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Biomedical subjects

W Tang

Publications and source records attributed to W Tang.

At least 127 records · Page 7Linked to original sources
PubMed1999

Coronary calcium does not accurately predict near-term future coronary events in high-risk adults.

BACKGROUND: Prognostic risk models have had limited success in predicting coronary events in subjects with multiple risk factors. We and others have proposed an alternative approach using radiographically detectable coronary calcium. We evaluated and compared the predictive value of these 2 approaches for determining coronary event risk in asymptomatic adults with multiple coronary risk factors. In addition, we assessed the predictive value of a risk model that included calcium score and cardiac risk-factor data. METHODS AND RESULTS: We recruited 1196 asymptomatic high-coronary-risk subjects who then underwent risk-factor assessment and cardiac electron-beam CT (EBCT) scanning and were followed up for 41 months with a 99% success rate. We applied the Framingham model and our data-derived risk model to determine the 3-year likelihood of a coronary event. The mean age of our cohort was 66 years, and mean 3-year Framingham risk was 3.3+/-3.6%. Sixty-eight percent (818 subjects) had detectable coronary calcium. There were 17 coronary deaths (1.4%) and 29 nonfatal infarctions (2. 4%). The receiver operating characteristic (ROC) curve areas calculated from the Framingham model, our data-derived risk model, and the calcium score were 0.69+/-0.05, 0.68+/-0.05, and 0.64+/-0.05, respectively (P=NS). When calcium score was included as a variable in the data-derived model, the ROC area did not change significantly (0.68+/-0.05 to 0.71+/-0.04; P=NS). CONCLUSIONS: Neither risk-factor assessment nor EBCT calcium is an accurate event predictor in high-risk asymptomatic adults. EBCT calcium score does not add significant incremental information to risk factors, and its use in clinical screening is not justified at this time.

Aged↗
PubMed1999

The ability of p53 to activate downstream genes p21(WAF1/cip1) and MDM2, and cell cycle arrest following DNA damage is delayed and attenuated in scid cells deficient in the DNA-dependent protein kinase.

scid mouse embryonic fibroblasts are deficient in DNA-dependent protein kinase activity due to a mutation in the C-terminal domain of the catalytic subunit (DNA-PKcs). When exposed to ionizing radiation, the increase in levels of p53 was the same as in normal mouse embryonic fibroblasts. However, the rise in p21(WAF1/cip1) and mdm2 was found to be delayed and attenuated, which correlated in time with delayed onset of G1/S arrest by flow cytometric analysis. The p53-dependent G1 checkpoint was not eliminated: inactivation of p53 by the E6 protein in scid cells resulted in the complete loss of detectable G1/S arrest after DNA damage. Immunofluorescence analysis of normal cells revealed p53 to be localized predominantly within the cytoplasm prior to irradiation and then translocate to the nucleus after irradiation. In contrast, scid cells show abnormal accumulation of p53 in the nucleus independent of irradiation, which was confirmed by immunoblot analysis of nuclear lysates. Taken together, these data suggest that loss of DNA-PK activity appears to attenuate the kinetics of p53 to activate downstream genes, implying that DNA-PK plays a role in post-translational modification of p53, without affecting the increase in levels of p53 in response to DNA damage.

Animals↗
PubMed1999

Apoptosis and growth inhibition in malignant lymphocytes after treatment with arsenic trioxide at clinically achievable concentrations.

BACKGROUND: Arsenic trioxide (As2O3) can induce clinical remission in patients with acute promyelocytic leukemia via induction of differentiation and programmed cell death (apoptosis). We investigated the effects of As2O3 on a panel of malignant lymphocytes to determine whether growth-inhibitory and apoptotic effects of As2O3 can be observed in these cells at clinically achievable concentrations. METHODS: Eight malignant lymphocytic cell lines and primary cultures of lymphocytic leukemia and lymphoma cells were treated with As2O3, with or without dithiothreitol (DTT) or buthionine sulfoximine (BSO) (an inhibitor of glutathione synthesis). Apoptosis was assessed by cell morphology, flow cytometry, annexin V protein level, and terminal deoxynucleotidyl transferase labeling of DNA fragments. Cellular proliferation was determined by 5-bromo-2'-deoxyuridine incorporation into DNA and flow cytometry and by use of a mitotic arrest assay. Mitochondrial transmembrane potential (delta psi(m)) was measured by means of rhodamine 123 staining and flow cytometry. Protein expression was assessed by western blot analysis or immunofluorescence. RESULTS: Therapeutic concentrations of As2O3 (1-2 microM) had dual effects on malignant lymphocytes: 1) inhibition of growth through adenosine triphosphate (ATP) depletion and prolongation of cell cycle time and 2) induction of apoptosis. As2O3-induced apoptosis was preceded by delta psi(m) collapse. DTT antagonized and BSO enhanced As2O3-induced ATP depletion, delta psi(m) collapse, and apoptosis. Caspase-3 activation, usually resulting from delta psi(m) collapse, was not always associated with As2O3-induced apoptosis. As2O3 induced PML (promyelocytic leukemia) protein degradation but did not modulate expression of cell cycle-related proteins, including c-myc, retinoblastoma protein, cyclin-dependent kinase 4, cyclin D1, and p53, or expression of differentiation-related antigens. CONCLUSIONS: Substantial growth inhibition and apoptosis without evidence of differentiation were induced in most malignant lymphocytic cells treated with 1-2 microM As2O3. As2O3 may prove useful in the treatment of malignant lymphoproliferative disorders.

Adenosine Triphosphate↗
PubMed1999

Transcriptional activation of the murine CTP:phosphocholine cytidylyltransferase gene (Ctpct): combined action of upstream stimulatory and inhibitory cis-acting elements.

CTP:phosphocholine cytidylyltransferase plays a key role in regulating the rate of phosphatidylcholine biosynthesis. However, the proximal regulatory elements for the gene (Ctpct) that encode this enzyme and the cognate transcription factors involved have not been characterized. Ctpct promoter activities were deduced from promoter deletion constructs linked to a luciferase reporter and transiently transfected into C3H10T1/2 and McArdle RH7777 cells. Positive regulatory elements were located between -130 and -52 bp from the transcription start site. Basal expression resided downstream between -52 and +38 bp. DNase I protection and electromobility-shift assays indicated that Sp1-related nuclear factors bind to a stimulatory, a possible inhibitory and minimal promoter element. Gel-shift assays confirmed that all three regulatory regions bound Sp1. Sp1 was further implicated when Sp1-deficient Drosophila cells were co-transfected with promoter-reporter constructs and an Sp1 construct. DNase I assays also indicated that the Ap1 binding elements could be occupied in the proximal activator and minimal promoter regions. Gel-shift assays demonstrated that the distal activator region could bind Ap1 and an unknown transcription factor. We conclude that Sp1, Ap1 and an unknown transcription factor have important roles in regulating expression of the Ctpct gene.

Animals↗
PubMed1999

Protection against chronic cadmium toxicity by caloric restriction.

Exposure to cadmium (Cd) can result in nephrotoxicity and osteotoxicity. Because Cd-induced nephrotoxicity involves oxidative stress and caloric restriction decreases oxidative stress, we examined whether reduced caloric intake will protect against Cd-induced nephrotoxicity. In addition, the protection against the osteotoxicity was also examined. Male and female Sprague-Dawley rats were provided drinking water containing 100 mg Cd/l. Since fluid intake relative to the body weight was higher in females as compared to the males, the Cd concentration in their water was reduced to 80 mg/l after 3 months and 65 mg/l after 6.5 months. During the 27 month exposure period the males and females consumed a total of about 5 g Cd/kg body weight. Food was restricted to 20 g/day after the first 3 months. During the unrestricted food intake period Cd exposure reduced the bone density in females by 23%, with a partial recovery and stabilization during the caloric restriction phase. Hepatic and renal Cd accumulation and corresponding metallothionein (MT) levels were very similar in both sexes. The reported critical Cd concentration for nephrotoxicity was reached by 9 months. Renal MT levels were maximum at this time. Despite a 1.5-fold increase in renal Cd concentration over the next 18 months, there was no significant increase in renal MT levels. In spite of high renal Cd levels and lack of availability of sufficient MT, there was no sign of nephrotoxicity, as measured by urinary protein and glucose excretion. It is concluded that caloric restriction prevents Cd-induced nephrotoxicity and also appears to control the osteotoxicity of Cd.

Animals↗
PubMed1999

Macrophage-targeted CTP:phosphocholine cytidylyltransferase (1-314) transgenic mice.

CTP:phosphocholine cytidylyltransferase (CT) is a rate-limiting and complexly regulated enzyme in phosphatidylcholine (PC) biosynthesis and is important in the adaptation of macrophages to cholesterol loading. The goal of the present study was to use transgenesis to study the CT reaction in differentiated macrophages in vivo. We successfully created macrophage-targeted transgenic mice that overexpress a truncated form of CT, called CT-314. Sonicated homogenates of peritoneal macrophages overexpressing CT-314 protein demonstrated a two-fold increase in CT activity in vitro compared with homogenates from nontransgenic macrophages. CT-314 macrophages, however, demonstrated no increase in CT activity or PC biosynthesis in vivo. This finding could not be explained simply by intracellular mistargeting of CT-314, by the inability of CT-314 to associate with cellular membranes, or by substrate limitation. To further probe the mechanism, an in vitro assay using intact nuclei was developed in an attempt to preserve interactions between CT, which is primarily a nuclear enzyme in macrophages, and other nuclear molecules. This intact-nuclei assay faithfully reproduced the situation observed in living macrophages, namely, no significant increase in CT activity despite increased CT-314 protein. In contrast, CT activity in sonicated nuclei from CT-314 macrophages was substantially higher than that from nontransgenic macrophages. Thus, a sonication-sensitive interaction between excess CT and one or more nuclear molecules may be responsible for the limitation of CT activity in CT-314 macrophages. These data represent the first report of a CT transgenic animal and the first study of a differentiated cell type with excess CT.

Animals↗
PubMed1999

Protection against chronic cadmium toxicity by glycine.

A Japanese drug containing glycine, glycyrrhizin, and cysteine (Stronger Neo-Minophagen C) has been reported to protect against chronic cadmium (Cd) toxicity. The present study was conducted to evaluate which of the three constituents of this drug was the main antagonist for Cd toxicity and whether the mechanism of protection involved antioxidant action. Adult female Sprague-Dawley rats were injected sc with 5 micromol CdCl2/kg per day, five times per week, for 15 weeks. Four groups of Cd-injected animals received co-treatments with either 10 mg glycyrrhizin/kg, 100 mg glycine/kg, 5 mg cysteine/kg, or with a mixture of all three compounds, five times per week, starting from week 7. An additional Cd-injected group was co-treated with vitamin E (100 mg/kg, five times per week, starting from week 7) as a positive control. Only those animals that received vitamin E, Minophagen mixture, or glycine were protected against Cd-induced hepatotoxicity as well as nephrotoxicity. All three co-treatments suppressed Cd-induced hepatic and renal lipid peroxidation. We conclude that the reported beneficial effects of Stronger Neo-Minophagen C are due to glycine, which appears to protect against chronic Cd toxicity by reducing oxidative stress.

Animals↗
PubMed1999

Treatment of chronic cadmium nephrotoxicity by N-acetyl cysteine.

Chronic cadmium (Cd)-induced nephrotoxicity is believed to be irreversible at advanced stages and no treatment is currently available. This study examined the beneficial effect of N-acetyl cysteine (NAC) on Cd-induced nephrotoxicity. Female Sprague-Dawley rats were injected s.c. with 5 micromol CdCl2/kg per day, five times/week for up to 26 weeks. Nephrotoxicity was detected after 10 weeks by elevation in urinary lactate dehydrogenase activity and protein. NAC co-administration from week 13 prevented the progression of nephrotoxicity. In these animals, with low-level nephrotoxicity, discontinuation of Cd exposure at the end of week 22 resulted in gradual recovery over the next several weeks, without the need for treatment with NAC. On the other hand, discontinuation of NAC co-treatment at the end of week 22 resulted in quick progression of nephrotoxicity, indicating that NAC protection was short-lived. Resumption of NAC treatment and cessation of Cd exposure after 26 weeks resulted in rapid recovery from advanced nephrotoxicity. It is concluded that protection from Cd-induced nephrotoxicity is possible by continued co-administration of NAC and that recovery from advanced nephrotoxicity can also be achieved with NAC, provided that Cd exposure is stopped.

Acetylcysteine↗
PubMed1999

Racial differences in the significance of coronary calcium in asymptomatic black and white subjects with coronary risk factors.

OBJECTIVES: To compare the significance of a specific feature of coronary atherosclerosis--coronary calcium--in asymptomatic black and white subjects with coronary risk factors. BACKGROUND: The natural history and clinical evolution of coronary atherosclerosis differs between blacks and whites. Differences in the underlying pathobiology of atherosclerosis may be one determinant of the ethnic variability in the clinical manifestation of coronary atherosclerosis. METHODS: In 1,375 high-risk but asymptomatic subjects (93 blacks [6.8%] and 1,282 whites [93.2%]) with at least one risk factor but no prior evidence of coronary disease, we assessed coronary risk factors, calculated Framingham risk of a coronary event and evaluated coronary calcium with digital subtraction fluoroscopy. We then followed these subjects clinically for 70 +/- 13 months, noting the occurrence of the following coronary events: death due to coronary heart disease (CHD); myocardial infarction (MI); angina pectoris; and performance of coronary bypass or angioplasty. RESULTS: Risk factor profiles were similar in black and white subjects (6-year Framingham risk 15 +/- 7% in blacks, 14 +/- 8% in whites [NS]). Coronary calcium was present in 59.9% of white subjects but only 35.5% of black subjects (p = 0.0001). Nevertheless, after 70 months of follow-up, more blacks than whites (22 blacks [23.7%] vs. 190 whites [14.8%]; p = 0.04) suffered one of the following end points: CHD death, MI, angina or revascularization. The age, gender and coronary risk-adjusted odds ratio of black race for at least one event was 2.16 (95% CI 1.34 to 3.48). CONCLUSIONS: Despite having a lowered prevalence of coronary calcium than high risk whites, high risk blacks suffer more CHD events. Coronary calcium therefore does not carry the same pathobiologic significance in blacks that it does in whites, consistent with the concept that there are specific racial differences in the natural history of CHD and its evolution into clinically manifest events.

Aged↗
PubMed1999

The effects of biphasic and conventional monophasic defibrillation on postresuscitation myocardial function.

OBJECTIVES: The purpose of this study was to compare the effects of biphasic defibrillation waveforms and conventional monophasic defibrillation waveforms on the success of initial defibrillation, postresuscitation myocardial function and duration of survival after prolonged ventricular fibrillation (VF). BACKGROUND: We have recently demonstrated that the severity of postresuscitation myocardial dysfunction was closely related to the magnitude of the electrical energy of the delivered defibrillation shock. In the present study, the effects of fixed 150-J low-energy biphasic waveform shocks were compared with conventional monophasic waveform shocks after prolonged VF. METHODS: Twenty anesthetized, mechanically ventilated domestic pigs were investigated. VF was induced with an AC current delivered to the right ventricular endocardium. After either 4 or 7 min of untreated ventricular fibrillation (VF), the animals were randomized for attempted defibrillation with up to three 150-J biphasic waveform shocks or conventional sequence of 200-, 300- or 360-J monophasic waveform shocks. If VF was not reversed, a 1-min interval of precordial compression preceded a second sequence of up to three shocks. The protocol was repeated until spontaneous circulation was restored or for a total of 15 min. RESULTS: Monophasic waveform defibrillation after 4 or 7 min of untreated VF resuscitated eight of 10 pigs. All 10 pigs treated with biphasic waveform defibrillation were successfully resuscitated. Transesophageal echo-Doppler, arterial pressure and heart rate measurements demonstrated significantly less impairment of cardiovascular function after biphasic defibrillation. CONCLUSIONS: Lower-energy biphasic waveform shocks were as effective as conventional higher energy monophasic waveform shocks for restoration of spontaneous circulation after 4 and 7 min of untreated VF. Significantly better postresuscitation myocardial function was observed after biphasic waveform defibrillation.

Analysis of Variance↗
PubMed1999

Roles of human hepatic cytochrome P450s 2C9 and 3A4 in the metabolic activation of diclofenac.

Recently, it was shown that diclofenac was metabolized in rats to reactive benzoquinone imines via cytochrome P450-catalyzed oxidation. These metabolites also were detected in human hepatocyte cultures in the form of glutathione (GSH) adducts. This report describes the results of further studies aimed at characterizing the human hepatic P450-mediated bioactivation of diclofenac. The reactive metabolites formed in vitro were trapped by GSH and analyzed by LC/MS/MS. Thus, three GSH adducts, namely, 5-hydroxy-4-(glutathion-S-yl)diclofenac (M1), 4'-hydroxy-3'-(glutathion-S-yl)diclofenac (M2), and 5-hydroxy-6-(glutathion-S-yl)diclofenac (M3), were identified in incubations of diclofenac with human liver microsomes in the presence of NADPH and GSH. The formation of the adducts was taken to reflect the intermediacy of the corresponding putative benzoquinone imines. While M2 was the dominant metabolite over a substrate concentration range of 10-50 microM, M1 and M3 became equally important products at >/=100 microM diclofenac. The formation of M2 was inhibited by sulfaphenazole or an anti-P450 2C9 antibody (5-10% of control values). The formation of M1 and M3 was inhibited by troleandomycin, ketoconazole, or an anti-P450 3A4 antibody (30-50% of control values). In studies in which recombinant P450 isoforms were used, M2 was generated only by P450 2C9-catalyzed reaction, while M1 and M3 were produced by P450 3A4-catalyzed reaction. Good correlations were established between the extent of formation of M2 and P450 2C9 activities (r = 0.93, n = 10) and between the extent of formation of M1 and M3 and P450 3A4 activities (r = 0.98, n = 10) in human liver microsomal incubations. Taken together, the data suggest that the biotransformation of diclofenac to M2 is P450 2C9-dependent, whereas metabolism of the drug to M1 and M3 involves mainly P450 3A4. Although P450s 2C9 and 3A4 both catalyze the bioactivation of diclofenac, P450 2C9 is capable of producing the benzoquinone imine intermediate at lower drug concentrations which may be more clinically relevant.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1999

A novel endonuclease mechanism directly visualized for I-PpoI.

A novel mechanism of DNA endonucleolytic cleavage has been visualized for the homing endonuclease I-PpoI by trapping the uncleaved enzyme-substrate complex and comparing it to the previously visualized product complex. This enzyme employs a unique single metal mechanism. A magnesium ion is coordinated by an asparagine residue and two DNA oxygen atoms and stabilizes the phosphoanion transition state and the 3'oxygen leaving group. A hydrolytic water molecule is activated by a histidine residue for an in-line attack on the scissile phosphate. A strained enzyme-substrate-metal complex is formed before cleavage, then relaxed during the reaction.

Amino Acid Substitution↗
PubMed1999

Indirubin, the active constituent of a Chinese antileukaemia medicine, inhibits cyclin-dependent kinases.

Indirubin is the active ingredient of Danggui Longhui Wan, a mixture of plants that is used in traditional Chinese medicine to treat chronic diseases. Here we identify indirubin and its analogues as potent inhibitors of cyclin-dependent kinases (CDKs). The crystal structure of CDK2 in complex with indirubin derivatives shows that indirubin interacts with the kinase's ATP-binding site through van der Waals interactions and three hydrogen bonds. Indirubin-3'-monoxime inhibits the proliferation of a large range of cells, mainly through arresting the cells in the G2/M phase of the cell cycle. These results have implications for therapeutic optimization of indigoids.

Animals↗
PubMed1999

Electrocardiographic prediction of the success of cardiac resuscitation.

OBJECTIVES: To identify a method for predicting the success or failure of a defibrillatory shock such as to avoid potentially detrimental interruptions of cardiopulmonary resuscitation (CPR). Such a method would also guide more optimal programming of automated external defibrillators. DESIGN: Prospective, observational animal study. SETTING: Medical research laboratory in a university-affiliated research and educational foundation. SUBJECTS: Domestic pigs. INTERVENTIONS: Ventricular fibrillation (VF) was electrically induced in 66 domestic pigs. After an interval of between 3 and 5 mins of untreated VF, precordial compression was begun. Electrocardiographic lead 2 was monitored and artifacts produced during precordial compression were removed by digital filtering. MEASUREMENTS AND MAIN RESULTS: In the derivation study, electrical defibrillation restored spontaneous circulation in 30 of the 66 animals. Successfully resuscitated animals had significantly greater coronary perfusion pressure, maximum VF amplitude, mean VF amplitude, and dominant VF frequency. No animals were resuscitated if the coronary perfusion pressure was <8 mm Hg, maximum amplitude was <0.48 mV, mean amplitude was <0.25 mV, or dominant frequency <9.9 Hz independently of the duration of untreated VF. When mean amplitude and dominant frequency were combined, the predictability was further improved. In an additional validation study of 14 animals, consecutive defibrillations were uniformly unsuccessful if the combination of mean amplitude and dominant frequency did not exceed the threshold values obtained in derivation study. CONCLUSION: Mean VF amplitude alone or in combination with dominant frequency of VF was expressed as a numerical score. It served as an objective noninvasive measurement on a par with that of coronary perfusion pressure for predicting the success of defibrillation. As such, it minimizes the detriment of repetitively interrupting mechanical interventions during CPR for electrical defibrillation when an electrical shock predictably fails to restore an effective rhythm.

Animals↗
PubMed1999

Sublingual capnometry: a new noninvasive measurement for diagnosis and quantitation of severity of circulatory shock.

OBJECTIVE: To investigate the feasibility and predictive value of sublingual Pco2 (P(SL)CO2) measurements as a noninvasive and early indicator of systemic perfusion failure. DESIGN: A prospective, criterion study. SETTING: Emergency department and medical and surgical intensive care units of an urban community medical center. PARTICIPANTS AND PATIENTS: Five normal human volunteers and 46 patients with acutely life-threatening illness or injuries. INTERVENTIONS: Intra-arterial or automated cuff blood pressure and arterial blood lactate (LAC) were measured concurrently with P(SL)CO2. RESULTS: P(SL)CO2 in five healthy volunteers was 45.2 +/- 0.7 mm Hg (mean +/- sD). Twenty-six patients with physical signs of circulatory shock and LAC >2.5 mmol/L had a P(SL)CO2 of 81 +/- 24 mm Hg. This contrasted with patients admitted without clinical signs of shock and LAC of <2.5 mmol/L who had a P(SL)CO2 of 53 +/- 8 mm Hg (p < .001). The initial P(SL)CO2 of 12 patients who died before recovery from shock was 93 +/- 27 mm Hg, and this contrasted with 58 +/- 11 mm Hg (p < .001) in hospital survivors. Increases in P(SL)CO2 were correlated with increases in LAC (r2 = .84; p < .001). When P(SL)CO2 exceeded a threshold of 70 mm Hg, its positive predictive value for the presence of physical signs of circulatory shock was 1.00. When it was <70 mm Hg, it predicted survival with a predictive value of 0.93. CONCLUSION: P(SL)CO2 may serve as a technically simple and noninvasive clinical measurement for the diagnosis and estimation of the severity of circulatory shock states.

Adolescent↗
PubMed1999

Effects of hyper- and hypoventilation on gastric and sublingual PCO(2).

We investigated the effects of hyper- and hypoventilation on gastric (Pg(CO(2))) and sublingual (Psl(CO(2))) tissue PCO(2) before, during, and after reversal of hemorrhagic shock. Pg(CO(2)) was measured with ion-sensitive field-effect transistor sensor and Psl(CO(2)) with a CO(2) microelectrode. Under physiological conditions and during hemorrhagic shock, decreases in arterial (Pa(CO(2))) and end-tidal (PET(CO(2))) PCO(2) induced by hyperventilation produced corresponding decreases in Pg(CO(2)) and Psl(CO(2)). Hypoventilation produced corresponding increases in Pa(CO(2)), PET(CO(2)), Pg(CO(2)), and Psl(CO(2)). Accordingly, acute decreases and increases in Pa(CO(2)) and PET(CO(2)) produced statistically similar decreases and increases in Pg(CO(2)) and Psl(CO(2)). No significant changes in the tissue PCO(2)-Pa(CO(2)) gradients were observed during hemorrhagic shock in the absence or in the presence of hyper- or hypoventilation. Acute changes in Pg(CO(2)) and Psl(CO(2)) should, therefore, be interpreted in relationship with concurrent changes in Pa(CO(2)) and/or PET(CO(2)).

Animals↗
PubMed1999

Studies on cytochrome P-450-mediated bioactivation of diclofenac in rats and in human hepatocytes: identification of glutathione conjugated metabolites.

The nonsteroidal anti-inflammatory drug diclofenac causes a rare but potentially fatal hepatotoxicity that may be associated with the formation of reactive metabolites. In this study, three glutathione (GSH) adducts, namely 5-hydroxy-4-(glutathion-S-yl)diclofenac (M1), 4'-hydroxy-3'-(glutathion-S-yl)diclofenac (M2), and 5-hydroxy-6-(glutathion-S-yl)diclofenac (M3), were identified by liquid chromatography-tandem mass spectrometry analysis of bile from Sprague-Dawley rats injected i.p. with a single dose of diclofenac (200 mg/kg). These adducts presumably were formed via hepatic cytochrome P-450 (CYP)-catalyzed oxidation of diclofenac to reactive benzoquinone imines that were trapped by GSH conjugation. In support of this hypothesis, M1, M2, and M3 were generated from diclofenac in incubations with rat liver microsomes in the presence of NADPH and GSH. Increases in adduct formation were observed when incubations were performed with liver microsomes from phenobarbital- or dexamethasone-treated rats. Adduct formation was inhibited by polyclonal antibodies against CYP2B, CYP2C, and CYP3A (40-50% inhibition at 5 mg of IgG/nmol of CYP) but not by an antibody against CYP1A. Maximal inhibition was obtained when the three inhibitory antibodies were used in a cocktail fashion (70-80% inhibition at 2.5 mg of each IgG/nmol of CYP). These data suggest that diclofenac undergoes biotransformation to reactive metabolites in rats and that CYP isoforms of the 2B, 2C, and 3A subfamilies are involved in this bioactivation process. With respect to CYP2C isoforms, rat hepatic CYP2C7 and CYP2C11 were implicated as mediators of the bioactivation based on immunoinhibition studies using antibodies specific to CYP2C7 and CYP2C11. Screening for GSH adducts also was carried out in human hepatocyte cultures containing diclofenac, and M1, M2, and M3 again were detected. It is possible, therefore, that reactive benzoquinone imines may be formed in vivo in humans and contribute to diclofenac-mediated hepatic injury.

Animals↗