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Amplification of cci4 toxicity by chlordecone: Destruction of rat hepatic microsomal cytochrome p‐450 subpopulation a

Shibani Chaudhury & Harihara M. Mehendale

b

a

Department of Pharmacology and Toxicology , University of Mississippi Medical Center , Jackson, Mississippi b

Department of Pharmacology and Toxicology , University of Mississippi Medical Center , Jackson, MS, 39216–4505 Published online: 20 Oct 2009.

To cite this article: Shibani Chaudhury & Harihara M. Mehendale (1991) Amplification of cci4 toxicity by chlordecone: Destruction of rat hepatic microsomal cytochrome p‐450 subpopulation, Journal of Toxicology and Environmental Health: Current Issues, 32:3, 277-294, DOI: 10.1080/15287399109531482 To link to this article: http://dx.doi.org/10.1080/15287399109531482

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AMPLIFICATION OF CCI4 TOXICITY BY CHLORDECONE: DESTRUCTION OF RAT HEPATIC MICROSOMAL CYTOCHROME P-450 SUBPOPULATION Shibani Chaudhury, Harihara M. Mehendale

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Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi

Previous work has established marked amplification of CCI4 hepatotoxicity by prior exposure to chlordecone (CD). Since CCI4 is toxic by virtue of its bioactivation by the hepatomicrosomal cytochrome P-450 (cyt P-450) system, which is in turn destroyed, our first interest was to determine if cyt P-450 isozymes were selectively destroyed in this interaction. CoCI2 also decreased hepatic P-450 contents, so our other interest was to observe whether CoCI2 selectively decreased or spared CCI4 metabolizing P-450 enzymes. Solubilized hepatic microsomes from variously treated rats were used. The treatment protocol was dietary CD (10 ppm, for 15 d), and CCI4 (100 µl/kg, ip). The treatments were CD alone, CCI4 alone, CD + CCI4 and with or without CoCI2 (60 mglkg/d, sc for 2 d) treatment on d 13 and 14 of the dietary protocol. The control group received normal diet and corn oil vehicle. The key mixed-function oxidase (MFO) parameters measured were microsomal protein, cyt P-450 content, and aminopyrine demethylase (APD). Decrease of P-450 levels ranged from 2.2-fold (CD + CCI4 to 1.3-fold (CD + CoCI2). APD activity decreased by 48 and 26.6% in CD + CCI4 and CD + CoCI2 treatments, respectively. Using an anion-exchange high-performance liquid chromatography (HPLC) column, solubilized microsomal hemoproteins were resolved into five peaks. The P-450 content associated with each peak was determined. In CD rats there was slight increase in peak heights, whereas peak heights in CCI4 and control treatments were similar. CoCI2 decreased all peaks, the decrease of peak I being maximal. In CD + CCI4 treatment, absence of peaks II and III was noted. Microsomal proteins stained for heme showed decreased staining intensity of hemoprotein bands, particularly band 4 (MW 52,000), which was absent in CD + CCI4 interaction. These findings suggest that (1) CoCI2 does not selectively decrease or spare any P-450 isozymes and (2) CD + CCI4 interaction does destroy specific P-450 isozymes.

Preliminary findings of these studies were presented at the Society of Toxicology meetings, Atlanta, Georgia, The Toxicologist 9:191, 1989. The study was supported in part by the Harry G. Armstrong Aerospace Medical Research Laboratory as part of the U.S. Air Force Installation, restoration program funded under the defense environmental restoration account and EPA award CR 814053. The research described in this article has been reviewed by the Health Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the agency nor does mention of trade names of commercial products constitute endorsement or recommendation for use. The authors acknowledge S. Makena and D. Welch for their technical assistance. Requests for reprints should be sent to Dr. Harihara M. Mehendale, Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, MS 39216-4505. 277 Journal of Toxicology and Environmental Health, 32:277-294, 1991 Copyright © 1991 by Hemisphere Publishing Corporation

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INTRODUCTION A decrease in hepatic microsomal cytochrome (cyt) P-450 is observed as one of the early responses in rats treated with CCI4 (Smuckler et al., 1967; Castro et al., 1968). Cobalt is known to inhibit heme biosynthesis in mammalian liver and spleen, thereby altering the biosynthesis of hemoproteins in these and other organs (Woods and Carver, 1977). Cobalt administration to rats results in a profound decrease in microsomal concentration of heme (Bell et al., 1988) and hemoproteins, specifically cyt P450 in the liver. Bell et al. (1988) employed CoCI2 in vivo to decrease the hepatic microsomal cyt P-450 content by 60-65%. More recent studies indicate that metabolism of ™CCI4 in rats pretreated with CoCI2 is not altered significantly (Young and Mehendale, 1989), suggesting that even significantly decreased cyt P-450 content does not influence CCI4 metabolism. Since the effect is mediated through the hindered heme synthesis, one may anticipate that the resultant decrease in cyt P-450 would not be selective against any particular isozyme. The above interpretations are consistent with this presumption. Chlordecone (Kepone; CD) is a ketonic organochlorine pesticide that greatly potentiates the hepatotoxicity and lethality of CCI4 at individually nontoxic or only marginally toxic doses (Curtis and Mehendale, 1979; Curtis et al., 1979; Klingensmith and Mehendale, 1982; Agarwal and Mehendale, 1983). Since CD is an inducer of the hepatic microsomal mixedfunction oxygenase system (Fabacher and Hodgson, 1976; Mehendale et al., 1977; Kaminsky et al., 1978), it is possible that the enhancement of CCI4 toxicity by CD may be related to enhanced bioactivation of CCI4 (Curtis et al., 1979). Previous studies have established a decrease in cyt P450 in the CD + CCI4 interaction (Klingensmith and Mehendale, 1983) and also in the CD + CoCI2 combination (Bell et al., 1988). It has been shown that microsomal cyt P-450 represents several unique hemeproteins with similar reduced CO absorption spectra (Lu and Levin, 1974; Guengerich, 1977). Cytochrome P-450 consists of a family of closely related isozymes (Lu and Levin, 1974; Guengerich, 1977) that catalyze the oxidative metabolism of a wide variety of endogenous and exogenous substrates. These isozymes are regulated not only by hormonal and dietary factors (Levin et al., 1975; Wascman et al., 1985), but also by exposure to xenobiotics that can induce or suppress specific cyt P-450 isozymes. The pioneering studies by Kotake and Funae (1980) and the more recent reports (Bansal et al., 1985; Bornheim et al., 1985; Holm and Kupfer, 1985; Iverson and Franklin, 1985; Bornheim and Correia, 1986) have demonstrated that ion-exchange HPLC may be employed not only for separation and purification of multiple cyt P-450 isozymes, but also for screening the inductive or suppressive effects of xenobiotics on particular isozyme species. The present study was undertaken to determine whether (1) CoCI2

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selectively decreases or spares CCI4 metabolizing enzymes; (2) specific forms of cyt P-450 were destroyed or spared in the CD + CCI4 interaction; and (3) the pretreatment with CoCI2 alters the effect of CD + CCI4 treatments. The anion-exchange HPLC of solubilized microsomes according to the method originally developed by Kotake and Funae (1980) was employed for this investigation. MATERIALS AND METHODS

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Animals

Male Sprague-Dawley rats (176-225 g) obtained from Charles River Breeding Laboratories (Wilmington, Mass.) were housed under 12-h photoperiod in our central animal facilities (room temperature 21 ± 1°C; relative humidity, 50-80%). Groups of four to six rats were used for various treatments. Animal Treatment

Table 1 presents the eight treatment groups employed in this study. Control or experimental diets were provided ad libitum for 15 d. The experimental diet consisted of a powdered rat chow (Purina Chow Co.; St. Louis, Mo.) containing 10 ppm chlordecone (CD; Chem Service, West Chester, Pa.) prepared as previously described (Klingensmith and Mehendale, 1982). The control diet consisted of powdered standard Purina rat chow prepared similarly without the addition of chlordecone. On d 13 and 14 of the dietary protocol, CoCI2 (60 mg/kg/d) was injected subcutaneously to groups 3, 4, and 7, 8 (Table 1). On d 15, CCI4 (100 jtl/kg, in corn oil) was injected intraperitoneally to groups 2 and 5. On d 15, CCI4 was administered to groups 4 and 8, which received CoCI2 for 2 successive days; group 1 received corn oil alone. Preparation of Microsomes

The animals were killed on d 16 (24 h after CCI4 or corn oil injection) under light diethyl ether anesthesia and the livers were dissected out, rinsed in ice-cold saline, and homogenized with 3 volumes of 1.15% KG using a Potter-Elvehjem glass homogenizer. Microsomes were prepared according to the method of Funae et al. (1986). Microsomes suspended at 20 mg protein/ml were solubilized with 10% Emulgen 911 (Kao Chemicals, Tokyo, Japan; final concentration 1%) with magnetic stirring for 30 min. Anion-Exchange HPLC of Solubilized Microsomes

High-pressure liquid chromatography of the solubilized microsomes was done according to the method of Iversen and Franklin (1985). Protein

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to 05 O

TABLE 1. Experimental Protocol Used in This Study Control diet for 15 d

10 ppm Chlordecone diet for 15 d

Groups

1

2

3

4

5

6

7

8

On d 13 On d 14 On d 15

Saline Saline Corn oil

Saline Saline CCI4

CoCI2 CoCI2 Corn oil

CoCI2 CoCI2 CCI4

Saline Saline Corn oil

Saline Saline CCI 4

CoCI2 CoCI2 Corn oil

CoCI2 CoCI2 CCI4

Note. For groups 3, 4, 7, and 8, CoCI2 (60 mg/kg) was injected subcutaneously in 0.9% NaCI on d 13 and 14 of the dietary protocol, while the CCI4 (100 /il/kg, single ip dose) treatment was as described under Methods.

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separation was done using a stainless-steel column (250 x 4.6 mm ID), equipped with a guard column (30 x 4.6 mm ID) and packed with Synchropak AX-300 (Anspec, Warrenville, III.). The eluate was monitored for absorbance at 417 nm with an LDC Spectro Monitor III and collected in 1-min aliquots for subsequent analysis. For ease of cyt P-450 quantitation, the aliquots were pooled into 5 fractions, allocation being made from injection points in the continuous 417-nm tracing. In doing this there might be some overlapping of the P-450 subpopulations in each fraction. The pooled fractions were concentrated in a Speed Vac Concentrator (Savant) for quantitation of P-450.

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Assays The cyt P-450 was quantitated by the method of Omura and Sato (1964) from the CO reduced difference spectra, using an extinction coefficient of 91 mJV)"1 cm" 1 . NADPH cyt c reductase activity was measured as described by Mazel (1971). Aminopyrine demethylase (APD) activity was measured as described previously (Klingensmith and Mehendale, 1982). The reaction was assayed by determining formaldehyde, a product of aminopyrine demethylation, by the method of Nash (1953). Protein concentrations were determined by the method of Lowry et al. (1951), with bovine serum albumin as the standard. Sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (PAGE) was done by the method of Laemmli (1970), and the gels were stained for hemoproteins with Coomassie brilliant blue. Statistics Student's Mest was used to compare the controls with the various treatments. In all cases, differences between mean values of various groups were judged to be significant when p < .01.

RESULTS Liver Weight There was no significant change in liver weight of the rats by the 15-d dietary exposure to CD (Fig. 1). This dietary protocol is known not to alter liver or body weight or show any toxicity (Curtis et al., 1979; Curtis and Mehendale, 1980; Mehendale, 1984). Significant increase in liver weight was observed in the animals maintained on control diet or on CD diet only upon challenge with CCI4 or CoCI2 (Fig. 1). Rats fed normal diet receiving CoCI2 + CCI4 showed significant decrease in liver weight as compared to control. However, the CoCI2 + CCI4 combination in the CDpretreated rats did not alter the liver weight.

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25

**

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10

0

Cont

CD

CD CoClo CD CC1 4 CoCl 2 CD + 2 CC1 4 CoCl 2 CC1 4 CoCl

ecu ecu FIGURE 1. Male Sprague-Dawley rats were maintained on either dietary chlordecone (10 ppm) or normal diet for 15 d. On d 13 and 14 some groups were challenged with CoCI2 (60 mg/kg/d, sc). CCI4 (100 /il/kg, ip in corn oil) was administered on d 15 to some groups. Controls received the corn oil vehicle only. On d 16 (24 h after CCI4 or corn oil injection) the livers were removed under diethyl ether anesthesia. Details of the treatments are described in the text. Changes in the liver weights of the rats under these treatments were recorded. Results are mean ± SD for three rats. Asterisks represent statistical difference from control at *p < .01 and **p < .001, respectively.

Hepatic Mixed Function Oxidase Parameters

In contrast to the liver weight data, the effects of CD pretreatment and CCI4 or CoCI2-induced decrease/increase of mixed-function oxidase (MFO) were variable and equivocal. The most effective treatment in terms of decreasing hepatic microsomal protein was CD followed by CoCI2 + CCI4 injections (Fig. 2). Microsomal protein contents were similar in control and CD groups. However, decreases in microsomal protein were observed in the CD + CCL, and CD + CoCI2 treatments, by 33 and 23.5%, respectively. Thus CD + CCL,-induced decrease in microsomal protein was greater than observed with the CD + CoCI2 combination. The microsomal cyt P-450 content in the livers of the rats, receiving the various treatments, is depicted in Figure 3. Hepatic cyt P-450 in the CD-treated rats increased by 83% as compared to the control rats, as anticipated (Curtis et al., 1979). The decrease in cyt P-450 contents caused by CoCI2 was similar in the rats maintained on normal diet (Fig. 3, control vs. CoCI2, 64%) and in the rats maintained on the 10 ppm CD diet (Fig. 3,

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CD vs. CD + CoCI2, 58%). As has been reported (Agarwal and Mehendale, 1983,1984), CCI4 administration to rats receiving normal diet did not result in any significant change in P-450 level (Fig. 3). There was a 76% decrease in the cyt P-450 content in the CD + CCI4 interaction as compared to CD treatment alone. NADPH cyt c reductase activity did not alter significantly among various treatments (Fig. 4). Although some increase and decrease in activity of this enzyme was noted in the CD + CoCI2 and CoCI2 + CCI4 treatments, respectively, these changes were statistically insignificant. Significant differences in APD activity were noted among the various treatments alone or in combination (Fig. 5) with CoCI2 or CCI4. APD activity was significantly decreased in all treatments except in the rats receiving CD pretreatment alone. Administration of 100 n\ CCI4/kg to rats maintained on the normal or the CD diet caused a decrease in the enzyme activity by 35 and 48%, respectively. However, the APD activity decreased by 44% in normal rats challenged with CoCI2, which was reduced by only 26.6% in the CD + CoCI2 interaction. Thus, CCI4 administration was more effective in the destruction of this cyt P-450-dependent reaction than CoCI2, in the CD-pretreated animals.

25 >

20 15

J_

* *

T

T

10

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CD

CCU CoCl? CD CD CoCU CD CC1 4 CoCl 2 CC1 4 CoCl 2

cci FIGURE 2. Rats were treated as in Figure 1. Hepatic microsomes were prepared from the rats after various treatments. Microsomes were solubilized and the protein concentration of the solubilized microsomes was determined. Results are mean ± SD for three rats. Asterisks represent statistical difference from control at *p < .01 and **p < .001, respectively.

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-450 (

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1.4

* * • *

0.4

T

T

_L

0.2 0.0

Cont

CD

CC1 4 CoCl 2 CD

CD

CoCl 2 CD

CC1 4 CoCl 2 CC1 4 CoCl 2 CC1 4 FIGURE 3. Rats were treated as in Figure 1. The cyt P-450 content of the solubilized, hepatic microsomes was determined as described in the text. Results are mean ± SD for three rats. Asterisks represent statistical difference from control at *p < .01 and **p < .001, respectively.

SDS-PAGE Analysis

Slab gel electrophoresis of the hepatic solubilized microsomes from the rats exposed to various treatments is shown in Figure 6. When equal amounts (8 /ig) of protein from the solubilized microsomes of each treatment group were separated by SDS-PAGE, we were able to define eight discrete bands in the molecular weight range of 66,000 to 45,000. From the migration distance of the standards, the apparent approximate molecular weights of these eight bands were 60,500, 56,500, 54,300, 52,000, 50,500, 48,500, 46,000, and 44,500, respectively. In the gel from animals pretreated with CD (well 6), band 5 had a higher staining intensity than the other bands. Normal rats treated with CCI4 (well 3) do not show any change in the staining intensity of the bands as compared to control (well 2). However, CD-pretreated rats challenged with CCI4 showed decreased staining intensity for all the bands, and the band migrating with a molecular weight of 52,000 was absent (well 7). CoCI2 treatment decreased the staining intensity of all the bands except band 6 (well 4), whereas CoCI2 challenge to CD pretreated rats resulted in increased staining intensity of all bands (well 8). CD + CoCl2 + CCI4 combination markedly decreased the staining intensity of all bands (well 9), and band

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4 (MW 52,000) was absent in this treatment, as was observed in the CD + CCI4 interaction.

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HPLC Analysis of Solubilized Rat Hepatic Microsomes

The anion-exchange HPLC profiles of hepatic solubilized microsomes from control rats and rats exposed to various treatments are shown in Figure 7. The HPLC profile contained mainly 5 peaks with elution times ranging from 3.2 to 30.9 min. The peaks at 3.20, 18.57, 20.97, 22.63, and 30.90 min were collected. These five fractions are designated by Roman numerals as in Figure 7. The CO reduced difference spectra of these collected peaks were recorded to determine the presence of cyt P-450. Specific P-450 content of these fractions are given in Figure 8 (Table 2). Rats pretreated with CD induced P-450 by 83%; the maximum increase in P-450 content and peak height was observed in fraction IV of the HPLC eluate (Table 2, Figs. 7B and 8). CCI4 administration to normal rats decreased the level of P-450 in fractions I and II, while in CD + CCI4 interactions there was significant decrease in P-450 content in the first four fractions. On the other hand, the HPLC profile of CD + CCI4

S 125

S oo 120

P6 110 105

o

100 o

6

95

Cont

CD

CC1 4 CoCl 2 CD CCL

CD

CoClo CD CCl

FIGURE 4. Rats are treated as in Figure 1. The NADPH cytochrome c reductase activity of the solubilized, hepatic microsomes from the variously treated groups was determined. Results are mean ± SD for three rats. Asterisks represent statistical difference from control at *p < .01 and **p < .001, respectively.

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T

0.4

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CCI4 CoClo CD CD CoCl 2 CD 4 d CC1 + 4 CoCl 2 CC1 4 CoCl 2

cci 4 FIGURE 5. Rats are treated as in Figure 1. Aminopyrine demethylase activity of the solubilized hepatic microsomes from the variously treated rats was determined. Results are mean ± SD of three rats. Asterisks represent significantly different from control and *p < .01 and **p < .001, respectively.

combination (Fig. 7C) showed the total absence of fractions II and III with a significant reduction in peak height of fraction IV. Although CoCI2 alone and CD + CoCI2 combination significantly decreased the levels of cyt P450 in each fraction (Fig. 8, Table 2), the HPLC profile showed the presence of all five peaks with slightly variable decrease in the peak heights. We observed a significant decrease in the peak height of fraction I in the CoCI2 exposure alone (Fig. 7D). Absence of peaks II and III and reduction of peak height of fraction IV was also noted in the hepatic microsomes of CD-pretreated rats challenged with CoCI2 + CCI4 (Fig. 7B), as was seen during CD + CCI4 interaction. Thus, the CD-induced form of cyt P-450 (peak IV) was selectively destroyed by CCI4 along with peaks II and III, while CoCl2 administered to normal or CD-pretreated animals uniformly decreased P-450 subpopulations, with slightly variable uniformity. DISCUSSION Previous studies have demonstrated the propensity of CD to potentiate CCI4 hepatotoxicity (Agarwal and Mehendale, 1983, 1984; Bell et al.,

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1988; Curtis and Mehendale, 1979; Curtis et al., 1979, 1981; Davis and Mehendale, 1980; Klingensmith and Mehendale, 1981, 1982, 1983, 1984; Klingensmith et al., 1983; Mehendale, 1984; Mehendale and Klingensmith, 1988). Acute or subchronic dietary pretreatment of male rats with 10 ppm CD, followed by a single low dose of CCI4 (50-200 /*l/kg), leads to dramatic elevation in serum enzymes, sharply impaired hepatobiliary functions, decreased hepatic mixed-function oxidase activity, and marked histological alteration in the liver morphology (Mazel, 1971; Curtis et al., 1979, 1981; Davis and Mehendale, 1980; Klingensmith and Mehendale, 1981,1982,1983; Klingensmith et al., 1983). The toxicity of CCI4 is dependent upon its bioactivation by the cyt P-450 system to one or more toxic intermediates that are believed to attack membrane lipids giving rise to toxic peroxides. These peroxides initially destroy the microsomal environment and eventually damage more cellular organelles culminating in cell death. Unlike CCI4, chlordecone is not metabolized in the rat. CD is an inducer of hepatic cyt P-450 (Fabacher and Hodgson, 1976; Mehendale et al., 1977; Kaminsky et al., 1978), and it was suggested ear-

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FIGURE 6. The effect of various treatments on rat hepatic microsomal protein as separated by SDS-PACE. Treatments as described in Table 1 (Methods). Equal amounts (8 ng) of microsomal proteins separated SDS-PAGE with 8 discrete bands in the molecular weight range of 45,000-66,000 were noted. Well 1: Protein standard with molecular weights 66,000, 45,000, 36,000, 29,000, and 24,000 from top to bottom. Wells 2-9 are microsomes from control, CCI4, CoCI2, CoCI2 + CCI4, CD, CD + CCI4, CD + CoCI2, and CD + CoCI2 + CCI4 treated rats, respectively.

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Amplification of CCl4 toxicity by chlordecone: destruction of rat hepatic microsomal cytochrome P-450 subpopulation.

Previous work has established marked amplification of CCl4 hepatotoxicity by prior exposure to chlordecone (CD). Since CCl4 is toxic by virtue of its ...
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