Time course of cytochromes P450 decline during rat hepatocyte isolation and culture: effect of l-NAME

Binda, Delphine; Lasserre-Bigot, Dominique; Bonet, Alexandre; Thomassin, Mireille; Come, M.P; Guinchard, C.; Bars, R.; Jacqueson, A.; Richert, Lysiane

doi:10.1016/s0887-2333(02)00118-2

Cited by 36 publications

(21 citation statements)

References 35 publications

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“…The data of the present communication thus confirm these observations and highlight that some other P450 isoforms are, in contrast, up-regulated after plating. We Binda et al, 2003) have previously suggested that the decline in P450 expression could be related to oxidative stress occurring during time in culture. Several genes associated with a cellular oxidative stress response, such as superoxide dismutase 2, glutathione reductase, p53, and peroxiredoxin, are overexpressed upon plating of human hepatocytes (data not shown).…”

Section: Genomic Expression In Human Hepatocytesmentioning

confidence: 98%

Gene Expression in Human Hepatocytes in Suspension After Isolation Is Similar to the Liver of Origin, Is Not Affected by Hepatocyte Cold Storage and Cryopreservation, but Is Strongly Changed After Hepatocyte Plating

Richert¹,

Liguori²,

Abadie³

et al. 2006

Drug Metab Dispos

120

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ABSTRACT:Isolated primary human hepatocytes are a well accepted system for evaluating pharmacological and toxicological effects in humans. However, questions remain regarding how culturing affects the liver-specific functions of the hepatocytes. In addition, cryopreservation could also potentially affect the differentiation state of the hepatocytes. The first aim of the present study was to compare gene expression in freshly isolated primary hepatocytes to that of the liver of origin and to evaluate the expression changes occurring after cryopreservation/thawing, both when maintained in suspension and after plating. The second aim of the present study was to evaluate gene expression in hepatocytes after cold storage of suspensions up to 24 h compared with freshly isolated hepatocytes in suspension. Our results show that the gene expression in freshly isolated human hepatocytes in suspension after isolation is similar to that of the liver of origin. Furthermore, gene expression in primary human hepatocytes in suspension is not affected by hepatocyte cold storage and cryopreservation. However, the gene expression is profoundly affected in monolayer cultures after plating. Specifically, gene expression changes were observed in cultured relative to suspensions of human hepatocytes that are involved in cellular processes such as phase I/II metabolism, basolateral and canalicular transport systems, fatty acid and lipid metabolism, apoptosis, and proteasomal protein recycling. An oxidative stress response may be partially involved in these changes in gene expression. Taken together, these results may aid in the interpretation of data collected from human hepatocyte experiments and suggest additional utility for cold storage and cryopreservation of hepatocytes.The liver serves as the primary site of detoxification of exogenous and endogenous compounds in the systemic circulation. Other biological and physiological functions include the production and secretion of critical blood and bile components, such as albumin, bile salts, and cholesterol. The liver is also involved in protein, steroid, and fat metabolism, as well as vitamin, iron, and sugar storage.One of the most complex functions specific to liver is its ability to metabolize an enormous range of xenobiotics. Many drugs present in the blood are absorbed by hepatocytes, where they can be metabolized via phase I and II biotransformation reactions. Information concerning hepatic drug uptake and metabolism, phase I and phase II induction, drug interactions affecting hepatic metabolism, and hepatotoxicity are essential for the pharmacology and toxicology of a given drug. Because of major species differences both in the catalytic activities and regulation of enzymes involved in drug metabolism, many of these evaluations can only be accurately investigated with human tissue. Because intact cells more closely reflect the environment to which drugs are exposed in the liver, isolated primary human hepatocytes are a well accepted system for evaluating pharmacological and to...

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Section: Genomic Expression In Human Hepatocytesmentioning

confidence: 98%

Gene Expression in Human Hepatocytes in Suspension After Isolation Is Similar to the Liver of Origin, Is Not Affected by Hepatocyte Cold Storage and Cryopreservation, but Is Strongly Changed After Hepatocyte Plating

Richert¹,

Liguori²,

Abadie³

et al. 2006

Drug Metab Dispos

120

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“…The expression of many liver-specific genes decreases during the initial stage of cultivation of PHHs (Boess et al, 2003;Richert et al, 2006), often associated with declines in metabolic capacities (Richert et al, 2002;Binda et al, 2003;Madan et al, 2003). They are, however, maintained at acceptable levels over time in longterm cultures using sandwich configuration and an appropriate medium (Hewitt et al, 2007;Tuschl et al, 2009;Mueller et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Transcriptomic Hepatotoxicity Signature of Chlorpromazine after Short- and Long-Term Exposure in Primary Human Sandwich Cultures

et al. 2013

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Drug-induced liver injury is the most frequent reason for market withdrawal of approved drugs, and is difficult to predict in animal models. Here, we analyzed transcriptomic data derived from shortand long-term cultured primary human hepatocytes (PHH) exposed to the well known human hepatotoxin chlorpromazine (CPZ). Samples were collected from five PHH cultures after short-term (1 and 3 days) and long-term (14 days) repeat daily treatment with 0.1 or 0.2 mM CPZ, corresponding to C max . Two PHH cultures were additionally treated with 1 mM CPZ, and the three others with 0.02 mM CPZ. Differences in the total number of gene changes were seen between donors and throughout treatment. Specific transcriptomic hepatotoxicity signatures were created for CPZ and consisted of inflammation/hepatitis, cholestasis, and liver proliferation in all five donors, as well as fibrosis and steatosis, which were observed in four of five donors. Necrosis was present in three of five donors, and an indicative signature of cirrhosis was observed after long-term 14-day repeat treatment, also in three of five donors. The inter-donor variability in the inflammatory response to CPZ treatment was associated with variability in the strength of the response of the transcriptomic hepatotoxicity signatures, suggesting that features of inflammation could be related to the idiosyncratic hepatotoxic effects of CPZ in humans.

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“…These assays focus mainly on ligandreceptor interactions in which activation of a specific receptor is linked to a transcription reporter mechanism. Assays based on mammalian cell lines are in general more sensitive; however, the metabolic capacity of both mammalian and yeast cellbased assays is rather limited [11][12][13]. The latter are relatively easy to use and very robust, making them suitable for screening of samples from practice without complex sample cleanup procedures.…”

Section: Introductionmentioning

confidence: 99%

Evidence of the indirect hormonal activity of prohormones using liver S9 metabolic bioactivation and an androgen bioassay

Rijk¹,

Bovee²,

Groot³

et al. 2008

Anal Bioanal Chem

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Prohormones such as dehydroepiandrosterone (DHEA) are steroid precursors that do not show hormonal activity by themselves. Abuse of these prohormones in cattle fattening is hard to prove because of strong in vivo metabolism and the difficulty to detect metabolites which are not significantly above endogenous levels. The aim of the present work was to develop an in vitro assay capable of detecting the indirect hormonal activity of prohormones that might be present in feed supplements and injection preparations. Sample extracts were incubated with a bovine liver S9 fraction in order to mimic the in vivo metabolic activation. Subsequently incubated extracts were exposed to a highly androgen-specific yeast bioassay to detect hormonal activity. Metabolic activation of DHEA, 4-androstene-3,17-dione (4-adione) and 5-androstene-3,17-diol (5-adiol) resulted in an increased androgenic activity caused by the formation of the active androgen 17β-testosterone (17β-T), as shown by ultra-performance liquid chromatography and time-of-flight mass spectrometry with accurate mass measurement. The developed in vitro system successfully mimics the hydroxysteroid dehydrogenase (HSD)-and cytochrome P450-mediated in vivo metabolic transitions, thus allowing assessment of both bioactivity and chemical identification without the use of animal experiments. Screening of unknown supplement samples claimed to contain DHEA resulted in successful bioactivation and positive screening results according to the androgen yeast biosensor.

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Time course of cytochromes P450 decline during rat hepatocyte isolation and culture: effect of l-NAME

Cited by 36 publications

References 35 publications

Gene Expression in Human Hepatocytes in Suspension After Isolation Is Similar to the Liver of Origin, Is Not Affected by Hepatocyte Cold Storage and Cryopreservation, but Is Strongly Changed After Hepatocyte Plating

Gene Expression in Human Hepatocytes in Suspension After Isolation Is Similar to the Liver of Origin, Is Not Affected by Hepatocyte Cold Storage and Cryopreservation, but Is Strongly Changed After Hepatocyte Plating

Transcriptomic Hepatotoxicity Signature of Chlorpromazine after Short- and Long-Term Exposure in Primary Human Sandwich Cultures

Evidence of the indirect hormonal activity of prohormones using liver S9 metabolic bioactivation and an androgen bioassay

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