Effects of phenobarbital on biliary excretion of organic ...Effects of phenobarbital on biliary...

AMERICAN JOURNAL OF PHYSIOLOGY VoI. 217, No. I, July 1969. Printed in

Effects of phenobarbital on biliary excretion of

organic acids in male and female rats

LARRY G. HART, ANTHONY M. GUARINO, AND RICHARD H. ADAMSON Laboratory of Chemical Pharmacology, fVational Cancer Institute, National Institutes of Heal& Bethesda, Maryland 20014

HART, LARRY G., ANTHONY M. GUARINO, AND RICHARD H, ADAMSON. Effects of phenobarbital on biliary excretion of organic acids 2*lz male and female rats. Am. J. Physiol. 2 17( 1) : 46-52. 1969.-The effects of phenobarbital pretreatment on the biliary excretion of four organic anions, indocyanine green (ICG), chlorothiazide (CTZ), phenol red, and probenecid, were investigated. Indo- cyanine green and CT2 are not metabolized in the rat, whereas phenol red and probenecid are excreted into the bile largely as glucuronides. Phenobarbital-pretreated male rats excreted more ICG and probenecid, but not more phenol red or CTZ, than controls. Females pretreated with phenobarbital excreted more CT2 and probenecid, but not greater amounts of ICG or phenol red. In the induced animals the glucuronides of phenol red and probenecid were not excreted in greater amounts than in control animals. Where increased biliary excretion of these compounds were observed, it was concluded to be due primarily to the increased bile flow in phenobarbital-treated animals.

indocyanine green; chlorothiazide; phenol red; probenecid differences; phenobarbital induction ; glucuronide excretion

; sex

A VARIETY OF DRUGS and other foreign compounds are elim- inated from the circulation entirely or in part by excretion into the bile (4, 30, 35, 36). Many of these same compounds are first metabolized and/or conjugated by the liver, with glucuronide conjugation usually the most important in biliary excretion (35).

There have been numerous studies on the effects of drugs as inducers of the hepatic microsomal drug-metabolizing enzymes and these have been currently reviewed (8). How- ever, relatively few reports have appeared on the effects of these enzyme inducers on the hepatic excretory pathways. Schellhas et al. (31) have reported that phenobarbital- treated rats excreted twice as much sulfobromophthalein (BSP) in the bile as did controls, and this effect was corre- lated with a marked enhancement of BSP conjugation in vitro. Fujimoto et al. (10) observed more rapid disappearance of BSP from plasma in mice pretreated with phenobarbital. The enhanced plasma clearance was reversed by simul- taneous administration of ethionine suggesting that adaptive effects on the conjugating enzyme were involved in the enhanced clearance. Roberts and Plaa (29) demonstrated a significantly enhanced disappearance of bilirubin from plasma of phenobarbital-treated mice, and an increase in the maximal rate of bilirubin excretion in rats. However, in the induced rats there was no change in bilirubin concentra-

tion in the bile, due to a concomitant increase in bile volume. Thus, the increase in bilirubin excretion appeared to be related primarily to the increase in bile volume although increases in uptake and conjugating capacity might also have been involved.

The purpose of the present report was to investigate the effects of phenobarbital pretreatment on the hepatic excretory patterns of four foreign organic acids or anions which are known to be actively transported into the bile. Both bilirubin and BSP are organic anions, as are the principal endogenous components of bile, the bile acids (7, 36). Two of the acids in the present study, chlorothiazide (15) and indocyanine green (ICG) (6), are taken up and excreted by the liver in rats as the unchanged compound. The other two compounds, phenolsulfonphthalein (phenol red) and probenecid, are extensively conjugated to glucuronides prior to excretion into the bile (13, 16). It has been reported that barbiturate pretreatment increases both the hepatic glucuronide conjugation of bilirubin in mice and rats (5, 9), and the glucuronylating activity for p-nitrophenol in rats

(42) Ai a second phase of this project the hepatic excretory pat-

terns in female rats were compared with those of males. Among the usual laboratory species, rats most characteristically exhibit marked sex differences in hepatic drug metabolism (I 1).

METHODS

Male Sprague-Dawley rats (NIH Colony), weighing 250- 350 g, and females, weighing 220-275 g, were used and were maintained on laboratory chow and tap water. Phenobar- bital sodium, 40 mg/kg, was injected intraperitoneally twice daily for 3 days. Control animals received identical volumes of saline (2 ml/kg). A nimals were used about 18 hr after the final injection. Rats were anesthetized with barbital, 250 mg/kg, followed in 15-30 min by pentobarbital 30-45 mg/kg; both drugs were given intraperitoneally. Through a midline incision, the renal pedicles were ligated and the com- mon bile duct was cannulated with a short-beveled 23-gauge hypodermic needle shaft attached to 10 inches of polyethyl- ene tubing. Unless otherwise indicated, bile was collected for 30 min prior to drug administration, followed by three 30-min collection periods. Animals were kept under a warm- ing lamp for the duration of the experiment, and it was observed that the presence of such a lamp prevented any significant decreases in bile flow. A blood sample was then

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PHENOBARBITAL AND SEX DIFFERENCES IN BILE EXCRETION 47

drawn by cardiac puncture and the liver was excised, blotted free of blood, and weighed. Drugs were dissolved in water or saline and were administered intravenously (femoral vein).

Indocyanine green in plasma and bile was diluted with water and assayed by measuring its absorption at 805 rnp as described by Wheeler et al. (39). Hepatic content of ICG was estimated as follows: Liver was homogenized with a volume, in milliliters, of distilled water equal to the wet weight, in grams, of the liver. To 1 ml of the resulting 50 % homogenate in a 13-ml stoppered centrifuge tube 9 ml absolute methanol were added. The contents were mixed well by inversion and the tubes were centrifuged 15 min at 4 C. An aliquot of the clear supernatant was read at 790 rnp against appropriate tissue and reagent blanks. Under the conditions of this assay, ICG is very stable, the recovery of added dye is quantitative, and the tissue blanks are neg- ligible. Chlorothiazide was estimated calorimetrically in 4 % p-toluenesulfonic acid filtrates of bile and plasma after al- kaline hydrolysis in a boiling water bath for 30 min as described by Baer et al. (1). Phenol red and its glucuronide in bile were measured as previously described (16) as were the protein-free filtrates of plasma prepared by the method of Marshall and Vickers (24). Probenecid and its glucuronide were measured by the radioassay described previously (13).

Statistical comparisons were made with the Student f test (P < 0.05 for rejection of the null hypothesis).

Indocyanine green (Cardio-Green) was generously sup- plied by Hynson, Westcott & Dunning, Inc., Baltimore, Md., while grateful acknowledgment for probenecid-14C (ring labeled) (SA 3. I &mg), unlabeled probenecid, and chlorothiazide (Diuril) is made to the Merck Institute for Therapeutic Research, West Point, Pa. Phenol red was pur- chased in its water-soluble form from the Hartman-Led- don Co., Philadelphia, Pa.

RESULTS

Indocyanine green excretion. It can be seen in Table 1 that there was a significant increase (P < O*Ol) of about 60 % over control in total 90-min biliary excretion by male rats pretreated with phenobarbital for 3 days (65 vs. 41 %). Like- wise, the pattern of excretion differed between phenobarbital-treated and control animals in that peak biliary excretion occurred during the first 30-min period for the former, while in the controls absolute excretion rose and fell more

gradually over the course of the experimental period. A possible explanation for the increased biliary clearance

of ICG may be provided by the effects of phenobarbital on bile flow as also seen in Table 1. There was a significantly greater (P < 0.01) average bile flow in the phenobarbital- treated animals at all time periods. (Bile flow rates were about 50 % greater (significant at P < 0.05) in all phenobarbital-pretreated rats, male and female, with the singIe exception of some time periods of the female rats which received ICG (Table 1); such differences may be cited in the text, but are actually enumerated only in Tables 1 and 9.) The bile Aow was greater even before ICG was injected. Cal- culation of the average percent increase in bile flow produced by phenobarbital gives a range of from 52 to 60 % for the three 30-min time periods and this increase corre- sponds with the overall increase over control excretion of

TABLE 1. Efects of phenobarbital on bile juza, and excrelion of inducyanine green in male and female rats

Time, min

-30-O

O-30

30-60

60-90

-30-O

O-30

30-60

60-90

Sal Pb

Sal Pb

Sal Pb

Sal Pb

Sal Pb

Sal Pb

Sal Pb

Sal Pb

152*10* 219zt 16

154&12* 234& 12

148zt13* 231ztx3

146& 15* 233+ 16

131*15* 189zt 14$

16511~5 189% 14

177+7 199&l 1

174&l 1 zoo*9

14&l* 31&3

16&l* 23&l

11-41 41.0+2.2* 12&l 65.4&2.5

55+3t 52+4$

25&l? Z&t2

7zt1t 86.8+2.2t 7&Q 82.4&l .2§

Rats were pretreated with phenobarbital (Pb) 40 mg/kg ip, twice daily for 3 days, or with equivalent volumes of saline (Sal). On the 4th day, animals were anesthetized, bile ducts cannulated, and renal pedicIes were ligated. Indocyanine green, 1 mg/kg, was given intravenously after collecting bile for a control (-30- to 0-min) interval and bile was then collected for three 30-min periods. Results are expressed as mean + SE for 3-6 animals. * P (sal vs. Pb) < 0.05. t p (&al vs. &al) < 0.05. 8 p (Fpb vs. Mph) < 0.05.

ICG. Roberts and Plaa (29) observed parallel increases in bile flow and maximal excretion of bilirubin after phenobarbital pretreatment.

Moreover, in their experiments there were no significant differences in bile bilirubin concentrations. It should be noted, that the single injections of ICG which were used in our experiments were not Tm doses, while Roberts and Plaa (29) employed a constant infusion of bilirubin at a Tm dose. In the present report the average bile concentration was higher during the first 30 min in the phenobarbital-treated group (133 vs. 93 pg/ml; P < 0.05) with the reverse being true during the 30- to 60-min period (100 vs. 119 pg/ml; P > 0.5) and 60- to 90-min period (51 vs. 78 pg/ml; P < 0.05). (B 1 i e concentrations (pg/ml) of the various drugs studied are cited in the text where deemed necessary but to conserve space are not listed among the tabular data.) These trends in the biliary excretion profile may be misleading if it is not understood that in the phenobarbital-treated animals the greatest portion of the ICG dose was excreted during the 0- to 30-min period. Hence, when given at this dose, the excretion of ICG during the next two 30-min periods may have fallen off because the load presented to the liver was decreasing.

Table 1 also gives data on the biliary excretion of ICQ in male and female rats and biliary flow in female rats


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48 HART, GUARINO, AND ADAMSQN

treated with KG. The saline-treated females excreted the drug more rapidly than did either phenobarbital-treated or control male rats, and the pattern of excretion showed an even sharper decline than was seen in male rats (Table 1). There was no apparent enhancement of ICG excretion in female rats following phenobarbital treatment. The phenobarbital-treated and control females excreted from 82 to 87 % of the injected dose in 90 min as compared with 65 % in phenobarbital-treated and 41 % in control male rats. Ap- parently there is a choleretic response to ICG in females, but not in males at this dose.

TABLE 3. Efect of phenobarbital pretreatment on bihry excredion of bhenol red and its glucuronide in male rats

J A - - I

Time After Pretreat- Injection, min ment

Excretion as y0 of Injected Dose

Phenol red Glucuronide

Excretion of Conjugate as y0

of Total Colored Material Excreted

O-30 Sal Pb

31.4~112.2 39.2A3.4

11.4+1.3 lL4&1.8

26.8zt3.5 22.8+3.7

30-60 Sal Pb

16.4zkO.8 14.8H.5

7.7~111.2 6.4hO.8

31.7It4.4 29.8+3.5

ChZorothia& excretion. It can be noted (Table 2) that the absolute excretion per 30 min of chlorothiazide (in control males) was greater than in phenobarbital-treated males during all three collection periods. The average bile concentrations in the controls were also markedly greater (P < 0.01) at each 30-min interval. Bile volumes were again greater in the phenobarbital-treated animals.

60-90 Sal Pb

8.3M.4 7.4zto.4

4.6ztO.5 3.6hO.5

35.4It3.4 32.6k3.9

Total Sal Pb

56,2 61.4

23.7 21.3

-

In Table 2 it also can be observed that phenobarbital treatment of female rats resulted in nearly a twofold increase over controls in both absolute excretion of chlorothiazide at each 30-min interval and in total percent of the dose excreted in 90 min. Excretion of the drug was also significantly less in the control female than in either control or phenobarbital-treated male rats. In the female rats treated with phenobarbital the greater excretion appeared to be related directly to increased bile flow, about 50 % above control, since there were no significant differences in biliary concentration of chlorothiazide.

See footnotes for Table 1. Phenol red, 20 mg/kg, was gfven intravenously, and bile was collected for three 3O-min periods. Results are expressed as mean + SE for 4 animals.

TABLE 4. Efect of phenobarbital pretreatment on bihary excretion of phenol red and its ghuronide in female ruts

Excretion of Conjugate as v0

of Total Colored Material Excreted

14.2~12.3 11.5zt2.7

Phenol red excretion. These experiments failed to indicate any significant effects of phenobarbital on either total biliary excretion or excretion of phenol red as its glucuronide in male rats (Table 3). The percent of the injected dose excreted as conjugate in the phenobarbital-treated rats was somewhat lessduringthe 30- to 60- and 60- to 90-min periods than in the controls, although these differences were not significant. At all times, the bile concentrations (pg/ml) were considerably lower in the phenobarbital-treated animals.

Excretion as y0 of Injected Dose Time After Pretreat-

Injection, min ment Phenol red Glucuronide

O-30 Sal 36.4+2.3 6.0&l .2 Pb 44.2+3.4 5.7&l .4

30-60 Sal 18.3+0.7” 5.OhO.2 Pb 16.lztO.5 3.9zto.7

60-90 Sal 10.2+0.4* 2.9ztO. 1 Pb 8. ho.3 2.2zkO.4

Total

21.4zt1.2 19.2+3.0

22.2~1~0.8 20.6zt2.7

Sal Pb

64.8 68.3

13,Y 11.8

See footnotes for Table 3* Results are expressed as mean & SE

for 4-5 animals. *P (Sal vs, Pb) < 0.05.

The effects of phenobarbital on the biliary excretion of phenol red and its glucuronide in females can be seen in Table 4m The percent of the dose excreted as the parent compound was significantly greater for the controls in both the 30- to 60- and60- to 90-min periods. Neither the total amount of the dose excreted nor the proportion excreted as the glucuronide was altered by phenobarbital treatment. There

were, however, significant differences between the sexes with regard to the percent of the dose excreted as the conjugate (Table 5). Phenobarbital-treated and control female rats excreted only about 55 % as much as the glucuronide as did the males.

TABLE 2. Efects of phenobarbitu/ on bihry excretion of chlorothiasfde in male und female rats

One possible reason for the apparent lack of phenobarbital effects on phenol red excretion might be that the glucuronyl transferase which conjugates phenol red may be less sensitive to phenobarbital induction than are the microsomal oxidative enzymes.

Probenecid excretion. Probenecid has been shown to inhibit the hepatic excretion of carboxylic and sulfonic acids (15, 23, 37). Like phenol red, probenecid is itself excreted primarily as a glucuronide conjugate (13). Unlike phenol red, however, phenobarbital pretreatment of male rats resulted in a marked enhancement of the biliary excretion of probenecid (Table 6). Th e excretion of unchanged probenecid and conjugate were increased about equally with the net effect being that the percentage of the excreted radioactivity in the form of the conjugate remained essentially the same, Bile concentrations of total drug were significantly different only during the 60- to 90-min period, with the bile from

Absolute Excretion, &lo0 g per 30 min Cumulative 7C of Dose Excreted

6&90 in 90 min

-~

Sex Pretreat- ment

M Sal 199+ 16* 147+9*t 26.0&1.6* Pb 182*22* 105*9* 20.7&Z .2*

F Sal 112+12 13.8zt1.3 Pb 213xW 24.1*1.9*

- See footnotes for Table 1. Chlorothiazide, 20 mg/kg, was given

intravenously over a I-min period and bile was collected for three 30-min periods. Results are expressed as mean & SE for 6-7 animals. * P (Sal-F vs. Pb-F, Sal-M, Pb-M) < 0.05. t P (Sal-M vs. Pb-M) < 0.05.


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TAmx 5. Sex di$erence in biliary excretion of indocyanine green. In the current study, the biliary excretion phenol red glucuronide of indocyanine green (ICG) in male rats was markedly en-

t hanced by phenobarbital pretreatment, while that of chloro- Cumulative TO of Dose Excreted in 90 min thiazide was not, although both are anions at physiological

Pretreatment

Sal

Pb

Sex

M F

M F

Phenol red Glucuronide

I 56.2zt3.2 j 23.7&3.0* 64-.8+2.8 i 13*9&l l 3 b

I 61 l 4zt3.6 1 21.3+3.0* 68.3+3.5 1 11.8jZ.3

pH and are excreted unchanged, presumably by the hepatic process for organic acids (15, 36). In male rats with ligated renal pedicles, chlorothiazide, 50 mg/kg (168 pmoles/kg), was injected into a femoral vein followed 10 min later by injection of KG, 1 mg/kg (1.3 pmoles/kg), in the other femoral vein. As can be seen in Table 8, a 130-fold greater molar dose of chlorothiazide did not inhibit bile secretion

See footnotes for Table 4. * P (M vs. F) < 0.05. of ICG, but rather enhanced it since 68 % of the dose of ICG was excreted in 90 min vs. 48 % excreted in the saline- treated controls. An explanation for these paradoxical results may be provided by the data on bile flow rates (Table 8). At the dose used, chlorothiazide produced a marked increase in bile flow which lasted throughout the experimental period. This choleretic effect of the drug has been reported (15). It is of interest to compare Table 8 with Table 1 wherein it can be noted that the effects seen on total ICG excretion, on the pattern of ICG excretion, and on the magnitude of increase in bile flow were very similar. It might be reasonably concluded from these parallel effects

TABLE 6. Effect of phenobarbital pretreatment on biliary excretion of probenecid and its glucuronide in male rats ___

Excretion of Conjugate as yO of

Total Radioactwity Excreted

Excretion as yO of Injected Dose Time After Injection,

min

Pretreat- ment

Probenecid Glucuronide

Sal 2.7ztO.6” 5.4ztO.8* 67.4+1.8 Pb 5.5ztO.2 8.6hO.7 60.9X2.2

Sal 3.6&O-7* 7.2ztl l 5 66.7h3.3 Pb 5.8+0.3 10.7zto.7 64.7zt2.3

Sal 3.7zto.4 8.5ztO.5 69.7zt2.2 Pb 5.lzfzO.3 8.8ztO.4 63.5zt1.4

Sal 10.0+1.7* 21.1h2.8

Pb 16.4&0.7 28.1zU.6

O-30

30-60

60-90

Total

TABLE 7. Efect of phenobarbital @treatment on biliary excretion of probenecid and its glucuronide in female rats

Excretion as T ; of Injected Dose Excretion of Conjugate as s of

Total Radioactivity Excreted

61.5+1.8 61.2+3.1

62.7+X .6 66.3zt2.5

60.2&l 2 63.6zt2.2

Time-After Injection,

min

Pretreat- ment

Glucuronide Probenecid

2.8&O-3* 5.3zto.5

4.0+0.2* 6.1ztO.5

4.4*0.4* 5.8zt0.3

See footnotes for Table 1. ProbenecidJ4C, 50 mg/kg, was in- O-30 4.6&O-8* 8.3+0.2

6.9+0.9* 11.8ztO.4

6.5&0.4* 10.1+0.4

18,0+2 .O*

30.2ztO.9

Sal Pb

Sal I’b

Sal Pb

Sal

Pb

jetted iv, Results are expressed as mean & SE for 3 animals. (Sal vs. Pb) < 0+05.

* P

30-60

control animals having a greater concentration of drug (3.09 vs. 2.31 mg/ml). As before, the phenobarbital-treated rats had a significantly higher flow of bile than did controls, even before injection of probenecid. Furthermore, the choleretic effect of probenecid was seen about equally in both groups. Thus, as with the nonmetabolized drug ICG, the increased excretion of probenecid in phenobarbital-

60-90

Total

treated rats appears to De We primarily to an increase in See footnote for Table 1. * P (Sal vs. Pb) < 0.05. bile volume, rather than to an increase in metabolism.

There did not appear to be any detectable sex differences in the Dattern of excretion for probenecid and its metabolite TABLE 8. Effect of chhrothaside on bihary excretion of J . . . . 1 .

Cumulative 70 of Dose Ex-

creted in 90 min

48.0 68.3

(Table’ 7). Bile fl ow rates an; choleretic response to pro- zndocyanzne green zn male rats -i benecid in phenobarbital-treated and control female . rats

Bile Flow, pliters/lOO

g per 30 min

-

Pretreatment

Sal 157 Chloro 228

Sal 169 Chloro 243

Sal 161 Chloro 237

Absolute Excretion, pg/lOO

g per 30 min

18 36

20 22

10 10

were comparable to the same parameters in males. As in males, the excretion of glucuronide conjugate as a percentage of the total radioactivity excreted in the bile was not altered by phenobarbital pretreatment. Comparing Table 6 with Table 7, there does seem to be a more pronounced effect of phenobarbital in female rats on the percentage of the dose of probenecid excreted, particularly during the 30- to 60- and 60- to 90-min periods. Concentrations of total drug in bile were not different at any time period,

Time, min

O-30

30-60

60-90

while bile flow was increased by about 50 % in phenobar- I

bital-treated female rats. Thus, the marked &crease in Rats with ligated renal pedicles received chlorothiazide (chloro),

probenecid excretion in female rats also appears to be due 50 mg/kg, intravenously over a 2-min period. Controls received

to increased bile flow. an equivalent volume of saline. Indocyanine green, 1 mg/kg, was

E$ects 03’ chlorothia~ide administration on biliary excretion of injected 10 min later and bile was then collected for three 30-min periods. Results are expressed as the mean for 2 animals.


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50

Time, min Pretreat- ment

Plasma. Con;;/~ltlon,

Liver Concentration,

&ml

Absolute Excretion, .pg/kg

per m m

Sal Pb

Sal Pb

o-5 1.2 23.5 1 .o 1.1 17.6 1.8

O-10 0.7 22.0 4.3 0.5 16.5 10.1

o-15 0.5 19*4 9.9 0.5 15.0 12 .o

O-30 15.2 12.8 13.9 13.1

Sal Pb

Sal Pb

0.5 0.6

TABLE 9. Effects of phenobarbital on plasma disappearance, heputic concentration, and bile excretion of indocyunine green at early times after administration to mule ruts

of phenobarbital pretreatment and chlorothiazide administration that the enhanced excretion of ICG in both cases was due largely to the increases in bile volume. Attempts to study the reverse effect, i.e., prior administration of ICG on chlorothiazide excretion, were inconclusive due to the large blanks in bile and plasma from the intense color of ICG.

Efects of phenobarbital treatment on @mu disa~~earunce, hepatic uptake, and bile excretion of indocyanine green at early times after injection. In an attempt to further elucidate the mechanism of action of phenobarbital on ICG excretion, different groups of rats were injected with ICG as before, bile samples were collected for 5, 10, 15, and 30 min, and animals were sacrificed at the end of each of these times and liver and plasma samples were removed for assay as described in METHODS. Analysis of the data in Table 9 shows sharp differences at the early times (5, 10, 15 min) in the parameters examined and little difference between treated and controls by 30 min after ICG administration. After 10 min, in the phenobarbital-treated rats, total bile excretion was 2 to 2.5fold greater, plasma levels were lower, and bile-to- plasma concentration ratios were correspondingly greater. Conversely, liver levels (pg/g) were about 33 % lower in the phenobarbital-treated animals at 5, 10, and 15 min. These results suggest that the phenobarbital effect is exerted primarily on the excretory phase although a somewhat more rapid uptake and/or plasma disappearance may also be in .volved. The decrease in hepatic concentration mili- tates against a possible enhancement of storage by pheno- barbi tal. Klaassen and Plaa (22) reported an enhanced plasma disappearance of ICG in phenobarbital-treated rats.

DISCUSSION

Pretreatment of rats with phenobarbital resulted in significant increases in the biliary excretion of indocyanine green in male rats, chlorothiazide in females, and probenecid in both sexes. There was no enhancement of the excretion of phenol red. Recently, Plaa and co-workers (22, 29) have reported enhanced plasma disappearance and excretion, in terms of increased apparent transport maximums (Tms),

HART, GUARINO, AND ADAMSON

for BSP, bilirubin, and phenol-3,6-dibromphthalein disul- fonate (DBSP).

Our results do not lend themselves to any simple inter- pretation. Phenol red and probenecid are biotransformed to glucuronides, yet the biliary excretion of the former was not altered by phenobarbital, while probenecid excretion was increased in both sexes. With probenecid, there were increases in the percentage of the dose excreted as the conjugate. However, there were parallel increases in excretion of the aglycone, such that the proportion of the total material excreted in the bile as the conjugate did not change after phenobarbital treatment. These data suggest that the effects of the inducer per se were not directly on the conjugating enzyme but were nonspecific perhaps allowing more rapid availability of substrate or more rapid removal of the glucuronide product into the bile. The glucuronyl transferases are apparently less sensitive to phenobarbital induction than are microsomal oxidases (42). DeLeon et al. (9) reported that phenobarbital treatment for 4 days did not increase hepatic glucuronyl transferase activity with bilirubin or O-aminophenol as glucuronide acceptors, while 14- day pretreatment did significantly increase formation of bilirubin glucuronide. A 3- to 4-day pretreatment with phenobarbital is optimal for induction of most hepatic drug metabolizing enzymes (8). Furthermore, both phenol red and probenecid form O-glucuronides, and Remmer (27) observed increased urinary excretion of N-glucuronides but not of O-glucuronides after treatment with phenobarbital.

Liver microsomes of adult male rats characteristically me- tabolize drugs at a greater rate than do microsomes from females (11). Inscoe and Axelrod (18) showed that in vitro glucuronyl transferase activity for O-aminophenol glucuroni- dation was about 4 times as great in liver microsomes from adult male rats as in microsomes from females. On the other hand, transferase activity in females was much more respon- sive to benzpyrene induction, with increases in activity of more than 250 % occurring after benzpyrene compared with only a 40 % increase in males. In our experiments, the percent of the dose of phenol red in females that was excreted as the glucuronide was only, 50-60 % that in males, but phenobarbital pretreatment was ineffective in either sex in increasing the proportion excreted as conjugate. There were no differences between the sexes for excretion of probenecid. Most of the cited references on the effects of inducers on glucuronyl transferases have been in vitro studies and not directly comparable with the present in vivo results. Re- cently, however, Goldstein and Taurog (12) reported markedly enhanced biliary excretion of thyroxine in both female and male rats after a single pretreatment with 3,4- benzpyrene. There was no increase in bile flow and their data indicated that the increases in excretion were due to increases in hepatic glucuronyl transferase activity. These authors observed smaller increases in thyroxine excretion in both sexes after phenobarbital pretreatment which could be accounted for by increased bile flow. Analogous to our studies with probenecid, they found no increases in the proportion of the biliary thyroxine present as glucuronides in phenobarbital-treated animals.

Indocyanine green and chlorothiazide are not metabolized prior to their biliary excretion. Thus, the enhanced excretion of ICG in males and of chlorothiazide in females must have


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been mediated by an effect of phenobarbital other than its acceleration of hepatic drug metabolism. It has been estab- lished that phenobarbital does not cause increased biliary uptake or excretion by displacement from plasma protein binding sites (3, 29). As suggested by Plaa and co-workers (22,29), increases in biliary excretion seem to be due primarily to increases in bile Aow, which we also consistently observed in both sexes after phenobarbital. Phenobarbital is well known to cause an increase in liver weight (8). In our studies, we observed about a 25 % increase in weight which is similar to increases observed by others, and appears to be a parenchymal hypertrophy and not a hyperplasia (2).

The proposed mechanism of bile formation might be examined in relation to the phenobarbital-induced choleresis and hepatocellular hypertrophy. It has been proposed that the primary event in the formation of bile is the active transport of bile acid anions from parenchymal cells into the bile canaliculi (36). The osmotic effect of these substances results in a passive flow of water and solutes into the canaliculi. Virtually all the bile acids excreted into mammalian bile are first conjugated with taurine and glycine, with taurine predominating in rat bile (17). The bile acids appear to be derived almost entirely from the metabolism of cholesterol. In the rat, Siperstein and Chaikoff (33) and Siper- stein et al. (34) demonstrated that 80-90 % of the body cholesterol was converted to bile acids which were then excreted into bile as conjugates. Recently, Jones and Arm- strong (19) and Wada et al. (38) have shown that phenobarbital pretreatment produced 4- to 5-fold increases in cholesterol synthesis from acetate or mevalonate. Jones and Arm- strong (19) did not find an increased accumulation of cholesterol in either liver or plasma, suggesting perhaps that the formed compound was rapidly metabolized and/or excreted. Thus, it seems reasonable to propose that the large increase in bile flow seen after phenobarbital is due primarily to an increased formation and excretion of taurocholate and other bile acids. If true, this effect could be termed an cLendogenous” choleresis in that the choleretic solute is formed in situ. Direct evidence for such a mechanism is lacking at present. Furthermore, it is not known whether the rate-limiting step lies in the conversion of cholesterol to bile acids or in the enzyme conjugation of the latter with taurine or glycine. With reference to bile pigment excretion, a twofold increase in bile bilirubin excretion has been reported after phenobarbital treatment (32).

The parallel enhancing effects on ICG excretion of both phenobarbital (Table 1) and chlorothiazide (Table 8) are in agreement with Sperber’s contention (36) that factors which augment bile flow (i*e., choleretic agents) exert their effects in the same manner as those initiating bile formation.

REFERENCES

1. BAER, J. E., H. L. LEIDY, A.V, BROOKS, AND K. H. BEYER. The physiological disposition of chlorothiazide (Diuril) in the dog. J. Pharmacol. Exptl. Therap. 125 : 295-302, 1959.

2. BARKA, T., AND H. POPPER, Liver enlargement and drug toxicity. Medicine 46 : 103-l 17, 1967.

3. BERNSTEIN, G., S. A. ARTZ, J. HASEN, AND J. H. OPPENHEIMER. Hepatic accumulation of lz51-thyroxine in the rat: augmentation by phenobarbital and chlordane. Endocrinology 82 : 406-409, 1968.

4. BRAUER, R. MT. Mechanisms of bile secretion. J. Am. Med. Assoc.

6. CHERRICK, G. R., S. W. STEIN, AND C. M. LEEVY. Indocyanine green: observations on its physical properties, plasma decay and hepatic extraction. J. Clin. Invest. 39: 592-600, 1960.

7, COMBES, B. Excretory function of the liver. In: The Liver, edited by C. Rouiller. New York: Academic, 1964, vol. II, p. l-35.

8. CONNEY, A. H. Pharmacological implications of microsomal enzvme induction. Pharmacol. Rev. 19: 3 17-366. 1967. 169: 1462-1466, 1959. , ,

It has been shown that administration of taurocholate and dehydrocholate will increase the apparent transport maxi- mum (Tm) for BSP threefold (26, 28). Above a certain level of taurocholate, competition by the bile salt for carrier sites outweighed its choleretic effects and the Tm for BSP de- clined. In our studies, the choleretic effects of chlorothiazide also appeared to predominate over its possible competition with ICG for the transport process (Table 8). However, it must be noted that Hargreaves and Lathe (14) observed marked inhibition of ICG excretion with only lo- to lOO- fold greater molar amounts of several compounds. Millburn et al. (25) have speculated that the physiological role of the hepatic anion transport process is to secrete endogenous anions, primarily the bile acids or salts, and to a quantitatively smaller degree, the bile pigments such as bilirubin. If, as proposed, the mechanism by which phenobarbital increases bile flow is to increase taurocholate synthesis and excretion, then it is reasonable to suppose that the excess bile acid molecules resulting may compete favorably with those foreign substrates possessing a lower affinity for the remaining carrier sites. In animals with normal renal circulation, phenol red (2 1) and chlorothiazide (1) are pre- dominantly excreted in the urine with only a minor fraction in the bile. Indocyanine green, however, is quantitatively cleared by the liver into the bile even in animals with normal renal function (20). Thus, competition by the endogenous acids for the carrier sites might offset their choleretic effects with the result being no net increase in excretion of drugs in those cases when there was no enhancement of biliary excretion after phenobarbital.

It must be pointed out that the speculative discussion above does not exclude two other possible mechanisms of increased bile formation; namely, induced secretion of bicarbonate (Wheeler and Ramos (40)) or the second canalicLzlar mechanism reported by Wheeler, Ross, and Bradley (4 1).

In conclusion, it appears that phenobarbital may be a useful tool in exploring not only the mechanisms for the biliary excretion of both foreign and endogenous compounds, but also those involved in bile formation itself.

The authors gratefully acknowledge the skillful technical assistance of Miss Jacqueline Call.

Present address of L. G. Hart: Bio-Medical Research Laboratories, Atlas Chemical Industries, Inc., Wilmington, Del. 19899.

A. M. Guarino is a Research Associate in the Pharmacology- Toxicology Program, National Institute of General Medical Sciences, National Institutes of Health.

A preliminary account of portions of this work has appeared previously (Federation Proc. 27: 302, 1968).

Received for publication 22 August 1968.

5. CATZ, C., AND S. J. YAFFE. Pharmacology modification of bil’ irubin conjugation in the newborn. Am. J. Diseases Children 104: 516-517, 1962.


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52 HART, GUARINQ AND ADAMSON

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