- Email: [email protected]
Effect of preexposure to kepone on the biliary excretion of imipramine and sulfobromophthalein
TOXICOLOGY
AND APPLIEDPHARMACOLOGYqO,247-259(1977)
Effect
of Preexposure of Imipramine
to Kepone on the Biliary and Sulfobromophthalein’
Excretion
H. M. MEHENDALE Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi 39216 Received September l&1976;
accepted December 20,1976
Effect of Preexposure to Kepone on the Biliary Excretion of Imipramine and Sulfobromophthalein. MEHENDALE, H. M. (1977). Toxicol. Appl. Pharmacol. 40, 247-259. The effect of kepone on the biliary excretion of [Wlimipramine (IMP) was studied using isolated perfused rat liver preparations obtained from control and kepone-pretreated animals. Biliary excretion of endogenously formed polar metabolites of IMP was markedly suppressed (70 %) by preexposure to kepone at 200 ppm in daily ration for 8 days. In vitro addition ofkepone at 5 x 10F4M concentration to the perfusate of untreated liver preparations elicited a 20 ‘A decreasein the biliary excretion of endogenously formed metabolites. At concentrations up to 5 x 10e5 M, kepone was without any discernible effect. The marked suppression of biliary elimination of endogenously formed metabolites is unrelated to the rate of metabolism of IMP. Furthermore, the biliary excretion of exogenously provided polar metabolites of IMP was also suppressed (61%) by preexposure to kepone. This suppression of biliary excretion of IMP metabolites was accompanied by an increase in the bile flow suggesting a dichotomy between bile secretion and biliary excretion. In vitro addition of kepone also resulted in a suppresion (48 %) of biliary elimination of exogenously provided metabolites of IMP. In the experiments in which kepone wasadded, bile flow was unaffected. Biliary excretion of sulfobromophthalein (BSP) was unaffected by preexposure to kepone, suggesting that kepone-induced impairment of hepatobiliary function may not be detected by BSP clearance tests. In addition, different mechanisms might be involved in the transport of IMP metabolites and BSP across the biliary canaliculi. From these results, it is suggested that the kepone-induced impairment of hepatobiliary function may be located at the site of transfer of metabolites across the bile canaliculi. Kepone is the trade name for a commercially available pesticide, decachlorooctahydro1,3,4-metheno-2H-cyclobuta (cd) pentalen-Zone (common name, chlordecone). First introduced in 1958, it has been used as an insecticide against leaf-eating insects, ants, and cockroaches and as a larvicide against flies (Martin, 1971). Kepone has caused human ailment after occupational exposure in a manufacturing facility and has been the 1This investigationwas supported by Grant No. GRSG RR05386. A preliminary report was presentedat the Fall Meeting of the American Societyof Pharmacologyand Experimental Therapeutics, New Orleans,Louisiana, 1976 [Pharmacologist Z8,195 (1976)]. Copyright IQ 1977 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
247 ISSN 0041-008X
248
H. M. MEHENDALE
subject of concern as an environmental contaminant in the area (Anonymous, 1976a). Kepone is a carbonyl analog of mirex (Fig. l), an insecticide used for the control of fire ants in the southeastern United States. Both mirex (Innes et al., 1969) and kepone (Anonymous, 1976b) are reported to cause hepatocarcinomas in experimental animals. Mirex causes liver enlargement and proliferation of the endoplasmic reticulum associated with an increase in the mixed function oxidases (Baker et al., 1972; Byard et al., 1975; Gaines and Kimbrough, 1970; Mehendale et al., 1973). In a previous study from this laboratory, it was shown that mirex pretreatment causes an increase in bile flow (Mehendale, 1976a). However, despite the above changes, the biliary excretion of 4chlorobiphenyl (l-CB) was suppressed markedly after preexposure of rats to mirex (Mehendale, 1976a). This mirex-induced impairment of hepatobiliary functions was unrelated to either the bile flow or the slightly decreased rate of metabolism of l-CB. Since the biliary excretion of exogenously provided, readily excretable metabolites of MIREX
_
KEPONE
491 WT. 546 FIG. 1. Structures of mirex and kepone.
MOL.
l-CB was also markedly suppressed after preexposure to mirex, it was suggested that the locus of mirex-induced suppression was at the site of transport of metabolites from the hepatccytes to the bile canaliculi. Preexposure to phenobarbital caused a slight enhancement of the metabolism of [14C]1-CB, the bile flow, and biliary excretion of the metabolites of l-CB, observations consistent with the known effects of phenobarbital (Klaassen, 1970; Levine, 1973). Another example of suppression of biliary excretion comes from the experiments with the hypolipidemic drug nafenopin (Levine, 1974a; Levine and Bognacki, 1976; Uesugi and Levine, 1976). Bile flow was increased after nafenopin in all the above experiments. The present investigations with kepone were conducted in order to determine its effect on hepatobiliary function. It was of interest to investigate whether or not the substitution of two chlorine atoms of mirex with the comparatively polar carbonyl group would change the effect of this compound on bile flow and biliary excretory function. Isolated perfused rat liver preparations obtained from control and treated animals were utilized to investigate the biliary excretion of the tricyclic antidepressant imipramine and the dye sulfobromophthalein. METHODS Animals Male Sprague-Dawley rats (Charles River Breeding Labs, Wilmington, Massachusetts) were maintained in the central animal facilities away from any known inducers. The bedding used for these animals was made from untreated ground corn cobs. Ani-
KEPONE
AND
HEPATOBILIARY
FUNCTION
249
mals were provided with water and a standard diet of Purina rat chow blocks ad Iibitum until used for an experiment as a liver or blood donor. Animals from which the blood was collected weighed 250-350 g, whereas the animals used as liver donors weighed 200-250 g. During all surgical procedures animals were held under ether anesthesia. Drugs and Chemicals
Kepone was a gift of Allied Chemical Corporation (Union Texas Petroleum, Agr. Dept., Baltimore, Maryland). [14C]Imipramine hydrochloride was purchased from Amersham/Searle Corporation (Des Plaines, Illinois). Sulfobromophthalein (BSP) and unlabeled imipramine hydrochloride were obtained from Sigma Chemical Company (St. Louis, Missouri) and Geigy Pharmaceuticals (Ardsley, New York), respectively. Rats were pretreated with kepone at the 200-ppm level in the Purina rat chow meal ration. The desired amount of kepone was dissolved in acetone and this was mixed with the ground rat chow (50 ml of solution/kg of food). Control diet was prepared similarly but without kepone. The acetone was allowed to evaporate in the hood by spreading the final diet in a large pan in the hood. Two treated or control rats were maintained per cage and allowed to eat only either the control or treated meal ration and water ad Zibitum. On Day 9 control and treated animals were used as liver donors for isolated perfused rat liver preparations. Experimental
Isolated perfused rat liver preparations were obtained by using previously described procedures (Mehendale, 1976a and b). Livers were perfused with 30% rat blood obtained by mixing heparinized whole rat blood with Krebs-Ringer bicarbonate-buffered solution containing bovine serum albumin (45 g/liter) and glucose (3.2 g/liter). The recirculating perfusate volume (150 or 115 ml) depended on whether or not perfusate samples were required for extraction ofmetabolites. After the initial equilibration period of 30 min, experiments were commenced by adding a bolus amount of [14C]imipramine at an initial perfusate concentration of 2 x 10e4 M. Blood samples (5 ml) were withdrawn at 5, 10,15,30,60, and 120 min. Appropriate corrections were made in calculations for the amount of radiolabel removed at every time point. Bile was collected in 2.5-ml graduated centrifuge tubes at 15,30,60, and 120 min. At the end of the 2-hr experiment, the liver was removed, weighed, and homogenized in three parts of ice-cold distilled water and the homogenate was either kept ice-cold until extraction or frozen and stored at -20°C until later use. In addition to preexposure experiments, the effect of in vitro addition of kepone to the isolated perfused rat liver preparation was also studied. After the equilibration period of 30 min, kepone was added to the perfusate to obtain a final concentration of 5 x low4 M. Kepone was dissolved in a mixture of Emulphor-620 (gift of GAF Corp., New York, New York) (Mehendale, 1976b) (1: 1) and water (8 parts) and 0.5 ml of this solution was added to the perfusate. The liver was then allowed to equilibrate another 15 min before commencing an experiment. In some experiments, polar metabolites of imipramine were added to the perfusate of control and treated isolated perfused rat liver preparations. Previously collected bile from [i4C]imipramine experiments was used as the source of polar metabolites. Bile collected from similar isolated perfused liver experiments with [14C]imipramine con-
250
H. M. MEHENDALE
tamed less than 2 % of 14C as imipramine and desmethyl imipramine (Moldowan and Bellward, 1974), the balance being polar metabolites of imipramine. Glucuronides of various hydroxylated metabolites (G), the hydroxylated metabolites (OH), and Noxides comprise the polar metabolites of which glucoronides predominate (Moldowan and Bellward, 1974). These experiments were conducted exactly as described above. In some other experiments, 5 mg of BSP was added to the perfusate (115 ml) of control and treated rat liver preparations. Blood (0.5 ml) was collected at 5, 10, 15, 30, and 60 min and bile at 15, 30, and 60 min after the addition of BSP. BSP was quantitated according to the method of Kutob and Plaa (1962). The concentration of BSP in the plasma or bile was determined spectrophotometrically. An aliquot of the plasma (0.1 ml) or bile (0.025 ml) was diluted with an appropriate volume of 0.1 N NaOH and read at 580 nm. Extraction and Analyses Blood, bile, and liver were extracted and analyzed for imipramine and metabolites using the procedures described by Moldowan and Bellward (1974). Blood (0.1 ml of plasma), bile (0.025 ml), and liver homogenate (0.2 ml) samples were counted in 10 ml of Aquasol (New England Nuclear, Boston, Massachusetts). Metabolites were determined by “C-counting after extraction of samples. All radioassays were carried out in a Beckman LD-250 scintillation spectrometer using internal standard for quench correction. Statistical analyses were done using Student’s t test for significance at p < 0.05 level. RESULTS
Biliary Excretion of [‘4C]Zmipramine Metabolites The pharmacokinetics of biliary excretion of imipramine metabolites by isolated perfused rat liver preparations reported here are in good agreement with those reported by Moldowan and Bellward (1974), e.g., the 1-hr biliary excretion value of 27.9 + 3.3 % reported here is indistinguishable from their value of 28.5 + 1.7 ‘A, In Fig. 2 are biliary excretion data for [14C]imipramine-derived radioactivity from control liver preparations and rat liver preparations from kepone-pretreated animals. The biliary excretion of the metabolites of imipramine was suppressed by 70% at the termination of 2-hr perfusion experiments. This marked suppression of biliary excretory function was evident at every time point examined (Fig. 2). In the preliminary experiments it was found that the addition of up to 5 x 10d5 M kepone in the perfusate was without any discernible effect on the biliary excretion of radiolabel from [14C]imipramine (Mehendale, 1976~). These experiments were designed for the purpose of examining the effect of the presence of kepone in the perfusion system on the biliary excretion of [14C]imipramine metabolites. At increased concentrations (5 x low4 M), kepone produced a statistically significant depression of the biliary excretion of [‘4C]imipramine metabolites (Fig. 2). Although this inhibition by added kepone was considerably less marked than that in keponepretreated livers, the effect was prevalent at all time points examined. Bile Flow Biliary excretion of some compounds has been directly associated with the rate of bile flow (Goldstein and Taurog, 1968 ; Hart et al., 1969 ; Klaassen, 1970; Klaassen and
KEPONE
: : 8
SO so.
AND
HEPATOBILIARY
0
1
; d
251
FUNCTION
CONTROL
A KEPONE
ADDED
A KEPONE
TREATED
60
30
TIME.
90
120
MIN.
FIG. 2. Effect ofkepone on biliaryexcretion of [%]imipramine by isolated perfusedratliver. Keponepretreated animals (A) received 200 ppm of kepone in daily rations for 8 days and controls (0) received similar diet without kepone. In other experiments (A) kepone was added (5 x 10m4M) to the perfusate of liver preparations obtained from untreated animals. The ordinate gives the cumulative biliary excretion as a percentage of the total [%]imipramine added to the perfusate. The abscissa shows the time at which bile samples were assayed. Results are the means & SE of three to four experiments. The asterisk indicates the values are significantly different from controls (p < 0.05).
Plaa, 1968; Roberts and Plaa, 1967). Hence, the effect of kepone on the volume of bile secretion was investigated using perfused liver preparations. Figure 3 illustrates the effect of kepone pretreatment and added kepone (5 x 10e4 M) on the rate of bile flow. In kepone-pretreated rat liver preparations an increase in bile flow was noted (p < 0.05). Although added kepone produced a 17 % depression initially, no statistically significant effects were noted at specific times or for the cumulative values. C.
100 1
0 CONTROL
A KEPONE
ADDED
A KEPONE
TREATTED
i0
d0
9b
120
TIME.MIN.
FIG. 3, Effect of kepone on bile flow from isolated perfused rat liver preparations. Kepone-pretreated animals (A) received 200 ppm of kepone in daily rations for 8 days and controls (0) received similar diets without kepone. In other experiments (A) kepone was added (5 x 10e4 M, 15 min prior to [WJimipramine) to the perfusate of liver preparations obtained from untreated animals. The ordinate gives the bile flow as microliters per minute kilogram body weight. The abscissa shows the time at which the bile volume was measured. Results are the means _+ SE of six to eight experiments. The asterisk indicates that the values are significantly different (p < 0.05) from controls.
H.M.MEHENDALE
A
30
60
TIME.
90
min.
0
120
TIME.
min.
FIG. 4. Pharmacokinetics of [r4C]imipramine in the perfusate of isolated perfused livers obtained from controls (A), kepone-pretreated (B) and kepone-added (C) experiments. Kepone-pretreated animals received 200 ppm of kepone in daily rations for 8 days and controls received similar diets without kepone. In other experiments kepone was added (5 x 10W4M, 15 min prior to [%]imipramine) to the perfusate of liver preparations obtained from untreated animals. Total radioactivity (0; total t4C) was separated into imipramine (n ; IMP), desmethyl imipramine (0; DMI) and polar metabolites (m; G, OH, N-O: glucuronides, free hydroxylated metabolites, and N-oxides). The ordinate shows the concentration of these substances in the perfusate (pg/ml) and the abscissa gives the time points at which perfusate samples were assayed. Results are the means + SE of three to four experiments. The asterisk indicates the values are significantly (p < 0.05) different from controls.
Disposition of [‘4C]lmipramine The data on the disappearance of imipramine and appearance of various metabolites in the perfusate in the control liver preparations (Fig. 4A) are in good agreement with those of Moldowan and Bellward (1974). Initially the concentration of [14C]imipramine dropped rapidly, representing rapid hepatic extraction and this was followed by a slower decline representing metabolism and biliary elimination. The metabolites appeared in the perfusate as early as 5 min. The concentration of desmethylimipramine peaked at 15 min. The concentration of polar metabolites (G, OH, and N-O) increased steadily before beginning to level off. The most pronounced effect of kepone pretreatment (Fig. 4B) was on the concentration of polar metabolites which accumulated in the perfusate at increasing concentrations. This resulted from an efflux of polar metabolites from the liver as indicated by increasing concentration of the total radioactivity in the perfusate after the 15min time period, at which time the net hepatic uptake of the radiolabel had peaked. The results would suggest that although the polar metabolites are available for biliary elimination, the markedly suppressed biliary excretion (Fig. 2)
KEPONE
AND
HEPATOBILIARY
3b
d0
TIME.
(FIG.
253
FUNCTION
li0
min.
4. Continued.)
results in the mobilization of these metabolites from the liver for efllux into the blood circulation. When kepone was added in vitro to the perfused liver preparations obtained from untreated animals (Fig. 4C), the disposition of [14C]imipramine and its metabolites in the perfusate appeared to be disturbed only slightly. Despite a significant decrease in the biliary excretion of imipramine metabolites in the bile (Fig. 2), there were no discernible differences in either the concentration of metabolites or in the total radiolabel in the perfusate. The data in Fig. 5 show that kepone-pretreated livers retained a significantly higher quantity of [14C]imipramine-derived radioactivity both in the liver and blood compared to the controls. However, when kepone was added in vitro to the liver preparations in sufficient concentrations to cause a decrease in biliary excretion (Fig. 2), the liver alone retained almost a corresponding quantity of the compound (Fig. 5) since no significant difference was found in the perfusate concentrations. The quantity of imipramine remaining in the perfusate and liver at the end of the 2-hr experiment was determined in order to examine the effect of kepone on the cumulative metabolism of imipramine. The results (Table 1) indicate that there was a statistically significant increase in the amount of the parent compound remaining. However, since only a small percentage of the total compound remained unmetabolized (I .8 and 3.6 % for control and kepone-pretreated, respectively), this difference is unlikely to be of mechanistic relevance. On the other hand, the increase in the parent compound remaining after the in vitro addition of kepone (1.8 and 14% for control and keponeadded, respecitvely) may be at least partially adequate to account for the slightly decreased rate of biliary excretion (Fig. 2).
254
H. M. MEHENDALE
100 ,, 1
CONTROL
n q
KEPONE
TREATED
KEPONE
ADDED
BILE
BLOOD
LIVER
FIG. 5. Effect of kepone on the distribution of radioactivity from [14C]imipramine in three compartments, bile, liver, and blood. Kepone-pretreated (8) animals received ZOOppm of kepone indailyrations for 8 days and controls (0) received similar diets without kepone. In other experiments (a) kepone was added (5 x 10e4 M, 15 min prior to [14C]imipramine) to the perfusate of liver preparations obtained from untreated animals. The bar diagram shows the distribution of the total radiolabel in the three compartments of the perfusion model at the end of the 2-hr perfusion experiment. Results are the means +SE of three to four experiments. The asterisk indicates that the values are significantly different (p < 0.05) from controls.
Biliary Excretion of Exogenous [14C]Imipramine Metabolites
Since, from the above experiments, it appeared that the kepone-induced impairment of biliary excretory function could not be accounted for by the differences in the rate of imipramine metabolism, the following experiments were conducted. It was postulated that, if the effects of kepone were unrelated to the rate of metabolism, the kepone effect should also manifest itself on the biliary excretion of the polar metabolites of imipramine. Readily excretable polar metabolites of imipramine were added to the perfusate of liver preparations obtained from control and kepone-pretreated animals. The results (Fig. 6) show that control livers excreted over 88 ‘A of the exogenously provided metabolites in the bile in 2 hr. Preexposure to kepone resulted in a marked suppression (61%) of biliary TABLE I EFFECT
OF KEPONE
Treatment Control Kepone-treated Kepone-added
ON METABOLISM
OF [14C]I~~~~~~~~~
BY ISOLATED
Distribution of [14C]imipramine after a 2-hr perfusion h3) Blood Liver 20.1 + 8.8
131.3 + 35.8
46.4 + 20.1 91.5 it 27.7
262.0 +_ 25.4' 1205.4 + 330.8b
PERFUSED
RAT
LIVER’
Imipramine remaining as a percentage of total dose 1.8 3.6 14.4
OKepone-pretreated rats received 200 ppm of kepone in their daily rations for 8 days and controls received similarly prepared rations without kepone. Isolated perfused liver preparations were obtained on Day 9 and [%]imipramine was added (2 x 10m4M) to the perfusate. In other experiments, kepone was added to the perfusate (5 x 10e4 M) 15 min prior to [‘*C]imipramine. Results are the means +_SE of three experiments. * The values are significantly different from controls (p < 0.05).
KEPONE
u
100 loo-
1
AND
HEPATOBILIARY
255
FUNCTION
0 CONTROL b KEPONE ADDED A KEPONE
TREATED
3b
d0 TIME.
9b
6 120
MIN.
FIG. 6. Effect of kepone on biliary excretion of exogenously provided polar metabolites of [%]imipramine by isolated perfused rat liver. Kepone-pretreated animals (A) received 200 ppm of kepone in daily rations for 8 days and controls (0) received similar diets without kepone. In other experiments (A), kepone was added (5 x low4 M, 15 min prior to [Wlimipramine) to the perfusate of rat liver preparations obtained from untreated animals. Bile collected from previous [WJimipramine experiments was used as the exogenous source of polar metabolites (G, OH, N-O) of imipramine. The ordinate shows cumulative biliary excretion as a percentage of the total radiolabel and the abscissa gives the time points at which the bile samples were assayed. Results are means +SE of four experiments. The asterisk indicates the values are significantly different from controls (p < 0.05).
Although less marked, biliary excretion was also suppressed (48%) when kepone was added to the perfusate of control liver preparations. Figure 7 illustrates how the radioactivity from the polar metabolites was distributed in the three compartments (bile, liver, and blood) at the end of the 2-hr experiment. Although there was a slight tendency for increased accumulation of these metabolites in the liver, the predominant material remained in the blood, in the presence of added kepone as well as with pretreated livers.
excretion.
13CONTROL m KLP~NE TREATED a KEPONE ADDEb
BILE
LIVER
BLOOD
7. Effect of kepone on the distribution of polar metabolites of [‘%]imipramine in the three compartments, bile, liver, and blood. Kepone-pretreated animals (m) received 200 ppm of kepone in daily rations for 8 days and the controls (0) received similar diets without kepone. In other experiments (a), kepone was added (5 x 10e4 M, 15 min prior to the polar metabolites of [“C]imipramine) to the perfusate of liver preparations obtained from untreated animals. Results are means ?SE of four experiments. The asterisk indicates the values are significantly different from controls (p < 0.05). FIG.
256
H. M. MEHENDALE
Biliary Excretion of BSP
It was of interest to seeif the kepone-induced impairment of biliary excretory function would also affect the biliary excretion of another anion, BSP. The results indicated that the biliary excretion of BSP in 1 hr was unaffected by kepone pretreatment (78.6 + 6.7 % vs 86.0 + 3.9 ‘A). Although there was a tendency for slightly increased biliary excretion following kepone pretreatment, this difference was not statistically significant. Plasma concentration-time curves were quantitatively indistinguishable indicating that the areas under the curve where similar. Also the biliary excretion time curves for control and kepone-pretreated liver preparations were quantitatively similar. In view of these results this kinetic information is not presented.
DISCUSSION
The immediate objective of these experiments was to investigate the effect of preexposure to kepone on biliary excretory function. In addition, it was of interest to gain some insight into the possible mechanism(s) by which kepone-induced modification of hepatobiliary function is manifested. The results point out that kepone, like its structural analog mirex (Mehendale, 1977), causes marked suppression of hepatobiliary function as judged by the impairment of biliary excretion of imipramine metabolites. However, this aberration of hepatobiliary function could not be detected by a BSP clearance test. In fact, preexposure to kepone did not alter the biliary excretion of BSP. On the other hand, preexposure to mirex (Mehendale, 1977) caused an impairment of biliary excretion of imipramine metabolites as well as hepatic clearance of BSP. These observations suggest the existence of two different mechanisms for biliary excretion of imipramine metabolites and BSP. Furthermore, while there are similarities in the mirex- and kepone-induced suppression of hepatobiliary function, there may be certain differences in the mechanism by which the effects are manifested. These differences might be utilized as experimental probes in the dissection of the mechanisms by which these and other environmental agents modify hepatobiliary function. The immediate implication of this lack of kepone-induced effect upon BSP clearance is that clinical BSP clearance tests may not be relied upon solely as an index of hepatobiliary function. Another peculiar aspect of the kepone- and mirex-induced modification of hepatobiliary function is the separation of biliary excretory function and secretion of bile. Despite the marked suppression of biliary excretion of imipramine metabolites, bile secretion is enhanced by preexposure to kepone. In this regard kepone and mirex are similar (Mehendale, 1976a). One reason for the increased bile flow may be the bile duct proliferation associated with liver hypertrophy due to preexpsosure to mirex (Mehendale et al., 1973) and kepone (Atwal, 1973; Klein et al., 1976). At any rate, kepone- and mirex-induced modification of biliary function appears to be unrelated to the bile flow. An additional mechanism for drug-induced modification of hepatobiliary function is altered metabolism (Levine, 1972, 1974b). Although the mechanism of altered metabolism may be questioned on the grounds that agents which modify biliary excretion of metabolizable substrates also modify the biliary excretion of substances which are not biotransformedprior to biliary elimination (Klaassen, 1970), it is still a plausible mecha-
KEPONE
AND
HEPATOBILIARY
FUNCTION
257
nism for the substrates that need prior biotransformation (Levine, 1972, 1974b). Since imipramine has to be metabolized for biliary excretion (Moldowan and Bellward, 1974), and the metabolism involves at least six different kinds of microsomal enzymes (Bickel and Weder, 196g), altered metabolism was considered as a possible mechanism for the kepone-induced suppression of biliary excretion. Although there was statistically significant difference in the amount of the parent compound remaining (Table 1) between control and kepone-pretreated liver experiments, the net difference is inadequate to account for a 70% decrease in the biliary excretion (Fig. 2). Furthermore, a similar kind of decrease in the biliary excretion was observed (61%) when readily excretable metabolites were exogenously provided to the kepone-pretreated livers (Fig. 6). In the experiments in which kepone was added the difference in the total metabolism of the parent compound might at least partly explain the decrease in the endogenously formed metabolites of imipramine (Table 1 and Fig. 2). However, the persistent and slightly greater suppression of biliary excretion of exogenously provided metabolites of imipramine by in vitro addition of kepone (Fig. 6) makes the above explanation incomplete and unsatisfactory. One question raised by the experiments in which kepone was added is the difference in the effect of added kepone on the biliary excretion of endogenously formed metabolites and the exogenously provided metabolites of imipramine. These results tend to support the existence of two different pools of metabolites: one representing the synthetic phase of metabolism of imipramine and the other being the recirculating pool of metabolites representing the postsynthetic phase (Gessner and Hamada, 1974; Mehendale, 1976b). Robinson et al. (1971) have suggested an anatomical continuity between canalicular membranes and certain sites of the endoplasmic reticulum responsible for the transformation of the parent species into polar metabolites. The metabolites that escape into the perfusate form a separate pool which is more susceptible to added kepone than the synthetic phase. Kepone-induced suppression of biliary excretion of both endogenously formed, as well as exogenously provided, metabolites may be explained by the susceptibility of the transport of metabolites from both the pools. The exact mechanism of kepone-induced impairment of hepatobiliary function is far from clear. Competitive interference by kepone at the site of transfer of imipramine metabolites to the bile canaliculi is one possibility. At the outset this appears unlikely in view of the very small quantity of kepone excreted in the bile(less than 0.8 ‘A of the dose in 3 hr) of bile duct-cannulated rats (H. B. Matthews, personal communication). In view of the above observations and arguments, it is suggested that the mechanism of kepone-induced effect is related to the transfer of the metabolites from the hepatocytes to the bile canaliculi. Microsomal ATPase (Schwartz et al., 1972) may be involved in the transport of various substances across cellular membranes. The primary source for such energy requiring transport of substances is mitochondrial ATP. Kepone is an effective inhibitor of native hepatic mitochondrial ATPase as well as DNP stimulated ATPase activity (Desaiah et al., 1977a). This inhibitory effect of kepone on ATP utilization as well as ATP production can be demonstrated by kepone pretreatment studies, in perfused liver preparations, and by in vitro addition of kepone to hepatic mitochondria (Desaiah et al., 1977a and b). A relationship between the interference ofkepone in energy production and impairment of hepatobiliary function is a matter of conjecture at this moment.
258
H. M. MEHENDALE
Further work with various model transport compounds would be useful in characterizing the etiology of kepone-induced impairment of hepatobiliary function.
REFERENCES ANONYMOUS (1976a). Senate panel probes kepone disaster. Chem. Eng. News 1,17-18. ANONYMOUS (1976b). Report on carcinogenesis bioassay of technical grade chlordecone
(kepone). Carcinogenesis Program, Division of Cancer Cause and Prevention, National Cancer Institute. ATWAL, 0. S.(1973). Fatty changes and hepatic cell excretion in avian liver. An electron microscopical study of kepone toxicity. J. Comp. Pathol. 83, 115-124. BAKER,R. C., COONS,L. B., MAILMAN, R. B., AND HODGSON, E. (1972). Induction of hepatic mixed function oxidases by the insecticide, mirex. Environ. Res. 5,418-424. BICKEL, M. H., AND WEDER, J. H. (1968). The total fate of a drug. Kinetics of distribution, excretion and formation of 14 metabolites in rats treated with imipramine. Arch. Int. Pharmacodyn. 173,433-463. BYARD, J. L., KOKEPE, U. C., ABRAHAM, L., GOLBERG, L., AND COULSTON, F. (1975). Biochemical changes in the liver of mice fed mirex. Toxicol. Appl. Pharmacol. 33,70-77. DESAIAH, D., Ho, I. K., AND MEHENDALE, H. M. (1977a). Inhibition of mitochondrial Mg++ ATPase activity in isolated perfused rat liver by kepone. Biochem. Pharmacol. 26, in press. DESAIAGH, D., Ho, I. K., AND MEHENDALE, H. M. (1977b). Effects of kepone and mirex on mitochondrial Mg2+-ATPase activity in rat liver. Toxicol. Appl. Pharmacol. 39,219-228. GAINES, T. B., AND KIMBROUGH, R. D. (1970). Oral toxicity of mirex in adult and suckling rats. Arch. Environ. Health 21, 7-14. GESSNER, T., AND HAMADA, N. (1974). High biliary and systemic excretion of a glucuronide during its hepatic synthesis.Life Sci. 15, 83-94. GOLDSTEIN, J. A., AND TAUROG, A. (1968). Enhanced biliary excretion of thyroxine glucuronide in rats pretreated with benzpyrene. Biochem. Pharmacol. 17, 1049-1065. HART, L. G., GUARINO, A. M., AND ADAMSON, R. H. (1969). Effects of phenobarbital on biliary excretion of organic acids in male and female rats. Amer. J. Physiol. 217,46-52. INNES, J. R. M., ULLAND, B. M., VALERIO, M. G., PETRUCELLI, L., FISHBEIN, L., HART, E. R., PALLATTA, A. J., BATES, R. R., FALK, H. L., CART,J. J. KLEIN, M., MITCHELL,J., ANDPETERS,
J. (1969). Bioassay of pesticides and industrial chemicals for tumorigenicity in mice. A preliminary report. J. Nat. Cancer Inst. 42, 1101-1114. KLAASSEN,C. D. (1970). Effects of phenobarbital on the plasma disappearance and biliary excretion of drugs in rats. J. Pharmacol. Exp. Ther. 168,289-300. KLAASSEN, C. D., AND PLAA, G. L. (1968). Studies on the mechanisms of phenobarbital enhanced sulfobromophthalein disappearance. J. Pharmacol. Exp. Ther. 161, 361-366. KLEIN, R. L., THURESON-KLEIN, A., AND MEHENDALE,H. M. (1976). Ultrastructural effects of acute kepone treatment in rats. Proceedings. Electron Microscopy Society of America. Thirty-fourth Annual Meeting (G. W. Bailey, ed.), Vol. 34, p. 286. KUTOB,S. D., ANDPLAA,G. L. (1962). Assessmentof liver function in mice with BSP.J. Appl. Physiol. 17, 123-125. LEVINE,W. G. (1972). Biliary excretion of 3-methylcholanthrene as controlled by its metabolism. J. Pharmacol. Exp. Ther. 183,42@426. LEVINE,W. G. (1973). Effect of phenobarbital and 3-amino-1,2,4-triazole on the metabolism and biliary excretion of 3,4-benzpyrene in the rat. Life Sci. 13, 723-732. LEVINE,W. G. (1974a). Effect of hypolipidemic drug nafenopin (2-methyl-2-[p-(1,2,3,4-tetrahydro-1-naphthyl) phenoxyl] propionic acid; TPIA; SU-13,437) on hepatic disposition of foreign compounds in the rat. Drug Metabol. Disp. 2, 178-186. LEVINE,W. G. (1974b). Hepatic uptake, metabolism and biliary excretion of 7,12-demethylbenzanthracene. Drug Metabol. Disp. 2, 169-177. LEVINE,W. G., AND BOGNACKI,J. (1976). Biliary excretion of 3,4-benzyprene in nafenopintreated rats. J. Pharmacol. Exp. Ther. 196,486-492.
KEPONEAND HEPATOBILIARYFUNCTION
259
MARTIN, H. (ed.) (1971). Pesticide Manual,
2nd Ed., p. 90. British Crop Protection Council. MEHENDALE,H. M. (1976a). Mirex-induced suppression of biliary excretion of polychlorinated biphenyl compounds. Toxicol. Appl. Pharmacol. 36,369-381. MEHENDALE,H. M. (1976b). Uptake and disposition of chlorinated biphenyls by isolated perfused rat liver. Drug Metabol. Disp. 4, 124-132. MEHENDALE, H. M. (1976c). Kepone-induced suppression of hepatobiliary function. Pharmacologist 18, 195. MEHENDALE, H. M. (1977). Mirex-induced impairment of hepatobiliary function. Drug Metabol. Disp. 5, 56-62 MEHENDALE, H. M., CHEN, P. R., FISHBEIN, L., AND MATTHEWS, H. B. (1973). Effect of mirex on the activities of various rat hepatic mixed function oxidases.Arch. Environ. Cont. Toxicol. 1,245-2X MOLDOWAN, M., AND BELLWARD, G. D. (1974). Studies on [14C]imipramine metabolism in the isolated perfused rat liver. Canad. J. Physiol. Pharmacol. 52,441-&O. ROBERTS, R. J., AND PLEA, G. L. (1967). Effect of phenobarbital on the excretion of an exogenous bilirubin load. Biochem. Pharmacol. 16, 827-835. ROBINSON, S. H., YANNONI,C., AND NAGASAWA, S. (1971). Bilirubin excretion in rats with normal and impaired bilirubin conjugation: Effect of phenobarbital. J. Clin. Invest. 50, 2606-
2613. SCHWARTZ, A., LINDENMAYER, G. E., AND ALLEN, J. C. (1972). The Na+, K+- ATPase membrane transport system: Importance in cellular function. In Current Topics in Membranes and Transport (F. Bonner and A. Kleinzeller, eds.), pp. l-82. Academic Press, New York. UESUGI, T., AND LEVINE, W. G. (1976). Effect of nafenopin (SU-13437) on liver function.
Influence on the hepatic transport of phenolphthalein glucuronide and chlorothiazide. Drug Metabol. Disp. 4, 107-l 11.
AND APPLIEDPHARMACOLOGYqO,247-259(1977)
Effect
of Preexposure of Imipramine
to Kepone on the Biliary and Sulfobromophthalein’
Excretion
H. M. MEHENDALE Department of Pharmacology and Toxicology, University of Mississippi Medical Center, Jackson, Mississippi 39216 Received September l&1976;
accepted December 20,1976
Effect of Preexposure to Kepone on the Biliary Excretion of Imipramine and Sulfobromophthalein. MEHENDALE, H. M. (1977). Toxicol. Appl. Pharmacol. 40, 247-259. The effect of kepone on the biliary excretion of [Wlimipramine (IMP) was studied using isolated perfused rat liver preparations obtained from control and kepone-pretreated animals. Biliary excretion of endogenously formed polar metabolites of IMP was markedly suppressed (70 %) by preexposure to kepone at 200 ppm in daily ration for 8 days. In vitro addition ofkepone at 5 x 10F4M concentration to the perfusate of untreated liver preparations elicited a 20 ‘A decreasein the biliary excretion of endogenously formed metabolites. At concentrations up to 5 x 10e5 M, kepone was without any discernible effect. The marked suppression of biliary elimination of endogenously formed metabolites is unrelated to the rate of metabolism of IMP. Furthermore, the biliary excretion of exogenously provided polar metabolites of IMP was also suppressed (61%) by preexposure to kepone. This suppression of biliary excretion of IMP metabolites was accompanied by an increase in the bile flow suggesting a dichotomy between bile secretion and biliary excretion. In vitro addition of kepone also resulted in a suppresion (48 %) of biliary elimination of exogenously provided metabolites of IMP. In the experiments in which kepone wasadded, bile flow was unaffected. Biliary excretion of sulfobromophthalein (BSP) was unaffected by preexposure to kepone, suggesting that kepone-induced impairment of hepatobiliary function may not be detected by BSP clearance tests. In addition, different mechanisms might be involved in the transport of IMP metabolites and BSP across the biliary canaliculi. From these results, it is suggested that the kepone-induced impairment of hepatobiliary function may be located at the site of transfer of metabolites across the bile canaliculi. Kepone is the trade name for a commercially available pesticide, decachlorooctahydro1,3,4-metheno-2H-cyclobuta (cd) pentalen-Zone (common name, chlordecone). First introduced in 1958, it has been used as an insecticide against leaf-eating insects, ants, and cockroaches and as a larvicide against flies (Martin, 1971). Kepone has caused human ailment after occupational exposure in a manufacturing facility and has been the 1This investigationwas supported by Grant No. GRSG RR05386. A preliminary report was presentedat the Fall Meeting of the American Societyof Pharmacologyand Experimental Therapeutics, New Orleans,Louisiana, 1976 [Pharmacologist Z8,195 (1976)]. Copyright IQ 1977 by Academic Press, Inc. All rights of reproduction in any form reserved. Printed in Great Britain
247 ISSN 0041-008X
248
H. M. MEHENDALE
subject of concern as an environmental contaminant in the area (Anonymous, 1976a). Kepone is a carbonyl analog of mirex (Fig. l), an insecticide used for the control of fire ants in the southeastern United States. Both mirex (Innes et al., 1969) and kepone (Anonymous, 1976b) are reported to cause hepatocarcinomas in experimental animals. Mirex causes liver enlargement and proliferation of the endoplasmic reticulum associated with an increase in the mixed function oxidases (Baker et al., 1972; Byard et al., 1975; Gaines and Kimbrough, 1970; Mehendale et al., 1973). In a previous study from this laboratory, it was shown that mirex pretreatment causes an increase in bile flow (Mehendale, 1976a). However, despite the above changes, the biliary excretion of 4chlorobiphenyl (l-CB) was suppressed markedly after preexposure of rats to mirex (Mehendale, 1976a). This mirex-induced impairment of hepatobiliary functions was unrelated to either the bile flow or the slightly decreased rate of metabolism of l-CB. Since the biliary excretion of exogenously provided, readily excretable metabolites of MIREX
_
KEPONE
491 WT. 546 FIG. 1. Structures of mirex and kepone.
MOL.
l-CB was also markedly suppressed after preexposure to mirex, it was suggested that the locus of mirex-induced suppression was at the site of transport of metabolites from the hepatccytes to the bile canaliculi. Preexposure to phenobarbital caused a slight enhancement of the metabolism of [14C]1-CB, the bile flow, and biliary excretion of the metabolites of l-CB, observations consistent with the known effects of phenobarbital (Klaassen, 1970; Levine, 1973). Another example of suppression of biliary excretion comes from the experiments with the hypolipidemic drug nafenopin (Levine, 1974a; Levine and Bognacki, 1976; Uesugi and Levine, 1976). Bile flow was increased after nafenopin in all the above experiments. The present investigations with kepone were conducted in order to determine its effect on hepatobiliary function. It was of interest to investigate whether or not the substitution of two chlorine atoms of mirex with the comparatively polar carbonyl group would change the effect of this compound on bile flow and biliary excretory function. Isolated perfused rat liver preparations obtained from control and treated animals were utilized to investigate the biliary excretion of the tricyclic antidepressant imipramine and the dye sulfobromophthalein. METHODS Animals Male Sprague-Dawley rats (Charles River Breeding Labs, Wilmington, Massachusetts) were maintained in the central animal facilities away from any known inducers. The bedding used for these animals was made from untreated ground corn cobs. Ani-
KEPONE
AND
HEPATOBILIARY
FUNCTION
249
mals were provided with water and a standard diet of Purina rat chow blocks ad Iibitum until used for an experiment as a liver or blood donor. Animals from which the blood was collected weighed 250-350 g, whereas the animals used as liver donors weighed 200-250 g. During all surgical procedures animals were held under ether anesthesia. Drugs and Chemicals
Kepone was a gift of Allied Chemical Corporation (Union Texas Petroleum, Agr. Dept., Baltimore, Maryland). [14C]Imipramine hydrochloride was purchased from Amersham/Searle Corporation (Des Plaines, Illinois). Sulfobromophthalein (BSP) and unlabeled imipramine hydrochloride were obtained from Sigma Chemical Company (St. Louis, Missouri) and Geigy Pharmaceuticals (Ardsley, New York), respectively. Rats were pretreated with kepone at the 200-ppm level in the Purina rat chow meal ration. The desired amount of kepone was dissolved in acetone and this was mixed with the ground rat chow (50 ml of solution/kg of food). Control diet was prepared similarly but without kepone. The acetone was allowed to evaporate in the hood by spreading the final diet in a large pan in the hood. Two treated or control rats were maintained per cage and allowed to eat only either the control or treated meal ration and water ad Zibitum. On Day 9 control and treated animals were used as liver donors for isolated perfused rat liver preparations. Experimental
Isolated perfused rat liver preparations were obtained by using previously described procedures (Mehendale, 1976a and b). Livers were perfused with 30% rat blood obtained by mixing heparinized whole rat blood with Krebs-Ringer bicarbonate-buffered solution containing bovine serum albumin (45 g/liter) and glucose (3.2 g/liter). The recirculating perfusate volume (150 or 115 ml) depended on whether or not perfusate samples were required for extraction ofmetabolites. After the initial equilibration period of 30 min, experiments were commenced by adding a bolus amount of [14C]imipramine at an initial perfusate concentration of 2 x 10e4 M. Blood samples (5 ml) were withdrawn at 5, 10,15,30,60, and 120 min. Appropriate corrections were made in calculations for the amount of radiolabel removed at every time point. Bile was collected in 2.5-ml graduated centrifuge tubes at 15,30,60, and 120 min. At the end of the 2-hr experiment, the liver was removed, weighed, and homogenized in three parts of ice-cold distilled water and the homogenate was either kept ice-cold until extraction or frozen and stored at -20°C until later use. In addition to preexposure experiments, the effect of in vitro addition of kepone to the isolated perfused rat liver preparation was also studied. After the equilibration period of 30 min, kepone was added to the perfusate to obtain a final concentration of 5 x low4 M. Kepone was dissolved in a mixture of Emulphor-620 (gift of GAF Corp., New York, New York) (Mehendale, 1976b) (1: 1) and water (8 parts) and 0.5 ml of this solution was added to the perfusate. The liver was then allowed to equilibrate another 15 min before commencing an experiment. In some experiments, polar metabolites of imipramine were added to the perfusate of control and treated isolated perfused rat liver preparations. Previously collected bile from [i4C]imipramine experiments was used as the source of polar metabolites. Bile collected from similar isolated perfused liver experiments with [14C]imipramine con-
250
H. M. MEHENDALE
tamed less than 2 % of 14C as imipramine and desmethyl imipramine (Moldowan and Bellward, 1974), the balance being polar metabolites of imipramine. Glucuronides of various hydroxylated metabolites (G), the hydroxylated metabolites (OH), and Noxides comprise the polar metabolites of which glucoronides predominate (Moldowan and Bellward, 1974). These experiments were conducted exactly as described above. In some other experiments, 5 mg of BSP was added to the perfusate (115 ml) of control and treated rat liver preparations. Blood (0.5 ml) was collected at 5, 10, 15, 30, and 60 min and bile at 15, 30, and 60 min after the addition of BSP. BSP was quantitated according to the method of Kutob and Plaa (1962). The concentration of BSP in the plasma or bile was determined spectrophotometrically. An aliquot of the plasma (0.1 ml) or bile (0.025 ml) was diluted with an appropriate volume of 0.1 N NaOH and read at 580 nm. Extraction and Analyses Blood, bile, and liver were extracted and analyzed for imipramine and metabolites using the procedures described by Moldowan and Bellward (1974). Blood (0.1 ml of plasma), bile (0.025 ml), and liver homogenate (0.2 ml) samples were counted in 10 ml of Aquasol (New England Nuclear, Boston, Massachusetts). Metabolites were determined by “C-counting after extraction of samples. All radioassays were carried out in a Beckman LD-250 scintillation spectrometer using internal standard for quench correction. Statistical analyses were done using Student’s t test for significance at p < 0.05 level. RESULTS
Biliary Excretion of [‘4C]Zmipramine Metabolites The pharmacokinetics of biliary excretion of imipramine metabolites by isolated perfused rat liver preparations reported here are in good agreement with those reported by Moldowan and Bellward (1974), e.g., the 1-hr biliary excretion value of 27.9 + 3.3 % reported here is indistinguishable from their value of 28.5 + 1.7 ‘A, In Fig. 2 are biliary excretion data for [14C]imipramine-derived radioactivity from control liver preparations and rat liver preparations from kepone-pretreated animals. The biliary excretion of the metabolites of imipramine was suppressed by 70% at the termination of 2-hr perfusion experiments. This marked suppression of biliary excretory function was evident at every time point examined (Fig. 2). In the preliminary experiments it was found that the addition of up to 5 x 10d5 M kepone in the perfusate was without any discernible effect on the biliary excretion of radiolabel from [14C]imipramine (Mehendale, 1976~). These experiments were designed for the purpose of examining the effect of the presence of kepone in the perfusion system on the biliary excretion of [14C]imipramine metabolites. At increased concentrations (5 x low4 M), kepone produced a statistically significant depression of the biliary excretion of [‘4C]imipramine metabolites (Fig. 2). Although this inhibition by added kepone was considerably less marked than that in keponepretreated livers, the effect was prevalent at all time points examined. Bile Flow Biliary excretion of some compounds has been directly associated with the rate of bile flow (Goldstein and Taurog, 1968 ; Hart et al., 1969 ; Klaassen, 1970; Klaassen and
KEPONE
: : 8
SO so.
AND
HEPATOBILIARY
0
1
; d
251
FUNCTION
CONTROL
A KEPONE
ADDED
A KEPONE
TREATED
60
30
TIME.
90
120
MIN.
FIG. 2. Effect ofkepone on biliaryexcretion of [%]imipramine by isolated perfusedratliver. Keponepretreated animals (A) received 200 ppm of kepone in daily rations for 8 days and controls (0) received similar diet without kepone. In other experiments (A) kepone was added (5 x 10m4M) to the perfusate of liver preparations obtained from untreated animals. The ordinate gives the cumulative biliary excretion as a percentage of the total [%]imipramine added to the perfusate. The abscissa shows the time at which bile samples were assayed. Results are the means & SE of three to four experiments. The asterisk indicates the values are significantly different from controls (p < 0.05).
Plaa, 1968; Roberts and Plaa, 1967). Hence, the effect of kepone on the volume of bile secretion was investigated using perfused liver preparations. Figure 3 illustrates the effect of kepone pretreatment and added kepone (5 x 10e4 M) on the rate of bile flow. In kepone-pretreated rat liver preparations an increase in bile flow was noted (p < 0.05). Although added kepone produced a 17 % depression initially, no statistically significant effects were noted at specific times or for the cumulative values. C.
100 1
0 CONTROL
A KEPONE
ADDED
A KEPONE
TREATTED
i0
d0
9b
120
TIME.MIN.
FIG. 3, Effect of kepone on bile flow from isolated perfused rat liver preparations. Kepone-pretreated animals (A) received 200 ppm of kepone in daily rations for 8 days and controls (0) received similar diets without kepone. In other experiments (A) kepone was added (5 x 10e4 M, 15 min prior to [WJimipramine) to the perfusate of liver preparations obtained from untreated animals. The ordinate gives the bile flow as microliters per minute kilogram body weight. The abscissa shows the time at which the bile volume was measured. Results are the means _+ SE of six to eight experiments. The asterisk indicates that the values are significantly different (p < 0.05) from controls.
H.M.MEHENDALE
A
30
60
TIME.
90
min.
0
120
TIME.
min.
FIG. 4. Pharmacokinetics of [r4C]imipramine in the perfusate of isolated perfused livers obtained from controls (A), kepone-pretreated (B) and kepone-added (C) experiments. Kepone-pretreated animals received 200 ppm of kepone in daily rations for 8 days and controls received similar diets without kepone. In other experiments kepone was added (5 x 10W4M, 15 min prior to [%]imipramine) to the perfusate of liver preparations obtained from untreated animals. Total radioactivity (0; total t4C) was separated into imipramine (n ; IMP), desmethyl imipramine (0; DMI) and polar metabolites (m; G, OH, N-O: glucuronides, free hydroxylated metabolites, and N-oxides). The ordinate shows the concentration of these substances in the perfusate (pg/ml) and the abscissa gives the time points at which perfusate samples were assayed. Results are the means + SE of three to four experiments. The asterisk indicates the values are significantly (p < 0.05) different from controls.
Disposition of [‘4C]lmipramine The data on the disappearance of imipramine and appearance of various metabolites in the perfusate in the control liver preparations (Fig. 4A) are in good agreement with those of Moldowan and Bellward (1974). Initially the concentration of [14C]imipramine dropped rapidly, representing rapid hepatic extraction and this was followed by a slower decline representing metabolism and biliary elimination. The metabolites appeared in the perfusate as early as 5 min. The concentration of desmethylimipramine peaked at 15 min. The concentration of polar metabolites (G, OH, and N-O) increased steadily before beginning to level off. The most pronounced effect of kepone pretreatment (Fig. 4B) was on the concentration of polar metabolites which accumulated in the perfusate at increasing concentrations. This resulted from an efflux of polar metabolites from the liver as indicated by increasing concentration of the total radioactivity in the perfusate after the 15min time period, at which time the net hepatic uptake of the radiolabel had peaked. The results would suggest that although the polar metabolites are available for biliary elimination, the markedly suppressed biliary excretion (Fig. 2)
KEPONE
AND
HEPATOBILIARY
3b
d0
TIME.
(FIG.
253
FUNCTION
li0
min.
4. Continued.)
results in the mobilization of these metabolites from the liver for efllux into the blood circulation. When kepone was added in vitro to the perfused liver preparations obtained from untreated animals (Fig. 4C), the disposition of [14C]imipramine and its metabolites in the perfusate appeared to be disturbed only slightly. Despite a significant decrease in the biliary excretion of imipramine metabolites in the bile (Fig. 2), there were no discernible differences in either the concentration of metabolites or in the total radiolabel in the perfusate. The data in Fig. 5 show that kepone-pretreated livers retained a significantly higher quantity of [14C]imipramine-derived radioactivity both in the liver and blood compared to the controls. However, when kepone was added in vitro to the liver preparations in sufficient concentrations to cause a decrease in biliary excretion (Fig. 2), the liver alone retained almost a corresponding quantity of the compound (Fig. 5) since no significant difference was found in the perfusate concentrations. The quantity of imipramine remaining in the perfusate and liver at the end of the 2-hr experiment was determined in order to examine the effect of kepone on the cumulative metabolism of imipramine. The results (Table 1) indicate that there was a statistically significant increase in the amount of the parent compound remaining. However, since only a small percentage of the total compound remained unmetabolized (I .8 and 3.6 % for control and kepone-pretreated, respectively), this difference is unlikely to be of mechanistic relevance. On the other hand, the increase in the parent compound remaining after the in vitro addition of kepone (1.8 and 14% for control and keponeadded, respecitvely) may be at least partially adequate to account for the slightly decreased rate of biliary excretion (Fig. 2).
254
H. M. MEHENDALE
100 ,, 1
CONTROL
n q
KEPONE
TREATED
KEPONE
ADDED
BILE
BLOOD
LIVER
FIG. 5. Effect of kepone on the distribution of radioactivity from [14C]imipramine in three compartments, bile, liver, and blood. Kepone-pretreated (8) animals received ZOOppm of kepone indailyrations for 8 days and controls (0) received similar diets without kepone. In other experiments (a) kepone was added (5 x 10e4 M, 15 min prior to [14C]imipramine) to the perfusate of liver preparations obtained from untreated animals. The bar diagram shows the distribution of the total radiolabel in the three compartments of the perfusion model at the end of the 2-hr perfusion experiment. Results are the means +SE of three to four experiments. The asterisk indicates that the values are significantly different (p < 0.05) from controls.
Biliary Excretion of Exogenous [14C]Imipramine Metabolites
Since, from the above experiments, it appeared that the kepone-induced impairment of biliary excretory function could not be accounted for by the differences in the rate of imipramine metabolism, the following experiments were conducted. It was postulated that, if the effects of kepone were unrelated to the rate of metabolism, the kepone effect should also manifest itself on the biliary excretion of the polar metabolites of imipramine. Readily excretable polar metabolites of imipramine were added to the perfusate of liver preparations obtained from control and kepone-pretreated animals. The results (Fig. 6) show that control livers excreted over 88 ‘A of the exogenously provided metabolites in the bile in 2 hr. Preexposure to kepone resulted in a marked suppression (61%) of biliary TABLE I EFFECT
OF KEPONE
Treatment Control Kepone-treated Kepone-added
ON METABOLISM
OF [14C]I~~~~~~~~~
BY ISOLATED
Distribution of [14C]imipramine after a 2-hr perfusion h3) Blood Liver 20.1 + 8.8
131.3 + 35.8
46.4 + 20.1 91.5 it 27.7
262.0 +_ 25.4' 1205.4 + 330.8b
PERFUSED
RAT
LIVER’
Imipramine remaining as a percentage of total dose 1.8 3.6 14.4
OKepone-pretreated rats received 200 ppm of kepone in their daily rations for 8 days and controls received similarly prepared rations without kepone. Isolated perfused liver preparations were obtained on Day 9 and [%]imipramine was added (2 x 10m4M) to the perfusate. In other experiments, kepone was added to the perfusate (5 x 10e4 M) 15 min prior to [‘*C]imipramine. Results are the means +_SE of three experiments. * The values are significantly different from controls (p < 0.05).
KEPONE
u
100 loo-
1
AND
HEPATOBILIARY
255
FUNCTION
0 CONTROL b KEPONE ADDED A KEPONE
TREATED
3b
d0 TIME.
9b
6 120
MIN.
FIG. 6. Effect of kepone on biliary excretion of exogenously provided polar metabolites of [%]imipramine by isolated perfused rat liver. Kepone-pretreated animals (A) received 200 ppm of kepone in daily rations for 8 days and controls (0) received similar diets without kepone. In other experiments (A), kepone was added (5 x low4 M, 15 min prior to [Wlimipramine) to the perfusate of rat liver preparations obtained from untreated animals. Bile collected from previous [WJimipramine experiments was used as the exogenous source of polar metabolites (G, OH, N-O) of imipramine. The ordinate shows cumulative biliary excretion as a percentage of the total radiolabel and the abscissa gives the time points at which the bile samples were assayed. Results are means +SE of four experiments. The asterisk indicates the values are significantly different from controls (p < 0.05).
Although less marked, biliary excretion was also suppressed (48%) when kepone was added to the perfusate of control liver preparations. Figure 7 illustrates how the radioactivity from the polar metabolites was distributed in the three compartments (bile, liver, and blood) at the end of the 2-hr experiment. Although there was a slight tendency for increased accumulation of these metabolites in the liver, the predominant material remained in the blood, in the presence of added kepone as well as with pretreated livers.
excretion.
13CONTROL m KLP~NE TREATED a KEPONE ADDEb
BILE
LIVER
BLOOD
7. Effect of kepone on the distribution of polar metabolites of [‘%]imipramine in the three compartments, bile, liver, and blood. Kepone-pretreated animals (m) received 200 ppm of kepone in daily rations for 8 days and the controls (0) received similar diets without kepone. In other experiments (a), kepone was added (5 x 10e4 M, 15 min prior to the polar metabolites of [“C]imipramine) to the perfusate of liver preparations obtained from untreated animals. Results are means ?SE of four experiments. The asterisk indicates the values are significantly different from controls (p < 0.05). FIG.
256
H. M. MEHENDALE
Biliary Excretion of BSP
It was of interest to seeif the kepone-induced impairment of biliary excretory function would also affect the biliary excretion of another anion, BSP. The results indicated that the biliary excretion of BSP in 1 hr was unaffected by kepone pretreatment (78.6 + 6.7 % vs 86.0 + 3.9 ‘A). Although there was a tendency for slightly increased biliary excretion following kepone pretreatment, this difference was not statistically significant. Plasma concentration-time curves were quantitatively indistinguishable indicating that the areas under the curve where similar. Also the biliary excretion time curves for control and kepone-pretreated liver preparations were quantitatively similar. In view of these results this kinetic information is not presented.
DISCUSSION
The immediate objective of these experiments was to investigate the effect of preexposure to kepone on biliary excretory function. In addition, it was of interest to gain some insight into the possible mechanism(s) by which kepone-induced modification of hepatobiliary function is manifested. The results point out that kepone, like its structural analog mirex (Mehendale, 1977), causes marked suppression of hepatobiliary function as judged by the impairment of biliary excretion of imipramine metabolites. However, this aberration of hepatobiliary function could not be detected by a BSP clearance test. In fact, preexposure to kepone did not alter the biliary excretion of BSP. On the other hand, preexposure to mirex (Mehendale, 1977) caused an impairment of biliary excretion of imipramine metabolites as well as hepatic clearance of BSP. These observations suggest the existence of two different mechanisms for biliary excretion of imipramine metabolites and BSP. Furthermore, while there are similarities in the mirex- and kepone-induced suppression of hepatobiliary function, there may be certain differences in the mechanism by which the effects are manifested. These differences might be utilized as experimental probes in the dissection of the mechanisms by which these and other environmental agents modify hepatobiliary function. The immediate implication of this lack of kepone-induced effect upon BSP clearance is that clinical BSP clearance tests may not be relied upon solely as an index of hepatobiliary function. Another peculiar aspect of the kepone- and mirex-induced modification of hepatobiliary function is the separation of biliary excretory function and secretion of bile. Despite the marked suppression of biliary excretion of imipramine metabolites, bile secretion is enhanced by preexposure to kepone. In this regard kepone and mirex are similar (Mehendale, 1976a). One reason for the increased bile flow may be the bile duct proliferation associated with liver hypertrophy due to preexpsosure to mirex (Mehendale et al., 1973) and kepone (Atwal, 1973; Klein et al., 1976). At any rate, kepone- and mirex-induced modification of biliary function appears to be unrelated to the bile flow. An additional mechanism for drug-induced modification of hepatobiliary function is altered metabolism (Levine, 1972, 1974b). Although the mechanism of altered metabolism may be questioned on the grounds that agents which modify biliary excretion of metabolizable substrates also modify the biliary excretion of substances which are not biotransformedprior to biliary elimination (Klaassen, 1970), it is still a plausible mecha-
KEPONE
AND
HEPATOBILIARY
FUNCTION
257
nism for the substrates that need prior biotransformation (Levine, 1972, 1974b). Since imipramine has to be metabolized for biliary excretion (Moldowan and Bellward, 1974), and the metabolism involves at least six different kinds of microsomal enzymes (Bickel and Weder, 196g), altered metabolism was considered as a possible mechanism for the kepone-induced suppression of biliary excretion. Although there was statistically significant difference in the amount of the parent compound remaining (Table 1) between control and kepone-pretreated liver experiments, the net difference is inadequate to account for a 70% decrease in the biliary excretion (Fig. 2). Furthermore, a similar kind of decrease in the biliary excretion was observed (61%) when readily excretable metabolites were exogenously provided to the kepone-pretreated livers (Fig. 6). In the experiments in which kepone was added the difference in the total metabolism of the parent compound might at least partly explain the decrease in the endogenously formed metabolites of imipramine (Table 1 and Fig. 2). However, the persistent and slightly greater suppression of biliary excretion of exogenously provided metabolites of imipramine by in vitro addition of kepone (Fig. 6) makes the above explanation incomplete and unsatisfactory. One question raised by the experiments in which kepone was added is the difference in the effect of added kepone on the biliary excretion of endogenously formed metabolites and the exogenously provided metabolites of imipramine. These results tend to support the existence of two different pools of metabolites: one representing the synthetic phase of metabolism of imipramine and the other being the recirculating pool of metabolites representing the postsynthetic phase (Gessner and Hamada, 1974; Mehendale, 1976b). Robinson et al. (1971) have suggested an anatomical continuity between canalicular membranes and certain sites of the endoplasmic reticulum responsible for the transformation of the parent species into polar metabolites. The metabolites that escape into the perfusate form a separate pool which is more susceptible to added kepone than the synthetic phase. Kepone-induced suppression of biliary excretion of both endogenously formed, as well as exogenously provided, metabolites may be explained by the susceptibility of the transport of metabolites from both the pools. The exact mechanism of kepone-induced impairment of hepatobiliary function is far from clear. Competitive interference by kepone at the site of transfer of imipramine metabolites to the bile canaliculi is one possibility. At the outset this appears unlikely in view of the very small quantity of kepone excreted in the bile(less than 0.8 ‘A of the dose in 3 hr) of bile duct-cannulated rats (H. B. Matthews, personal communication). In view of the above observations and arguments, it is suggested that the mechanism of kepone-induced effect is related to the transfer of the metabolites from the hepatocytes to the bile canaliculi. Microsomal ATPase (Schwartz et al., 1972) may be involved in the transport of various substances across cellular membranes. The primary source for such energy requiring transport of substances is mitochondrial ATP. Kepone is an effective inhibitor of native hepatic mitochondrial ATPase as well as DNP stimulated ATPase activity (Desaiah et al., 1977a). This inhibitory effect of kepone on ATP utilization as well as ATP production can be demonstrated by kepone pretreatment studies, in perfused liver preparations, and by in vitro addition of kepone to hepatic mitochondria (Desaiah et al., 1977a and b). A relationship between the interference ofkepone in energy production and impairment of hepatobiliary function is a matter of conjecture at this moment.
258
H. M. MEHENDALE
Further work with various model transport compounds would be useful in characterizing the etiology of kepone-induced impairment of hepatobiliary function.
REFERENCES ANONYMOUS (1976a). Senate panel probes kepone disaster. Chem. Eng. News 1,17-18. ANONYMOUS (1976b). Report on carcinogenesis bioassay of technical grade chlordecone
(kepone). Carcinogenesis Program, Division of Cancer Cause and Prevention, National Cancer Institute. ATWAL, 0. S.(1973). Fatty changes and hepatic cell excretion in avian liver. An electron microscopical study of kepone toxicity. J. Comp. Pathol. 83, 115-124. BAKER,R. C., COONS,L. B., MAILMAN, R. B., AND HODGSON, E. (1972). Induction of hepatic mixed function oxidases by the insecticide, mirex. Environ. Res. 5,418-424. BICKEL, M. H., AND WEDER, J. H. (1968). The total fate of a drug. Kinetics of distribution, excretion and formation of 14 metabolites in rats treated with imipramine. Arch. Int. Pharmacodyn. 173,433-463. BYARD, J. L., KOKEPE, U. C., ABRAHAM, L., GOLBERG, L., AND COULSTON, F. (1975). Biochemical changes in the liver of mice fed mirex. Toxicol. Appl. Pharmacol. 33,70-77. DESAIAH, D., Ho, I. K., AND MEHENDALE, H. M. (1977a). Inhibition of mitochondrial Mg++ ATPase activity in isolated perfused rat liver by kepone. Biochem. Pharmacol. 26, in press. DESAIAGH, D., Ho, I. K., AND MEHENDALE, H. M. (1977b). Effects of kepone and mirex on mitochondrial Mg2+-ATPase activity in rat liver. Toxicol. Appl. Pharmacol. 39,219-228. GAINES, T. B., AND KIMBROUGH, R. D. (1970). Oral toxicity of mirex in adult and suckling rats. Arch. Environ. Health 21, 7-14. GESSNER, T., AND HAMADA, N. (1974). High biliary and systemic excretion of a glucuronide during its hepatic synthesis.Life Sci. 15, 83-94. GOLDSTEIN, J. A., AND TAUROG, A. (1968). Enhanced biliary excretion of thyroxine glucuronide in rats pretreated with benzpyrene. Biochem. Pharmacol. 17, 1049-1065. HART, L. G., GUARINO, A. M., AND ADAMSON, R. H. (1969). Effects of phenobarbital on biliary excretion of organic acids in male and female rats. Amer. J. Physiol. 217,46-52. INNES, J. R. M., ULLAND, B. M., VALERIO, M. G., PETRUCELLI, L., FISHBEIN, L., HART, E. R., PALLATTA, A. J., BATES, R. R., FALK, H. L., CART,J. J. KLEIN, M., MITCHELL,J., ANDPETERS,
J. (1969). Bioassay of pesticides and industrial chemicals for tumorigenicity in mice. A preliminary report. J. Nat. Cancer Inst. 42, 1101-1114. KLAASSEN,C. D. (1970). Effects of phenobarbital on the plasma disappearance and biliary excretion of drugs in rats. J. Pharmacol. Exp. Ther. 168,289-300. KLAASSEN, C. D., AND PLAA, G. L. (1968). Studies on the mechanisms of phenobarbital enhanced sulfobromophthalein disappearance. J. Pharmacol. Exp. Ther. 161, 361-366. KLEIN, R. L., THURESON-KLEIN, A., AND MEHENDALE,H. M. (1976). Ultrastructural effects of acute kepone treatment in rats. Proceedings. Electron Microscopy Society of America. Thirty-fourth Annual Meeting (G. W. Bailey, ed.), Vol. 34, p. 286. KUTOB,S. D., ANDPLAA,G. L. (1962). Assessmentof liver function in mice with BSP.J. Appl. Physiol. 17, 123-125. LEVINE,W. G. (1972). Biliary excretion of 3-methylcholanthrene as controlled by its metabolism. J. Pharmacol. Exp. Ther. 183,42@426. LEVINE,W. G. (1973). Effect of phenobarbital and 3-amino-1,2,4-triazole on the metabolism and biliary excretion of 3,4-benzpyrene in the rat. Life Sci. 13, 723-732. LEVINE,W. G. (1974a). Effect of hypolipidemic drug nafenopin (2-methyl-2-[p-(1,2,3,4-tetrahydro-1-naphthyl) phenoxyl] propionic acid; TPIA; SU-13,437) on hepatic disposition of foreign compounds in the rat. Drug Metabol. Disp. 2, 178-186. LEVINE,W. G. (1974b). Hepatic uptake, metabolism and biliary excretion of 7,12-demethylbenzanthracene. Drug Metabol. Disp. 2, 169-177. LEVINE,W. G., AND BOGNACKI,J. (1976). Biliary excretion of 3,4-benzyprene in nafenopintreated rats. J. Pharmacol. Exp. Ther. 196,486-492.
KEPONEAND HEPATOBILIARYFUNCTION
259
MARTIN, H. (ed.) (1971). Pesticide Manual,
2nd Ed., p. 90. British Crop Protection Council. MEHENDALE,H. M. (1976a). Mirex-induced suppression of biliary excretion of polychlorinated biphenyl compounds. Toxicol. Appl. Pharmacol. 36,369-381. MEHENDALE,H. M. (1976b). Uptake and disposition of chlorinated biphenyls by isolated perfused rat liver. Drug Metabol. Disp. 4, 124-132. MEHENDALE, H. M. (1976c). Kepone-induced suppression of hepatobiliary function. Pharmacologist 18, 195. MEHENDALE, H. M. (1977). Mirex-induced impairment of hepatobiliary function. Drug Metabol. Disp. 5, 56-62 MEHENDALE, H. M., CHEN, P. R., FISHBEIN, L., AND MATTHEWS, H. B. (1973). Effect of mirex on the activities of various rat hepatic mixed function oxidases.Arch. Environ. Cont. Toxicol. 1,245-2X MOLDOWAN, M., AND BELLWARD, G. D. (1974). Studies on [14C]imipramine metabolism in the isolated perfused rat liver. Canad. J. Physiol. Pharmacol. 52,441-&O. ROBERTS, R. J., AND PLEA, G. L. (1967). Effect of phenobarbital on the excretion of an exogenous bilirubin load. Biochem. Pharmacol. 16, 827-835. ROBINSON, S. H., YANNONI,C., AND NAGASAWA, S. (1971). Bilirubin excretion in rats with normal and impaired bilirubin conjugation: Effect of phenobarbital. J. Clin. Invest. 50, 2606-
2613. SCHWARTZ, A., LINDENMAYER, G. E., AND ALLEN, J. C. (1972). The Na+, K+- ATPase membrane transport system: Importance in cellular function. In Current Topics in Membranes and Transport (F. Bonner and A. Kleinzeller, eds.), pp. l-82. Academic Press, New York. UESUGI, T., AND LEVINE, W. G. (1976). Effect of nafenopin (SU-13437) on liver function.
Influence on the hepatic transport of phenolphthalein glucuronide and chlorothiazide. Drug Metabol. Disp. 4, 107-l 11.