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The present study was performed in order to obtain the thiopurine methyltransferase (TPMT) activity frequency distribution histogram in a Spanish population. A total of 3640 Spanish clinical laboratory samples were evaluated, which included 1249 patients with Crohn's disease, 589 with ulcerative colitis, 348 with multiple sclerosis (MS), 487 with several autoimmune diseases different from the above-mentioned diseases and 967 a donor group. We have measured the TPMT activity in red blood cells (RBCs) by a radiochemical method, using S-adenosyl-L-[methyl-3H]methionine as methyl donor. The different groups present in their entirety a normal distribution histogram and a wide range of TPMT activity from 0 to 41 U/ml RBCs. The differences found between the Spanish population TPMT activity frequency distribution histogram and the pattern previously described in a North American population were not due to azathioprine treatment or gender. The effect of autoimmune diseases on TPMT activity was evaluated: the enzymatic activity was similar in the donor group (19.9 +/- 6.3 U/ml RBCs) and in the patients with Crohn's disease (20.0 +/- 5.8 U/ml RBCs) and ulcerative colitis (19.7 +/- 6.1 U/ml RBCs); however, it decreased significantly (p<0.0001) in MS patients (17.1 +/- 6.1 U/ml RBCs) with respect to the donor group. In conclusion, our results show that the Spanish population TPMT distribution is closer to that of the Jewish population of Israel than to North American populations, and that in MS the enzymatic activity of TPMT decreases significantly. This observation may take into account the usage of azathioprine as therapeutic agent in Spanish MS patients.

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The current article describes a new assay to measure thiopurine methyltransferase (TPMT) activity from red blood cells. This method is based on the measurement of the reaction product 6-methylmercaptopurine (6-MMP) by high-performance liquid chromatography (HPLC). 6-MMP is extracted by ethyl acetate with recoveries of 85%, 80%, 80%, and 92% for 50, 250, 500, and 1,000 ng/100 microL packed red blood cells, respectively. 6-MMP was identified and measured by a Zorbax CN column installed in an HPLC system. The chromatograms were resolved using a mobile phase consisting of 40 mmol/L sodium phosphate buffer (pH 3) and methanol in a gradient from 1% to 20% of methanol. Under these conditions 6-MMP is well resolved from substrates (6-mercaptopurine and S-adenosyl-L-methionine) and endogenous peaks. When the TPMT activity from 20 patients was measured by the HPLC-linked assay and the classic radiochemical method, a linear correlation was obtained between both procedures ( y = 0.99x + 0.33; x-axis, radiochemical assay; y-axis, HPLC-linked assay; r = 0.98). In conclusion, the current report describes a new, reliable, safe, and nonradioactive method to measure TPMT activity that is shorter and simpler than the previously described ones.

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The effect of the antiandrogen flutamide on the prostatic vasoactive intestinal peptide (VIP) receptor/effector system was studied in rats. Rats were s.c. injected with a daily dose of flutamide (15 mg/kg B.W.) or vehicle for 14 days. Drug treatment resulted in histological evidence of gland involution and increased plasma membrane fluidity as estimated by fluorescence spectroscopy. The number of VIP receptors and the stimulatory effect of VIP on adenylate cyclase activity in prostatic membranes decreased in flutamide-treated rats. However, the pattern of forskolin stimulation of the enzyme activity was not modified by this drug. Androgen-receptor blockade by flutamide also decreased the prostatic levels of alpha(s,) alpha(i1/2), and alpha(i3/0) G-protein subunits, as estimated by an immunological procedure. Whereas apoptotic DNA fragmentation was evidenced in prostate from 3-day castrated animals, a heterogeneous electrophoretic pattern was observed after flutamide treatment. Thus, androgen-receptor blockade by flutamide results in an important impairment of the components of the VIP receptor/effector system in rat prostate as well as in a modification of their coupling extent, which is presumably due to differences observed in plasma membrane fluidity. These results represent a crosstalk in the prostate between two mechanisms of signal transduction involved in cell proliferation.

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Cyclosporin A (CsA) at concentrations up to 1 microM induced apoptosis in a dose-dependent manner in cultured rat hepatocytes for 48 h in the presence of insulin and epidermal growth factor (EGF). The effect of CsA was evidenced by the DNA fragmentation pattern constituted by fragments of multiples of 180-200 base pairs, which is a characteristic of programmed cell death. The metabolic activity did not change significantly in the presence of 0.1 microM CsA and diminished to 49% of control in the presence of 1 microM CsA. Changes in the metabolic activity were correlated with a decrease in both [methyl-3H]thymidine uptake and DNA content, which reflects a decrease in the cell number. The treatment of cells with CsA (1 microM) decreased the metabolic activity/DNA content ratio by 24% with respect to dimethyl sulphoxide (DMSO) control, which also suggests, under these conditions, that the necrosis achieved is at most only 24%. In addition, the changes observed (apoptotic process, arrest of the cell cycle and apparition of a necrotic process) were correlated with an increase in the high-affinity guanosine triphosphatase (GTPase) enzymes.

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Treatment of cultured rat hepatocytes with cyclosporin A (0.01-1 microM) for 24, 48, or 72 h in the presence of insulin and epidermal growth factor induced an inhibition on cell proliferation in a time- and concentration-dependent manner, with an IC50 = 0.05 microM CsA corresponding to 48-h treatment. The inhibitory effect of CsA at < or = 0.1 microM doses for 48 h on [3H]thymidine uptake was reversed after withdrawal of the drug and subsequent addition of insulin plus EGF or serum; however, at 1 microM CsA the effect was irreversible and numerous bright small vesicles were observed. The molecular mechanism involved in CsA action in hepatocytes seems to be independent on cAMP and pertussis-toxin sensitive G proteins.

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Isolated rat hepatocyte couplets were used to study the effect of S-adenosyl-L methionine (SAMe) treatment on disruption of canalicular function caused by cyclosporin A (CyA). Canalicular function was assessed by counting the percentage of couplets that were able to accumulate the fluorescent cholephile choly-lysyl-fluorescein (CLF) into the canalicular vacuole between the two cells, i.e., canalicular vacuole accumulation (CVA). Cotreatment with 1 mmol/L SAMe prevented the inhibition of canalicular vacuole accumulation caused by CyA (75 nmol/L and 100 nmol/L), whereas treatment with it after CyA was unsuccessful. SAMe prevented the dose dependent reduction caused by CyA (5 nmol/L-1 mumol/L) both on CVA and on retention of CLF preaccumulated within the canaliculus, the effect on retention being complete. No difference in intracellular content of reduced glutathione (GSH) between the control and any dose level of the immunosuppressor, with or without SAMe treatment was observed, suggesting that changes in intracellular reduced GSH levels are not involved in the effects of SAMe. F-actin was stained with fluorescein-isothiocyanate phalloidin and fluorescence measurements were performed by confocal microscopy. The ratio of the percanalicular area fluorescence/total couplet fluorescence, indicative of F-actin distribution, significantly decreased with CyA. However, cotreatment of CyA with SAMe protected the integrity of the pericanalicular cytoskeleton, suggesting that this beneficial effect on canalicular function may maintain canalicular contractions and/or preserve tight junction function. Results are discussed in relation to possible involvement of the transmethylation pathway, modifications in membrane fluidity, effects on bile acid transport, and of inhibition of uptake of CyA. They suggest that SAMe could be a good candidate for protecting against CyA-induced membrane dysfunction.

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Acid-base disturbances were examined during experimentally induced infection in the rabbit with Eimeria stiedai, a parasite that profoundly modifies liver morphology and physiology, resulting in anatomical and functional alterations similar to those appearing in different human hepatic diseases. Over 28 days of infection, bicarbonate and lactate concentrations, partial pressures of O2 and CO2, and pH values were determined in the blood and bile of infected animals and compared with the values obtained in noninfected rabbits. The plasma activity of several liver-indicator enzymes was also evaluated. Under our experimental conditions we observed an uncompensated metabolic acidosis that developed with elevated levels of lactate and reduced concentrations of bicarbonate in blood and bile and tended to be compensated by respiratory and biliary mechanisms.

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The plant saponin, diosgenin, is known to induce a marked increase in biliary cholesterol/phospholipid ratio. We reasoned that putative biliary lipid supply vesicles might be similarly enriched with cholesterol. Seven-day diosgenin feeding to rats resulted in significantly increased biliary cholesterol and cholesterol/phospholipid ratio, but had no effect on total cholesterol or phospholipid content of the liver. Subcellular fractionation of livers showed no selective increase in any fraction (nuclear, mitochondrial, lysosomal, microsomal) of the homogenate. Further subfractionation of microsomal or nuclear (plasma membrane) fractions also showed no difference between control and diosgenin groups. Thus, no intracellular vesicle fraction has been identified with the provision of the enhanced biliary cholesterol and the results are discussed in terms of the possible involvement of cytosolic lipid-binding proteins as putative lipid carriers to the canalicular membrane as an alternative to the presence of the lipid in lipid supply vesicles.

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Isolated rat hepatocyte couplets were used to study the effects of different concentrations of cyclosporin A in relation to canalicular function. Canalicular function was assessed by counting the percentage of couplets which were able to accumulate the fluorescent cholephile cholyl lysyl fluorescein (CLF) into the canalicular vacuole between the two cells, i.e. canalicular vacuole accumulation (CVA). At lower doses, the immunosuppressor increased the CVA, reaching 121 +/- 3.86% of control at 25 nM cyclosporin A. However, higher doses of cyclosporin A induced a concentration-dependent inhibition of CVA to 64.0 +/- 3.51% of control at 100 nM. Modifications in canalicular area (as % couplet area) were also observed. Image analysis of the fluorescent image showed that cyclosporin A (25 nM) increased canalicular area by 25% (of control); however, this parameter decreased to 36% of control at 100 nM cyclosporin A. In addition, at 100 nM, cyclosporin A reduced the proportion of couplets retaining CLF within the canaliculus to 75.0 +/- 6.59% of control. Treatment of couplets with cyclosporin A (0-2 microM) for 15 min revealed that reduced glutathione (GSH) intracellular content does not change significantly at these doses. However, alteration in pericanalicular F-actin at 100 nM cyclosporin A may be an important factor in the disruption of the canalicular function induced by higher doses of the immunosuppressor.

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We investigated the effect of cyclosporine A (CyA) administered as a single i.v. dose of 20 and 40 mg/kg body wt, on biliary secretion of cholesterol, phospholipid, bile acid, and lysosomal marker and canalicular plasma membrane marker enzymes in anaesthetized Wistar rats. CyA reduced the concentration and biliary secretion of cholesterol, phospholipid and bile acid to a considerable extent; the inhibitory effect of CyA on the biliary secretion of phospholipid and bile acid was greater than that on cholesterol. The biliary outputs of acid phosphatase (AcP) and gamma-glutamyltransferase (gamma-GT) were also diminished by the drug, all these effects being dose-dependent. Maximum decreases in bile acid secretion were observed 10 min after administration, whereas those of cholesterol and phospholipid were delayed. Bile acid concentrations and secretion returned to pretest values at 30-50 min after CyA injection whereas those of cholesterol and phospholipid remained significantly reduced at this time point. The greater inhibitory effect of CyA on the biliary outputs of phospholipid and bile acid relative to cholesterol secretion together with the asynchronous fall and recovery of bile acid, cholesterol and phospholipid concentrations and secretion alter the cholesterol/bile acid, phospholipid/bile acid and cholesterol/phospholipid molar ratios as well as the lithogenic index, thus suggesting that CyA would uncouple biliary lipid secretion from bile acid secretion. Since under physiological conditions biliary lipid and gamma-GT secretion is related to and dependent upon bile acid secretion, we propose that the CyA-induced inhibition on lipid and gamma-GT secretion is, at least partly, secondary to the fall in bile acid output caused by the drug. However, since CyA inhibits secretory processes independent of the hepatobiliary flux of bile acid, such as the exocytic discharge of AcP, and because it also uncouples biliary lipid from bile acid secretion, other mechanisms and factors involved in lipid and protein secretion (such as intracellular transport, canalicular membrane fluidity and/or intracanalicular events) might also be altered by this drug.

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The effect of cyclosporin A on the maximal secretion of bilirubin was investigated in male Wistar rats. Following a saturating load of the pigment, intravenous administration of cyclosporin A as a single bolus (20 mg/kg) significantly reduced bile flow and bile acid secretion. The bilirubin secretory transport maximum was alos significantly reduced. The decreased excretion of the bile pigment corresponded both to bilirubin mono- and di-conjugates, the excretion of unconjugated bilirubin being slightly and non-significantly reduced. Plasma and liver bilirubin concentrations were significantly increased, while liver UDP-glucuronosyltransferase activity did not significantly differ between control and cyclosporin A-treated rats. Our data indicate that, although in cyclosporin A-treated rats there are no changes in the activities of others markers of hepatocellular dysfunction, the overall plasma to bile transfer of bilirubin is altered. The reduced hepatobiliary transport of the pigment is mainly caused by an impairment of its intracellular/canalicular transport.

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In an attempt to understand the hepatotoxicity associated with immunosuppressive therapy with cyclosporin A, we investigated the effects of acute cyclosporin A administration on biliary secretion, serum bile acid and bilirubin levels and the histological changes in the hepatic parenchyma in anesthetized male Wistar rats. The animals were divided into three experimental groups that received equal volumes (1 ml, intravenously) of physiological saline (controls), cyclosporin A vehicle (a fat emulsion, Intralipid, mixed with absolute ethanol) or cyclosporin A dissolved in the aforementioned mixture. In another series of assays, horseradish peroxidase was coinjected with cyclosporin A vehicle or with the solution containing cyclosporin A. Only after cyclosporin A administration was an immediate inhibition in bile flow and in the biliary concentrations and secretion of bile acids and bilirubin found. In addition, a delay in the peak time of the appearance of horseradish peroxidase together with a reduction in the biliary excretion rate and in the total amount of horseradish peroxidase excreted were observed during cholestasis. At 40 to 50 min after drug administration, all biliary parameters evaluated had returned to the pretest values. The relationship between bile flow and bile acid secretion showed that cyclosporin A-induced cholestasis is related to a decrease of both the bile acid-dependent and bile acid-independent fractions of bile flow. At the end of the cyclosporin A assays, the serum bile acid, total bilirubin and conjugated bilirubin concentrations were greater than those observed in the controls and Intralipid-treated animals. These effects were dose-dependent. Light microscopy and transmission electron microscopy studies did not reveal architectural hepatic abnormalities.(ABSTRACT TRUNCATED AT 250 WORDS)

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The effect of cyclosporine vehicle, Cremophor EL, on bile flow and biliary bile acids and bilirubin output was studied in anesthetized male Wistar rats. Intravenous administration of Cremophor EL or castor oil as a single bolus reduced bile flow and the biliary output of bile acids and bilirubin. The Cremophor EL-induced cholestasis was an immediate and reversible phenomenon, since at 30-35 min after drug injection all parameters evaluated had returned to control values. A slight increase in serum bilirubin concentrations was observed. Our data indicate that the observed cholestasis is related to a reduction in both bile acid-dependent and bile acid-independent bile flow, probably due to a transitory hepatotoxic effect of Cremophor EL. We conclude that the clinically used vehicle for i.v. administration of cyclosporine, Cremophor EL, has adverse effects on hepatobiliary physiology in the rat and suggest that an alternative vehicle should be used.

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