TOXICOLOGY AND APPLIED PHARMACOLOGY 58, 510-519 (1981)

The Effect of Diethylstilbestrol Treatment on the Metabolism and Biliary Excretion of [14C]Phenytoin in the

Isolated Perfused Rat Liver1s2

CHRISTOPHER MONTGOMERY AND MARY VORE

Department of Pharmacology, College of Medicine, and the Graduate Center for Toxicology University of Kentucky, Lexington, Kentucky 40536

Received October 20, 1980; accepted December 30, 1980

The Effect of Diethylstilbestrol Treatment on the Metabolism and Biliary Excretion of [Y]Phenytoin in the Isolated Perfused Rat Liver. MONTGOMERY, C., AND VORE, M. (1981). Toxicol. Appl. Pharmacol. 58, 510-519. The metabolism and biliary excretion of [‘4C]phe- nytoin (DPH) were examined in isolated perfused livers taken from Sprague-Dawley rats pretreated with 0.01,0.05,0.1,0.5, and 1.0 mgiday diethylstilbestrol (DES) SC for 6 days. No difference was seen in the rate of disappearance of DPH from the perfusate or in the per- fusate levels of its hydroxylated metabolite, 5-phenyl-5-para-hydroxyphenylhydantoin (HPPH). The biliary excretion of HPPH-glucuronide, however, was significantly depressed in livers from DES-treated rats and resulted in a significant increase in the amount of HPPH- glucuronide appearing in the perfusate. A linear relationship existed between the percentage decrease in biliary excretion of HPPH-glucuronide and the log of the pretreatment dose of DES. Bile flow was significantly depressed at all pretreatment doses of DES such that bile flow was 53.7 and 10.9% of bile flow in controls after 0.01 and 1.0 mg/day DES, respectively. The low bile flow appeared to limit secretion of HPPH-glucuronide in the bile since the maxi- mal concentration of HPPH-glucuronide in bile was greater in livers from DES-treated rats than controls and no significant differences were found in the maximal bile/perfusate con- centration ratios of HPPH-glucuronide.

Diethylstilbestrol (DES) is a potent syn- thetic estrogen that has been used in live- stock as a feed additive and therapeutically for estrogen replacement. Its use has been restricted in recent years because of vari- ous genitourinary abnormalities arising in humans following in utero exposure.

A few reports have appeared indicating that chronic DES treatment induces hepatic

i This work was supported by BRSG RR 05374, Public Health Service Grant HD 13250, and Grant 1 P30 CA 23154 from NIH to the Ephraim McDowell Community Cancer Network.

* Preliminary reports of this work were presented at the Society of Toxicology Meetings in March 1980 (Abstract No. 39).

dysfunction. Treatment of male mice with 1 mg DES/day SC for 13 days resulted in 33% mortality and severe hepatotoxicity along with an increase in liver size in all surviving mice (Selye, 1939). When patients with a history of jaundice following estrogen ther- apy were given 1 mg DES/day for several months, the jaundice in three out of four pa- tients increased significantly (Beam er al., 1956). Gallagher et al. (1966) demonstrated that treatment of rats with natural estrogens or the synthetic estrogen, DES, increased retention of sulfobromophthalein (BSP), a dye used as an indicator of liver function due to its quantitative elimination in bile. Treatment with DES has also been shown

0041-008X/81/060510-10$02.00/0 510 Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

DES EFFECTS ON PHENYTOIN ELIMINATION 511

to inhibit mixed-function oxidase and other oxidoreductase enzymes in hepatic micro- somes (Fahim et al., 197 1; Einarsson et al.,

1975). We have demonstrated that pregnancy, a

physiological state in which estrogen levels are increased, or chronic estrogen treat- ment, had no effect on the hydroxylation of phenytoin (DPH) to 5-phenyl-Qaru-hy- droxyphenylhydantoin (HPPH) or on the subsequent conjugation of HPPH to glu- curonic acid, but markedly inhibited the bil- iary excretion of HPPH-glucuronide (Vore et al., 1978, 1979; Vore and Montgomery, 1980). These studies indicated that in- creased estrogen levels could lead to reten- tion of glucuronide conjugates of drugs.

Until its recent ban, several thousand pounds of DES were used annually in live- stock. Results of environmental impact studies on DES have been inadequate to date but suggest the persistence of DES in the environment (Zondek and Sulman, 1943; Gregers-Hansen, 1964). Because of the use of DES clinically, the indication that it induced hepatic dysfunction and its presence in the environment, the following studies were designed to characterize the effects of chronic DES treatment on he- patic excretory function. DPH was chosen as a model compound because its elimina- tion involves hydroxylation by the mixed- function oxidase system, glucuronidation by glucuronyltransferase and biliary excre- tion.

METHODS

Female Sprague-Dawley rats (Harlan Industries, Indianapolis, Ind.) weighing 200 ?Z 20 g were pre- treated with DES for 6 days SC using 0.2 ml/dose pro- pylene glycol as the vehicle. Rats were pretreated with either 0.01, 0.05, 0.1, 0.5, or 1.0 mg/day DES corre- sponding to approximately 0.05, 0.25, 0.52, 2.40, or 4.95 mglkglday. Weight loss (6.4 f 3.1% of initial body weight; p > 0.05) was observed only in rats treated with 1 mg/day DES; the milligram per kilogram dose in this group was calculated by averaging the milligram per kilogram dose on the first and last day of treat-

ment. Three to five rats were studied at each dose. Nonpretreated rats (n = 3) and rats pretreated with propylene glycol vehicle (0.2 ml/day SC for 6 days, n = 4) were found to have no difference in any of the pa- rameters examined by Student’s t test. These two groups were therefore pooled (n = 7) and used as con- trols. All data were analyzed for significant differences using Student’s t test.

Twenty-four hours after the last pretreatment dose, livers were surgically removed and perfused via the portal vein with 80 ml of perfusate at an approximate rate of 2 ml/min/g liver. Perfusate contained 20% male rat donor blood in Krebs-Ringer bicarbonate buffer with 1% (w/v) glucose. Pertusate was oxygenated with 95% O*-5% CO:, while passing through 14 fi of Silastic medical-grade tubing (0.058 in. i.d., 0.77 in. o.d., Dow Coming, Midland, Mich.) in a separate lung apparatus similar to that described by Hamilton et al. (1974). Per- fusions were performed in a Plexiglas box maintained at 37°C.

Liver and perfusate were equilibrated for 30 min be- fore adding 1.6 &i of [14C]DPH (4 &Umg, l&i/~mol) to the perfusate to give an initial concen- tration of 5 &ml (20 PM). One-milliliter perfusate samples were taken at 0, 10, 30, 50, 70, and 90 min. After the IO-min sample collection, 1 ml of 1 M

NaHCOs was added to the perfusate to maintain the pH at 7.4. One milliliter of fresh perfusate was added to the reservoir after each of the other sample collec- tions to maintain perfusate volume. Bile was collected in tared scintillation vials via a PE-10 (Clay Adams, Parsippany, N.J.) cam&a placed in the bile duct, and bile volume was determined gravimetrically.

The levels of [14C]DPH, [“C]HPPH, and [Y]HPPH-glucuronide in the perfusate were deter- mined according to the method of Vore et al. (1978). Bile samples were incubated with 30% H,Oz for 1 hr at 50°C before addition of scintillation cocktail and quan- titation for the conjugate of [‘*C]HPPH. Efficiencies were determined using internal standard ([14C]to- luene). No attempt was made to quantitate the forma- tion of S-(3,4-dihydroxy-1,5-cyclohexadien-l-yl)-5- phenylhydantoin dihydrodiol. The dihydrodiol has been shown to account for less than 10% of the total metabolites appearing in the bile (Gerber et al., 1971).

At the end of the perfusion period, livers were weighed and stored at - 20°C. At the time of analysis they were thawed, homogenized in a total volume of 50 ml of 0.1 M phosphate buffer (pH 6.8), and a l-ml aliquot was analyzed for DPH, HPPH, and HPPH-glu- curonide in exactly the same manner as the perfusate samples (Vore et a/., 1978). Total recovery of radioac- tivity in bile, perfusate, and liver for all experiments was 88.3 2 0.8%.

Additional experiments were performed to deter- mine whether the nature of the metabolites of DPH ap- pearing in the bile or perfusate were altered by DES treatment. Two female rats were used; one pretreated

512 MONTGOMERY AND VORE

for 6 days with 1 mg DES/day, and one control. Bile was collected in a single tared tube for 90 min and a portion analyzed by thin-layer chromatography (tic) in a chloroform:methanol:acetic acid (70:29.5:0.5) sol- vent system. Authentic DPH and HPPH were used as reference standards. In the control liver, 37% of [l*C]DPH added to the perfusate appeared in the bile as the [14C]HPPH conjugate while 0.1,0.3, and 0.8% of the dose were identified in bile as the dihydrodiol (I?, = 0.6; Gerber et al., 1971), HPPH, and DPH, respec- tively. In the DES-treated rat, 6.3% of the dose ap- peared in the bile as the [“C]HPPH conjugate while 0.02, 0.06, and 0.2% of the dose were identified as the dihydrodiol, HPPH, and DPH, respectively. A portion of each bile sample was then incubated with @-glu- curonidase (Type 1X; Sigma Chemical Co., St Louis, MO.), for 48 hr at 37°C and then extracted four times with 8 ml of diethylether (ether), which removed greater than 98% of all radioactivity. The pooled ether fractions were dried under Np, the residue was dis- solved in acetone, and a portion analyzed by tic. Greater than 96% of the radioactivity was identified as [14C]HPPH. Therefore, the metabolite in the bile of both the control and DES-treated rat was identified as the glucuronide conjugate of HPPH. In a separate ex- periment, 5 ml of perfusate was taken at the end of a liver perfusion (90 min) from a control rat and a rat pretreated with 1 mg DES/day for 6 days. The perfu- sate was extracted twice with ether and the ether ex- tracts discarded. The remaining aqueous layer was made 3 M phosphate (pH 6.8 at 0.1 M) by addition of equimolar amounts of KIHPOl and NaH,PO, and ex- tracted twice with 10 ml of acetone to remove 99% of the remaining radioactivity. The acetone extracts were pooled, dried under nitrogen, and incubated for 48 hr at 37°C with p-glucuronidase in 0.05 M phosphate buffer (pH 6.8). Two extractions with 4 ml of ether re- moved greater than 98% of the radioactivity from this aqueous layer. Ether extracts were pooled, dried under nitrogen, and dissolved in acetone. A portion was then analyzed by tic as described above. Greater than 97% of the radioactivity appearing on the chroma- togram was identified as [*4C]HPPH. Therefore, the polar metabolite in the perfusate of both the control and the DES-treated rat was identified as the glucuron- ide conjugate of HPPH.

Materials

Ready-Solv scintillation cocktail was obtained from Beckman (Fullerton, Calif.). DPH and HPPH (Gold Label, 99+% purity) were obtained from Aldrich Chemical Company, Inc. (Milwaukee, Wise.). [“C]DPH (48.5 &i/pmol) was obtained from New En- gland Nuclear Corporation (Boston, Mass.). The ra- dioactive purity (>98%) of the [‘*C]DPH was estab-

lished by thin-layer chromatography in two solvent

systems. The solvents used for extractions were l- chlorobutane (Burdick and Jackson, Muskegon, Mich.) and diethylether (Mallinckrodt Chemical Works, St. Louis, MO.).

RESULTS

Pretreatment of rats with DES increased liver weight, expressed as percentage body weight (mean + SE), from 3.1 +- 0.1 in con- trol rats to 3.4 * 0.2, 3.5 k 0.1, 4.3 -+ 0.1, 4.0 f 0.2, and 3.8 + 0.1 in rats treated with 0.01, 0.05, 0.1, 0.5, and 1.0 mg DES/day, respectively. These data are in agreement with previous studies which demonstrated that treatment with DES (Selye, 1939) or endogenous estrogens (Griffiths er al., 1941) increased liver weight.

The elimination of DPH from the perfus- ate in livers from control rats and from rats pretreated with DES is shown in Fig. 1A. Only three of the five DES pretreatment doses are shown in this and other figures for clarity. DES treatment had no effect on the concentration of DPH in the perfusate. The half-life of DPH (mean +. SE) in the perfus- ate was 16.2 + 6.2, 17.1 f 4.8, 28.7 + 7.0, 24.7 + 3.2, 18.0 k 3.5, and 13.8 + 2.3 min in control rats and rats treated with 0.01, 0.05,O. 1,0.5, and 1 .O mg DES/day, respec- tively, and were not significantly different, thus indicating an equal ability by all livers to remove DPH from the perfusate. The levels of HPPH in the perfusate (Fig. 1B) increased during the first 30 min followed by a slight decline (control and 0.01 mg/day DES) or leveling off (1.0 and 0.1 mg/day DES). There was no significant accumula- tion of HPPH in the perfusate with time in DES-treated rats even though the rate of elimination of HPPH appeared to be slower in rats treated with 0.1 and 1 .O mg DES/day and at one point the concentration of HPPH was significantly elevated.

The appearance of HPPH-glucuronide in the perfusate (A), and bile (B) and bile flow (C) are shown in Fig. 2. At the 0.1 and 1.0 mg/day DES pretreatment doses, a 2.5-fold

DES EFFECTS ON PHENYTOIN ELIMINATION 513

10.0-r A

0 ConIrol ta 8.mtng DOS

5.@- . 0.1 ” ” . 1.0 ” ”

?a

l.O- $$zj$

0.5 1 1 I 1 I 10 30 I8 70 80

TIME (mln)

FIG. 1. The concentration of DPH (A) and HPPH (B) in the pertkate of livers from control rats and livers from rats pretreated with DES (0.01, 0.1 and 1.0 mg/day) SC for 6 days. (*p < 0.05.)

higher concentration of HPPH-glucuronide was found in the perfusate (A) correspond- ing to a 2.7- to 4.7-fold lesser amount of HPPH-glucuronide appearing in the bile (B). At the lowest pretreatment dose (0.01 mg DES/day) the appearance of HPPH-glu- curonide in the perfusate and bile was not different from control. When the percent- age inhibition of cumulative biliary excre- tion of HPPH-glucuronide (from Fig. 2B) was plotted against the logarithm of the pre- treatment dose of DES, a linear relation- ship was obtained (Fig. 3). These data dem- onstrate a dose-dependent inhibition by DES pretreatment on biliary excretion of HPPH-glucuronide .

Bile flow was significantly decreased at

all pretreatment doses such that there was a 46.3 and 89.1% decrease in bile flow relative to control after 0.01 and 1.0 mg/day DES, respectively. At the highest pretreatment dose (1.0 mg DES/day), three out of seven rats were eliminated from the study due to complete bile stasis.

The concentration of HPPH-glucuronide in the bile was significantly lower at 10 min in rats pretreated with 0.1 and 1.0 mg DES/day (Fig. 4), however, at later time pe- riods, the concentration of the glucuronide in bile was significantly increased in DES- treated rats. Calculation of the bile/perfus- ate concentration ratio of HPPH-glucuro- nide provides a measure of the ability of the liver to excrete this metabolite in bile

514 MONTGOMERY AND VORE

Control HPPH-G

OOO- B

ooo-

HPPH-G

C 1.4-

BILE FLOW

1.0. I T I T

IO io i0 io O-0 TIME (mln)

FIG. 2. The concentration of HPPH-glucuronide in the perfusate (A), cumulative excretion of HPPH-glucuronide in the bile (B), and bile flow in livers from control rats and livers from rats pre- treated with DES (C) at the indicated doses. (*p < 0.05, **p < 0.01.)

from control and DES-treated rats (data not shown).

against a concentration gradient. As shown in Fig. 4, only at the 10 and 30-min time pe- riods at the higher pretreatment doses of DES was there a significant decrease in bile/perfusate concentration ratios of HPPH-glucuronide. The maximal concen- tration ratios achieved at 30 min in control rats were not significantly different from those ratios achieved at 70 min in DES- treated rats (1.0 and 0.1 mg DES/day). Thus, although the appearance of the maxi- mum bile/perfusate concentration ratio was delayed in DES-treated rats, the maximum ability of the liver to concentrate HPPH- glucuronide in the bile was not impaired by DES treatment. Bile/liver concentration ratios of HPPH-glucuronide determined at 90 min did not differ significantly in livers

DISCUSSION

The metabolism of DPH to HPPH and of HPPH to HPPH-glucuronide are catalyzed by the microsomal mixed-function oxidase system and glucuronyltransferase, respec- tively. Treatment of animals with endoge- nous estrogens or the synthetic estrogen DES has been shown to inhibit mixed-func- tion oxidase and glucuronyltransferase ac- tivity when measured in microsomal prepa- rations (Fahim et al., 1971; Einarsson et al., 1975; Inscoe and Axelrod, 1960; Jori et al., 1969). This inhibition of enzyme activ-

DES EFFECTS ON PHENYTOIN ELIMINATION 515

loo-1

oo-

ZO-

1 I 1 1 f .o* .05 0.1 0.5 1.0 5 10

DOSE (mgf kg)

FIG. 3. The percentage inhibition of the cumulative excretion of HPPH-glucuronide in the bile as a function of the log of the pretreatment dose of DES in mg/kg. (r = 0.98; p < 0.01.)

ity has been attributed, at least in part, to the fact that estrogens and DES are also substrates of the mixed-function oxidase system (Kuntzman et af., 1964) and glu- curonyltransferase (Lucier and McDaniel, 1977) and may therefore compete for me- tabolism. Although kinetic studies were not done, addition of DES to microsomal incu- bations at concentrations of 0.5 and 5 mM inhibited the hydroxylation of DPH to HPPH by 63 and 95%, respectively (Kutt and Verebely, 1970). One of the questions addressed in the present studies was

WPPtl-G

whether chronic treatment with DES inhib- ited the metabolism of DPH to HPPH in the isolated perfused rat liver, a system which measures the metabolic capacity of the in- tact organ under near physiological condi- tions. Since the only route of elimination of DPH in the perfused liver is via hydroxyla- tion to HPPH, the rate of elimination, or the half-life of DPH in the perfusate, is directly proportional to its rate of metabo- lism. The data clearly indicate that chronic DES treatment, at doses of 0.01 to 1.0 mglday, had no significant effect on the me-

0 Control

a 0.01 mg De5

kg 0.1 ” ”

l 1.0 ” ”

-aalL 10 30 SO 70 so

FIG. 4. The concentration of HPPH-glucuronide in the bile of livers from control and DES-treated rats. (*p < 0.05, **p < 0.01.)

516 MONTGOMERY AND VORE

120 HPPH -G Cl Control

@ 0.01 rg DOS

I 0.1 ” ” l 1.0 ” ”

20 SO 70 00

TIME (mln)

FIG. 5. Bile to perfusate concentration ratios of HPPH-glucuronide in livers from control and DES- treated rats. (*p 2 0.05, **p < 0.01.)

tabolism of DPH to HPPH by the mixed- function oxidase system. Since there was no effect of DES treatment on the rate of formation of HPPH nor a significant accu- mulation of HPPH in the perfusate, and the only route of elimination of HPPH in the perfused liver is via metabolism to HPPH- glucuronide, it can be inferred that DES treatment had no profound effect on glu- curonyltransferase activity. However, since the rate-limiting step in the metabo- lism of DPH to HPPH-glucuronide is the hydroxylation to HPPH (Inaba and Umeda, 1975), it is likely that small changes in glu- curonyltransferase activity would go unde- tected in the present system.

Chronic treatment with DES or other es- trogens decreases hepatic excretory func- tion as measured by an increase in BSP re- tention (Gallagher et al., 1966; Sandberg et al., 1967; Kreek et al., 1969). Despopoulos (1970) reported irreversible decreases in bile flow and transient decreases in BSP bil- iary excretion acutely after addition of 18 pm01 (4.8 mg) of DES to the isolated per-

fused rat liver. In the present studies, chronic DES treatment significantly de- creased bile flow at all pretreatment doses with maximum inhibition, approximately %, seen at the highest treatment dose of 1.0 mg DES/day. Maximum inhibition, about 81%, of the cumulative biliary excre- tion of HPPH-glucuronide was also at- tained at this treatment dose. Because of the greater decrease in bile flow relative to biliary excretion of HPPH-glucuronide seen at all treatment doses of DES, the con- centration of HPPH-glucuronide in bile was higher in DES-treated rats than in controls, particularly at later time periods (Fig. 4). Consequently, there was no significant dif- ference in the maximum bileiperfusate con- centration ratio of HPPH-glucuronide in control vs DES-treated rats. The maximum values were delayed in DES-treated rats (70 min) vs controls (30 min) indicating a de- crease in the initial rates of excretion of HPPH-glucuronide into bile as is also clear from Fig. 2B. The bile/liver concentration ratios of HPPH-glucuronide at 90 min were

DES EFFECTS ON PHENYTOIN ELIMINATION 517

also not different in control and DES-treated animals.

The mechanism by which DES inhibited the excretion of HPPH-glucuronide into bile is not known. One possible explanation could be direct competition for biliary ex- cretion between HPPH-glucuronide and DES-glucuronide. DES is metabolized pri- marily to the monoglucuronide which is ex- creted extensively in the bile in the rat; 94% of an ip dose of [14C]DES was eliminated in the bile in 24 hr in bile-cannulated rats (Fischer et al., 1966; Clark et al., 1969). DES underwent extensive enterohepatic circulation so that in the intact rat 3 days were required for excretion of 73 and 5% of the dose in feces and urine, respectively (Fischer et al., 1966). The acute toxicity of DES was markedly increased in bile duct-li- gated rats, thus indicating the importance of a patent bile duct for its elimination (Klaassen, 1973). In order to determine if daily treatment of rats with DES, coupled with decreased biliary excretory function could lead to significant accumulation of DES, two rats were treated with 1 mg/day [14C]DES (3.73 pmol/&i) SC for 6 days. The livers were perfused in the usual fash- ion on Day 7 and total radioactivity in per- fusate, liver, and bile analyzed at 90 min. Less than 0.1% of the total dose of [14C]DES was excreted in bile and less than 1% of the total dose remained in the liver and perfusate. It is unlikely that this small amount (-250 nmol) of DES-related ma- terial remaining in the liver could competi- tively inhibit the biliary excretion of 1600 nmol of DPH, particularly since significant inhibition of biliary excretion was present at much lower pretreatment doses of DES.

The decrease in bile flow appears to be largely responsible for the decrease in bili- ary excretion of HPPH-glucuronide since neither the maximum concentration in bile nor the maximum bile/perfusate concentra- tion ratio were decreased by DES treat- ment. There was no dose of DES which in- hibited biliary excretion without inhibiting

bile flow. This is in contrast to the effect of pregnancy in which the biliary excretion of HPPH-glucuronide was inhibited to a much greater extent than was bile flow so that maximum bile/perfusate concentration ratios were decreased from 150 in livers from control rats to 6 in livers from rats at 21 days of gestation (Vore et al., 1978). Thus DES appears to decrease bile flow preferentially whereas in pregnancy, biliary excretion is preferentially inhibited.

The mechanism by which DES treatment decreases bile flow remains unclear, largely because the determinants of bile flow are not well understood. The formation of bile is believed to be due to the active transport of bile acids into the canaliculus with subse- quent osmotic flow of water and electro- lytes (bile acid-dependent flow) and the ac- tive transport of inorganic solutes (probably sodium ion via Na+, K+-ATPase) also with subsequent osmotic flow of water and other solutes (bile acid-independent flow) (Boyer and Klatskin, 1970; Erlinger and Dhumeaux, 1974). Estrogen treatment has been shown to inhibit Na+, K+-ATPase in canalicular-membrane-enriched fractions of liver and this decrease in enzyme activity is correlated with a decrease in bile acid-in- dependent flow (Gumucio and Valdivieso, 1971; Reichen and Paumgartner, 1977; Davis et al., 1978). The limited supply of bile acids available in the isolated perfused liver (Boyer and Klatskin, 1970; Boyer, 1971) along with inhibition of bile acid-inde- pendent flow may account for the profound inhibition of bile flow seen at all treatment doses of DES.

The data presented clearly indicate that in contrast to previous studies which exam- ined mixed-function oxidase activity in vitro, treatment with doses of DES as high as 1 mg/day did not alter DPH microsomal metabolism when determined in the per- fused liver preparation. These studies have also characterized DES as a potent choles- tatic agent in that even at the lowest dose studied (0.01 mg/day) a 50% reduction in

518 MONTGOMERY AND VORE

bile flow was obtained. The low but signifi- cant cholestatic doses of DES given to rats in the present studies (0.1-0.3 mg/kg; 0.3- 1.5 mg/m2) are similar to doses of DES given to humans (0.1-0.4 mg/kg; 5.2-13.3 mg/m2) for treatment of cancer (Noller and Fish, 1974) and may therefore result in in- creased retention of xenobiotics excreted in the bile in these patients.

In summary, the elimination of DPH by the liver involves (1) metabolism of DPH to HPPH and HPPH-glucuronide, (2) forma- tion of the bile, and (3) transport of the HPPH conjugate into the bile against a con- centration gradient. The data presented in- dicates that DES does not affect the metab- olism of DPH but inhibits the biliary excretion’ of HPPH-glucuronide primarily by interfering with the formation of bile al- though direct inhibition of the transport of the HPPH-conjugate into the bile may also be a contributory factor. Further studies are under way to delineate the mechanism by which DES decreases bile flow and in- hibits biliary excretion.

ACKNOWLEDGMENTS

We thank Ms. Diane Reed for her help in the prepa- ration of this manuscript.

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