Marc Thevenin

Modulation of energy status and cytotoxicity induced by FK506 and cyclosporin A in a renal epithelial cell line

Abstract FK506 and cyclosporin A (CsA) are two potent immunosupressants with similar toxicity profile. Nephrotoxicity is the main adverse effect of both compounds. The aim of this study is to compare the in vitro nephrotoxic effects on renal epithelial cell line LLC-PK1 by measuring cell viability and energy status as evaluated by concentrations of ATP and ATP metabolites. Cell viability (expressed as IC50 was assessed via thiazolyl blue (MTT) assay after incubation for 4–24 h with FK506 or CsA. ATP and its metabolites were determined by HPLC after 4 and 6 h incubation with FK506 or CsA alone at the respective IC50. Both FK506 and CsA decreased cell viability to similar extents, in a dose- and time-dependent manner. After 4 h incubation, both drugs decreased ATP levels (−25%) and increased uric acid levels. However, the latter percentage increase was twofold higher with CsA (18%) than with FK506 (9%). The energy charge, calculated according to levels of adenine nucleotides, was decreased by 10% in FK506-treated cells and by 27% in CsA-treated cells. At the end of 6-h incubation, FK506-treated cells maintained ATP levels coupled with energy charge at near control levels whereas the levels were 32% lower in CsA treated cells. Compared to the 4 h-incubation, the increase in uric acid was similar for FK506 but was doubled with CsA. The decrease in cell integrity and ATP depletion induced by CsA in LLC-PK1 cells was only transiently observed with FK506. By preserving energy status, FK506 leads to fewer metabolic disturbances than CsA in the renal epithelial cell line LLC-PK1, demonstrating a minor potential nephrotoxicity.

Separation of oxazepam, lorazepam, and temazepam enantiomers by HPLC on a derivatized cyclodextrin-bonded phase: application to the determination of oxazepam in plasma

The enantioselective high-performance liquid chromatography (HPLC) of three racemic 3-hydroxybenzodiazepines, oxazepam (Oxa), lorazepam (Lor), and temazepam (Tem), is a difficult operation because of the spontaneous chiral inversion in polar solvent. To solve this problem, we have developed an HPLC method based on a chiral Cyclobond I-2000 RSP column, maintained at 12 degrees C, and a reversed mobile phase (acetonitrile in 1% triethylamine acetate buffer, TEAA) at a flow rate of 0.4 ml/min. Peaks were detected by a photodiode-array detector at 230 nm for quantification and by an optical rotation detector for identification of (+) and (-) enantiomers. The results showed that peak resolutions of Oxa, Lor, and Tem enantiomers, analyzed under the same conditions, were 3.2, 2.0, and 1.8, respectively. For the determination of Oxa enantiomers in plasma of rabbits, extraction with diethyl ether at pH 1.5, a polar organic mobile phase, and a Cyclobond I-2000 SP column were used. Other analytical conditions were the same as previously described. Blood samples were immediately cooled at 4 degrees C and centrifuged at 0 degrees C for the collection of plasma. The results showed a difference in plasma S(+)- and R(-)-oxazepam concentrations in rabbits. No racemization of S(+)- or R(-)-Oxa enantiomers, added alone to blank plasma, was observed after extraction and enantioselective HPLC analysis.

Effect of age and photoperiodic conditions on metabolism and oxidative stress related markers at different circadian stages in rat liver and kidney

It has been shown that some cytochrome P450-dependent enzyme activities could present daily fluctuations, particularly CYP3A isoenzymes which are enhanced during the dark period. The aim of this study was to investigate whether age and photoperiodic conditions at different circadian stages could influence these fluctuations. Young mature (10 weeks) and old (22 months) Wistar rats were initially exposed to light-dark cycles 12:12 during 4 weeks, and secondly 18:6 for either one week or six weeks. Erythromycin N-demethylase (CYP3A-dependent), 7-ethoxycoumarin O-deethylase (CYP1A-dependent) and aniline 4-hydroxylase (CYP2E-dependent) activities were determined in liver and kidney microsomes at different hours after darkness onset (HADO). In addition, liver and kidney GSH, GSHPx, ATP, TBARS were determined. During the LD 12:12 cycle, while no significant modification was observed in CYP1A- and 2E-dependent enzyme activities as functions of HADO, erythromycin N-demethylase activity (CYP3A-dependent) showed a significant increase during the second third of the dark period in both young and old rats. After switching to a LD 18:6 cycle, this variation was still observed during second third of the dark period, to a lesser but still significant degree, with no difference between one week and six weeks exposure to the new photoperiod. It can be noted that the old rats showed a significantly lower level of erythromycin N-demethylase activity than the young rats, in parallel to a decrease in GSH, GSHPx and ATP, and an increase in TBARS. These results confirm the lower resistance of old animals to oxidative stress. The observed variations in metabolism parameters underline the need for study designs in pharmaco-toxicology taking into account the possible risks induced by circadian changes, especially in aged subjects.

[34] Cold light fluorimetry: A microtitration technology for cell culture to evaluate anethole dithiolethione and other biothiols

Publisher Summary Biothiols are compounds of growing biological interest. This chapter describes screening these drugs using a new microtitration technology, cold light fluorimetry (CLF), and comparing it to classic fluorimetric methods. Until now, in vitro screening of such molecules was done using photometric or fluorimetric microtitration methods which are not sufficiently sensitive and/or specific. The only fluorimetric methodology available for cell culture is flow cytometry, which is not adapted to screening because the equipment is too heavy and expensive. The classic microtitration fluorimetric methods are reviewed and the cold light concept is discussed. The three main microplate fluorescence readers have been tested. These fluorimeters read correctly in ultraviolet (UV) or visible spectra but rarely in both, and they have low sensitivity. Therefore a new fluorimetric technology has been developed which proved to be sensitive and could be adapted to direct screening in 96-well microplates. To improve the fluorescence signal, the choice of plastic microplates is decisive. Black microplates used for fluorimetric biochemistry assays are not adapted for cell biology, because they are neither treated for cell culture nor sterilized.

Implication of CYP 3A in the toxicity of cyclosporin G (CSG), cyclosporin A (CSA) and FK506 on rat hepatocytes in primary culture

Abstract FK506, cyclosporin A (CsA), and its structural analog cyclosporin G (CsG) are immunosuppressant drugs mainly metabolized by hepatic cytochrome P-450 3A (CYP 3A) oxygenase. FK506 metabolites exhibit greater toxicity than the parent drug, while CsA metabolites are far less toxic than CsA itself. The aim of our study was to compare the toxicity of CsG with CsA and FK506 as a function of CYP 3A induction. Hepatocytes from Wistar rats with or without dexamethasone (DEX) induction (200 mg/kg per day, p.o for 4 days) were used in primary culture. The DEX-inductive effect on CYP 3A was assessed by SDS-PAGE. After 6 h incubation with CsG, CsA or FK506 (5 to 200 μM), cell viability (expressed as IC50), intracellular calcium content and apoptosis were evaluated. Concerning cytotoxicity, IC50 values for CsG, CsA and FK506 were 75, 50 and 180 μM respectively in non-induced cultures, and 150, 120 and 25 μM in induced cultures. For intracellular calcium content, a dose-dependent increase was observed in all cultures. However this increase is more important for CsG and CsA in non-induced cultures (150%) compared to induced cultures (110%) at 150 μM. Conversely for FK506, this increase is greater in induced cultures (150%) than in non-induced cultures (127%). Estimation of the percentage of apoptotic cells shows similar variations. Our results show that the toxicity of the three drugs in rat hepatocytes is dependent on CYP 3A induction: increased for FK506, decreased for CsA and CsG. Moreover, with regard to the three tests used, the toxic effects of CsG are close to those of CsA, indicating that CsG metabolites are also less toxic than the parent drug.

FK506 (Tacrolimus) decreases the cytotoxicity of cyclosporin A in rat hepatocytes in primary culture: implication of CYP3A induction

Abstract Tacrolimus (FK506) and cyclosporin A (CsA) are two potent immunosuppressants mainly metabolized by hepatic cytochrome P-450 3A (CYP3A) monooxygenase. The aim of this study was to compare the toxic effects of the two drugs on hepatocytes in primary culture as a function of their metabolism and to explore the variations of cytotoxicity when both drugs are associated. The cytotoxicity of FK506 and CsA, as expressed by their IC50 values, was of the same order but with a switch according to whether hepatocytes were induced or uninduced by dexamethasone, CsA being more toxic in its native form and FK506 through its metabolism. Similar results were obtained with the intracellular calcium content. When both drugs were associated at their IC50 values, the expected additive cytotoxic effect was not observed. Moreover, when small quantities of FK506 were added to CsA at its IC50, cell viability improved in the induced cultures. It is hypothesized that the interaction between the two drugs relies on a mechanism involving both competition of FK506 and CsA for CYP3A and of their immunophilin complexes for a common site on the calcineurin-calmodulin complex.

Effects of a new cephalosporin, cefpirome (HR 810), on ATPase activities of rabbit renal proximal tubule suspensions: Comparison with cephaloridine and cefotaxime.

The effect of cefpirome (HR 810), a new cephalosporin, on ATPase activities of rabbit renal proximal tubules has been measured and compared with that of cephaloridine and cefotaxime. Only cephaloridine, the nephrotoxicity of which is well established in the rabbit, produced after 60 min treatment a dose-dependent decrease in Na(+)/K(+)- and Mg(2+)-ATPase activities. Cefotaxime and cefpirome, which have a low nephrotoxic potential in the rabbit, did not exert any effect on ATPase activities.

Ex vivo study on ATPase activities of rabbit renal proximal tubules as a predictive model for nephrotoxicity: Comparison with an in vitro study on a new cephalosporin (cefpirome).

An ex vivo study on adenosine triphosphatase (ATPase) activities of rabbit renal proximal tubules was conducted with a new cephalosporin, cefpirome (HR 810), a positive control, cephaloridine, and a reference third-generation cephalosporin, cefotaxime. Compared with controls, CPH caused a significant time-dependent decrease in ATPase activities [12%, 2 hr after treatment (P < 0.01) and 75%, 48 hr after treatment (P < 0.001)]. This decrease was accompanied by a significant loss in the energy charge of the adenylate pool [27%, 2 hr after treatment (P < 0.001)]. Neither cefotaxime nor cefpirome caused such decreases. The results confirmed those of a previously published in vitro study. The advantages and disadvantages of these two experimental procedures as predictive models for nephrotoxicity are discussed.