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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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The effect of arachidonic acid (AA; 60 μM) on oxidative stress in HepG2 and HepG2 cells overexpressing CYP2E1. Oxidative stress was assessed 1, 3, 6, 12, and 24 h after AA treatment using flow cytometry detection of dichlorofluorescein diacetate fluorescence. The same AA treatment was done in cells grown prior to the treatment for 1 week in culture medium supplemented with 1 mM acetate (Acetate + AA) or 100 mM ethanol (Ethanol + AA). Mean DCF values after 24 h of AA treatments are also shown in Table 1
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Fig6: The effect of arachidonic acid (AA; 60 μM) on oxidative stress in HepG2 and HepG2 cells overexpressing CYP2E1. Oxidative stress was assessed 1, 3, 6, 12, and 24 h after AA treatment using flow cytometry detection of dichlorofluorescein diacetate fluorescence. The same AA treatment was done in cells grown prior to the treatment for 1 week in culture medium supplemented with 1 mM acetate (Acetate + AA) or 100 mM ethanol (Ethanol + AA). Mean DCF values after 24 h of AA treatments are also shown in Table 1

Mentions: Ethanol itself only slightly but not significantly increased DCF fluorescence in nontransduced and transduced cells (Fig. 6, Table 1) while AA greatly increased oxidative stress only in cells overexpressing CYP2E1 reaching more than fourfold increase (P < 0.01) after 24 h of treatment. In transduced cells but not in HepG2 cells, DCF fluorescence was increased already after 3 h of AA treatment (Fig. 6). In contrast to naïve cells, in transduced cells treated with AA a double peak fluorescence histograms appeared (Fig. 7, histogram C) representing two cell subpopulations—dead cells in the left part of histogram C and alive cells on the right side of the histogram C. AA-induced oxidative stress in transduced cells was reduced by 4MP and slightly less decreased by cell pretreatment with TSA (Table 1) but the fluorescence was not normalized. In cells overexpressing CYP2E1 grown with ethanol or with acetate prior to AA treatment, oxidative stress induced by AA was lower than in naïve cells, respectively, by 25 % (P < 0.01) and by 19 % (P < 0.01).Fig. 6


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)

The effect of arachidonic acid (AA; 60 μM) on oxidative stress in HepG2 and HepG2 cells overexpressing CYP2E1. Oxidative stress was assessed 1, 3, 6, 12, and 24 h after AA treatment using flow cytometry detection of dichlorofluorescein diacetate fluorescence. The same AA treatment was done in cells grown prior to the treatment for 1 week in culture medium supplemented with 1 mM acetate (Acetate + AA) or 100 mM ethanol (Ethanol + AA). Mean DCF values after 24 h of AA treatments are also shown in Table 1
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3927057&req=5

Fig6: The effect of arachidonic acid (AA; 60 μM) on oxidative stress in HepG2 and HepG2 cells overexpressing CYP2E1. Oxidative stress was assessed 1, 3, 6, 12, and 24 h after AA treatment using flow cytometry detection of dichlorofluorescein diacetate fluorescence. The same AA treatment was done in cells grown prior to the treatment for 1 week in culture medium supplemented with 1 mM acetate (Acetate + AA) or 100 mM ethanol (Ethanol + AA). Mean DCF values after 24 h of AA treatments are also shown in Table 1
Mentions: Ethanol itself only slightly but not significantly increased DCF fluorescence in nontransduced and transduced cells (Fig. 6, Table 1) while AA greatly increased oxidative stress only in cells overexpressing CYP2E1 reaching more than fourfold increase (P < 0.01) after 24 h of treatment. In transduced cells but not in HepG2 cells, DCF fluorescence was increased already after 3 h of AA treatment (Fig. 6). In contrast to naïve cells, in transduced cells treated with AA a double peak fluorescence histograms appeared (Fig. 7, histogram C) representing two cell subpopulations—dead cells in the left part of histogram C and alive cells on the right side of the histogram C. AA-induced oxidative stress in transduced cells was reduced by 4MP and slightly less decreased by cell pretreatment with TSA (Table 1) but the fluorescence was not normalized. In cells overexpressing CYP2E1 grown with ethanol or with acetate prior to AA treatment, oxidative stress induced by AA was lower than in naïve cells, respectively, by 25 % (P < 0.01) and by 19 % (P < 0.01).Fig. 6

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