Haem oxygenase 1 expression is associated with prognosis in cholangiocarcinoma patients and with drug sensitivity in xenografted mice.

OBJECTIVE: Haem oxygenase-1 (HO-1) plays important roles in cytoprotection and tumour growth. Cholangiocarcinoma (CCA) is a deadly malignancy with very poor prognosis. The role of HO-1 in tumour progression in CCA up to now has been relatively unexplored, thus, its possible therapeutic implications in CCA have been investigated here. MATERIALS AND METHODS: HO-1 expression in tumour tissues from 50 CCA patients was determined by immunohistochemical analysis and its association with survival time was evaluated using the Kaplan-Meier method. Its role in CCA cells in vitro was evaluated by transwell and wound healing assays and suppression of HO-1 expression by siRNA. Effects of HO-1 inhibition on gemicitabine (GEM)-mediated tumour suppression was evaluated in nude mice xenografted with CCA cells. RESULTS: HO-1 expression was inversely associated with median overall survival time. Hazard ratio of patients with high HO-1 expression was 2.42 (95% CI: 1.16-5.08) with reference to low expression and HO-1 knock-down expression inhibited transwell cell migration. Suppression of HO-1 by Zn-protoporphyrin (ZnPP) enhanced cytotoxicity to GEM in CCA cells, validated in CCA xenografts. Treatment with GEM and ZnPP almost completely arrested tumour growth, whereas treatment with only a single reagent, retarded it. Tumour inhibition was associated with reduction in expression of Ki-67 and microvascular density, and enhanced p53 and p21 immunohistochemical staining. CONCLUSION: High HO-1 expression was associated with poor prognosis of CCA. Synergistic role of HO-1 inhibition in chemotherapy of CCA is a promising insight for treatment of this tumour and warrants further investigation.

Inhibitory effects of polyphenolic compounds on human arylamine N-acetyltransferase 1 and 2.

Arylamine N-acetyltransferases (NAT) are important enzymes involved in the metabolic activation of aromatic and heterocyclic amines and inhibitors of NAT enzymes may be valuable as chemopreventive agents. Phytochemicals including cinnamic acid derivatives, various classes of flavonoids and coumarins were tested for the inhibitory activity on NAT1 and NAT2 from human liver and the human cholangiocarcinoma cell line: KMBC cells. Assays were performed using p-aminobenzoic acid and sulfamethazine as selective substrates for NAT1 and NAT2, respectively. NAT1 and NAT2 activities were present in liver cytosol. However, the KMBC cells showed only NAT1 activity. There was a marked difference in the ability of the test chemicals to inhibit NAT1 and NAT2. Caffeic acid, ferulic acid, gallic acid and EGCG inhibited NAT1 but not NAT2, whereas scopuletin and curcumin inhibited NAT2 but not NAT1. Quercetin, kaemferol and other flavonoids, except epicatechin and silymarin, inhibited both enzymes. The kinetics of inhibition of NAT1 by caffeic acid, EGCG and quercetin were of the non-competitive type, whereas that of NAT2 by quercetin, curcumin and kaemferol was also of the non-competitive type. The most potent inhibitor was quercetin, which has the inhibitory constants for NAT1 and NAT2 of 48.6 +/- 17.3 and 10.0 +/- 1.8 microM, respectively.

Association between genetic polymorphisms of CYP1A2, arylamine N-acetyltransferase 1 and 2 and susceptibility to cholangiocarcinoma.

Human CYP1A2 and arylamine N-acetyltransferases, which are encoded by the polymorphic CYP1A2 and NAT genes respectively, have been shown to have wide interindividual variations in metabolic capacity and may be potential modifiers of an individual's susceptibility to certain types of cancers. The present study aimed to evaluate the relationship between CYP1A2, NAT1 and NAT2 polymorphisms and cholangiocarcinoma (CCA), the most prevalent cancer in the north-east of Thailand. A total of 216 CCA patients and 233 control subjects were genotyped by polymerase chain reaction with restriction fragment length polymorphism based assays. Two CYP1A2 alleles (CYP1A2*1A wild-type and *1F), six NAT1 alleles (NAT1*4 wild-type, *3, *10, *11, *14A and *14B) and seven NAT2 alleles (NAT2*4 wild-type, *5, *6A, *6B, *7A, *7B and *13), which are the major alleles found in most populations, were analysed. Although CYP1A2*1A allele, NAT1*10 allele, and the NAT2 slow acetylator alleles were not associated with CCA risk, among the male subjects, the genotype CYP1A2*1A/*1A conferred a decreased risk of the cancer (adjusted odds ratio (OR) 0.28, 95% confidence interval (CI) 0.08-0.94) compared with CYP1A2*1F/1*F. Frequency distributions of rapid NAT2*13 and two slow alleles (*6B and *7A), but not the other major alleles, were associated with lower CCA risk. Adjusted OR of the genotypes consisting of at least one of these alleles significantly decreased the cancer risk compared with none of them (OR 0.26, 95% CI 0.15-0.44). This study suggests that the NAT2 polymorphism may be a modifier of individual risk to CCA.

The antiulcerative effect of Thai Musa species in rats.

Bananas are reported to have an antipeptic ulcer effect, however, the beneficial action can be affected by many factors, including the variety. Our study was undertaken to investigate the antipeptic ulcer effect of the Palo and Horn varieties of banana, grown and consumed in the northeast of Thailand. Indomethacin and acetic acid-induced gastric lesions in rats were employed as models of peptic ulcer disease. The lengths of gastric lesions in the glandular part of the stomach were measured for the assessment of the protective effect of bananas. The healing effect was studied by histological examination of the ulcerated area. The lesions in rats treated with the extract of banana were significantly less dominant than those of the control. The average length of total lesions of rats treated with an extract of Palo or Horn bananas at a dose of 1.0 g/kg/d for 3 days prior to indomethacin administration were 4.47+/-1.2 and 1.87+/-0.44 mm, respectively, whereas those observed in the control rats were 14.56+/-2.43 mm. In the ulcer-healing model, only the Hom-banana-extract-treated group showed a beneficial effect which manifested as a milder degree of histological change than that of the indomethacin-induced-chronic-ulcer control group. However, in acetic acid-induced ulcers, the histological changes of every group were similar. The present findings indicate that bananas of different varieties have varying antipeptic ulcer effects. The extracts of Palo and Hom bananas have a prominent gastroprotective effect, whereas only the extract of Hom banana had an observed ulcer-healing effect.

Alteration of drug kinetics in rats following exposure to trichloroethylene.

The effect of trichloroethylene (TCY) was investigated to determine whether repeated exposure alters the pharmacokinetics of some drugs. Sprague-Dawley rats were given intraperitoneal injections of TCY (5 mmol/kg) in corn oil once daily for 3 days, while the control group received only corn oil. Four hours after the last dose, theophylline, quinidine, or pentobarbital were administered. Blood samples were collected at appropriate intervals for drug analyses. There was a small decrease in plasma clearance of theophylline, with no change in volume of distribution (V(d)) as compared with controls. For quinidine, the elimination half-life was unchanged, and the V(d) was decreased by 40%. The clearance of pentobarbital was decreased by 40% in male rats, but not in the females. Nonetheless, the duration of the sleeping time for both sexes was remarkably prolonged as compared with the control group. There was a decrease in the cytochrome P-450 content only in male rats. In conclusion, exposure to TCY causes changes in some drug kinetics, probably resulting from differential effects on the drug-metabolizing enzymes.

Mechanism of ketoconazole-induced elevation of individual serum bile acids in the rat: relationship to the effect of ketoconazole on bile acid uptake by isolated hepatocytes.

Ketoconazole, an imidazole derivative, has been implicated in a number of hepatic dysfunctions. The aim of the present study was to determine the effect of in vivo treatment of rats with ketoconazole on individual serum bile acid levels and the in vitro effects of ketoconazole on the hepatocellular uptake of two bile acids and two other model substrates transported by liver cells. Male Sprague-Dawley rats were treated i.p. with a single injection of ketoconazole of 25 mg/kg (n = 4) or 50 mg/kg (n = 4); the control group (n = 4) received the vehicle only at a dose of 1 ml/kg. Blood samples were collected at 4 hr after dosing. With high-performance liquid chromatography, the serum was assayed for individual serum bile acids. At the higher dose, ketoconazole produced a significant increase in serum levels of cholic acid, taurocholic acid, chenodeoxycholic acid, glycocholic acid, glycochenodeoxycholic acid, glycodeoxycholic acid, deoxycholic acid and taurochenodeoxycholic acid compared with the control group (P < .05). Cholic acid, taurocholic acid and chenodeoxycholic acid levels were significantly raised in rats treated with the lower dose. In vitro, ketoconazole strongly inhibited the hepatocellular uptake of [14C]cholic acid, [14C]taurocholic acid and [3H]ouabain but not [14C]2-aminoisobutyric acid, which indicated that the effect is relatively specific. The kinetics of inhibition were competitive and the inhibition constants for taurocholate and ouabain were 6 and 1 microM, respectively. Ketoconazole inhibited by both Na(+)-dependent taurocholate uptake and stimulated bile acid countertransport of preloaded hepatocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of cyclosporin A on bilirubin uptake by isolated rat and human hepatocytes.

Several studies have reported that cyclosporin A (CsA) causes elevated serum bilirubin and bile acid levels, suggesting hepatobiliary dysfunction. This study examines the effects of CsA on the uptake of radiolabelled bilirubin by rat and human hepatocyte suspensions. The initial uptake rates of [(3)H]bilirubin, over a concentration range of 0.05 to 2.0 mum, were determined in isolated hepatocytes that were preincubated with or without CsA. A saturable process for uptake of bilirubin was observed and kinetic constants calculated. Isolated human hepatocytes showed bilirubin uptake rates similar to those of rodent cells. CsA inhibited bilirubin uptake by both rat and human isolated hepatocytes. The apparent inhibition constants for the effect of CsA on bilirubin uptake were 39 and 26 mum, for rat and human hepatocytes, respectively. This inhibitory effect was evident at a dose of 1 mum, which is similar to CsA blood levels seen in those cases in which hyperbilirubinaemia has been reported. However, hepatocytes isolated from rats administered CsA (10 mg/kg/day ip, in Cremophore, for 5 days) exhibited uptake rates similar to those of cells isolated from control animals treated with only the Cremophore vehicle. Overall, the data are consistent with a receptor-mediated uptake of bilirubin by hepatocytes from rats and humans. The inhibition of bilirubin uptake by CsA may explain, at least in part, the hyperbilirubinaemia observed clinically.

Chemical-induced interference with hepatocellular transport. Role in cholestasis.

Transport of endogenous chemicals both into (at the basolateral membrane) and out of (at the canalicular membrane) hepatocytes plays an important role in bile formation. Hence, interference with these processes, for example by chemicals, may result in reduced bile output. Several different systems are available for the study of transport and hence chemicals that may interfere with the process. These have been used to varying degrees with isolated hepatocytes probably being the most popular over recent years. It is likely that hepatocyte couplets and highly purified plasma membrane vesicles will be increasingly employed over the ensuing years. The inhibitory effects of several chemicals on the transport of bile acids have been demonstrated with indications that this may help to account for some aspects of chemical-induced hepatobiliary dysfunction. For example, the inhibition of transport of bile acids by cyclosporin A is consistent with the reported pattern of liver dysfunction in patients on high doses of this immunosuppressant. Investigation into chemical-induced interference with electrolyte transport has yet to receive the same degree of attention. This and other aspects have been suggested as deserving of and likely to be subjected to more intensive experimentation over the next few years.

Kinetics of taurocholate efflux from freshly isolated suspensions and primary cultures of rat hepatocytes.

Isolated rat hepatocytes have been advocated as a model to study aspects of mechanism of chemical-induced interference with biliary excretory function. Some technical problems do exist in studying efflux, such as the reuptake of the previously excreted substrate. Another concern is the loss of liver-specific functions in hepatocytes with continuing time in culture. It is important to address such technical aspects and to determine whether the process of efflux is compromised in primary cultures of hepatocytes. In the presence of Na+ the apparent efflux of taurocholate from hepatocytes was shown to be significantly confounded by reuptake of substrate. The unidirectional efflux was best demonstrated in buffer where choline replaced Na+. A comparison of efflux kinetics for cultured cells to those in suspension showed that both apparent affinity for transport carriers and transport capacity were greater in the former. The simple diffusion component for efflux increased with the time in culture, but affinity for transport carriers and transport capacity remained unchanged over 6 to 24 hr. However, it was not possible to determine meaningful kinetic constants after 24 hr in culture because the uptake of taurocholate was so low. Primary cultured hepatocytes may therefore be of limited value in the study of efflux of bile salts in the longer term, mainly because of the inability of cells to take up and accumulate a sufficiently high level of bile salts.

Interference with hepatocellular substrate uptake by 1,1,1-trichloroethane and tetrachloroethylene.

The effects of chlorinated aliphatic hydrocarbon solvent exposure on hepatocellular transport of some model substrates have been investigated. Exposure of isolated hepatocytes to 1,1,1-trichloroethane or tetrachloroethylene resulted in suppression of uptake of taurocholate, ouabain, and 2-aminoisobutyric acid but not CdCl2 or 3-O-methyl-D-glucose. The effect was clearly evident at noncytotoxic concentrations, as indicated by the lack of intracellular enzyme leakage and unaltered intracellular K+ ion concentration. Moreover, the ultrastructure of solvent-exposed hepatocytes was similar to that of control cells, except for a reduction in membrane microvilli. The suppression of uptake was reversible provided that sufficient time was allowed for the cells to recover. The mechanism of this inhibition may be associated with energy-linked processes, as uptake of taurocholate, ouabain, and 2-aminoisobutyric acid is energy requiring while uptake of CdCl2 and 3-O-methyl-D-glucose is not. Cellular ATP was reduced in a dose-dependent manner, but a marked depletion occurred only at cytotoxic concentrations. Na(+)-K(+)- and Mg2(+)-ATPase activities in hepatocyte plasma membrane preparations were also inhibited by solvent exposure. The data suggest that 1,1,1-trichloroethane and tetrachloroethylene interfere specifically with energy-dependent hepatic transport functions and that a decrease in ATP levels and/or inhibition of cell membrane ATPases may be the mechanism.

Comparison of uptake kinetics in freshly isolated suspensions and short-term primary cultures of rat hepatocytes.

The apparent kinetics of uptake of various model substrates were examined for hepatocytes in suspension and primary culture up to 72 h. The ability of hepatocytes to take up taurocholate and ouabain was decreased in culture. Vmax for uptake of both substrates diminished rapidly with increasing time in culture. An increase in Km was observed in cultures 6 h after plating, but there was no further change with prolongation of culture time. The decrease of uptake of taurocholate and ouabain during culture may be due to the reduction in the number of transport carriers plus a decrease of affinity of the carrier to substrates. The nonsaturable component of cadmium uptake was much reduced in cultured cells compared with the suspensions. The saturable process was lower in 6 h culture but increased to a level comparable with the fresh cells at longer culture time. No significant change was found in the Km between suspensions and cultures. Uptake of alpha-aminoisobutyric acid was greater in culture while that of 3-O-methyl-D-glucose was relatively stable but about one-half that found in cell suspension. Thus, uptake of two substrates, taurocholate and ouabain, is clearly compromised with increasing time in primary culture, while uptake of the other substrates does not reflect such a dramatic decrease. It is therefore apparent that the cell preparation of choice in uptake studies depends on the substrate and the nature of the experiments.

Inhibition of taurocholate transport by cyclosporin A in cultured rat hepatocytes.

Cyclosporin A, a powerful immunosuppressant, has been shown to interfere with the transport of bile salts and other substrates in isolated rat liver cells and to suppress bile flow and bile salt secretion in situ. Cyclosporin A was added to primary hepatocyte cultures just before taurocholate to study the immediate effects on transport. In prolonged exposure experiments cyclosporin A was dissolved in culture media. Efflux was studied by preloading cultured cells with [14C]taurocholate and then changing to sodium-free buffer containing no taurocholate. Cyclosporin A inhibited the uptake and efflux of taurocholate in a competitive manner when added just before determination of transport. Hepatocyte cultures which were pre-exposed to cyclosporin A (1-25 microM) for 18 hr and then washed, also showed a significantly lower taurocholate uptake. The longer the pre-exposure time the greater was the suppression. However, the inhibition could be reversed gradually by incubation of cultured cells in fresh media, complete reversal being attained within 3 hr. These results indicate that cyclosporin A competitively inhibits the taurocholate transport system. The interaction between cyclosporin A and transport components is rapid and long lasting but reversible.

N-acetyltransferase polymorphism in Thailand.

A series of 222 individuals from the northeastern provinces of Thailand were studied with respect to acetylator phenotypes. Among individuals of pure Thai descent 55.5% were rapid acetylators. The corresponding figure for Chinese was 66.0%. There were no significant differences between Thais and Chinese. The result shows a lower frequency of rapid acetylators in Thais than in previous reports on Thais.

Some parameters of affinity chromatography in the purification of antibody against Naja naja siamensis toxin 3.

Some parameters in the purification of antibodies against Naja naja siamensis toxin 3 by affinity chromatography were studied on toxin Sepharose, toxin-succinylaminoethyl Sepharose, toxin-albumin Sepharose and toxin succinylaminoethyl Biogel adsorbents. Immunologically pure antibody with 10-12-fold increase in potency was obtained by chromatography of horse refined globulin on all these adsorbents. The maximum antibody binding capacities were higher for adsorbents containing linear spacers but represented only 7-16% of the theoretical binding capacity. The operational half-lives of the adsorbents ranged from 19 to 108 days with toxin-albumin Sepharose showing the highest stability. The recovery of neutralizing capacity was about 30-356% for any of the 4 adsorbents. It is concluded that improvements regarding the antibody binding capacity and the recovery of neutralizing capacity should be made before large scale purification of antibody for therapeutic purposes can be attempted.