The reactive W-O-sulfate conjugate of W-hydroxy-2-acetylaminofluorene is believed to be one of its ultimate carcinogenic metabolites. The role of W-O-sulfation in the metabolism of this compound was investigated in the perfused rat liver and in the rat in vivo. In isolated rat livers perfused with 1.2 HIMinorganic sulfate, 43% of the dose of /V-hydroxy-2-acetylaminofluorene was converted to the A/-O-glucuronide conjugate. After omis sion of sulfate from the perfusion medium, this increased to 76% of the dose. A major fraction of W-hydroxy-2-acetylaminofluorene was converted to as yet unidentified water-soluble metabolites that were not glucuronides or sulfates. This fraction decreased from 38% in control perfusions to 19% when sulfate was omitted. These findings indicate that conjugation with glucuronic acid takes over when sulfation of W-hydroxy-2-acetylaminofluorene is impaired. As a result, less of the watersoluble metabolites was formed, presumably because these are, in part, derived from the A/-O-sulfate conjugate. When the sulfation inhibitor pentachlorophenol was added to the perfusion 15 minutes before W-hydroxy-2-acetylaminofluorene, the fraction that was converted to the N-O-glucuronide increased, and the water-soluble metabolites decreased. Therefore, inhibition of sulfation by this inhibitor and by omis sion of sulfate give similar results. When rats were pretreated i.p. with pentachlorophenol 45 min before i.v. treatment with /V-hydroxy-2-acetylaminofluorene (60 /Â?mol/kg),47% of the dose was excreted in bile and urine as the W-O-glucuronide; in untreated controls, this was 27% of the dose. In pretreated rats, the water-soluble metabolites comprised 27% of the dose; in untreated controls, they made up 45% of the dose. These findings indicate that at least 20% of an i.v. dose of 60 jumol of /V-hydroxy-2-acetylaminofluorene per kg is sulfated in the rat. Omission of sulfate in the perfused liver decreased covalent binding of [9-t4C]-/V-hydroxy-2-acetylaminofluorene to DMA, RNA, and protein by 70 to 80% and addition of pentachloro phenol to the perfusion medium resulted in a similar decrease. Pretreatment with pentachlorophenol in vivo resulted in a de creased binding to protein and RNA in the liver. These findings show that /V-O-sulfation of /V-hydroxy-2-acetylaminofluorene is responsible for most of the reactive metabolite(s) that react with protein and RNA at the doses used in this study.

The influence of bile salts on hepatic transport of the organic anion dibromosulphthalein (DBSP) was investigated in rats. Bile salts influence the hepatic uptake, intracellular binding, and biliary excretion of DBSP. The overall effect depends on the administered dose of bile salts and DBSP. High doses of bile salts inhibited hepatic uptake of DBSP, whereas low doses of bile salts stimulated bile flow and simultaneously increased maximal biliary excretion of DBSP. The uncharged nonbile salt choleretic ouabain also stimulated biliary DBSP excretion. In contrast, the anionic nonbile salt choleretics, ethacrynic acid and theophylline, did not stimulate biliary excretion of DBSP. Because DBSP inhibited biliary excretion of ethacrynic acid and its metabolites, the lack of a stimulatory effect of ethacrynic acid choleresis might be explained by concomitant inhibition of biliary excretion of DBSP, masking the stimulatory effect of ethacrynic acid. Biliary transport maximum of DBSP was highly correlated with bile flow. The biliary clearance (Vmax/Km) was only moderately changed by bile salt administration, whereas the increase in the maximal biliary excretion rate was more pronounced, implying that the apparent Km for biliary excretion of DBSP was also increased by the bile salts. It is inferred that the stimulation of net biliary excretion of DBSP by bile salts may be due to a diminished transport from bile into the hepatocytes as the consequence of the decreased biliary concentration caused by the choleresis.

1. In freely moving, unanesthetized rats bile flow was measured continuously over the whole day--night cycle. Bile composition was analysed and the influence of food intake on bile flow was investigated. 2. In both sexes a distinct circadian variation of bile production was observed. The mean night-time production was 50% higher than the day-time value for female rats and 38% for male rats. In the morning when the light was switched on, a sharp decrease in secretion rate was prominent and bile flow gradually increased in the afternoon. 3. The pattern of food intake was positively correlated with the pattern of food bile secretion. During fasting only the general level of bile flow decreased, but the circadian variation persisted. Refeeding again increased the mean level of bile flow. 4. The chenodeoxycholate/cholate ratio in these rats with permanent bile fistulae was higher than in rats with "acute" bile fistulae and changed during the day--night cycle. The ratio decreased from 1.01 at 05.00 hours to a minimum of 0.41 at 15.00 hours. 5. During the day--night cycle the sodium, potassium, calcium and cholesterol concentrations were relatively constant. The total bile salt concentration was only slightly changed, so that both the bile salt-dependent fraction and the bile salt-independent fraction were subject to about the same circadian variations.

1. A new and rapid continuous assay of rat liver microsomal UDP-glucuronyltransferase (EC 2.4.1.17) has been developed. It is based on measurement of UDP production from UDP-glucuronate during the glucuronidation reaction; UDP production was continuously measured by coupling it to the conversion of NADH into NAD+ through pyruvate kinase and lactate dehydrogenase. This assay is independent of the acceptor substrate used; several findings confirm its applicability. 2. The glucuronidation rate of a series of phenol derivatives was determined with this assay, by using a Triton X-100-activated microsomal preparation as enzyme source. Conjugation of a series of nitrophenol derivatives was also investigated by the 'classical' assay (measurement of disappearance of the yellow colour of the nitrophenol during glucuronidation). The substrate with the highest conversion rate was 3-methyl-2-nitrophenol. 3. Both electron releasing and electron withdrawing ring substituents increased the glucuronidation rate of the phenol derivatives, as compared with phenol. 4. Lipid solubility seems important for determining the conversion rate: poorly lipid-soluble substrates were glucuronidated only at a low rate and high lipid solubility seems to be a prerequisite for high conversion rate. Glucuronidation of poorly lipid-soluble compounds may be limited by diffusion. 5. The consequences of these findings for the interpretation of studies on heterogeneity of the enzyme are discussed.