Western blot analysis for nitrotyrosine protein adducts in livers of saline-treated and acetaminophen-treated mice.
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The hepatic centrilobular necrosis produced by the analgesic/antipyretic acetaminophen correlates with metabolic activation of the drug leading to its covalent binding to protein. However, the molecular mechanism of the toxicity is not known. Recent immunohistochemical analyses using an antinitrotyrosine antiserum indicated that nitrotyrosine protein adducts co-localized with the acetaminophen-protein adducts in the centrilobular cells of the liver. Nitration of proteins is believed to occur by peroxynitrite, a substance formed by the rapid reaction of superoxide with nitric oxide. Nitric oxide and superoxide may be formed by activated Kupffer cells or by other cells. Because we were unable to successfully utilize the commercial antiserum in Western blot analyses of liver fractions, we developed a new antiserum. With our antiserum, liver fractions from saline-treated control and acetaminophen-treated mice were successfully analyzed for nitrated proteins. The immunogen for this new antiserum was synthesized by coupling 3-nitro-4-hydroxybenzoic acid to keyhole limpet hemocyanin. A rabbit immunized with this adduct yielded a high titer of an antiserum that recognized BSA nitrated with peroxynitrite. Immunoblot analysis of nitrated BSA indicated that nitrotyrosine present in a protein sample could be easily detected at levels of 20 pmoles. Immunohistochemical analyses indicated that nitrotyrosine protein adducts were detectable in the centrilobular areas of the liver. Immunoblot analysis of liver homogenates from both saline-treated and acetaminophen-treated mice (300 mg/kg) indicate that the major nitrotyrosine protein adducts produced have molecular weights of 36 kDa, 44 kDa, and 85 kDa. The 85-kDa protein stained with the most intensity. The hepatic homogenates of the acetaminophen- treated mice showed significantly increased levels of all protein adducts. (+info)
Frequent paracetamol use and asthma in adults.
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BACKGROUND: The pulmonary antioxidant glutathione may limit airway inflammation in asthma. Since paracetamol (acetaminophen) depletes the lung of glutathione in animals, a study was undertaken to investigate whether frequent use in humans was associated with asthma. METHODS: Information was collected on the use of analgesics as part of a population based case-control study of dietary antioxidants and asthma in adults aged 16-49 years registered with 40 general practices in Greenwich, South London. The frequency of use of paracetamol and aspirin was compared in 664 individuals with asthma and in 910 without asthma. Asthma was defined by positive responses to questions about asthma attacks, asthma medication, or waking at night with shortness of breath. The association between analgesic use and severity of disease amongst asthma cases, as measured by a quality of life score, was also examined. RESULTS: Paracetamol use was positively associated with asthma. After controlling for potential confounding factors the odds ratio for asthma, compared with never users, was 1.06 (95% CI 0.77 to 1.45) in infrequent users (+info)
Intrabursal injection of clodronate liposomes causes macrophage depletion and inhibits ovulation in the mouse ovary.
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To investigate the role of the ovarian macrophage population in ovulation, we examined the effect of depleting this population using liposome-encapsulated clodronate. Clodronate liposomes, saline liposomes, or saline alone was injected under the ovarian bursa in gonadotropin-primed adult mice, either 84 h (Day -3) or 36 h (Day -1) before ovulation. Ovulation rates were determined by counting the number of oocytes released. The numbers of graafian follicles and corpora lutea were also counted immediately before and after ovulation. Macrophage distribution within the theca and stroma of preovulatory ovaries was examined by immunohistochemistry with specific monoclonal antibodies to the macrophage antigens macrosialin, major histocompatability complex class II (Ia), and F4/80. Injection of clodronate liposomes on Day -1 did not affect ovulation rates, whereas administration on Day -3 caused a significant reduction in ovulation rate (mean oocytes ovulated = 5. 25 +/- 0.6 from clodronate liposome-treated ovaries and 9.13 +/- 0.9 from saline-treated ovaries, respectively, P < 0.05). The numbers of macrosialin-positive macrophages present in the theca at ovulation were reduced by treatment with clodronate liposomes on Day -1, and treatment on Day -3 reduced the numbers of Ia-positive and macrosialin-positive macrophages present in the theca. When the subsequent ovarian cycles were examined by vaginal smearing, the metestrous-2/diestrous stage was found to be extended in clodronate liposome-treated animals (7.5 +/- 1.3 days vs. 3.4 +/- 0.4 days for saline liposome-treated animals, P < 0.05). These results suggest that thecal macrophages may be involved in the regulation of follicular growth and rupture, as well as being important for the normal progression of the estrous cycle. (+info)
Effect of continuous epidural 0.2% ropivacaine vs 0.2% bupivacaine on postoperative pain, motor block and gastrointestinal function after abdominal hysterectomy.
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We have investigated the effect of 24-h postoperative continuous epidural infusion of 0.2% ropivacaine or 0.2% bupivacaine 8 ml h-1 on pain, request for supplementary analgesics, motor block and gastrointestinal function, in a double-blind, randomized study in 60 patients undergoing open hysterectomy. There were no significant differences between groups in pain, number of patients requesting supplementary analgesics, motor block, ability to walk or time to first flatus or stool. In the subgroup of patients who received supplementary analgesics, patients in the ropivacaine group received significantly more ketorolac than patients in the bupivacaine group. Time to discharge from hospital was similar with ropivacaine and bupivacaine. (+info)
Effect of meloxicam on postoperative pain after abdominal hysterectomy.
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We studied 36 patients, allocated randomly to receive meloxicam 15 mg rectally (n = 18) or placebo suppository (n = 18) before total abdominal hysterectomy in a double-blind study. Visual analogue scores for pain at rest (P < 0.005), on movement (P < 0.05) and on coughing (P < 0.05) were significantly decreased in the meloxicam group during the first 24 h after surgery. Mean 24-h PCA morphine requirements were 33.2 (SD 16.9) mg and 38.2 (20.8) mg in the meloxicam and placebo groups, respectively (ns). There was no difference in the incidence of nausea, vomiting or sedation between groups. (+info)
Paracetamol, alcohol and the liver.
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It is claimed that chronic alcoholics are at increased risk of paracetamol (acetaminophen) hepatotoxicity not only following overdosage but also with its therapeutic use. Increased susceptibility is supposed to be due to induction of liver microsomal enzymes by ethanol with increased formation of the toxic metabolite of paracetamol. However, the clinical evidence in support of these claims is anecdotal and the same liver damage after overdosage occurs in patients who are not chronic alcoholics. Many alcoholic patients reported to have liver damage after taking paracetamol with 'therapeutic intent' had clearly taken substantial overdoses. No proper clinical studies have been carried out to investigate the alleged paracetamol-alcohol interaction and acute liver damage has never been produced by therapeutic doses of paracetamol given as a challenge to a chronic alcoholic. The paracetamol-alcohol interaction is complex; acute and chronic ethanol have opposite effects. In animals, chronic ethanol causes induction of hepatic microsomal enzymes and increases paracetamol hepatotoxicity as expected (ethanol primarily induces CYP2E1 and this isoform is important in the oxidative metabolism of paracetamol). However, in man, chronic alcohol ingestion causes only modest (about twofold) and short-lived induction of CYP2E1, and there is no corresponding increase (as claimed) in the toxic metabolic activation of paracetamol. The paracetamol-ethanol interaction is not specific for any one isoform of cytochrome P450, and it seems that isoenzymes other than CYP2E1 are primarily responsible for the oxidative metabolism of paracetamol in man. Acute ethanol inhibits the microsomal oxidation of paracetamol both in animals and man. This protects against liver damage in animals and there is evidence that it also does so in man. The protective effect disappears when ethanol is eliminated and the relative timing of ethanol and paracetamol intake is critical. In many of the reports where it is alleged that paracetamol hepatotoxicity was enhanced in chronic alcoholics, the reverse should have been the case because alcohol was actually taken at the same time as the paracetamol. Chronic alcoholics are likely to be most vulnerable to the toxic effects of paracetamol during the first few days of withdrawal but maximum therapeutic doses given at this time have no adverse effect on liver function tests. Although the possibility remains that chronic consumption of alcohol does increase the risk of paracetamol hepatotoxicity in man (perhaps by impairing glutathione synthesis), there is insufficient evidence to support the alleged major toxic interaction. It is astonishing that clinicians and others have unquestion-ingly accepted this supposed interaction in man for so long with such scant regard for scientific objectivity. (+info)
In vitro glucuronidation using human liver microsomes and the pore-forming peptide alamethicin.
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The UDP-glucuronosyltransferases (UGTs) are a superfamily of membrane-bound enzymes whose active site is localized inside the endoplasmic reticulum. Glucuronidation using human liver microsomes has traditionally involved disruption of the membrane barrier, usually by detergent treatment, to attain maximal enzyme activity. The goals of the current work were to develop a universal method to glucuronidate xenobiotic substrates using microsomes, and to apply this method to sequential oxidation-glucuronidation reactions. Three assays of UGT catalytic activity estradiol-3-glucuronidation, acetaminophen-O-glucuronidation, and morphine-3-glucuronidation, which are relatively selective probes for human UGT1A1, 1A6, and 2B7 isoforms, respectively, were developed. Treatment of microsomes with the pore-forming peptide alamethicin (50 microg/mg protein) resulted in conjugation rates 2 to 3 times the rates observed with untreated microsomes. Addition of physiological concentrations of Mg(2+) to the alamethicin-treated microsomes yielded rates that were 4 to 7 times the rates with untreated microsomes. Optimized assay conditions were found not to detrimentally affect cytochrome P450 activity as determined by effects on testosterone 6beta-hydroxylation and 7-ethoxycoumarin deethylation. Formation of estradiol-3-glucuronide displayed atypical kinetics, and data best fit the Hill equation, yielding apparent kinetic parameters of K(m)(app) = 0.017 mM, V(max)(app) = 0.4 nmol/mg/min, and n = 1.8. Formation of acetaminophen-O-glucuronide also best fit the Hill equation, with K(m)(app) = 4 mM, V(max)(app) = 1.5 nmol/mg/min, and n = 1.4. Alternatively, morphine-3-glucuronide formation displayed Michaelis-Menten kinetics, with K(m)(app) = 2 mM and V(max)(app) = 2. 5 nmol/mg/min. Finally, alamethicin treatment of microsomes was found to be effective in facilitating the sequential oxidation-glucuronidation of 7-ethoxycoumarin. (+info)
The hepatic inflammatory response after acetaminophen overdose: role of neutrophils.
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Acetaminophen overdose induces severe liver injury and hepatic failure. There is evidence that inflammatory cells may be involved in the pathophysiology. Thus, the aim of this investigation was to characterize the neutrophilic inflammatory response after treatment of C3Heb/FeJ mice with 300 mg/kg acetaminophen. A time course study showed that neutrophils accumulate in the liver parallel to or slightly after the development of liver injury. The number of neutrophils in the liver was substantial (209 +/- 64 PMN/50 high-power fields at 12 h) compared to baseline levels (7 +/- 1). Serum levels of TNF-alpha and the C-X-C chemokines KC and MIP-2 increased by 28-, 14-, and 295-fold, respectively, over levels found in controls during the injury process. In addition, mRNA expression of MIP-2 and KC were upregulated in livers of acetaminophen-treated animals as determined by ribonuclease protection assay. However, none of these mediators were generated in large enough quantities to account for neutrophil sequestration in the liver. There was no upregulation of Mac-1 (CD11b/ CD18) or shedding of L-selectin on circulating neutrophils. Moreover, an anti-CD18 antibody had no protective effect against acetaminophen overdose during the first 24 h. These results indicate that there is a local inflammatory response after acetaminophen overdose, including a substantial accumulation of neutrophils in the liver. Because of the critical importance of beta2 integrins for neutrophil cytotoxicity, these results suggest that neutrophils do not contribute to the initiation or progression of AAP-induced liver. The inflammation observed after acetaminophen overdose may be characteristic for a response sufficient to recruit neutrophils for the purpose of removing necrotic cells but is not severe enough to cause additional damage. (+info)