The metabolism of 4-aminobiphenyl in rat. II. Reaction of N-hydroxy-4-aminobiphenyl with rat blood in vitro

1. N-Hydroxy-4-aminobiphenyl (N-hydroxy-ABP) reacts with HbFe2+ of rat blood in vitro at a molar ratio of 1:47 to produce 20% HbFe3+ within 1 min; N-hydroxy-ABP oxidized 9.4 equiv. of HbFe2+. N-hydroxy-ABP rapidly disappeared and HbFe3+ was reduced at a rate of 44 microM/min. 2. On titration of rat blood in vitro with N-hydroxy-ABP up to 0.81 mM, 4-nitrosobiphenyl (nitroso-BP) disappeared within 5 min; with concn of N-hydroxy-ABP greater than 0.81 mM, N-hydroxy-ABP was present also as nitroso-BP, indicating saturation of reactive binding sites. When N-hydroxy-ABP reacted with HbFe2+ at a molar ratio of 1:103 to 1:1.9, 13 to 1.3 equiv. of HbFe3+ were formed per mol of N-hydroxy-ABP in 5 min, indicating that with increasing N-hydroxy-ABP concn side-reactions increased. 3. After incubation of N-hydroxy-ABP (1.72 mM) with rat Hb (7.66 mM HbFe2+), nitroso-BP disappeared with a half-life of 1 min, maximal HbFe3+ of 72% occurred at 47 min, and the concn of 4-aminobiphenyl (ABP) increased at a rate of 51 nmol/ml per h. 4. In rats injected with 0.24 mmol/kg ABP, HbFe3+ concn plateaued at 56% after 75 min, indicating an equilibrium between HbFe3+ formation and HbFe3+ reduction. Such equilibrium was simulated by titrating rat blood in vitro with N-hydroxy-ABP for 1 h. 5. The long-lasting HbFe3+ formation by ABP in rat results from a cycle of activation of ABP to N-hydroxy-ABP, its rapid co-oxidation with HbFe2+ to form HbFe3+ and nitroso-BP, and binding of nitroso-BP to erythrocyte thiol groups. ABP is released from the Hb adduct and enters a new cycle of activation and inactivation, until terminated by ring-hydroxylation.     

The metabolism of 4-aminobiphenyl in rat. I. Reaction of N-hydroxy-4-aminobiphenyl with rat blood in vivo

1. ³H-4-Aminobiphenyl (ABP, 5?mg) given i.p. to rat had elimination half-lives of 15.6, 17 and 17?h, respectively, for urinary, faecal and total ³H elimination. ¹⁴C-ABP administered orally to rats at 100?mg/kg gave elimination half-lives of 31, 36.7 and 34?h, respectively, for urinary, faecal and total ¹⁴C elimination.

2. Semi-log plots of percentage dose remaining in the body versus time indicated that: (i) 82% of ³H activity was excreted in 36?h with a half-life of 14.4?h and 18% with a half-life of 46.2?h, and (ii) 77% of ¹⁴C activity was excreted in 48?h with a half-life of 15?h and 23% with a half-life of 180?h.

3. After i.p. injection of 10?mg/kg ¹⁴C-ABP to rats, ferrihaemoglobin (HbFe³⁺) concn increased to 60% in 2h, accompanied by accumulation of ¹⁴C activity in erythrocytes, indicating that the active metabolite, N-hydroxy-4-aminobiphenyl (N-hydroxy-ABP) had oxidized haemoglobin-Fe²⁺ (HbFe²⁺) and was bound to the erythrocyte.

4. ABP given i.p. to rats at 0.24 mmol/kg rapidly appeared in blood, disappeared with a half-life of 30?min, and blood concn plateaued at 30 nmol/ml. The concn of 4-acetyl-aminobiphenyl (AABP) plateaued at 17 nmol/ml after 15?min, indicating a dynamic equilibrium between N-acetylation of ABP and N-deacetylation of AABP. The concn of 4′-hydroxy-4-acetylaminobiphenyl (4′-hydroxy-AABP) increased slowly at 1.65 nmol/h.

5. AABP given i.p. to rats at 0.88 mmol/kg slowly appeared in the blood, accompanied by the appearance of ABP and 4′-hydroxy-AABP and formation of HbFe³⁺. After 4?h the concn of AABP and ABP was 27.35 mmol/ml, indicating a dynamic equilibrium between N-deacetylation of AABP and acetylation of ABP. Neither N-hydroxy-ABP nor N-hydroxy-4-acetylaminobiphenyl (N-hydroxy-AABP) were found.     

The metabolism of 4-aminobiphenyl in rat. IV. Ferrihaemoglobin formation by 4-aminobiphenyl metabolites

1. Rats dosed with nitrosobenzene (56 μmol/kg), 4-chloronitrosobenzene (53 μmol/kg), 3,4-dichloronitrosobenzene (53 μmol/kg), 4-ethoxynitrosobenzene (86 μmol/kg), 4-nitrosobiphenyl(nitroso-BP, 55 μmol/kg) or 2-nitrosofluorcne (256 μmol/kg) had maximal ferrihaemoglobin (HbFe³⁺) concn of 69, 68, 69, 67, 55 and 42% after 15, 25, 48, 35, 80 and 115 min, respectively, indicating differences in solubility of the nitrosoarenes in body fluids.

2. Nitroso-BP and 3-hydroxy-4-aminobiphenyl (3-hydroxy-ABP) catalytically oxidized HbFe²⁺ in bovine erythrocytes in vitro; nitroso-BP was three times as active as 3-hydroxy-ABP. 3′,4′-Dihydroxy-4-aminobiphenyl (3′,4′dihydroxy-ABP) showed only low catalytic activity, and seven other ABP metabolites exhibited only marginal activity.

3. Nitroso-BP was inactive in solutions of purified human Hb, but 3-hydroxy-ABP catalytically oxidized HbFe²⁺, indicating that nitrosoarenes oxidize HbFe²⁺ in erythrocytes in vitro and in vivo by a mechanism different from that of o-aminophenols. The second-order rate constant for HbFe²⁺ oxidation by 3-hydroxy-ABP at 37°C was k₂ = 19.1±1.31/mol per s.     

The metabolism of 4-aminobiphenyl in rat. IV. Ferrihaemoglobin formation by 4-aminobiphenyl metabolites.

1. Rats dosed with nitrosobenzene (56 mumol/kg), 4-chloronitrosobenzene (53 mumol/kg), 3,4-dichloronitrosobenzene (53 mumol/kg), 4-ethoxynitrosobenzene (86 mumol/kg), 4-nitrosobiphenyl(nitroso-BP, 55 mumol/kg) or 2-nitrosofluorene (256 mumol/kg) had maximal ferrihaemoglobin (HbFe3+) concn of 69, 68, 69, 67, 55 and 42% after 15, 25, 48, 35, 80 and 115 min, respectively, indicating differences in solubility of the nitrosoarenes in body fluids. 2. Nitroso-BP and 3-hydroxy-4-aminobiphenyl (3-hydroxy-ABP) catalytically oxidized HbFe2+ in bovine erythrocytes in vitro; nitroso-BP was three times as active as 3-hydroxy-ABP. 3',4'-Dihydroxy-4-aminobiphenyl (3',4'dihydroxy-ABP) showed only low catalytic activity, and seven other ABP metabolites exhibited only marginal activity. 3. Nitroso-BP was inactive in solutions of purified human Hb, but 3-hydroxy-ABP catalytically oxidized HbFe2+, indicating that nitrosoarenes oxidize HbFe2+ in erythrocytes in vitro and in vivo by a mechanism different from that of o-aminophenols. The second-order rate constant for HbFe2+ oxidation by 3-hydroxy-ABP at 37 degrees C was k2 = 19.1 +/- 1.31/mol per s.     

The in vitro/in vivo comparative metabolism of 4-aminobiphenyl using isolated hepatocytes

The metabolism of 4-aminobiphenyl by isolated hepatocytes from various species was compared with urinary metabolite profiles in the same species. Radioactive compounds in concentrates of ether extracts from hepatocytes or urine following hydrolysis were analysed by TLC and reversed phase HPLC in conjunction with radioactivity monitoring and synthetic standards.The major metabolites from hepatocytes and in urine were 4-acetamidobiphenyl, 3-hydroxy-4-aminobiphenyl 4-hydroxy-4-aminobiphenyl and 4-hydroxy-4-acetamidobiphenyl. Oxidation of the amine nitrogen gave hydroxylamino, nitroso and nitro compounds. Minor metabolites were 2-hydroxy amine and amide, the hydroxamic acid and the oxamic acid. The urinary metabolite profiles correlated well with those from hepatocytes for each species.Abbreviations Used 4-ABP 4-aminobiphenyl - AA 4-acetamidobiphenyl - A3-OH 3-hydroxy-4-aminobiphenyl - A4-OH 4-hydroxy-4-aminobiphenyl - A2-OH 2-hydroxy-4-aminobiphenyl - AA4-OH 4-hydroxy-4-acetamidobiphenyl - AA3-OH 3-hydroxy-4-acetamidobiphenyl - AA2-OH 2-hydroxy-4-acetamidobiphenyl - AAN-OH N-hydroxy-4-acetamidobiphenyl - NBP 4-nitrobiphenyl - AN-OH 4-hydroxylaminobiphenyl - NOBP 4-nitrosobiphenyl Dedicated to Professor Dr. med. Herbert Remmer on the occasion of his 65th birthday     

N-hydroxy-N-arylacetamides. V. Differences in the mechanism of haemoglobin oxidation in vitro by N-hydroxy-4-chloroacetanilide and N-hydroxy-4-chloroaniline

1. Autoxidation of N-hydroxy-4-chloroaniline(I) in buffer pH 7.4 was rapid and yielded 4,4'-azoxybischlorobenzene, 4-chloronitrosobenzene, 4-chloronitrobenzene, and 4-chlorophenyl nitroxide. In contrast, autoxidation of N-hydroxy-4-chloroacetanilide(II) was very slow, since in ether and water 78 and 92%, respectively, had decomposed in six months. 2. Haemoglobin(HbO2)-catalysed autoxidation of (I) occurred at a molar ratio of haemoglobin-Fe2+ to (I) of less than 0.25 and was accompanied by ferrihaemoglobin(HbFe3+)-formation and oxygen consumption. Coupled oxidation of HbO2 with (I) occurred at a molar ratio of greater than 0.2 and was accompanied by liberation of oxygen and the formation of HbFe3+, haemoglobin-4-chloronitrosobenzene complex, HbO2, desoxyhaemoglobin, 4-chloronitrosobenzene, 4-chloronitrobenzene, 4-chloroaniline, 4,4'-azoxybischlorobenzene, and 4-chlorophenyl nitroxide. At an equimolar ratio of 10(-3) M haemoglobin-Fe2+ to (I), 96% HbO2 was converted into HbFe3+ (50%) and haemoglobin-4-chloronitrosobenzene complex in the initial fast phase of the reaction, but only 34% of the bound oxygen was liberated, the rest was sequentially reduced to water. (I) completely disappeared, and 4-chloronitrosobenzene was the major metabolite, mainly bound to haemoglobin. 3. Chemical oxidation of (II) by PbO2 in benzene produced acetyl 4-chlorophenyl nitroxide, whose spontaneous decomposition gave 38% 4-chloronitrosobenzene, 33% N-acetoxy-4-chloroacetanilide, 10% 4-chloroacetanilide, and 8% 4-chloronitrobenzene. Its spontaneous decomposition in water also followed second order kinetics, K = 350 l mol-1 sec-1 and yielded N-(2-acetylamino-5-chlorophenyl)-p-benzo-quinoneimine-N-oxide in addition. 4. In the coupled oxidation of 10(-3) M haemoglobin-Fe2+ with 10(-3) M (II), 75% HbFe3+ was formed after 1 h, but only one third of the equivalent of oxygen was released, and two thirds were reduced to water. Concentration of (II) decreased by 5% only, indicating that one mol of (II) had catalysed the oxidation of 15 equivalents of haemoglobin-Fe2+. The identity of the product pattern formed with HbO2 with that produced by chemical one-electron oxidation indicated that oxygen bound to haemoglobin also functions as an acceptor for electrons from (II) as from (I), but the different redox potentials can explain why the secondary aromatic nitroxide was catalytically active and the primary nitroxide was not.     

The oxidation of 4-aminobiphenyl by horseradish peroxidase.

The oxidation of the carcinogen 4-aminobiphenyl (4-ABP) catalyzed by the model peroxidase enzyme horseradish peroxidase (HRP) was investigated. 4-ABP served as a reducing cosubstrate for HRP during the enzyme-catalyzed reduction of the synthetic hydroperoxide, 5-phenyl-4-penten-1-yl hydroperoxide, to its corresponding alcohol. Spectral analysis during the incubation of HRP, 4-ABP, and H2O2 showed an increase in absorbance at 230 and 325 nm and decrease at 270 nm, suggesting metabolite formation. Oxygen consumption was not detected in incubations of HRP, 4-ABP, and H2O2. However, oxygen uptake was observed after the addition of glutathione, which indicated that a free radical metabolite of 4-ABP was formed by the peroxidase. The 4-ABP free radical reacted with glutathione forming a glutathionyl radical which, in turn, reacted with and consumed oxygen. HPLC analysis of organic extracts of incubations with HRP, [3H]-4-ABP, and H2O2 showed the formation of one major peak identified by mass spectroscopy as 4,4'-azobis(biphenyl). The addition of glutathione to the incubations decreased the formation of 4-ABP metabolites, suggesting a reduction of the 4-ABP free radical and/or the formation of glutathione conjugates. Subsequent HPLC analysis of incubations including [35S]glutathione indicated formation of several unidentified 4-ABP-glutathione conjugates as well as recovery of parent compound. These studies suggest that HRP metabolizes 4-ABP by a one-electron oxidation mechanism, resulting in formation of a free radical. This radical can either react with a second radical to form azobis(biphenyl), be reduced by glutathione back to parent, or react with glutathione to form glutathione conjugates.     

N-hydroxy-N-arylacetamides. III: Mechanism of haemoglobin oxidation by N-hydroxy-4-chloroacetanilide in erythrocytes in vitro

N-Hydroxy-4-chloroacetanilide(N-hydroxy-4C1AA) was the most active, and N-hydroxy-2-acetylaminofluorene(N-hydroxy-2AAF) the least active compound among six N-hydroxy-N-arylacetamides, in forming ferrihaemoglobin(HbFe3+) in bovine erythrocytes in the presence of 11 mM glucose. N-Hydroxy-4C1AA oxidized 25 equiv. of HbFe2+, both in the presence and absence of glucose or lactate. Therefore, its catalytic properties did not depend on metabolic regeneration by the NADPH- or NADH-dependent erythrocyte reductases. In contrast, N-hydroxy-4-chloroaniline(N-hydroxy-4C1A) oxidized 760 equiv. of HbFe2+ in the presence of glucose, but only 81 equiv. of HbFe2+ in the presence of lactate. These results indicate that the catalytic activity depended on the metabolic regeneration from 4-chloronitrosobenzene(4-C1NOB) by NADPH-dependent erythrocyte reductases. A relationship was established between HbFe3+ concn. and the concn. of N-hydroxy-4C1A and 4-C1NOB(determined together), 4-chloroacetanilide(4-C1AA) and 4-chloroaniline(4-C1A), indicating co-oxidation of N-hydroxy-4C1AA and oxyhaemoglobin in erythrocytes and partial reduction of the newly formed 4-C1NOB to 4-C1A. In rat blood in vitro incubated with N-hydroxy-4C1AA, 4-C1NOB concn. increased with increasing HbFe3+ concn., indicating that 4-C1NOB was formed by co-oxidation of oxyhaemoglobin and N-hydroxy-4C1AA, and not by enzymic N-deacetylation.     

N-Hydroxy-N-arylacetamides. III: Mechanism of haemoglobin oxidation by N-hydroxy-4-chloroacetanilide in erythrocytes in vitro

1. N-Hydroxy-4-chloroacetanilide(N-hydroxy-4ClAA) was the most active, and N-hydroxy-2-acetylaminofluorene(N-hydroxy-2AAF) the least active compound among six N-hydroxy-N-arylacetamides, in forming ferrihaemoglobin(HbFe³⁺) in bovine erythrocytes in the presence of 11 mM glucose.

2. N-Hydroxy-4ClAA oxidized 25 equiv. of HbFe²⁺, both in the presence and absence of glucose or lactate. Therefore, its catalytic properties did not depend on metabolic regeneration by the NADPH- or NADH-dependent erythrocyte reductases.

3. In contrast, N-hydroxy-4-chloroaniline(N-hydroxy-4ClA) oxidized 760 equiv. of HbFe²⁺ in the presence of glucose, but only 81 equiv. of HbFe²⁺ in the presence of lactate. These results indicate that the catalytic activity depended on the metabolic regeneration from 4-chloronitrosobenzene(4-CINOB) by NADPH-dependent erythrocyte reductases.

4. A relationship was established between HbFe³⁺ concn. and the concn. of N-hydroxy-4ClA and 4-CINOB(determined together), 4-chloroacetanilide(4-ClAA) and 4-chloroaniline(4-CIA), indicating co-oxidation of N-hydroxy-4ClAA and oxyhaemoglobin in erythrocytes and partial reduction of the newly formed 4-CINOB to 4-CIA.

5. In rat blood in vitro incubated with N-hydroxy-4CIAA, 4-CINOB concn. increased with increasing HbFe³⁺ concn., indicating that 4-CINOB was formed by co-oxidation of oxyhaemoglobin and N-hydroxy-4CIAA, and not by enzymic N-deacetylation.     

The N-hydroxylation and ring-hydroxylation of 4-aminobiphenyl in vitro by hepatic mono-oxygenases from rat,mouse, hamster,rabbit and guinea-pig

1. An analytical?h.p.l.c. method has been developed which permits the separation and quantification of the in vitro metabolites of 4-aminobiphenyl (4-ABP). The method employs gradient elution from a reverse phase column.

2. The major metabolite in vitro of 4-ABP in liver fractions from rat, mouse, guinea-pig, rabbit and hamster was N-hydroxy-4-aminobiphenyl.

3. The observation that liver fractions from the guinea-pig are very effective in the N-hydroxylation of 4-ABP is in excellent agreement with the 1966 report from Kiese's laboratory, showing that the N-hydroxylation of 4-ABP is an important metabolic pathway in vivo in this species.

4. The ortho-phenol, 3-hydroxy-4-aminobiphenyl was also an important metabolite in each species except guinea-pig and rabbit.

5. Hydroxylation at the 4′ and 2′ positions was a minor pathway in all species studied.

6. Aroclor 1254 was a potent inducer of N-hydroxylation in rat, mouse and guinea-pig but not hamster and rabbit. Phenobarbital induced N-hydroxylation in rabbit and guinea-pig but not rat, while methylcholanthrene induced in rat and guinea-pig but not rabbit.     

In vitro metabolism study of combretastatin A-4 in rat and human liver microsomes.

The phase I biotransformation of combretastatin A-4 (CA-4) 1, a potent tubulin polymerization inhibitor with antivascular and antitumoral properties, was studied using rat and human liver subcellular fractions. The metabolites were separated by high-performance liquid chromatography and detected with simultaneous UV and electrospray ionization (ESI) mass spectrometry. The assignment of metabolite structures was based on ESI-tandem mass spectrometry experiments, and it was confirmed by comparison with reference samples obtained by synthesis. O-Demethylation and aromatic hydroxylation are the two major phase I biotransformation pathways, the latter being regioselective for phenyl ring B of 1. Indeed, incubation with rat and human microsomal fractions led to the formation of a number of metabolites, eight of which were identified. The regioselectivity of microsomal oxidation was also demonstrated by the lack of metabolites arising from stilbenic double bond epoxidation. Alongside the oxidative metabolism, Z-E isomerization during in vitro study was also observed, contributing to the complexity of the metabolite pattern. Moreover, when 1 was incubated with a cytosolic fraction, metabolites were not observed. Aromatic hydroxylation at the C-6' of phenyl ring B and isomerization led to the formation of M1 and M2 metabolites, which were further oxidized to the corresponding para-quinone (M7 and M8) species whose role in pharmacodynamic activity is unknown. Metabolites M4 and M5, arising from O-demethylation of phenyl ring B, did not form the ortho-quinones. O-Demethylation of phenyl ring A formed the metabolite M3 with a complete isomerization of the stilbenic double bond.     

In vivo and in vitro pharmacokinetics and metabolism of vincaalkaloids in rat. II. Vinblastine and vincristine

Vinblastine and Vincristine pharmacokinetics, including tissue distribution, metabolism and biliary excretion, were investigated, using both "in vitro" and "in vivo" models, after i.v. injections in rats. Plasma kinetic curves were best fitted to a two-compartment open model. The average terminal half-lives of VLB and VCR were 14.3 h and 7.5 h, respectively. The systemic clearance and apparent distribution volume for VLB, respectively 1.49 l/h/kg and 11.46 l/kg, were significantly greater than those of VCR, 0.12 l/h/kg and 0.41 l/kg. VCR was found to be widely distributed in tissues after i.v. injections in rats. The highest drug accumulation site was the intestine (122.0 ng/g wet tissue at 24 h). Liver and kidneys also retained high proportions of drug (respectively, 47.0 ng/g and 44.4 ng/g at 24 h). Biliary excretion was more rapid for VCR (42.7% of total radioactivity excreted over 24 h) than VLB (28.2% of total dose over 24 h). For both molecules, the percentage of radioactivity excreted in bile over 30-48 h ranged between 40-50% of total dose. At high doses, either biliary excretion rate or cumulated excretion was reduced. High performance liquid chromatography analysis of bile samples revealed four biotransformation products for VLB and three for VCR. When incubated in freshly isolated rat hepatocytes, VLB penetrated more rapidly and intensely into the cells (more than 90% of total dose taken up over 20 min) than VCR (only about 40% accumulated), probably through a passive diffusion mechanism followed by tight cellular binding. "In vitro" metabolism patterns were similar to those found "in vivo", except for the most polar metabolites observed "in vitro". Two anti-Vinca monoclonal antibodies with different specificities were used to test VCR metabolite immunoreactivities. The results suggested that some structural modifications occurred in the catharantine moiety of the molecule but that the dimeric structure seemed to be well conserved after biotransformation.     

Felbamate in vitro metabolism by rat liver microsomes.

Incubation of [14C]felbamate at 37 degrees C for 60 min with liver microsomes from untreated Sprague-Dawley rats converted 10% of the drug to the p-hydroxy (6%) and 2-hydroxy (4%) metabolites. With microsomes from phenobarbital-pretreated rats, 21% of the drug was metabolized to the p-hydroxy (7.5%) and 2-hydroxy (13.5%) metabolites. With microsomes from felbamate-pretreated rats, up to 25% of drug was metabolized to the p-hydroxy (5%) and 2-hydroxy (20%) metabolites. In addition, a small amount of the monocarbamate metabolite was also present, but no other metabolites were formed. Coincubations of [14C]phenytoin with felbamate had no effect on the metabolism of phenytoin, whereas the amount of [14C]felbamate metabolized in the presence of phenytoin decreased by 30-38%.     

Induction of phase I and II drug metabolism in rat small intestine and colon in vitro.

The aim of this study was to evaluate drug metabolism in rat small intestinal and colon precision-cut slices during 24 h of incubation and the applicability of these slices for enzyme induction studies. Various parameters were evaluated: intracellular levels of ATP (general viability marker), alkaline phosphatase activity (specific epithelial marker), villin expression (specific epithelial marker), and metabolic rates of 7-ethoxycoumarin (CYP1A), testosterone (CYP3A and CYP2B), and 7-hydroxycoumarin (glucuronide and sulfate conjugation) conversions. ATP and villin remained constant up to, respectively, 5 and 8 h in small intestine and up to 24 h in colon. The metabolic rate remained constant in small intestinal slices up to 8 h and decreased afterward to 24 to 92%, depending on the substrate studied. The inducibility of metabolism in small intestinal and colon slices was tested with several inducers at various concentrations and incubation times. The following inducers were used: 3-methylcholanthrene, beta-naphthoflavone, indirubin, and tert-butylhydroquinone (aryl hydrocarbon receptor ligands), dexamethasone (glucocorticoid receptor/pregnane X receptor ligand) and phenobarbital (constitutive androstane receptor ligand). After incubation with inducers, metabolic rates were evaluated with 7-ethoxycoumarin and testosterone (phase I) and 7-hydroxycoumarin (phase II) as substrate. All inducers elevated the metabolic rates consistent with the available published in vivo induction data. Induction of enzyme activity was already detectable after 5 h (small intestine) and after 8 h (colon) for 3-methylcholanthrene and beta-naphthoflavone and was clearly detectable for all tested inducers after 24 h (up to 20-fold compared with noninduced controls). In conclusion, small intestinal and colon precision-cut slices are useful for metabolism and enzyme induction studies.     

The metabolism of canrenone in vitro by rat liver preparations

1. The metabolism of [1-³H]canrenone, a primary metabolite of spironolactone and potassium canrenoate, by rat liver preparations in vitro has been investigated.

2. Canrenone was metabolized by 3-oxo-δ⁴-reduction to give 3α-hydroxy-5β-spirolactones, and also by a number of O₂ and NADPH-dependent microsomal hydroxylation reactions.

3. A major metabolic route requiring the presence of a microsomal fraction, but apparently independent of oxygen and NADPH, led to the formation of a number of compounds tentatively identified as trihydroxy-spirolactones.     

Bromobenzene metabolism in vivo and in vitro. The mechanism of 4-bromocatechol formation

The metabolism of bromobenzene has been examined in isolated hepatocytes and liver microsomes from phenobarbital-induced rats and in phenobarbital-induced rats in vivo. The metabolite profile produced upon incubation of isolated rat hepatocytes with bromobenzene differed with the hepatocyte concentration. At a low hepatocyte concentration (0.5 x 10(6) cells/ml), 4-bromophenol was the major metabolite, while at higher hepatocyte concentrations (2.0 and 5.0 x 10(6) cells/ml) bromobenzene-3,4-dihydrodiol was the major metabolite. 4-Bromophenol was the primary metabolite in incubations with rat liver microsomes. In vivo, 3- and 4-bromophenol were more predominant, with very little dihydrodiol formed. 4-Bromocatechol, a potentially toxic metabolite of bromobenzene, was formed in vivo as well as in isolated hepatocytes and microsomes. However, the mechanism of catechol formation differed, as determined by the retention of a deuterium label at the para position of bromobenzene. In microsomes, 4-bromophenol was the predominant precursor metabolite of 4-bromocatechol. In isolated hepatocytes, although the relative contribution of 4-bromophenol as the bromocatechol precursor differed with hepatocyte concentration, bromobenzene-3,4-dihydrodiol was the predominant precursor at all concentrations. In vivo, as in isolated hepatocytes, 4-bromocatechol was formed primarily via bromobenzene-3,4-dihydrodiol.     

The metabolism of 5-hydroxytryptamine and beta-phenylethylamine in perfused rat lung and in vitro.

1 Metabolism of 5-hydroxytryptamine (5-HT) and beta-phenylethylamine (PHE) by monoamine oxidase (MAO) was investigated in rat isolated lungs and in mitochondrial preparations from rat lung. 2. In perfused lungs 5-HT metabolism had an apparent Km of 2 microgram and PHE metaoblism a Km of 54 microgram, whereas in vitro the Km values were 330 microgram and 28 microgram respectively. 3 In vitro, MAO activity had substrate and inhibitor specificities compatible with the presence of A and B types of MAO. 4 In perfused lung, metabolism of 5-HT but not that of PHE was inhibited by desmethylimipramine. 5 These results show that PHE metabolism in perfused lung, unlike that of other metabolized amines, is not limited by transport and the transport process for PHE is unlike that of 5-HT or noradrenaline. 6 These results also show that the kinetic parameters obtained for MAO activity in vitro do not generally apply to the isolated lung where transport of substrate can be the deciding factor. This discrepancy emphasizes that the enzymic properties of the whole organ cannot relaibly be deduced from its enzymic content.