.8; 16.0 20.2 +- 1.5 24.7 + 2.8

168; 320 277 +- 3 2 206 -+ 28

78; 70 61 + 4d 79 + 4

13; 28 29 +

2

+

+

14 2

+

2.4; 4.4 5.7 0.5 3.4 0.1

a C a l c u l a t e d as b o u n d d p m / m g p r o t e i n divided b y t o t a l d p m / m g b C a l c u l a t e d as d p m / t o t a l e x t r a c t d i v i d e d b y d p m / 0 . 5 ml, t i m e s 100. CMean *~ S.E. o f 3, e x c e p t 2 values f o r h i g h d o s e c o n t r o l s . dSignificantly different from control or chlordecone.

±

p r o t e i n , t i m e s 100.

to protein was unaffected by chlordecone and decreased by phenobarbital (Table II). The proportion of binding to lipid and protein was affected by the dose of CC14 administered. For controls, the hepatotoxic dose of CC14 yielded more binding of label to lipid and less to protein. This pattern was also observed with the chlordecone group and there were no differences from controls. In the phenobarbital group the same proportion of radioactivity was bound to protein at the low and high CCL doses, and, although more of the label was associated with lipid in the high CC14 dose group, this increase was less than that observed in the control group. In the high dose groups, the absolute amount bound to protein was greater in the phenobarbital group but the amount of 14C in the tissue was also increased. DISCUSSION

The results presented here extend previous work from this laboratory demonstrating that exposure to the pesticide chlordecone results in a dramaticaUy increased hepatotoxic response to CCL. This interaction is believed to be enhanced CC14 hepatotoxicity since the biochemical and histopathological observations are more consistent with CC14 than chlordecone toxicity 99

[3]. It should be noted that in the present studies the dose of chlordecone and duration of exposure were both much less than previously used, yet resulted in greatly increased CC14 hepatotoxicity. Possible mechanisms underlying this interaction were examined. While CC14 itself does not deplete hepatic GSH, depletion of this free radical scavenger by other agents, such as diethyl meleate, can result in increased susceptibility to CC14 induced hepatotoxicity [9].The hypothesis that a chlordecone induced depletion of GSH could account for the increased toxicity observed with CC14 was tested. Treatment with chlordecone had no effect on GSH; neither depletion nor a "rebound" increase, often associated with more subtle alterations of GSH metabolism, was observed. Hence chlordecone enhanced CC14 hepatotoxicity can not be accounted for by alterations in hepatic GSH levels. Carbon tetrachloride-induced hepatic necrosis has been attributed both to peroxidative damage to lipid components of cellular membranes [10] and to alkylation of tissue macromolecules by a CC14 metabolite [9]. In the present studies, the effects of chlordecone treatment on CC14 induced lipid peroxidation and on binding of label from ~4CC14 were determined as possible mechanisms initiating hepatotoxicity, rather than as an index of hepatotoxicity. Therefore, for the in vivo studies, the rats were sacrificed and livers removed 1 h after CC14 administration. Impaired hepatic function (i.e., loss of ribosomes from the rough endoplasmic reticulum, decreased demethylation of antipyrine, decreased protein synthesis and appearance of diene conjugates in lipids) has been observed as early as 1 h following CC14 administration [ 32]. While CCl4-stimulated lipid peroxidation in vitro was not significantly enhanced, the tendency for increased malondialdehyde formation in the chlordecone pretreated animals suggested that chlordecone might enhance the susceptibility of liver cell membranes to peroxidative damage. Therefore, the effect of chlordecone on lipid peroxidation was determined in vivo. While phenobarbital pretreatment resulted in more CC14-induced peroxidation, in agreement with the results of Suarez et al. [33] and Lindstrom and Anders [17], chlordecone pretreatment did not. Since chlordecone pretreatment resulted in increased hepatotoxicity but not in increased lipid peroxidation, the present results do not support the view that lipid peroxidation plays a causal role in chlordecone enhancement of CC14 induced hepatic necrosis. In addition, phenobarbital pretreatment was associated with milder CC14-induced hepatotoxicity than chlordecone pretreatment. Dissociation of lipid peroxidation and hepatotoxicity has been reported based on species differences [34]. Further, while pretreat~ ment with 3-methylcholanthrene results in protection against CC14 toxicity, CC14 induced lipid peroxidation is not decreased [33]. The ~4CC14 binding experiments provided further, albeit indirect, evidence that there is not a greater effect of CC14 on membrane lipids in chlordecone pretreated animals. The greater binding to lipid at the high dose of CC14, at the expense of binding to protein, suggests that membrane lipids were

100

preferentially damaged at the high dose o f CC14. This sensitivity, however, was n o t affected b y chlordecone pretreatment. The increased CC14 hepatotoxicity in phenobarbital treated rats is generally attributed to increased activation of CC14 to the toxic intermediate secondary to induction o f the hepatic mixed function oxidase system [15,16,17,33]. Chlordecone is also a p o t e n t inducer o f this system, if administered in higher doses [35,36]. While the pentobarbital sleepingtime studies suggested that the chlordecone treatment used here did not result in HMFO induction, Kaminsky et al. [36] have shown that the pattern of enzyme activities and c y t o c h r o m e s induced b y chlordecone depends on both dose and duration of treatment. Therefore, the effect of chlordecone treatment on activation of CC14 was studied, using irreversible binding of label from 14CC14 as a measure of activation. These experiments failed to demonstrate an increase o f the relative proportion or absolute a m o u n t of the label bound. Thus, the extent of binding did n o t correlate with hepatotoxicity. Other investigators have also shown a lack of correlation between binding of ~4C from 14CC14 and toxicity [37,38]. Binding o f label from 14CC14 in vitro can occur without concomittant loss of HMFO activity or cytochrome P-450 content [37]. Pretreatment with 3-methylcholanthrene (which protects against CC14 hepatotoxicity) increases, rather than decreases, binding of label from ~4CC14 in vitro [38]. Similarly, Roberts and Jollow [39] have shown that, while 3-hydroxyacetanilide and 4-hydroxyacetanilide (acetaminophen) bind equally well to hepatic protein, only the latter is hepatotoxic. The present studies also suggest that mechanism(s) other than enhanced bioactivation should be considered to explain enhancement of CC14 hepatotoxicity. While phenobarbital treatment increased the amount of ~4C associated with protein, the a m o u n t of label present in the liver was also increased. While increased metabolism of ~4CC14 would cause some increase of the concentration of label, the amount of label in the pellet was insufficient to account for that increase as a result of formation o f reactive metabolites. Thus, the increased CC14-induced hepatotoxicity typically observed after phenobarbital treatment may be due to increased concentration of substrate at the enzyme. Conversely, protection against CC14 hepatotoxicity b y SKF-525A is associated with decreased CC14 in the liver [40,41]. We have examined several mechanisms often associated with CC14 hepatotoxicity, in an a t t e m p t to unravel the underlying causes of chlordecone enhancement of CC14 hepatotoxicity. The results indicated that neither increased lipid peroxidation nor depletion of hepatic GSH can account for increased toxicity. The chlordecone treatment used did not result in induction of the H M F O system. It is possible that chlordecone induces the specific c y t o c h r o m e which activates CC14 and that irreversible binding of label from ~4CC14 is n o t a sensitive index of that activation. It is also possible that chlordecone, by some unkno~m mechanism, renders the hepatic tissue more susceptible to CC14 damage. Although direct evidence is n o t available for such a mechanism, this concept obviates the 101

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