Lassina Badolo

Effect of cryopreservation on the activity of OATP1B1/3 and OCT1 in isolated human hepatocytes

Drug metabolism in liver is the major pathway for xenobiotic elimination from the body. Access to intracellular metabolising enzymes is possible through passive diffusion of lipophilic drugs through cell membrane or active uptake of more polar drugs by specific uptake transporters. Organic Anion Transporting Polypeptides (OATP/SLCO) and Organic Cation Transporters (OCT/SLC22A) are among the most important transporters involved in xenobiotic transport into hepatocytes. Isolated hepatocytes are the model of choice for drug metabolism and drug transport investigations. These primary cells are used either as fresh directly after isolation from liver biopsies, or after subsequent cryopreservation in liquid nitrogen. While cryopreserved hepatocytes are a more convenient and flexible tool for in vitro investigations, information on the functionality of transporter activity after cryopreservation is still sparse. The present study investigated the effect of cryopreservation of human hepatocytes on the uptake of [(3)H]-estradiol-17β-glucuronide (E(2)17βG, substrate of OATP1B1/3/SLCO1B1/3) and [(3)H]-1-methyl-4-phenylpyridinium (MPP+, substrate of OCT1/SLC22A1) into hepatocytes from 6 and 5 human donors, respectively. The results showed that cryopreserved human hepatocytes display carrier-mediated uptake of E(2)17βG and MPP+. While the affinity of E(2)17βG for OATP1B1/3/SLCO1B1/3 was not affected by cryopreservation (Km unchanged, the Wilcoxon signed pair t test gave p=1), V(max) and CL(uptake) values decreased in average by 47% (p=0.06). The passive diffusion of E(2)17βG decreased significantly after cryopreservation (p=0.03). Cryopreservation did not affect Km, V(max) or the passive diffusion of MPP+ in human hepatocytes. In conclusion, the present study showed that cryopreserved human hepatocytes are useful tool to investigate hepatic uptake mediated by OATP1B1/3/SLCO1B1/3 or OCT1/SLC22A1, two of the most important hepatic uptake transporters.

LC-ICP-MS and LC-ESI-(MS)n identification of Se-methylselenocysteine and selenomethionine as metabolites of methylseleninic acid in rat hepatocytes

The metabolism of methylseleninic acid in isolated rat hepatocytes was investigated. Selenium containing metabolites excreted from the cells were detected in the supernatant of the incubation sample by LC-ICP-MS. After pre-treatment of the supernatant by preparative chromatography and pre-concentration by lyophilisation, a major metabolite was identified by molecular mass spectrometry as Se-methylselenocysteine by LC-ESI-MS, MS2 and MS3 and a minor metabolite was identified as selenomethionine by LC-ESI-MS2 and MS3 and LC-ESI-MS2(SRM). This is the first time these metabolites have been identified in hepatocytes. Complementary data from ion trap and triple quadrupole MS instruments provided solid proof of metabolite identities. A time course study showed that S-(methylseleno)cysteine and S-(methylseleno)glutathione were intermediates in the formation of the major metabolite. It is questioned if methylseleninic acid is a relevant model compound for methylated Se-amino acids in vitro.

In situ identification of dimethyl diselenide in hepatocytes treated with methylseleninic acid by membrane inlet mass spectrometry

The advantages and drawbacks of membrane inlet mass spectrometry (MIMS) for identification of volatile selenium compounds were investigated. Hepatocytes were incubated with methylseleninic acid (MeSeA) directly in the MIMS reaction cell and dimethyl diselenide (DMeDSe) was identified as the major volatile metabolite. This compound was also identified as the major reaction product after incubation of glutathione (GSH) with MeSeA without the presence of hepatocytes. One advantage of MIMS is the possibility of in situ monitoring of volatile metabolites with minimum risk of sample loss and without the need for trapping devices. Another advantage is the possibility of concurrent monitoring of cell viability by simultaneous detection of oxygen consumption and CO2 production. Although ideal for screening, the technique suffers from a relative high detection limit (in the 1 µM range) and lack of robustness. This paper presents for the first time in situMS data on the formation of DMeDSe.

Simple and rapid enzymatic assay of ornithine decarboxylase activity.

Since the induction of putrescine synthesis by ornithine decarboxylase (ODC) is observed in many pathological and physiological processes, a useful and simple method to assay this enzyme activity should be an interesting tool to quantify the biological importance of its induction. An enzymatic method to assay ODC is reported here. This method is based on the reaction between putrescine and soya diamine oxidase. The reaction releases H(2)O(2), which is measured by a colorimetric method. The validation of this method showed good accuracy (98+/-5% of recovery). High precision and reproducibility were obtained. A linearity with a correlation coefficient of 0.999 in the range of 2.5-25 nmol was obtained. This method is also rugged and specific. The application of the assay of ODC activity showed that it is useful as a rapid and simple tool for assaying ODC activity in vitro. Comparison with the HPLC determination of ODC activity shows strong correlation along with the high accuracy of the two methods.

Cognitive enhancing effects of an AMPA receptor positive modulator on place learning in mice

This study presents an in vivo investigation of the arylpropylsulfonamide α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor positive modulator (R,R)-N,N-(2,20-[biphenyl-4-40-diyl]bis[propane-2,1-diyl])dimethanesulfonamide (PIMSD). The pharmacokinetics of the drug were examined in male C57BL/6J mice and the drug concentration in blood plasma determined after subcutaneous injection of 1mg/kg b.w. This analysis revealed a rapid increase of the plasma concentration, peaking within 30min after administration with a T(1/2) of approximately 30min and a peak plasma concentration of about 2μM. Analysis of brain tissue homogenates also indicated blood-brain barrier permeability of the compound. Cognitive enhancing effects of the drug were then studied on place learning in male C57BL/6J mice in a water maze. In order to elucidate the potential positive effects of PIMSD on spatial learning the muscarinergic antagonist scopolamine was utilized, which is known to impair spatial learning ability. The mice were divided into four groups and subjected to two sequential subcutaneous injections administered 25min prior to behavioural testing: (1) vehicle/vehicle; (2) PIMSD/vehicle; (3) scopolamine/vehicle; (4) PIMSD/scopolamine. PIMSD at a dose of 3mg/kg b.w. was able to partially reverse the impairment given by 0.5mg/kg b.w. scopolamine. These results suggest that arylpropylsulfonamides such as PIMSD may have a therapeutic use in the enhancement of cognitive function and support the hypothesis that AMPA receptor potentiation is one mechanism that can be targeted for diseases of cognitive impairment.

Inhibition of ornithine decarboxylase by ifenprodil.

Ifenprodil (NMDA receptor antagonist) was tested as an inhibitor of ornithine decarboxylase. It was found that ifenprodil inhibited ornithine decarboxylase activity with the same potency as alpha-difluoromethylornithine, a major inhibitor of ornithine decarboxylase. This result suggests that ifenprodil could target either the polyamine site on the NMDA receptor complex or/and polyamine biosynthesis.

Ability of bisbenzylputrescine and propanediamine analogs to modulate the intracellular polyamine pool of P388D1 cells

The effects of a series of bisbenzyldiamine analogs have been tested on P388D1 cell line in vitro. Their effects on cell growth, polyamine oxidase (PAO) activity and intracellular polyamine content were determined. The cytotoxicity tests were performed in culture medium supplemented with 100 μmol/L aminoguanidine (I), 100 μmol/L aminoguanidine and 100 μmol/L N,N′-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72,527) (II), and finally 100 μmol/L aminoguanidine and 200 μmol/L D,L-difluoromethylornithine (DFMO) (III). The IC50 values under conditions I and III were similar, suggesting that inhibition of ornithine decarboxylase by DFMO did not affect the biological effect of our derivatives. Spermine and spermidine remained nontoxic in conditions I and III. However in the condition II, the toxicity of all tested compounds (excepted spermidine) was increased, suggesting that the inhibition of cellular PAO increased their toxicity.The enzymatic test of PAO showed that at high doses inhibition of this enzyme by putrescine analogs occurred, while the N-methylated propanediamine derivative increased the enzyme activity; however, these results do not correlate with cytotoxicity tests. When these derivatives were incubated for 48 h with the cells, all of them increased the cell content in putrescine (∼160%) and spermine (∼145%) and decreased the spermidine content (∼75%) without any modification of the total amount of polyamine.The correlation between the cytotoxic results and the intracellular polyamine determination shows that the increase in spermine content along with the inhibition of retroconverting PAO enzyme increases the toxic effect of tested compounds (including spermine), suggesting that spermine toxicity is more important in the absence of intracellular oxidation processes.