Phase I and phase II xenobiotic biotransformation in cultures and co-cultures of adult rat hepatocytes

Rogiers, Vera; Vandenberghe, Yves; Callaerts, A.; Verleye, Gino; Cornet, Miranda; Mertens, Karin; Sonck, Walter; Vercruysse, Antoine

doi:10.1016/0006-2952(90)90345-l

Cited by 78 publications

(24 citation statements)

References 32 publications

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“…These two features could be related because in pure hepatocyte cultures the addition of proteoglycans improves gap junction activity (137). The enhanced survival and function of hepatocytes cocultured with rat liver epithelial cells have been confirmed by many investigators (134,(138)(139)(140)(141)(142)(143); the induction of CYP2B1 and CYP2B2 by phenobarbital has also been demonstrated (137,144). Moreover, other cells have been effective, including both liver endothelial cells and nonhepatic cells (145)(146)(147).…”

Section: However Survival and Metabolic Compe-mentioning

confidence: 87%

Liver cell models in in vitro toxicology.

Guillouzo

1998

Environ Health Perspect

300

125

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In vitro liver preparations are increasingly used for the study of hepatotoxicity of chemicals. In recent years their actual advantages and limitations have been better defined. The cell models, slices, and mainly primary hepatocyte cultures, appear to be the most powerful in vitro systems, as liver-specific functions and responsiveness to inducers are retained either for a few days or several weeks depending on culture conditions. Maintenance of phase and phase 11 xenobiotic metabolizing enzyme activities allows various chemical investigations to be performed, including determination of kinetic parameters, metabolic profile, interspecies comparison, inhibition and induction effects, and drug-drug interactions. In vitro liver cell models also have various applications in toxicology: screening of cytotoxic and genotoxic compounds, evaluation of chemoprotective agents, and determination of characteristic liver lesions and associated biochemical mechanisms induced by toxic compounds. Extrapolation of the results to the in vivo situation remains a matter of debate. Presently, the most convincing applications of liver cell models are the studies on different aspects of metabolism and mechanisms of toxicity. For the future, there is a need for better culture conditions and differentiated hepatocyte cell lines to overcome the limited availability of human liver tissues. In addition, strategies for in vitro analysis of potentially toxic chemicals must be better defined. Environ Health Perspect 106(Suppl 2):511-532 (1998). http.//ehpnetl.niehs.nih.gov/docs/1998/Suppl-2/51 1-532guillouzo/abstract.html

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Section: However Survival and Metabolic Compe-mentioning

confidence: 87%

Liver cell models in in vitro toxicology.

Guillouzo

1998

Environ Health Perspect

300

125

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“…Loss of P450 activity on culturing has been reported repeatedly using conventional (monolayer) cultures of isolated hepatocytes (Sherratt and Damani, 1989;Rogiers et al, 1990;Wortelboer et al, 1990;McMillan et al, 1991;Bayliss et al, 1994) and has been ascribed to activation of NO-synthesis as a result of collagenase treatment (Lopez-Garcia, 1998) and dedifferentiation of hepatocytes by loss of cell-to-cell or cell-to-matrix interactions. Indeed, culturing of hepatocytes in cocultures with other liver-derived cells and/or in combination with extracellular matrix has expanded the time course in which hepatocytes stay viable (Rogiers et al, 1990) and to some extent has also led to better preserved P450 levels (Koebe et al, 1994;Evans, 1995;Kern et al, 1997). However, damage by collagenase, loss of intercellular communication, and cell-matrix interactions cannot play a role in the loss of P450 activity in the present study and those of others (Wright and Paine, 1992;VandenBranden et al, 1998;Renwick et al, 2000) because no disruptive isolation procedure is used to prepare slices and cellular interactions are presumably maintained.…”

Section: Fig 4 Intrinsic Clearance Of Model Compounds 7-hc (A) Tesmentioning

confidence: 86%

Empirical Validation of a Rat in Vitro Organ Slice Model as a Tool for in Vivo Clearance Prediction

Graaf¹,

Kanter²,

Jager³

et al. 2006

Drug Metab Dispos

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ABSTRACT:Tissue slices have been shown to be a valuable tool to predict metabolism of novel drugs. However, besides the numerous advantages of their use for this purpose, some potential drawbacks also exist, including reported poor penetration of drugs into the inner cell layers of slices and loss of metabolic capacity during prolonged incubation, leading to underprediction of metabolic clearance. In the present study, we empirically identified (and quantified) sources of underprediction using rat tissue slices of lung, intestine, kidney, and liver and found that thin liver slices (؎100 m) metabolized model substrates (7-hydroxycoumarin, testosterone, warfarin, 7-ethoxycoumarin, midazolam, haloperidol, and quinidine) as rapidly as isolated hepatocytes. Furthermore, it was found that organ slices remain metabolically active for sufficient periods of incubation, enabling study of the kinetics of low clearance compounds. In addition, we determined the influence of albumin on the clearance prediction of six model substrates. For three of these substrates, the intrinsic clearance in the presence of albumin was approximately 3 times higher than that obtained from incubations without albumin, but corrected for unbound fraction. This resulted in a much more accurate prediction of in vivo whole body metabolic clearance for these compounds. Collectively, these results show that drawbacks of the use of slices for clearance prediction are largely surmountable. Provided that thin liver slices and physiological albumin concentration are used, whole body metabolic clearance is predicted with acceptable (2-fold) accuracy with organ slices. These results emphasize the applicability of organ slices in this field of research.

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“…We are aware that P4501A2 may not be sufficient for the activation of aromatic amines to ultimate mutagenic metabolites. For example, activity of acetyltransferase, an important enzyme in the production of ultimate mutagens and carcinogens from aromatic amines (1 31, is usually very low in V79 cells (FJ Wiebel, personal communication). However, a V79 variant cell strain (V79-NH) that constitutively expresses the N-acetyltransferase enzyme was made available to us by Dr. Wiebel and is being engineered for expression of cytochrome P4501A2.…”

Section: Discussionmentioning

confidence: 97%

Stable expression of rat cytochrome P450IA2 cDNA and hydroxylation of 17β‐estradiol and 2‐aminofluorene in V79 Chinese hamster cells

Wölfel

Platt

Dogra

et al. 1991

Molecular Carcinogenesis

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In continuation of our work toward the establishment of a working cell bank for metabolic and toxicological studies, V79 Chinese hamster cells were genetically engineered for stable expression of rat cytochrome P450IA2. Full-length cDNA encoding rat P450IA2 was obtained by searching a cDNA library made from Aroclor 1254-induced rat liver mRNA and by joining a small 5'-end fragment to a fragment containing the rest of the cDNA. The sequence of the cDNA was confirmed by DNA sequencing and comparison to a previously published cDNA sequence. The reconstructed full-length cDNA was inserted into a simian virus 40 early promoter-containing eukaryotic expression vector and cotransferred with the neomycin phosphotransferase gene as a selective marker into V79 cells by the calcium/phosphate-coprecipitation technique. G418-resistant V79 cell clones were checked for chromosomal integration of the cDNA by Southern blotting, for expression of authentic mRNA and protein by northern and western blotting, and for P450IA2-specific enzymatic activities such as hydroxylation of 17 beta-estradiol and 2-aminofluorene.

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Phase I and phase II xenobiotic biotransformation in cultures and co-cultures of adult rat hepatocytes

Cited by 78 publications

References 32 publications

Liver cell models in in vitro toxicology.

Liver cell models in in vitro toxicology.

Empirical Validation of a Rat in Vitro Organ Slice Model as a Tool for in Vivo Clearance Prediction

Stable expression of rat cytochrome P450IA2 cDNA and hydroxylation of 17β‐estradiol and 2‐aminofluorene in V79 Chinese hamster cells

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