PubMed Health⌕ Search

Biomedical subjects

S G Hart

Publications and source records attributed to S G Hart.

9 recordsLinked to original sources
PubMed1995

Immunohistochemical localization of acetaminophen in target tissues of the CD-1 mouse: correspondence of covalent binding with toxicity.

Administration of hepatotoxic doses of acetaminophen (APAP) to mice results in necrosis, not only of liver cells but of renal proximal tubules and bronchiolar and olfactory epithelium. In the liver, covalent binding is localized to the centrilobular hepatocytes which later undergo necrosis. This study was undertaken to compare the cellular distribution of bound APAP in all four major target tissues with that of cytochrome P4502E1 (a P450 isoenzyme commonly associated with APAP bioactivation), with emphasis on the cell types which later undergo necrosis. Tissues were collected from mice at selected times after APAP administration (600 mg/kg, po) and fixed by microwave irradiation for immunohistochemistry, or in formalin for histopathological study. Immunohistochemical localization of bound APAP was performed on 5-microns paraffin sections using an affinity-purified anti-APAP antibody. Similar tissues from naive mice were used for immunohistochemical localization of cytochrome P4502E1 (using a polyclonal sheep anti-P4502E1 antibody). Positive staining with both the anti-APAP and the anti-P4502E1 antibodies was similar in distribution, being present in the cell types which become damaged by APAP in all four target tissues. These results demonstrate that covalent binding and subsequent necrosis are localized in common with cytochrome P4502E1, suggesting that, as in the liver, toxicity in extrahepatic targets is also related to the ability of these tissues to activate APAP in situ.

Acetaminophen↗
PubMed1995

Gender-related differences in susceptibility to acetaminophen-induced protein arylation and nephrotoxicity in the CD-1 mouse.

Acetaminophen (APAP) is a commonly used analgesic and antipyretic agent which, in high doses, causes liver and kidney necrosis in man and animals. Damage in both target organs is greatly dependent upon biotransformation. However, in the CD1 mouse only males exhibit cytochrome P450-dependent nephrotoxicity and selective protein covalent binding. The lack of renal toxicity in female mice may reflect the androgen dependence of renal CYP2E1. To study this, female mice were pretreated with testosterone propionate and then challenged 6 days later with APAP. Groups of control males and females were similarly challenged with APAP for comparison. All groups exhibited hepatotoxicity after APAP with similar glutathione (GSH) depletion, covalent binding, centrilobular necrosis, and elevation of plasma sorbitol dehydrogenase activity. By contrast, APAP-induced nephrotoxicity occurred only in males and in the females pretreated with testosterone. No nephrotoxicity was evident in APAP-challenged control females. The selective pattern of hepatic and renal protein arylation previously reported for male mice was similarly observed in testosterone-pretreated female mice. Western blot analysis of microsomes showed that testosterone increased renal CYP2E1 levels without altering hepatic CYP2E1. Testosterone pretreatment, in vivo, also resulted in increased activation of APAP in vitro in kidney microsomes with no effect on the in vitro activation of APAP in liver microsomes. These data suggest that APAP-mediated GSH depletion, covalent binding, and toxicity in the kidneys of testosterone-pretreated females results from increased APAP activation by the testosterone-induced renal CYP2E1. This further suggests that renal, rather than hepatic, biotransformation of APAP to a toxic electrophile is central to APAP-induced nephrotoxicity in the mouse.

Acetaminophen↗
PubMed1994

Acetaminophen nephrotoxicity in CD-1 mice. I. Evidence of a role for in situ activation in selective covalent binding and toxicity.

Acetaminophen (APAP) administration (600 mg/kg, ip) to 18-hr-fasted, 3-month-old male CD-1 mice results in necrosis of the renal convoluted proximal tubules. To selectively inhibit APAP activation in the kidney but not in the liver, 10-week-old male CD-1 mice were castrated under ether anesthesia and allowed to recover for a minimum of 2 weeks before use. Microsomal activation of APAP in vitro was monitored by trapping the APAP-derived electrophile as an N-acetylcysteine conjugate. Production of the conjugate was significantly decreased in renal microsomes from castrated mice. By contrast, hepatic microsomal production of the APAP conjugate was not changed by castration. Castration also did not alter APAP covalent binding in liver or APAP hepatotoxicity. Also, the overall metabolism of APAP was not altered by castration, as there were no significant differences in the 24-hr excretion of APAP urinary metabolites between castrated and sham-operated mice. However, castration did significantly protect mice against APAP nephrotoxicity, as evidenced by decreases in plasma urea nitrogen levels and in the severity of histopathologic damage assessed 12 hr after APAP. Castration also reduced the level of selective covalent binding of APAP metabolites to renal proteins as detected on Western blots with affinity-purified anti-APAP antibodies. These findings suggest that, for the mouse, intrarenal biotransformation of APAP to a reactive electrophile significantly contributes to the APAP covalent binding within the kidneys and the subsequent nephrotoxicity.

Acetaminophen↗
PubMed1994

Clofibrate pretreatment diminishes acetaminophen's selective covalent binding and hepatotoxicity.

Peroxisome proliferators have been shown to diminish acetaminophen (APAP) hepatotoxicity (Biochem. Pharmacol. 43, 1395, 1992). To investigate the mechanistic basis for this protection CD-1 male mice were given corn oil or 500 mg clofibrate (CFB)/kg, ip, daily for 10 days. They were then fasted overnight and either killed without challenge or at 4 or 12 hr after challenge with 800 mg APAP/kg (in 50% propylene glycol). At 12 hr, hepatotoxicity was evidenced by elevated plasma sorbitol dehydrogenase and histopathology in corn oil but not in CFB-pretreated mice. At 4 hr after APAP treatment, hepatic glutathione (GSH) depletion and selective arylation of the major APAP target proteins were both greatly diminished by CFB pretreatment. Western blot analysis with the anti-58 antibody of liver cytosol from unchallenged mice showed no apparent changes in the levels of the 58-kDa major APAP target protein with CFB treatment. These findings suggest that protection could be the result of diminished net availability of generated electrophile. In vitro, measurements indicated that the specific activity in microsomes for APAP oxidation by cytochrome P450 was not changed by CFB treatment; whereas GSH S-transferase activity in cytosol was decreased by 25%. Pretreatment with CFB also produced a significant elevation in hepatic GSH. These studies indicate that protection by CFB might result from increased availability of hepatic GSH which could trap APAP electrophile nonenzymatically, thereby decreasing covalent binding and preventing toxicity.

Acetaminophen↗
PubMed1990

Acetaminophen hepatotoxicity: correspondence of selective protein arylation in human and mouse liver in vitro, in culture, and in vivo.

Human and mouse liver were exposed to an APAP-activating system, in vitro. Subsequent immunochemical analysis of electrophoretically separated proteins with an affinity-purified anti-APAP antibody indicated that when a cytosolic fraction from human liver was incubated with APAP, an NADPH-regenerating system, and mouse microsomes selective APAP binding occurred predominantly to proteins of approximately 38, 58, and 130 kDa. To evaluate whether similar proteins are targeted in situ, primary cultures of human hepatocytes were treated with 10 mM APAP for 4 hr prior to immunochemical analysis. APAP binding was again detected in protein bands of approximately 38, 58, and 130 kDa. In addition, selective binding was also noted to other cytosolic protein bands, e.g., approximately 52 and 62 kDa. For mouse liver, the majority of the binding, in vitro or in culture, was to proteins of approximately 44 and 58 kDa with lesser binding to proteins of approximately 33 and 130 kDa among others. By contrast, at the times monitored, little covalent binding was detected in the 44-kDa region in the human liver experiments. Most noteworthy was the finding that when the protein arylation patterns on liver samples from a human APAP fatality were compared to those from a mouse given a hepatotoxic dose of APAP, the binding patterns were similar to those detected after the in vitro and the culture experiments with mouse and human livers. Furthermore, an immunohistochemical analysis revealed that as with the mouse, APAP covalent binding in the human liver exhibited a distinct zonal pattern consistent with centrilobular binding. That APAP arylation of the 58- and 130-kDa proteins was observed in livers from both mice and humans suggests that the mouse provides a valid model for studying the mechanistic importance of covalent binding. Elucidation of the identities and functions of the common targeted proteins may clarify their toxicological significance.

Acetaminophen↗
PubMed1989

Selective acetaminophen metabolite binding to hepatic and extrahepatic proteins: an in vivo and in vitro analysis.

Acetaminophen (APAP) administration (600 mg/kg, po) to fasted male CD-1 mice resulted in cellular damage to liver, lung, and kidney. An affinity purified antibody against covalently bound APAP was used to identify APAP-protein adducts in microsomal and cytosolic extracts from these target organs. The proteins were resolved on SDS-PAGE, transblotted to nitrocellulose membranes, and analyzed immunochemically. Covalent binding of APAP to intracellular proteins was only observed in those organs which exhibited cellular damage; no APAP adducts were detected in tissues which did not undergo necrosis. In all target tissues the arylation of proteins was not random but highly selective with two adducts of 44 and 58 kDa accounting for the majority of the total APAP-bound proteins which were detected immunochemically. In addition, a third major APAP-protein adduct of 33 kDa was also observed in kidney cytosol. The severity of tissue damage and the amount of adducts present in these tissues could be significantly reduced when mice were pretreated with the mixed function oxidase inhibitor, piperonyl butoxide, prior to APAP dosing. Immunochemical analysis of plasma from APAP-treated animals indicated the presence of several protein adducts by 4 hr following drug administration. These adducts did not appear to be of plasma origin. Incubation of cytosolic proteins from liver, lung, kidney, spleen, brain, and heart with an APAP metabolite generating liver microsomal system demonstrated that the cytosolic 58-kDa protein target was native to all tissues tested. By contrast, the 58-kDa protein target did not appear to be endogenous to plasma since it was not detected when plasma was incubated in vitro with the liver microsomal system. These studies indicate that, although the 58-kDa proteins appear to be endogenous to both target and nontarget tissues, the 58-kDa APAP-protein adducts are detectable only in tissues which become damaged by APAP.

Acetaminophen↗
PubMed1989

Measuring moment-to-moment pilot workload using synchronous presentations of secondary tasks in a motion-based trainer.

A simulation was conducted to determine whether the sensitivity of secondary task measures of pilot workload could be improved by synchronizing their presentation to the occurrence of specific events or pilot actions. This synchronous method of presentation was compared to the more typical asynchronous method, where secondary task presentations are independent of pilot's flight-related activities. Twelve pilots flew low- and high-difficulty scenarios in a motion-base trainer with and without concurrent secondary tasks (e.g., choice reaction time, time production). The difficulty of each scenario was manipulated by the addition of 21 flight-related tasks superimposed on a standard approach and landing sequence. Secondary task performance did reflect workload differences between scenarios and among flight segments within scenarios, replicating the results of an earlier study in which the secondary tasks were presented asynchronously. In addition, the choice reaction time secondary task was also sensitive to the workload of specific activities within flight segments. Workload ratings were virtually identical between this and the earlier study.

Aerospace Medicine↗
PubMed1987

Inflight application of three pilot workload measurement techniques.

Three measures of workload were tested during 11 routine missions conducted by the NASA Kuiper Airborne Observatory: communications performance, subjective ratings, and heart rate. The activities that contributed to crewmember workload varied; the commander was responsible for aircraft control and navigation whereas the copilot handled communications with ATC and the astronomers. Ratings of workload, stress, and effort given by the two crewmembers were highly correlated and varied across flight segments, peaking during takeoff and landing. Since the pilots performed different tasks during each segment, their ratings appeared to reflect overall crew workload, rather than experiences specific to each pilot. Subjective fatigue increased significantly from takeoff to landing for all flights, although the increase was significantly greater as landing times shifted from 10:00 p.m. to 9:00 a.m. The type, source, number, and frequency of communications varied significantly across flight segments, providing an objective indicator of pilot workload. Heart rate was significantly higher for the aircraft commander than for the copilot. Although heart rate peaked for both positions during takeoff and landing, the amount of change was significantly greater for the aircraft commander. Subjective ratings of stress, workload, and mental effort were significantly correlated with heart rate and communications frequency but were unrelated to mission duration, rated fatigue, or pilot evaluation of performance.

Aerospace Medicine↗