Prediction of enthalpy and standard Gibbs energy of vaporization of haloaromatics from atomic properties.

Halogenated benzenes form a class of pollutants with a huge number of members - 1504 distinct benzene compounds, where one or more hydrogen atoms are replaced by halogens, may exist theoretically. This study presents a user friendly method for accurate prediction of vapor pressures and enthalpies of vaporization, at 298.15 K, of any mono or poly halobenzene compound. The derived equations for the prediction of those vaporization properties depend just on the number of each constituent halogen atom. This is a consequence of the absence of intramolecular interactions between the halogen atoms, revealed after examining vaporization results of ca. 40 halogenated benzenes. In order to rationalize the estimation equations, the contribution of the halogen atoms for the referred to above properties of vaporization was decomposed into two atomic properties - the volume and electron affinity. Extension of the applicability of the estimation method to substituted benzenes containing other substituent groups beyond halogen atoms as well as to some polycyclic aromatic species was tested with success.

FXR-dependent and -independent interaction of glucocorticoids with the regulatory pathways involved in the control of bile acid handling by the liver.

Treatment with glucocorticoids (GCs) may cause adverse effects, including cholestasis. The ability of dexamethasone, prednisolone and budesonide to affect the liver handling of bile acids (BAs) has been investigated. In rats treated with GCs for 4 days, altered serum and bile BA levels, changed conjugation pattern, and delayed and decreased ability to conjugate/secrete exogenously administered deoxycholate, were found using HPLC-MS/MS. RT-QPCR analyses revealed that GC treatment also induced a down-regulation of liver nuclear receptors (Fxr, Gr and Shp), transporters (Ntcp, Mrp4 and Bcrp) and enzymes (Cyp7a1 and Baat), whereas Bsep, Mrp2 and Cyp27a1 were up-regulated. Human HepG2 and Alexander cell lines were used as in vitro models of liver cells with and without constitutive FXR expression, respectively. In HepG2 cells, GCs induced a decreased expression of FXR and SHP, and inhibited the regulatory effect of GW4064 on FXR-target genes. In Alexander cells, only when they were transfected with FXR+RXR, GW4064 caused up-regulation of SHP and OSTß, and a down-regulation of CYP27A1. GCs had the opposite effect on these genes, both in the absence and in the presence of FXR expression. Co-transfection of Alexander cells with IR-1-Luc and FXR+RXR revealed that GCs did not inhibit but moderately enhanced FXR activity. Moreover, GCs have a synergistic effect on GW4064-induced FXR activation, whereas chenodeoxycholate and GW4064 have an additive effect. In conclusion, GCs are able to directly or indirectly activate FXR but they also antagonize, through FXR-independent mechanisms, the expression of FXR and FXR target genes involved in the hepatic handling of BAs.

Genetic variants in genes involved in mechanisms of chemoresistance to anticancer drugs.

Refractoriness to the pharmacological treatment of cancer is dependent on the expression levels of genes involved in mechanisms of chemoresistance and on the existence of genetic variants that may affect their function. Thus, changes in genes encoding solute carriers may account for considerable inter-individual variability in drug uptake and the lack of sensitivity to the substrates of these transporters. Moreover, changes in proteins involved in drug export can affect their subcellular localization and transport ability and hence may also modify the bioavailability of antitumor agents. Regarding pro-drug activation or drug inactivation, genetic variants are responsible for changes in the activity of drug-metabolizing enzymes, which affect drug clearance and may determine the lack of response to anticancer chemotherapy. The presence of genetic variants may also decrease the sensitivity to pharmacological agents acting through molecular targets or signaling pathways. Recent investigations suggest that changes in genes involved in DNA repair may affect the response to platinum-based drugs. Since most anticancer agents activate cell death pathways, the evasion of apoptosis plays an important role in chemoresistance. Several genetic variants affecting death-receptor pathways, the mitochondrial pathway, downstream caspases and their natural modulators, and the p53 pathway, whose elements are mutated in more than half of tumors, and survival pathways, have been reported. The present review summarizes the available data regarding the role of genetic variants in the different mechanisms of chemoresistance and discusses their potential impact in clinical practice and in the development of tools to predict and overcome chemoresistance.

Strategies for overcoming chemotherapy resistance in enterohepatic tumours.

When considered together, enterohepatic tumours, i.e., those affecting the liver, the biliary tree and gallbladder and the intestine, constitute the first cause of death due to cancer. Although in many cases surgery and radiotherapy are efficacious, these therapeutic strategies cannot always be implemented. Moreover, even when the removal of tumours is possible, pre- and post-operative pharmacological adjuvant regimens are often needed. However, one important limitation to the use of cytostatic drugs to treat enterohepatic tumours is that they generally exhibit marked refractivity to currently available pharmacological approaches. In addition, most of them increase their chemoresistance during treatment. In view of the high refractivity of these tumours to anti-cancer drugs and the existence of undesirable side effects, both of which are drawbacks in the available chemotherapy, several novel therapeutic approaches have been devised. The purpose of the present review is to offer some insight into the different types of strategies that have already been evaluated and incorporated into clinical practice, such as therapies based on the use of molecular targets, as well as into the approaches that are still under experimental development, such as the chemosensitization of cancer cells, genetic manipulation of tumour or host cells, and cell-specific enhancement of intracellular concentrations of the active agent by efficient targeting of pro-drugs or by using inhibitors of efflux pumps.

Foetal 'flat' bile acids reappear during human liver regeneration after surgery.

BACKGROUND: Changes in bile acid (BA) pool, such as the reappearance of typically foetal-type molecular species with a 'flat' structure at the steroid ring, occur during hepatocarcinogenesis, both in humans and rodents. Moreover flat-BAs also appear during rat liver regeneration. These changes can be detected in urine. The aim of the present study was to investigate whether flat-BAs also reappear during human liver regeneration, and whether this change correlates with the magnitude of liver resection. MATERIALS AND METHODS: Patients undergoing partial hepatectomy were divided in two groups: major hepatectomy group (> 50% of hepatic tissue resection, n = 17) and minor hepatectomy group (< 50%, n = 13). BAs were extracted from serum and urine (collected over 24 h) and analysed by gas chromatography-mass spectrometry. Samples were obtained before surgery (day 0) and on the third and seventh days after hepatectomy. RESULTS: In serum, total BAs significantly increased on day seven after hepatectomy, but only a moderate increase in flat-BA concentrations was observed. By contrast, urinary excretion of total as well as flat-BAs significantly increased at day three and day seven after hepatectomy. Moreover, the amount of flat-BAs excreted in urine during the first week after partial hepatectomy correlated with the magnitude of the resection. CONCLUSIONS: Urinary BA output increases and flat-BAs reappear in urine during human liver regeneration. These results suggest that determination of BAs in urine may be an interesting parameter obtained by non-invasive techniques whose actual clinical value during human liver regeneration warrants further evaluation.

Expression in human trophoblast and choriocarcinoma cell lines, BeWo, Jeg-3 and JAr of genes involved in the hepatobiliary-like excretory function of the placenta.

Using cytokeratin-7-positive trophoblast cells (hTr) isolated from human term placentas and the choriocarcinoma cell lines (hCC) BeWo, Jeg-3 and JAr, the expression of genes involved in the hepatobiliary excretion of cholephilic compounds was investigated by RT-PCR/sequencing followed by measurement of the absolute abundance of mRNA by real-time RT-PCR. Although mRNA of BSEP was detectable and its expression confirmed by Western blotting, its very low expression (higher in hTr than in whole placenta and hCC) did not permit its detection by immunohistochemistry. In hTr, the expression was high for OATP-B/2B1, OATP-8/1B3, MRP1, MRP3, BCRP, FIC1, RARalpha, FXR and SHP, low for OSTalpha, MRP2, MRP4, MRP8, MDR1, CAR and SXR, very low for OATP-A/1A2 and MDR3, and not detectable for OATP-C/1B1, HNF1alpha and HNF4. Expression patterns in hCC mimicked those in hTr, although some important cell line-specific differences were found. The functionality of transporters expressed in hCC was confirmed by their ability to take up and export estradiol 17beta-d-glucuronide in a self-inhibitable and temperature-sensitive manner. In conclusion, several transporters, export pumps, and nuclear receptors involved in the liver excretory function may play a similar role in the placenta, whose specific aspects can be studied by selectively using BeWo, Jeg-3 or JAr cells.

Effect of maternal cholestasis and treatment with ursodeoxycholic acid on the expression of genes involved in the secretion of biliary lipids by the neonatal rat liver.

In juvenile rats born from mothers with obstructive cholestasis during pregnancy (OCP), transient latent cholestasis together with alterations in the secretion of biliary lipids have been reported. Here we investigated whether the expression of genes involved in this function is already modified at birth and examined the effect of treating pregnant rats with ursodeoxycholic acid (UDCA; i.g., 60 microg/100 g b.w./day). Cholanemia was markedly higher in mothers with OCP, and was further increased by UDCA. In the Control pups, cholanemia increased after birth, whereas in OCP and OCP+UDCA pups, hypercholanemia decreased after birth. Steady-state mRNA levels in neonatal liver were measured by real-time quantitative RT-PCR. The expression of basolateral bile acid transporters was not affected by OCP and was unchanged (Oatp1/1a1 and Oatp4/1b2) or moderately increased (Ntcp and Oatp2/1a4) by UDCA. In both groups, the expression of ABC proteins was either not modified (Bsep, Bcrp and Mrp2) or enhanced (Mrp1 and Mrp3), that of phospholipid flippase Mdr2 was not changed, whereas that of cholesterol transporter Abcg5/Abcg8 was impaired. The expression of the nuclear receptor FXR was not affected by OCP or UDCA, whereas that of SHP and key enzymes in bile acid synthesis (Cyp7a1, Cyp8b1 and Cyp27) was increased in both groups. In conclusion, OCP affects the expression in the neonatal liver of genes involved in hepatobiliary function, which cannot be prevented, at this stage, by treating pregnant rats with UDCA, even though this treatment has been found to partially restore normal lipid secretion later during post-natal development.

Chronic renal failure-induced changes in serum and urine bile acid profiles.

Although the kidney is believed to play a minor role in bile acid (BA) excretion, chronic renal failure (CRF) has been reported to be accompanied by alterations in the BA balance. The aim of the present work was to evaluate the changes in BA serum concentrations and renal excretion in patients with different stage of CRF or after kidney transplantation and to elucidate whether these might play a role in the development of pruritus, a common symptom in this disease. This study was carried out on 125 patients. None of them had a history or signs of hepatobiliary malfunction. They belonged either to a control group (N = 31) or to one of the three following CRF groups: patients maintained only on a low-protein diet (diet group, N = 23); the same, together with periodic sessions of hemodialysis (dialysis group, N = 42); and patients who had undergone a kidney transplant more than 1 and less than 2 years before (transplanted group, N = 29). Serum and urine BA concentrations were assayed by gas chromatography-mass spectrometry. Pruritus was quantified by means of a questionnaire answered at the time of sample collection. A marked hypercholanemia together with a reduction in BA output into urine and profound alterations in the profiles of these compounds in both serum and urine in patients with CRF were observed. The levels of total BAs in serum, but not the proportions of molecular species, were corrected by hemodialysis. By contrast, kidney transplant reversed BA serum patterns to normality but, due to immunosuppressive therapy with cyclosporin A, total serum BA concentrations were consistently elevated in this group. Pruritus was more frequent in patients with impaired kidney function and hypercholanemia, although no significant correlation between the degree of this symptom and the magnitude of the serum concentrations of total or individual BAs were found. By contrast, in spite of hypercholanemia, once renal function had been restored by kidney transplantation, none of the patients suffered from pruritus. These results suggest that the kidney plays an important role in determining the serum BA pool size and composition and that hypercholanemia may be a contributing factor, but not the only one, determining the development of pruritus in patients with CRF.

Changes in the pattern of bile acids in the nuclei of rat liver cells during hepatocarcinogenesis.

Bile acids reach the nuclei of hepatocytes, where they may play an important role in controlling gene expression by binding to nuclear receptors. In previous studies, changes in the amounts of the different molecular species of bile acids in the hepatocyte nucleus during rat liver regeneration have been reported. The aim of the present work was to investigate whether this also occurs during rat hepatocarcinogenesis. Liver cell nuclei were isolated after homogenization of livers from healthy adult rats (controls) and from rats at different time points during chemically induced hepatocarcinogenesis, corresponding to the stages of foci (12 weeks), hepatoma (20 weeks) and carcinoma (32 weeks). Bile samples from the cannulated common bile duct were collected for 1h from different sets of animals undergoing hepatocarcinogenesis. Bile acids in bile, liver homogenates and isolated nuclei were measured by GC-MS. Because the yield of nuclei isolated changed during the course of hepatocarcinogenesis (control, 20.1%; 12 weeks, 23.6%; 20 weeks, 7.8%; 32 weeks, 5.1%), amounts of bile acids in nuclei were corrected for the amount of DNA in each preparation. During hepatocarcinogenesis, bile acid concentrations in liver homogenates were reduced to approximately half the values obtained in control livers, while the levels of bile acids in both isolated nuclei and bile were not decreased. Hepatocarcinogenesis induced changes in the composition of bile acid pools. These were manifest as an increase in the proportion of cholic acid and a decrease in that of ursodeoxycholic acid in both bile and liver. These modifications differed from the changes seen in the nuclear bile acid pool, where a decrease in the proportion of cholic acid together with an increase in that of ursodeoxycholic acid were the major changes observed during hepatocarcinogenesis. With regard to the 'flat' bile acids (allo-cholic acid plus Delta(5)- or Delta(4)-unsaturated bile acids), a marked hepatocarcinogenesis-induced increase in the output of these species in bile was found. However, these bile acids were only found in liver homogenates at the hepatoma stage, whereas they were not detected in isolated nuclei at any stage of hepatocarcinogenesis. In summary, these results support the existence of a bile acid pool in hepatocyte nuclei whose composition differs from that of the extranuclear bile acid pool. Moreover, they indicate that, during hepatocarcinogenesis, the composition of the nuclear pool undergoes important alterations.

Predominance of human versus rat phenotype in the metabolic pathways for bile acid synthesis by hybrid WIF-B9 cells.

The rat hepatoma-human fibroblast hybrid cell line WIF-B9 stably exhibits the structural and functional characteristics of normal differentiated hepatocytes. The abilities of these cells to synthesize bile acids and amidate them with glycine and taurine were investigated. The release of bile acids into the culture media over 72 h was assessed by gas chromatography-mass spectrometry. WIF-B9 cells were able to synthesize bile acids (1.10+/-0.17 nmol/mg protein) but less efficiently than rat hepatocytes in primary culture (2.19+/-0.19 nmol/mg protein; P<0.01). The patterns of major bile acid species produced by both types of cells were also different. Cholic acid (CA; 72%) and beta-muricholic acid (19%) were the major bile acids produced by rat hepatocytes, while chenodeoxycholic acid (CDCA) accounted for only 4.5% of total bile acids. In contrast, muricholic acids were absent, while CA (62%) and CDCA (34%) were the most abundant bile acids synthesized by WIF-B9 cells. Using reverse transcription-polymerase chain reaction and gene- and species-specific primers for key enzymes involved in bile acid synthesis, the expression of human, but not rat, orthologues of CYP7A1, CYP27, CYP8B and CYP7B1 was found in WIF-B9 cells. Induction of cell stress by serum deprivation did not change the amount of total bile acids synthesized by these cells, but an inversion of the CA-to-CDCA ratio from 1.8 to 0.3 together with a marked increase in the proportion of intermediate metabolites related to the acidic pathway was found. Using 500 microM radiolabeled CA and 2 mM of taurine or glycine, the ability to amidate CA over 48 h was determined by high performance liquid chromatography. Rat hepatocytes conjugated more than 90% CA with either amino acid, whereas this ability was very poor (< 2%) in WIF-B9 cells. Regarding the expression of enzymes and the products of bile acid synthesis, it may be concluded that the human phenotype predominates over that of the rat in WIF-B9 cells. Moreover, these cells are almost completely unable to further conjugate primary bile acids, which facilitates the manipulation of these steroids in analytical procedures. These characteristics make WIF-B9 cells a suitable in vitro model to carry out studies on bile acid synthesis by 'human-like' metabolic pathways.

Liver organotropism and biotransformation of a novel platinum-ursodeoxycholate derivative, Bamet-UD2, with enhanced antitumour activity.

BACKGROUND/AIMS: Several members of a novel family of bile acid derivatives with cytostatic and virostatic activity have been synthesized and characterized. The aim of this work was to investigate the liver organotropism and biotransformation of two novel compounds with enhanced DNA-reactivity: Bamet-D3, in which a glycine-polyamine tandem was used as a spacer to separate the glycocholic acid moiety from the platinum(II) atom, and Bamet-UD2, in which cisplatin was directly bound to the carboxylate group of two ursodeoxycholic acid moieties. METHODS: Drug uptake and "in vitro" toxicity were investigated using rat hepatocytes in primary culture. Following i.v. administration of 0.5 mumol cisplatin, Bamet-D3 or Bamet-UD2, bile output, urinary and fecal excretion, organ distribution and pharmacokinetic parameters were determined in short-term (3 h) and long-term (14 days) experiments carried out on anaesthetized and conscious rats, respectively. Liver biotransformation was investigated by HPLC analysis of bile samples. Total platinum was measured by flameless atomic absorption spectroscopy. Using Nude mice, antitumour activity was investigated in subcutaneously implanted Hepa 1-6 mouse hepatoma cells. RESULTS: Uptake by rat hepatocytes was Bamet-UD2 (11.3 nmol/mg protein) > Bamet-D3 (5.6 nmol/mg protein) > cisplatin (2.1 pmol/mg protein). Bamet-UD2 induced "in vitro" cell toxicity, which was not observed for Bamet-D3 or cisplatin. On the contrary, no toxicity "in vivo" for Bamet-UD2 was found which was observed for cisplatin and Bamet-D3. This may be related with the fact that bile output of Bamet-UD2, which occurs with no major biotransformation, was > 10 fold higher than that of cisplatin and 3-fold higher than that of Bamet-D3, which was previously transformed into at least three different metabolites. Fecal excretion was Bamet-UD2 > Bamet-D3 > cisplatin, whereas urinary output was Bamet-D3 > cisplatin > Bamet-UD2. Accordingly, a marked liver- and a reduced kidney-vectoriality for Bamet-UD2, but not for Bamet-D3, was observed. Bamet-UD2 and cisplatin, but not Bamet-D3, were efficient in inhibiting tumour growth whereas, only Bamet-UD2 significantly prolonged survival time. CONCLUSIONS: There results indicate that Bamet-UD2 is a cisplatin-ursodeoxycholate derivative with strong antitumour activity, marked hepatobiliary organotropism, and reduced toxic side-effects as compared to the parent drug cisplatin.

Low in vivo toxicity of a novel cisplatin-ursodeoxycholic derivative (Bamet-UD2) with enhanced cytostatic activity versus liver tumors.

Cisplatin-bile acid derivatives belonging to the Bamet-family maintain both liver organotropism and cytostatic activity. "In vivo" toxicity and usefulness as chemotherapeutic agent versus liver tumors of a novel drug, Bamet-UD2 [cis-diamminechlorocholylglycinate platinum (II)], with enhanced "in vitro" cytostatic activity was investigated. Using orthotopically implanted mouse Hepa 1-6 hepatoma in the liver of Nude mice, the antitumor effect of Bamet-UD2 was compared with that of a previously characterized compound of this family, Bamet-R2 [cis-diamminebis-ursodeoxycholate platinum(II)], and cisplatin. Life span was significantly prolonged in mice treated with both Bamets (Bamet-UD2 > Bamet-R2), compared with animals receiving saline or cisplatin. All these drugs inhibit tumor growth (Bamet-UD2 = cisplatin > Bamet-R2). However, toxicity-related deaths only occurred under cisplatin treatment. Using rats maintained in metabolic cages, organ-specific toxicity and drug accumulation in tissues were investigated. The amount of both Bamets in the liver was severalfold higher than that of cisplatin. By contrast, a significantly higher amount of cisplatin in kidney and nerve was found. In lung, heart, muscle, brain, and bone marrow the amount of drug was small and also significantly lower in animals receiving Bamets. Signs of neurotoxicity (altered nerve conduction velocity), nephrotoxicity (increased serum urea and creatinine concentrations and decreased creatinine clearance), and bone marrow toxicity (decreased platelet and white blood counts) in animals treated with cisplatin but not with the Bamets were found. These results indicate that, owing to strong antitumor activity together with absence of side effects, Bamet-UD2 may be useful in the treatment of liver tumors.

Increased levels of typically fetal bile acid species in patients with hepatocellular carcinoma.

The aim of this work was to investigate the reappearance during liver neoplasia of bile acids (BAs) species, which are unusual in healthy adults, but common in fetuses. Serum and urine samples were collected from patients with hepatocellular carcinoma (HCC; n=27), and for comparative purposes, with liver cirrhosis (n=49), liver metastasis (n=19), chronic viral hepatitis (n=11) and healthy volunteer (control group; n=26) groups. BAs were identified and measured by GC--MS. Hypercholanaemia was found in all groups of patients. In HCC, this was characterized by a marked increase in the chenodeoxycholate/cholate ratio in both serum and urine. Although increased levels of BAs, with hydroxylations at unusual positions, and oxo-BAs were found in HCC, these were not significantly different from those observed in other groups. However, BAs with a flat structure, i.e. Delta(4)-unsaturated- and 5 alpha- or allo-BAs, which were almost absent in healthy subjects, were markedly increased in the serum and urine of HCC patients. They were also detected, although in much lower amounts, in liver metastasis and liver cirrhosis, but not in viral hepatitis. Flat-BAs were better detected in urine than in serum. Urinary Delta(4)-unsaturated-BA output was significantly lower in patients with small tumours (<3 cm) compared with those with higher size tumours. No correlation between flat-BA output into urine and serum alpha-fetoprotein or total BAs was found. These results suggest that Delta(4)- and/or allo-BAs are particularly elevated in patients with HCC, which may be a potentially useful complementary, rather than alternative, marker for early detection of liver neoplasia.

Bile acid secretion during rat liver carcinogenesis.

Retro-differentiation of liver parenchyma during neoplastic processes is characterized by the expression of tumor antigens, such as alpha-fetoprotein and the placental isoenzyme of glutathione-S-transferase (GST-P). To investigate whether this may also affect a typical liver function such as bile acid secretion was the aim of this work. Rat hepatocarcinogenesis was induced by diethylnitrosamine (i.p., 200 mg/Kg body weight at day 0) and promoted by two-thirds partial hepatectomy (at day 21) plus 2-acetamidofluorene administration (50 mg/Kg body weight, subcutaneously, twice a week from day 14 to day 35). In order to carry out planimetric measurements of neoplastic tissue after immunohistochemical staining, a novel monoclonal antibody (MAb 14.1.3) against GST-P with no cross-reactivity against the major liver isoform of GST (GST-H) was raised. Analysis of total biliary bile acid output using the 3alpha-hydroxysteroid dehydrogenase method indicated that a significant reduction (-26%) occurred during the formation of GST-P-positive foci (12 wk). This was restored to normal values during adenoma formation (16-20 wk), but decreased again during carcinoma transformation (32 wk). These changes were not parallel to that observed in bile flow, which was progressively but slightly decreased throughout the whole period under study. HPLC analysis of bile samples collected for 1 h at different time points during hepatocarcinogenesis revealed that in contrast to what happens during cholestatic disease, a continuous and progressive increase in the cholic acid-to-chenodeoxycholic acid ratio (from 4.4+/-0.5 in control animals to 15.1+/-1.9 in rats with hepatocellular carcinoma) occurs. A significant and transient increase at 16 wk (+120%) in the proportion of bile acids amidated with glycine as compared to those conjugated with taurine was also observed. These results indicate that the mechanisms accounting for the secretion of major bile acids are modified differently at various steps of rat liver tumor development.

Comparison of the effects of bile acids on cell viability and DNA synthesis by rat hepatocytes in primary culture.

Bile acid-induced inhibition of DNA synthesis by the regenerating rat liver in the absence of other manifestation of impairment in liver cell viability has been reported. Because in experiments carried out on in vivo models bile acids are rapidly taken up and secreted into bile, it is difficult to establish steady concentrations to which the hepatocytes are exposed. Thus, in this work, a dose-response study was carried out to investigate the in vitro cytotoxic effect of major unconjugated and tauro- (T) or glyco- (G) conjugated bile acids and to compare this as regards their ability to inhibit DNA synthesis. Viability of hepatocytes in primary culture was measured by Neutral red uptake and formazan formation after 6 h exposure of cells to bile acids. The rate of DNA synthesis was determined by radiolabeled thymidine incorporation into DNA. Incubation of hepatocytes with different bile acid species - cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), in the range of 10-1000 microM - revealed that toxicity was stronger for the unconjugated forms of CDCA and DCA than for CA and UDCA. Conjugation markedly reduced the effects of bile acids on cell viability. By contrast, the ability to inhibit radiolabeled thymidine incorporation into DNA was only slightly lower for taurodeoxycholic acid (TDCA) and glycodeoxycholic acid (GDCA) than for DCA. When the effect of these bile acids on DNA synthesis and cell viability was compared, a clear dissociation was observed. Radiolabeled thymidine incorporation into DNA was significantly decreased (-50%) at TDCA concentrations at which cell viability was not affected. Lack of a cause-effect relationship between both processes was further supported by the fact that well-known hepatoprotective compounds, such as tauroursodeoxycholic acid (TUDCA) and S-adenosylmethionine (SAMe) failed to prevent the effect of bile acids on DNA synthesis. In summary, our results indicate that bile acid-induced reduction of DNA synthesis does not require previous decreases in hepatocyte viability. This suggests the existence of a high sensitivity to bile acids of cellular mechanisms that may affect the rate of DNA repair and/or proliferation, which is of particular interest regarding the role of bile acids in the etiology of certain types of cancer.

Further evidence of the usefulness of bile acids as molecules for shuttling cytostatic drugs toward liver tumors.

BACKGROUND/AIMS: To use bile acids as shuttles for directing cytostatic drugs toward liver tumors, the ability of the tumor to take up these compounds must be maintained. Thus, we investigated whether glycocholate (GC) derivatives such as the fluorescent FITC-GC and the cytostatic Bamet-R2 are taken up by neoplastic tissue at different stages of chemically-induced rat liver carcinogenesis. METHODS: Placental glutathione-S-transferase (GST-P) was immunohistochemically detected. Uptake studies were carried out on pure GST-P-positive cell cultures, obtained by treatment with ethacrinic acid. FITC-GC, Bamet-R2 or cisplatin was administered (i.v.) to anaesthetized rats. Platinum in culture cells, liver and kidney was measured by flameless atomic absorption. RESULTS: Co-localization after FITC-GC i.v. administration revealed that only 15% (20 weeks) and 30% (32 weeks) of GST-P-positive tissue was not able to take up FITC-GC. GC uptake was lower in GST-P-positive cells than in normal hepatocytes. Bamet-R2, uptake was lower than that for GC, but similar in both cell types. The amount of Bamet-R2 or cisplatin retained by GST-P-positive tissue after in vivo administration was progressively increased during carcinogenesis. Moreover, this amount was higher for Bamet-R2 than for cisplatin. By contrast, in the kidney, it was higher for cisplatin than for Bamet-R2. CONCLUSION: These results indicate that at the different stages of rat hepatocarcinogenesis most GST-P-positive tissue is able to take up bile acid derivatives, such as Bamet-R2.

Cholephilic characteristics of a new cytostatic complex of cisplatin with glycocholate (Bamet-R2).

The aim of this work was to investigate both the existence of enterohepatic circulation of cisplatin-cholylglycinate complex, Bamet-R2, and the relevance of biliary versus urinary excretion of this compound. Two experimental models were used: (i) intraluminal perfusion of 'in situ' ileum in anaesthetized rats bearing a biliary catheter that permitted bile sample collection and (ii) conscious rats in which a permanent intraarterial catheter had been implanted to carry out sequential blood sampling after intravenous (i.v.) or intragastric (i.g.) drug administration. Total platinum in serum, bile, ileum, liver, urine and feces was measured by flameless atomic absorption spectroscopy. Serum concentration versus time curves obtained after i.v. administration of 1 micromol Bamet-R2 or cisplatin revealed that the area under the curve was significantly higher for Bamet-R2 than for cisplatin (+48%). Non-ultrafiltrable platinum accounted for 54.8 and 48.4% of serum platinum 168 h after cisplatin and Bamet-R2 i.v. administration, respectively. When the animals received i.g. 1 micromol cisplatin or Bamet-R2, serum concentrations of total platinum were markedly higher (three-fold) after Bamet-R2 than after cisplatin administration. The area under the curve was, also in this case, significantly higher for Bamet-R2 than for cisplatin (+28%). This was in part due to the enhanced intestinal absorption of Bamet-R2, as confirmed in experiments on perfused rat ileum, where a markedly higher amount of the drug was found in ileum tissue and bile after perfusion with media containing Bamet-R2 as compared with experiments where cisplatin instead of Bamet-R2 was added to perfusion media. Moreover, after i.v. administration to conscious rats, excretion of Bamet-R2 by the kidney was three-fold lower than that of cisplatin, while elimination of the former compound into feces was four-fold higher than that of the latter. In summary, these results indicate that in addition to the previously reported cytostatic activity of Bamet-R2, this complex has interesting cholephilic characteristics typical of bile acids, such as low urinary excretion together with enhanced intestinal absorption and biliary secretion, probably endowed by the cholylglycyl moiety included in the Bamet-R2 molecule.

DNA interaction and cytostatic activity of the new liver organotropic complex of cisplatin with glycocholic acid: Bamet-R2.

The aim of this study was to investigate the ability of the new liver organotropic complex of cisplatin with glycocholate (GC), Bamet-R2, to interact with DNA, inhibit its replication and hence reduce tumor-cell proliferation. Changes in the electrophoretic mobility of the open and covalently closed circular forms of the pUC18 plasmid DNA from Escherichia coli, a shift in the denaturation temperature of double-stranded DNA, and ethidium-bromide displacement from DNA binding, were induced by Bamet-R2 and cisplatin, but not by GC. Neutral-red retention was used to measure the number of living cells in culture after long-term (72-hr) exposure to these compounds and to evaluate the effect on cell viability after short-term (6-hr) exposure. Bamet-R2 and cisplatin, but not GC, induced significant inhibition of cell growth. This effect ranged from mild to strong, depending upon the sensitivity of the different cell types as follows: cisplatin, rat hepatocytes in primary culture < rat hepatoma McA-RH7777 cells (rH) < human colon carcinoma LS 174T cells (hCC) < mouse hepatoma Hepa 1-6 cells (mH); Bamet-R2, rat hepatocytes < mH approximately equal to hCC < rH. DNA synthesis was measured by radiolabeled-thymidine incorporation into DNA. Bamet-R2 and cisplatin, but not GC, significantly inhibited the rate of DNA synthesis by these cells. After short-term exposure to Bamet-R2 or GC, no acute cell toxicity was observed, except on hCC cells. By contrast, acute toxicity was induced by cisplatin for all cell types studied. The in vivo anti-tumoral effect was investigated in 3 different strains of mice following s.c. implantation of tumor cells (mouse sarcoma S-18011 cells in Swiss and B6 mice and hCC cells in nude mice). In all 3 models, tumor growth was inhibited by Bamet-R2 and cisplatin to a similar degree. However, signs of toxicity (increases in blood urea concentrations and decreases in packed blood cell volume and in liver, kidney and body weight) and a reduction in survival rate were observed only during cisplatin administration. In sum, these results indicate that this bile-acid derivative can be considered as a cytostatic drug whose potential usefulness deserves further investigation.

Transport and biotransformation of the new cytostatic complex cis-diammineplatinum(II)-chlorocholylglycinate (Bamet-R2) by the rat liver.

Rat liver uptake and bile output of the cytostatic complex cis-diammineplatinum(II)-chlorocholylglycinate (Bamet-R2) were studied. Up to 100 microM, Bamet-R2 uptake by rat hepatocytes in primary culture followed saturation kinetics (Vmax = 0.65 +/- 0.12 nmol/5 min per mg protein; K(M) = 45.2 +/- 10.7 microM). Bamet-R2 uptake was lower than that of cholylglycinate (CG) but higher than that of cisplatin. Replacement of 116 mM NaCl by 116 mM choline chloride did not significantly reduce Bamet-R2 uptake. Addition of 500 microM CG, cholic acid, estrone sulfate, or ouabain to 50 microM Bamet-R2-containing incubation media inhibited Bamet-R2 uptake. No liver biotransformation of Bamet-R2 occurred, as indicated by HPLC analysis of bile collected from anesthetized rats after intravenous administration of the drug. Bamet-R2 uptake and secretion into bile by isolated rat livers exceeded those of cisplatin but were lower than those of CG. Differences between Bamet-R2 and CG were more marked for bile output than for liver uptake. Thus, higher Bamet-R2 than CG or cisplatin liver content was found. Co-administration of Bamet-R2 and CG revealed that CG induced a slight reduction in Bamet-R2 uptake and a marked inhibition in Bamet-R2 bile output. By contrast, Bamet-R2 had no effect on CG on either liver uptake or bile output. In sum, the present data indicate that Bamet-R2 is efficiently taken up and secreted into bile by the rat liver by mechanisms shared in part by natural bile acids.

In vitro test to determine the effect of cytostatic drugs on co-cultured rat hepatocytes and hepatoma cells.

Using uptake of the fluorescent bile acid derivative cholylglycylamido-fluorescein (FITC-GC) as a measurement of liver cell population size and function, the antiproliferative and toxic effects of the well known cytostatic drug, cisplatin was evaluated on rapidly growing rat hepatoma McA-RH7777 cells and rat hepatocytes in primary culture under non-proliferating conditions. Co-culture set up to mimic the in vivo situation of tumour and extratumoural liver tissue exposed to cytostatic chemotherapy does not markedly affect the survival or the growth dynamics of both cell types. FITC-GC uptake as corrected for DNA and protein contents in the dish was significantly lower in hepatoma cells than in rat hepatocytes throughout the experimental period (96 h). Effect of 0.1-100 microM cisplatin exposure from 24 to 96 h of culture on cell population size, as measured by protein and DNA contents in the culture dishes, were consistent with changes observed in total FITC-GC uptake. Cisplatin concentrations lower than 50 microM did not affect FITC-GC uptake by rat hepatocytes. By contrast, a progressively increasing effect on hepatoma cells as from 2 microM cisplatin was observed. Two phases in the decay of FITC-GC uptake versus cisplatin concentrations were found in co-cultures exposed to this drug. The first segment, between 2 microM and 50 microM, was characterized by a slow decay that matched the response of hepatoma cells to cisplatin exposure. This was considered to be due to the antiproliferative effect of cisplatin. The second segment, with a steeper decay, matched the effect of cisplatin on hepatocytes. This was interpreted as being due to non-specific toxicity. These results suggest that FITC-GC uptake by co-culture of hepatocytes and tumour cells provides a useful experimental model to explore the mechanism of action and the size of beneficial effect window for new drugs in vitro.