P. C. Hirom

Bile and Urine as Complementary Pathways for the Excretion of Foreign Organic Compounds

1. The urinary and biliary excretion in the rat of 30 aromatic compounds with mol. wt. of 100-850, and largely excreted unchanged, has been studied. 2. These compounds fall into three groups as regards their pattern of elimination, which is related to mol. wt: group 1, with mol. wt. less than 350 and the major route of elimination the urine. When urinary excretion is prevented by ligating the renal pedicles the biliary excretion remains low. group 2, with mol, wt. of 450-850 which are excreted predominantly in bile. Even when the bile duct is obstructed, only small amounts of these compounds are found in urine. group 3, with mol. wt. of 350-450, which are eliminated extensively in both urine and bile. When one of these routes is blocked excretion by the other increases. 3. These studies emphasize the interrelationship of urine and bile as excretory routes for organic compounds. Urine and bile are complementary pathways; the extent of urinary excretion is greatest for the compounds of lowest mol. wt. and tends to decrease as mol. wt. increases and biliary excretion becomes more extensive.

The biliary excretion and enterohepatic circulation of benzo (a)pyrene and its metabolites in the rat

Abstract The enterohepatic circulation of benzo( a )pyrene (BP) has been investigated in the rat with a view to determining the availability of potentially toxic metabolities to tissues within this cycle. Some 60% of the dose of [ 14 C]-BP (3 μmoles kg −1 , i.v.) is excreted in bile in 6 hr, with less than 3% in urine. The biliary metabolities are mainly polar conjugates; only 8% of the 14 C in 2 hr bile samples can be directly extracted into ethyl acetate. However, following hydrolysis by β-glucuronidase some 40% of the 14 C is extractable at pH 7. The extract consisted of polar metabolites (polyhydroxylated and/or conjugated; 37.5%), BP 4,5-diol (16.8%), BP 3,6-quinone (5.9%). 9-hydroxy BP (5.4%) and 3-hydroxy BP (5.3%) as indicated by co-chromatography with authentic standards on reversed phase HPLC, together with several unidentified metabolites. The proximate carcinogen BP 7, 8-diol was not detected. Biliary metabolites of [ 14 C]-BP undergo enterohepatic recirculation in the rat; following the intraduodenal infusion of bile containing metabolites of [ 14 C]-BP into bile duct cannulated rats, approximately 20% of the dose is absorbed and excreted in bile in 30 hr, with only 1% in urine. The pattern of metabolites in this bile is very similar to that in bile from rats administered [ 14 C]-BP i.v. Following a single i.v. dose of [ 14 C]-BP (3 μmoles kg −1 ) to rats with re-entrant bile duct cannulae, which allowed intermittent collection of bile over a period of several days with minimal interference to the enterohepatic circulation, the proximate carcinogen BP 7, 8-diol was detected in recirculating bile. Biliary metabolites of BP, which have recently been shown to be mutagenic, can thus traverse the intestine to undergo enterohepatic circulation in the rat.

Enterohepatic recycling of phenolphthalein, morphine, lysergic acid diethylamide (LSD) and diphenylacetic acid in the rat Hydrolysis of glucuronic acid conjugates in the gut lumen

1. Biliary elimination in female Wistar albino rats 3 h after i.p. injection of [3H]phenolphthalein, [3H]morphine, 14C-LSD and [14C]diphenylacetic acid was 90%, 45%, 75% and 57% respectively, predominantly as glucuronides. 2. Infusion of 3 h bile from the previous experiments into the duodena of bile-duct-cannulated animals demonstrated enterohepatic circulation, amounting in 24 h to 85%, 41%, 28% and 66% of the infused doses of the conjugates of phenolphthalein, morphine, LSD and diphenylacetic acid respectively. 3. Pretreatment with antibiotics to suppress intestinal microflora decreased this enterohepatic recirculation to 22%, 8.6% and 21% in 24 h for phenolphthalein, morphine and diphenylacetic acid glucuronides respectively. Antibiotic pretreatment did not influence the absorption and re-excretion of infused doses of the free aglycones, thus demonstrating the importance of bacterial beta-glucuronidase hydrolysis of the biliary conjugates. 4. The extent of intestinal absorption of the aglycones after bacterial beta-glucuronidase hydrolysis of the conjugates is related to their lipid-solubility as estimated by octan-1-ol:0.1 M phosphate buffer partition ratios (P-values). 5. The persistence of compounds in the enterohepatic circulation is determined by the faecal and urinary elimination of the circulating compounds. Faecal elimination is governed by the extent of intestinal absorption of the circulating compounds, which is influenced by the efficacy of intestinal hydrolysis of the conjugates and the relative lipophilicity of the aglycones released.

The Influence of Dose on the Pattern of Conjugation of Phenol and 1-Naphthol in Non-Human Primates

1. The pattern of conjugation of phenol and 1-naphthol was investigated in several primates; three Old World species (rhesus, cynomolgus, patas monkeys), two New World species (capuchin, tamarin), and two prosimians (bushbaby, tree shrew). 2. Following intra-muscular phenol or 1-naphthol (10 mg/kg), sulphation was the major conjugation in the Old World monkeys and prosimians, whereas glucuronidation predominated in the New World species. 3. In rhesus and cynomolgus monkeys, sulphation decreased as dose increased, but remained the major conjugation with both substrates at dose levels of 0.01 to 25 mg/kg. 4. In the capuchin, the conjugation pattern of phenol changed markedly as dose increased; at 0.01 and 1 mg/kg sulphation was the major conjugation, whereas at 10 and 25 mg/kg glucuronidation predominated. With 1-naphthol only small amounts of sulphate were excreted; glucuronic acid conjugation was the major metabolism at all four dose levels. 5. The importance of considering both substrate and dose when making inter-species comparisons, particularly with man, is discussed.

Molecular Weight and Chemical Structure as Factors in the Biliary Excretion of Sulphonamides in the Rat

1. Biliary excretion in biliary-cannulated female rats of 23 derivatives of sulphanilamide, in which the N4-position is substituted with various carboxyacyl groups and the N1-position with acetyl or 2-thiazole, has been studied.2. Six compounds having molecular weights in the range 172–314 were poorly excreted in the bile ( 5–10% of dose).3. Above the threshold mol. wt., biliary excretion is high (20–70% of dose) but there is no direct relationship between the two, as the extent of biliary excretion is not directly proportional to mol. wt.4. There is no direct relationship between the extent of biliary excretion and the relative lipid solubilities as measured by distribution ratios between buffer pH 7.4 and organic solvents. Nevertheless th...

Sex and species differences in the biliary excretion of tartrazine and lissamine fast yellow in the rat, guinea-pig and rabbit. The influence of sex hormones on tartrazine excretion in the rat.

A sex difference in the biliary excretion of tartrazine, which occurs in the rat, has been found not to occur in the guinea‐pig and rabbit. Male and female rats excrete 13 and 29% respectively of an intravenous dose of tartrazine (50 μmol/kg) in the bile in 3 h. The corresponding figures for male and female guinea‐pigs and rabbits were 33 and 39%, and 5 and 6%, respectively. The sex difference in the rat was unrelated to any differences in renal function, for when the renal pedicles were ligated 22 and 63% of a dose was excreted in the bile of males and females respectively. Treatment of male and female rats with oestradiol and testosterone respectively influenced this sex difference in hepatic function but males were not affected by treatment with progesterone. Thus the extent of biliary excretion of tartrazine in males was increased by oestradiol pretreatment from 14 to 33% of the dose whereas testosterone pretreatment of females decreased excretion from 31 to 16% of the dose. The related dye lissamine fast yellow does not exhibit marked sex or species differences, 75–90% of a dose being excreted in the bile of males and females of all three species. Both dyes are excreted unchanged in the bile and urine of the three species.

Metabolism and excretion of benzo[a]pyrene in the rabbit

1. Following i.v. administration of [14C]benzo[a]pyrene (3 mumol/kg) to rabbits, 30% of the 14C dose appeared in bile and 12% in urine, within six hours. 2. Biliary and urinary metabolites were mainly conjugated; less than 12% of the 14C was extractable with ethyl acetate, but after treatment with beta-glucuronidase or aryl sulphatase 30-40% became extractable. 3. H.p.l.c. analysis of the extracts indicated that the major non-polar metabolite was benzo[a]pyrene, 9,10-diol (18% of 14C in bile and 24% of 14C in urine, mainly conjugated with glucuronic acid). Smaller amounts of the 4,5-diol, the 3,6-quinone, and the 9-hydroxy- and 3-hydroxybenzo[a]pyrene were also found in bile (total less than 10%), together with 9-hydroxybenzo[a]pyrene and two unknown metabolites (X and Y) in urine (total less than 4%). 4. The proximate carcinogen, the 7,8-diol, was not detected in any extract. 5. After intraduodenal administration of biliary metabolites of [14C]benzo[a]pyrene (approx. 0 X 3 mumol), 14C was excreted in the bile (21% dose) and urine (14%) within 23 h, indicating that metabolites can undergo enterohepatic circulation in the rabbit.

Metabolism and enterohepatic circulation of benzo(a)pyrene-4,5-epoxide in the rat

After i.v. administration of 3H-benzo(a)pyrene-4,5-epoxide (32.5 mumol/kg) to rats, 76% of the 3H appeared in bile within 3 h. The glutathione conjugate of benzo(a)pyrene-4,5-epoxide was the major biliary metabolite (33% of dose), together with a glucuronic acid conjugate of benzo(a)pyrene-4,5-diol (18%) and an unidentified metabolite (10%). The glutathione and glucuronic acid conjugates both undergo extensive enterohepatic circulation. Thus, following the intraduodenal administration of the 3H-labelled conjugates, 26% of the radioactivity was excreted in the bile after 24 h in the case of the glutathione derivative, and 40% in the case of the glucuronide. The benzo(a)pyrene-4,5-diol glucuronide, on enterohepatic circulation, appears in the bile in the same form as the conjugate administered with no evidence of further metabolism of the polycyclic hydrocarbon moiety. The glutathione conjugate of benzo(a)pyrene-4,5-epoxide, on recirculation, is reexcreted in bile as one unidentified metabolite, which is susceptible to the action of arylsulphatase.

The biliary excretion of tartrazine. Sex differences in the rat and species differences in the rat, guinea-pig and rabbit

The excretion of tartrazine in the bile and urine has been studied in biliary cannulated rats, rabbits and guinea‐pigs. This dye is excreted unchanged by these species. In the rat a sex difference in the relative amounts of tartrazine excreted in bile and urine has been found. Male rats excrete in 3 h about 17 % of an intravenous dose (50 μmol/kg) in the bile and about 70% in the urine, whereas females excrete about 40 and 45 % respectively. The biliary excretion of tartrazine in the rat appears to be influenced by dose level, for at the lower level of 4.5 μmol/kg male rats excrete about 9 % of an intravenous dose in the bile and 64% in the urine in 3 h, the corresponding values for female rats being 30% and 50%. There is also a species difference in the extent of biliary excretion of tartrazine (50 μmol/kg intravenously). The female rat and female guinea‐pig excrete in 3 h about 40 % of the dose in the bile and a similar amount in the urine whereas the female rabbit excretes only 6% in the bile and nearly 70% in the urine. Previous work in this laboratory has shown that molecular weight is an important factor in the biliary excretion of foreign compounds and the present results fit in with this view.

3-Hydroxy-Trans-7,8-Dihydro-7,8-Dihydroxy-Benzo(a)Pyrene, a Metabolite of 3-Hydroxybenzo(a)Pyrene

3-Hydroxybenzo(a)pyrene(3-OH-BP) (I) is a major metabolite of the environmental pro-carcinogen BP. It is produced in a wide variety of biological systems (1–4) and is excreted as a glucuronide in bile (5, 6). Under appropriate conditions it binds to DNA (7) but it is not carcinogenic (8) and is a poor tumour initiator (9).