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S D Cohen

Publications and source records attributed to S D Cohen.

At least 55 records · Page 3Linked to original sources
PubMed1989

Selective protein arylation by acetaminophen and 2,6-dimethylacetaminophen in cultured hepatocytes from phenobarbital-induced and uninduced mice. Relationship to cytotoxicity.

To evaluate the mechanistic importance of covalent binding in acetaminophen (APAP)-induced hepatotoxicity, we compared the effects of 2,6-dimethylacetaminophen (2,6-DMA) to those of APAP in primary cultures of mouse hepatocytes. Immunochemical analysis of electrophoretically separated proteins has shown that the majority of covalent binding after a cytotoxic dose of APAP occurs on two major bands of 44 and 58 kD (Bartolone et al., Biochem Pharmacol 36: 1193-1196, 1987). At equimolar concentrations, 2,6-DMA bound proteins only 15% as extensively as did APAP and was not cytotoxic in hepatocytes from uninduced mice. However, when the hepatocytes were obtained from phenobarbital-induced mice, APAP administration resulted in increased protein arylation and a more rapid onset of cytotoxicity. Furthermore, in the cells from phenobarbital-induced mice, 2,6-DMA not only resulted in increased binding but also in overt cytotoxicity. Since our affinity-purified anti-APAP antibody did not cross-react with 2,6-DMA, a new antibody specific for 2,6-DMA was prepared and, after affinity purification, was used to detect 2,6-DMA protein adducts by Western blotting. Results indicated that, in hepatocytes from both phenobarbital-induced and non-induced mice, the binding of 2,6-DMA was also highly selective with the most prominent target being the 58 kD cytosolic protein. However, by contrast to APAP, only minimal binding to the 44 kD protein was detected after 2,6-DMA treatment. Although several additional protein adducts were increased in treated cells from phenobarbital-induced mice, the 58 kD protein was clearly the most prominently arylated target associated with both APAP and 2,6-DMA cytotoxicity. These data suggest that both the specificity of covalent binding as well as the extent of binding to the major targets may play an important role in the ensuing toxicity.

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

Selective protein arylation and the age dependency of acetaminophen hepatotoxicity in mice.

Male CD-1 mice 1, 1.5, 2, and 3 months old were given 600 mg of acetaminophen (APAP)/kg, po, and liver damage was assessed 12 hr later. The most severe hepatotoxicity was in 3-month-old mice, while the other age groups exhibited little damage. The onset of susceptibility to APAP hepatotoxicity did not correlate with the level of activity of the mixed-function oxidase system as assessed in vitro, since drug metabolizing capability was similar between 2- and 3-month-old mice. Through 4 hr after administration of APAP to 2- and 3-month-old mice in vivo, glutathione (GSH) depletion and both plasma and liver APAP concentrations were similar between ages. Additionally, 24 hr after dosing, 3-month-old mice excreted marginally more APAP-glucuronide conjugate and parent compound in urine than 2-month-old animals, while both age groups excreted similar amounts of the APAP-sulfate and GSH-derived conjugates. Even though the extent of binding of radioactive APAP to macromolecules at 4 hr was similar between 2- and 3-month-old animals, the pattern of immunochemically targetted cytosolic and microsomal proteins was different. Thus, in APAP exposure the extent of binding to specific proteins rather than the overall amount of covalent binding may be the critical determinant of the hepatotoxic response. In the present study, the age-related differences in susceptibility to APAP-induced hepatotoxicity were related to the differences in selective protein arylation.

Acetaminophen↗
PubMed1989

Immunohistochemical localization of acetaminophen-bound liver proteins.

A sensitive immunofluorescence assay was developed for localizing acetaminophen (APAP) protein adducts in liver sections from treated mice. Affinity-purified anti-APAP antibodies, when applied to liver sections from mice given 600 mg APAP/kg, po, were preferentially localized in cells of the centrilobular region. At 30 min after dosing, covalently bound APAP was detected only in those cells most proximal to the central vein. Thereafter, binding spread throughout the centrilobular zone. However, by 8 hr the overall intensity of staining decreased and binding appeared more diffuse. Western blot analysis of electrophoretically resolved proteins from similarly treated mice revealed a corresponding temporal arylation of cytosolic proteins by APAP and indicated that the fluorescence detected at 30 min was associated with arylation of protein(s) of 44 kDa. The findings demonstrate the sensitivity and utility of immunohistochemical techniques in the study of covalently bound toxicants and emphasizes the temporal link between selective protein arylation in individually targetted cells to the development of APAP hepatotoxicity.

Acetaminophen↗
PubMed1988

Immunochemical analysis of acetaminophen covalent binding to proteins. Partial characterization of the major acetaminophen-binding liver proteins.

A sensitive immunoassay for detecting acetaminophen (APAP) bound to proteins was developed using an affinity purified antibody directed against the N-acetylated end of the APAP molecule. Western blots of electrophoretically resolved liver proteins taken from mice given an hepatotoxic dose of APAP demonstrated that nearly 85% of the total detectable protein-bound APAP was covalently associated with proteins of 44 and 58 kD. Pretreatment of liver extracts with the sulfhydryl-specific reagent, N-ethylmaleimide (NEM), prior to derivatization with the reactive metabolite of APAP, N-acetyl-p-benzoquinone imine (NAPQI), greatly reduced immunochemically detectable APAP-protein adducts and indicated that the antibody detects protein-thiol conjugates of APAP. To investigate the basis of the binding selectivity in vivo, a variety of systems which yielded APAP-protein adducts were analyzed. Systems which activate APAP enzymatically, as in hepatocyte suspensions or in post-mitochondrial (S9) fractions fortified with an NADPH-regenerating system, resulted in a protein binding profile similar to that produced in vivo. Conversely, when extracts or cells were treated with chemically synthesized NAPQI, an alternative protein binding profile was obtained. Two-dimensional electrophoretic analysis of the reduced protein thiol (PSH) content of liver proteins using [3H]NEM labeling revealed that the 58 kD APAP-binding proteins were rich in PSH, whereas the major 44 kD binding protein had virtually no detectable PSH. Many PSH-rich proteins that were not arylated in vivo did bind NAPQI in vitro. However, the 44 kD proteins were not arylated when chemically synthesized NAPQI was added to homogenates or cell suspensions. The present data further suggest that, in addition to the amount and reactivity of free protein sulfhydryls, the cellular localization with respect to the cytochrome P-450 activation site may influence the susceptibility of proteins to NAPQI binding. These findings signal the need for caution in interpreting studies of APAP mechanisms that rely solely on NAPQI addition.

Acetaminophen↗
PubMed1988

Antidotal effectiveness of N-acetylcysteine in reversing acetaminophen-induced hepatotoxicity. Enhancement of the proteolysis of arylated proteins.

The post-arylative mechanisms by which N-acetylcysteine (NAC) reduces the severity of the hepatotoxicity induced by acetaminophen (APAP) were investigated in primary cultures of mouse hepatocytes. When administered at selected times immediately following removal of medium containing 10 mM APAP, 2.0 mM NAC was shown to restore glutathione levels through 16 hr of APAP pretreatment and to minimize the leakage of glutamate-oxaloacetate transaminase resulting from the first 8 hr of drug exposure. This temporal difference defined a critical period in which cells were responsive to NAC and permitted the investigation of potential post-arylative mechanisms of the antidote. In the absence of NAC during the recovery period, the cellular loss of covalently-bound APAP could be accounted for by the appearance of arylated proteins in the medium without any apparent degradation of APAP-bound proteins. By contrast, when NAC was present during the recovery period, there was a decrease in intracellular protein-bound APAP which could not be accounted for by that detected in the medium. Since during the recovery period the low residual intracellular concentration of APAP could not contribute significantly to any additional covalent binding in this system, NAC could not merely be acting as a nucleophilic trap for the reactive electrophile. Furthermore, NAC is not likely to dissociate covalently bound APAP from proteins. Hence, the overall decrease in covalent binding observed in cultures previously exposed to APAP for up to 8 hr must have arisen from an NAC-dependent enhancement of the degradation of the arylated proteins. However, after a more prolonged exposure to APAP, the ineffectiveness of NAC may have resulted from APAP-induced irreparable damage to the intracellular proteolytic system. These data suggest that the post-arylative efficacy of NAC may reside in the ability of the antidote to restore the functional capacity of the proteolytic system to rid the cells of arylated proteins.

Acetaminophen↗
PubMed1988

Dissociation of covalent binding from the oxidative effects of acetaminophen. Studies using dimethylated acetaminophen derivatives.

The cytotoxic effects of 10 mM acetaminophen (APAP) in primary cultures of non-induced mouse hepatocytes are accompanied by depletion of intracellular glutathione (GSH), arylation of protein, and loss of protein sulfhydryl (PSH) groups. Investigation of the stoichiometry of the covalent binding and PSH loss after APAP exposure demonstrated a greater loss in PSH than could be accounted for by covalent binding to proteins and suggests that APAP exhibits both oxidative and arylative actions in cell culture. Subcellular fractionation revealed that the PSH oxidation induced by APAP was greatest in the microsomal fraction. Exposure of the hepatocytes to 10 mM 3,5-dimethyl-acetaminophen (3,5-DMA) or 2,6-dimethyl-acetaminophen (2,6-DMA) permitted dissociation of the oxidative and arylative properties of APAP. Even though treatment of cultured hepatocytes with 3,5-DMA did not result in covalent binding, there was a more rapid depletion of intracellular GSH, oxidation of PSH, and cytotoxicity compared to APAP. This investigation also provides the first evidence that the cytotoxic effects of both APAP and 3,5-DMA are accompanied by the formation of protein aggregates of high molecular weight that are not disulfide linked. The aggregates probably reflect the oxidative properties of these drugs and may be a mediator of their toxic effects. By contrast, 2,6-DMA, which did bind to cellular proteins and deplete GSH, did not lead to PSH loss, protein aggregation, or cytotoxicity. Since PSH oxidation and protein aggregation correlated well with cytotoxicity, these data suggest that the oxidative component of APAP and 3,5-DMA can play a significant role in eliciting cellular damage in cultured hepatocytes.

Acetaminophen↗
PubMed1988

Acetaminophen-induced inhibition of hepatic mitochondrial respiration in mice.

Morphological changes are observed in mitochondria early in the course of acetaminophen (APAP) hepatotoxicity. In order to determine if functional deficits also occur, this study examined the effect of APAP, in vivo and in vitro, on mitochondrial respiration in fasted, male CD-1 mice (3-4 months old). After a hepatotoxic dose of APAP (600 mg/kg, po), when glutamate was used as the respiratory substrate, state 3 respiration (ADP-stimulated) was inhibited and this was reflected in a decreased respiratory control ratio (RCR). In contrast, when succinate was the respiratory substrate, the decreased RCR was reflective of an increase in state 4 (resting) respiration. There was no detectable effect after a nonhepatotoxic dose of APAP (300 mg/kg, po). These APAP-induced respiratory effects and hepatotoxicity were prevented by piperonyl butoxide pretreatment, and were absent in 1- and 2-month-old mice, which are resistant to APAP-induced damage. Since the APAP-induced inhibition of mitochondrial respiration, in vivo, correlated with age-related and piperonyl butoxide-dependent differences in toxicity, the data suggest that the in vivo effects result, at least in part, from a mixed-function oxidase generated metabolite. In vitro, both state 3 and state 4 respiration, as well as the RCR, were inhibited by APAP in a concentration-dependent manner with glutamate as substrate. However, no effects were observed with succinate as substrate, thereby contrasting with results obtained following in vivo exposure. Therefore the in vitro effects of APAP are different from those observed in vivo and may result from a direct insult of the parent compound. These studies suggest that early alterations in mitochondrial function may be mechanistically important in APAP hepatotoxicity.

Acetaminophen↗
PubMed1988

Rat IL-3 stimulates the growth of rat mucosal mast cells in culture.

Rat mast cells with the properties of mucosal mast cells (MMC) proliferate in cultures of haemopoietic tissue in the presence of conditioned medium (CM) from antigen- or mitogen-activated T lymphocytes. The present study shows that recombinant rat interleukin-3 (rIL-3) both stimulated the development of MMC from bone marrow (BM) precursors and maintained the proliferation of rat MMC lines in an identical manner to that of CM. The content per cell of the MMC granule-specific proteinase RMCPII was similar in both IL-3- and CM-stimulated cultures. Passage of CM through DEAE-cellulose separated two active peaks that stimulated autologous MMC proliferation. The biochemical properties of peak 1 were similar to those of murine IL-3 and stimulated multi-potential stem cell development in soft agar cultures of BM cells from rats treated with 5-fluorouracil (which enriches for haemopoietic stem cells). RIL-3 was also active in this assay whereas peak 2 was not, demonstrating that peak 1 contained IL-3 activity. The presence of MMC in the majority of multi-potential colonies in the soft agar cultures confirmed the early stem cell origin of the MMC lineage. The cultured BM-derived mast cells in the rat are analogues of the MMC subset that is most readily observed proliferating in the gastrointestinal tract in response to helminth parasite infection. The demonstration that IL-3 is responsible for the development and proliferation of MMC should lead to a better understanding of the functional roles of these cells.

Animals↗
PubMed1987

Antiviral effect of 9-(1,3-dihydroxy-2-propoxymethyl)guanine against cytomegalovirus infection in a guinea pig model.

The antiviral activity of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) against guinea pig cytomegalovirus (GPCMV) was evaluated in guinea pig cell cultures and in Hartley guinea pigs. The 50% effective dose of DHPG against GPCMV replication in cell cultures was 71 microM. Ultrastructural studies revealed that DHPG inhibited the formation of viral cores and the production of nucleocapsids, enveloped virions and dense bodies, but the drug did not prevent the formation of virus induced intranuclear tubular structures. In vivo, guinea pigs inoculated intraperitoneally with GPCMV were treated with DHPG, 25 mg/kg subcutaneously, twice daily. Treatment was initiated 24 h after infection and continued for 7 days. During the acute infection, the average body weights of DHPG-treated, virus infected guinea pigs were approximately 14% lower than the sham-treated counterparts on day 10, 11 and 13 post-virus inoculation. Virus infectivity titers were higher in the lungs of DHPG-treated guinea pigs on day 10 than the sham-treated ones. Although there was no significant difference on histopathologic lesions in the spleen, liver and lungs of the drug-treated and the sham-treated guinea pigs, DHPG treated animals appeared to have fewer virus-induced lesions or inclusions in the kidneys and salivary glands than the sham-treated ones. In addition, virus infectivity titers in the salivary gland of DHPG treated guinea pigs were consistently lower than the sham-treated animals.

Acyclovir↗
PubMed1987

Relationship of skin conductance activity to clinical features in obsessive-compulsive ritualizers.

Before treatment 49 obsessive-compulsive (o-c) ritualizers were presented with two series of brief stimuli--15,100db tones (brief neutral) and 15 presentations of a ritual-evoking object (brief aversive). Habituation of skin conductance (SC) responses to the tones was reduced compared with that previously found in normal subjects. Neither habituation rates to tones nor aversive stimuli were related to coexisting anxiety or depression or to the severity of o-c symptoms. The increased arousal induced by the aversive stimuli was sustained, that induced by the tones was short-lived and SC level and subjective anxiety had returned to resting levels by the end of the tone series. Concordance between SC activity and subjective anxiety was much greater during and after the presentations of ritual evoking stimuli than of tones. There were few correlations between SC and clinical measures, though patients who strongly resisted and were able to control their compulsive urges were more aroused.

Anxiety Disorders↗
PubMed1987

Comparison of fluorescent-antibody-to-membrane-antigen test, indirect immunofluorescence assay, and a commercial enzyme-linked immunosorbent assay for determination of antibody to varicella-zoster virus.

The demand for sensitive and specific assays to determine immune status to varicella can be expected to increase with the anticipated availability of a varicella-zoster virus vaccine for use in nonimmune adults, especially health care personnel, and in immunosuppressed children. Although the fluorescent-antibody-to-membrane-antigen (FAMA) test remains the reference standard to which other tests are compared, simpler alternative assays are needed. In this study, the FAMA was compared with a simple indirect immunofluorescence assay (IFA) and a commercially available enzyme-linked immunosorbent assay (ELISA) for the detection of antibody to varicella-zoster virus. One hundred and twelve serum samples were screened by the FAMA test and IFA at a 1:5 dilution, and 100% agreement was found. Of these samples, 101 were available for testing by ELISA, and identical results were obtained with 97 samples (96% agreement). When the samples were screened at a 1:2 dilution, 99 of 101 results agreed. In addition, 31 spinal fluid samples were tested by all three methods. When screening was at a 1:2 dilution, there was 96.8% agreement between the FAMA test and IFA. When the cutoff value established for sera was used for the spinal fluid samples, there was 90.3% agreement between the ELISA and the FAMA test. Thus, both IFA and ELISA can be considered sensitive and specific alternatives to the FAMA test, and in addition, both use commercially available reagents.

Antibodies, Viral↗
PubMed1987

Extrahepatic lesions induced by acetaminophen in the mouse.

Acetaminophen in acute overdose is primarily recognized as potentially hepatotoxic with few descriptions of extrahepatic lesions other than nephrotoxicity. Fasted adult, male mice, both standard and germ-free, were given acetaminophen orally and killed at selected times, from 30 minutes to 48 hours after treatment. In addition to the expected hepatic effects after 600 mg acetaminophen/kg, degenerative and necrotic changes were found in four non-hepatic tissues. Nephrosis developed 2 to 4 hours after treatment and paralleled the course of hepatic damage. Necrosis of bronchiolar epithelium in the absence of inflammation was evident 4 to 6 hours after acetaminophen administration as was onset of testicular changes. Spermatidic degeneration with early development of spermatidic multinucleated giant cells were characteristic features. Areas of lymphoid necrosis were also visible in splenic follicles and Peyer's patches 18 to 24 hours after treatment. These observations have demonstrated that other tissues in addition to liver and kidney are damaged by acetaminophen toxicity and should be considered in cases of acetaminophen overdosage.

Acetaminophen↗
PubMed1987

Ultrastructural changes during acute acetaminophen-induced hepatotoxicity in the mouse: a time and dose study.

This study was undertaken to evaluate the early ultrastructural changes during the development of acetaminophen hepatotoxicity. Doses at or near the threshold for hepatotoxicity were selected to permit comparison of early reversible effects to those which ultimately progressed to necrosis in the absence of early agonal effects or drug-induced mortality. Both 300- and 600-mg/kg doses resulted in similar declines in hepatic glutathione levels to 14 and 22% of control values, respectively, by 2 hours, with more rapid recovery after the low dose. Plasma sorbitol dehydrogenase activity was elevated after 600 mg/kg but not after 300 mg/kg. During the first 2 hours after acetaminophen there was cytomegaly with rapid progression to necrosis after 600 mg/kg but minimal progression after 300 mg/kg. Ultrastructurally, vesiculation, vacuolation and mitochondrial and plasma membrane degeneration culminated in scattered single cell death by 4 hours and widespread centrilobular necrosis by 8 hours after 600 mg/kg. The time course of lesion development was slower after 300 mg/kg with damage restricted to the first two to three rows of centrilobular cells and limited numbers of isolated necrotic cells by 8 hours. By 18 to 24 hours livers of mice given 300 mg/kg appeared normal. Results are consistent with the endoplasmic reticulum being the site of acetaminophen activation and initial attack. However, early ultrastructural changes in mitochondria and plasma membrane observed after the high dose were not prominent after the low dose. This suggests that early acetaminophen damage to these organelles may play a critical role in acetaminophen hepatotoxicity.

Acetaminophen↗
PubMed1986

Alteration of in vivo and in vitro effects of heroin by esterase inhibition.

Selective inhibition of peripheral esterases by tri-ortho-tolyl phosphate in the mouse resulted in an increase in the analgetic activity of heroin, without affecting the activity of morphine. In vitro inhibition of esterases by paraoxon reduced the affinity of heroin for the opiate receptor, while that of morphine was unaffected. These results suggest that both central and peripheral esterases are involved in the metabolism of heroin and that interference with critical esterases can alter its pharmacologic and toxicologic effects.

Acetates↗
PubMed1985

Differential cholinesterase inhibition and muscarinic receptor changes in CD-1 mice made tolerant to malathion.

The lethality of organophosphate (OP) cholinesterase (CHE) inhibitors is thought to result from depression of the respiratory center in the brain stem, constriction of and increased secretion by the airways, and paralysis of the respiratory musculature. While tolerance to the cholinergic toxicity of OPs has been well documented, such studies have not included investigations of the brain stem and extrapulmonary airways. In this report tolerance to the insecticide malathion is demonstrated. At 24 hr after 14 daily doses of malathion (400 mg/kg, ip), CHE activities were 27, 26, and 28% of control in striatum (ST), hippocampus (HI), and cortex (CX), respectively, while brain stem CHE activity was 41% of control. In addition, the numbers of muscarinic receptors (Bmax) decreased 30, 20, and 22% in ST, HI, and CX, respectively. In contrast, there was no change in brain stem Bmax. The lack of parallelism between CHE inhibition and Bmax effects in brain stem as compared to all other tissues tested raises questions as to the mechanism(s) by which the respiratory system adapts during repeated OP exposure.

Animals↗