Biomedical subjects

P A Wilce

Publications and source records attributed to P A Wilce.

At least 37 records · Page 2Linked to original sources

PubMed1997

Ethanol feeding enhances inflammatory cytokine expression in lipopolysaccharide-induced hepatitis.

Elevated concentrations of plasma tumour necrosis factor (TNF)-alpha, interleukin (IL)-1 and IL-6 have been detected in patients with alcoholic hepatitis and have been implicated in the pathogenesis of hepatocyte necrosis. The present study used a rat model to conduct a detailed histological and biochemical examination of the expression of various pro-inflammatory cytokines and associated liver pathology in ethanol-potentiated lipopolysaccharide (LPS)-induced liver injury. Male Wistar rats were pair-fed either the control or ethanol-containing (36% of caloric intake as ethanol) form of the Lieber-DeCarli liquid diet for 6 weeks. Liver injury was induced by the i.v. injection of LPS (1 microgram/g bodyweight), with animals being killed at 0, 1, 3, 6, 12 and 24 h after injection. At the later time points, plasma transaminase and transpeptidase activities were significantly elevated in ethanol-fed LPS-treated rats compared with control-fed LPS-treated animals. At these times after LPS treatment, hepatocytes in ethanol-fed animals displayed fatty change and necrosis with an associated neutrophil polymorph infiltrate. Time course analysis revealed that plasma TNF-alpha (1-3 h post-LPS) and IL-6 (3 h post-LPS) bioactivity was significantly elevated in ethanol-fed compared with control-fed animals. No difference was seen in plasma IL-1 alpha concentration (maximal in both groups 6 h post-LPS). The expression of TNF-alpha, IL-1 alpha, IL-1 beta and IL-6 mRNA were elevated between 1 and 6 h post-LPS in the livers of both control and ethanol-fed rats. However, ethanol-fed LPS-treated animals exhibited significantly higher maximal expression of IL-1 and IL-6 mRNA. Comparison of the appearance of cytokine mRNA and plasma bioactivity indicated an effect of ethanol feeding on post-transcriptional processing and/or the kinetics of the circulating cytokines. Elevated levels of both hepatic cytokine mRNA expression and the preceding plasma cytokines are presumably a necessary prerequisite for hepatic injury seen in this model and, therefore, possibly for the damage seen in human alcoholics. Further studies using this model may lead to significant advances in our understanding of the pathogenic mechanisms of alcoholic liver disease in humans.

Alanine Transaminase↗

PubMed1997

Differential expression of mitochondrial NADH dehydrogenase in ethanol-treated rat brain: revealed by differential display.

The technique of polymerase chain reaction (PCR) differential display was used to detect alterations in gene expression after chronic alcohol administration. Male Wistar rats were treated with ethanol vapor for 14 days. The cDNA generated from mRNA isolated from the hippocampi of ethanol-treated and control animals was compared by PCR differential display. A differentially expressed cDNA fragment was used to screen mRNA samples by Northern analysis. The level of a mRNA was significantly elevated (x 2.5) in the hippocampus, but not the cortex of alcohol-treated rats up to 48 hr after withdrawal. Sequence analysis of the cDNA fragment revealed an almost perfect homology to rat mitochondrial NADH dehydrogenase subunit 4 mRNA. The selective induction of this mRNA in alcohol-treated rat brain areas suggests altered metabolic processes and possible dysfunction of the mitochondria. The technique of PCR differential display may prove useful in further analysis of gene expression during alcohol dependence and withdrawal.

Alcoholism↗

PubMed1996

FOS and JUN as markers for ethanol-sensitive pathways in the rat brain.

The expression of proteins coded by the immediate early genes of the fos family and c-jun was used to study the effect of acute ethanol administration on convulsant-induced neuronal activity in rat brain. Immunoreactivity for both types of protein was induced by either SC injection of pentylenetetrazole or by IP injection of N-methyl-D-aspartic acid. Both agents elicited distinct patterns of behaviour and a high level of FOS-immunoreactivity in the cerebral cortex and hippocampus. Acute IP doses of ethanol (1.0-3.0 g/kg) significantly reduced the behaviours and FOS-immunoreactivity induced in the cerebral cortex by both pentylenetetrazole and N-methyl-D-aspartic acid. Pentylenetetrazole-induced FOS-immunoreactivity in the hippocampus was also inhibited by ethanol. In contrast, N-methyl-D-aspartic acid-induced FOS-immunoreactivity in the hippocampus was not inhibited by any dose of ethanol. c-JUN immunoreactivity showed a distinct pattern of induction in the hippocampus after injection of N-methyl-D-aspartic acid. Ethanol (3.0 g/kg) inhibited N-methyl-D-aspartic acid-induced c-JUN-immunoreactivity in the hippocampus and cerebral cortex. The differences in inhibition of immunoreactivity suggest that the sensitivity of the NMDA- and GABAA-related neuronal pathways to ethanol varies among different anatomical structures.

Animals↗

PubMed1996

Decreased severity of ethanol withdrawal behaviors in kainic acid-treated rats.

The involvement of kainate (KA)-sensitive regions in ethanol withdrawal behaviors was investigated in male Wistar rats given three intraperitoneal (IP) injections of KA (12 mg/kg) or saline each followed by recovery at 4 degrees C for 5 h and room temperature for 3 days and a final KA or saline injection at room temperature. Some animals received MK-801 (1 mg/kg, IP) 30 min after each injection and one group received saline only. The saline/saline, saline/MK-801, and KA/MK-801 groups displayed typical ethanol withdrawal behaviors 8-12 h after ethanol withdrawal. These behaviors were attenuated in the KA/saline group. Audiogenic seizures could be induced in all treatment groups 12 h after withdrawal. There was severe neuronal degeneration in the hippocampal CA region and the piriform cortex of the KA/saline-treated animals that was reduced by MK-801 treatment. The inferior colliculus remained intact. These results suggest that the N-methyl-D-aspartate receptor mediates KA-induced damage in limbic structures and that these regions may play an important role in typical, but not audiogenically induced ethanol-withdrawal behaviors.

Animals↗

PubMed1996

Relationship between alcohol intake and immunoglobulin a immunoreactivity with acetaldehyde-modified bovine serum albumin.

Acetaldehyde, the main metabolite of ethanol, is a highly reactive species that reacts with macromolecules to produce unstable and stable adducts. Acetaldehyde-modified proteins are immunogenic and have been detected in the liver and blood of alcoholics. Furthermore, antibodies reactive with acetaldehyde-modified proteins have been detected in the plasma of social drinkers and alcoholics. However, the class distribution of immunoglobulins reactive with modified proteins was different in the two groups, being predominantly immunoglobulin (Ig)M in social drinkers, but IgM and IgA in alcoholics. In this study, we demonstrate that heavy drinkers (alcohol intake > 130 g/week for females and 150 g/week for males) also exhibit IgA reactivity with acetaldehyde-modified proteins. The IgA adduct-specific reactivity (IgA reactivity with acetaldehyde-modified bovine serum albumin-reactivity with native bovine serum albumin) showed a moderate correlation with self-reported alcohol intake, but did not correlate with markers such as plasma transaminase, gamma-glutamyltransferase activity, or mean corpuscular volume. IgA adduct-specific reactivity had similar specificity to the conventional tests of alcohol abuse, but had higher sensitivity than the other tests, especially with heavy drinkers. Data presented herein demonstrate that elevated IgA reactivity with acetaldehyde-modified epitopes is associated with heavy drinking and is a potential marker for high alcohol intake.

Acetaldehyde↗

PubMed1996

A preliminary study of the effects of laser radiation on collagen metabolism in cell culture.

A low power Ga-As pulse laser was used to stimulate cultured human embryonic fibroblast cells. Energy fluencies varied from 0-1 J/cm2 over a period of 1-4 days. Fibroblast procollagen production was monitored by the synthesis of [3H] hydroxyproline, and DNA replication was assessed by [3H] thymidine incorporation. Following laser treatment, controlled pepsin digestion measured the increase in cell biostimulation. Maximum increase in collagen production and cell biostimulation occurred after 4 episodes of laser treatment at 24-hour intervals. Laser doses between 0.099 and 0.522 J/cm2 had the most significant stimulatory effects on fibroblast function. Clinical efficacy of the low power Ga-As pulse laser may be related to enhanced connective tissue repair.

Arsenic↗

PubMed1995

In vivo pharmacological study of spermine-induced neurotoxicity.

Spermine-induced neurotoxicity and its pharmacological manipulation was studied in the rat striatum in vivo. Spermine (50, 100, 250 nmol) was injected into the striatum and the volume of damage quantified by computer-based image analysis. Spermine produced a dose-dependent increase in the volume of damage. Co-administration of MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate; dizocilpine, 60 nmol), 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline (25, 40 nmol) and pretreatment with pentobarbital (40 mg/kg, i.p.) significantly reduced the volume of damage induced by 100 nmol spermine. MK-801 (30 nmol) was also effective in reducing the damage induced by 50 nmol spermine. Treatment with a specific inhibitor of nitric oxide synthase, N omega-nitro-L-arginine methyl ester (50 mg/kg, i.p., twice daily for 10 days) was ineffective. These results suggest an involvement of both N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors in the cascade of spermine-induced neurotoxicity.

Animals↗

PubMed1995

Immediate early gene expression during morphine withdrawal.

The expression of immediate early genes (IEG)s c-fos, c-jun and zif/268 was studied during naloxone-precipitated morphine withdrawal in various organs of the rat. Dependence was induced over a period of 6 days by a graded regimen of 6-hourly injections. Northern analysis revealed peak expression of all IEGs occurred in the forebrain plus cerebellum at 20 min and at 60 min in the brain stem following morphine withdrawal. Increased levels of c-fos and c-jun mRNA were observed in the spinal cord at 40 min of morphine withdrawal. An increase in c-fos and c-jun but not zif/268 mRNAs was seen in the jejunum between 20 and 60 min. Elevated levels of the IEG protein products in the cerebral cortex, hippocampus, thalamus, cerebellum, brain stem and spinal cord were observed at 60 min following morphine withdrawal. These data emphasize the temporal and spatial variation in IEG expression in different tissues during opiate withdrawal.

Animals↗

PubMed1995

Expression of the genes coding for ornithine decarboxylase and its regulatory protein antizyme in the developing rat brain.

The ontogenic expression of the genes coding for ornithine decarboxylase (ODC) and its inhibitory protein, antizyme (AZ), was studied in the rat brain between embryonic day (E) 16 and postnatal day (P) 66. The level of ODC mRNA in whole brain was maximal at P2 and rapidly declined by P7 to a low level that was maintained into the adult. Levels of AZ mRNA also peaked at P2, and high levels were sustained into the adult. Regional studies indicated that between P2 and P60 ODC mRNA levels declined in the cerebral cortex, hippocampus and brainstem. These changes reflect the ontogenic pattern of protein levels and enzyme activity suggesting control of this enzyme may occur at the level of transcription. The level of AZ mRNA markedly increased in the cerebellum between P10 and P60. The level of ODC and AZ mRNA at P5 was not altered after continuous suppression of ODC enzyme activity by alpha-difluoromethylornithine between P0 and P5. This suggests that ODC gene expression is not subject to product-related feedback inhibition during this period. Immunohistochemical localisation of ODC protein at P5 revealed ubiquitous distribution of immunoreactivity with higher levels in the hippocampus, cerebral cortex and discrete nuclei in the brainstem. ODC protein was undetectable in the adult rat brain.

Animals↗

PubMed1994

Anti-acetaldehyde adduct antibodies generated by ethanol-fed rats react with reduced and unreduced acetaldehyde-modified proteins.

We have previously shown that rats fed ethanol for prolonged periods generate antibodies reactive with proteins modified by acetaldehyde in vitro. In this report we demonstrate that these antibodies react with two groups of adducts: those formed when acetaldehyde reacts with proteins at 37 degrees C for 24 hr ('unreduced' adducts) and those formed by a 1 hr incubation followed by the addition of sodium cyanoborohydride (a reducing agent specific for Schiff bases) to the reaction mixture ('reduced' adducts). These data suggest that adducts from both of these groups are formed in vivo as a result of ethanol ingestion by rats.

Acetaldehyde↗

PubMed1994

Distribution and turnover of acetaldehyde-modified proteins in liver and blood of ethanol-fed rats.

Previous studies have shown that acetaldehyde (AcH)-modified proteins are formed in the liver and blood of rats fed ethanol-containing diets. In this study we report the application of ELISA techniques to study the subcellular distribution and turnover of AcH-modified proteins in ethanol-fed and control rats. Modified proteins were found in liver mitochondrial, crude membrane and cytosolic fractions, as well as in plasma from ethanol-fed rats. No adducts were detected by our assay in haemolysates from the same animals. The rate of decline of AcH-modified proteins after cessation of ethanol feeding was also examined. Modified liver cytosolic proteins were shown to decline with a half-life of 2.3 weeks, whereas modified plasma adducts declined with a half-life of 4.8 weeks.

Acetaldehyde↗

PubMed1994

Elevated AP-1 DNA-binding activity in rat brain during ethanol withdrawal.

The DNA-binding activity of the transcription factors AP-1, CREB and OCT was investigated in nuclear extracts of brains from rats undergoing ethanol withdrawal. AP-1 DNA-binding activity but not that of CREB or OCT was increased in the cerebral cortex, hippocampus and cerebellum 15 and 17 hr after ethanol withdrawal. A similar increase in AP-1 binding activity was observed in subcortical structures at 17 hr of ethanol withdrawal. No change in any transcription factor DNA-binding activity was observed in the brain stem. Our results suggest that increased expression of immediate early genes is coupled with an increase in DNA-binding function, indicating that they may play a role in the long-term molecular response to ethanol withdrawal.

Animals↗

PubMed1994

The effect of chronic ethanol feeding on cytokines in a rat model of alcoholic liver disease.

There is a growing body of evidence to suggest that cytokines may be involved in the aetiology of alcoholic hepatitis. To study the effects of chronic ethanol feeding on cytokine production we have maintained rats on the control and ethanol-containing forms of the Lieber-DeCarli liquid diet for six weeks. The animals were then given an i.v. injection of endotoxin to induce hepatitis. It was found that the ethanol-fed animals had biochemical and histological evidence of mild to severe liver damage whereas control-fed animals had minimal liver damage. When plasma levels of cytokines were measured, it was found that the ethanol-fed rats produced much higher levels of tumour necrosis factor and interleukin 6 bioactivity than the control-fed rats. However, elevated levels of interleukin 1 protein were not seen in the ethanol-fed animals.

Alcoholism↗

PubMed1994

Studies on the usefulness of acetaldehyde-modified proteins and associated antibodies as markers of alcohol abuse.

Acetaldehyde, an intermediate in ethanol metabolism, has been shown to react with proteins to produce both stable and unstable adducts. These modified proteins are immunogenic, leading to the production of anti-adduct antibodies. In this report we describe studies carried out to determine whether anti-adduct antibodies could be used as a marker of alcohol abuse. We have used ELISAs to measure plasma immunoreactivity with unmodified and acetaldehyde-modified bovine serum albumin in groups of female and male social drinkers, heavy drinkers and alcoholics. When total immunoreactivity with the unmodified and acetaldehyde-modified proteins was measured it was found that each of the groups exhibited elevated reactivity with the modified protein as compared to the unmodified. Similar results were obtained when IgM immunoreactivity was measured. However, when IgA immunoreactivity was measured the reactivity with the modified protein was more greatly elevated in the heavy drinking and alcoholic groups than in the social drinking groups. There was no evidence of IgG reactivity with modified protein in any of the groups. These data suggest that elevated IgA reactivity with acetaldehyde-modified proteins may be a marker for high alcohol intake.

Acetaldehyde↗

PubMed1993

Polyamine-enhanced NMDA receptor activity: effect of ethanol.

The effect of ethanol on spermidine-enhanced, N-methyl-D-aspartate (NMDA)-induced seizures and c-fos expression was investigated in the rat brain. The latency of tonic-clonic convulsions induced by i.p. administration of NMDA (50 mg/kg) was decreased by prior i.c.v. injection of spermidine (0.1-2.5 mumol) in a dose-dependent manner. Neither NMDA (50 mg/kg) nor spermidine (up to 2.5 mumol) alone induced c-fos mRNA expression in the brain. When both agents were administered, significant induction of c-fos expression occurred 30 min after the convulsion. Prior treatment with ethanol did not alter the curve of spermidine dose-dependency over most of the range. The c-fos expression induced by a combination of NMDA (50 mg/kg) and spermidine (1.0 mumol) was unaffected by ethanol. Only at a high dose of ethanol (2.0 g/kg) and at minimal spermidine enhancement was NMDA-induced seizure and c-fos expression inhibited. These results suggest that polyamines may have an important role in modulating NMDA receptor function in vivo and that polyamine enhancement of NMDA receptor function is relatively insensitive to the inhibitory effects of ethanol.

Animals↗

PubMed1993

Expression of the gene coding for the NR1 subunit of the NMDA receptor during rat brain development.

Expression of the gene coding for the NR1 subunit of the N-methyl-D-aspartate (NMDA)-type of glutamate receptor was investigated in the developing rat brain. Peak NR1 gene expression in the whole brain occurred at approximately postnatal day (P) 10 with a second increase in the adult. To determine the ontogenic expression in the various brain regions, the expression of NR1 at P2, P10 and P60 was compared. The regional studies indicated increased expression at P60 in the cerebellum. In the midbrain and diencephalon, levels of expression at P10 and P60 were higher than at P2, while in the hippocampus, expression at P10 was significantly higher than at either P2 or P60. Expression in the other brain regions was constant over the period studied. These data indicate a region-specific expression of NR1 in the central nervous system during ontogeny.

Animals↗

PubMed1993

Ethanol and protein kinase C in rat brain.

The effect of chronic ethanol consumption on the catalytic activity of protein kinase C isolated from rat brain was studied in two different ways. Enzyme activity was first measured by phosphorylation of Histone IIIS in vitro. There was no change in the activity of the cytosolic enzyme. Membrane-associated enzyme activity was reduced in the ethanol-treated animal. This difference was not evident if the enzyme was stimulated by arachidonate. The reduction in enzyme activity was confirmed by analysis of the phosphorylation of endogenous substrates in intact synaptosomes. When the binding of the ligand [3H]phorbol dibutyrate was measured by quantitative autoradiography, increased binding to membrane-associated protein kinase C was observed in the CA1 region of the hippocampus but not in other brain regions. These results indicate that ethanol treatment results in a general reduction in membrane-associated protein kinase C activity as measured in vitro but the effect may not be consistent in all brain regions. The differential effect in the CA1 region of the hippocampus may be a reflection of a disruption in the normal regulation of protein kinase C activity in this area and may indicate that this region is a sensitive target for the action of ethanol.

Animals↗

PubMed1993

Antibodies from alcoholics, ethanol-fed rats and a rabbit immunised with proteins modified by acetaldehyde in vitro react with liver cytosolic proteins from ethanol-fed rats.

In previous studies we have shown that ethanol-fed rats generate antibodies reactive with proteins modified by acetaldehyde in vitro and that their livers contain proteins modified by acetaldehyde. In this study we demonstrate that the antibodies from these animals react with the modified proteins found in their livers. Furthermore, when the antibodies reactive with specific proteins were isolated, they were found to react with all of the modified proteins detected by the whole serum. This suggests that all of the proteins modified by acetaldehyde in vivo carry the same or similar epitope(s). In addition, antibodies from alcoholics and a rabbit immunised with proteins modified by acetaldehyde in vitro also reacted with the liver cytosolic proteins from ethanol-fed rats. Therefore it appears that similar epitopes are generated in alcoholics as a result of ethanol misuse, in rat liver due to prolonged ethanol feeding and by the in vitro modification procedure used to produce the immunogen for the rabbit.

Acetaldehyde↗