RESUMEN
WHAT IS KNOWN AND OBJECTIVE: Dosage adjustment of 500 mg ertapenem daily is recommended for patients with advanced kidney disease. 30% of ertapenem is cleared by a session of haemodialysis (HD). However, because most published carbapenems studies have excluded patients on dialysis, little is known about the dosing of ertapenem to avoid central nervous system (CNS) toxicity in regular HD patients. We report of four patients who developed CNS toxicity in such patients. CASE SUMMARY: The 4 HD patients developed unexplained CNS toxicity manifested as seizures, hallucination and cognitive dysfunction after receiving 3-7 consecutive recommended doses of ertapenem. Their symptoms of CNS toxicity were completely resolved within 8 days after discontinuation of ertapenem. In one of our presented cases, we demonstrated the very high level of plasma ertapenem accumulating with several consecutive doses. Cognitive function gradually recovered in line with a corresponding decline in blood level of ertapenem. WHAT IS NEW AND CONCLUSIONS: This is the first report of ertapenem-associated CNS toxicity in patients on regular HD and utilizing the plasma ertapenem concentration to demonstrate the causal relationship. The recommended dosage of 500 mg ertapenem daily may be still too high in regular HD patients, especially in Asians, owing to their relatively small body size. An increased awareness of ertapenem-associated CNS toxicity would avoid unnecessary examinations, hospitalization, and potentially catastrophic complications.
Asunto(s)
Antibacterianos/efectos adversos , Antibacterianos/farmacocinética , Enfermedades del Sistema Nervioso Central/inducido químicamente , Diálisis Renal , beta-Lactamas/efectos adversos , beta-Lactamas/farmacocinética , Anciano , Pueblo Asiatico , Relación Dosis-Respuesta a Droga , Ertapenem , Femenino , Humanos , Masculino , Insuficiencia RenalRESUMEN
The effects of carboxyfullerene on a well-known neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite 1-methyl-4-phenyl-pyridinium (MPP+) were investigated. In chloral hydrate-anesthetized rats, cytosolic cytochrome c was elevated in the infused substantia nigra 4 h after an intranigral infusion of MPP+. Five days after local application of MPP+, lipid peroxidation (LP) was elevated in the infused substantia nigra. Furthermore, dopamine content and tyrosine hydroxylase (TH)-positive axons were reduced in the ipsilateral striatum. Concomitant intranigral infusion of carboxyfullerene abolished the elevation in cytochrome c and oxidative injuries induced by MPP+. In contrast, systemic application of carboxyfullerene did not prevent neurotoxicity induced by intraperitoneal injection of MPTP. In mice, systemic administration of MPTP induced a dose-dependent depletion in striatal dopamine content. Simultaneous injection of carboxyfullerene (10 mg/kg) actually potentiated MPTP-induced reduction in striatal dopamine content. Furthermore, systemic administration of carboxyfullerene (30 mg/kg) caused death in the MPTP-treated mice. An increase in the striatal MPP+ level and reduction in hepatic P450 level were observed in the carboxyfullerene co-treated mice. These data showed that systemic application of carboxyfullerene appears to potentiate MPTP-induced neurotoxicity while local carboxyfullerene has been suggested as a neuroprotective agent. Furthermore, an increase in striatal MPP+ level may contribute to the potentiation by carboxyfullerene of MPTP-induced neurotoxicity.
Asunto(s)
1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Ácidos Carboxílicos/farmacología , Dopaminérgicos/toxicidad , Compuestos de Piridinio/toxicidad , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/administración & dosificación , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/antagonistas & inhibidores , Animales , Apoptosis/efectos de los fármacos , Axones/enzimología , Axones/metabolismo , Western Blotting , Sistema Enzimático del Citocromo P-450/metabolismo , Grupo Citocromo c/metabolismo , Citosol/efectos de los fármacos , Citosol/metabolismo , Dopamina/metabolismo , Electroquímica , Fulerenos , Peroxidación de Lípido/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/enzimología , Masculino , Ratones , Microinyecciones , Neostriado/citología , Neostriado/enzimología , Neostriado/metabolismo , Estrés Oxidativo/fisiología , Compuestos de Piridinio/administración & dosificación , Compuestos de Piridinio/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Sustancia Negra/efectos de los fármacos , Sustancia Negra/metabolismo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
1. The results of homology modelling of human cytochrome P4501A2 (CYP1A2) based on the CYP2C5 crystal structure are reported. It exhibits improved sequence homology relative to that of CYP102. 2. It was demonstrated that many selective substrates for CYP1A2 could fit within the putative active site of the enzyme, and in orientations which agree with documented evidence for CYP1A2-mediated metabolism. 3. Furthermore, a number of amino acid residues lining the haem pocket have been shown, via site-directed mutagenesis, to have an influence on substrate metabolism, and these experimental findings from the literature are consistent with the modelled interactions for selective substrates. 4. The binding affinities of several CYP1A2 substrates have also been calculated from the CYP1A2 active site interactions and they agree closely with experimental values.
Asunto(s)
Citocromo P-450 CYP1A2/química , Sistema Enzimático del Citocromo P-450/química , Esteroide 21-Hidroxilasa/química , Secuencia de Aminoácidos , Animales , Fenómenos Químicos , Química Física , Cricetinae , Cristalografía por Rayos X , Familia 2 del Citocromo P450 , Peces , Humanos , Lípidos/química , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conejos , Especificidad de la EspecieRESUMEN
Rutaecarpine is one of the main alkaloids of an herbal remedy, Evodia rutaecarpa, which has been used for the treatment of gastrointestinal disorder and headache. Effects of rutaecarpine on hepatic and renal cytochrome P450 (CYP)-dependent monooxygenase were studied in C57BL/6J mice. Treatment of mice with rutaecarpine by gastrogavage at 50 mg/kg/day for three days resulted in 57%, 41%, 6-, and 6-fold increases of hepatic microsomal benzo(a)pyrene hydroxylation, 7-ethoxycoumarin O-deethylation, 7-ethoxyresorufin O-deethylation, and 7-methoxyresorufin O-demethylation activities, respectively. However, the treatment had no effects on hepatic oxidation activities toward benzphetamine, N-nitrosodimethylamine, nifedipine, and erythromycin. In the kidney, rutaecarpine-treatment resulted in 2-fold and 42% increases of microsomal benzo(a)pyrene hydroxylation and 7-ethoxycoumarin O-deethylation activities, respectively. The treatment also increased renal 7-ethoxyresorufin O-deethylation activity to a detectable level. Immunoblot analysis of microsomal proteins showed that rutaecarpine-treatment increased the protein levels of CYP1A1 and CYP1A2 in the liver, whereas hepatic level of CYP3A-immunoreacted protein was not affected by rutaecarpine. These CYPs were not detectable in the immunoblot analyses of control and rutaecarpine-treated mouse kidney microsomes. These results indicated that rutaecarpine was a CYP1A inducer and showed potent inductive effects on both CYP1A1 and CYP1A2 in the liver.
Asunto(s)
Alcaloides/farmacología , Sistema Enzimático del Citocromo P-450/biosíntesis , Medicamentos Herbarios Chinos/farmacología , Riñón/enzimología , Hígado/enzimología , Administración Oral , Alcaloides/administración & dosificación , Animales , Relación Dosis-Respuesta a Droga , Medicamentos Herbarios Chinos/administración & dosificación , Inducción Enzimática , Immunoblotting , Alcaloides Indólicos , Riñón/efectos de los fármacos , Hígado/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , QuinazolinasRESUMEN
To evaluate the protective effects of baicalein and wogonin against benzo[a]pyrene- and aflatoxin (AF) B(1)-induced toxicities, the effects of these flavonoids on the genotoxicities and oxidation of benzo[a]pyrene and AFB(1) were studied in C57BL/6J mice. Baicalein and wogonin reduced benzo[a]pyrene and AFB(1) genotoxicities as monitored by the umuC gene expression response in Salmonella typhimurium TA1535/pSK1002. Baicalein added in vitro decreased liver microsomal benzo[a]pyrene hydroxylation (AHH) activity with an ic(50) of 33.9 +/- 1.4 microM at 100 microM benzo[a]pyrene. Baicalein also inhibited AFQ(1) and AFB(1)-epoxide formation from AFB(1) (50 microM) oxidation (AFO) with ic(50) values of 22.8 +/- 1.4 and 5.3 +/- 0.8 microM, respectively. However, the in vitro inhibitory effects of wogonin on AHH and AFO activities in liver microsomes were less than those of baicalein as inhibition by 500 microM wogonin was only about 51-65%. Treatment of mice with liquid diets containing 5 mM baicalein and wogonin resulted in 22 and 49% decreases in hepatic AHH activities, respectively. Baicalein treatment resulted in 39 and 32% decreases in AFQ(1) and AFB(1)-epoxide formation from liver microsomal AFO, respectively. Wogonin treatment resulted in 39 and 47% decreases in AFQ(1) and AFB(1)-epoxide formation, respectively. A 1-week pretreatment with wogonin significantly decreased hepatic DNA adduct formation in mice treated with 200 mg/kg of benzo[a]pyrene via gastrogavage. These in vitro and in vivo effects suggested that baicalein and wogonin might have beneficial effects against benzo[a]pyrene- and AFB(1)-induced hepatic toxicities and that wogonin had a stronger protective effect in vivo.
Asunto(s)
Aflatoxina B1/toxicidad , Benzo(a)pireno/toxicidad , Flavanonas , Flavonoides/farmacología , Microsomas Hepáticos/efectos de los fármacos , Sustancias Protectoras/farmacología , Animales , Dieta , Interacciones Farmacológicas , Hidroxilación , Masculino , Ratones , Ratones Endogámicos C57BL , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , Pruebas de Mutagenicidad , NADPH-Ferrihemoproteína Reductasa/efectos de los fármacos , NADPH-Ferrihemoproteína Reductasa/metabolismoRESUMEN
Effects of baicalein and wogonin, the major flavonoids of Scutellariae radix, on cytochrome P450 (CYP), UDP-glucuronosyl transferase (UGT), and glutathione S-transferase (GST) were studied in C57BL/6J mice. One-week treatment of mice with a liquid diet containing 5 mM baicalein resulted in 29%, 14%, 36%, 28%, and 46% decreases of hepatic benzo(a)pyrene hydroxylation (AHH), benzphetamine N-demethylation (BDM), N-nitrosodimethylamine N-demethylation (NDM), nifedipine oxidation (NFO), and erythromycin N-demethylation (EMDM) activities, respectively. Treatment with a liquid diet containing 5 mM wogonin resulted in 43%, 22%, 21%, 24%, and 35% decreases of hepatic AHH, BDM, NDM, NFO, and EMDM activities, respectively. However, hepatic 7-methoxyresorufin O-demethylation (MROD) activity was increased and decreased by baicalein- and wogonin-treatments, respectively. Similar modulation was observed with caffeine 3-demethylation (CDM) activity. Immunoblot analysis showed that the levels of hepatic CYP2E1 and CYP3A proteins were decreased by both baicalein- and wogonin-treatments. Hepatic CYP1A2 protein level was increased by baicalein but decreased by wogonin. In extrahepatic tissues, renal AHH activity was decreased by wogonin whereas pulmonary AHH, 7-ethoxyresorufin O-deethylation (EROD), and MROD activities were increased by both flavonoids. Both baicalein and wogonin strongly increased CYP1A protein level in mouse lung. Hepatic and renal UGT activities toward p-nitrophenol were suppressed by baicalein- and wogonin-treatments. However, cytosolic GST activity was not affected by flavonoids. These results suggest that ingestion of baicalein or wogonin can modulate drug-metabolizing enzymes and the modulation shows tissue specificity.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Flavanonas , Flavonoides/farmacología , Glucuronosiltransferasa/metabolismo , Animales , Glutatión Transferasa/metabolismo , Isoenzimas/metabolismo , Riñón/enzimología , Hígado/enzimología , Pulmón/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Especificidad de Órganos , Receptores de Hidrocarburo de Aril/fisiologíaRESUMEN
In vitro and in vivo effects of naringin on microsomal monooxygenase were studied to evaluate the drug interaction of this flavonoid. In vitro addition of naringin up to 500 microM had no effects on benzo(a)pyrene hydroxylase (AHH) activity of mouse liver microsomes. In contrast, the aglycone naringenin at 300 to 500 microM decreased AHH activity by 50% to 60%. Analysis of Lineweaver-Burk and Dixon plots indicated that naringenin competitively inhibited AHH activity with an estimated Ki of 39 microM. Naringenin at 100 microM also reduced metabolic activation of benzo(a)pyrene to genotoxic products as monitored by umuC gene expression response in Salmonella typhimurium TA1535/pSK1002. In the presence of equimolar naringenin and benzo(a)pyrene, umuC gene expression presented as beta-galactosidase activity was reduced to a level similar to the control value. Administration of a liquid diet containing 10 mg/ml naringin for 7 days caused 38% and 49% decreases of AHH and 7-methoxyresorufin O-demethylase activities, respectively. In contrast, the administration had no effects on cytochrome P450 (P450)-catalyzed oxidations of 7-ethoxyresorufin, 7-ethoxycoumarin, N-nitrosodimethylamine, nifedipine, erythromycin and testosterone. Microsomal P450 and cytochrome b5 contents and NADPH-P450 reductase activity were not affected. Immunoblot analysis using MAb 1-7-1, which immunoreacted with both P450 1A1 and 1A2, revealed that the level of P450 1A2 protein was decreased by 38%. These results demonstrate that naringenin is a potent inhibitor of AHH activity in vitro and naringin reduces the P450 1A2 protein level in vivo. These effects may indicate a chemopreventive role of naringin against protoxicants activated by P450 1A2.
Asunto(s)
Antioxidantes/farmacología , Hidrocarburo de Aril Hidroxilasas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Flavanonas , Flavonoides/farmacología , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Animales , Antioxidantes/farmacocinética , Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Benzopireno Hidroxilasa/antagonistas & inhibidores , Benzopireno Hidroxilasa/metabolismo , Inhibidores Enzimáticos del Citocromo P-450 , Citocromos b5/antagonistas & inhibidores , Citocromos b5/metabolismo , Relación Dosis-Respuesta a Droga , Antagonistas de Estrógenos/farmacocinética , Antagonistas de Estrógenos/farmacología , Flavonoides/farmacocinética , Cinética , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Aflatoxin B1 (AFB1) is a potent hepatocarcinogen in experimental animals and a hazard to human health in several parts of the world. Implementation of rational intervention plans requires understanding of aspects of the roles of individual chemical steps involved in its disposition. AFB1 is activated to AFB1 exo-8,9-epoxide primarily by cytochrome P450 (P450) enzymes, particularly P450 3A4. However, P450 3A4 and other P450s also oxidize AFB1 to less dangerous products. The exo-epoxide is unstable in H2O (t1/2 1 s at 25 degreesC, k=0.6 s-1) and the diol product undergoes base-catalyzed rearrangement to a dialdehyde that reacts with protein lysine residues. AFB1 exo-8, 9-epoxide reacts with DNA to give adducts in high yield (>98%). This interaction is characterized by a Kd of approximately 1.4 mM, intercalation between base pairs, and rapid reaction with the guanyl N7 atom (k approximately 40 s-1). A proton field on the periphery of DNA is postulated to catalyze hydrolysis and also conjugation. Rat and especially human epoxide hydrolase show very little rate acceleration of hydrolysis of AFB1 exo- or endo-8,9-epoxide. However, glutathione transferases (GSTs) can catalyze AFB1 exo-8,9-epoxide conjugation. Kinetic analysis indicates a range of ratios of kcat/Kd varying from 10 to 1700 s-1 M-1, with the polymorphic GST M1-1 having the highest activity of the human GSTs. Studies with human hepatocytes indicate a major role for GST M1-1 in AFB1 conjugation and that the model chemoprotective agent oltipraz can act by both inducing GSTs and inhibiting P450s.
Asunto(s)
Aflatoxina B1/farmacocinética , Carcinógenos/farmacocinética , Animales , Biotransformación , Sistema Enzimático del Citocromo P-450/metabolismo , ADN/efectos de los fármacos , Epóxido Hidrolasas/metabolismo , Humanos , Inactivación MetabólicaRESUMEN
Cytochrome P450 3A7 is the major P450 form present in fetal liver tissue and may be responsible for the detoxification of many drugs that reach the fetal circulation. We report the development of bacterial expression systems for P450 3A7. Maximal yields (up to 50 nmol P450/liter culture) were obtained with a construct in which the 5'-terminus of the 3A7 cDNA was modified to include the MALLLAVFL N-terminal sequence of recombinant bovine P450 17A (H. J. Barnes, M. P. Arlotto, and M. R. Waterman, Proc. Natl. Acad. Sci. USA 88, 5597-5601, 1991) and to incorporate several downstream amino acid substitutions derived from the P450 3A5 sequence. This sequence also appeared optimal for expression of P450 3A4 and 3A5. Recombinant P450 3A7 was partially purified using ion-exchange and hydroxylapatite chromatography and reconstituted with NADPH-cytochrome P450 reductase, cytochrome b5, and lipids. Activity comparable to that of P450 3A4 was demonstrated toward a number of procarcinogens. An alternative approach was used to further characterize recombinant 3A7 due to low yields of recombinant protein in the expression and poor recovery in the purification. P450 3A7 was subcloned into a bicistronic vector containing human NADPH-cytochrome P450 reductase and expressed in bacteria. Recombinant P450 3A7 coexpressed in bacterial membranes with NADPH-cytochrome P450 reductase showed similar levels of activity toward erythromycin (N-demethylation) and ethylmorphine (N-demethylation) to P450 3A4 and 3A5 expressed in the same system, whereas 3A7 was less active toward midazolam (1'- and 4-hydroxylation) and nifedipine (oxidation).
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Sistema Enzimático del Citocromo P-450/biosíntesis , NADH NADPH Oxidorreductasas/biosíntesis , Proteínas Recombinantes/biosíntesis , Secuencia de Aminoácidos , Apoproteínas/biosíntesis , Apoproteínas/genética , Secuencia de Bases , Biotransformación , Carcinógenos/metabolismo , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Escherichia coli/genética , Genes , Vectores Genéticos , Humanos , Oxigenasas de Función Mixta/biosíntesis , Oxigenasas de Función Mixta/genética , Datos de Secuencia Molecular , Mutagénesis , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/metabolismo , NADPH-Ferrihemoproteína Reductasa , Especificidad por SustratoRESUMEN
Aflatoxin B1 (AFB1) must be activated to the electrophilic AFB1 exo-8,9-epoxide to be genotoxic and carcinogenic. A role for epoxide hydrolase in detoxication has been suggested but never directly addressed. In light of recent studies determining the instability of AFB1 exo-8,9-epoxide in H2O, a role for epoxide hydrolase appears dubious. Rat liver or recombinant rat epoxide hydrolase provided an enhancement to the already fast hydrolysis rate of up to 22%. Purified human epoxide hydrolase provided no detectable enhancement to the rate of chemical hydrolysis. Some reduction in the genotoxicity of AFB1 was observed when the ratio of rat epoxide hydrolase to cytochrome P450 was high (approximately 50-fold). An 80-fold excess of human epoxide hydrolase over cytochrome P450 only produced an effect of approximately 25% inhibition. It appears, therefore, that there is little evidence to support a role for epoxide hydrolase in the detoxication of AFB1.
Asunto(s)
Aflatoxina B1/análogos & derivados , Epóxido Hidrolasas/metabolismo , Hígado/enzimología , Aflatoxina B1/metabolismo , Animales , Humanos , Hidrólisis , Cinética , Pruebas de Mutagenicidad , Conejos , Ratas , Respuesta SOS en GenéticaRESUMEN
Aflatoxin B1 (AFB1) exo-8,9-epoxide, the reactive product of the hepatocarcinogen AFB1, is stable in aprotic solvents but hydrolyzes rapidly in H2O at 25 degrees C and pH 7 (t1/2 = 1 s). However, it is also known that some glutathione (GSH) transferases can conjugate the epoxide with GSH to give the adduct in high yield. We developed an approach to estimating kinetic parameters for reactions involving this epoxide or other substrates that are unstable to H2O. Varying concentrations of the (anhydrous) epoxide and GSH transferase were mixed and the GSH conjugates were measured. The final concentrations of product were known for each set of the starting epoxide and enzyme concentrations in a modeling approach, where the competition with the hydrolysis reaction is considered with two variables, a K for binding of the enzyme and epoxide and a rate k2, which includes microscopic steps following complex formation and resulting in conjugate formation. The ratio k2/K, a measure of enzyme efficiency, varied among individual recombinant GSH transferases in the the order (rat) 10-10 >> 3-3 > (human) M1-1 > T1-1 > A1-1 > P1-1 > A2-2, from 3 x 10(6) to 10 M(-1) s(-1). The high ratio of M1-1 among the human GSH transferase enzymes tested is consistent with other work in which GSH-AFB1 conjugates were not detected in hepatocytes with an M1 null polymorphism. This general kinetic approach should be applicable to estimation of kinetic parameters involved in the interaction of other unstable substrates with enzymes.
Asunto(s)
Aflatoxina B1/análogos & derivados , Glutatión Transferasa/metabolismo , Glutatión/metabolismo , Aflatoxina B1/metabolismo , Animales , Humanos , Cinética , Ratas , Proteínas Recombinantes/metabolismoRESUMEN
Cytochrome P450 (P450) 3A4 is the most abundant human P450 and oxidizes a diversity of substrates, including various drugs, steroids, carcinogens, and macrolide natural products. In some reactions, positive cooperativity has been reported in microsomal studies. Flavonoids, e.g., 7,8-benzoflavone (alpha-naphthoflavone, alpha NF), have been shown to stimulate some reactions but not others. In systems containing purified recombinant bacterial P450 3A4, positive cooperativity was seen in oxidations of several substrates, including testosterone, 17 beta-estradiol, amitriptyline, and most notably aflatoxin (AF) B1. With these and other reactions, alpha NF typically reduced cooperativity (i.e., the n value in a Hill plot) while either stimulating or inhibiting reactions. With the substrate AFB1, alpha NF both stimulated 8,9-epoxidation and inhibited 3 alpha-hydroxylation. The same patterns were seen with AFB1 in a fused P450 3A4-NADPH-P450 reductase protein. alpha NF did not alter patterns of activity plotted as a function of NADPH-P450 reductase concentration in systems containing the individual proteins. The patterns of AFB1 oxidation to the two products were modified considerably in systems in which NADPH-P450 reductase was replaced with a flavodoxin or ferredoxin system, iodosylbenzene, or cumene hydroperoxide. AFB2, which differs from AFB1 only in the presence of a saturated 8,9-bond, was not oxidized by P450 3A4 but could inhibit AFB1 oxidation. These and other results are considered in the context of several possible models. The results support a model in which an allosteric site is involved, although the proximity of this putative site to the catalytic site cannot be ascertained as of yet. In order to explain the differential effects of alpha NF and reduction systems on the two oxidations of AFB1, a model is presented in which binding of substrate in a particular conformation can facilitate oxygen activation to enhance catalysis.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Microsomas Hepáticos/enzimología , Oxigenasas de Función Mixta/metabolismo , Aflatoxina B1/metabolismo , Amitriptilina/metabolismo , Benzoflavonas/farmacología , Carbamazepina/metabolismo , Cromatografía Líquida de Alta Presión , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Citocromos b5/metabolismo , Diazepam/metabolismo , Estradiol/metabolismo , Humanos , Hidroxilación , Cinética , Cloruro de Magnesio/farmacología , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/aislamiento & purificación , Modelos Químicos , Oxidación-Reducción , Especificidad por Sustrato , Testosterona/metabolismoRESUMEN
Many catalytic activities of cytochrome P450 (P450) 3A4, the major human liver P450 enzyme, require cytochrome b5 (b5) for optimal rates. The stimulatory effect of b5 on P450 reactions has generally been thought to be due to transfer of electrons from ferrous b5 to the ferrous P450-O2-substrate complex. We found that apo-b5, devoid of heme, could substitute for b5 in stimulating two prototypic activities, testosterone 6beta hydroxylation and nifedipine oxidation. The stimulatory effect was not seen with albumin, hemoglobin, catalase, or cytochrome c. Apo-b5 could not substitute for b5 in a testosterone 6beta hydroxylation system composed of NADH-b5 reductase and P450 3A4. Rates of electron transfer from NADPH-P450 reductase to ferric P450 3A4 were too slow (<2 min-1) to support testosterone 6beta hydroxylation ( approximately 14 min-1) unless b5 or apo-b5 was present, when rates of approximately 700 min-1 were measured. The oxidation-reduction potential (Em,7) of the ferric/ferrous couple of P450 3A4 was unchanged ( approximately -310 mV) under different conditions in which the kinetics of reduction were altered by the addition of substrate and/or apo-b5. Rapid reduction of P450 3A4 required substrate and a preformed complex of P450 3A4, NADPH-P450 reductase, and b5; the rates of binding of the proteins to each other were 2-3 orders of magnitude less than reduction rates. We conclude that b5 can facilitate some P450 3A4-catalyzed oxidations by complexing with P450 3A4 and enhancing its reduction by NADPH-P450 reductase, without directly transferring electrons to P450.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Citocromos b5/metabolismo , Oxigenasas de Función Mixta/metabolismo , Animales , Apoproteínas/metabolismo , Catálisis , Bovinos , Clonación Molecular , Citocromo P-450 CYP3A , Sistema Enzimático del Citocromo P-450/química , Grupo Citocromo b/metabolismo , Citocromos b , Citocromos b5/química , Transporte de Electrón , Escherichia coli , Humanos , Cinética , Microsomas Hepáticos/enzimología , Oxigenasas de Función Mixta/química , NADPH-Ferrihemoproteína Reductasa/metabolismo , Conejos , Proteínas Recombinantes/metabolismo , Espectrofotometría , Especificidad por Sustrato , Testosterona/metabolismoAsunto(s)
Cadmio/toxicidad , Carcinógenos/toxicidad , Cloruros/toxicidad , Sistema Enzimático del Citocromo P-450/metabolismo , Contaminantes Ambientales/toxicidad , Metalotioneína/metabolismo , 7-Alcoxicumarina O-Dealquilasa/metabolismo , Animales , Benzopireno Hidroxilasa/metabolismo , Cloruro de Cadmio , Citocromo P-450 CYP1A1 , Citosol/efectos de los fármacos , Citosol/enzimología , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Branquias/efectos de los fármacos , Branquias/enzimología , Branquias/metabolismo , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , NADPH-Ferrihemoproteína Reductasa/metabolismo , Oxidorreductasas/metabolismo , Espectrofotometría Atómica , Taiwán , TilapiaRESUMEN
Levels and catalytic activities of cytochrome P450 (P450) enzymes involved in the oxidation of drugs and carcinogens were determined in human adult lungs and fetal livers and compared with those in microsomes from adult livers. P450s immunoreactive with anti-human P4501A1 and anti-human P4503A antibodies were detected in fetal liver microsomes by immunoblotting analysis, and P450s related P4501A1, 2A6, 2C9, 2E1, and 3A4 were determined in adult lung microsomes; all of these P450 enzymes were detected in much higher amounts in adult liver microsomes except that P4501A2 was only the 1A subfamily of P450 found in adult livers. Drug oxidation activities with the substrates ethoxyresorufin, coumarin, 7-ethoxycoumarin, bufuralol, and testosterone were determined in these microsomes, and we found that none of the activities were higher in microsomes of adult lungs and fetal livers than in adult livers. Activation of procarcinogens to reactive metabolites that induce umu gene expression in Salmonella typhimurium TA1535/pSK1002 or NM2009 was also examined and it was found that activities with (+)- and (-)-enantiomers of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene were higher in fetal liver microsomes than adult lung or liver microsomes. The adult liver and lung activities for these two procarcinogens were similar on the basis of microsomal protein contents despite the fact that p450 contents are higher in liver than lung microsomes. alpha-Naphthoflavone, a known inhibitor of P4501A-related activities, did not affect these procarcinogen activation in fetal liver microsomes. Fetal liver microsomes catalyzed activation of aflatoxin B1 and sterigmatocystin, two procarcinogens known to be activated by P4503A4/7 in humans, although activation of carcinogenic arylamines that are good substrates for P4501A2 was much lower in microsomes of fetal livers and adult lungs than in adult livers. These results suggest that in human fetal livers at least two P450 enzymes, a form of P450 that is immunoreactive P4501A1 and P4503A7, are actually expressed and these enzymes are suggested as being involved in the activation of the (+)- and (-)-enantiomers of 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene and the carcinogenic mycotoxins, respectively. The exact nature of the former enzyme in fetal livers is unknown. In adult human lungs, several P450 enzymes are expressed, although the precise roles of these enzymes in the oxidation of xenobiotics were not determined due to the low level of expression of these P450s.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Microsomas/enzimología , Xenobióticos/metabolismo , Anticuerpos/inmunología , Anticuerpos/metabolismo , Carcinógenos/metabolismo , Inhibidores Enzimáticos del Citocromo P-450 , Feto/metabolismo , Regulación Bacteriana de la Expresión Génica/genética , Humanos , Immunoblotting , Hígado/metabolismo , Pulmón/metabolismo , Microsomas/química , Oxidación-Reducción , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Salmonella typhimurium/metabolismo , Testosterona/metabolismoRESUMEN
Rabbit cytochrome P450 1A2 was modified with succinimidyl carbonate poly(ethylene glycol) monomethyl ether, purified by size exclusion high performance liquid chromatography, and lyophilized. Modification of cytochrome P450 1A2 caused no structural deformation of the heme as evidenced by the similarity of the spectral signatures for both the ferric form and the ferrous-CO complex to the respective forms for the unmodified enzyme. Ethoxyresorufin O-deethylation activity in the presence of iodosobenzene for the modified enzyme was comparable to that of the native enzyme.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Microsomas Hepáticos/enzimología , Oxidorreductasas/metabolismo , Animales , Cromatografía Líquida de Alta Presión , Citocromo P-450 CYP1A2 , Sistema Enzimático del Citocromo P-450/biosíntesis , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Liofilización , Cinética , Oxidorreductasas/biosíntesis , Oxidorreductasas/química , Oxidorreductasas/aislamiento & purificación , Polietilenglicoles , Conejos , EspectrofotometríaRESUMEN
In recent years there has been considerable interest in the effect of variations in activities of xenobiotic-metabolizing enzymes on cancer incidence. This interest has accelerated with the development of methods for analyzing genetic polymorphisms. However, progress in epidemiology has been slow and the contributions of polymorphisms to risks from individual chemicals and mixtures are often controversial. A series of studies is presented to show the complexities encountered with a single chemical, aflatoxin B1 (AFB1). AFB1 is oxidized by human cytochrome P450 enzymes to several products. Only one of these, the 8,9-exo-epoxide, appears to be mutagenic and the others are detoxication products. P450 3A4, which can both activate and detoxicate AFB1, is found in the liver and the small intestine. In the small intestine, the first contact after oral exposure, epoxidation would not lead to liver cancer. The (nonenzymatic) half-life of the epoxide has been determined to be approximately 1 sec at 23 degrees C and neutral pH. Although the half-life is short, AFB1-8,9-exo-epoxide does react with DNA and glutathione S-transferase. Levels of these conjugates have been measured and combined with the rate of hydrolysis in a kinetic model to predict constants for binding of the epoxide with DNA and glutathione S-transferase. A role for epoxide hydrolase in alteration of AFB1 hepatocarcinogenesis has been proposed, although experimental evidence is lacking. Some inhibition of microsome-generated genotoxicity was observed with rat epoxide hydrolase; further information on the extent of contribution of this enzyme to AFB1 metabolism is not yet available.
Asunto(s)
Aflatoxina B1/metabolismo , Carcinógenos/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Epóxido Hidrolasas/metabolismo , Glutatión Transferasa/metabolismo , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/enzimología , Aflatoxina B1/toxicidad , Animales , Carcinógenos/toxicidad , Aductos de ADN/metabolismo , Escherichia coli/enzimología , Humanos , Hidrólisis , Neoplasias Hepáticas/epidemiología , Oxidación-Reducción , Ratas , Proteínas Recombinantes/metabolismoRESUMEN
CYP3A4 represents the most important form of human cytochrome P450 active in drug metabolism. Reconstitution of this enzyme has in the past been a major problem. Using purified cDNA-expressed CYP3A4 incorporated into membranous vesicles made from microsomal phospholipids, rates of nifedipine and testosterone oxidation of about 60 nmol/nmol P450/min were achieved, whereas similar reconstitution into dilauroyl-phosphatidylcholine micelles was unsuccessful. A higher Vmax for nifedipine oxidation was obtained in negatively charged vesicles as compared to neutral membranes, whereas the membrane charge did not influence the Km. It is concluded that the native function of CYP3A4 requires a negatively charged microsomal membrane.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Oxigenasas de Función Mixta/metabolismo , Animales , Membrana Celular/enzimología , Citocromo P-450 CYP3A , Humanos , Hidroxilación , Lípidos de la Membrana/metabolismo , Potenciales de la Membrana , NADH NADPH Oxidorreductasas/metabolismo , NADPH-Ferrihemoproteína Reductasa , Nifedipino/metabolismo , Ratas , Especificidad por Sustrato , Testosterona/metabolismoRESUMEN
Alfentanil, sufentanil, and fentanyl are synthetic opioids that are metabolized by oxidative N-dealkylation in the liver. We have previously shown that cytochrome P-450 3A4 (CYP3A4) contributes significantly to human liver microsomal alfentanil oxidation. Since identification of specific drug-metabolizing enzymes allows prediction of the variables affecting drug metabolism, the purpose of the present study was to identify the P-450 enzymes responsible for sufentanil and fentanyl metabolism in human liver microsomes. Microsomal preparations fortified with a reduced nicotinamide-adenine dinucleotide phosphate-generating system were incubated with 0.25 microM 3H-fentanyl or 3H-sufentanil. Rates of N-dealkylated metabolite formation significantly correlated with nifedipine oxidation activity (a marker of CYP3A4 activity) for fentanyl and sufentanil (r = 0.93 and 0.87, n = 18, respectively), but not with the oxidation activity for ethoxyresorufin (CYP1A2), S-mephenytoin (CYP2C19), bufuralol (CYP2D6), or chlorzoxazone (CYP2E1). Gestodene and troleandomycin (chemical inhibitors of CYP3A4) and antibody to CYP3A4 inhibited N-dealkylation of fentanyl and sufentanil. Chemical inhibitors of CYP2C, 2E1, and 2D6 did not inhibit N-dealkylation of fentanyl and sufentanil. Recombinant CYP3A4 expressed in Escherichia coli showed N-dealkylation activity of fentanyl and sufentanil, while expressed CYP1A2, 2C10, and 2E1 enzymes did not. We conclude that CYP3A4 is responsible for fentanyl and sufentanil N-dealkylation in vitro.