Use of Primary Rat and Human Hepatocyte Sandwich Cultures for Activation of Indirect Carcinogens: Monitoring of DNA Strand Breaks and Gene Mutations in Co-cultured Cells

Fahrig, Rudolf; Rupp, Martin; Steinkamp-Zucht, Angela; Bader, Augustinus

doi:10.1016/s0887-2333(98)00005-8

Cited by 17 publications

(5 citation statements)

References 25 publications

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“…The inadequate representation of drug metabolising enzymes is a general problem which is also encountered in acute toxicity and genotoxicity experiments with stable cells lined and various attempts were made to solve it. For example, the cultivation conditions of primary hepatocytes which possess a broad spectrum of xenobiotic drug metabolising enzymes have been improved (561,562) , other solutions may be the establishment of hepatoma cell lines which have retained the activities of several enzymes in an inducible form, (for review see (299) ) and the construction of genetically engineered lines which express human phase I and phase II enzymes (563,564) . The cultivation conditions of the cells may have a strong impact on the outcome of antioxidant studies.…”

Section: Use Of -Omics Based Approaches For the Investigation Of Oxidmentioning

confidence: 99%

Use of conventional and -omics based methods for health claims of dietary antioxidants: a critical overview

et al. 2008

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This article describes the principles and limitations of methods used to investigate reactive oxygen species (ROS) protective properties of dietary constituents and is aimed at providing a better understanding of the requirements for science based health claims of antioxidant (AO) effects of foods. A number of currently used biochemical measurements aimed of determining the total antioxidant capacity and oxidised lipids and proteins are carried out under unphysiologcial conditions and are prone to artefact formation. Probably the most reliable approaches are measurements of isoprostanes as a parameter of lipid peroxidation and determination of oxidative DNA damage. Also the design of the experimental models has a strong impact on the reliability of AO studies: the common strategy is the identification of AO by in vitro screening with cell lines. This approach is based on the assumption that protection towards ROS is due to scavenging, but recent findings indicate that activation of transcription factors which regulate genes involved in antioxidant defence plays a key role in the mode of action of AO. These processes are not adequately represented in cell lines. Another shortcoming of in vitro experiments is that AO are metabolised in vivo and that most cell lines are lacking enzymes which catalyse these reactions. Compounds with large molecular configurations (chlorophylls, anthocyans and polyphenolics) are potent AO in vitro, but weak or no effects were observed in animal/human studies with realistic doses as they are poorly absorbed. The development of -omics approaches will improve the scientific basis for health claims. The evaluation of results from microarray and proteomics studies shows that it is not possible to establish a general signature of alterations of transcription and protein patterns by AO. However, it was shown that alterations of gene expression and protein levels caused by experimentally induced oxidative stress and ROS related diseases can be normalised by dietary AO.

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Section: Use Of -Omics Based Approaches For the Investigation Of Oxidmentioning

confidence: 99%

Use of conventional and -omics based methods for health claims of dietary antioxidants: a critical overview

et al. 2008

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“…For example, general expression of CYP211 enzymes is lost in cell culture, but we found higher metabolites in our organotypical model than in freshly isolated cells in our previous study [8]. Additionally, we have demonstrated in our previous work that an organotypical sandwich model is always superior to the conventional collagen‐coated single gel model for drug biotransformation [8,21,41]. Further, we recently reported a new two‐compartment model of diazepam biotransformation in an organotypical culture with mathematical modeling of parameters [26].…”

Section: Discussionmentioning

confidence: 69%

Biotransformation of diazepam in a clinically relevant flat membrane bioreactor model using primary porcine hepatocytes

Maringka¹,

Giri²,

Nieber

et al. 2011

Fundamemntal Clinical Pharma

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In vitro biotransformation of drug using commercial culture medium with serum may not be the ideal culture medium for clinical application in extracorporeal bioartificial liver support (BAL) systems. In these systems, patient's blood or plasma is plumbed to primary hepatocytes within a seeded bioreactor, creating interaction between plasma and seeded hepatocytes. To address this situation, we investigated the biotransformation potential of diazepam in primary porcine hepatocytes with a flat membrane bioreactor (FMB); we used human plasma exposure and serum-free media in organotypical double gel culture model for long-term culture. We investigated diazepam clearance and all major metabolites of diazepam, such as oxazepam, temazepam, and desmethyldiazepam, in conventional single gel and organotypical sandwich models and compared them to the FMB model. Diazepam elimination was higher in double gel cultures with exposure to both SF 3 medium conditions and plasma, when compared to the single gel model in a Petri dish. It was observed that in the FMB, diazepam elimination was stable at about 3 pg/h/cell in plasma and SF 3 exposure. Oxazepam synthesis in the bioreactor was approximately one quarter less than in the Petri dish, but there were no differences between N-desmethyldiazepam and temazepam synthesis in double gel culture. In the flat membrane bioreactor, there was no decrease in the biotransformation of diazepam in plasma exposure compared with the control group. Our results suggest that this plasma exposure bioreactor may offer a useful approach in clinical use of extracorporeal BAL, as well as for drug metabolite investigation into toxicological research.

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“…The RR fruit extract had a remarkably high total antioxidant capacity of 1154 mM TE, considering values of commonly known fruit juices that range between 3 and 30 mM TE (Prior et al, 2003). This is most likely due to the exceptionally high amount of polyphenols (64.9 g of GAE/L 382 F. H. VAN DER WESTHUIZEN ET AL. and cytochrome P450 were significantly inhibited by perfusion and isolation (Richert et al, 2001;Fahrig et al, 1998). This suggests that primary hepatocytes are not the ideal cellular system to investigate the contribution of a putative modulator to GSH metabolism and redox fluxes in vitro.…”

Section: Discussionmentioning

confidence: 99%

In vitro antioxidant, antimutagenic and genoprotective activity of Rosa roxburghii fruit extract

Westhuizen

Rensburg

Rautenbach

et al. 2007

Phytotherapy Research

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The antioxidant properties of the fruit of the Rosa roxburghii (RR) plant have been associated with several putative health promoting effects. The possible cytotoxic, mutagenic/antimutagenic and genotoxic effects of RR fruit extract were investigated. The effect on antioxidant status and protection against induced oxidative stress were also investigated using primary rat hepatocytes. A RR fruit extract containing 45 g/L total ascorbic acid and 65 g/L total polyphenols was used in this study. Dilutions up to 0.08% (v/v) increased significantly the antioxidant status in primary rat hepatocytes. The glutathione redox state was decreased with RR treatment but was increased in Chang liver cells and MT-2 lymphoblast. No cyto-or genotoxicity were observed at levels of up to 5% (v/v) of the fruit extract. In addition, a significant protection against t-BHP induced oxidative stress was observed in primary rat hepatocytes. The Ames test revealed no mutagenic activity using the Salmonella typhimurium strains TA98, TA100 and TA102. A significant antimutagenic effect of the extract was observed against the metabolic activated mutagens 2-acetylaminofluorene and aflatoxin B1 and to a lesser extent against methyl methanesulfonate. It is concluded that these results support the associated health promoting potential of Rosa Roxburghii fruit and in particular against oxidative stress.

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Use of Primary Rat and Human Hepatocyte Sandwich Cultures for Activation of Indirect Carcinogens: Monitoring of DNA Strand Breaks and Gene Mutations in Co-cultured Cells

Cited by 17 publications

References 25 publications

Use of conventional and -omics based methods for health claims of dietary antioxidants: a critical overview

Use of conventional and -omics based methods for health claims of dietary antioxidants: a critical overview

Biotransformation of diazepam in a clinically relevant flat membrane bioreactor model using primary porcine hepatocytes

In vitro antioxidant, antimutagenic and genoprotective activity of Rosa roxburghii fruit extract

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