Glutathione status in primary cultures of rat hepatocytes and its role in cell attachment to collagen

Morrison, Maria H.; Monte, Donato Di; Jernström, Bengt

doi:10.1016/s0009-2797(85)80079-x

Cited by 19 publications

(4 citation statements)

References 20 publications

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“…This effect can be totally attributed to the actions of trypsin, because when tested alone this agent caused the identical changes of thiol homeostasis in HBF (data not shown) as the PET solution used in this study. A decreased GSH content following trypsinization has also been shown in primary hepatocyte cultures (Morrison et al, 1985). This decline was hypothesized to be dependent on thiol-disulfide exchange reactions leading to the formation of mixed disulfides, a phenomenon clearly demonstrated in the present study.…”

Section: Discussionsupporting

confidence: 81%

Growth‐associated modifications of low‐molecular‐weight thiols and protein sulfhydryls in human bronchial fibroblasts

Atzori

Dypbukt

Sundqvist

et al. 1990

Journal Cellular Physiology

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The thiol redox status of cultured human bronchial fibroblasts has been characterized at various growth conditions using thiol-reactive monobromobimane, with or without the combination of dithiotreitol, a strong reducing agent. This procedure has enabled measurement of the cellular content of reduced glutathione (GSH), total glutathione equivalents, cysteine, total cysteine equivalents, protein sulfhydryls, protein disulfides, and mixed disulfides. Passage of cells with trypsin perturbs the cellular thiol homeostasis and causes a 50% decrease in the GSH content, whereas the total cysteine content is subsequently increased severalfold during cell attachment. During subsequent culture, transient severalfold increased levels of GSH, protein-bound thiols, and protein disulfides are reached, whereas the total cysteine content gradually declines. These changes in the redox balance of both low-molecular-weight thiols and protein-bound thiols correlate with cell proliferation and mostly precede the major growth phase. When the onset of proliferation is inhibited by maintenance of cells in medium containing decreased amounts of serum, the GSH content remains significantly increased. Subsequent stimulation of growth by addition of serum results in decreased GSH levels at the onset of proliferation. In thiol-depleted medium, proliferation is also inhibited, whereas GSH levels are increased to a lesser extent than in complete medium. Exposure to buthionine sulfoximine inhibits growth, prevents GSH synthesis, and results in accumulation of total cysteine, protein-bound cysteine, and protein disulfides. For extracellular cystine, variable rates of cellular uptake correlate with the initial increase in the total cysteine content observed following subculture and with the GSH peak that precedes active proliferation. The results strongly suggest that specific fluctuations in the cellular redox balance of both free low-molecular-weight thiols and protein sulfhydryls are involved in growth regulation of normal human fibroblasts.

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Section: Discussionsupporting

confidence: 81%

Growth‐associated modifications of low‐molecular‐weight thiols and protein sulfhydryls in human bronchial fibroblasts

Atzori

Dypbukt

Sundqvist

et al. 1990

Journal Cellular Physiology

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“…The depletion of GSH in the early period of culture is probably due to its regulatory function of thiol/disulfide groups in protein and/or its involvement in the synthesis of essential cytoskeletal proteins. 29 Our results agree with this report, but after 24 h of culture, all groups showed a similar pattern of GSH level decrease. In that study, 29 a period of cell culture longer than 24 h was not investigated.…”

Section: Discussionsupporting

confidence: 93%

“…This ARE could represent a 50% clearance in plasma ammonia concentration to values around 50 mM, values compatible with normal ammonium levels in human plasma (19-60 mM). 28 Morrison et al 29 have found that in primary cultures of hepatocytes the intracellular levels of GSH decline within 1 h of culture, to about 50% of that observed in freshly isolated cells. At that point, between 4 and 24 h the GSH levels are replenished when the cells are exposed to medium containing amino acids.…”

Section: Discussionmentioning

confidence: 97%

Primary Culture of Rat Hepatocytes After Cold Storage in the University of Wisconsin Solution: A Tool to Study the Effects of Hypothermic Preservation

Pazo¹,

Rodríguez²,

Vega³

et al. 2002

Cell Preservation Technology

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The effect of long-term hypothermic preservation on the function and morphology of rat hepatocytes cultured up to 120 h was studied. Cold storage was performed using modified University of Wisconsin solution, originally developed for organ preservation. After different times of cold storage of hepatocytes, a primary culture system was used to evaluate their ability to attach and form a monolayer of cells on a Petri dish, as well as to perform metabolic functions: capacity of maintaining intracellular enzymes, ammonium removal, and presence of albumin expression. The primary culture system is good enough to evaluate the effects on cells maintained in hypothermia when these cells are placed in normothermic conditions in an oxygenated medium. The results showed that preserved hepatocytes maintained relatively good functions (expressed as attachment, ammonia removal, and albumin expression) when they were preserved up to 72 h in modified University of Wisconsin solution at 0°C and then cultured for up to 120 h. This work raises the possibility of efficiently studying the functional response of cold-stored cells and provides the potential of having viable and functional cells to be applied in different systems such as bioartificial liver devices or cellular transplantation, overcoming, in part, the scarcity of liver donors. 189

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“…50% of that in freshly isolated hepatocytes with 1 h culture probably due to its regulatory function of thiol/disulfide groups in protein and/or its involvement in the synthesis of essential cytosol proteins, although the GSH content could be recovered between 4 and 24 h in the presence of amino acids in culturing medium. In addition, the attaching of the hepatocytes to collagen in the plate would be attenuated by inhibition of GSH synthesis, which possibly led to a non-cytotoxic loss on the viability (Morrison et al, 1985). Therefore, BSO was perhaps not optimal for the depletion of GSH based on the inconsistent results from various studies.…”

Section: Glutathione Reductase-dtnb Recycling Assaymentioning

confidence: 99%

Structure‐dependent relative toxic potencies of selected pyrrolizidine alkaloids

Gao¹,

Schrenk²

2023

Lebensmittelchemie

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Pyrrolizidine alkaloids are naturally occurring secondary plant metabolites mainly found in plant families of Asteraceae, Boraginaceae, and Fabaceae. Chemically, Pas consist of a pyrrolizidine core bearing hydroxyl groups, the so‐called necine base, and mono‐ or dicarboxylic necine acids bound to the pyrrolizidine core via ester linkages. 1,2‐unsaturated PAs are hepatotoxic, genotoxic, and carcinogenic due to the highly reactive pyrrolic metabolites formed by cytochrome P450 monooxygenases (CYPs) primarily in the liver. The presence of PAs as frequent contaminants in the wide variety of food and feed products has to be considered a relevant safety issue.Based on the currently available data, the risk assessment of PAs was mainly approached using the two most toxic potent congeners, i.e., lasiocarpine and riddelliine. However, it is well recognized that toxicity is differing significantly between the congeners related to their structural features. The risk of PA‐containing products is indeed overestimated, and a comprehensive risk assessment should take these differences into account.After analyzing the data of many PAs, Merz and Schrenk derived interim Relative Potency (iREP) factors to present the differences in their toxicity between the sub‐groups of PA congeners concerning their structural features. The use of such iREP factors could probably provide a more scientific basis for PA risk assessment until sufficient experimental analysis of the toxicities of individual congeners is applied. To obtain a better understanding of the relationship between structure and toxicity of PA congeners and provide more evidence for further refinement of relative (toxic) potency factors, data of the in vitro cytotoxicity, genotoxicity, and mutagenicity of diverse individual PA congeners (lasiocarpine, monocrotalineφ, retrorsine, senecionine, seneciphyllineφ, echimidineφ, europineφ, heliotrineφ, indicine, and lycopsamine) has been generated in our project supported by Kooperation Phytopharmaka. Among them, lasiocarpine, retrorsine, senecionine, indicine and lycopsamine have been investigated on my part.Cytotoxicity was assessed using the Alamar blue assay in primary rat hepatocytes, HepG2 cells, and the HepG2 (CYP3A4) cell line. In HepG2 cells, none of the selected PAs exhibited cytotoxic effects, probably due to the lack of CYPs. In primary rat hepatocytes as well as in HepG2(CYP3A4) cells, a clear structure dependent cytotoxicity could be demonstrated. The role of CYP450 enzymes in metabolic activation was further confirmed using an inhibition assay. A kinetic assay analyzing 7‐benzyloxyresorufin‐O‐ dealkylation (BROD) was used for measuring the activity of CYP450 enzymes. Furthermore, the utilization of a glutathione‐reductase‐DTNB recycling assay indicated that glutathione might not play a critical role in PA‐induced cytotoxicity. A micronucleus test was used for determining the PA‐induced clastogenic genotoxicity. All selected PA congeners exhibited concentration‐dependent toxicity in the HepG2 (CYP3A4) cells. The relative potencies of PA congeners estimated from Alamar blue assay and micronucleus assay are generally consistent with the following ranking: lasiocarpine > senecionine > seneciphylline > retrorsine > heliotrine (?) echimidine > europine = indicine = lycopsamine = monocrotaline. The relative toxic potencies evaluated based on our findings were not completely consistent with the iREP classification previously reported by Merz and Schrenk. Monocrotaline in both assays exhibited considerably lower toxic potency. Echimidine, however, was more toxic than expected. On the other hand, mutagenicity was measured in Ames fluctuation assay with Salmonella typhimurium strains TA98 and TA100. None of the selected PA congeners up to 300 μM showed mutagenic effects despite metabolic activation with S9‐mix.

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Glutathione status in primary cultures of rat hepatocytes and its role in cell attachment to collagen

Cited by 19 publications

References 20 publications

Growth‐associated modifications of low‐molecular‐weight thiols and protein sulfhydryls in human bronchial fibroblasts

Growth‐associated modifications of low‐molecular‐weight thiols and protein sulfhydryls in human bronchial fibroblasts

Primary Culture of Rat Hepatocytes After Cold Storage in the University of Wisconsin Solution: A Tool to Study the Effects of Hypothermic Preservation

Structure‐dependent relative toxic potencies of selected pyrrolizidine alkaloids

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