Skin disorders and moisture in incontinent nursing home residents: intervention implications.

OBJECTIVE: To provide data needed to design an intervention trial to prevent or treat skin disorders in a high risk, incontinent nursing home population. DESIGN: The incidence and prevalence of nine common skin disorders were measured prospectively over a 60-day period using trained observers. Urinary and fecal incontinence frequency were measured over 24 hours, and mobility was measured with subjects both in and out of bed. Direct measures of skin moisture were taken with an impedance device in the presence and absence of urinary incontinence. Multiple regression analyses were used to relate the incontinence and mobility variables to the presence and development of skin disorders. SETTING: Four nursing homes. PARTICIPANTS: One hundred incontinent nursing home residents. MAIN OUTCOME MEASURES: Prospective measures of nine common skin disorders and skin moisture in four perineal regions under continent and incontinent conditions. RESULTS: All subjects had at least one skin condition identified during the 60-day data collection period. The most commonly observed skin condition was blanchable erythema, which occurred in 94% of the subjects, predominantly in the front and back regions that were closest to the urethra and rectum. Twenty-one percent of residents developed either a Stage 1 (nonblanchable erythema) or 2 pressure ulcer. All skin conditions were transient when measured every 3 weeks with the exception of blanchable erythema, which showed stability. Stage 3 or greater pressure ulcers and edema were not observed, and interrater reliability for the measure of papules was poor. Measures of urinary and fecal incontinence severity were correlated with blanchable erythema severity, and blanchable erythema and low bed mobility were predictive of pressure ulcer severity. Blanchable erythema severity was also predictive of Stage 1 and 2 pressure ulcers. Skin moisture levels in the back perineal farthest from the rectum (peripheral) were affected most by urinary incontinence. CONCLUSION: A trial to detect a 50% preventive effect on Stage 1 and 2 pressure ulcers would require that 167 subjects be monitored for 60 days. The transient nature of the skin effects require that skin be monitored at least once a week. Because blanchable erythema is so prevalent and appears to be associated with more severe skin conditions, it would make an excellent marker for beginning to assess the potential preventive effects of various interventions on the incidence of pressure ulcers and other related skin disorders in incontinent patients. It is likely that the back area peripheral to the urethra and rectum would experience the greatest benefit from an intervention trial to reduce moisture caused by incontinence.

Phospholipase A2 activation in cultured mouse hepatocytes exposed to tumor necrosis factor-alpha.

High concentrations of tumor necrosis factor alpha (TNF alpha) are cytotoxic to cultured hepatocytes. Impairment of energy metabolism and generation of an intracellular oxidant stress are important events in the pathogenesis of this toxicity (6). In the present study, we have examined the role of phospholipase A2 activation in TNF alpha-induced toxicity in mouse hepatocytes, since it has been reported to play a key role in TNF alpha cytolytic activity in other cell types. Recombinant murine TNF alpha (0.1 microgram/mL) caused a dose-dependent increase in PLA2 activity in cultured mouse hepatocytes. The increase in PLA2 activity was observed after only 0.5 hour of exposure (152 +/- 10% of control), and continued to increased over the first 4 hours of exposure (292 +/- 32%). However, TNF alpha-induced GSSG efflux and ATP depletion did not occur until after 2 hours of exposure. Furthermore, a small level of cytotoxicity was observed after a 24 hour incubation period. Putative PLA2 inhibitors, chlorpromazine (CPZ) and 4-bromophenacyl bromide (BPB), both prevented the TNF alpha-induced increase in PLA2 activity. They also reduced ATP depletion, GSSG efflux, and cytotoxicity. The PLA2 inhibitor, manoalide (a natural marine product), completely prevented PLA2 activation and cytotoxicity induced by TNF alpha. Pretreatment of hepatocytes with cycloheximide, to inhibit protein synthesis, increased TNF alpha-induced cytotoxicity. Cycloheximide pretreatment also potentiated PLA2 activation, ATP depletion, and GSSG efflux. CPZ and BPB both reduced the extent of PLA2 activation, ATP depletion, GSSG formation, and cytotoxicity in the cycloheximide pretreated cells exposed to TNF alpha. Taken together, these results demonstrate that TNF alpha activates PLA2, which occurs prior to other deleterious events in hepatocytes, and that inhibition of PLA2 activity reduces cell injury by TNF alpha. This suggests that PLA2 activation may lead to impairment of energy metabolism, an oxidant stress, and cytotoxicity in cells exposed to TNF alpha. Additionally, protein synthesis inhibition potentiates TNF alpha induction of PLA2 and toxicity, suggesting that there is a protein-synthesis-dependent protective mechanism in hepatocytes which ameliorates the effects induced by PLA2. These findings provide strong evidence that PLA2 activation plays an important role in the pathogenesis of toxicity induced by TNF alpha in cultured mouse hepatocytes.

Tumor necrosis factor: receptor binding and expression of receptors in cultured mouse hepatocytes.

Recombinant murine tumor necrosis factor (TNF-alpha) was labeled with 125I and used to determine the binding characteristics, internalization and intracellular degradation in cultured mouse hepatocytes. [125I]TNF-alpha bound specifically to hepatocytes and Scatchard analysis of the data indicated binding to both a low-affinity (Kd = 20 nM) high capacity (51225 sites/cell) component and high-affinity component (Kd = 4 pM), with low capacity (290 sites/cell). The extent of TNF-alpha binding to hepatocytes correlated closely with its biological activity in hepatocytes, as indexed by depletion of intracellular ATP. At concentrations lower than 0.06 nM there was minimal binding and no effect on cellular ATP, whereas maximal binding at concentrations greater than 45 nM caused 80% depletion (in comparison to controls) of hepatocyte ATP. Incubation at 37 degrees C resulted in rapid uptake, internalization and degradation of [125I]TNF-alpha. This was followed by release of degraded material from hepatocytes. Examination, by reverse transcriptase/polymerase chain reaction technology, of hepatocyte RNA extracted after the 4-hr adherence period revealed that mouse hepatocytes expressed mRNA for both TNF-alpha receptor 1 and TNF-alpha receptor 2, and that the relative abundance of TNF-alpha receptor 1 was approximately 7-fold greater than that for TNF-alpha receptor 2. Because it has been shown that these receptors have different affinities for TNF-alpha, this may explain the high- and low-affinity binding sites present on cultured mouse hepatocytes.

The role of xanthine oxidase in oxidative damage caused by cytokines in cultured mouse hepatocytes.

In the present study we have examined the potential role of xanthine oxidase (XO) in the intracellular oxidative stress induced by combinations of recombinant murine TNF alpha (rMuTNF alpha) and murine interferon-gamma (IFN gamma) in cultured mouse hepatocytes. IFN gamma alone and the combination of rMuTNF alpha and IFN gamma increased XO activity after a 4 hr exposure period. rMuTNF alpha alone increased XO activity only after 24 hr. At the later time point, the increased XO activity was accounted for by decreased XDH activity. However, the apparent conversion of XDH to XO cannot account for the early effects of rMuTNF alpha on hepatocyte function, particularly the onset of an oxidative stress (as indicated by efflux of GSSG from the hepatocytes). This effect is observed after two hours, and it is temporally the earliest sign of alteration of cellular function caused by rMuTNF alpha. Increased XO activity was not observed until 4 hr after treatment with rMuTNF alpha/IFN gamma. In addition, inhibition of XO activity with allopurinol did not ameliorate GSSG efflux from hepatocytes treated with the cytokines. However, the ATP depletion caused by the combination of rMuTNF alpha and IFN gamma and the cytotoxicity observed with the combined cytokines in cells pre-treated with BCNU (to inhibit glutathione reductase) was inhibited by allopurinol. These results show that the onset of oxidative stress in cultured mouse hepatocytes is not due to conversion of XDH to XO. However, events which follow the efflux of GSSG, such as ATP depletion and cytotoxicity in cells with impaired anti-oxidant defenses, may be partially due to increased XO activity, especially in cells treated with both rMuTNF alpha and IFN tau.

Oxidative stress in cultured hepatocytes exposed to acetaminophen.

The effect of acetaminophen (APAP) exposure on the formation of oxidized glutathione (GSSG) was investigated in cultured mouse hepatocytes to determine if oxidative damage is involved in the toxicity of this drug. Incubations of hepatocytes for 24 hr with 1 mM APAP produced a time-dependent loss of cell viability which was preceded by depletion of reduced glutathione (GSH) and an increase in GSSG formation. Pretreatment with 1,3-bis(chloroethyl)-1-nitrosourea (BCNU) (0.1 mM) for 30 min, which irreversibly inhibited glutathione reductase (GSSG-Rd) activity, increased the extent of GSSG formation produced by APAP exposure and potentiated its cell killing. Pretreatment of hepatocytes with 20 mM deferoxamine (DFO) for 1 hr to chelate ferric iron decreased GSSG formation and cell killing produced by APAP. Pretreatment with BCNU or DFO did not affect APAP oxidation as determined by the formation of the APAP-GSH conjugate or the covalent binding of APAP metabolites to cellular protein. Hence, increasing the susceptibility of hepatocytes to an oxidative stress with BCNU increased both the formation of GSSG and cell killing produced by APAP. Conversely, decreasing their susceptibility to an oxidative stress by chelating iron with DFO decreased GSSG formation and cell injury. It follows that APAP toxicity involves oxidative processes that occur early in the poisoning process and are a major factor contributing to injury in these cells.

Cytokine toxicity and induction of NO synthase activity in cultured mouse hepatocytes.

Interferon-gamma (IFN-gamma) has been shown to exacerbate tumor necrosis factor alpha (TNF alpha)-induced hepatotoxicity in vivo as well as act synergistically with TNF alpha in a variety of biological actions. In the present study we have examined interactions of IFN-gamma with TNF alpha and the role of nitric oxide synthase (NOS) activity in the generation of an intracellular oxidant stress in isolated mouse hepatocytes. Exposure to either IFN-gamma or TNF alpha significantly increased NOS activity. In combination, TNF alpha and IFN-gamma markedly increased NOS activity beyond that expected for a merely additive effect. IFN-gamma potentiated TNF alpha-induced effects on the hepatocyte glutathione pool, increasing the extent of GSH depletion and GSSG efflux. Furthermore, IFN-gamma exacerbated TNF alpha-induced ATP depletion. Exposure to both TNF alpha and IFN-gamma resulted in significant cytotoxicity in hepatocytes, whereas neither cytokine alone produced any toxicity. TNF alpha-induced cytotoxicity in hepatocytes pretreated with 1,3-bis (chloroethyl)-1-nitrosourea (BCNU, a glutathione reductase inhibitor) was potentiated by IFN-gamma. TNF alpha/IFN-gamma-induced GSSG efflux was prevented when hepatocytes were treated with the antioxidant mannitol. Furthermore, mannitol reduced the extent of ATP depletion as well as cytotoxicity induced by TNF alpha and IFN-gamma in either BCNU- or non-BCNU-treated hepatocytes. In contrast, mannitol abolished cytotoxicity in BCNU-treated cells exposed to TNF alpha alone. Thus, mannitol provides significant protection against deleterious oxidative effects induced by IFN-gamma and TNF alpha. However, IFN-gamma also appears to potentiate the deleterious effects of TNF alpha, at least in part, by mechanisms other than an increase in oxygen radical generation. Using the methylated analog of arginine, NG-monomethyl-L-arginine, to inhibit NOS activity, it was demonstrated that TNF alpha/IFN-gamma-induced ATP depletion, GSSG efflux, and cytotoxicity were not dependent upon the stimulation of NOS. Furthermore, significant increases in NOS activity did not occur until after 4 hr of exposure to either cytokine, whereas GSSG efflux and ATP depletion occurred during the first 4 hr of incubation. Taken together, these results indicate that IFN-gamma acts synergistically with TNF alpha, resulting in the potentiation of an intracellular oxidative stress, inhibition of energy metabolism, and cytotoxicity. However, these events do not appear to be related to an increase in NOS activity.

Protection from oxidative damage in mouse liver cells.

Paracetamol toxicity in mouse hepatocytes involved oxidative stress initiated by the formation of NAPQI. This oxidative component of paracetamol injury is associated with the latter stages of the poisoning process. Ebselen, a drug with GSH-peroxidase activity, was effective in ameliorating these oxidative events.

Tumor necrosis factor induced oxidative stress in isolated mouse hepatocytes.

Tumor necrosis factor alpha (TNF alpha) is a macrophage-derived cytokine which participates in homeostatic tissue repair. It is also a potentially useful antitumor agent. Liver toxicity, however, limits TNF alpha's clinical utility and suggests that it may play a role in liver toxicity of various etiologies. To determine the direct effects of TNF alpha on hepatocytes, in the absence of infiltrating leukocytes and other inflammatory mediators, an isolated mouse hepatocyte model has been used in the present study. Hepatocytes exposed to recombinant human TNF alpha (1-10 micrograms/ml) exhibited intracellular GSH depletion and GSSG efflux during the first 2 hr of exposure, but no cytotoxicity was observed. However, TNF alpha was toxic to hepatocytes pretreated with 1,3-bis(chloroethyl)-1-nitrosourea to inhibit GSSG-reductase activity. Furthermore, these cells exhibited a greater efflux of GSSG upon exposure to TNF alpha. TNF alpha also caused a marked decrease in cellular ATP concentrations, which occurred after initiation of effects on the glutathione pool. These findings indicate that high concentrations of TNF alpha induce an oxidant stress in isolated hepatocytes. The antioxidants mannitol and benzoate, as well as the iron chelator deferoxamine, reduced the extent of TNF alpha-induced oxidant effects in hepatocytes, which indicates that the oxidant stress may involve hydroxyl radical generation. Hepatocytes treated with ruthenium red or fructose were less susceptible to TNF alpha-induced ATP depletion, which suggests that mitochondrial calcium cycling may be involved in disruption to energy metabolism.

Postnatal mice have low susceptibility to paracetamol toxicity.

The hepatotoxicity of paracetamol in mice of 2, 3, 8-10, 24-26, 32-34, and 52-54 wk of age was determined by lethality data, histopathologic examination of the liver, and appearance of glutamate-pyruvate transaminase and glutamate-oxaloacetate transaminase activities in the plasma over an 8-h exposure period. At a dose of 300 mg/kg, there was evidence of hepatocytic necrosis and transaminase leakage in the 32- to 34- and 52- to 54-wk-old mice, but lethality was only recorded in the oldest age group. At 500 mg/kg, paracetamol produced 30% lethality in 3-wk-old mice and between 50 and 90% lethality in the adult age groups. There was histologic evidence of hepatocytic necrosis at all of these ages and its extent increased with age. Similarly, there were increases in plasma transaminases in each of these age groups. However, in 2-wk-old mice there was no lethality, no hepatocytic necrosis, and no increase in plasma transaminases. The lack of susceptibility of 2-wk-old mice to paracetamol toxicity was not due to immaturity of the cytochrome P-450 enzymes responsible for metabolism of paracetamol to its reactive metabolite (N-acetyl-p-benzoquinone imine). In fact, the activity of this enzyme pathway in 2-wk-old mice was greater than that in adults. The partial clearance of the glutathione-derived metabolites of paracetamol after a nontoxic (50 mg/kg) dose was 80% greater in 2-wk-old mice than in 8- to 10-wk-old mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Postnatal development of enzyme activities associated with protection against oxidative stress in the mouse.

A number of enzyme systems are important in the protection of cells from chemical-induced oxidative damage. Little is known of the relative importance of these enzymes during postnatal development and its is possible that changes in their activity during this period may alter the susceptibility to toxic agents. This study investigated the activities of glutathione peroxidase, glutathione reductase, catalase, superoxide dismutase, gamma-glutamyl-cysteine synthetase and glutathione synthetase in the liver, lung and kidney of postnatal and adult mice. The first 3 postnatal weeks are characterized by marked changes in the activities of enzymes that protect against oxidative stress (glutathione peroxidase/reductase, catalase and superoxide dismutase). Overall, the activity of these enzymes suggests that the mouse has a higher level of protection against peroxides at various stages during this period but lower capacity to detoxify superoxide anions. The activities of the glutathione-synthetic enzymes (gamma-glutamylcysteine synthetase and glutathione synthetase) were significantly lower in the kidney of the postnatal mice, but the liver and lung had levels similar to those in the adult. Glutathione turnover in the liver of 2-week-old mice was not different from that in adults. The results indicate a complex pattern of development in the activities of detoxification enzyme systems during postnatal development.

A role for the glutathione peroxidase/reductase enzyme system in the protection from paracetamol toxicity in isolated mouse hepatocytes.

The role of the glutathione peroxidase/reductase (GSH-Px/GSSG-Rd) enzyme system in protection from paracetamol toxicity was investigated in isolated mouse hepatocytes in primary culture. The effect of inhibitors of these enzymes on the toxicity of paracetamol and on t-butylhydroperoxide (t-BOOH), used as a positive control, was determined. 1,3-Bis(chloroethyl)-1-nitrosourea (BCNU) was used to inhibit GSSG-Rd, and goldthioglucose (GTG) used to inhibit GSH-Px. Both these inhibitors increased cell membrane damage in response to oxidative stress initiated by t-BOOH. However, they also increased the susceptibility of hepatocytes to paracetamol toxicity, indicating that a component of paracetamol's toxic effect involves formation of species that are detoxified by the GSH-Px/GSSG-Rd enzymes. To further examine the role of these enzymes, age-related differences in their activity were exploited. Hepatocytes from two-week-old mice were less susceptible to both t-BOOH and paracetamol toxicity than were those from adult mice. This corresponds to higher activity of cytosolic GSH-Px/GSSG-Rd in this age group. However, after inhibition of GSSG-Rd with BCNU, hepatocytes from these postnatal mice were more susceptible to paracetamol toxicity. This suggests that the higher activity of GSH-Px/GSSG-Rd in hepatocytes from two-week-old mice is responsible for their reduced susceptibility to paracetamol toxicity. The data indicate that the GSH-Px/GSSG-Rd enzymes contribute to protection from paracetamol toxicity and suggest that formation of peroxides contributes to this drug's hepatotoxic effects.

Responses of erectile tissue from impotent men to pharmacological agents.

Erectile tissue was removed from the corpora cavernosa of 25 impotent men undergoing surgery for insertion of penile prostheses. Strips, set up in an organ bath, were contracted by the alpha-adrenergic agonist phenylephrine. There was no significant difference between tissue taken from men with diabetes, alcoholism, Peyronie's disease or men with no obvious condition causing the impotence. The sensitivity of tissues from hypertensive patients was significantly reduced but this was probably due to drugs being taken for hypertension. Precontracted tissues could be relaxed by acetylcholine or isoprenaline. The responses, however, were inconsistent, so that no difference between the different groups of patients was apparent.

Comparison of the susceptibility of hepatocytes from postnatal and adult mice to hepatotoxins.

Age-related changes of susceptibility to hepatotoxicity induced by four hepatotoxic compounds were investigated using an isolated mouse hepatocyte model. Hepatocytes isolated from 2-week-old mice and adult mice (8-10 weeks old) were exposed to different concentrations (including toxic concentrations) of paracetamol, furosemide, iodoacetic acid and t-butylhydroperoxide for incubation times up to 24 hr. Cell damage was assessed by leakage of lactate dehydrogenase. Analysis of variance indicated that the hepatocytes from the 2-week-old mice were less susceptible to the toxic effects of all four hepatotoxins. The activities of catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase were determined in both hepatocytes and whole liver from the two age groups. While catalase was significantly greater in adults, glutathione peroxidase, glutathione reductase and superoxide dismutase were all higher in the 2-week-old mice. Since these three enzymes are involved with protection against oxidative stress, it is likely that the higher activity in hepatocytes from 2-week-old mice is responsible for the reduced susceptibility to damage induced by the four hepatotoxins.