Development of haemostatic decontaminants for the treatment of wounds contaminated with chemical warfare agents. 1: evaluation of in vitro clotting efficacy in the presence of certain contaminants.

The treatment of penetrating, haemorrhaging injuries sustained within a hazardous environment may be complicated by contamination with toxic chemicals. There are currently no specific medical countermeasures for such injuries. Haemostats with an absorbent mechanism of action have the potential to simultaneously stop bleeding and decontaminate wounds. However, a primary requirement of a 'haemostatic decontaminant' is the retention of clotting function in the presence of chemical contaminants. Thus, the aim of this study was to investigate the haemostatic efficacy of seven commercially available haemostats in the presence of toxic chemicals (soman, VX, sulphur mustard, petrol, aviation fuel and motor oil). Clot viscosity was assessed ex vivo using thrombelastography following treatment of pig blood with: (i) toxic chemical; (ii) haemostat; or (iii) haemostat in combination with toxic chemical. Several contaminants (VX, petrol and GD) were found to be pro-haemostatic and none had an adverse effect on the rate with which the test products attained haemostasis. However, the total clot strength for blood treated with certain haemostats in the presence of sulphur mustard, soman and petrol was significantly decreased. Three test products failed to demonstrate haemostatic function in this ex vivo (thrombelastography) model; this was tentatively ascribed to the products achieving haemostasis through a tamponade mechanism of action, which can only be replicated using in vivo models. Overall, this study has identified a number of commercial products that may have potential as haemostatic decontaminants and warrant further investigation to establish their decontaminant efficacy.

Development of haemostatic decontaminants for the treatment of wounds contaminated with chemical warfare agents. 2: evaluation of in vitro topical decontamination efficacy using undamaged skin.

The risk of penetrating, traumatic injury occurring in a chemically contaminated environment cannot be discounted. Should a traumatic injury be contaminated with a chemical warfare (CW) agent, it is likely that standard haemostatic treatment options would be complicated by the need to decontaminate the wound milieu. Thus, there is a need to develop haemostatic products that can simultaneously arrest haemorrhage and decontaminate CW agents. The purpose of this study was to evaluate a number of candidate haemostats for efficacy as skin decontaminants against three CW agents (soman, VX and sulphur mustard) using an in vitro diffusion cell containing undamaged pig skin. One haemostatic product (WoundStat™) was shown to be as effective as the standard military decontaminants Fuller's earth and M291 for the decontamination of all three CW agents. The most effective haemostatic agents were powder-based and use fluid absorption as a mechanism of action to sequester CW agent (akin to the decontaminant Fuller's earth). The envisaged use of haemostatic decontaminants would be to decontaminate from within wounds and from damaged skin. Therefore, WoundStat™ should be subject to further evaluation using an in vitro model of damaged skin.

Silver and nanoparticles of silver in wound dressings: a review of efficacy and safety.

Wound infections present a significant clinical challenge, impacting on patient morbidity and mortality, with significant economic implications. Silver-impregnated wound dressings have the potential to reduce both wound bioburden and healing time. The silver ion Ag+ is the active antimicrobial entity; it can interfere with thiol (-SH) groups and provoke the generation of reactive oxygen species (ROS), a major contributor to its antibacterial efficacy. Recently, silver nanoparticles have gained considerable interest in wound bioburden reduction and in anti-inflammation, as they can release Ag+ ions at a greater rate than bulk silver, by virtue of their large surface area. If released from dressings, they also have the potential to cross biological compartments. This review aims to consolidate recent findings as to the efficacy and safety of different formulations of silver used as an antiseptic agent in dressings, summarising the features of silver nanomaterials, with particular attention to the dose-dependencies for biological effects, highlighting the need for information on their uptake and potential biological effects.

Induction and progression of cholangiofibrosis in rat liver injured by oral administration of furan.

Cholangiofibrosis is a structural anomaly that precedes the development of cholangiocarcinoma in some rodent models. In this article, the authors examine the contribution of the epithelial and mesenchymal cells in the pathogenesis of this complex lesion. Furan was administered to rats by gavage in corn oil at 30 mg/kg b.w. (five daily doses per week) and livers were sampled between eight hr to three months. Characteristically the administration of furan caused centrilobular injury, and restoration was accomplished by proliferation of hepatocytes. Some areas of the liver were, however, more severely affected, and here, injury extended into portal and capsular areas, which resulted in a rapid proliferation of ductular cells that extended into the parenchyma accompanied by a subtype of liver fibroblasts. These ductules either differentiated into hepatocytes, with loss of the associated fibroblasts, or progressed to form tortuous ductular structures that replaced much of the parenchyma, leading to cholangiofibrosis. Although it is unclear what determines the difference in the hepatic response, a loss of micro-environmental cues that instigate hepatocyte differentiation and termination of the hepatocyte stem cell repair response may be perturbed by continual furan administration that results in an irreversible expansile lesion that may mimic the features of cholangiocarcinoma.

Evidence of oxidative stress and associated DNA damage, increased proliferative drive, and altered gene expression in rat liver produced by the cholangiocarcinogenic agent furan.

Furan is a potent cholangiocarcinogen in rat by an as yet undefined mechanism. The risk to man remains unclear. Using a time-course stop study design, we have investigated the potential of furan to induce oxidative stress and DNA damage associated with inflammatory and regenerative responses in rat liver. Furan was administered via oral gavage (30 mg/kg b.w. 5 daily doses per week), and livers were analyzed at time points between eight hr and three months. A one-month recovery group previously treated for three months was also included. There was a marked association between CYP2E1 expression and DNA oxidation (8-oxo-dG) in areas of centrilobular hepatocyte necrosis seen after a single dose. After one-month recovery from three-month treatment, 8-oxo-dG was still observed in areas of furan-induced cholangiofibrosis. Furan-induced changes in the expression of various genes associated with oxidative stress, DNA damage, and cell cycle control were identified during treatment and recovery. We propose that furan-induced cholangiocarcinomas emerge from areas of cholangiofibrosis as a result of a combination of chronic, persistent indirect damage to DNA through oxygen radicals coupled with persistent proliferative signals, including loss of connexin 32, that act to convert this DNA damage to fixed mutations.

Fluorescent choleretic and cholestatic bile salts take different paths across the hepatocyte: transcytosis of glycolithocholate leads to an extensive redistribution of annexin II.

We have used fluorescent derivatives of the choleretic bile salts cholate and chenodeoxycholate, the cholestatic salt lithocholate, and the therapeutic agent ursodeoxycholate to visualize distinct routes of transport across the hepatocyte and delivery to the canalicular vacuole of isolated hepatocyte couplets. The cholate and chenodeoxycholate derivatives produced homogeneous intracellular fluorescence and were rapidly transported to the vacuole, while the lithocholate analogue accumulated more slowly in the canalicular vacuole and gave rise to punctate fluorescence within the cell. Fluorescent ursodeoxycholate showed punctate intracellular fluorescence against a high uniform background indicating use of both pathways. Inhibition of vesicular transport by treatment with colchicine and Brefeldin A had no effect on the uptake of any of the compounds used, but it dramatically impaired delivery of both the lithocholate and the ursodeoxycholate derivatives to the canalicular vacuole. We conclude that while the chenodeoxycholate and cholate analogues traverse the hepatocyte by a cytoplasmic route, lithocholate and ursodeoxycholate analogues are transported by vesicle-mediated transcytosis. Treatment of couplets with glycine derivatives of lithocholate and ursodeoxycholate, but not cholate or chenodeoxycholate, led to a marked relocalization of annexin II, which initially became concentrated at the basolateral membrane, then moved to a perinuclear distribution and finally to the apical membrane as the incubation progressed. This suggests that lithocholate and ursodeoxycholate treatment leads to a rapid induction of transcytosis and that annexin II exchange occurs upon membrane fusion at all stages of the hepatocyte transcytotic pathway. These results indicate that isolated hepatocyte couplets may provide an inducible model system for the study of vesicle-mediated transcytosis.

Multicenter study to assess potential hazards from exposure to lipid peroxidation products in soya bean oil from Trilucent breast implants.

In response to a Hazard Notice by the Medical Devices Agency of the UK in 2000 regarding the Trilucent breast implant (TBI), an expert panel was convened to implement a research program to determine whether genotoxic compounds were formed in the soybean oil filler (SOF) of TBIs and whether these could be released to produce local or systemic genotoxicity. The panel established a research program involving six laboratories. The program recruited 47 patients who had received TBIs (9 patients had received silicone implants previously). A reference group (REBI) of 34 patients who had exchanged either silicone (17 patients) implants (REBI-E) or patients (17) who were to receive primary implantation augmentation with silicone (REBI-PIA), and who were included as needed to increase either the pre- or post-explantation sample number. Of the 17 REBI-E patients, 5 had silicone implants and 12 had saline implants previously (prior to the last exchange). Investigation was undertaken before and after replacement surgery in the TBI patients and before and after replacement or augmentation surgery in the REBI patients. The pre- to post-operative sample interval was 8-12 weeks. Pre-operative samples were collected within 7 days prior to the operation. Information on a variety of demographic and behavioral features was collected. Biochemical and biological endpoints relating to genotoxic lipid peroxidation (LPO) products potentially formed in the SOF, and released locally or distributed systemically, were measured. The SOF of explanted TBIs was found to have substantial levels of LPO products, particularly malondialdehyde (MDA), and low levels of trans-4-hydroxy-2-nonenal (HNE) not found in unused implants. Mutagenicity of the SOF was related to the levels of MDA. Capsules that formed around TBIs were microscopically similar to those of reference implants, but MDA-DNA adducts were observed in capsular macrophages and fibroblasts of only TBI capsules. These cell types are not progenitors of breast carcinoma (BCa) and the location of the implants precludes LPO products reaching the mammary epithelial cells which are progenitors of BCa. Blood levels of LPO products were not increased in TBI patients compared to REBI patients and did not change with explantation. In TBI patients, white blood cells did not show evidence of increased levels of LPO-related aldehyde DNA adducts. In conclusion, based on a number of measured parameters, there was no evident effect that would contribute to breast or systemic cancer risk in the TBI patients, and the recommended treatment of TBI patients involving explantation was judged appropriate.

Activity of OGG1 variants in the repair of pro-oxidant-induced 8-oxo-2'-deoxyguanosine.

Cells are continuously exposed to damaging reactive oxygen species (ROS), which are produced from both endogenous and exogenous sources. 8-Oxodeoxyguanosine (8-oxodG) is an abundant base lesion formed during oxidative stress which, if not repaired, can give rise to G:C-->T:A transversions in DNA. The 8-oxoguanine DNA glycosylase-1 (OGG1)-initiated base excision repair (BER) pathway operates to remove 8-oxodG lesions. Ogg1 deletion and polymorphism may result in a hypermutator phenotype and susceptibility to oxidative pathologies including cancer. Limited and conflicting evidence exists regarding the repair capacity of a prevalent human OGG1 (hOGG1) polymorphism, the Cys326-hOGG1 variant. The formamidopyrimidine DNA glycosylase (FPG)-modified comet assay was used to investigate the ability of sodium dichromate, potassium bromate and Ro19-8022 (+light) to induce DNA damage in mogg1(-/-) null (KO) and wild-type (WT) mouse embryonic fibroblasts (MEFs) and to assess hOGG1 variant-initiated BER capacities under conditions of oxidative stress. Treatment of WT MEFs with these pro-oxidant agents induced direct DNA strand breaks in a concentration-dependent manner, whereas, identical treatment of KO MEFs produced no effect. In contrast, KO MEFs accumulated significantly more FPG-sensitive sites than WT MEFs. Expression of hOGG1 in KO MEFs restored the WT phenotype in response to all pro-oxidants tested. The results suggest OGG1-initiated BER generates direct DNA strand breaks detected by the conventional comet assay, thus it is important that researchers do not interpret these as direct damage per se but rather a reflection of the repair process. The data also indicate Cys326-hOGG1-initiated BER is transiently impaired with respect to Ser326-hOGG1 (wild-type)- and Gly326-hOGG1 (artificial)-initiated BER following pro-oxidant treatment, possibly via hOGG1 cysteine 326 oxidation. This finding suggests the homozygous cys326/cys326 genotype may be classified as a biomarker of disease susceptibility, which is in support of a growing body of epidemiological evidence.

Different mechanisms of modulation of gap junction communication by non-genotoxic carcinogens in rat liver in vivo.

This is a comparative study of the mechanisms by which three different rodent non-genotoxic carcinogens modulate connexin-mediated gap junction intercellular communication in male rat liver in vivo. In the case of the peroxisome proliferating agent Wy-14,643, a non-hepatotoxic dose of 50mg/kg led to a marked loss of inter-hepatocyte dye transfer associated with a loss of both Cx32 and Cx26 protein expression. In contrast, p,p'-dichlorodiphenyltrichloroethane (DDT) at a non-hepatotoxic dose (25mg/kg) was not found to alter Cx32 or Cx26 expression or to produce a measurable Cx32 serine phosphorylation but did give a small, significant reduction of cell communication. Carbon tetrachloride (CCl(4)) did not affect cell communication (despite a small significant reduction of Cx32 content) at a non-hepatotoxic dose. Both loss of communication and Cx32 expression was observed only at a dose that caused hepatocyte toxicity as evidenced by increased serum alanine aminotransferase activity. Overall, the findings emphasise that loss of gap junctional communication in vivo can contribute to carcinogenesis by non-genotoxic carcinogens through different primary mechanism. In contrast to Wy-14,643 and DDT, the results with CCl(4) are consistent with a requirement for hepatotoxicity in its carcinogenic action.

Macromolecule oxidation and DNA repair in mussel (Mytilus edulis L.) gill following exposure to Cd and Cr(VI).

The oxidation of DNA and lipid was analysed in the marine mussel (Mytilus edulis) in response to exposure (10microg/l and 200microg/l) to cadmium (Cd) and chromium [Cr(VI)]. Concentration dependent uptake of both metals into mussel tissues was established and levels of gill ATP were not depleted at these exposure levels. DNA strand breakage in gill cells (analysed by the comet assay) was elevated by both metals, however, DNA oxidation [measured by DNA strand breakage induced by the DNA repair enzyme formamidopyrimidine glycosylase (FPG)] was not elevated. This was despite a statistically significant increase in both malondialdehyde and 4-hydroxynonenal - indicative of lipid peroxidation - following treatment with Cd. In contrast, both frank DNA stand breaks and FPG-induced DNA strand breaks (indicative of DNA oxidation) were increased following injection of mussels with sodium dichromate (10.4microgCr(VI)/mussel). The metals also showed differential inhibitory potential towards DNA repair enzyme activity with Cd exhibiting inhibition of DNA cutting activity towards an oligonucleotide containing 8-oxo-7,8-dihydro-2'-deoxyguanosine and Cr(VI) showing inhibition of such activity towards an oligonucleotide containing ethenoadenosine, both at 200microg/l. The metals thus show DNA damage activity in mussel gill with distinct mechanisms involving both direct and indirect (oxidative) DNA damage, as well as impairing different DNA repair capacities. A combination of these activities can contribute to adverse effects in these organisms.

The multiple influences of glutathione on bromate genotoxicity: implications for the dose-response relationship.

Glutathione displays multiple roles in the oxidative genotoxicity of potassium bromate. On the one hand, reduced glutathione has a demonstrated role in the activation of bromate to species capable of oxidising DNA. However, if this activation should occur within the gut or extracellularly once bromate is absorbed, this may limit the ability of the chemical to oxidise cellular DNA in vivo. Moreover, glutathione may offer protection against the damaging species produced by its interaction with bromate. Finally, if bromate exposure of cells is sufficiently high to deplete glutathione, a secondary oxidative stress and associated DNA damage may occur. These observations would suggest non-linearity in the dose-response to DNA damage in vivo.

Oxidative damage produced by Cr(VI) and repair in mussel (Mytilus edulis L.) gill.

This study has assessed DNA damage induced by oxidative stress and its subsequent repair in mussels. Gill was obtained from mussels collected from New Brighton, UK within 24 h and also after 1 month maintenance under laboratory conditions. The pro-oxidant sodium dichromate produced a statistically significant increase in DNA strand breaks (DSB) in these gill cells at both time points as measured by the COMET assay. The response was higher at 1 month in association with a higher concentration of GSH which is known to activate Cr(VI) producing reactive oxygen species. DSB were shown, through studies in wild type and OGG-1-null mouse fibroblasts, to be produced by repair enzymes in response to Cr(VI). In support of evidence for repair of oxidative DNA damage, we have also demonstrated for the first time repair activity in mussel gill towards 8-oxo-dG using an oligonucleotide cutting assay.

Regulation of c-fos transcription by chemopreventive isoflavonoids and lignans in MDA-MB-468 breast cancer cells.

Isoflavonoids and lignans are diet constituents with chemopreventive properties. We compared the ability of the isoflavonoids genistein and equol, the lignans enterodiol, enterolactone and nordihydroguaiaretic acid (NDGA) and the lignan metabolite methyl p-hydroxyphenyllactate to interfere with mitogenic and tumour promotional signal transduction pathways. Their effects on c-fos mRNA levels after induction by either epidermal growth factor (EGF) or the tumour promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was measured in human breast cancer-derived MDA-MB-468 cells. Of the six agents, only genistein decreased EGF-induced, c-fos transcription (by 63% compared to control at 100 mumol/l). In contrast, both genistein and equol at 100 mumol/l decreased TPA-induced c-fos levels, by 75 and 67%, respectively. NDGA and methyl p-hydroxyphenyllactate did not inhibit TPA mediated c-fos transcription and enterolactone and enterodiol had only a weak inhibitory effect. NDGA at 0.1-10 mumol/l increased c-fos mRNA levels. None of the agents inhibited protein kinase C and only genistein inhibited EGF receptor-linked protein tyrosine kinase obtained from MDA-MB-468 cells, with an IC50 of 60 mumol/l. NDGA and genistein arrested cell colony formation potently, genistein was 15-fold more growth-inhibitory than equol. The results suggest that both genistein and equol interfere similarly with TPA-induced signal transduction pathways. Inhibition by genistein of EGF-induced c-fos mRNA transcription is probably related to its interruption of EGF receptor-linked protein tyrosine kinase, whereas genistein-induced growth arrest is not. If ability to antagonise phorbol ester effects is important for chemopreventive efficacy, equol and genistein might be equi-efficacious chemopreventors, whereas enterolactone, enterodiol and NDGA should be much less potent. If phorbol ester antagonism together with antimitogenic activity determine optimal chemopreventive activity of this type of agent, genistein would be more potent than equol.

The formation and metabolism of N-hydroxymethyl compounds--IX. N-(acetoxymethyl)-4-chlorobenzamide: an electrophile but not a mutagen in Salmonella typhimurium.

The electrophilic properties of 4-chloro-N-(hydroxymethyl)benzamide as a model compound of carbinolamides formed during the metabolic oxidation of N-methylamides were investigated. 4-Chloro-N-(hydroxymethyl)benzamide did not react with nucleophiles such as cyanide or glutathione under physiological conditions. In contrast, N-(acetoxymethyl)-4-chlorobenzamide yielded the cyanomethylamide with KCN and S-(4-chlorobenzamidomethyl)glutathione with glutathione. Under nonaqueous conditions, N-(acetoxymethyl)-4-chlorobenzamide reacted avidly with ethanethiol, with methanol and with diethylamine in the presence of base, whereas 4-chloro-N-(hydroxymethyl)benzamide did not afford products under these conditions. These results show clearly that N-(acetoxymethyl)-4-chlorobenzamide is the precursor of reactive electrophilic methyleneimines. 4-Chloro-N-(hydroxymethyl)benzamide was not biotransformed to such electrophilic species when incubated with mouse hepatic microsomes or a microsomal supernatant with acetyl-CoA or a PAPS generating system. Neither 4-chloro-N-(hydroxymethyl)benzamide nor its acetate ester were mutagenic in the short term bacterial assay using Salmonella typhimurium. Nevertheless, esters of carbinolamides, such as N-(acetoxymethyl)-4-chlorobenzamide, might possess toxic or carcinogenic properties.

Bleomycin-induced unscheduled DNA synthesis in non-permeabilized human and rat hepatocytes is not paralleled by 8-oxo-7,8-dihydrodeoxyguanosine formation.

The genetic toxicity of the antitumour antibiotic bleomycin (BLM) is thought to involve the formation of a reactive oxygen intermediate. 8-Oxo-7,8-dihydrodeoxyguanosine (oxo8dG), an oxidation product of deoxyguanosine, is one of the major products formed when isolated DNA is exposed to oxygen radical generating systems. Gamma-irradiation (10-500 Gy 60Co; 10 Gy/min) or BLM and Fe2+ (37.5-150 U/L and 0.5 mM, respectively) treatment of isolated DNA (0.25 mg/mL) increased oxo8dG above background. In the latter case, the effect was greater than that with Fe2+ (0.5 mM) alone and was dependent on the dose of BLM. When DNA was irradiated with 500 Gy60Co, deoxyguanosine oxidation was inhibited by antioxidants (ethanol: 37.5 and 98% inhibition at 2 and 20 mM, respectively; mannitol: 20.5, 60 and 92% inhibition at 0.1, 1.0 and 10 mM, respectively). Similarly the BLM-induced production of oxo8dG was inhibited (64%) by mannitol (10 mM). BLM also caused production of base propenals on interaction with isolated DNA. In contrast, oxo8dG was not induced above background concentration (27 mol oxo8dG/10(6) mol dG) in permeabilized (37 degrees) and non-permeabilized (4 degrees and 37 degrees) rat hepatocytes treated with BLM (260 U/L). Despite this, there was extensive BLM-induced unscheduled DNA synthesis (10 and 100 U/L) in non-permeabilized rat and human hepatocytes in the absence of hydroxyurea. These findings, in accord with other observations, draw into question the role of .OH in BLM-induced DNA damage and the mimicry of ionizing radiation in cellular systems.

Effect of oxidative stress and disruption of Ca2+ homeostasis on hepatocyte canalicular function in vitro.

Isolated rat hepatocyte couplets were used to study the effects of menadione and a rise in the intracellular concentration of calcium on biliary canalicular function. Canalicular function was assessed by counting the percentage of couplets which were able to accumulate the fluorescent cholephile, cholyl lysyl fluorescein (CLF) into the canalicular vacuole between the two cells. Menadione induced a concentration-dependent inhibition of the canalicular vacuole accumulation (CVA) of CLF reaching 7.6 +/- 1.8% of control at 100 microM menadione. This disruption was not prevented by blocking receptor-operated calcium channels with Ni2+ (300 microM). The concentration range of menadione used did not deplete cellular ATP content. In contrast glutathione content was reduced to 52% of its control value by 100 microM menadione. A rise in cytosolic calcium induced by the calcium ionophore, A23187 (up to 30 microM) also disrupted CVA in a concentration-dependent manner. Release of endoplasmic reticulum calcium stores by thapsigargin (50 nM) affected the retention of canalicular contents to a much lesser extent, although it was able to stimulate a reduction in canalicular area to 40% of its original value, assumed to be due to canalicular contraction. Menadione (30 and 100 microM) reduced the fluorescence of phalloidin-FITC-labelled F-actin in both the total and pericanalicular cytoskeleton. Canalicular function was therefore disrupted by non-lethal concentrations of menadione via a mechanism which does not appear to involve ATP depletion or the entry of extracellular calcium, but is associated with a depletion of both cellular glutathione and F-actin. An increase in the concentration of intracellular calcium can stimulate canalicular contraction, and at relatively high concentrations calcium can also disrupt canalicular function.

Evidence for direct-acting oxidative genotoxicity by reduction products of azo dyes.

The intestinal flora forms a complex ecosystem that metabolizes dietary and endogenous nutrients under primarily anaerobic conditions. The ingestion of azo dyes has been proposed as one source of potential genotoxic agents. Many intestinal bacteria are able to reduce the azo bond (termed azofission), which liberates the substituted naphthol compounds. The standard Ames test has not demonstrated mutagenicity either by various common food colorings or by their reduced end products in Salmonella typhimurium strains TA98 and TA100. In contrast, genetic toxicity was demonstrated in the Escherichia coli differential kill assay and in S. typhimurium TA102 for the reduced dyes. The superoxide free radical was produced by the azo dyes only after reduction by the intestinal bacteria Enterococcus faecalis and Bacteroides thetaiotaomicron.

Nafenopin causes protein kinase C-mediated serine phosphorylation and loss of function of connexin 32 protein in rat hepatocytes without aberrant expression or localization.

The characteristics and mechanism of the inhibition of connexin-mediated gap junctional communication by the non-genotoxic rodent hepatocarcinogen, nafenopin, has been studied in rat hepatocytes. Nafenopin caused a time- and concentration-dependent inhibition of dye coupling in hepatocytes as assessed by transfer of microinjected lucifer yellow. A half-maximum inhibitory effect of nafenopin occurred at approximately 50 microM, which was not cytotoxic. The inhibitory effect was reversible since a significant recovery of communication was observed 3 h after removal of the chemical. The protein kinase inhibitor Gö6976 prevented the inhibition of dye coupling, but a tyrosine kinase inhibitor (genistein) did not. Connexin 32 and 26 protein expression, as assessed by immunoblotting, was similar in nafenopin-treated hepatocytes compared to controls, with the exception that in a 10-h culture with nafenopin, the level of connexin 26 was elevated compared to controls. Immunohistochemistry indicated that the localization of plaques containing connexin 32 was not affected in hepatocytes by nafenopin. Immunoprecipitated connexin 32 was, however, detected by an anti-phosphoserine antibody following nafenopin treatment, but not in controls. This serine phosphorylation was prevented in the presence of Gö6976. The results give further support for a role of protein kinase C in the post-translational inactivation of connexin 32 function in rat hepatocytes by nafenopin.

ANIT-induced disruption of biliary function in rat hepatocyte couplets.

alpha-Naphthylisothiocyanate (ANIT) induces intrahepatic cholestasis in rats, involving damage to biliary epithelial cells; our study aims to investigate whether disruption of biliary function in hepatocytes can contribute to early stages of ANIT-induced intrahepatic cholestasis. Isolated rat hepatocyte couplets were used to investigate biliary function in vitro by canalicular vacuolar accumulation (cVA) of a fluorescent bile acid analogue, cholyl-lysyl-fluorescein (CLF), within the canalicular vacuole between the two cells. After a 2-h exposure to ANIT, there was a concentration-dependent inhibition of cVA (cVA-IC50; 25 microM), but no cytotoxicity (LDH leakage or [ATP] decline) within this ANIT concentration range. There was no loss of cellular [GSH] at low ANIT concentrations, but, at 50 microM ANIT, a small but significant loss of [GSH] had occurred. Diethylmaleate (DEM) partially depleted cellular [GSH], but addition of 10 microM ANIT had no further effect on GSH depletion. Reduction in cVA was seen in DEM-treated cells; addition of ANIT to these cells reduced cVA further, but the magnitude of this further reduction was no greater than that caused by ANIT alone, indicating that glutathione depletion does not enhance the effect of ANIT. F-actin distribution (by phalloidin-FITC staining) showed an increased frequency of morphological change in the canalicular vacuoles but only a small, non-significant (0.05 < p < 0.1) increase in proportion of the F-actin in the region of the pericanalicular web. The results are in accord with a disruption of hepatocyte canalicular secretion within two h in vitro, at low, non-cytotoxic concentrations of ANIT, and the possible involvement of a thiocabamoyl-GSH conjugate of ANIT (GS-ANIT) in this effect.