Disruption of polarized function in hepatocytes.
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Animals↗
Biomedical subjects
J K Chipman
Publications and source records attributed to J K Chipman.
CYP2E1-mediated mechanism of anti-genotoxicity of the broccoli constituent sulforaphane.
The broccoli constituent sulforaphane (1-isothiocyanate-4-methylsulfinylbutane) has previously been shown to protect rats against 9,10-dimethyl-1,2-benz[a]anthracene tumorigenesis, thought to be due, at least in part, to induction of phase II detoxification. We investigated the ability of sulforaphane to also inhibit the phase I enzyme cytochrome P450 isoenzyme 2E1 (CYP2E1), which is responsible for activation of several carcinogens, including dialkylnitrosamines. Using the p-nitrophenol hydroxylation assay in microsomes from livers of acetone-treated Sprague-Dawley rats, sulforaphane was shown to be a potent competitive inhibitor of CYP2E1 with a Ki of 37.0 +/- 4.5 microM. In view of this result, we studied the capacity of sulforaphane to inhibit the genotoxicity of N-nitrosodimethylamine (NDMA). Sulforaphane at concentrations of > 0.8 microM inhibited the mutagenicity of NDMA (4.4 mg/plate) in Salmonella typhimurium strain TA100 after pre-incubation for 45 min with cytosol extract from livers of Balb/c mice pre-treated with acetone. Unscheduled DNA synthesis induced by NDMA (33.5 microM) in mouse hepatocytes was inhibited in a dose-dependent manner by sulforaphane at 0.064-20 microM. Sulforaphane was unable to inhibit mutagenicity of sodium azide (5 micrograms/plate), a direct acting mutagen, in the Salmonella assay. It was not itself genotoxic in hepatocytes, as measured by unscheduled DNA synthesis, or mutagenic in the strain of Salmonella employed and cytotoxic only at high concentrations (> or = 0.5 mM). These findings suggest that inhibition of CYP2E1 by sulforaphane may offer chemoprotection against carcinogenic substrates of this enzyme.
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Hepatobiliary function and toxicity in vitro using isolated hepatocyte couplets.
1. Hepatocyte couplets can be routinely prepared from rat liver to produce a suitable in vitro model for polarized primary cells. 2. Centrifugal elutriation provides a means of producing enriched subpopulations of periportal and perivenous couplets from the same liver, thus providing a means of studying the influence of zonal heterogeneity on hepatobiliary function. 3. The maintenance of structural and secretory polarity demonstrated by hepatocyte couplets provides a convenient in vitro system for mechanistic studies of factors both regulatory and adversely affecting hepatobiliary functions. 4. Couplets are also uniquely appropriate for specific studies of regulation at the biliary pole, on the performance of junctions and on the maintenance and rate of transcytotic movement. 5. The possibility also exists that effects of an in vivo pre-exposure to agents causing hepatobiliary dysfunction can be assessed in couplets ex vivo.
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Absorption of 3-chloro-4-(dichloromethyl)-5-hydroxy-2-[5H] furanone (MX) through rat small intestine in vitro.
The intestinal absorption of 3-chloro-4-(dichloromethyl)-5-hydroxy-2-[5H] furanone (MX), a highly mutagenic furanone found in chlorinated waters, was studied using an in vitro everted rat gut sac system, using reverse mutation in Salmonella typhimurium to detect mutagens transported from the mucosal to the serosal compartments. Absorption was measurable, but limited, with significant increase in bacterial revertants (serosal compartment) noted at a dose of 50 micrograms/ml MX (mucosal compartment, p < 0.05). Gut sac incubation with MX and glutathione (GSH, 1.0 mM) resulted in no detectable absorption of mutagens. Preincubation with diethylmaleate to deplete mucosal GSH resulted in increased absorption of MX-derived mutagens compared to controls (a significant induction of revertant colonies was noted at a dose of 25 micrograms/ml MX p < 0.05). Gut sac incubation with chlorinated fulvic acids resulted in no detectable absorption of mutagens. In vitro studies to assess the possibility of beta-lyase activation of the postulated MX-GSH conjugate showed no mutagenic activation.
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Connexin genes as tumour suppressor genes?
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Animals↗
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.
Actins↗
Induction of DNA strand breaks by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone and humic substances in relation to glutathione and calcium status in human white blood cells.
3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) induced DNA strand-breakage (measured by fluorometric analysis of DNA unwinding) in human white blood cells at sub-cytotoxic doses (1.0-1000 microM; 60 min exposure). Although a dose-dependent decrease in glutathione levels was observed with MX, this is not necessarily the causative factor in the observed DNA damage, since no strand breakage was seen on depletion of cellular glutathione to 23% of control by diethylmaleate. In addition, the strand scission does not appear to be mediated by elevation of intracellular calcium as no MX-induced release of calcium stores was observed. Strand breakage may, however, be Ca(2+)-dependent as evidenced by inhibition following deprivation of Ca2+ by Quin-2. Chlorinated fulvic acids (> 3 micrograms/ml) also depleted glutathione and induced strand breaks at sub-cytotoxic doses (up to 300 micrograms/ml) on prolonged exposure (60 min). The unchlorinated material was, however, equally able to cause DNA strand breakage (without glutathione depletion). MX appears, therefore, to be only one of a number of components of chlorinated humic substances able to induce DNA strand breakage.
Calcium↗
Absence of unscheduled DNA synthesis in rat hepatocytes treated with mutagenic and cytotoxic chlorinated humic substances.
Chlorinated hydrophilic macromolecular humic acids (CHMA) (0.07-3.0 mg per plate) were mutagenic in Salmonella typhimurium strain TA100. In contrast, the chlorinated derivatives did not induce unscheduled DNA synthesis (nuclear incorporation of [3H]thymidine) in cultured rat hepatocytes even after depletion of intracellular glutathione with buthionine sulphoximine (0.001 mM) and at concentrations of CHMA up to 3.0 mg per plate eliciting cytotoxicity. Glutathione depletion did however potentiate cytotoxicity and hepatocyte glutathione concentrations were lowered by CHMA treatment indicating reactivity of CHMA in the cell.
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Propane 2-nitronate is the major genotoxic form of 2-nitropropane.
The mutagenicity of 2-nitropropane in Salmonella typhimurium (strain TA100) was proportional to the pH (range 6.1-9.1) of the medium used for pre-incubation of the agent and for incubation of the agent with the Salmonella. The mutagenicity correlated with an enhanced rate of tautomerase to propane 2-nitronate at relatively high pH as measured by high performance liquid chromatography. Both the mutagenicity in Salmonella typhimurium (strains TA100 and TA102) and the rate of tautomerisation to the nitronate was lower with 2-deutero-2-nitropropane than with non-deuterated 2-nitropropane. Furthermore, 2-deutero-2-nitropropane was less potent in the induction of unscheduled DNA synthesis in rat hepatocytes over a 4-h period. Propane 2-nitronate therefore appears to be pivotal in the causation of the genetic toxicity of 2-nitropropane. The presence of hepatocytes enhanced nitronate production from 2-nitropropane suggesting a contribution from hepatic enzymes in the tautomerisation reaction.
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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.
Actins↗
Modulation of phenobarbitone-induced loss of gap junctional intercellular communication in hepatocyte couplets.
Phenobarbitone (PB) produced a dose- and time-dependent decrease in gap junctional intercellular communication (GJIC) (up to 25.0 +/- 5.3% inhibition) in rat hepatocyte couplets (4 h cultures). The effect was reversible and independent of protein synthesis. This inhibition was exacerbated (to 53.3 +/- 5.4% inhibition) by depletion of intracellular glutathione following pretreatment with diethylmaleate (0.5 microM, 15 min). Inhibition was also significantly enhanced by addition of the cytochrome P450 inhibitors SKF 525A (25 microM) and metyrapone (20 nM). In contrast, hepatocyte couplets derived from rats pretreated with PB (0.1% w/v in drinking water) for up to 28 days were fully functional regarding GJIC and were found to be refractory to the effects of PB added in vitro. This, coupled with the lack of effect of p-hydroxy-PB, suggests that an active metabolite of PB is not involved in the inhibition of GJIC which may, instead, be through an oxidative stress, which is prevented by glutathione.
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Evaluation of the relative sensitivity of chromosome painting (FISH) as an indicator of radiation-induced damage in human lymphocytes.
Fluorescence in situ hybridization with whole chromosome libraries, also known as chromosome "painting", is an easy and rapid method for detection of chromosome aberrations. To evaluate the sensitivity of this in radiation dosimetry we have made comparisons with G-banding analysis and also with physicochemical measurements of radiation induced DNA damage (DNA strand breaks and 8-hydroxydeoxyguanosine formation). Heparinised human blood was irradiated at room temperature with a range (0-10 Gy) of gamma irradiation from a cobalt 60 source. Chromosome spreads prepared from phytohaemagglutinin stimulated "whole blood" lymphocyte cultures were hybridized in situ with the whole chromosome 1 library, coded, and scored for aberrant cells. Dose response curves plotted as percent abnormal cells obtained by the two cytogenetic methods were similar and it would appear that chromosome "painting" compared favourably with G-banding for the detection of aberrations. The measurement of DNA strand breaks by a fluorimetric alkaline unwinding assay showed similar sensitivity to chromosome "painting" whereas the formation of 8-hydroxydeoxyguanosine did not correlate with aberration frequencies.
Adult↗
Glutathione deficiency does not elevate susceptibility of bacteria to the mutagenicity of chlorinated humic acids.
1. Rat liver 9,000 g supernatant protected against the mutagenic effect of chlorinated hydrophilic macromolecular humic acids (CHMA) in Salmonella typhimurium strain TA100. 2. Protection against mutagenicity of CHMA was mediated by glutathione and was partially dependent on glutathione S-transferase activity. 3. In contrast to the above findings, CHMA showed lower mutagenicity in Salmonella typhimurium and Escherichia coli strains of bacteria that are deficient in glutathione compared to their mutagenicity in parental (glutathione-rich) bacterial strains. 4. Glutathione-deficient cells do not provide test systems with elevated sensitivity for the detection of mutagenic chlorinated humic substances.
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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.
Azo Compounds↗
Periportal- and perivenous-enriched hepatocyte couplets: differences in canalicular activity and in response to oxidative stress.
Unlike isolated single hepatocytes, hepatocyte couplets retain their apical polarity, and, during short-term culture form an enclosed canalicular space or vacuole between the two adjacent cells into which biliary secretion is initiated. Hepatocyte couplets were prepared after partial collagenase perfusion of rat liver. Centrifugal elutriation was used to fractionate the preparation into six couplet-containing suspensions. Image analysis was used to determine the size of cultured couplets. The size of the couplets ranged from 34.1 +/- 0.76 microns and 684 +/- 24.1 microns 2 (mean length and area respectively +/- S.E.M.) in Fraction 2, to 43.7 +/- 0.57 microns and 1033 +/- 33.8 microns 2 length and area respectively in Fraction 7. Glutamine synthetase activity was assessed in each freshly eluted fraction and was shown to be predominant in Fractions 6 and 7. Pretreatment of rats with CCl4, which selectively destroys perivenous hepatocytes, decreased the proportion of couplets in these fractions by over 67%, and their glutamine synthetase activity by over 97%. It was concluded that Fractions 2 and 3 contained predominantly couplets of Zone 1 (periportal) origin, Fractions 4 and 5 those from Zone 2, and Fractions 6 and 7 predominantly couplets of Zone 3 (perivenous) origin. The development of canalicular secretory activity was assessed in the couplets after a 15 min incubation with a fluorescent bile acid, cholyl-lysyl-fluorescein (CLF). This was sigmoidal in all fractions, but slower in the periportal couplets, taking 5.1 h for 50% to show secretory activity in Fraction 2, compared with 2.7 h for Fraction 7. Incubation of hepatocyte couplets with 1 or 10 microM taurodehydrocholate, a non-toxic bile acid analogue, did not influence the rate of development of accumulation of CLF by the couplets or the area of the canalicular vacuole in any fraction. However, it did decrease the CLF content of couplets incubated with CLF for 15 min to a greater extent in those of perivenous origin. After subjecting the couplets to oxidative stress by incubation with 20 microM menadione (2-methyl-1,4-naphthoquinone), it was evident that periportal couplets were less able to maintain canalicular secretory activity than perivenous couplets.
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Hexavalent chromium produces DNA strand breakage but not unscheduled DNA synthesis at sub-cytotoxic concentrations in hepatocytes.
Rat hepatocytes were used to investigate the possible induction of unscheduled DNA synthesis (UDS) and the extent of DNA strand breaks induced by sodium dichromate (a representative chromium(VI) compound) and chromium acetate hydroxide (chromium(III)) in vitro. Cytotoxicity, measured using tetrazolium salt (MTT) reduction assay, was found at a much higher dose of chromium(III), (> 50 microM), compared to that of chromium(VI), (> 2.5 microM), in cultured hepatocytes over 20 h treatment at 37 degrees C. Chromium(VI), but not chromium(III), stimulated minimal UDS in hepatocytes at sub-cytotoxic concentrations. A positive UDS response was only observed at cytotoxic concentration. DNA strand breaks in hepatocytes were induced by chromium(VI) following incubation at 37 degrees C for 1 h at doses of 10, 20 and 40 microM sodium dichromate. The subsequent ligation of such strand breaks in hepatocytes treated with 40 microM chromium(VI) for 1 h at 37 degrees C was demonstrated. The majority of strand breaks was repaired within 30 min following removal of the chromate. In conclusion, chromate-induced DNA strand breakage, possibly involving the formation of oxygen radicals and lack of significant UDS have some analogy to those produced by ionizing radiation.
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Stability and optimization of canalicular function in hepatocyte couplets.
An enriched preparation of rat hepatocyte couplets was obtained by collagenase perfusion and subsequent elutriation (> 85 per cent couplets and triplets; viability of over 95 per cent). Canalicular secretory activity (the ability to accumulate cholyl-lysyl-fluorescein, CLF) was first apparent after 2 h of culture at 37 degrees C and was present in over 80 per cent of the total population after 5-6 h. This remained almost constant for at least 4 h in both elutriated and directly plated cells. Initial storage of freshly prepared couplets at 4 degrees C for up to 6 h prior to incubation had no adverse effect upon secretory function. Reduction of canalicular secretory activity occurred at a concentration of the hepatotoxic agent menadione (IC50 17 microM) that was lower than that required to induce mild plasma-membrane blebbing (IC50 43 microM). This study has optimized and characterized the canalicular secretory effectiveness and stability of an enriched preparation of hepatocyte couplets, and established the feasibility of studies of toxic agents on hepatobiliary function in a heterogeneous population of hepatocytes. In this preparation other biochemical parameters can be assessed, thus complementing previous techniques using individual couplets.
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Effects of 1,1-dichloroethene and of some of its metabolites on the functional viability of mouse hepatocytes.
1,1-Dichloroethene (DCE) is hepatotoxic in rodents, and the expression of its toxicity involves probably its metabolism. In this study the role of DCE metabolites in the generation of the hepatotoxic lesion was investigated. Hepatocytes from male BALB/c mice in suspension were used as the experimental model. Cells were incubated with DCE for up to 5 hr and cellular viability was assessed by measurement of the release of lactate dehydrogenase into the medium and by alterations in the reduction of the dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. After incubation for 3 hr DCE at 0.5 mM caused maximal toxicity, whereas at 0.1 mM DCE was only marginally toxic. Cytotoxicity was exacerbated by pretreatment of mice with buthionine sulfoximine (1.6 g/kg), an inhibitor of glutathione biosynthesis, given 4 hr prior to hepatocyte isolation. Inclusion of N-acetylcysteine (10 mM) into the incubate protected cells against DCE-induced cytotoxicity. Coincubation with octylamine (0.5 mM), an inhibitor of cytochrome P450, abolished the cytotoxic potential of 0.5 mM DCE during incubation for 3 hr. DCE toxicity was increased in hepatocytes from mice which had received ethanol or acetone in their drinking water, both of which induce levels of the hepatic cytochrome P450 isozyme P450 2E1. Incubation of cells with the P450 2E1 inhibitors N,N-dimethylformamide (10 mM) or diethyldithiocarbamate (100 microM) protected liver cells against the detrimental effect of DCE. Pretreatment of animals with phenobarbital, which induces the P450 2B subfamily, or 3-methylcholanthrene, which induces P450 1A1, did not affect the degree of hepatocytotoxicity elicited DCE.(ABSTRACT TRUNCATED AT 250 WORDS)
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