Hepatocytes studies, isolated

Digoxin uptake into rat hver shces showed a temperature-dependent component, compatible with the involvement of carrier-mediated uptake mechanisms. Quinine markedly inhibited the uptake of digoxin, in contrast to its diastereomer quinidine, which only slightly inhibited the digoxin uptake in rat liver slices. This stereoselective inhibition is in line with results obtained in isolated rat hepatocytes and isolated perfused rat hvers [90,91]. These results were also found after cryopreservation of the slices, indicating that carrier-specific phenomena can be studied after cryopreservation [92]. 

Hepatocytes isolated from male Wistar rats (180-250 g) were treated with 0.2 mM mono(2-ethylhexyl) phthalate or 1 mM 2-ethylhexanol for 48 h (Gray et al., 1982). Both di(2-ethylhexyl) phthalate metabolites increased carnitine acetyltransferase activity about nine-fold. In studies with hepatocytes from male Sprague-Dawley rats (180-220 g), treatment with 0.2 mM mono(2-ethylhexyl) phthalate and 1.0 mM 2-ethylhexanol for 48 h resulted in induction of carnitine acetyltransferase activity about 15-fold and six-fold, respectively (Gray et al., 1983). Mono(2-ethylhexyl) phthalate was also shown to induce cyanide-insensitive palmitoyl-CoA oxidation and, by ultra-structural examination, to increase numbers of peroxisomes. Hepatocytes were isolated from Wistar-derived rats (180-220 g) and treated for 72 h with 0-0.5 mM mono(2-ethylhexyl) phthalate and some mono(2-ethylhexyl) phthalate metabolites (Mitchell etal., 1985). Treatment with mono(2-ethylhexyl) phthalate and metabolites VI and IX (see Figure 1) resulted in a concentration-dependent induction of cyanide-insensitive palmitoyl-CoA oxidation. In addition, 0-0.5 mM mono(2-ethylhexyl) phthalate and 0-1.0 mM metabolite VI produced concentration-dependent increases in lauric acid hydroxylation. Treatment with metabolites I and V resulted in only small effects on the enzymatic markers of peroxisome proliferation. In another study with hepatocytes from Wistar-derived rats (180-220 g), metabolite VI was shown by subjective ultrastructural examination to cause proliferation of peroxisomes (Elcombe Mitchell, 1986). 

Primary hepatocyte cultures may also be employed to study species differences in hepatic peroxisome proliferation (lARC, 1995 Doull et al, 1999). Hepatocytes were isolated from male Sprague-Dawley rats (180-220 g), male Syrian hamsters (70-80 g) and male Dunkin-Hartley guinea-pigs (400-450 g). Treatment with 20-200 0,M mono(2-ethylhexyl) phthalate for 70 h caused strong induction of cyanide-insensitive palmitoyl-CoA oxidation activity in rat hepatocytes (up to 600% of control levels), while no marked effect was observed in Syrian hamster (up to 120% of control) or guinea-pig (down to 80% of control) hepatocytes (Lake et al., 1986). 

Cultured mammalian cells and isolated hepatocyte studies. There have been several recent reports of the damaging effects of visible light exposure (>400 nm) on various microorganisms (9,10) and cultured mammalian cells (11-13). We have reported that human diploid cells on exposure to visible light (14) and oxygen (>10%) (15) lost the ability to proliferate, while ultrastructural studies showed the presence of numerous damaged mitochondria in the illuminated cells (16). WI-38 human fibroblasts show a grad-... 

The challenge is to predict systemic clearance, volume of distribution and oral bioavailability in humans from a combination of in vitro and in vivo preclinical data. If this prediction can become reliable, then phase I studies become more confirmatory. The use of human hepatocytes and isolated enzymes can form a critical part of the in vitro database. 

In vitro cytotoxicity assays using isolated cells have been applied intermittently to cyanobacterial toxicity testing over several years." Cells investigated for suitability in cyanobacterial toxin assays include primary liver cells (hepatocytes) isolated from rodents and fish, established permanent mammalian cell lines, including hepatocytes, fibroblasts and cancerous cells, and erythrocytes. Earlier work suggested that extracts from toxic cyanobacteria disrupted cells of established lines and erythrocytes," but studies with purified microcystins revealed no alterations in structure or ion transport in fibroblasts or erythrocytes,... 

In a study of the metabolism of methyl parathion in intact and subcellular fractions of isolated rat hepatocytes, a high performance liquid chromatography (HPLC) method has been developed that separates and quantitates methyl parathion and six of its hepatic biotransformation products (Anderson et al. 1992). The six biotransformation products identified are methyl paraoxon, desmethyl parathion, desmethyl paraoxon, 4-nitrophenol, />nitrophenyl glucuronide, and /wiitrophenyl sulfate. This method is not an EPA or other standardized method, and thus it has not been included in Table 7-1. 

In vitro studies on isolated cells including hepatocytes, erythrocytes, fibroblasts, and alveolar cells continue to demonstrate the specificity of action that these toxins have for liver cells (83,86,93). This specificity has led Aune and Berg (94) to use isolated rat hepatocytes as a screen for detecting hepatotoxic waterblooms of cyanobacteria. 

Of particular interest in brevetoxin research are the diagnosis of intoxication and identification of brevetoxins and their metabolites in biological fluids. We are investigating the distribution and fate of radiolabeled PbTx-3 in rats. Three model systems were used to study the toxicokinetics and metabolism of PbTx-3 1) rats injected intravenously with a bolus dose of toxin, 2) isolated rat livers perfused with toxin, and 3) isolated rat hepatocytes exposed to the toxin in vitro. 

In vitro metabolism of pH]PbTx-3 was studi in isolated rat hepatocytes (25). Hepatocyte monolayers cultured in 6-well plates containing 1 ml modified Williams E medium were incubated with 0.1 fig radiolabeled toxin at 37 C for 24 hr. The... 

These studies represent the first report of the metabolism of brevetoxins by mammalian systems. PbTx-3 was rapidly cleared from the bloodstream and distributed to the liver, muscle, and gastrointestinal tract. Studies with isolated perfused livers and isolated hepatocytes conflrmed the liver as a site of metabolism and biliary excretion as an important route of toxin elimination. [ H]PbTx-3 was metabolized to several compounds exhibiting increased polarity, one of which appeared to be an epoxide derivative. Whether this compound corresponds to PbTx-6 (the 27,28 epoxide of PbTx-2), to the corresponding epoxide of PbTx-3, or to another structure is unknown. The structures of these metabolites are currently under investigation. 

Data from both in vivo and in vitro systems showed PbTx-3 to have an intermediate extraction ratio, indicating in vivo clearance of PbTx-3 was equally dependent upon liver blood flow and the activity of toxin-metabolizing enzymes. Studies on the effects of varying flow rates and metabolism on the total body clearance of PbTx-3 are planned. Finally, comparison of in vivo metabolism data to those derived from in vitro metabolism in isolated perfused livers and isolated hepatocytes suggested that in vitro systems accurately reflect in vivo metabolic processes and can be used to predict the toxicokinetic parameters of PbTx-3. 

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. 

Chemiluminescence has been used to show increased free-radical production in I/R injury in isolated hepatocytes and in isolated rat livers (Caraceni et al., 1992 Nunes et al., 1992). Studies in isolated rat liver have shown that ischaemia results in increased conversion of... 

Thor, H., Smith, M.T., Hartzell, P., Bellomo, G., Jewell, S.A. and Orrenius, S. (1982). The metabolism of menadione (2-methyl-1,4-naphthoquinone) by isolated hepatocytes. A study of the impact of oxidative stress in the intact cell. J. Biol. Chem. 257, 12419-12425. 

Sandker, G. W., Weert, B., Olinga, P., Wolters, H., Slooff, M. J., Meijer, D. K., Groothuis, G. M., Characterization of transport in isolated human hepatocytes. A study with the bile add taurocholic add, the uncharged ouabain and the organic... 

Guillouzo, A. (1986). Use of isolated and cultured hepatocytes for xenobiotic metabolism and cytotoxicity studies. In Isolated and Cultured Hepatocytes (Guillouzo, A. and Guguen-Guillouzo, C., Eds.). John Libbey, London, pp. 313-332. 

McQueen, C.A. and Williams, G.M. (1987). Toxicology studies in cultured hepatocytes from various species. In The Isolated Hepatocyte Use in Toxicology and Xenobiotic Biotransformation (Rauckman, E.J. and Padilla, G.M., Eds.). Academic Press, Orlando, FL, pp. 51-67. 

OHara TM, Borzelleca JF, Clarke EC, et al. 1989. A CC14/CHC13 interaction study in isolated hepatocytes Selection of a vehicle. Fundam Appl Toxicol 13 605-615. 

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