An In Vitro Assay to Assess Transporter-Based Cholestatic Hepatotoxicity Using Sandwich-Cultured Rat Hepatocytes (original) (raw)
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Toxicological …, 2003
and Mg 2؉ from the incubation buffer destroyed canalicular junctions, resulting in bile acid efflux into the incubation buffer. Canalicular transport was calculated based on the difference between the amount of bile acid effluxed into the Ca/Mg 2؉ -free and regular buffers with linear efflux up to 10 min. Hepatocytes cultured in the nonsandwich configuration also transported taurocholic acid, but at 50% the rate in sandwiched cultures. Cyclosporin A, bosentan, CI-1034, glyburide, erythromycin estolate, and troleandomycin inhibited efflux in a concentration-dependent manner. In contrast, new generation macrolide antibiotics with lower incidence of clinical hepatotoxicity were much less potent inhibitors of efflux. An in vivo study was conducted whereby glyburide or CI-1034, administered iv to male rats, produced a 2.4-fold increase in rat total serum bile acids. A synergistic 6.8-fold increase in serum total bile acids was found when both drugs were delivered together. These results provide methods to evaluate inhibitory effects of potentially cholestatic compounds on bile-acid transport, and to rank compounds according to their hepatotoxic potential.
Transport of drugs in isolated hepatocytes the influence of bile salts
Biochemical Pharmacology, 1978
The influence of bile salts on hepatic transport of drugs was studied using isolated hepatocyte suspensions. Upra~e of the organic anions, dibromosulphthalein (DBSP), indcrcyanine green (ICG) and an organic cation, N4-acetyi procainamide ethobromide (APAEB) was measured. Afte; 60 min incubation the amount of DBSP, ICG and APAEB nresent in the cells was 17. 41 and 4.5 ner cent of the added amount respectively. The release of DBSP, ICG and APAEB from the hepatocytes preincubated with the agents under study, after 60 min incubation in fresh medium was 80.5, 12.5 and 48.9 per cent of the amount initially present respectively. The presence of bile canalicular membranes in the isolated hepatocytes was demonstrated by enzymehistochemistry: 5'nucleotidase activity showed sharp branched bands over the cell surface. When bile salts were present in the incubation medium. the cellular content of DBSP, ICG and APAEB was diminished. The taurocholate concentration which caused 50 per cent of the maximal effect was 0.07mM. O.lOmM and 0.06mM in experiments with DBSP, ICG and APAEB respectively. Pharmacokinetic analysis revealed that the influence of bile salts on cellular content of the three compounds was due to inhibition of the uptake into the isolated hepatocytes, rather than stimulation of release from the cells. The hypothesis, that stimulation of biliary output of organic anions in viw is due to a modifying effect of bile salts on the canalicular membranes. instead of being the result of the increased bile flow, is not supported by this study.
Journal of Gastroenterology and Hepatology, 1996
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 54kDa 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 'X+-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.
Pharmacology of bile acids and their derivatives: Absorption promoters and therapeutic agents
European Journal of Drug Metabolism and Pharmacokinetics, 2006
The role of bile acids in pharmacotherapy is reviewed in this article. The therapeutic use of bile has been recognized since ancient times. Previously bile acids were the standard treatment for gallstones where chenodeoxycholic acid and ursodeoxycholic acid were effective in promoting the dissolution of cholesterol gallstones. Today their therapeutic role looks set to expand enormously. Bile acids as absorption promoters have the potential to aid intestinal, buccal, transdermal, ocular, nasal, rectal and pulmonary absorption of various drugs at concentrations that are non-toxic. Keto derivatives of cholic acid, such as 3a,7a,dihydroxy-12-keto-5a-cholic acid (sodium salt and methyl ester) are potential modifiers of blood-brain barrier transport and have been shown to promote quinine uptake, enhance the analgesic effect of morphine and prolong the sleeping time induced by pentobarbital. They have also been shown to be hypoglycaemic. Bile acids as therapeutic agents have the potential to produce beneficial effects in sexually transmitted diseases, primary biliary cirrhosis, primary sclerosing cholangitis, gallstones, digestive tract diseases, cystic fibrosis, cancer and diabetes.
Bile Acids in Physiology, Pathology and Pharmacology
Current Drug Metabolism, 2015
Bile acids, synthesized by hepatocytes from cholesterol, are specific and quantitatively important organic components of bile, where they are the main driving force of the osmotic process that generates bile flow toward the canaliculus. The bile acid pool comprises a variety of species of amphipathic acidic steroids. They are not mere detergent molecules that play a key role in fat digestion and the intestinal absorption of hydrophobic compounds present in the intestinal lumen after meals, including liposoluble vitamins. They are now known to be involved in the regulation of multiple functions in liver cells, mainly hepatocytes and cholangiocytes, and also in extrahepatic tissues. The identification of nuclear receptors, such as farnesoid X receptor (FXR or NR1H4), and plasma membrane receptors, such as the G protein-coupled bile acid receptor (TGR5, GPBAR1 or MBAR), which are able to trigger specific and complex responses upon activation (with dissimilar sensitivities) by different bile acid molecular species and synthetic agonists, has opened a new and promising field of research whose implications extend to physiology, pathology and pharmacology. In addition, pharmacological development has taken advantage of advances in the understanding of the chemistry and biology of bile acids and the biological systems that interact with them, which in addition to the receptors include several families of transporters and export pumps, to generate novel bile acid derivatives aimed at treating different liver diseases, such as cholestasis, biliary diseases, metabolic disorders and cancer. This review is an update of the role of bile acids in health and disease.
INFLUENCE OF BILE ACIDS ON THE BILIARY TRANSPORT MAXIMUM OF PHENOLSULPHONPHTHALEIN IN THE RAT
Clinical and Experimental Pharmacology and Physiology, 1988
1. The effect of changes in bile acid secretion induced by cholestyramine treatment or taurocholate infusion on the biliary transport maximum (Tm) of phenolsulfonphthalein (PSP) was studied in Wistar rats. 2. Five hours after oral administration of cholestyramine (1.5 g/kg bodyweight) the biliary output of bile acids decreased to 51% and bile flow to 76% of control values. The percentage of conjugated and unconjugated PSP excreted into bile and the Tm of the dye were not significantly modified by cholestyramine pretreatment. 3. Administration of sodium taurocholate at increasing rates (60-480 nmol/100 g bodyweight per min) enhanced bile flow and the biliary output of bile acids in a linear dose-related fashion. The Tm of PSP increased progressively until a maximum of 29% above the control values was reached at a taurocholate dose of 240 nmol/100 g bodyweight per min). The enhancement corresponded mainly to the unconjugated dye, the excretion of conjugated PSP not being significantly modified by the infusion of the bile acid. 4. The results indicate that bile acids can influence to some extent biliary excretion of PSP in the rat, although this component is of minor importance at low bile acid secretory rates.
Antibiotic-induced Elevations of Plasma Bile Acids in Rats Independent of Bsep Inhibition
Toxicological Sciences, 2017
Drug-induced liver injury (DILI) is a common toxicity observed in drug development and can lead to withdrawal of approved drugs from the market. To better understand the numerous mechanisms of DILI, recent efforts have focused on transporter inhibition, specifically liver canalicular bile salt export pump (Bsep) as one mechanism of DILI, and on the potential use of plasma bile acids as monitorable mechanism-based biomarkers of Bsep inhibition. To explore alternative mechanisms of bile acid increases in plasma, 6 antibiotic and 2 nonantibiotic drugs unlikely to be Bsep inhibitors were evaluated in rat studies. Surprisingly, all 6 antibiotics demonstrated 2-to 14-fold increases of plasma taurocholic acid (TCA). Also, unconjugated primary bile acids and secondary bile acids (both taurine-conjugated and unconjugated) were decreased in rat plasma after antibiotic treatments, but not with the nonantibiotic drugs. These results suggest alternative mechanisms of bile acids regulation such as attenuation of bacterial deconjugation of bile acids following reduction of gut microflora by antibiotics. Measurements of TCA transport in rat hepatocytes and Bsep-containing membrane vesicles suggest that inhibition of uptake into hepatocytes could also contribute to increases in plasma bile acid concentrations, while excluding inhibition of Bsep as a mechanism. These studies further demonstrate that there are several mechanisms that can lead to conjugated bile acid increases in plasma. By carefully considering the time course and magnitude of changes of individual bile acids relative to any changes seen in transaminases and bilirubin, interpretations and conclusions of the involvement of Bsep inhibition are enabled.
Bile acid-induced liver toxicity: Relation to the hydrophobic-hydrophilic balance of bile acids
Medical Hypotheses, 1986
Hypertransaminasemia is a frequent side effect during chenodeoxycholic administration for gallstone dissolution. Evidence suggests that this effect is not mediated by lithocholic acid, the intestinal metabolite of chenodeoxycholic acid, but that toxicity is due to the chenodeoxychol~c acid itself. In vitro cytotoxicity of bile salts is positively proportional to their detergent effect, which is, on the other hand, related to their hydrophobic-hydrophilic balance. We hypothesize that in vivo also liver injury can occur when the liver is perfused by an high proportion of strongly detergent bile salts. The more detergent bile salts are unconjugated or glycine conjugated, while the lesser are taurine conjugated and sulfated. Within each class the following order of decreasing detergent power can be indicated: lithocholic) deoxycholic) chenodeoxycholic > cholic > ursodeoxycholic acid. Besides chronic exogenous administration of chenodeoxycholic or deoxycholic acids, conditions in which the liver is perfused by an high mass of highly detergent bile salts are those characterized by an enhanced intestinal biodegradation of bile salts. These conditions, which are common features of some chronic inflammatory bowel diseases, are frequently associated with liver damage. On the other hand, a normally detergent bile salt pool can become hepatotoxic for liver cells which have already been injured. In this respect, as already reported for increased sulfation, the increased proportion of taurine conjugates and the reduced formation of deoxycholic acid in liver cirrhosis can be regarded as protective mechanisms. Liver toxicity induced by bile salts' detergency can be prevented by favouring tauroconjugation or reducing the intestinal degradation of bile salts or by administering poorly detergent bile salts.
Comparative Biochemistry and Physiology Part A: Physiology, 1996
Dose-response curves for taurocholate and tauroursodeoxycholate were performed in rat and rabbit livers to get more insight into species differences in the hepatic bile acid uptake. The bile acids showed saturation kinetics in both animals, the V,,, in rat being higher than in rabbit and the K, being lower in the rat than in the rabbit for both the bile acids, with no significant difference in the hepatic cells morphometric parameters. Therefore, it is possible that differences in the kinetic parameters are related to the number and, to a lesser extent, to the affinity of the transporters on the sinusoidal plasma membranes. COMP BIOCHEM PHYSIC),. 113A:2:157-164. 1996.
Hepatic uptake and biliary secretion of bile acids in the perfused rat liver
Pharmacological Research, 1992
Hepatic uptake and biliary secretion have been evaluated in the isolated perfused rat liver for cholic, chenodeoxycholic, ursodeoxycholic acid, both free and taurine-conjugated; the physicochemical properties of the bile acids have also been calculated and related to these experimental parameters. Cholic acid disappearance rate from the perfusate was the fastest, followed by that of ursodeoxycholic and chenodeoxycholic; it was also faster for taurine-conjugated bile acids than for their respective unconjugated forms. The recovery in bile was higher for conjugated than for unconjugated bile acids, and, among each class, was higher for cholic than for chenodeoxycholic and ursodeoxycholic. The hepatic uptake correlated negatively (r=-0.99) with the bile acid lipophilicity, while the biliary secretion correlated with the solubility of the molecules. These results show the effect of the physicochemical properties of BA on their hepatic handling, at the physiological concentration of BA in the portal blood.