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Histone acetylation and arachidonic acid cytotoxicity in HepG2 cells overexpressing CYP2E1.

Holownia A, Mroz RM, Wielgat P, Jakubow P, Jablonski J, Sulek J, Braszko JJ - Naunyn Schmiedebergs Arch. Pharmacol. (2013)

Bottom Line: Arachidonic acid altered cell proliferation and was cytotoxic mostly to cells engineered to overexpress CYP2E1 but both effects were significantly lower in cells pretreated with ethanol or acetate.Cytotoxicity was also significantly decreased by 4-methylpyrazole--a CYP2E1 inhibitor and by trichostatin--an inhibitor of histone deacetylases.In cells pretreated with acetate or ethanol, the oxidative stress induced by arachidonic acid was also significantly lower.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Pharmacology, Medical University of Bialystok, Waszyngtona 15A, 15-274, Bialystok, Poland, Holow_sinai@hotmail.com.

ABSTRACT
The aim of this work was to assess the role of ethanol-derived acetate and acetate-mediated histone acetylation in arachidonic acid-induced stress in HepG2 cells and cells overexpressing CYP2E1. Cells were grown for 7 days with 1 mM sodium acetate or 100 mM ethanol; their acetylated histone proteins and histone deacetylase 2 expression was quantified using Western blot. Ethanol- or acetate-pretreated cells were also treated for 24 h with 60 μM arachidonic acid to induce oxidative stress. Cytotoxicity was estimated by lactate dehydrogenase release, 3-[4,5-dimethylthiazolyl-2] 2,5-diphenyltetrazolium bromide test, and by DNA damage, while oxidative stress was quantified using dichlorofluorescein diacetate. Cells grown with ethanol or acetate had increased acetylated histone H3 levels in both cell types and elevated acetylated histone H4 levels in cells overexpressing CYP2E1 but not in naïve cells. In cells overexpressing CYP2E1 grown with ethanol, expression of histone deacetylase 2 was reduced by about 40 %. Arachidonic acid altered cell proliferation and was cytotoxic mostly to cells engineered to overexpress CYP2E1 but both effects were significantly lower in cells pretreated with ethanol or acetate. Cytotoxicity was also significantly decreased by 4-methylpyrazole--a CYP2E1 inhibitor and by trichostatin--an inhibitor of histone deacetylases. In cells pretreated with acetate or ethanol, the oxidative stress induced by arachidonic acid was also significantly lower. Our data indicate that histone hyperacetylation may in some extent protect the cells against oxidative stress. It is possible that acetate may act as an antioxidant at histone level. This mechanism may be relevant to alcohol-induced liver injury.

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Histograms of DCF fluorescence reflecting oxidative stress in HepG2 cells overexpressing CYP2E1 (A) and the same cells treated for 6 (B) or 24 h (C) with 60 μM arachidonic acid (AA). After 6 h of cell treatment, increased oxidative stress (right shifted single peak fluorescence histogram B vs. A) was observed and after 24 h DCF fluorescence histograms were broad and double-peak representing (left part of histogram C) dead cell and (right part of histogram C) alive cells with high levels of oxidative stress
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Fig7: Histograms of DCF fluorescence reflecting oxidative stress in HepG2 cells overexpressing CYP2E1 (A) and the same cells treated for 6 (B) or 24 h (C) with 60 μM arachidonic acid (AA). After 6 h of cell treatment, increased oxidative stress (right shifted single peak fluorescence histogram B vs. A) was observed and after 24 h DCF fluorescence histograms were broad and double-peak representing (left part of histogram C) dead cell and (right part of histogram C) alive cells with high levels of oxidative stress

Mentions: Arachidonic acid (AA; 60 μM, 24 h)-induced lactate dehydrogenase (LDH) release, cytotoxicity, alterations in cell proliferation (S + G2/M cells), and oxidative stress (DCF) in HepG2 cells and HepG2 cells overexpressing CYP2E1 and in the same cell types grown for 1 week prior to AA treatment in the culture medium supplemented with 100 mM ethanol or 1 mM acetate. In some experiments, cells were additionally treated for 24 h with both AA and with CYP2E1 inhibitor, 4-methylpyrazole (4MP; 5 mM) or pretreated for 24 h before AA with trichostatin (TSA, 100 ng/ml) to inhibit histone deacetylases. LDH activity in the culture medium was measured using a LDH cytotoxicity kit and results are expressed as a fraction of total enzyme activity acquired after cell sonication. To quantify oxidative stress, the cells were stained with dichlorofluorescein diacetate (DCF) and green fluorescence was captured in viable cells only using flow cytometry. Alterations in cell growth and AA cytotoxicity were tested in flow cytometry after cell staining with propidium iodide (PI) and analysis of DNA-PI fluorescence histograms using multicycle software. Table shows fractions of damaged cells (subdiploid—GO/G1zone of the DNA fluorescence histograms; Fig. 7—“early G0/G1 cells”) and percentages of proliferating cells in S(DNA synthesis) + G2/M (post-DNA-synthesis/mitosis) phases of cell cycle (Fig. 7). Each PI fluorescence distribution histogram was derived from analysis of at least 5,000 cells and six samples were analyzed in each group. Table shows mean values ± SD


Histone acetylation and arachidonic acid cytotoxicity in HepG2 cells overexpressing CYP2E1.

Holownia A, Mroz RM, Wielgat P, Jakubow P, Jablonski J, Sulek J, Braszko JJ - Naunyn Schmiedebergs Arch. Pharmacol. (2013)

Histograms of DCF fluorescence reflecting oxidative stress in HepG2 cells overexpressing CYP2E1 (A) and the same cells treated for 6 (B) or 24 h (C) with 60 μM arachidonic acid (AA). After 6 h of cell treatment, increased oxidative stress (right shifted single peak fluorescence histogram B vs. A) was observed and after 24 h DCF fluorescence histograms were broad and double-peak representing (left part of histogram C) dead cell and (right part of histogram C) alive cells with high levels of oxidative stress
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3927057&req=5

Fig7: Histograms of DCF fluorescence reflecting oxidative stress in HepG2 cells overexpressing CYP2E1 (A) and the same cells treated for 6 (B) or 24 h (C) with 60 μM arachidonic acid (AA). After 6 h of cell treatment, increased oxidative stress (right shifted single peak fluorescence histogram B vs. A) was observed and after 24 h DCF fluorescence histograms were broad and double-peak representing (left part of histogram C) dead cell and (right part of histogram C) alive cells with high levels of oxidative stress
Mentions: Arachidonic acid (AA; 60 μM, 24 h)-induced lactate dehydrogenase (LDH) release, cytotoxicity, alterations in cell proliferation (S + G2/M cells), and oxidative stress (DCF) in HepG2 cells and HepG2 cells overexpressing CYP2E1 and in the same cell types grown for 1 week prior to AA treatment in the culture medium supplemented with 100 mM ethanol or 1 mM acetate. In some experiments, cells were additionally treated for 24 h with both AA and with CYP2E1 inhibitor, 4-methylpyrazole (4MP; 5 mM) or pretreated for 24 h before AA with trichostatin (TSA, 100 ng/ml) to inhibit histone deacetylases. LDH activity in the culture medium was measured using a LDH cytotoxicity kit and results are expressed as a fraction of total enzyme activity acquired after cell sonication. To quantify oxidative stress, the cells were stained with dichlorofluorescein diacetate (DCF) and green fluorescence was captured in viable cells only using flow cytometry. Alterations in cell growth and AA cytotoxicity were tested in flow cytometry after cell staining with propidium iodide (PI) and analysis of DNA-PI fluorescence histograms using multicycle software. Table shows fractions of damaged cells (subdiploid—GO/G1zone of the DNA fluorescence histograms; Fig. 7—“early G0/G1 cells”) and percentages of proliferating cells in S(DNA synthesis) + G2/M (post-DNA-synthesis/mitosis) phases of cell cycle (Fig. 7). Each PI fluorescence distribution histogram was derived from analysis of at least 5,000 cells and six samples were analyzed in each group. Table shows mean values ± SD

Bottom Line: Arachidonic acid altered cell proliferation and was cytotoxic mostly to cells engineered to overexpress CYP2E1 but both effects were significantly lower in cells pretreated with ethanol or acetate.Cytotoxicity was also significantly decreased by 4-methylpyrazole--a CYP2E1 inhibitor and by trichostatin--an inhibitor of histone deacetylases.In cells pretreated with acetate or ethanol, the oxidative stress induced by arachidonic acid was also significantly lower.

View Article: PubMed Central - PubMed

Affiliation: Department of Clinical Pharmacology, Medical University of Bialystok, Waszyngtona 15A, 15-274, Bialystok, Poland, Holow_sinai@hotmail.com.

ABSTRACT
The aim of this work was to assess the role of ethanol-derived acetate and acetate-mediated histone acetylation in arachidonic acid-induced stress in HepG2 cells and cells overexpressing CYP2E1. Cells were grown for 7 days with 1 mM sodium acetate or 100 mM ethanol; their acetylated histone proteins and histone deacetylase 2 expression was quantified using Western blot. Ethanol- or acetate-pretreated cells were also treated for 24 h with 60 μM arachidonic acid to induce oxidative stress. Cytotoxicity was estimated by lactate dehydrogenase release, 3-[4,5-dimethylthiazolyl-2] 2,5-diphenyltetrazolium bromide test, and by DNA damage, while oxidative stress was quantified using dichlorofluorescein diacetate. Cells grown with ethanol or acetate had increased acetylated histone H3 levels in both cell types and elevated acetylated histone H4 levels in cells overexpressing CYP2E1 but not in naïve cells. In cells overexpressing CYP2E1 grown with ethanol, expression of histone deacetylase 2 was reduced by about 40 %. Arachidonic acid altered cell proliferation and was cytotoxic mostly to cells engineered to overexpress CYP2E1 but both effects were significantly lower in cells pretreated with ethanol or acetate. Cytotoxicity was also significantly decreased by 4-methylpyrazole--a CYP2E1 inhibitor and by trichostatin--an inhibitor of histone deacetylases. In cells pretreated with acetate or ethanol, the oxidative stress induced by arachidonic acid was also significantly lower. Our data indicate that histone hyperacetylation may in some extent protect the cells against oxidative stress. It is possible that acetate may act as an antioxidant at histone level. This mechanism may be relevant to alcohol-induced liver injury.

Show MeSH
Related in: MedlinePlus