Hepatocytes incubation

Isolated hepatocytes incubated with ionic iron rapidly undergo lipid peroxidation. Some studies have not shown a consequent decrease in viability (as indicated by uptake of trypan blue or release of enzymes). This is probably a result of short incubation times, as changes in viability lag behind increases in lipid peroxidation, and may not occur for more than 2 h after lipid peroxidation begins (Bacon and Britton, 1990). Recent studies have shown strong correlations between increased lipid peroxidation [production of thiobarbituric acid (TBA) reactants] and loss of cell viability (trypan blue staining) (Bacon and Britton, 1989). The significance of the lag between lipid peroxidation and decreases in cell viability is as yet uncertain. 

As in the case in the analysis of food samples, the introduction of relatively inexpensive MS detectors for GC has had a substantial impact on the determination of methylxanthines by GC. For example, in 1990, Benchekroun published a paper in which a GC-MS method for the quantitation of tri-, di-, and monmethylxanthines and uric acid from hepatocyte incubation media was described.55 The method described allows for the measurement of the concentration of 14 methylxanthines and methyluric acid metabolites of methylxanthines. In other studies, GC-MS has also been used. Two examples from the recent literature are studies by Simek and Lartigue-Mattei, respectively.58 57 In the first case, GC-MS using an ion trap detector was used to provide confirmatory data to support a microbore HPLC technique. TMS derivatives of the compounds of interest were formed and separated on a 25 m DB-% column directly coupled to the ion trap detector. In the second example, allopurinol, oxypurinol, hypoxanthine, and xanthine were assayed simultaneously using GC-MS. 

Benchekroun, Y., Desage, M., Ribon, B., and Brazier, J.L., Gas chromatographic-mass spectrometric quantition of tri-, di- and monomethylxanthines and uric acid from hepatocyte incubation media, J. Chromatogr., 532,261,1990. 

Biological characterization of the nanoparticles was carried out by monitoring in vitro interactions with hepatocytes isolated from rat liver [12], Haemagglutination inhibition test of erythrocytes with ricine agglutinin (RCA120) was carried out [13]. The fluorescence of the hepatocytes incubated with the FlTC-labeled nanoparticles was determined by means of a FACS Star Becto-Dickinson instmment. 

Figure 6 Correlation between testosterone 6P-hydroxylation and CYP3A protein levels, as determined by Western blot, in human hepatocytes incubated with several prototypical inducers.

Figure 12 Dose- and time-dependent induction of CYP3A activity in human cultured hepatocytes incubated with rifampicin. Cells cultured on Matrigel-coated 96-well plates were incubated with increasing doses of rifampicin and CYP3A was determined by probing the cells with DFB prior to RNA isolation. Abbreviation DFB, [3-[(3,4-difhiorobenzyl)oxy]-5,5-dimethyl-4-[4-(methylsulfonyl)phenyl]furan-2(5/F)-one].

Fig. 12. Effect of extracellular Ca2 concentration on the accumulation of Ca2+ by isolated rat hepatocytes incubated for 5 min with 10 nM vasopressin plus 10 mM glucagon. Reproduced from Ref. 160 by permission of the authors and publisher.

In a study by Orzechowski et al. (1995), hepatocytes from adult male Wistar rats and NMRI mice were incubated for 1 hour with 0.5 mM 14C-benzene, and the supernatant analyzed for metabolites. Formation of sulfate conjugates of benzene, hydroquinone, and 1,2,4-benzenetriol was also studied in a separate experiment. Mouse hepatocytes produced two metabolites (1,2,4-trihydroxybenzene sulfate and hydroquinone sulfate) that were not found in rat hepatocyte incubations. These sulfate metabolites were found in incubations including benzene, or the metabolites themselves, hydroquinone and 1,2,4-benzenetriol. Mouse hepatocytes were almost three times more effective in metabolizing benzene, compared to rat hepatocytes. This difference was accounted for in the formation of hydroquinone, hydroquinone sulfate, and 1,2,4-trihydroxybenzene sulfate. These in vitro experiments indicate there are both quantitative and qualitative differences in rodent metabolism of benzene. 

For example, Liu and Hop [19] described the hepatocytic incubation of a drug candidate possessing a methoxy substituent (RCH2OCH3). The resultant observation of a compound isobaric (at low resolution) with the parent compound was resolved with high-resolution mass measurements determined using a quadrupole-time-of-flight mass spectrometer (Q-TOF). The accurate mass of the observed peak was 0.0362 amu lower than the parent compound and correlated to the formation of a carboxylic acid moiety (RCOOH) via demethylation and subsequent oxidation. 

The use of intact human hepatocytes may allow a more accurate extrapolation of in vitro results to in vivo. The study is performed using intact human hepatocytes incubated with isoform-specific substrate and the test article. The intact plasma membrane and the presence of all hepatic metabolic pathways and cofactors allow distribution and metabolism of the test article. The resulting inhibitory effect therefore should be physiologically more relevant to the in vivo situation than results with cell-free systems. 

FIGURE 4.6 Mass chromatograms showing a metabolite generated in hepatocyte incubations as indicated by comparing the 0, 10, and 30min incubations. 

With these innovative sample introduction techniques, 96 samples can be analyzed in less than 20 min, which is compared to >2.5 h for traditional LC-MS/ MS. However, as in the case for the BioTrove s RapidFire system where no LC separation is involved, both techniques rely solely on the specificity of MS/ MS to differentiate different compounds. To take full advantage of FlashQuant and LDTD MS on complex system (e.g., hepatocyte incubation assay or in vivo studies), matrix effects, isobaric interferences, metabolic interferences, and MS cross-talk need to be carefully investigated prior to applying those techniques in samples analysis. 

Biotransformation of Sulfamethazine and B-Nortestosterone by Pig Hepatocytes. Incubation of pig hepatocytes, isolated from livers of a number of different animals, with the antibacterial agent sulfamethazine resulted in the formation of a single... 

EquiUbrium of the a-oxoglutarate-malate exchange implies that (a-oxo-glutarate/malate), /(a-oxoglutarate/malate)j.y, = 1, since is not involved in this exchange. From data published by Siess et al. [35,37] it can be calculated that this condition is approximately fulfilled in hepatocytes incubated with lactate. 

It has been proposed that fatty acyl-CoA esters, which are potent inhibitors of ADP transport in isolated mitochondria, also control flux through the translocator in vivo and hence the production of ATP (see [4] for literature). However, as pointed out by Stubbs [7], such inhibition makes little physiological sense in situations like starvation or long-term exercise, when fatty acids are an important fuel especially under the latter conditions the translocator must operate at high capacity to provide the cytosol with ATP. Indeed, in isolated hepatocytes incubated with various concentrations of fatty acids no inhibition of ATP transport by fatty acyl-CoA could be observed [71]. Possibly this inhibition is prevented by a low-molecular weight cytosolic protein with a high affinity for fatty acyl-CoA [72]. 

In male rat hepatocytes incubated with 100 pg/ml of diterpenoids from skullcap, the compounds caused apoptosis. Reactive metabolites formed by GYP3A depleted cellular thiols, increasing cellular calcium and opening the mitochondrial permeability transition pore (MPTP). Cyclosporine A, an inhibitor of MPTP opening, prevented cytochrome c release, caspase activation, and apoptosis, and caspase inhibitors also prevented apoptosis (Haouzi et al. 2000). 

The metabolism of ochratoxin A was studied in cultured rat and human primary hepatocytes incubated with non-cytotoxic concentrations of PH]ochratoxin A ranging from 10 to 10 mol/l for 8 h. In rat hepatocytes, ochratoxin A was metabolized to small amounts of three products. In addition to 4-hydroxy-ochratoxin A, which is a known product of ochratoxin A biotransformation, two novel metabolites were detected and tentatively identified as hexose and pentose conjugates of ochratoxin A. In vitro induction with 3-methylcholanthrene increased the formation of 4-hydroxy-ochratoxin A but did not alter the formation of the conjugated metabolites (Gross-Steinmeyer et al., 2002). 

As found initially in human skin fibroblasts, " formate is also produced in other cell types such as rat hepatocytes, ° human HepG2 hepatoma cells and canine MDCK cells during the degradation of 3-MBFAs (unpublished data). The ratio of formate to CO2 production can differ, however, being high in fibroblasts (about and low in rat hepa-tocytes (about 0.3-0.4). ° As a consequence, both products should be determined in order to obtain valid data. Addition of unlabelled formate to isolated rat hepatocytes incubated with 3-methyl-[l- " C]heptadecanoate decreased the generation of radioactive CQ. This was accompanied by a compensatory increase in radioactive formate. This clearly indicates that formate is formed first and subsequently converted to CO2. 

FIGURE 5.5 Experimental model for determination of biliary uptake of test compounds in cultured hepatocytes. Incubation with calcium-free medium would release the compound from bile ducts into medium and allow determination of the amount of compound excreted in bile. 

As an approximation to a universal response detector, an ultraviolet (UV) detection with a high concentration of in vivo sample or in vitro incubation can be used to calibrate the mass spectrometric response of the metabolites. Josephs et al. (2009) recently reported the use of high-performance liquid chromatography (HPLC)-UV detection to get area responses of a 30-tiM in vitro microsome or hepatocyte incubation. The incubated sample was then diluted in matrix to create a single point calibration for mass spectrometric quantitation of the metabolites. The results from this method were successfully verified using buspirone and proprietary compounds for which the synthetic standards for their metabolites were available. 

Diethyl maleate (5 mM) and ethyl methanesul-fonate (35 mM) treatments rapidly depleted cellular reduced glutathione below detectable levels (1 nM/ 10 cells), and induced lipid peroxidation and necrotic cell death in freshly isolated rat hepatocytes (Tirmenstein etal. 2000). In hepatocytes incubated with 2.5 mM diethyl maleate and 10 mM ethyl methanesulfonate, however, the complete depletion of cellular GSH observed was not sufficient to induce lipid peroxidation or cell death. Instead, diethyl maleate- and ethyl methanesulfonate-induced lipid peroxidation and cell death were dependent on increased reactive oxygen species production as measured by increases in dichlorofluorescein fluorescence. The addition of antioxidants (vitamin E succinate and deferoxamine) prevented lipid peroxidation and cell death suggesting that lipid peroxidation is involved in the sequence of events leading to necrotic cell death induced by diethyl maleate and ethyl methanesulfonate. 

This hypothesis is supported by some in vitro studies in which cultured hepatocytes incubated with cheno have been shown to release lactic dehydrogenase and transaminases into the culture medium (11). The "direct toxicity against liver membrane was not observed when urso was tested using the same experimental design. This experimental evidence is of particular interest since it reflects the clinical observation that cheno in some cases produces hypertransaminasemia, but urso does not. 

---