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We have previously identified a silencer (negative enhancer) in glutathione transferase P (GST-P) gene which is strongly and specifically induced during hepatocarcinogenesis of the rat. At least three trans-acting factors bind to multiple cis-elements located in this silencer. One of these factors, SF-B (Silencer Factor B) specifically binds to GPS1 (GST-P Silencer 1) and has been cloned by a Southwestern protocol. Analysis of DNA and deduced amino acid sequence reveals that SF-B clone is most likely identical to an IL-6 inducible trans-activator LAP/IL6-DBP. Binding efficiency of SF-B to GPS1 is indistinguishable from that to IL6-responsive element found in C-reactive protein gene. The possibility that SF-B/LAP/IL6-DBP functions as a dual positive and negative regulator is discussed.

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.

We have detected glutathione transferase (GST) activity towards 1-chloro-2,4-dinitrobenzene (CDNB) and benzo[a]-pyrene 4,5-oxide (BPO) in freshly isolated peripheral lymphocytes, phytohaemagglutinin (PHA)-stimulated lymphocytes, interleukin-2 (IL-2)-dependent T-cells and lymphoblastoid B-cell lines. The detection of conjugating activity with BPO implies the expression of a neutral, 'mu-like' GST form by both resting and cultured lymphocytes. In a sample of 12 unrelated individuals, BPO conjugating activity of freshly isolated lymphocytes showed a polymorphic distribution. In comparison with freshly isolated cells, BPO activity was increased 2- to 5-fold in IL-2-dependent T-cells and 4- to 10-fold in B-cell lines. The CDNB activity of T-cells was not significantly different to that of freshly isolated cells (P greater than 0.05) but activity in B-cell lines showed a significant increase (P less than 0.01). These data indicate that measurement of BPO activity in freshly isolated lymphocytes may allow the study of the human GST-mu polymorphism. However, IL-2-dependent culture of T-cells or viral immortalization of resting lymphocytes appears to cause the activation or induction of a GST form or forms which conjugate BPO efficiently. In the T-cells this effect may be mediated through addition of IL-2 to the culture since PHA treatment alone did not elevate activity.

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.

& Patrick, 1969; Horton et al., 1970; Lapey & Gardner, 1971 ; Cole & Dirks, 1972). Glucose uptake by the cystic fibrosis fibroblast is not abnormal (Benke et al., 1972); under the influence of 50pM-ouabain, the decrease in glucose uptake (measured in pg of glucose/h per mg of protein) noted in five cystic fibrosis fibroblast cell lines is similar to that of six control (normal) skin fibroblast cell lines (Fig. 1 ) . This is evidence, albeit indirect, for a normal Na++K+-ATPase in cystic fibrosis. The support of the Cystic Fibrosis Research Trust is gratefully acknowledged. I should also like to thank Dr. A. P. Norman, TheHospitalfor SickChildren, LondonW.C.1, forhisco-operation during the work.

1. [14C]Prenazone administered to rats (60 mg/kg) as a suspension by stomach tube was excreted to the extent of 43% dose in the urine in 24 h with only 3% in the faeces. After four days the cumulative excretion amounted to 52% in the urine and 15% in the faeces.2. The two major urinary metabolites were trans-4-(3'-hydroxymethylbut-2'-enyl)-1,2-diphenylpyrazolidine-3, 5-dione (monohydroxyprenazone) and trans-4-(3'-hydroxymethylbut-2'-enyl)-1 -(p-hydroxyphenyl)-2-phenylpyrazolidine-3,5-dione- (dihydroxyprenazone). These did not appear to be conjugated.3. Only very small amounts of unchanged prenazone were found in the urine.4. The above monhydroxyprenazone and related compounds were synthesized.

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.

Since chlorpromazine hydrochloride [2-chloro-10-(3-dimethylaminopropyl)-phenothiazine hydrochloride] is commonly implicated in causing bile-secretory failure in man and is secreted into bile, we have studied the physicochemical interactions of the drug with the major components of bile in vitro. Chlorpromazine hydrochloride molecules are amphiphilic by virtue of possessing a polar tertiary amine group linked by a short paraffin chain to a tricyclic hydrophobic part. At pH values below the apparent pK (pK'a 7.4) the molecules are water-soluble cationic detergents. We show that bile salts in concentrations above their critical micellar concentrations are precipitated from solution by chlorpromazine hydrochloride as insoluble 1:1 salt complexes. In the case of mixed bile-salt/phosphatidylcholine micellar solutions, however, the degree of precipitation is inhibited by the phospholipid in proportion to its mole fraction. With increases in the concentration of chlorpromazine hydrochloride or bile salt, micellar solubilization of the precipitated complexes results. Sonicated dispersions of the negatively charged phospholipid phosphatidylserine were also precipitated, but dispersions of the zwitterionic phospholipid phosphatidylcholine were not. Chlorpromazine hydrochloride efficiently solubilized these membrane phospholipids as mixed micellar solutions when the drug:phospholipid molar ratio reached 4:1. Polarizing-microscopy and X-ray-diffraction studies revealed that the precipitated complexes were amorphous and potentiometric studies confirmed the presence of a salt bond. Some dissociation of the complex occurred in the case of the most polar bile salt (Ks 0.365). As canalicular bile-salt secretion determines much of bile-water flow, we propose that complexing and precipitation of bile salts by chlorpromazine hydrochloride and its metabolites may be physicochemically related to the reversible bile-secretory failure produced by this drug.

1. The excretion in the bile and urine after intravenous injection of 16 organic anions having molecular weights between 355 and 752 was studied in female rats, guinea pigs and rabbits. 2. These compounds were mostly excreted unchanged, except for three of them, which were metabolized to a slight extent (

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).

Various aspects of the species differences in the metabolism of drugs and other compounds foreign to the body have been reviewed on several occasions (see Williams, 1964, 1967a, b, 1969, 1971a, b, 1974a, b; Williams and Smith, 1965), and some general patterns have emerged. It appears that the basic pattern of xenobiotic metabolism is the same in most species, but within this pattern there are tremendous species variations. The basic pattern of metabolism is simply that a compound usually undergoes transformation in the body in two phases.

The polycyclic aromatic hydrocarbon benzo(a)pyrene* is metabolically activated by monooxygenase and epoxide hydrolase (EC. 4.2.1.63) to reactive intermediates, which are mutagenic, cytotoxic and carcinogenic (1–3). The metabolic profile obtained by incubation of BP with various mammalian systems in vitro has been investigated (4). However, the fate of BP in vivo has received relatively little attention. Metabolites of BP are excreted via the bile in the rat (5) and rabbit (6) and BP metabolites in rat bile have recently been shown to be mutagenic to tester strains of Salmonella typhimurium (7). Rat and human intestinal microflora are capable of hydrolyzing conjugates of BP metabolites (8) to release relatively non-polar aglycones.

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.

A classical compartmental pharmacokinetic model was developed to describe the systemic blood concentration-time profile of phenolphthalein and its glucuronide conjugate (total 3H) following a single intravenous bolus injection of [3H]phenolphthalein. The model incorporates a biliary transport system, including a finite lag time for the biliary phenophthalein glucuronide to be hydrolyzed in the intestine before absorption. Data obtained from bile duct-cannulated animals were fit to the same model excluding any component for intestinal absorption. Agreement between the rate constants obtained for both fits indicates that the model is internally consistent. The model was then used to simulate a 24-hour time-course of phenolphthalein-equivalent blood concentrations which indicates that the long apparent half-lives calculated during this period are artifacts of recirculation.

Feprazone is a recently synthesised non-steroidal anti-inflammatory drug (1). It is structurally closely related to phenylbutazone, the difference lying in the side chain which is dimethylallyl in feprazone (see formula in Fig. 1) and butyl in phenylbutazone. For the purposes of the study the drug was labelled with 14C (Fig. 1) starting with [ 14C] i-diethylmalonate which was condensed with dimethylallyl chloride to give diethyldimethylallyl [2–14C] Amalonate. This was further condensed with hydrazobenzene to yield [ 14c] feprazone (2).

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.

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.

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.

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...

1. The conjugation of [ 14 C]benzoic acid has been studied in a species of African fruit bat ( Epomops franqueti ). Hippuric acid was found as a metabolite of benzoic acid in this bat, in contrast to the Indian fruit bat ( Pteropus giganteus ) which does not make this metabolite. 2. Three conjugates of benzoic acid were found in the urine of Epomops franqueti ), namely benzoylglucuronide (32% of the 14 C excreted in 24 hr), benzoylglycine (hippuric acid; 31%) and benzoylglutamic acid (11%).