Investigation of a cluster of leukaemia in the Illawarra region of New South Wales, 1989-1996.

OBJECTIVES: To investigate a cluster of leukaemia among young people and assess the plausibility of a disease-exposure relationship. DESIGN: Descriptive analysis of population-based leukaemia incidence data, review of evidence related to the causation of leukaemia, assessment of environmental exposures to known leukaemogens, and resulting risks of leukaemia. SETTING: Illawarra region of New South Wales, Australia, focusing on suburbs between the Port Kembla industrial complex and Lake Illawarra (the Warrawong area). MAIN OUTCOME MEASURES: Standardised incidence ratios (SIRs) for leukaemia; current measured and past estimated ambient air benzene concentrations; and expected leukaemia cases attributable to estimates of ambient air benzene concentrations. RESULTS: In 1989-1996, 12 leukaemia cases among Warrawong residents aged less than 50 years were observed, more than the 3.49 cases expected from the rate in the rest of the Illawarra region (SIR, 343.8; 99% CI, 141.6-691.7). These people lived in suburbs immediately to the south-southwest of a coke byproducts plant (a major industrial source of benzene, one of the few known leukaemogens). The greatest excess was among 15-24-year-olds (SIR, 1085.6; 99% CI, 234.1-3072.4). In 1996, ambient air concentrations of benzene averaged less than 1 part per billion (ppb). Since 1970, ambient air concentrations of benzene were estimated to have averaged up to 3 ppb, about one-thousandth of the level at which leukaemia risk has been identified in occupational epidemiological studies. Using the risk assessment model developed by the US Environmental Protection Agency, we estimate that past benzene levels in the Warrawong area could have resulted in 0.4 additional cases of leukaemia in 1989-1996. CONCLUSIONS: The excess occurrence of leukaemia in the Warrawong area in 1989-1996 is highly unusual. Current environmental benzene exposure and the reconstructed past environmental benzene exposure level are too low to explain the large excess of leukaemia. The cause of the cluster is uncertain.

Toluene-induced elevation of serum bile acids: relationship to bile acid transport.

Raised concentrations of serum bile acids (SBA) following occupational exposure to a number of halogenated aliphatic hydrocarbon solvents and after in vivo exposure of experimental animals to these substances have been reported in several studies in recent years. However, the widely used nonchlorinated aromatic hydrocarbon solvent, toluene, has not been critically examined for its effect on serum bile acids. Accordingly, the effect of in vivo treatment with toluene on SBA and its direct in vitro effects on the transport of bile acids by isolated rat hepatocytes were investigated in this study. In vivo treatment with toluene (2.3 mmol/kg body weight, ip, on each of 3 consecutive days) resulted in a significant rise in the serum concentrations of total and some individual bile acids while other parameters of hepatobiliary function were unaltered. Administration of a higher dose of solvent (9.2 mmol/kg body weight, i.p.) resulted in a further increase in total SBA levels together with a significant rise in serum activities of some liver enzymes. In vitro application of noncytotoxic doses of toluene in the vapor phase to hepatocytes isolated from untreated rats resulted in a significant inhibition of the initial rate-(V0)-of uptake of cholic acid (CA). Similarly, accumulation of CA and taurocholic acid (TC) over an extended incubation time by hepatocytes exposed to toluene was significantly inhibited. Kinetic analysis revealed a noncompetitive inhibition of CA uptake as suggested by a decline in Vmax and an unaltered K(m). In contrast, the initial rate of efflux of these substates and their continuous efflux from preloaded cells were unaffected by exposure to toluene. Thus, toluene exposure inhibited the transport and accumulation of bile acids by hepatocytes in a manner largely similar to that of halogenated solvents, and this inhibition could explain the raised SBA concentrations following in vivo exposure to this solvent. These findings are consistent with and provide mechanistic data to support previous studies where increased SBA levels (in the absence of any evidence of liver injury as measured by liver enzyme tests) were reported in workers following occupational exposure to this solvent. Additionally, in full agreement with our previous investigations in which SBA levels were found to be a sensitive biological marker of exposure to halogenated aliphatic hydrocarbon solvents, the data support a similar role for SBA on exposure to toluene as well.

In vitro interference with hepatocellular uptake of bile acids by xylene.

Occupational exposure to a mixture of two widely used aromatic hydrocarbon solvents, xylene and toluene, has been associated with a significant rise in the concentrations of serum bile acids (SBA). We have recently shown that toluene interferes with the transport of bile acids by hepatocytes and this could explain elevated SBA after occupational exposure or following in vivo administration of this compound to experimental animals. However, it is not known if xylene, like its monomethylated homologue, toluene, could interfere with the processes of bile acid transport by hepatocytes. Therefore, the present studies were undertaken to examine this possibility. Direct addition of a non-cytotoxic dose (2.5 microliters/2.8 x 10(6) cells) of xylene (in vapour phase) to hepatocytes isolated from untreated rats significantly inhibited the initial rates (determined from slope of the lines in the linear range (20-80 s)) of uptake (V0) of 10 microM cholic acid (CA) and-taurocholic acid (TC) by 37 and 48%, respectively (P < 0.05). Similarly, accumulation of these substrates by hepatocytes over an extended incubation time up to 30 min was significantly inhibited to the same extent by xylene exposure. This inhibitory effect was found to be reversible when sufficient time was allowed for the cells to recover. In contrast, the initial rates (V0) of efflux (determined from slope of the lines in the linear range (1-5 min)) of these bile acids (25 microM) and their continuous efflux (up to 30 min) from preloaded cells incubated with a similar dose of xylene were not (except for the 1 min time point) significantly different from those of controls. In conclusion, xylene interferes with the transport of bile acids by hepatocytes in a manner largely similar to that of its monomethylated homologue, toluene. These findings extend our previous observations on aliphatic and aromatic hydrocarbon solvents and provide mechanistic data at a cellular level to support a causal role for xylene (as well as toluene) in raised SBA levels of exposed individuals.

Biochemical assay of serum bile acids: methods and applications.

Immunoassays and bioluminescence assays of bile acids in serum have provided relatively simple and sensitive methods for assessing the concentration of selected sub-groups of bile acids. However, these assays do not provide full data for each of the individual bile acids. The recent development of sensitive techniques such as high-performance liquid chromatography (HPLC) and gas-liquid chromatography-mass spectrometry (GLC-MS) have made possible the separation and quantification of free and conjugated bile acids in biological samples. Several studies have demonstrated the value of individual serum bile acid levels and bile acid ratios when assessing the hepatic function of experimental animals treated with various hepatoxic agents, and in humans with various hepatic disorders. Current data show that individual serum bile acids are more sensitive and specific as early predictors of hepatic injury, and are an accurate independent prognostic indicator. These studies have provided further insight into the various determinants of serum bile acid levels in physiological and pathological conditions affecting the liver. Future studies using these techniques and perhaps monoclonal antibodies, fast atom bombardment-mass spectrometry (FAB-MS) and nuclear magnetic resonance (NMR) for bile acid assays may provide both researcher and clinician with a reliable, sensitive and specific indicator of hepatic injury.

Raised concentration of serum bile acids following occupational exposure to halogenated solvents, 1,1,2-trichloro-1,2,2-trifluoroethane and trichloroethylene.

OBJECTIVES: The objectives of this study were threefold. First, to examine the hepatic effects of occupational exposure to 1,1,2-trichloro-1,2,2-trifluoroethane (FC 113) using conventional and newer tests (serum bile acids) of hepatobiliary function. Second, to assess the effects of altered work practices that included a reduced exposure to a different halogenated solvent (trichloroethylene) on the same parameters of liver function; and finally, to gather further data to support or refute the contention that serum bile acid (SBA) levels could provide a sensitive biological marker of exposure to these solvents. DESIGN: Two groups of workers (control and exposed) in an Australian steel industry participated in the study. The exposed group (n = 5-6) comprised individuals who had either exposure to FC 113 (68.2 +/- 12.6 ppm) or trichloroethylene (8.9 +/- 3.1 ppm) during the application of these solvents in a cleaning procedure, whereas the control group (n = 7-11) was composed of non-solvent-exposed office workers in the same company. The initial investigation involved exposure to FC 113 while a follow-up study was undertaken after changes in work practices were made including replacement of FC 113 with trichloroethylene (TRI). METHODS: Standard liver function tests and individual serum bile acids (ISBA) were measured before and after exposure to solvents and simultaneously in the control subjects by enzymatic methods and high performance liquid chromatography (HPLC), respectively. RESULTS: Statistical analysis of the data showed a significant increase in the concentration of total serum bile acids (TSBA), some of the subgroups of SBA, and a few of the ISBA in workers after a period of exposure to FC 113. After TRI replaced FC 113 together with other changes in work practices to give substantial reduction in exposure to solvent, a repeat study also found elevated SBA after the cleaning procedure but to a lesser extent. No other indications of adverse liver effects, as measured by conventional parameters of hepatobiliary function, were detected. CONCLUSION: Exposure to FC 113 was clearly associated with a significant rise in SBA levels, which are sensitive indicators of liver function. This finding is consistent with, and provides further support for, our previous investigations on chlorinated aliphatic hydrocarbon solvents which showed that SBA levels are a sensitive biological marker of exposure to these solvents. Changes in work practices including replacement of FC 113 resulted in a reduced effect on SBA, consistent with lower exposures.

Current concepts of hepatic uptake, intracellular transport and biliary secretion of bile acids: physiological basis and pathophysiological changes in cholestatic liver dysfunction.

Hepatic sinusoidal uptake of bile acids is mediated by defined carrier proteins against unfavourable concentration and electrical gradients. Putative carrier proteins have been identified using bile acid photoaffinity labels and more recently using immunological probes, such as monoclonal antibodies. At the sinusoidal domain, proteins with molecular weights of 49 and 54 kDa have been shown to be carriers for bile acid transport. The 49 kDa protein has been associated with the Na(+)-dependent uptake of conjugated bile acids, while the 54 kDa carrier has been involved in the Na(+)-independent bile acid uptake process. Within the hepatocyte, cytosolic proteins, such as the glutathione S-transferase (also designated the Y protein), the Y binders and the fatty acid binding proteins, are able to bind bile acids and possibly facilitate their movement to the canalicular domain. At the canalicular domain a 100 kDa carrier protein has been isolated and it has been shown by several laboratories that this particular protein is concerned with canalicular bile acid transport. The system is ATP-dependent and follows Michaelis-Menten kinetics. Interference with bile acid transport has been demonstrated by several chemicals. The mechanisms by which these chemicals inhibit bile acid transport may explain the apparent cholestatic properties observed in patients and experimental animals treated with these agents. Several studies have shown that Na+/K(+)-ATPase activity is markedly decreased in cholestasis induced by ethinyloestradiol, taurolithocholate and chlorpromazine. However, other types of interference have been described and the cholestatic effects may be the result of several mechanisms. Cholestasis is associated with several adaptive changes that may be responsible for the accumulation of bile acids and other cholephilic compounds in the blood of these patients. It may be speculated that the nature of these changes is to protect liver parenchymal cells from an accumulation of bile acids to toxic levels. However, more detailed quantitative experiments are necessary to answer questions with regard to the significance of these changes and the effect of various hepatobiliary disorders in modifying these mechanisms. It is expected that the mechanisms by which bile acid transport is regulated and efforts to understand the molecular basis for these processes will be among the areas of future research.

Sequential changes in serum levels of individual bile acids in patients with chronic cholestatic liver disease.

In order to determine the value of serum bile acids in predicting the course of chronic cholestatic liver diseases, we measured individual serum bile acids serially, using high-performance liquid chromatography, over a 4 year observation period in 12 patients with primary biliary cirrhosis and six patients with primary sclerosing cholangitis. The changes in individual serum bile acids and the ratios thereof, conventional liver tests and Child-Turcotte and Mayo scores were compared between survivors (n = 10) and patients who underwent liver transplantation for (n = 3) or died of the liver disease (n = 5). Patients with a serum total chenodeoxycholic acid concentration at study entry that exceded 15 mumol/L were 10 times more likely to die or need a liver transplant in the following 4 years than those with chenodeoxycholic acid levels < 15 mumol/L (P < 0.05). None of the other biochemical parameters or clinicopathological scores could similarly discriminate between the two groups at entry. Time-dependent analyses for the cholic acid/chenodeoxycholic acid ratio, serum total bilirubin and albumin concentrations and Child-Turcotte and Mayo scores were able to differentiate between primary sclerosing cholangitis patients who died or were transplanted and those who were not, whereas age of the patients and other parameters did not. The taurocholic acid/taurochenodeoxycholic acid ratio fell during progression of primary biliary cirrhosis but rose in temporal relationship with primary sclerosing cholangitis. This differential pattern of change was unique compared with other clinical and laboratory indices. In conclusion, serum chenodeoxycholic acid levels and the cholic acid/chenodeoxycholic acid ratio in both diseases were independent indices that allowed for the prediction of survival or the need for liver transplantation. These indices are worthy of further examination in a larger group of patients as prognostic criteria for chronic cholestatic liver disease and in the assessment of the efficacy of therapeutic interventions, including liver transplantations.

Alpha-naphthylisothiocyanate-induced elevation of serum bile acids: lack of causative effect on bile acid transport.

In recent years chemicals including chlorinated solvents have been found to interfere with the transport of bile acids (BA) by hepatocytes, which probably accounts for the raised serum bile acids (SBA) after exposure. However, the known cholestatic agent, alpha-naphthylisothiocyanate (ANIT) has never been fully examined for its effect on these processes. Accordingly, the direct effects in vitro and the effects of in vivo treatment on bile acid transport have been investigated in this study. Direct addition of ANIT (5-100 microns) to hepatocytes isolated from untreated rats did not result in any change in uptake or efflux of taurocholic acid (TC), one of the most obviously elevated SBA in ANIT-treated rats. Additionally, accumulation of TC over an extended incubation period was not affected by ANIT. In vivo treatment with ANIT (50 mumol/kg i.p. on each of 3 consecutive days) resulted in a marked elevation of total serum bile acids (TSBA) and a slight increase in the activity of serum alkaline phosphatase (ALP) and a very mild hyperbilirubinemia, while other markers of liver injury were unaltered. In hepatocytes isolated from these rats, Km and Vmax for uptake and V0 for efflux were no different between ANIT and vehicle-treated animals. In conclusion, ANIT showed no effects on transport of BA on in vitro exposure or after treatment in vivo where SBA were clearly elevated. The lack of effects of ANIT on transport of bile acids is consistent with other postulated mechanisms of action. Furthermore, this indicates that the effects noted with solvents are not necessarily replicated by substances known to cause histopathological cholestasis.

Formation and persistence of DNA adducts in different target tissues of rats after multiple administration of benzo[a]pyrene.

Exposure of humans to polycyclic aromatic hydrocarbons is an ongoing concern because of the carcinogenicity of these substances. DNA adducts are being increasingly used as indicators of carcinogen exposure. While considerable experimental evidence exists to support their use there are aspects that require further attention, especially after repeated exposure, which has led to this series of experiments. Male Sprague-Dawley rats were dosed with 10 mg/kg benzo[a]pyrene (B[a]P) i.p., 3 times/week for 2 weeks. At 1, 3, 7, 14, 28 and 56 days after the last treatment liver, lung, spleen and peripheral blood mononuclear cells (PBMNs) were sampled. The DNA adduct levels, as measured by the 32P-postlabelling technique, were significantly increased in all tissues, with lung having the highest levels. At day 14 total DNA adducts in lung, spleen and PBMNs were still > 50% of the level at day 1. The removal of total DNA adducts was found to be fastest in liver > spleen > PBMNs > lung. A consistent correlation of total adducts between the lung and PBMNs was observed. A major adduct, designated adduct 1, was detected in all tissues, while adduct 4 was only found in liver and lung. Adduct 5 was detected only in lung, where it constituted approximately 38-49% of total adducts and persisted at a higher level than either adduct 1 or adduct 4 for the entire post-exposure period. These results indicate that B[a]P induced a significant increase in DNA adduct levels in all tissues tested and that total adducts in PBMNs reflect total lung adduct levels. DNA adducts were still readily detectable 56 days after exposure ceased. Thus the results support the use of PBMNs as surrogates for estimation of B[a]P exposure in lung, the primary target organ, and indicate that samples taken days or weeks after repeated exposure will still yield DNA adducts at detectable levels. The role and significance of adduct 5 deserves further investigation, as it was detected only in the primary target organ for B[a]P-induced cancer.

Individual serum bile acids in apprentice spray painters in association with solvent exposure.

The effects of exposure to solvents on serum bile acids were investigated by comparing a group of apprentice vehicle spray painters (exposed group) with one of apprentice electricians. Apprentice spray painters from the study were subdivided into high- and low-solvent-exposure groups. Concentrations of individual serum bile acids (SBA) were measured and compared with conventional liver function tests (LFTs). Total, free, glycine- and taurine-conjugated SBA were consistently found to be present at higher levels in the spray painters than in the electricians, even at the beginning of the apprenticeship. Total SBA tended to increase in spray painters with increasing years of exposure during the apprenticeship, but this was significant at only one time point. No rises were observed over the sampling period in electricians. The mean values of individual and total SBA concentrations were all found to be higher in the high-exposure group than in the low-exposure group, with some differences reaching statistical significance. None of the routine liver biochemistry parameters was different between spray painters and electricians. gamma-Glutamyl transferase (GGT) was the only enzyme found to be significantly different between the high- and low-exposure groups, but all values were within the normal range. This study suggests that occupational exposure even to low levels of solvent mixtures results in increases in SBA. The increased SBA may be indicative of a subclinical liver dysfunction. Alternatively, they may reflect solvent exposure only, with the raised levels having no pathologic implication or consequence.

In vitro interference with hepatocellular transport of taurocholate by 1,1,2-trichloro-1,2,2-trifluoroethane.

In recent years workers in our laboratory have shown that several industrial chlorinated aliphatic hydrocarbon solvents interfere with the transport of bile acids by hepatocytes and this interference may account for the raised concentration of serum bile acids that has been observed after occupational exposure to solvents. There has been concern about the effects on workers of a selective solvent used in degreasing electrical equipment, 1,1,2-trichloro-1,2,2-trifluoroethane (FC 113). However, this compound has not been investigated for effects on bile acid transport. Therefore we undertook the present study to examine the direct in vitro effects of FC 113 on uptake and efflux of bile acids by isolated rat hepatocytes. FC 113, at non-cytotoxic doses after a 20-min equilibration time, showed significant (P < 0.05) inhibitory effects on the initial rate of uptake of taurocholate (TC), whereas accumulation of TC over an extended incubation time was not affected. Kinetic analysis revealed a non-competitive inhibition of TC uptake as evidenced by a decline in V(max) and an unaltered K(m). The initial rate of efflux of TC and the continuous efflux of this model substrate from preloaded cells incubated with different doses of solvent were not significantly different from those of controls. However, the highest dose of solvent inhibited the process of efflux at the early time points. The data suggest that FC 113 interferes with bile acid transport in a reversible manner similar to that of the chlorinated aliphatic hydrocarbons. It would be expected, therefore, that this solvent would cause an increase in serum bile acids in exposed workers.

Inhibition by trichloroethylene and 1,1,2-trichloro-1,2,2-trifluoroethane of taurocholate uptake into basolateral rat liver plasma membrane vesicles.

It has previously been shown that trichloroethylene (TRI) and 1,1,2-trichloro-1,2,2-tri-fluoroethane (FC 113) interfere with the transport of bile acids by isolated human and rat hepatocytes in a dose-dependent and reversible manner. This finding may explain the rise in serum bile acids (SBA) following exposure to these chemicals. However, the effect of these compounds on the transport of bile acids across the cellular membrane in the absence of confounding variables, such as interference by intracellular metabolism, binding to cytosolic proteins and intracellular conjugation, has not been investigated. Accordingly, in vitro effects of TRI and FC 113 on uptake of [(3)H]taurocholate (TC) into purified basolateral (blLPM) and canalicular (cLPM) rat liver plasma membrane vesicles were examined by a rapid filtration technique. Both TRI and FC 113 caused a dose-dependent inhibition of TC uptake into blLPM vesicles at an approximate concentration of 3 mm and 72 mum, respectively. Initial rates of TC uptake in the presence of TRI and FC 113 were significantly inhibited by 69 and 61%, respectively (P < 0.05). In contrast, these chemicals had no effect on TC uptake into cLPM vesicles. This confirms studies in intact cells where these solvents were found to inhibit the uptake of bile acids by hepatocytes rather than interfere with the process of efflux. In conclusion, and consistent with the previous findings, the data suggest that the mechanism of TRI and FC 113-induced elevation of SBA may, in part, be due to selective inhibition of bile acid transport by the parent compounds at the basolateral domain of the hepatocyte plasma membrane.

Comparison of DNA adduct induction in vitro by PAHs and nitro-pahs in freshly isolated rat hepatocytes.

Polycyclic aromatic hydrocarbons (PAHs) and nitro-PAHs have been identified widely in occupational and environmental pollution, such as diesel engine emissions and other combustion products. In most cases, hepatic biotransformation is involved in converting these chemicals to their carcinogenic metabolites. It has been demonstrated that isolated hepatocytes possess substantial amounts of the enzymes responsible for metabolizing xenobiotics and are therefore a convenient model for studying chemicals that require activation to exert their carcinogenic effects. In this study, rat hepatocytes were isolated by collagenase digestion and then exposed to benzo[a]pyrene (B) [a]P), benzo[a]anthracene (B[a]A), 1-nitropyrene (1-NP) and 1,6-dinitropyrene (1,6-DNP) at different doses and/or times so that DNA adducts levels, as measured with the (32)P-postlabelling technique, could be compared. Each of the four compounds tested induced significant increases of total DNA adducts with clear dose-related responses. One or more individual adducts were identified as major adducts for each compound. Time-related increases of DNA adducts were also observed from 1 to 4 hr of incubation. Greater amounts of DNA adducts were induced by B[a]P or 1,6-DNP than by B[a]A or 1-NP, with potency being in the order 1,6-DNP > B[a]P > 1-NP B[a]A. These results demonstrate that freshly isolated hepatocytes can be used as an effective in vitro system for the detection of DNA adducts using (32)P-postlabelling, and have shown 1,6-DNP to be the most potent of the tested constituents of diesel emissions.

Hepatoprotection in ethinylestradiol-treated rats is provided by tauroursodeoxycholic acid, but not by ursodeoxycholic acid.

Ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA) have been suggested as potential treatments for drug-induced cholestasis. It was therefore decided to study the effects of administration of UDCA or TUDCA on individual serum bile acid concentration, conventional liver tests and associated hepatic ultrastructural changes in ethinylestradiol-treated (EE) rats mg/kg per day). Control rats were treated s.c. with propylene glycol. EE-treated rats were randomly assigned to receive daily i.p. injections of placebo, TUDCA or UDCA. Four rats in each group were treated for 4 consecutive days, and a second four for 14 days. After 4 days of treatment, the serum levels of cholic acid and taurocholic acid were significantly increased in EE-treated rats. None of the conventional liver tests were significantly different among the four groups. After 14 days of treatment the serum levels of cholic acid, chenodeoxycholic acid, glycocholic acid, glycochenodeoxycholic acid, taurocholic acid, taurochenodeoxycholic acid, bilirubin, alkaline phosphatase and gamma glutamyltransferase were significantly raised in EE and EE plus UDCA treated rats. EE plus TUDCA treated rats, however, had no significant changes in these individual serum bile acids or conventional liver tests. The ultrastructure of livers from EE plus TUDCA treated rats was similar to those of controls. On the other hand, EE and EE plus UDCA rats both showed a significant reduction in sinusoidal microvilli. These results show that treatment of rats for 4 days with EE induces significant rises in the serum concentrations of two individual bile acids and that TUDCA protects against this.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Daily determination of individual serum bile acids allows early detection of hepatic allograft dysfunction.

Acute graft rejection is still a major cause of morbidity after orthotopic liver transplantation, and its diagnosis necessitates an invasive liver biopsy. Our aim has been to determine whether changes in individual serum bile acid levels after engraftment are sensitive, specific and reliable indicators of graft function and whether these changes can antedate other biochemical indicators of hepatic allograft rejection. Individual bile acids in 200 serum samples taken serially from eight adult liver transplant patients were measured. Patients with biopsy-confirmed graft dysfunction due to rejection or nonrejection causes (n = 6 episodes) had significantly higher serum concentrations of glycocholate plus glycochenodeoxycholate and taurocholate/taurochenodeoxycholate ratios than did noncomplicated grafts (n = 3). These changes antedated any other conventional biochemical parameters by at least 48 hr and were 100% sensitive and specific. None of the conventional liver function tests could match this. Acute rejection episodes (n = 3) were then compared with nonrejection causes of graft dysfunction (n = 3). In acute rejection we noted a significant increase in the concentration of glycodeoxycholate plus deoxycholate and a significant decrease in the cholate/chenodeoxycholate ratio compared with that in nonrejection graft malfunction. Both of these changes antedated any other biochemical parameters by 24 hr. In conclusion, individual serum bile acid assays, after orthotopic liver transplantation, can detect graft dysfunction resulting from any cause at an earlier time than routine biochemical tests, and they are sensitive, specific and reliable for early detection of graft dysfunction. In addition, acute rejection can be distinguished from other causes of graft dysfunction.

Differential effects of cyclosporin A on transport of bile acids by rat hepatocytes: relationship to individual serum bile acid levels.

Cyclosporin A treatment has been reported to induce hepatotoxicity marked by a rise in total serum bile acid and total bilirubin. The mechanism of cyclosporin A-induced hepatotoxicity seems to be related to interference with hepatocellular transport of these substrates although this remains to be fully substantiated. The purpose of this study was to investigate whether the hepatocellular uptake of the different bile acids, in the presence of cyclosporin A, is consistent with the changes in their respective individual serum bile acid concentrations. High-performance liquid chromatography has been used to assay individual serum bile acids in cyclosporin A-treated rats at doses of 0.1, 1, and 10 mg/kg/day for 4 days. Control rats were treated with Cremophor (1 ml/kg/day). At the higher doses, cyclosporin A produced a significant increase in levels of cholic acid, taurocholic acid, chenodeoxycholic acid, and deoxycholic acid compared with controls. Serum glycocholate was unaffected even at the highest dose. Inhibition of initial rate of uptake and accumulation of [14C]cholic acid, [14C]chenodeoxycholic acid, and [14C]deoxycholic acid by isolated rat hepatocytes was consistent with the changes in their respective serum bile acids. Coincubation of rat hepatocytes with unlabeled cholic acid (100 microM), the major serum bile acid in cyclosporin A-treated rats, showed a further inhibitory effect on [14C]cholic acid and [14C]deoxycholic acid accumulation. The initial rate of uptake of [14C]glycocholate was also inhibited. However, accumulation of glycocholic acid did not show significant changes at the longer incubation times (2-30 min). In addition, coincubation of rat hepatocytes with unlabeled cholic acid (100 microM) plus cyclosporin A did not induce any inhibition of glycocholate accumulation. Together, these differences provide an explanation for the unchanged serum levels of glycocholate. In conclusion, the changes in individual serum bile acids in cyclosporin A-treated rats are consistent with the effect of this drug on their hepatocellular accumulation.

Isolation of sinusoidal and canalicular liver plasma membranes: Effects of frozen storage of human material.

Sinudoisal and canalicular plasma membranes were prepared from rat and fresh or frozen human liver and characterized using plasma membrane marker enzymes. The distributions of these enzymes were used as a means of comparison between the plasma membrane fractions and homogenates from the rat and fresh and frozen human livers. Compared with their homogenate, all the sinusoidal plasma membrane preparations were poorly enriched with Na(+)-K(+)-ATPase (2.6-8.4-fold), a sinusoidal marker enzyme. Activities of Na(+)-K(+)-ATPase and Mg(2+)-ATPase were much greater in rat membranes whereas leucine aminopeptidase and alkaline phosphatase activities were much greater in the human membranes. Consideration of the extent of enrichment showed that Mg(2+)-ATPase and alkaline phosphatase are better markers in rat canalicular plasma membranes and leucine aminopeptidase in human membranes from both fresh and frozen tissue. Intracellular organelle marker enzymes were not enriched in the plasma membranes, thus indicating no significant contamination. Importantly, the relative enrichments of the marker enzymes showed no difference between fresh and frozen human liver. Absolute activities of the enzymes were decreased, however (except alkaline phosphatase), after frozen storage. This work indicates that human liver tissue may be frozen and stored in liquid nitrogen before plasma membranes are isolated with no effect on the distribution of marker enzymes in the membranes.

Effects of chlorinated solvents on the natural lymphocytotoxic activities of human liver immune cells.

Inhibition of the natural immune system may be involved in the liver cancer caused by some non-genotoxic chemicals when they are administered at high doses to certain strains of animals. Previous studies have shown that some chlorinated solvents inhibit liver natural immune function in rodents. In this preliminary study, the effects of in vitro exposure to three commonly used chlorinated solvents were determined on three tumoricidal activities expressed by isolated human liver immune cells-natural killer (NK), natural cytotoxic (NC) and natural P815 killer (NPK) (Wright and Stacey, 1991) cell activities. The NK, NC and NPK cell activities of immune cells isolated from three human livers were 115, 45 and 53 lytic units (LU(20%)/10(6) effector cells), respectively. In vitro exposure to trichloroethylene (TRI) inhibited all three natural immune activities, and the ranking of sensitivity was NPK NC NK. Tetrachloroethylene (TET) inhibited NC and NPK cell activities, but had little effect on NK cell activity. 1,1,1-Trichloroethane (TCE) had little or no effect on the three tumoricidal activities examined. Overall, these data show clear similarities to the results obtained in vitro using cells from experimental animals.