RESUMEN
OBJECTIVE: The use of cyclosporin A (CSA) and tacrolimus (TAC) in organ transplantation and in the therapy of immune disorders is often hampered by adverse effects, mainly nephro-, hepato- and neurotoxicity. For the development of these side effects, among others, an increased formation of reactive oxygen species, probably generated by the cytochrome P450 (CYP) system, has been accused. Since in this respect literature data are inconsistent, in the present study possible pro- and/or antioxidant effects of CSA and TAC and the involvement of the CYP system were re-evaluated in vitro. METHODS: Effects of CSA and TAC were examined on CYP mediated oxidase functions by stimulated lipid peroxidation (LPO), H2O2 production, and lucigenin (LC) or luminol (LM) amplified chemiluminescence (CL) in liver microsomes of either untreated rats or of rats treated with beta-naphthoflavone (BNF), phenobarbital (PB) or dexamethasone (DEX) and in human liver microsomes. RESULTS: In rat liver microsomes, CSA displayed pro-oxidant properties (though only very slightly), whereas in human liver microsomes small antioxidant effects were seen. With TAC in both species the antioxidant capacity prevailed. Treatment of rats with BNF or DEX caused an increase in the pro-oxidant effects of CSA with respect to LPO or LM-CL, whereas in liver microsomes of DEX-treated rats H2O2 production and LC-CL were diminished. CONCLUSIONS: CSA seems to have both pro-oxidant and antioxidant properties, whereas with TAC mainly an antioxidant capacity was seen. The CYP system seems to be involved in the pro-oxidant influence of CSA. Whether pro-oxidant or antioxidant effects predominate may depend on the antioxidant capacity of a tissue and on the CYP isoforms mainly present.
Asunto(s)
Antioxidantes/farmacología , Ciclosporina/farmacología , Sistema Enzimático del Citocromo P-450/biosíntesis , Inmunosupresores/farmacología , Hígado/efectos de los fármacos , Oxidantes/farmacología , Tacrolimus/farmacología , Animales , Dexametasona/farmacología , Relación Dosis-Respuesta a Droga , Inducción Enzimática , Humanos , Peróxido de Hidrógeno/metabolismo , Técnicas In Vitro , Isoenzimas/biosíntesis , Peroxidación de Lípido , Hígado/enzimología , Masculino , Microsomas Hepáticos , Fenobarbital/farmacología , Ratas , Ratas Wistar , beta-naftoflavona/farmacologíaRESUMEN
Previous experiments have shown that both in vivo and in vitro pre-treatment with various hormones increases the renal transport capacity for weak organic acids, such as PAH, in rats. The aim of the present study was to test whether or not accumulation of the anticancer drugs methotrexate (MTX), cisplatin (CP), raltitrexed (Tomudex) and topotecan (Hycamtin) can be increased in intact, healthy rat and human renal cortical slices and in human renal cell carcinoma (RCC). Intact, healthy human tissue was obtained from tumour bearing kidneys of patients suffering from RCC. Experiments were intended as a new approach to overcome so-called multidrug resistance. Kidney tissue slices were incubated for 24 h in William's medium E containing various concentrations of dexamethasone, T(3), or EGF. Thereafter slices were placed in anticancer drug containing Cross-Taggart medium and the drug uptake into kidney tissue was measured for 2 h. In intact rat and human renal tissue slices, the uptake of p-aminohippurate (PAH = reference substance) increased significantly after incubation in dexamethasone containing medium (134% and 156%, respectively). There were no stimulating effects of either T(3) or EGF on PAH accumulation. On the other hand, only the accumulation of MTX, but not of CP, raltitrexed or topotecan, was significantly enhanced after hormone pre-treatment both in intact renal tissue and in RCC. A stimulation of renal PAH accumulation can be performed ex vivo, as reported previously, both in intact rat and human renal cortical slices and in RCC. Discrepancies between the effects of dexamethasone and T(3) or EGF indicate different modes of action of these substances at the cellular level. Unfortunately, with the exception of MTX, the uptake of anticancer drugs can not be stimulated effectively ex vivo in human RCC tissue by the substances used. Evidently the transport of these anticancer drugs out of the kidney cells is more effective than their uptake.
Asunto(s)
Antineoplásicos/farmacocinética , Carcinoma de Células Renales/metabolismo , Dexametasona/farmacología , Factor de Crecimiento Epidérmico/farmacología , Glucocorticoides/farmacología , Neoplasias Renales/metabolismo , Riñón/metabolismo , Triyodotironina/farmacología , Animales , Antimetabolitos Antineoplásicos/farmacocinética , Transporte Biológico/efectos de los fármacos , Cisplatino/farmacocinética , Femenino , Humanos , Metotrexato/farmacocinética , Quinazolinas/farmacocinética , Ratas , Ratas Wistar , Tiofenos/farmacocinética , Topotecan/farmacocinéticaRESUMEN
Interactions of 27 steroids, among them 17 derivatives such as ethers, sulfates and amidosulfonates derived from 17 beta- and 17 alpha-estradiol, from testosterone and alpha- and beta-dihydrotesosterone and from dehydroepiandrosterone with rat liver microsomal cytochromes P450 (P450) were investigated in vitro by assessing binding to P450 and effects on P450 mediated monooxygenase functions as measured by different model reactions: ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD) and ethylmorphine N-demethylation (EMND). With the exception of 17 alpha-estradiol-3-dimethylamidosulfonate, estrone, its -3-methylether and -3-amidosulfonate and testosterone, all other steroids displayed type I or reverse type I binding to P450. All steroids inhibited EROD activity in micromolar concentrations. An additional strong inhibition of ECOD and EMND activities was only demonstrated for the androgens and progestins. Estriol, estrone and mestranol displayed less inhibitory actions on the model reactions than estradiol. No major differences in comparison to the parent compounds were noted with the other derivatives. The only exceptions were 17 beta-(8,9-dehydro-14 alpha,15 alpha-methylene)estradiol, which displayed stronger effects than estradiol, and dehydroepiandrosterone-3-sulfate, which was less effective than dehydroepiandrosterone. Possible antioxidant properties of the steroids were examined by the stimulated lipid peroxidation (LPO), H2O2 production, and lucigenin (LC) and luminol (LM) amplified chemiluminescence (CL) using rat liver microsomes. Additionally, the influence on rat whole blood chemiluminescence (WB-CL) was assessed. All the estrogens, but not their methylethers and amidosulfonates inhibited LPO in micromolar concentrations. The effects on the other oxidase model reactions or on WB-CL were less distinct. Only ethinylestradiol and 17 beta-(8,9-dehydro-14 alpha,15 alpha-methylene)estradiol displayed a strong inhibitory action on all model reactions. With the exception of dehydroepiandrosterone-3-sulfate, which in general had only weak effects, the androgen and progestin derivatives, in contrast, strongly decreased H2O2 formation and LM- and LC-CL, but were mostly ineffective on LPO and WB-CL.
Asunto(s)
Androstenodiona/análogos & derivados , Sistema Enzimático del Citocromo P-450/metabolismo , Deshidroepiandrosterona/análogos & derivados , Estradiol/análogos & derivados , Microsomas Hepáticos/efectos de los fármacos , 7-Alcoxicumarina O-Dealquilasa/metabolismo , Androstenodiona/metabolismo , Androstenodiona/farmacología , Animales , Citocromo P-450 CYP1A1/metabolismo , Deshidroepiandrosterona/metabolismo , Deshidroepiandrosterona/farmacología , Estradiol/metabolismo , Estradiol/farmacología , Etilmorfina-N-Demetilasa/metabolismo , Hígado/metabolismo , Mediciones Luminiscentes , Masculino , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Testosterona/análogos & derivados , Testosterona/metabolismo , Testosterona/farmacologíaRESUMEN
In vitro melatonin binds to human and rat liver microsomal cytochrome P-450 (P450) according to a type II substrate. The affinity is similar to that of aniline with a general left-shift. Melatonin interferes with model monooxygenase reactions indicative of different P450 forms in humans and rats (in humans according to the lower specific P450 content less pronounced): the strongest inhibition was found for ethoxyresorufin O-deethylation, indicating the binding to P450 1A, the binding to P450 2B (ethoxycoumarin O-deethylation) was less pronounced, the least inhibition was found for P450 3A (ethylmorphine N-demethylation) reaction. The oxidase function was also inhibited: luminol amplified chemiluminescence was more inhibited than the lucigenin amplified one, hydrogen peroxide formation was inhibited at concentrations higher than 10(-4) M, microsomal NADPH/Fe stimulated lipid peroxidation was inhibited at concentrations higher than 10(5) M. In vivo melatonin prolonged hexobarbital sleeping time in rats in a dose dependent manner (ip. co-administration of 1, 5 and 20 mg/kg b.w. melatonin with 100 mg/kg hexobarbital). Immediately after awakening the animals were sacrificed: a small increase in P450 concentrations cannot be explained, no changes in P450 monooxygenase or oxidase activities nor in microsomal lipid peroxidation or GSH status could be observed.
Asunto(s)
Antioxidantes/metabolismo , Antioxidantes/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Hígado/metabolismo , Melatonina/farmacología , Microsomas Hepáticos/enzimología , Compuestos de Anilina/metabolismo , Compuestos de Anilina/farmacología , Animales , Hexobarbital/metabolismo , Hexobarbital/farmacología , Humanos , Hipnóticos y Sedantes/metabolismo , Hipnóticos y Sedantes/farmacología , Técnicas In Vitro , Peroxidación de Lípido/efectos de los fármacos , Hígado/efectos de los fármacos , Microsomas Hepáticos/efectos de los fármacos , NADP/metabolismo , Ratas , Ratas Wistar , Sueño/efectos de los fármacosRESUMEN
Antioxidants of the vitamin E family have protective effects against metal toxicity. We examined the protective effect of racemic LLU-alpha [2,7,8-trimethyl-2-(carboxyethyl)-6-hydroxychroman] a metabolite of gamma-tocopherol, in comparison to the effect of alpha- and gamma-tocopherol in rats treated with sodium dichromate (Cr) or thallium sulfate (Tl). We measured metal nephrotoxicity based on urinary protein excretion and discussed it with respect to the metal concentration in renal tissue. The ranking of antioxidant activity (iron stimulated lipid peroxidation, luminol and lucigenin amplified chemiluminescence) was determined in the following order: alpha-tocopherolAsunto(s)
Antioxidantes/farmacología
, Cromanos/farmacología
, Riñón/efectos de los fármacos
, Metales/toxicidad
, Propionatos/farmacología
, Vitamina E/farmacología
, Animales
, Antioxidantes/metabolismo
, Cromanos/sangre
, Cromatos/sangre
, Cromatos/toxicidad
, Femenino
, Mediciones Luminiscentes
, Metales/sangre
, Canales de Potasio/efectos de los fármacos
, Propionatos/sangre
, Ratas
, Ratas Wistar
, Talio/sangre
, Talio/toxicidad
, Vitamina E/sangre
RESUMEN
Carps, both sexes, 3 years old, weighing about 1 kg, and tenches of both sexes, 6 years old, weight about 250 g, were caught from a Thuringian lake without industrial pollution in November 1995 (fish without food uptake, water temperature at about 10 degrees C) and kept for 2 weeks in basins with clean water and addition of 0, 0.1, 1.0 or 10.0 mg/l phenobarbital-Na (PB). The concentration of PB was controlled during and at the end of the exposure period. The animals were fed pellets, but no food uptake was observed. After 24-48 h in fresh water the fish were sacrificed and the following hepatic parameters were immediately determined biochemically: monooxygenase functions: cytochrome P450 (P450) content, ethylmorphine N-demethylation (EN), ethoxycoumarin O-deethylation (ECOD), ethoxyresorufin O-deethylation (EROD), 7-benzyloxy-4-methyl-coumarin O-debenzylation (BCDB); oxidase function indicators: microsomal Fe2+/NADPH dependent hydrogen peroxide formation (H2O2), microsomal Fe2+/NADPH dependent luminol and lucigenin amplified chemiluminescence (LMCL, LCCL), microsomal Fe2+/NADPH dependent lipid peroxide formation (LPO); oxidative state: lipid peroxidation products (TBARS) and GSH and GSSG. Additionally, the expression of three P450 isoforms, 1A1, 2B and 3A, was assessed immunohistochemically in tissue samples from brain, gill, heart, spleen, liver, gut and ovary of both fish species and in kidney of tenches. PB did not influence body or liver weights, but increased liver P450 concentration in both species by 50-100%, though not significantly. Carp: PB increased both EN and EROD significantly, but not ECOD and BCDB; H2O2 and TBARS were enhanced significantly. LPO, LMCL and LCCL were not significantly influenced. Tench: PB increased all monooxygenase reactions (EN, ECOD, BCDB and EROD), though only significantly ECOD; H2O2 was elevated only after treatment with 0.1 mg/l PB, whereas LPO was decreased (!) after treatment by all three concentrations, though significantly only after 1.0 mg/l PB. LMCL was depressed (not significantly), but LCCL increased 5fold. TBARS were significantly enhanced. P450 1A1 subtype expression was concentration dependently elevated by PB in gill and liver of both fish and in the heart and kidney of tenches, P450 2B and 3A isoforms expression was induced in brain, gill, heart, liver and gut of both fish and in the kidney of tenches. In summary, the increased activities of the monooxygenase reactions tested and the elevated expression of all three P450 isoforms investigated in certain tissues indicate an induction of the P450 families 1, 2 and 3 by PB in fish.
Asunto(s)
Carpas/metabolismo , Sistema Enzimático del Citocromo P-450/biosíntesis , Glutatión/metabolismo , Isoenzimas/biosíntesis , Peroxidación de Lípido/efectos de los fármacos , Hígado/efectos de los fármacos , Fenobarbital/farmacología , Especies Reactivas de Oxígeno/metabolismo , Esteroide Hidroxilasas/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Inducción Enzimática , Femenino , Agua Dulce , Hígado/metabolismo , Masculino , Especificidad de la EspecieRESUMEN
BACKGROUND: Nitric oxide (NO) synthesis is inhibited by the ADMA that accumulates in the plasma of patients with renal failure; however, the concentration of SDMA also is enhanced. Therefore, it has been hypothesized that ADMA and SDMA may contribute to hypertension in these patients. METHODS: We measured the concentrations of ADMA, SDMA and 21 endogenous amino acids in 257 persons by high pressure liquid chromatography (HPLC). RESULTS: The plasma concentrations of both ADMA and SDMA were significantly elevated in patients with chronic renal failure (CRF). The increase was more pronounced for SDMA (2.05 +/- 0.1 micromol/L vs. 0.5 +/- 0.04 micromol/L), whereas it was only moderate for ADMA (0.85 +/- 0.03 micromol/L vs. 0.73 +/- 0.06 micromol/L). In dialysis patients, the concentrations were further increased (ADMA, 1.05 +/- 0.04 micromol/L; SDMA, 2.68 +/- 0.13 micromol/L). After kidney transplantation, the concentration of SDMA returned to the baseline value (1.15 +/- 0.11 micromol/L), but that of ADMA remained enhanced (0.99 +/- 0.06 micromol/L). CONCLUSIONS: In CRF, especially the concentration of SDMA is significantly increased. Not only ADMA, but also SDMA are likely to be responsible for hypertension. Competition for reabsorption between SDMA and arginine within the kidney has to be considered for the interpretation of changes in the ratio between dimethylarginines and arginine in renal failure. Hemodialysis is not suitable for a long-lasting removal of methylarginines. Whether the administration of arginine could have promising effects on hypertension and complications of CRF needs to be studied in prospective trials.
Asunto(s)
Arginina/análogos & derivados , Arginina/sangre , Fallo Renal Crónico/sangre , Adulto , Anciano , Aminoácidos/sangre , Presión Sanguínea , Estudios de Casos y Controles , Creatinina/sangre , Femenino , Humanos , Fallo Renal Crónico/fisiopatología , Fallo Renal Crónico/terapia , Trasplante de Riñón , Masculino , Persona de Mediana Edad , Diálisis RenalRESUMEN
Metabolic pathways of estrogens are the formation of catechol estrogens (CE; 2- and 4-hydroxy-estrogens), redox cycling of CE and free radical generation, mediated through cytochrome P450 (P450) oxidase/reductase activity. In previous investigations subchronic administration of estrogens showed prooxidative and antioxidative activities in rat liver microsomes (BARTH et al. 1999). To find out whether or not catechol metabolites are responsible for prooxidative activity, we checked 2- and 4-hydroxy-estradiol (2OH-E2 and 4OH-E2) and the non-catechol metabolite 6alpha-hydroxy-estradiol (6alpha-OH-E2) for formation of reactive oxygen species in liver microsomes of 30-day-old male Wistar rats after 5 days treatment (1, 10 mg/kg b. wt. orally, once a day). The results were compared with those after treatment of the rats with estradiol (E2), estradiol valerate (E2V) and ethinylestradiol (EE2). In liver homogenates glutathione and lipid peroxides were determined, in microsomes NADPH-Fe++-stimulated lipid peroxidation (LPO), H2O2 generation and lucigenin (LUC) and luminol (LUM) amplified chemiluminescence (CL) were investigated. In liver 9000 x g supernatants monooxygenase activities were measured. The two catechol estrogens did not show any antioxidative activity, whereas 6alpha-OH-E2 significantly diminished lipid peroxides in the liver as well as LPO and LUM-CL in liver microsomes. Among estrogens, only EE2 showed antioxidative activity. Both CE inhibited ethoxycoumarin O-deethylation. Peroxidative activity as enhanced LUC-CL was found after 2OH-E2 (1 mg/kg b.wt.) and E2, but 10 times higher doses of both CE did not change LUC-CL. Microsomal H2O2 generation was enhanced by E2, E2V and both CE, not by 6alpha-OH-E2. The lower level of H2O2 enhancement caused by CE in comparison to E2 and E2V together with unchanged LUC-CL after high CE doses did not unequivocally prove the CE to be mainly responsible for the prooxidative activities of E2 and E2V in liver microsomes, at least in 30-day-old male rats. Unchanged GSH in the liver after CE administration supports this hypothesis.
Asunto(s)
Antioxidantes/farmacología , Estrógenos de Catecol/farmacología , Especies Reactivas de Oxígeno , Administración Oral , Animales , Antioxidantes/administración & dosificación , Relación Dosis-Respuesta a Droga , Estrógenos de Catecol/administración & dosificación , Hígado/citología , Masculino , Microsomas/fisiología , RatasRESUMEN
This paper is the third of a long-term planned series of papers dealing with ex vivo investigations of drug transport in human kidney. The aims of this study are (a) to investigate whether or not human renal cell carcinoma (RCC) can actively accumulate p-aminohippurate (PAH) and (b) to test the response of RCC on dexamethasone or triiodothyronine (T3) using tissue slices ex vivo. By this approach, the accumulation capacity of RCC should be stimulated as a prerequisite for an increased uptake of anti-tumour drugs. Tissue slices of RCC samples of 30 patients were incubated for 24 h in Williams medium E containing 0.01-50 microM dexamethasone or T3. Thereafter, slices were placed in PAH-containing Cross-Taggart medium, and PAH uptake into kidney tissue was measured for 2h under standardised conditions as described previously. In intact human renal cortical slices, PAH uptake capacity, expressed as slice to medium ratio (QS/M), was about 2.8 +/- 0.16 after 24 h of incubation and increased significantly in dexamethasone-containing medium in a concentration-dependent manner, up to approximately 150%, whereas T3 did not influence PAH accumulation. On the other hand, in RCC the PAH accumulation capacity was completely abolished (QsM approximately 1). However, after administration of dexamethasone, the accumulated amount of PAH increased significantly in RCC tissue in a concentration-dependent manner, up to approximately 190%. T3 was without effect in RCC, too. Surprisingly, the dexamethasone-mediated stimulation could be differentiated into responders and non-responders, with maximal effects at different concentrations for each patient. Nevertheless, the maximal transport rates remained low in RCC, even under hormone influence. In conclusion, a moderate stimulation of tubular transport capacity can be shown ex vivo in human RCC. This phenomenon is only of a relatively low degree compared with intact renal tissue. However, in principle, the response of RCC on dexamethasone could form a basis for further therapeutic strategies to overcome multi-drug resistance in RCC patients. For this purpose, additional experiments analysing the expression of transporters of the ABC cassette-type are in progress.
Asunto(s)
Carcinoma de Células Renales/metabolismo , Dexametasona/farmacología , Glucocorticoides/farmacología , Neoplasias Renales/metabolismo , Túbulos Renales/metabolismo , Triyodotironina/farmacología , Ácido p-Aminohipúrico/farmacocinética , Transportadoras de Casetes de Unión a ATP/metabolismo , Transporte Biológico/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Túbulos Renales/efectos de los fármacos , Técnicas de Cultivo de ÓrganosRESUMEN
Metabolic pathways of oestrogens are the formation of catechol oestrogens (CE; 2- and 4-hydroxy-oestrogens), redox cycling of CE and free radical generation, mediated through cytochrome P450 (P450) oxidase/reductase activity. We checked the oestrogens oestradiol (E2), oestradiol valerate (E2V) and ethinyloestradiol (EE2) for formation of reactive oxygen species in vitro and ex vivo in male Wistar rats in dependence on age. In liver microsomes of 10-, 30-, 60- and 270-day-old rats the influence of E2, E2V and EE2 (10(-7)-10(-3) M) on NADPH-Fe(++)-stimulated lipid peroxidation (LPO), H2O2 generation and lucigenin (LUC) and luminol (LUM) amplified chemiluminescence (CL) was investigated. The same parameters, additionally P450 content and monooxygenase activities were measured in liver 9000 x g supernatants after subchronic administration of the oestrogens (1, 10 mg/kg b. wt. orally). The most important results are the strong inhibitory capacities of the oestrogens in vitro on LPO in the order of E2V < E2 < EE2, most pronounced in 10-, 60- and 270-day-old animals. In microsomes of 30-day-old rats with the highest control LPO the antioxidative effect of the oestrogens was lower. Whereas the H2O2 generation was not changed by E2, enhanced by E2V, but diminished by EE2 in all age groups, CL(LUC) and CL(LUM) were inhibited in the order of E2 < E2V < EE2. Also after subchronical treatment of the rats the antioxidative action of the oestrogens was evident, microsomal LPO was inhibited in the order of E2 < E2V < EE2. All oestrogens inhibited ethylmorphine N-demethylation. But enhanced H2O2 generation and increased CL(LUC) also indicate a formation of reactive oxygen species by these oestrogens. Obviously in vitro the antioxidative phenolic structure of the oestrogens dominates, whereas after in vivo administration the dose- and age-dependent biotransformation produces prooxidative in addition to antioxidative structures.
Asunto(s)
Envejecimiento , Estrógenos/farmacología , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Estradiol/análogos & derivados , Estradiol/farmacología , Etinilestradiol/farmacología , Compuestos Ferrosos/farmacología , Peróxido de Hidrógeno/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Mediciones Luminiscentes , Masculino , NADP/farmacología , Ratas , Ratas WistarRESUMEN
Both chlormezanone enantiomers, for the first time obtained by enantiospecific HPLC with a 100% yield, bind to oxidized cytochrome P-450 in rat liver microsomes with a binding curve according to type I, similar to hexobarbital but less pronounced. There are no differences between the binding curves of the two enantiomers. Ethylmorphine N-demethylation, ethoxycoumarin and ethoxyresorufin O-deethylation are inhibited by both chlormezanone enantiomers at 0.1-1 mM concentrations: no differences could be found. Luminol and lucigenin amplified chemiluminescence indicating the formation of reactive oxygen species was not influenced by either enantiomer in concentration ranges between millimolar and micromolar, whereas hydrogen peroxide formation was inhibited. NADPH/Fe stimulated lipid peroxidation was not influenced. Scavenger activity could not be demonstrated: the zymosan stimulated whole blood chemiluminescence was not influenced significantly.
Asunto(s)
Clormezanona/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Microsomas Hepáticos/metabolismo , Relajantes Musculares Centrales/metabolismo , Acridinas/química , Analgésicos Opioides/metabolismo , Animales , Anticoagulantes/metabolismo , Cromatografía Líquida de Alta Presión , Cumarinas/metabolismo , Etilmorfina/metabolismo , Peróxido de Hidrógeno/metabolismo , Indicadores y Reactivos/química , Peroxidación de Lípido/efectos de los fármacos , Mediciones Luminiscentes , Luminol/química , Masculino , Oxazinas/metabolismo , Unión Proteica , Ratas , Especies Reactivas de Oxígeno/metabolismo , Estereoisomerismo , Zimosan/químicaRESUMEN
Ciprofibrates (racemate and both enantiomers, Raccip, R- and Scip) were administered orally in doses of 1 and 10 mg/kg once daily over 28 days to male inbred Fischer 344 rats, age 90-110 days at the beginning of the experiment. Body mass gain was observed in all groups. The 1 mg groups showed almost no difference to the control group. The 10 mg groups exhibited less body mass gain, most pronounced in the Scip group. Liver masses were increased in a dose dependent manner up to more than 200%, only the 10 mg Scip group was not significantly different from the 1 mg group which exhibited an increase in liver weight to about 175%. Also the kidney weights increased to 130%, whereas thymus and spleen weights were decreased in the high dose groups. Liver microsomal cytochromes P450 (P450) concentrations were not altered in the 1 mg groups and distinctly lowered in the 10 mg groups. Ethoxyresorufin and ethoxycoumarin O-deethylations were lowered in all experimental groups in a dose dependent manner, after administration of the high doses down to 30% of the control levels or less. Pentoxyresorufin O-depentylation, however, was increased in all 1 mg groups. In the high dose groups it was not altered. Ethylmorphine N-demethylation was decreased after administration of the high doses by about 50%, but only Scip decreased this reaction also after administration of the low dose. NADPH/Fe2+-stimulated microsomal luminol and lucigenin amplified chemiluminescence was increased, whereas hydrogen peroxide formation was depressed even by the low doses to 50% of the normal values, to about 25% by the high doses. Microsomal lipid peroxidation, however, was only slightly or not influenced. Glutathion concentrations (in the reduced and the oxidized form) were increased in a dose dependent manner by about 20 to 30%, the concentration of lipid peroxides was not significantly influenced. Thus, the effects of the enantiomers were not different and were similar to those of the racemate. In serum, cholesterol and triglycerides were only moderately lowered. Albumin concentrations were significantly enhanced in all groups, total proteins after 1 mg/kg Raccip only. Serum bilirubins were not altered, and among the indicator enzymes for liver damage only ALAT, alkaline phosphatase and the dehydrogenases were increased, in no case higher than twofold. Histologically distinct effects were seen after administration of both doses, more pronounced after 10 mg/kg, but with no differences between the enantiomers and Raccip: marked hypertrophy of the hepatocytes, reduced staining of the nuclei, strongly acidophilic granulated cytoplama, no basophilia of the cell bodies, loss of glycogen. These changes were most pronounced around the central veins. Hepatocyte apoptoses also were observed. By immunohistochemistry an increased staining was seen for all P450 isoforms tested (1A1, 2B1, 2E1, 3A2 and 4A1), predominantly perivenously and most pronounced after administration of the high doses without differences between Rcip, Scip or Raccip (preliminary results). By electron microscopy a moderate proliferation of peroxisomes after treatment with 1 mg/kg Cips with a ratio between mitochondria and peroxisomes of about 1:1 (controls: 10:1) was observed, and the peroxisomes were a more heterogeneous population. The relative portions of glycogen and both forms of the ER decreased. Treatment with 10 mg/kg Rcip, Scip or Raccip led to a strong increase in the number of peroxisomes, in some hepatocytes the ratio between mitochondria and peroxisomes was 1:3 with an increased heterogeneity among the peroxisomes evidenced by a broad range of electron densities. Most peroxisomes lacked a nucleoid. Thus, the biochemical effects differed only slightly and the morphological effects of the enantiomers were not different and were similar to those of the racemate.
Asunto(s)
Ácido Clofíbrico/análogos & derivados , Hipolipemiantes/toxicidad , Administración Oral , Animales , Colesterol/sangre , Ácido Clofíbrico/química , Ácido Clofíbrico/toxicidad , Sistema Enzimático del Citocromo P-450/metabolismo , Relación Dosis-Respuesta a Droga , Ácidos Fíbricos , Hipolipemiantes/química , Riñón/efectos de los fármacos , Riñón/patología , Peroxidación de Lípido/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/enzimología , Hígado/patología , Mediciones Luminiscentes , Masculino , Microcuerpos/efectos de los fármacos , Microcuerpos/ultraestructura , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Endogámicas F344 , Albúmina Sérica/análisis , Estereoisomerismo , Triglicéridos/sangre , Aumento de Peso/efectos de los fármacosRESUMEN
The enantiomers of ciprofibrate may be achieved by enantioselective HPLC separation of its methylesters using a OD - Daicel column. Ciprofibrates (racemate and both enantiomers) bind to oxidized cytochrome P-450 in rat liver microsomes according type II like aniline or most probably as inversed type I, but less pronounced and with a general shift to the left. Ethylmorphine N-demethylation, ethoxycoumarin and ethoxyresorufin O-deethylation are all inhibited by the ciprofibrates, most effectively ethoxyresorufin O-deethylation by S(-)-ciprofibrate even in microM concentrations. Microsomal luminol and lucigenin amplified chemiluminescence indicating the formation of reactive oxygen species, microsomal hydrogen peroxide formation and NADPH/Fe stimulated lipid peroxidation were inhibited in a concentration dependent manner in concentration ranges between mM and microM. This might be due to distinct scavenger activities of all 3 compounds: the zymosan stimulated chemiluminescence of whole blood was completely inhibited in mM concentrations and influenced significantly down to concentrations of 10 microM, whereas burst and phagocytosis tests with human polynuclear leucocytes were not influenced.
Asunto(s)
Ácido Clofíbrico/análogos & derivados , Sistema Enzimático del Citocromo P-450/metabolismo , Hipolipemiantes/metabolismo , Microsomas Hepáticos/metabolismo , Compuestos de Anilina/metabolismo , Compuestos de Anilina/farmacología , Animales , Cromatografía Líquida de Alta Presión , Ácido Clofíbrico/análisis , Ácido Clofíbrico/metabolismo , Ácido Clofíbrico/farmacología , Interacciones Farmacológicas , Ácidos Fíbricos , Hexobarbital/metabolismo , Hexobarbital/farmacología , Humanos , Peróxido de Hidrógeno/metabolismo , Hipolipemiantes/análisis , Hipolipemiantes/farmacología , Técnicas In Vitro , Masculino , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Fagocitosis/efectos de los fármacos , Ratas , Especies Reactivas de Oxígeno , Estallido Respiratorio/efectos de los fármacos , EstereoisomerismoRESUMEN
Possible antioxidative properties of three N-methyl-D-aspartate (NMDA)-receptor antagonists, the anesthetic ketamine and the antiparkinson drugs memantine and amantadine were investigated in vitro on the microsomal cytochrome P450 (P450) system of rat livers and on rat whole blood chemiluminescence in comparison to nicanartine, a substance with known antiatherosclerotic, hypolipemic and antioxidative capacity. For this purpose, the effects on NADPH- and iron-stimulated lipid peroxidation (LPO), hydrogen peroxide (H2O2) production, and NADPH- and iron-stimulated lucigenin (LC) and luminol (LM) amplified chemiluminescence (CL) were examined using rat liver microsomes. Additionally, the influence on LM amplified whole blood chemiluminescence after zymosan activation of polymorphonuclear leukocytes (WB-CL) was investigated. Furthermore, binding to P450 and effects on P450 mediated monooxygenase function, as measured by the model reactions ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD), and ethylmorphine N-demethylation (END), were assessed. Nicanartine concentration dependently reduced LPO and H2O2 production already at a concentration of 1 microM, whereas LC and LM amplified CL and WB-CL were not affected. EROD and END were concentration dependently diminished starting at 1 microM, and ECOD already at 0.1 microM. Ketamine decreased LPO, H2O2 production and LM and LC amplified CL, starting at 100 microM. WB-CL was significantly diminished already at 10 microM. EROD and ECOD were inhibited at 10 and 100 microM and END at 100 microM. With memantine a concentration dependent inhibition of LPO and WB-CL was seen at 100 and 1000 microM and a reduction of LC and LM amplified CL only at 1000 microM. H2O2 production was not affected. EROD and ECOD were significantly diminished by a concentration of 100 microM. No effect was observed on END. Amantadine significantly reduced LPO and WB-CL, but only at 1000 microM. H2O2 production and LC and LM amplified CL were not affected. EROD was significantly diminished at 100 microM, whereas no influence was seen on ECOD and END. Nicanartine displayed type II or reverse type I, ketamine, memantine and amantadine type I substrate binding to P450. The highest binding affinity to P450 was seen with nicanartine, followed by ketamine, memantine and then amantadine. These results demonstrate, that all four substances seem to act as radical scavengers and/or as inhibitors of the oxidative function of P450. All four substances seem to interfere with the monooxygenase function of P450. This may result in a possible influence on the biotransformation of endogenous as well as of foreign compounds. The effects of nicanartine were much more pronounced than those of ketamine, memantine, and amantadine.
Asunto(s)
Amantadina/farmacología , Antioxidantes/farmacología , Ketamina/farmacología , Memantina/farmacología , Microsomas Hepáticos/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , 7-Alcoxicumarina O-Dealquilasa/metabolismo , Animales , Antiparkinsonianos/farmacología , Citocromo P-450 CYP1A1/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Relación Dosis-Respuesta a Droga , Peróxido de Hidrógeno/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Masculino , Microsomas Hepáticos/enzimología , Piridinas/farmacología , Ratas , Ratas Wistar , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
The chemiluminescence (CL) amplifiers luminol (LM) and lucigenin (LC) react with different reactive oxygen species (ROS) in dependence on the ROS generating system used. With liver microsomes LMCL indicates predominantly superoxide anion radicals, whereas LCCL is mainly a measure for hydroxyl radical formation or of reactive organic radicals. With brain microsomes only LCCL, but not LMCL could be measured. For both brain microsomes from newborn (both sexes) and 60 day-old (male) rats LCCL is dependent on protein and NADPH concentration, activity in newborns being only 15% compared with young adult rats. As compared with liver microsomes 10-fold higher protein concentrations are needed to obtain comparable LCCL, whereas the NADPH demand is the same as with liver. A distinct ontogenetic development was demonstrated: low activities in the fetus, in newborn and 10-day-old rat are followed by higher activities with increasing age, after a maximum at an age of 60 days a decline was observed. Microsomal lipidperoxidation (LPO) was measured as formation of thiobarbituric acid reactive substances (TBARS) and was also dependent on protein and NADPH concentration. Unexpectedly, LPO with brain microsomes from newborn rats did not show any developmental variation.
Asunto(s)
Acridinas/administración & dosificación , Encéfalo/crecimiento & desarrollo , Indicadores y Reactivos/administración & dosificación , Peroxidación de Lípido/fisiología , Luminol/administración & dosificación , Animales , Encéfalo/fisiología , Femenino , Mediciones Luminiscentes , Masculino , Microsomas , RatasRESUMEN
Evidence is provided that the amplifiers luminol and lucigenin react with different reactive oxygen species (ROS), depending on the ROS-generating system used. H2O2 is used to produce calibration curves for luminol- and lucigenin-amplified chemiluminescence. With this chemiluminescence generator we characterized the specificity and sensitivity of luminol- and lucigenin-amplified chemiluminescence and also studied penicillin G, a known enhancer of luminol-amplified chemiluminescence. The combination of luminol and lucigenin in reciprocally changing concentrations is effective in an additive manner, but the weak amplifier penicillin increases luminol-amplified chemiluminescence distinctly more than in an additive manner in different combinations. Lucigenin-amplified chemiluminescence is increased by penicillin at about 1% of the optimum concentration of penicillin; increasing concentrations of penicillin are less and less effective. On the other hand, low lucigenin concentrations enhance penicillin-amplified chemiluminescence at optimum penicillin concentrations more than in an additive manner. Fe2+ does not alter luminol-, lucigenin- or penicillin-amplified chemiluminescence. Co2+ increases luminol-amplified chemiluminescence by a factor of 100. Lucigenin- and penicillin-amplified chemiluminescence are minimally enhanced by Co2+. Cu2+ enhances luminol-amplified chemiluminescence with increasing concentrations by a factor of 1000. Lucigenin-amplified chemiluminescence increases also by the factor of 1000, but the concentration-reaction curve is not as steep. NaOCl enhances H2O2/Fe(2+)-driven luminol-amplified chemiluminescence in a concentration-dependent manner by a factor of 10(4) (in the highest concentration of 10 mmol/L) and lucigenin amplified chemiluminescence only by a factor of about 25. Catalase (CAT) abolishes luminol-, lucigenin- and penicillin-amplified chemiluminescence completely, whereas superoxide dismutase (SOD) has no effect on luminol- or penicillin-amplified chemiluminescence, but enhances lucigenin-amplified chemiluminescence five-fold increasingly with increasing SOD activity.
Asunto(s)
Acridinas , Peróxido de Hidrógeno , Mediciones Luminiscentes , Luminol , Penicilinas , Hipoclorito de Sodio , Calibración , Cationes , Aumento de la Imagen/métodos , Indicadores y Reactivos , Metales , Especies Reactivas de OxígenoRESUMEN
Nine natural and synthetic estrogens all derived from endogenous 17 beta-estradiol, were tested for their affinity to cytochrome P-450 (P450). Binding spectra of the estrogens with rat liver microsomal P450 and inhibition kinetics with characteristic monooxygenase model reactions (ethylmorphine N-demethylation, EN, and ethoxycoumarin O-deethylation, EO) were determined. In addition, uncoupling effects and/or free radical scavenger functions were analysed by NADPH/Fe2+ stimulated microsomal luminol- and lucigenin-amplified chemiluminescense (CL). 17 beta-Estradiol, 17 alpha-ethynylestradiol, and D-estradiol 3-methyl ether inhibited both monooxygenase reactions of cytochrome P-450, whereas L-estradiol 3-methyl ether inhibited EO only. 17 beta-Estradiol, 17 alpha-ethynylestradiol, and D-estradiol 3-methyl ether seem to act as free radical scavengers. From the results both structure activity relationships could be established and data on possible interferences with drug metabolism obtained. The enantiomers D- and L-estradiol 3-methyl ether differ in their effects on these systems.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Estrógenos/farmacología , Depuradores de Radicales Libres/farmacología , Animales , Biotransformación , Estrógenos/metabolismo , Masculino , Ratas , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Chlormezanone binds to oxidized cytochrome P450 in rat liver microsomes with a binding curve according to type I like hexobarbital but less pronounced and with a general shift to the left. Ethylmorphine N-demethylation, ethoxycoumarin and ethoxyresorufin O-deethylation are inhibited by chlormezanone in mM concentrations only whereas pentoxyresorufin O-depentylation is inhibited by about 50% in microM concentrations. Luminol and lucigenin amplified chemiluminescence indicating the formation of reactive oxygen species was not influenced in concentration ranges between mM and microM, whereas NADPH/Fe stimulated lipid peroxidation showed a tendency of inhibition. But scavenger activity could not be demonstrated: the zymosan stimulated chemiluminescence of whole blood was not influenced significantly. The degradation process of chlormezanone was elucidated. The first step involves ring opening by chemical hydrolysis with subsequent formation of an unstable acylhalfaminal which is the source of 4-chlorobenzaldehyde. This aldehyde undergoes enzymatically controlled oxidation to 4-chlorobenzoic acid which is the parent compound of following phase II reactions. The second degradation product is 2-carboxyethane-sulfinic-acid-N-methylamide, which is hydrolyzed very quickly. Neither oxidation of the sulfinic acid or its N-methylamide derivative could be observed nor N-demethylation of chlormezanone.
Asunto(s)
Clormezanona/metabolismo , Clormezanona/farmacología , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/metabolismo , Relajantes Musculares Centrales/metabolismo , Relajantes Musculares Centrales/farmacología , Animales , Biotransformación , Cumarinas/metabolismo , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/metabolismo , Interacciones Farmacológicas , Etilmorfina/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Mediciones Luminiscentes , Masculino , Microsomas Hepáticos/enzimología , Oxazinas/metabolismo , Oxigenasas/antagonistas & inhibidores , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Estimulación Química , Zimosan/farmacologíaRESUMEN
For the investigation of luminol (LM)-and lucigenin (LC)-amplified chemiluminescence (CL) in rat liver microsomes using both a liquid-scintillation counter (LKB/Wallac 1219 Rackbeta) and a Berthold luminometer (AutoLumat LB 953) optimal incubation mixtures and conditions and basic kinetics have been established. Whereas calibration curves for both LM- and LC-CL are performed with hydrogenperoxide (LC quantum yield is 6.25 fold higher as that of LM), distinct differences were revealed with microsomes, indicating that different reactive oxygen species (ROS) are determined: Both LM- and LC-CL follow the kinetics of enzymatic reactions in terms of dependence on protein and NADPH or NADH concentration, time course, temperature etc., but with differences. LM-CL does not work without addition of Fe2+, whereas LC-CL does. Both copper ions and copper bound in a complex abolish CL, LC-CL being much more sensitive. Isolated cytochrome P-450 (P450) and NADPH P450 reductase from liver of pheno-barbital treated rats alone proved to be inactive in LM-and LC-CL production, whereas te combination 1:1 without and with addition of lipid was highly active in both LM-and LC-CL. Ascorbic acid and glutathione as scavengers diminish both LM- and LC-CL in concentrations higher then 10(5). Dimethyl-sulfoxide (DMSO) was ineffective in LM-CL up to concentrations of 0.2 M, the very high concentration of 2 M diminished LM-CL only to 1/3. LC-CL was diminished starting at concentrations of 100 mM and at 2 M only 10% of maximum LC-CL was observed. The trap substance N-t-butyl-a-phenylnitrone (BNP) also diminished LC-CL more effectively than LM-CL. Clearcut differences were revealed by the addition of catalase and superoxide dismutase: both enzymes diminished LM-CL only, without any influence on LC-CL. Hexobarbital, a potent uncoupler of P450, enhances LM-CL fivefold, whereas LC-CL is barely influenced. Aniline (without uncoupling capability) decreased both LM-and LC-CL increasingly with increasing concentrations. Therefore the conclusion is drawn that LM-CL measures in liver microsomes predominantly superoxide anion radicals, whereas LC-CL is mainly a measure for microsomal hydroxyl radical formation or of reactive organic radicals. With microsomes of phenobarbital and beta-naphthoflavone treated rats CL was much higher but in principle the same kinetic characteristics could be shown. All results on microsomes were obtained uniformly with the liquid scintillation counter and the Berthold luminometer, the letter being much more effective and more sensitive.
Asunto(s)
Acridinas/farmacología , Luminol/farmacología , Microsomas Hepáticos/efectos de los fármacos , Compuestos de Anilina/metabolismo , Animales , Ácido Ascórbico/metabolismo , Catalasa/metabolismo , Cobre/metabolismo , Óxidos N-Cíclicos , Sistema Enzimático del Citocromo P-450/fisiología , Dimetilsulfóxido/metabolismo , Glutatión/metabolismo , Hexobarbital/metabolismo , Hierro/metabolismo , Cinética , Mediciones Luminiscentes , Masculino , NADPH Deshidrogenasa/fisiología , Óxidos de Nitrógeno/metabolismo , Proteínas/análisis , Ratas , Ratas Wistar , Superóxido Dismutasa/metabolismo , Temperatura , Factores de TiempoRESUMEN
In vitro influence of sodium selenite on cytochrome P-450-dependent formation of active oxygen species on lipid peroxidation (LPO) in rat liver microsomes was studied. Sodium selenite (10(-6)-10(-3) M) did not influence rates of NADPH/Fe-induced formation of active oxygen species (O2.-, OH., H2O2) and NAPDH-dependent LPO. Only at 10(-3) M selenite caused significant decrease in production of hydrogen peroxide in microsomes. Data obtained suggest that sodium selenite at physiological concentrations does not influence formation of active oxygen species by cytochrome P-450 and the rate of enzymatic LPO in rat liver microsomes.