A new acetylcholinesterase inhibitor with anti-PAF activity modulates oxidative stress and pro-inflammatory mediators release in stimulated RAW 264.7 macrophage cells. Comparison with tacrine.

Inflammatory injury and induction of oxidative stress have been implicated as causative factors in neurodegenerative diseases such as Alzheimer's disease (AD). Using LPS-stimulated RAW 264.7 macrophages as a model of inflammatory injury, LPS was found to stimulate ROS production (159%), GSH depletion (15%) and loss of mitochondrial activity (32%) as well as TNFalpha release (40%), and NO production (13.7 times), all parameters involved in AD. PMS777, a tetrahydrofuran derivative, designed as a dual PAF and acetylcholinesterase inhibitor, was found to decrease ROS (up to 32%) and NO production (up to 5 times), TNFalpha release (33%). PMS777 also prevents loss of mitochondrial activity, and GSH depletion. In contrast, tacrine was found to decrease ROS production (57% up to 102%) and TNFalpha level (up to 30%). It decreases NO release only at the highest concentrations without preventing loss of mitochondrial activity and GSH depletion. In this study, we show that PMS777 is strongly anti-inflammatory against LPS-induced responses in RAW 264.7. Differential effects between PMS777 and tacrine could be attributed to the anti-PAF activity of PMS777 which was able to fight inflammatory events and oxidative injury whereas tacrine only minimizes them. PMS777 could open a new approach in the treatment of AD.

Pharmacokinetics of oxaliplatin in patients with normal versus impaired renal function.

PURPOSE: The pharmacokinetics (PK) of platinum was investigated and compared in patients with normal (NRF) and impaired renal function (IRF), after they had received oxaliplatin at the recommended dose and delivery modality. METHODS: Oxaliplatin was administered at 130 mg/m(2) as a 2-h infusion without hydration. Patients were recruited and classified according to their creatinine clearance (CrCl > or < 60 ml/min), calculated using the Cockcroft and Gault formula. Blood was taken for PK analysis during and after the infusion. Twenty-three patients were included in the PK analysis (13 NRF and 10 IRF). At inclusion, the median CrCls were 70.5 ml/min (range 63-136) for the NRF group and 42 ml/min (range 27-57) for the IRF group. Three patients underwent a second course of treatment and additional blood sampling for analysis. Platinum levels in the plasma, ultrafiltrate and red blood cells (RBCs) were measured using flameless atomic absorption spectrophotometry (FAAS). RESULTS: Following the administration of oxaliplatin, platinum binding to plasma proteins and RBCs was rapid and extensive; at the end of the 2-h infusion, 27% of the platinum in the plasma remained free (40% bound to RBCs, 33% bound to plasma proteins). Neither the mean maximal concentration (C(max)) of total platinum in the plasma, the mean C(max) of ultrafilterable platinum in the plasma, nor the maximal platinum content in the RBCs differed significantly between the two groups (2.59 vs 2.58 microg/ml, 1.09 vs 1.28 microg/ml and 2. 06 vs 2.17 microg/ml, respectively, for patients with NRF vs IRF). After the end of the infusion, levels of total and free (ultrafilterable) platinum in the plasma declined biexponentially. The plasma clearance of both total and free platinum as well as the area under the curve (AUC) of the free platinum fraction correlate with the calculated CrCl (P=9 x 10(-3), P=3.1 x 10(-5) and P=9 x 10(-6), respectively). After a single course of oxaliplatin, toxicities reported in the two groups of patients were similar. CONCLUSIONS: Our results are in agreement with the in vitro data concerning the extensive binding of oxaliplatin to plasma proteins and RBCs. They also reveal a strong negative correlation between free drug plasma availability and renal function, with a corresponding positive correlation between clearance of the plasmatic platinum and renal function. Thus, renal impairment entails a greater overall exposure to platinum in the plasma. However, this study failed to elicit any relationship between moderate renal impairment and the acute toxicity associated with oxaliplatin.

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.

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.

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.

Acute paraquat poisoning: increased toxicity in one case with high alcohol intake.

Human paraquat poisoning from accidental or intentional ingestion is very often fatal. According to the amount of paraquat involved, death can occur within hours or weeks following ingestion. The inefficacy of the various treatments undertaken have led to determine prognostic factors based upon the evolution of plasma and urine paraquat concentrations or of usual biochemical parameters. We report one case of acute poisoning which, although the ingested dose of paraquat was not massive (< 50 mg/kg-1) and the severity indices were in favour of a delayed fatal outcome, has ended in an early death. The high blood alcohol level of the patient (3.34 g l-1) seems to be the main cause of the precocity of this death (86th hour).

Disturbances in toxicological experiments caused by Microsporum canis.

Cutaneous lesions which can lead to false positive results have been observed in several rabbits used for the determination of the cutaneous irritation capacity of a product (ITA, PII). The responsible agent was Microsporum canis. A preventive treatment by an antifungal agent did not modify toxicological experimental results.

[Dismutation as a metabolic pathway : transformation of the trimethyl 3,5,5, cyclohexanone (author's transl)]. / La dismutation comme voie de transformation métabolique - cas de la triméthyl 3,5,5, cyclohexanone.

Besides novel routes of biotransformation of xenobiotic substances recently reported, such as N-glucuronidation of teritary amines and quaternary ammonium componds, N-glucosylation, N-carbamylglucuronide formation from a primary amine, C-glucuronidation, conjugation with long-chain fatty acids, with polypeptides, carbon-chain elongation, stereospecific inversion of configuration at a satured carbon atom, formation of methylthio metabolites, the authors have described a new metabolic pathway: dismutation, in the case of 3,5,5, - trimethylcyclohexanone (dihydroisophorone). As a matter of fact, this compound leads, in vivo, to the formation of isophorone alpha and of cis and trans 3,5,5, - trimethyl cyclohexanols. These compounds, extracted from the urine of treated rats and rabbits, have been identified by gas-liquid chromatography and thin-layer chromatography.

Automated liquid-chromatographic analyzer used for toxicology screening in a general hospital: 12 months' experience.

We evaluated the clinical utility of an automated HPLC system (Remedi, Bio-Rad) for identification of drugs and metabolites in biological fluids. Serum or urine or both from 354 consecutive cases of poisoning were analyzed by the system and by a set of fluorescence polarization immunoassay (FPIA, Abbott) and thin-layer chromatographic (TLC) procedures. Antidepressants and most phenothiazines were recognized by the new system. Comparison of Remedi results with final clinical diagnoses yielded diagnostic specificity and sensitivity of 80% and 90%, respectively. Remedi detected 26 additional compounds that were neither reactive in the immunoassay screening tests nor detected by TLC procedures. Because the Remedi expands the range of drugs covered by the immunoassays and provides a rapid, preliminary report in emergency situations, we conclude that this system can be a useful complementary technique in the clinical toxicology laboratory. Although urine toxicological screening seemed adequate for a good toxicological report, blood analysis allows extra toxicokinetic data such as blood concentrations and half-life estimations.

Protective effect of nifedipine against cytotoxicity and intracellular calcium alterations induced by acetaminophen in rat hepatocyte cultures.

Alteration of calcium homeostasis has been proposed to play a major role in cell necrosis induced by a variety of chemical agents such as acetaminophen (APAP). In this study, a potential protective effect of the dihydropyridine calcium channel blocking agent, nifedipine, was investigated in vitro on acetaminophen-induced hepatocyte damage. Rat hepatocytes were exposed during 20 hours to various concentrations of APAP (0.50 to 4.00 mM). The following metabolic and functional parameters were investigated: -lactate dehydrogenase (LDH) release as an indicator of plasma membrane integrity, -cell viability evaluated by the colorimetric MTT assay, and intracellular calcium concentration as evaluated by two fluorimetric methods: a scanning laser cytometer using indo-1-AM as fluorescent probe and a fluorescence plate reader using fluo-3-AM as calcium indicator. Incubation of hepatocytes with APAP alone in the range 0.50 to 4.00mM resulted in a dose-response relationship with regard to LDH release (243% to 750% of control) and to the loss of cell viability (0 to 67% of control). Moreover these results were correlated with a significant increase in cytosolic calcium content (189 to 406 nM). Nifedipine treatment prior to APAP exposure, partially prevented LDH release, the plasma membrane blebbing, and thereby the loss of viability. In addition, intracellular calcium level progressively returned within the limits of the control values with increasing concentrations of nifedipine. It can be concluded that, in vitro conditions, nifedipine pretreatment exhibits a preventive effect against acetaminophen hepatocyte injury.

Effects of cyclosporin on kidney glutathione metabolism and cytochrome P-450 in the rabbit: possible implication of eicosanoid metabolism.

This study was designed to assess Cyclosporin A (CsA) nephrotoxicity in the rabbit-possibly a more sensitive species than the rat-and to explore the mechanism of this toxicity with special attention to glutathione metabolism disturbances and cytochrome P-450 level in the kidney. CsA given for three days at a daily dose of 50 mg/kg (s.c.) induced nephrotoxicity as assessed by histological abnormalities and by a significant increase in blood urea nitrogen and urinary enzyme activities: N-acetyl-beta-D-glucosaminidase and L-gamma-glutamyl-transferase. This observed renal injury was of the same order as that obtained in the rat. In addition, there was a significant increase in oxidized glutathione content (40%) while reduced glutathione level remained unchanged. Concurrently, there was a significant decrease in renal cortex glutathione reductase (49%) and to a lesser extent in glutathione peroxidase activities (16%) whereas that of glutathione-S-transferase was not modified. A significant increase in renal cortex cytochrome P-450 (3-fold versus controls) was also observed. The mechanism of CsA nephrotoxicity is to be related to a cytochrome P-450 induction. This event could induce the observed impairments in renal glutathione metabolism and Na+K(+)-ATPase activity, via a possible increase in eicosanoid metabolism.

Protection against cyclosporin-induced nephrotoxicity: 1. Effect of a PAF antagonist, BN50726.

The clinical usefulness of cyclosporin A (CsA) in organ transplantation is limited by its intrinsic nephrotoxicity, the mechanisms of which have not yet been completely elucidated. Various processes could be responsible for the CsA-induced nephrotoxicity including an interaction with glutathione metabolism, a lipoperoxidative process, or an interaction with platelet-activating factor (PAF). Therefore, the aim of this study was to assess the potential role of PAF in CsA-induced nephrotoxicity using a new PAF antagonist, BN50726. CsA alone provoked a significant increase in serum urea, urinary gamma-glutamyl transferase activity and oxidized glutathione level of kidney cortex in the rat. In contrast, when rats were treated with CsA plus BN50726, urinary gamma-glutamyltransferase activity was similar to that of controls and oxidized glutathione was significantly lower than that measured in rats treated with CsA alone. However, serum urea remained increased as it was in the CsA group. Our results suggest that BN50726 is able to prevent the damage of renal membranes probably by an antiperoxidative activity. These data indicate the potential therapeutic capacity of a PAF antagonist, BN50726, in reducing CsA nephrotoxicity and suggest interactions between PAF and CsA in the mechanism of renal injury.

Increased reduced glutathione and glutathione S-transferase activity in chronic cephaloridine nephrotoxicity studies in the rat.

The effect of repeated cephaloridine treatment on renal glutathione and related enzymes has been investigated in young adult male and female Sprague-Dawley rats. Animals were given intraperitoneally daily doses of either 750 mg/kg for two weeks or 500 or 750 mg/kg for three months. Measurement of blood and urinary parameters (electrolytes, urea, creatinine) did not reveal any renal function impairment and histological examination confirmed the absence of renal damage. By contrast, an increase in reduced glutathione (2 to 3-fold) and glutathione S-transferase activity (1.5 to 2-fold) was observed. These results are consistent with the development of an adaptative phenomenon to cephaloridine subchronic treatment in the rat, leading to a tolerance to high repeated doses.

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

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 microM), 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 microM respectively in non-induced cultures, and 150, 120 and 25 microM 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 microM. 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.

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.

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

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.

[Metabolic transformations of the trimethyl - 3,5,5, cyclohexene-2, one-1 (isophorone) (author's transl)]. / Transformations métaboliques de la triméthyl-3,5,5, cyclohexène-2, one-1 (isophorone).

In the rabbit, isophorone alpha is partly eliminated, unchanged in the expired air and in the urine and, for another part, metabolized in the organism and excreted in the urine. The metabolites which have been characterized are : dimethyl-5,5, cyclohexen-1, one-3, carboxylic-1 acid, deriving from isophorone by methyloxidation, isophorol (trimethyl-3,5,5 cyclohexen-2, ol-1) formed by the reduction of the ketonic group into a secondary alcohol and eliminated as a glucuronide, dihydroisophorone (trimethyl-3,5,5, cyclohexanone) proceeding from the hydrogenation of the cyclohexen cycle and cis and trans trimethyl-3,5,5, cyclohexanols-1. These latter compounds, found in small quantities, are very likely issued from dihydroisophorone which is transformed in the organism into isophorone alpha and cis and trans trimethyl-3,5,5, cyclohexanols-1, according to a process of dismutation.