Characterization of isolated Fe-loaded rat hepatocytes prepared by collagenase perfusion

Tyson, Charles A.; Green, Carol E.; LeValley, Susanna E.; Stephens, Robert J.

doi:10.1007/bf02796351

Cited by 8 publications

(4 citation statements)

References 14 publications

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“…Because actual cytosolic conditions can only be estimated, we regard the present method as very suitable for assessing relative changes to the chelatable iron pool in single cells or relative differences between different cell types/cell populations, but recommend that the limitations of ex situ calibration should be kept in mind when interpreting absolute values. We determined a total iron content of 4.7 Ϯ 1.5 nmol/10 6 rat hepatocytes-which is in line with the literature 12,48,49 -suggesting that in these cells, 1.0% Ϯ 0.3% of the total iron is chelatable, comparing well with the values given in the literature of 0.2% to 3.0%. 12,14,18,47 The concentration of chelatable iron in cultured hepatocytes determined with our method (9.8 Ϯ 2.9 µmol/L) differs markedly from the value determined in K562 cells (0.2-0.5 µmol/L) using the fluorescent indicator, calcein.…”

Section: Discussionsupporting

confidence: 89%

Determination of the Chelatable Iron Pool of Isolated Rat Hepatocytes by Digital Fluorescence Microscopy Using the Fluorescent Probe, Phen Green Sk

1999

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The intracellular pool of chelatable iron is considered to be a decisive pathogenetic factor for various kinds of cell injury. We therefore set about establishing a method of detecting chelatable iron in isolated hepatocytes based on digital fluorescence microscopy. The fluorescence of hepatocytes loaded with the fluorescent metal indicators, phen green SK (PG SK), phen green FL (PG FL), calcein, or fluorescein desferrioxamine (FL-DFO), was quenched when iron was added to the cells in a membrane-permeable form. It increased when cellular chelatable iron available to the probe was experimentally decreased by an excess of various membrane-permeable transition metal chelators. The quenching by means of the ferrous ammonium sulfate ؉ citrate complex and also the ''dequenching'' using 2,2Ј-dipyridyl (2,2Ј-DPD) were largest for PG. We therefore optimized the conditions for its use in hepatocytes and tested the influence of possible confounding factors. An ex situ calibration method was set up to determine the chelatable iron pool of cultured hepatocytes from the increase of PG SK fluorescence after the addition of excess 2,2Ј-DPD. Using this method, we found 9.8 ؎ 2.9 mol/L (mean ؎ SEM; n ‫؍‬ 18) chelatable iron in rat hepatocytes, which constituted 1.0% ؎ 0.3% of the total iron content of the cells as determined by atomic absorption spectroscopy. The concentration of chelatable iron in hepatocytes was higher than the one in K562 cells (4.0 ؎ 1.3 mol/L; mean ؎ SEM; n ‫؍‬ 8), which were used for comparison. This method allowed us to record time courses of iron uptake and of iron chelation by different chelators (e.g., deferoxamine, 1,10-phenanthroline) in single, intact cells. (HEPATOLOGY 1999;29: 1171-1179.)

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Section: Discussionsupporting

confidence: 89%

Determination of the Chelatable Iron Pool of Isolated Rat Hepatocytes by Digital Fluorescence Microscopy Using the Fluorescent Probe, Phen Green Sk

1999

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“…A relatively small size for the "free" iron pool has been inferred also from studies in vitro of liver homogenates, in which < 2% of the total iron was chelatable by desferrioxamine (2 1). The findings are in agreement with data indicating that the excess iron in hepatocytes exists as ferritin or hemosiderin (22) and that such storage iron is biologically sequestered (23). Thus, an important controlling factor in the size of the "free" pool may be release of iron from ferritin.…”

Section: Discussionsupporting

confidence: 92%

Iron mediates production of a neutrophil chemoattractant by rat hepatocytes metabolizing ethanol.

Hultcrantz¹,

Bissell²,

Roll³

1991

J. Clin. Invest.

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Ethanol metabolism in hepatocytes is accompanied by release of a potent lipid chemoattractant for neutrophils. Production of the factor may initiate the inflammation associated with alcoholic hepatitis. In previous studies with a cytosol system from liver, production was blocked by iron chelators as well as by catalase and superoxide dismutase, suggesting the involvement of oxyradicals in formation of the chemoattractant. These studies have examined the role of iron in intact hepatocytes using cells from rats fed an iron-deficient diet, a control diet or a diet containing 3% carbonyl iron. The iron content averaged 1.4 nmol/mg protein in iron-deficient cells, 6.3 in controls and 135.3 in iron-loaded cells. Hepatocytes from all groups were established in primary culture and incubated with ethanol (10 mM); the medium was assayed for chemoattractant activity for human neutrophils. Cultures from chow-fed or iron-loaded animals produced chemoattractant as previously reported. By contrast, chemoattractant production was undetectable in the irondeficient cultures. Addition of ferric citrate (10-M) restored chemoattractant production while increasing cellular iron in the deficient cells less than 50% (to 2.3 nmol/mg protein). Addition of desferrioxamine mesylate to cultures of iron-loaded cells ablated chemoattractant production. The data provide evidence for the importance of hepatocellular iron in production of this alcohol-related lipid chemoattractant and suggest that a small intracellular pool of "free" iron plays a critical role. (J. Clin.

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“…Cultured medium from control and treated oval cells were subjected to liver function analysis (Tyson et al 1982) using glutamate pyruvate transaminase (GPT) and glutamate oxaloacetate transaminase (GOT) assay kits (Span Diagnostics, Surat, India). Results were expressed as IU/L.…”

Section: Liver Function Testsmentioning

confidence: 99%

Mercuric chloride effects on adult rat oval cells-induced apoptosis

Chatterjee

Munshi

Chattopadhyay

et al. 2013

Toxicological & Environmental Chemistry

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In the present investigation, the toxicity of mercuric chloride (HgCl 2 ) was evaluated in adult oval cells isolated from rat utilizing the 2-acetylaminofluorene/partial hepatectomy technique. Isolated oval cells were incubated with 5 mM of HgCl 2 for 8 hr to elucidate in vitro cytotoxic responses. Recently, autophagic cell death was found in rat hepatocytes in vitro within 30 min of incubation with 5 mM of mercury (Hg) which triggered apoptosis and necroptosis in a time-dependent manner. Nuclear degradation occurred within 30 min of incubation and progressed with time until 8 hr. Apoptosis evidenced by activation of caspase-dependent pathway between 30 min to 8 hr of incubation was mediated via interchange of death domain signaling pathways. Receptor-interacting protein played a positive role to modulate the death domain receptors in the scenario of apoptotic death of oval cells until 6 hr. Autophagic marker proteins ATG12 and LC3B exerted a significant role in triggering apoptosis in 5 mM Hg-treated oval cells. No apparent expression of apoptosis-inducing-factor (AIF) and HMGB1 indicated absence of caspase-independent apoptosis and necrosis in the rat oval cells between 30 min and 8 hr. Thus a low concentration of Hg modulates programmed cell death in adult rat oval cells by altering expression of proteins involved in the molecular mechanisms of cellular functions.

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Characterization of isolated Fe-loaded rat hepatocytes prepared by collagenase perfusion

Cited by 8 publications

References 14 publications

Determination of the Chelatable Iron Pool of Isolated Rat Hepatocytes by Digital Fluorescence Microscopy Using the Fluorescent Probe, Phen Green Sk

Determination of the Chelatable Iron Pool of Isolated Rat Hepatocytes by Digital Fluorescence Microscopy Using the Fluorescent Probe, Phen Green Sk

Iron mediates production of a neutrophil chemoattractant by rat hepatocytes metabolizing ethanol.

Mercuric chloride effects on adult rat oval cells-induced apoptosis

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