Rat hepatocytes

A perfusion apparatus can be constructed and used to perfuse a rat liver with a solution of 0.05% collagenase in PBS-A (Seglen, 1976). Following treatment the released hepatocytes can be plated out onto collagen surfaces in the presence of 10% bovine serum, insulin and glucagon (10 /u,g/ml) and hydrocortisone (1 /iM). Attachment occurs within 3 h but no cell division occurs though liver specific enzymes 

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In rat liver mitochondria, in state 4, the AP was estimated to be about 220 mV, with the membrane potential representing about 90% of this (Nicholls, 1974 Appendix 3). Similar values have been reported for human and rat skeletal muscle mitochondria in state 4 (Stumpf et al., 1982). The control of the rate of electron transport is not only determined by the availability of ADP, but also of Pj oxidizable substrates, and oxygen. There is evidence for futile cycling of protons in intact normal rat hepatocytes (Brand et al., 1993). Recently, Porter and Brand (1993) found a correlation between the proton permeability of the inner membrane of liver mitochondria and body size in animals from the mouse (20 g) to horses (150 kg) with a decrease in permeability with increasing weight of several-fold at a constant... 

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. 

Anderson PN, Eaton DE, Murphy SD. 1992. Comparative metabolism of methyl parathion in intact and subcellular fractions of isolated rat hepatocytes. Fundam Appl Toxicol 18 221-226. 

Fetal rat hepatocytes DNA adducts + No data Dubois etal. 1996... 

Rat hepatocyte culture Unscheduled DNA synthesis No data - Hoechst 1984c... 

Fransson-Steen R, Warngard L. 1992. Inhibitory effects of endosulfan on gap junctional intercellular communication in WB-F344 rat liver cells and primary rat hepatocytes. Carcinogenesis 13(4) 657-662. 

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. 

The success of the soft ionization techniques (DCI, FAB, and LSIMS) presents several possibilities for detection of brevetoxins in complex matrices. Positive-ion DCI was used for the analysis of PbTx-3 metabolites generated in vitro by isolated rat hepatocytes (see below). Unmetabolized parent was conclusively identified and metabolites were tentatively identified, pending confirmation by alternate methods (see below). 

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... 

Rat (hepatocyte unscheduled DNA synthesis) breaks) DNA damage (unspecified) Mirsalis et al. 1989... 

Bai CL, Stacey NH. 1993. Mechanism of trichloroethylene-induced elevation of individual serum bile acids. II. In vitro and in vivo interference by trichloroethylene with bile acid transport in isolated rat hepatocytes. Toxicol Appl Pharmacol 121 296-302. 

Shimada T, Swanson AF, Leber P, et al. 1985. Activities of chlorinated ethane and ethylene compounds in the Salmonella microsome mutagenesis and rat hepatocyte/DNA repair assays under vapor phase exposure conditions. Cell Biol Toxicol 1 159-179. 

Ireson, C. et al.. Characterization of metabolites of the chemopreventive agent curcumin in human and rat hepatocytes and in the rat in vivo, and evaluation of their ability to inhibit phorbol ester-induced prostaglandin E2 production. Cancer Res., 61, 1058, 2001. 

The identification and quantification of potentially cytotoxic carbonyl compounds (e.g. aldehydes such as pentanal, hexanal, traw-2-octenal and 4-hydroxy-/mAW-2-nonenal, and ketones such as propan- and hexan-2-ones) also serves as a useful marker of the oxidative deterioration of PUFAs in isolated biological samples and chemical model systems. One method developed utilizes HPLC coupled with spectrophotometric detection and involves precolumn derivatization of peroxidized PUFA-derived aldehydes and alternative carbonyl compounds with 2,4-DNPH followed by separation of the resulting chromophoric 2,4-dinitrophenylhydrazones on a reversed-phase column and spectrophotometric detection at a wavelength of378 nm. This method has a relatively high level of sensitivity, and has been successfully applied to the analysis of such products in rat hepatocytes and rat liver microsomal suspensions stimulated with carbon tetrachloride or ADP-iron complexes (Poli etui., 1985). 

Poli, G., Dianzani, M.U., Cheeseman, K.H., Slater, T.F., Lang, J. and Esterbauer, H. (1985). Separation and characterization of the aldehydic products of lipid peroxidation stimulated by carbon tetrachloride or ADP-iron in isolated rat hepatocytes and rat liver microsomal suspensions. Biochem. J. 227, 629-638,... 

Garcia Ruiz, C., Fernandez Checa, J. and Kaplowitz, N. (1992). Bidirectional mechanism of plasma membrane transport of reduced glutathione in intact rat hepatocytes and membrane vesicles. J. Biol. Chem. 267, 2256-2264. 

Dianzani, M.U., Paradisi, L., Barrera, G., Rossi, M.A. and Parola, M. (1989). The action of 4-hydroxynonenal on the plasma membrane enzymes from rat hepatocytes. In Free Radicals, Metal Ions and Biopolymers (eds. P.C. Neaumont, D.J. Deeble, B.J. Parsons and C. Rice-Evans) pp. 329-346. RicheUeu Press, London. 

Reoxygenation following transient anoxia increases oxygen free radical formation by isolated rat hepatocytes. Hepatology 16, 159A. 

De Gray, J.A., Rao, D.N. and Mason, R.P. (1991). Reduction of paraquat and related bipyridylium compounds to free radical metabolites by rat hepatocytes. Arch. Biochem. Biophys. 289, 145-152. 

De Groot, H. and Brecht, M. (1991). Reoxygenation injury in rat hepatocytes, mediation by O2/H2O2 liberated fixjm sources other than xanthine oxidase. Biol. Chem. Hoppe. Seyler 372, 35-41. 

Ito, Y., Hiraishi, H., Razandi, M., Terano, A., Harada, T. and Ivey, K.J. (1992). Role of cellular superoxide dismutase against reactive oxygen metabolite-induced cell dam e in cultured rat hepatocytes. Hepatology 16, 247-254. 

Glende, E.A. and Pushpendian, C.K. (1986). Activation of phospholipase A2 by carbon tetrachloride in isolated rat hepatocytes. Biochem. Pharmacol. 35, 89-96. 

Morel, I., Lescoat, G., Cogrel, P., Sergent, O., Pasdeloup, N., Brissot, P., Cillard, P. and Cillard, J. (1993). Antioxidant and iron chelating activities of the flavonoids catechin, quercetin and diosmetin on iron-loaded rat hepatocyte cultures. Biochem. Pharmacol. 45, 13-19. 

FIGURE 10.2 Effects of alkylphenols on cell viability compared to the amounts of p-QMs formed in isolated rat hepatocytes. Source From Ref. 28, with permission from the American Chemical Society. 

Thompson, D. C. Constantin, T. D. Moldeus, P. Metabolism and cytotoxicity of eugenol in isolated rat hepatocytes. Chem.-Biol. Interact. 1991, 77, 137-147. 

Rat hepatocyte intrinsic clearance Human microsome intrinsic clearance Rat IV clearance... 

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