Glucocorticoid-dependent expression of O(6)-methylguanine-DNA methyltransferase gene modulates dacarbazine-induced hepatotoxicity in mice.

O(6)-methylguanine-DNA methyltransferase (MGMT) plays a crucial role in the defense against the alkylating agent-induced cytotoxic lesion O(6)-alkylguanine in DNA. Although a significant circadian variation in MGMT activity has been found in the liver of mice, the exact mechanism of the variation remains poorly understood. In this study, we present evidence that glucocorticoids were required for the 24-h oscillation of MGMT expression in mouse liver. The exposure of mouse hepatic cells (Hepa1-6) to dexamethasone (DEX) significantly increased the mRNA levels of MGMT in a dose-dependent manner. The DEX-induced increase in MGMT expression was reversed by concomitant treatment with RU486 [11beta-[p-(dimethylamino) phenyl]-17beta-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one], a glucocorticoid receptor antagonist. The mRNA levels of MGMT and its enzymatic activity in the liver of mice showed significant 24-h oscillations, which were not observed in adrenalectomized mice. A single administration of DEX to adrenalectomized mice significantly increased the mRNA levels of MGMT in the liver. These findings suggest that the 24-h oscillation in the hepatic expression of MGMT is caused by the endogenous rhythm of glucocorticoid secretion. Dacarbazine (DTIC), a potent O(6)-guanine-alkylating agent, causes serious hepatotoxicity accompanied by hepatocellular necrosis and hepatic vein thrombosis. DTIC-induced hepatotoxicity in mice was attenuated by administering the drug at the time of day when MGMT expression was abundant. The present findings suggest that glucocorticoid-regulated oscillation in the hepatic MGMT expression is the underlying cause of dosing time-dependent changes in DTIC-induced hepatotoxicity.

Inhibition of free radical-induced erythrocyte hemolysis by 2-O-substituted ascorbic acid derivatives.

Inhibitory effects of 2-O-substituted ascorbic acid derivatives, ascorbic acid 2-glucoside (AA-2G), ascorbic acid 2-phosphate (AA-2P), and ascorbic acid 2-sulfate (AA-2S), on 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative hemolysis of sheep erythrocytes were studied and were compared with those of ascorbic acid (AA) and other antioxidants. The order of the inhibition efficiency was AA-2S> or =Trolox=uric acid> or =AA-2P> or =AA-2G=AA>glutathione. Although the reactivity of the AA derivatives against AAPH-derived peroxyl radical (ROO(*)) was much lower than that of AA, the derivatives exerted equal or more potent protective effects on AAPH-induced hemolysis and membrane protein oxidation. In addition, the AA derivatives were found to react per se with ROO(*), not via AA as an intermediate. These findings suggest that secondary reactions between the AA derivative radical and ROO(*) play a part in hemolysis inhibition. Delayed addition of the AA derivatives after AAPH-induced oxidation of erythrocytes had already proceeded showed weaker inhibition of hemolysis compared to that of AA. These results suggest that the AA derivatives per se act as biologically effective antioxidants under moderate oxidative stress and that AA-2G and AA-2P may be able to act under severe oxidative stress after enzymatic conversion to AA in vivo.

A simple determination of mizoribine in human plasma by liquid chromatography with UV detection.

A simple liquid chromatography (LC) method was developed for determination of the therapeutic level of mizoribine in human plasma. After precipitation of plasma proteins with 6% perchloric acid, mizoribine was determined by LC with spectophotometric detection. The peak height for mizoribine was linearly related to its concentrations, which ranged from 0.09 to 3.13 microg/mL. Therefore, the limit of quantitation was considered to be 0.09 microg/mL. The accuracy was 104.96-107.37%. The intra- and interday relative standard deviation values were in the range of 1.10-3.25%. The detection limit was 0.025 microg/mL, defined as a signal-to-noise ratio of 3. The plasma concentrations of mizoribine were not related to the dosage. Because mizoribine was mainly excreted in the urine, the plasma concentrations of mizoribine might be affected by a change in renal function. Therefore, the mizoribine concentration in blood should be monitored and the dosage adjusted, depending on the condition of renal function. It was suggested that the present method may be applied well in the therapeutic drug monitoring for mizoribine.

Contrast media increase vascular endothelial permeability by inhibiting nitric-oxide production.

RATIONALE AND OBJECTIVES: Contrast media induce adverse effects including edema of the face, glottis, or lung. The endothelial function is maintained by nitric oxide (NO). The present study was designed to elucidate the role of NO in mediating endothelium-related adverse effects of contrast media. METHODS: Human microvascular endothelial cells grown on a Transwell membrane were incubated with iohexol or ioxaglate in the absence or presence of N(G)-monomethyl-L-arginine or sodium nitroprusside. After washing cells, the permeability of sodium fluorescein or Evans blue albumin and the accumulation of NO(2)(-) was examined. RESULTS: Contrast media (50-150 mgI/mL) dose-dependently increased the permeability coefficient by 30% to 230% and inhibited the formation of NO(2)(-) by 40% to 80%. Sodium nitroprusside and N(G)-monomethyl-L-arginine produced protective and aggravating effects on contrast media-increased permeability, respectively. CONCLUSIONS: The present study suggested that contrast media increase vascular endothelial permeability by inhibiting NO production, leading to vascular endothelium-related adverse effects of contrast media.

Protection by a radical scavenger edaravone against cisplatin-induced nephrotoxicity in rats.

Acute renal failure is a dose-limiting factor of cisplatin chemotherapy. Here, we show the protective effect of edaravone, a recently developed radical scavenger for clinical use, against cisplatin-induced renal dysfunction in rats. A marked increase in blood urea nitrogen and creatinine in serum, and histological changes including vacuolation, necrosis and protein casts were observed in proximal renal tubules at the fourth day after cisplatin injection (5-10 mg/kg). Repeated injection of edaravone (1-10 mg/kg, i.v. twice a day for 3 days) reversed the cisplatin-induced elevation of blood urea nitrogen and creatinine, and morphological changes in a dose-dependent manner. In particular, the protective effect of edaravone was almost complete at 10 mg/kg. Moreover, the compound was still fully effective, when it was administered only at the second day after cisplatin injection. On the other hand, the glutathione content in renal tissues lowered at the fourth day after cisplatin injection, which was reversed by the late treatment with edaravone. These findings suggest that the clinically available radical scavenger edaravone is potentially useful for the prevention of cisplatin-induced renal toxicity.

Cell-specific toxicity of fibrates in human embryonal rhabdomyosarcoma cells.

The effects of a variety of fibrates on the cell viability were examined in human embryonal rhabdomyosarcoma cells (HRMSC). Five fibrates, including fenofibrate, clofibrate, gemfibrozil, bezafibrate and ciprofibrate, all concentration-dependently reduced the cell viability determined by the mitochondrial enzyme activity. The cell injury occurred time-dependently and was marked at 24-48 h. The toxic action of fibrates was specific to HRMSC, since bezafibrate did not induce any marked changes in the viability of human microvascular endothelial cells or arterial smooth muscle cells. Synergistic cell injury was observed after a combined treatment with bezafibrate and simvastatin, although simvastatin alone reduced the cell viability. The cell injury was characterized by a typical nuclear damage, as evidenced by Hoechst 33342 staining and deoxynucleotidyl transferase dUTP nick-end label-positive staining. Similar cell-specific injury was induced by 8(S)-hydroxyeicosatetraenoic acid, a potent peroxisome proliferator-activated receptor alpha (PPARalpha) agonist. Consistent with these data, a marked expression for PPARalpha mRNA was observed in HRMSC but not in the endothelial or smooth muscle cells. Therefore, it is suggested that fibrates cause a cell-specific injury in HRMSC via activation of PPARalpha. Moreover, our present cell injury model using HRMSC may be useful for elucidating the mechanisms of clinical rhabdomyolysis induced by lipid-lowering agents.

Protective effects of amifostine and cyclooxygenase-1 inhibitor against normal human epidermal keratinocyte toxicity induced by methotrexate and 5-fluorouracil.

Our study aimed to find more effective protective agents against mucosa toxicity induced by methotrexate and 5-fluorouracil. We focused on the relationship between oral mucositis and keratinocyte injury and examined methotrexate and 5-fluorouracil-induced cytotoxicity in normal human epidermal keratinocyte cell lines. Cell viability and superoxide radical activity were measured based on converting WST-1 (4-[3-(4-indophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzen disulfonate) to a water-soluble formazan dye. DNA synthesis by 5-bromo-2'-deoxyuridine incorporation was measured as an indirect parameter of cell proliferation. Allopurinol and amifostine were used as the radical scavengers. l-glutamine was used as a mucosa-protective agent. A cyclooxygenase inhibitor interrupting the production of hydroxyl radicals in the arachidonic acid cascade was also examined. 5-fluorouracil and methotrexate caused cytotoxicity due to the activation of intracellular superoxide radicals specifically on normal human epidermal keratinocytes. From the electron spin resonance study, it was found that allopurinol was a superoxide radical scavenger, while amifostine was hydroxyl radical scavenger. Allopurinol showed no effect on the cytotoxicity due to 5-fluorouracil and methotrexate. The cell injury induced by methotrexate was restored by amifostine. However, the cell injury induced by 5-fluorouracil was markedly recovered by a selective cyclooxygenase-1 inhibitor compared to amifostine. It was suggested that amifostine and cyclooxygenase-1 inhibitor could be useful protective agents against methotrexate and 5-fluorouracil chemotherapeutic toxicity. Additionally, this in vitro cell injury model using normal human epidermal keratinocytes may be useful for understanding the pathophysiology of oral mucositis induced by chemotherapeutic agents.

Modulatory effects of 5-fluorouracil on the rhythmic expression of circadian clock genes: a possible mechanism of chemotherapy-induced circadian rhythm disturbances.

The circadian clock system is necessary to adapt endogenous physiological functions to daily variations in environmental conditions. Abnormality in circadian rhythms, such as the sleep-wake cycle and the timing of hormonal secretions, is implicated in various physiological and psychiatrical disorders. Recent molecular studies have revealed that oscillation in the transcription of specific clock genes plays a central role in the generation of 24h cycles of physiology and behavior. It has been noticed that patients receiving chemotherapeutic agents experience disturbances in their behavioral and physical performances, including circadian rhythms. To explore the underlying mechanism of chemotherapeutic agent-induced disturbance of these rhythms, we investigated the influence of 5-fluorouracil (5-FU), one of the most widely used chemotherapeutic agents for the treatment of cancers, on the expression of clock genes. Treatment of cultured NIH3T3 cells with 5-FU for 48 h resulted in a significant reduction of mRNA levels of Period1 (Per1) and Period2 (Per2) without affecting cell viability; however, treatment with the same amount of uracil, a structural analog of 5-FU, had little effect on the expression of clock genes. Consistent with its inhibitory actions, continuous administration of 5-FU (2 mg/kg/h) to mice attenuated the oscillation in the expressions of Per1 and Per2 in the liver and suprachiasmatic nuclei, the center of the mammalian circadian clock. These results reveal a possible pharmacological action by the chemotherapeutic agent 5-FU on the circadian clock mechanism, which is the underlying cause of its adverse effects on 24-h rhythms of physiology and behavior.

Determination of nonsteroidal anti-inflammatory drugs in human specimens by capillary zone electrophoresis and micellar electrokinetic chromatography.

Therapeutic drug monitoring of anti-inflammatory drugs is necessary for the identification of the agents that cause toxic events and for the decision on the treatment for intoxication. Recently, capillary electrophoresis (CE) has been developed for the simple and rapid analyses of a variety of chemical agents. Micellar electrokinetic chromatography (MEKC) can separate acidic, neutral and basic anti-inflammatory drugs in serum. Furthermore, serum samples are directly applied to the CE system without any pretreatments, and some anti-inflammatory drugs can be separated from serum albumin in the MEKC analysis. On the other hand, capillary zone electrophoresis (CZE) enables us to determine a few microg/mL levels of acidic anti-inflammatory drugs with simple running buffer and stacking technique. A rapid and simultaneous determination of several analgesic anti-inflammatory agents, including ibuprofen, acetaminophen, indomethacin, and salicylic acid in human serum has been developed by using CZE. Therefore, the CZE and MEKC analysis may become a potentially useful alternative to high-performance liquid chromatography (HPLC) and fluorescence polarization immunoassay (FPIA) for therapeutic drug monitoring, particularly in serum of patients suffering from intoxication by overdosage of nonsteroidal anti-inflammatory agents.

Simultaneous determination of sulfamethoxazole and trimethoprim in human plasma by capillary zone electrophoresis.

A capillary electrophoretic method for the simultaneous determination of sulfamethoxazole and trimethoprim in plasma was developed. Sulfamethoxazole and trimethoprim extracted from human plasma with ethyl acetate were analyzed at 20 kV and 25 degrees C using 15 mm phosphate buffer (pH 6.2) as the electrolyte. The detection was by UV at 220 nm. The run time was 8.0 min and the limit of quantification was 10.00 microg/mL for sulfamethoxazole and 2.00 microg/mL for trimethoprim. The recovery was >99% for both compounds. This method enabled the detection of sulfamethoxazole and trimethoprim in plasma of patients after oral ingestion of their combined formulation. The present simple and rapid method is applicable to drug monitoring in immunocompromised patients who are taking the combined formulation of these compounds for the treatment or prophylaxis of Pneumocystis carinii pneumonia.

A rapid and simultaneous determination of several analgesic antiinflammatory agents by capillary zone electrophoresis.

A rapid and simultaneous determination of several analgesic antiinflammatory agents--ibuprofen, acetaminophen, indomethacin, and salicylic acid--in human serum was developed by using capillary zone electrophoresis (CZE) coupled with diode-array ultraviolet detection. After precipitation of serum protein with acetonitrile containing 3-isobutyl-1-methylxanthine as the internal standard, an aliquot of deproteinized samples was applied directly to the CZE system. It enabled us to measure all of these four agents within 6 min, and there were no peaks interfering with the assay of these agents or 3-isobutyl-1-methylxanthine. Both the separation and quantification of these agents in human serum were reproducible after repeated analysis within a day or day-to-day analysis. In addition, there was a good correlation for each drug (r = 0.997-0.999) between the values in serum determined by CZE analysis and those measured either by high-performance liquid chromatography with ultraviolet detection (ibuprofen and indomethacin) or by fluorescence polarization immunoassay (acetaminophen and salicylic acid). Therefore, the present CZE analysis could provide a simple, rapid, and efficient method for the identification as well as monitoring of analgesic antiinflammatory agents, particularly in serum of patients suffering from intoxication by overdosage of these agents.