The differential cytotoxicity of methotrexate in rat hepatocyte monolayer and spheroid cultures

Walker, Tracy M.; Rhodes, Pauline; Westmoreland, Carl

doi:10.1016/s0887-2333(00)00036-9

Cited by 77 publications

(56 citation statements)

References 33 publications

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“…In addition, albumin secretion, urea synthesis, alanine aminotransferase, aspartate aminotransferase, intracellular glutathione levels, etc., are generally used as endpoints for in vitro hepatocellular function (Riss and Moravec, 2004;Wang et al, 2002;Hamid et al, 2004;Viluksela et al, 1996;Huhtala et al, 2002;Smet et al, 2000;Kang et al, 2004;Walker et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

Investigation of a Hepatotoxicity Screening System in Primary Cell Cultures-"What Biomarkers Would Need to Be Addressed to Estimate Toxicity in Conventional and New Approaches?"-

et al. 2005

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-High throughput toxicological estimation is required for safety evaluation in the early stage of drug discovery. In this context, establishment of an in vitro screening system reflecting in vivo toxicity is demanded for earlier safety assessment. We investigated LDH release and mitochondrial respiration (WST-1 reduction assay; WST-1) to detect cytotoxicity, morphological evaluation, and proteomics for estimating the reliable and sensitive biomarkers by using rat primary hepatocytes exposed to the compounds (acetaminophen, amiodarone, tetracycline and carbon tetrachloride) that are known to induce hepatotoxicity. In LDH release, no significant difference was detected between the control and compound exposed cells after exposure for 3 or 6 hr, but a dose-dependent increase was observed after exposure for 24 hr. Regarding the WST-1 assay, a dose-dependent reduction was detected after exposure for 6 and 24 hr to all of the compounds evaluated. In the proteomics analysis, 31 candidate proteins were identified from among the 103 demonstrating altered expression spots after exposure to acetaminophen. It was concluded that the cytotoxicity was detected earlier by measuring WST-1 than by measuring LDH release because the reduction of mitochondrial respiration is an expressions of earlier toxicity for cellular function, while the measured increase in the LDH release occurs after the failure of the cell membrane. Mitochondrial respiration ability was a useful parameter for cytotoxicity in in vitro hepato-toxicity screening, as cytotoxicity can be detected during the early stage of exposure. In addition to the conventional biomarkers, several protein biomarkers which relate to oxidative stress and metabolism-regulation were detected. Further comprehensive analysis of defined proteins would be necessary to estimate the more sensitive toxicology biomarker.

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Section: Introductionmentioning

confidence: 99%

Investigation of a Hepatotoxicity Screening System in Primary Cell Cultures-"What Biomarkers Would Need to Be Addressed to Estimate Toxicity in Conventional and New Approaches?"-

et al. 2005

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“…Liver spheroid culture, reported as early as the 1980s [Landry et al, 1985], was mainly employed in artificial liver studies [Lazar et al, 1995b;Hu et al, 1997]. It also showed potential applications in many other areas such as hepatotoxicology [Sipes et al, 1987;Dilworth et al, 2000;Walker et al, 2000], drug metabolism [Nakazawa et al, 1997], heat shock responses [Gosnell et al, 2002], and tumour studies [Roberts and Soames, 1993].…”

mentioning

confidence: 99%

“…These include self-assembly [Peshwa et al, 1994;Hansen et al, 1998;Yamada et al, 2001], magnetic bar rotationmediated [Yagi et al, 1993], gyrotatory-mediated [Walker et al, 2000], spinner-mediated [Sakai et al, 1996], and foam pore-trapping [Iijima et al, 1998] methods, and entrapping in collagen gel [Hansen et al, 1998] or alginated beads [Khalil et al, 2001]. Whatever the culture method, the liver cells in preparing threedimensional spheroid cultures experience a common process: intact liver tissue !…”

mentioning

confidence: 99%

Biochemical and functional changes of rat liver spheroids during spheroid formation and maintenance in culture: I. morphological maturation and kinetic changes of energy metabolism, albumin synthesis, and activities of some enzymes

Purcell

2003

J of Cellular Biochemistry

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In the process of isolated single liver cells coming together to form three-dimensional spheroids, cells undergo dramatic environmental changes. How liver cells respond to these changes has not been well studied before. This study characterized the functional and biochemical changes during liver spheroid formation and maintenance. Spheroids were prepared in 6-well plates from freshly isolated liver cells from male Sprague rats by a gyrotatory-mediated method. Morphological formation, and functional and biochemical parameters of liver spheroids were evaluated over a period of 21 days in culture. Liver spheroid formation was divided into two stages, immature (1-5 days) and mature (>5 days), according to their size and shape, and changes in their functionality. Galactose and pyruvate consumption was maintained at a relatively stable level throughout the period of observation. However, glucose secretion and cellular GPT and GOT activities were higher in immature spheroids, decreased upto day 5 and remained stable thereafter. Cellular gamma-glutamyltransferase (gamma-GT) and lactate dehydrogenase (LDH) activities were initially undetectable or low and increased as spheroids matured. Albumin secretion decreased rapidly within the first 2 days and increased as spheroids matured. It is concluded that cells undergo functional and biochemical changes during spheroid formation following isolation of liver cells from intact tissue. Functionality and biochemical properties recovered and were maintained in mature spheroids. A relatively stable period (6-15 days) of functionality in mature spheroids was identified and is recommended for applications of the model.

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“…Cells were grown in a humidified incubator at 37°C, with a 5% carbon dioxide atmosphere. For experiments investigating the presence of drugs, hydrocortisone, methotrexate or infliximab was added to the culture medium, diluted to the relevant concentration, as previously described [19,20,21]. …”

Section: Methodsmentioning

confidence: 99%

Gene Therapy for Pyoderma Gangrenosum: Optimal Transfection Conditions and Effect of Drugs on Gene Delivery in the HaCaT Cell Line Using Cationic Liposomes

Teagle¹,

Birchall

Hargest³

2016

Skin Pharmacol Physiol

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Background/Aims: Pyoderma gangrenosum (PG) is a rare ulcerative skin disease, currently treated empirically with immunosuppression. PG is a good target for gene therapy since the skin is easily accessible. This study used the FDA-approved vector Lipofectamine® 2000 to investigate in vitro transfection of skin keratinocytes. The aim was to determine an optimum transfection protocol, including the effect of drugs currently used to treat PG on the efficiency of gene transfer, since gene therapy is unlikely to be used as monotherapy. Methods: Cells of the HaCaT line were transfected with the lacZ reporter gene, and transgene expression was measured after a given time period. Conditions tested were: relative concentrations of DNA and Lipofectamine®, time from transfection to measurement of expression, pH, and exposure to clinically relevant drugs (hydrocortisone, methotrexate, infliximab). Results: The greatest levels of β-galactosidase expression were observed using a DNA:Lipofectamine® ratio of 1:5 (μg/μl) on day 3 after transfection, using culture medium at pH 7, and in the presence of hydrocortisone. Transfection efficiency was reduced by the presence of methotrexate and not significantly affected by infliximab. Conclusion: Gene therapy is a potential future strategy for the management of PG; this study is a step towards the development of a topical gene-based agent.

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The differential cytotoxicity of methotrexate in rat hepatocyte monolayer and spheroid cultures

Cited by 77 publications

References 33 publications

Investigation of a Hepatotoxicity Screening System in Primary Cell Cultures-"What Biomarkers Would Need to Be Addressed to Estimate Toxicity in Conventional and New Approaches?"-

Investigation of a Hepatotoxicity Screening System in Primary Cell Cultures-"What Biomarkers Would Need to Be Addressed to Estimate Toxicity in Conventional and New Approaches?"-

Biochemical and functional changes of rat liver spheroids during spheroid formation and maintenance in culture: I. morphological maturation and kinetic changes of energy metabolism, albumin synthesis, and activities of some enzymes

Gene Therapy for Pyoderma Gangrenosum: Optimal Transfection Conditions and Effect of Drugs on Gene Delivery in the HaCaT Cell Line Using Cationic Liposomes

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