M.D. Tingle

An investigation of the formation of cytotoxic, genotoxic, protein-reactive and stable metabolites from naphthalene by human liver microsomes

Chemically reactive epoxide metabolites have been implicated in various forms of drug and chemical toxicity. Naphthalene, which is metabolized to a 1,2-epoxide, has been used as a model compound in this study in order to investigate the effects of perturbation of detoxication mechanisms on the in vitro toxicity of epoxides in the presence of human liver microsomes. Naphthalene (100 microM) was metabolized to cytotoxic, protein-reactive and stable, but not genotoxic, metabolites by human liver microsomes. The metabolism-dependent cytotoxicity and covalent binding to protein of naphthalene were significantly higher in the presence of phenobarbitone-induced mouse liver microsomes than with human liver microsomes. The ratio of trans-1,2-dihydrodiol to 1-naphthol was 8.6 and 0.4 with the human and the induced mouse microsomes, respectively. The metabolism-dependent toxicity of naphthalene toward human peripheral mononuclear leucocytes was not affected by the glutathione transferase mu status of the co-incubated cells. Trichloropropene oxide (TCPO; 30 microM), an epoxide hydrolase inhibitor, increased the human liver microsomal-dependent cytotoxicity (19.6 +/- 0.9% vs 28.7 +/- 1.0%; P = 0.02) and covalent binding to protein (1.4 +/- 0.3% vs 2.8 +/- 0.2%; P = 0.03) of naphthalene (100 microM), and reversed the 1,2-dihydrodiol to 1-naphthol ratio from 6.6 (without TCPO) to 2.6, 0.6 and 0.1 at TCPO concentrations of 30, 100 and 500 microM, respectively. Increasing the human liver microsomal protein concentration reduced the cytotoxicity of naphthalene, while increasing its covalent binding to protein and the formation of the 1,2-dihydrodiol metabolite. Co-incubation with glutathione (5 mM) reduced the cytotoxicity and covalent binding to protein of naphthalene by 68 and 64%, respectively. Covalent binding to protein was also inhibited by gestodene, while stable metabolite formation was reduced by gestodene (250 microM) and enoxacin (250 microM). The study demonstrates that human liver cytochrome P450 enzymes metabolize naphthalene to a cytotoxic and protein-reactive, but not genotoxic, metabolite which is probably an epoxide. This is rapidly detoxified by microsomal epoxide hydrolase, the efficiency of which can be readily determined by measurement of the ratio of the stable metabolites, naphthalene 1,2-dihydrodiol and 1-naphthol.

Drug-protein conjugates—XI disposition and immunogenicity of dinitrofluorobenzene, a model compound for the investigation of drugs as haptens

The conjugation of drugs to autologous proteins is thought to be a key step in the hapten mechanism of drug hypersensitivity. We have studied the mild arylating agent dinitrofluorobenzene (DNP-F) as a model compound with which to investigate the relationship between the disposition and immunogenicity of drug haptens in two species, the rat and rabbit. Intramuscular administration of DNP-F (0.027-27 mumol/kg/day) for 4 days to male Wistar rats produced a dose-dependent (ED50 2.7 mumol/kg) IgG anti-DNP antibody response, measured by enzyme-linked immunosorbent assay. Subsequent monthly administrations (for 4 days) increased both the frequency and titre of antibody response. Intravenous administration of [3H]DNP-F (0.27 or 2.7 mumol/kg) for 9 days to male New Zealand White rabbits produced an IgG and IgM anti-DNP response in all animals from day 9 onwards. Formation of circulating (serum) DNP-protein conjugates was determined by radiometric analysis, and found to reach steady state (0.12-0.17% dose/ml) between days 6 and 8 and decline with a half-life of 7.4 days. The immunogenicity of fully characterized haptenated, autologous proteins was investigated in further experiments in which dinitrophenylated serum protein conjugates (DNP-RSP) and albumin conjugates (DNP-RSA) were prepared ex vivo and then administered (50 mg/kg; i.v.) to the rabbit from which the protein had been obtained. The plasma clearance and immunogenicity of DNP-RSA conjugates was dependent on epitope density. Anti-DNP antibodies were detected after administration of an RSA-DNP15 conjugate but not after either RSA-DNP0.5 or RSA-DNP5. Plasma concentrations of RSA-DNP15 conjugate declined slowly initially, but then fell rapidly between days 8 and 10. The plasma clearance of DNP-RSP conjugates showed a dependence on epitope density from day 1 onwards and anti-DNP antibodies were detectable after administration of all conjugates investigated (range of epitope densities 0.5-30 DNP/albumin equivalent). Thus conjugates derived from proteins other than albumin are likely to be the effective immunogens, for the model hapten DNP. These studies show that DNP-F is a useful model compound in studies of the disposition and immunogenicity of drugs acting as haptens, and may therefore be used as a positive control in experiments designed to assess the potential immunogenicity of drugs and other xenobiotics.

An Investigation into the Haematological Toxicity of Structural Analogues of Dapsone In-vivo and In-vitro

Abstract— With microsomes prepared from a single human liver, 4,4′‐diaminodiphenyl sulphone (DDS), 4‐acetyl‐4‐aminodiphenyl sulphone (MADDS), 4‐acetyl‐4‐aminodiphenyl thioether (MADDT) and 4,4′‐diacetyldiphenyl thioether (DADDT) caused significantly greater methaemoglobin formation compared with control. In‐vitro in the rat, the pattern of toxicity was slightly different: DADDT was not haemotoxic, whilst 3,4′‐diaminodiphenyl sulphone (3,4′DDS) and 3,3′‐diaminodiphenyl sulphone (3,3′DDS) as well as DDS, MADDS and MADDT were significantly greater than control. 4,4′ Acetyl diphenyl sulphone (DADDS), 4,4′ diaminodiphenyl thioether (DDT), 4,4′‐diaminodiphenyl ether (DDE) and 4,4′ diamino‐octofluorodiphenyl sulphone (F8DDS) did not cause significant methaemoglobinaemia in either human or rat liver microsomes. DDS, MADDS, and MADDT were not significantly different in haemotoxicity generation in‐vitro in the presence of human microsomes. In the rat in‐vitro, DDS, MADDS, and 3,4′DDS did not differ significantly in red cell toxicity, and were the most potent methaemoglobin formers. The 3,3′ DDS and MADDT derivatives were both significantly less toxic compared with DDS. None of the compounds tested caused haemoglobin oxidation in the absence of NADPH in‐vitro. In the whole rat, DDS, MADDS and MADDT caused significantly higher levels of methaemoglobin compared with control. None of the remaining compounds caused methaemoglobin formation which was significantly greater than control. DDS and MADDS were the most potent methaemoglobin formers tested, in‐vivo and in‐vitro. The 3,3′ and 3,4′DDS analogues caused no detectable haemotoxicity in‐vivo. However, the plasma elimination of the 3,4′ analogue was much more rapid compared with that of DDS. Overall, there was no correlation between log k0 and increasing haemotoxicity. The use of the two‐compartment system together with in‐vivo studies may be applied to the evaluation of the structural features required for bioactivation of candidate antiparasitic compounds to haemotoxic metabolites by cytochrome P450 enzymes.

Enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to protein-reactive drugs and metabolites: criteria for identification of antibody activity Detection and hapten specificity of anti-DNP, anti-captopril and anti- sulphanilamidobenzoic acid

Certain hypersensitivity reactions to drugs are thought to depend on coupling of reactive species (the drug itself or a metabolite) to macromolecules, leading to the formation of hapten-carrier conjugates. In assays for the detection of antibodies directed against such reactive species the drug or metabolite must be used in conjugated rather than free form. We describe ELISAs for the detection of anti-dinitrophenyl (DNP), anti-captopril (CP) and anti-sulphanilamidobenzoic acid (SABA) antibodies, in which the wells of microtitre plates are coated with hapten conjugated to protein. We define coating conditions and the following 3 criteria for identification of anti-hapten activity: Immunoglobulin in the test sample binds to the immobilised hapten-protein conjugate, but not to the immobilised protein alone. Binding is inhibited by preincubation of the test sample with protein conjugates incorporating the test hapten, but not by preincubation with the same unconjugated proteins, nor protein conjugates incorporating haptenic groups unrelated to the test hapten. The inhibitory hapten-protein conjugates are shown to be inactive in unrelated antigen-antibody interactions. Binding is blocked by preincubation of the test sample with low molecular weight chemical derivatives of the reactive hapten. The inhibitory derivatives must be shown to be inactive in unrelated antigen-antibody interactions. On the basis of these criteria, IgG anti-DNP and IgG anti-CP were detected in the sera of immunized rabbits. The IgG anti-DNP antibody recognised protein-conjugated DNP, DNP-lysine, N-acetyl-DNP-lysine and DNP-S-glutathione, whereas the IgG anti-CP antibody recognised CP-S-S-protein and CP-S-S-CP. By the same criteria IgG anti-SABA was detected in the sera of immunized mice. The antibody recognised free and protein-conjugated SABA, but not free sulphanilamide.

Gonadal influence on the metabolism and haematological toxicity of dapsone in the rat.

Abstract— Administration of dapsone (33 mg kg−1) to intact rats resulted in a marked elevation of methaemoglobin levels in male (435.0 ± 105.2% met Hb h) compared with female rats (59.0 ± 17.2% met Hb h). However, the clearance of dapsone was significantly faster in males compared with females. Female rats showed very low levels of methaemoglobin which were accompanied by significantly higher blood concentrations of parent drug. Clearance of dapsone in castrated animals was less than one‐third of that of the intact sham‐operated males (252.2 ± 67.2 vs 81.4 ± 33.0 mL h−1). Likewise, clearance of dapsone in ovarectomized rats was approximately half that of intact females. There were no significant differences in the disposition of dapsone between the ovarectomized (AUC, 431.0 ± 31.7 μg h mL−1; t 1/2, 15.62 ± 1.8 h) and castrated (AUC, 450.6 ± 150.9 μg h mL−1; t 1/2, 17.6 ± 7.9 h) animals. However, methaemoglobin levels in castrated males, although less than a third of those of intact males, significantly exceeded those of ovarectomized animals. There was no significant difference between the four groups of animals with respect to red cell sensitivity to the methaemoglobin‐forming capacity of the toxic metabolite of dapsone, the hydroxylamine. Metabolic conversion of dapsone to the hydroxylamine in the presence of NADPH was 7.6 ± 1.5% for liver microsomes from intact males and was significantly greater (P < 0.05) than the corresponding values for liver microsomes from castrated rats (5.3 ± 0.59%). Conversion of dapsone to dapsone‐NOH by liver microsomes from intact females and ovarectomized animals was below 1 % in both cases. This study illustrates the androgenic control of N‐hydroxylation in the rat.

Bioactivation and bioinactivation of drugs and drug metabolites: Relevance to adverse drug reactions

Adverse drug reactions that cannot be predicted from the pharmacological properties of the drug and which are not easily reproduced in laboratory animals are a major complication of drug therapy. It is necessary to investigate the mechanisms of such reactions in order to (1) define structural features within a given drug molecule which are responsible for causing toxicity and (2) to identify those individuals who are particularly sensitive to a given drug reaction. In theory, drug toxicity may arise by direct toxicity, genotoxicity or immune-mediated toxicity caused by either parent drug or chemical. In this respect chemically reactive metabolites are of particular importance and the balance between bioactivation and bioinactivation pathways of drug metabolism will be a critical factor in both the type and extent of toxicity. We have therefore developed in vitro techniques that incorporate human cells for the detection and characterization of stable, chemically reactive and cytotoxic metabolites. In such experiments bioactivation (by CYP1A, CYP2D6, CYP3A, etc.) can be investigated by use of a liver bank, while lymphocytes provide accessible human cells, which can be obtained from both patients and volunteers, genotyped and/or phenotyped for particular drug-metabolizing enzymes (eg. glutathione transferase mu). The relevance of in vitro experiments to drug toxicity observed in humans will be illustrated by reference to studies with anticonvulsants and antimalarials.

Bioactivation and inactivation of aflatoxin B1 by human, mouse and rat liver preparations: Effect on SCE in human mononuclear leucocytes

The purpose of this study was to investigate the use of human and animal subcellular liver fractions in an in vitro evaluation of carcinogenic risk. The bioactivation and bioinactivation of the known genotoxic carcinogen aflatoxin B1 by human, mouse and rat liver preparations was investigated using the SCE assay in human lymphocytes as a genotoxic endpoint. There was a 10-fold variation in SCE response (1.1-11.6 SCE/Cell) in human mononuclear leucocytes (MNLs) after aflatoxin B1 was activated by human liver microsomes (n = 6). Activation correlated with the CYP1A2 phenotype of livers (r = 0.8; p < 0.05), but there was no correlation with either GST M1 genotype or epoxide hydrolase phenotype. Mouse liver microsomes activated aflatoxin B1 to a greater extent [(1 micro M) 12.8 +/- 2.51 SCE/Cell] than either rat [(10 micro M) 12.0 +/- 3.84 SCE/Cell or human (L25) [(10 micro M) 8.8 +/- 2.00 SCE/Cell liver microsomes. The addition of mouse liver cytosol and reduced glutathione (GSH) significantly (p < 0.001) reduced aflatoxin B1-dependent genotoxicity, whereas the addition of either human or rat cytosol (+GSH) was without effect. These data indicate that species variation in both bioactivation and bioinactivation can exist. Therefore there is a necessity for careful selection of activation systems from species whose biochemical profile reflects that of man.

Inhibition of dapsone-induced methaemoglobinaemia by cimetidine in the rat during chronic dapsone administration

Abstract— Dapsone undergoes N‐acetylation to monoacetyl dapsone as well as N‐hydroxylation to a hydroxylamine which is responsible for the haemotoxicity (i.e. methaemoglobinaemia; Met Hb) of the drug. Since dapsone is always given chronically, we have investigated the ability of cimetidine to inhibit Met Hb formation caused by repeated dapsone administration. The drug was given (i.p.) to four groups (n = 6 per group) of male Wistar rats, 300–360 g. Group I received 10 mg kg−1 at 1, 24, 48 and 72 h. Group II received 10 mg kg−1 at 1, 8, 24, 32, 48, 56, 72 and 80 h. Groups III and IV received the drug as for groups I and II, respectively, as well as cimetidine (50 mg kg−1) 1 h before each dose of dapsone. Twice daily dapsone administration (Group II) resulted in a significantly greater (P < 0.05) Met Hb AUC (757 ± 135 vs 584 ± 115% Met Hb h), dapsone AUC (140 ± 17.5 vs 113 ± 130 μg h mL−1) and monoacetyl dapsone AUC (48.2 ± 18.3 vs 10.8 ± 4.6 μg h mL−1) compared with a single daily dapsone dose (group I). The administration of cimetidine before the once daily dose of dapsone (group III) resulted in a significant (P < 0.05) fall in Met Hb (302 ± 179 vs 584 ± 115% Met Hb h) and an increase in both the dapsone (151 ± 22.2 vs 113 ± 13.0 μg h mL−1) and monoacetyl dapsone AUC values (33.6 ± 5.8 vs 10.8 ± 4.0 μg h mL−1) compared with a single daily dose of dapsone (group I). Administration of cimetidine before the twice daily dose of dapsone (group IV) resulted in no significant change in Met Hb or monoacetyl dapsone levels, despite a marked increase in the AUC after dapsone compared with control (303 ± 53.2 vs 140 ± 17.5 μg h mL−1 P < 0.05; group II). The administration of a single dose of monoacetyl dapsone alone resulted in rapid production of methaemoglobinaemia (17.1 ± 7.2%) at 1 h; however, prior administration of cimetidine did not significantly affect methaemoglobin levels over 24 h (287.6 ± 77.9 vs 316.4 ± 120.2% Met Hb h). These studies indicate that although cimetidine may reduce Met Hb formation during chronic dapsone administration, dose reduction of dapsone is required to avoid haemotoxicity because of the increased accumulation of both parent drug and its monoacetyl metabolite.

The effect of fluorine substitution on the haemotoxicity of Primaquine

Abstract 5-Fluoro-6-methoxy-8-nitroquinoline was synthesised by a modified Skraup Reaction and was subsequently converted into 5-fluoroprimaquine in three steps. 5-Fluoroprimaquine 9 is less susceptible to in vitro bioactivation than primaquine 1 in a range of different species. However, significant bioactivation of 9 was observed with hepatic microsomes from two species including man.

Inhibition of Dapsone-induced Methaemoglobinaemia by Cimetidine in the Presence of Trimethoprim in the Rat

Abstract— Administration of dapsone in combination with trimethoprim and cimetidine to male rats resulted in a marked decrease (P < 0·05) in measured methaemoglobin levels (46·2±24% Met Hb h) compared with administration of dapsone alone (124·5 ± 24·4% Met Hb h). The elimination half‐life of dapsone (814 ± 351 min) was more than doubled in the presence of trimethoprim and cimetidine compared with control (355 ± 160 min, P < 0·05). However, there were no significant differences in AUC and clearance when dapsone was administered in combination with trimethoprim and cimetidine compared with dapsone alone. Co‐administration of trimethoprim with dapsone in the absence of cimetidine did not affect either methaemoglobin formation, AUCs, half‐lives, or clearance values of dapsone compared with control. There was a threefold increase in the AUC of trimethoprim (6296 ± 2249 μg min mL−1) in the presence of dapsone compared with trimethoprim alone (2122 ± 552 μg min mL−1). There was also a corresponding decrease in the clearance of trimethoprim in the presence of dapsone compared with control (19·1±6·9 vs 60·8 ± 21·0 mL min−1). However, there was no change in the elimination half‐life of trimethoprim between the two experimental groups (273 ± 120 vs 292 ± 54 min). The AUC of trimethoprim increased more than threefold in the presence of cimetidine (7100 ± 1501 μg min mL−1) compared with trimethoprim alone (2122 ± 552 μg min mL−1). There was also a corresponding reduction in the clearance of trimethoprim in the presence of cimetidine (61·2 ± 21·2 vs 17·8 ± 9·3 mL min−1) compared with control. However, there was no significant change in the elimination half‐life of trimethoprim after the administration of cimetidine (273 ± 136 vs 215 ± 109 min). Administration of either trimethoprim or cimetidine alone did not cause methaemoglobin levels to exceed control values. The administration of trimethoprim with dapsone and cimetidine resulted in a significant increase in AUC (2122 ± 552 vs 5744 ± 3289 μg min mL−1), a fall in clearance (17·8 ± 9·3 vs 60·8 ± 21 mL min−1), but no change in half‐life (252 ± 134 vs 273 ± 136 h) of trimethoprim. The co‐administration of cimetidine significantly reduced dapsone‐mediated methaemoglobin formation in the presence of trimethoprim, whilst the AUC of trimethoprim was significantly increased in the presence of both cimetidine and dapsone.