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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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Representative flow cytometry histograms of propidium iodide fluorescence distributions (MultiCycle transformation) in HepG2 cells overexpressing CYP2E1 (a) and in the same cells treated for 24 h with arachidonic acid (b). The cells were quantified by their relative distribution in the damaged-subdiploid GO/G1 zone of the DNA fluorescence histograms (early G0/G1 cells), diploid (G0/G1 zone)—pre-DNA synthesis/resting, S-phase—DNA synthesis, and G2/M—post-DNA-synthesis/mitosis phases. Each histogram was derived from analysis of 5,000 cells and six samples were analyzed in each group. Quantification of cytotoxicity (early G0/G1 cells) and proliferation (S + G2/M cells) is shown in Table 1
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Fig5: Representative flow cytometry histograms of propidium iodide fluorescence distributions (MultiCycle transformation) in HepG2 cells overexpressing CYP2E1 (a) and in the same cells treated for 24 h with arachidonic acid (b). The cells were quantified by their relative distribution in the damaged-subdiploid GO/G1 zone of the DNA fluorescence histograms (early G0/G1 cells), diploid (G0/G1 zone)—pre-DNA synthesis/resting, S-phase—DNA synthesis, and G2/M—post-DNA-synthesis/mitosis phases. Each histogram was derived from analysis of 5,000 cells and six samples were analyzed in each group. Quantification of cytotoxicity (early G0/G1 cells) and proliferation (S + G2/M cells) is shown in Table 1

Mentions: Table 1 shows flow cytometry data of PI fluorescence reflecting cytotoxicity and proliferation of HepG2 cells or HepG2 cells overexpressing CYP2E1, and the same cell types grown for 1 week with 100 mM ethanol or 1 mM acetate and then treated for 24 h with AA as described in Materials and methods. Damaged cells were assessed as “early” G0/G1 cells and cell proliferation was quantified as S + G2/M cells (Fig. 5). Twenty-four hours of cell treatment with AA resulted in cytotoxicity in both cell types, but significantly higher toxicity was again observed in cells overexpressing CYP2E1. In HepG2 cells, AA treatment produced about 11 % of dead cells while in cells overexpressing CYP2E1 approximately 35 % of cells were damaged. AA cytotoxicity was lower in CYP2E1-expressing cells, which prior to AA treatment had grown with ethanol (20 % of dead cells; P < 0.01) or with acetate (18 % of dead cells, P < 0.01), while in HepG2 cells, such protective effects were not observed. 4MP decreased AA cytotoxicity by about 35 % (P < 0.05) in transduced cells only and similar effect was observed when transduced cells were pretreated with TSA (P < 0.05).Fig. 5


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)

Representative flow cytometry histograms of propidium iodide fluorescence distributions (MultiCycle transformation) in HepG2 cells overexpressing CYP2E1 (a) and in the same cells treated for 24 h with arachidonic acid (b). The cells were quantified by their relative distribution in the damaged-subdiploid GO/G1 zone of the DNA fluorescence histograms (early G0/G1 cells), diploid (G0/G1 zone)—pre-DNA synthesis/resting, S-phase—DNA synthesis, and G2/M—post-DNA-synthesis/mitosis phases. Each histogram was derived from analysis of 5,000 cells and six samples were analyzed in each group. Quantification of cytotoxicity (early G0/G1 cells) and proliferation (S + G2/M cells) is shown in Table 1
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3927057&req=5

Fig5: Representative flow cytometry histograms of propidium iodide fluorescence distributions (MultiCycle transformation) in HepG2 cells overexpressing CYP2E1 (a) and in the same cells treated for 24 h with arachidonic acid (b). The cells were quantified by their relative distribution in the damaged-subdiploid GO/G1 zone of the DNA fluorescence histograms (early G0/G1 cells), diploid (G0/G1 zone)—pre-DNA synthesis/resting, S-phase—DNA synthesis, and G2/M—post-DNA-synthesis/mitosis phases. Each histogram was derived from analysis of 5,000 cells and six samples were analyzed in each group. Quantification of cytotoxicity (early G0/G1 cells) and proliferation (S + G2/M cells) is shown in Table 1
Mentions: Table 1 shows flow cytometry data of PI fluorescence reflecting cytotoxicity and proliferation of HepG2 cells or HepG2 cells overexpressing CYP2E1, and the same cell types grown for 1 week with 100 mM ethanol or 1 mM acetate and then treated for 24 h with AA as described in Materials and methods. Damaged cells were assessed as “early” G0/G1 cells and cell proliferation was quantified as S + G2/M cells (Fig. 5). Twenty-four hours of cell treatment with AA resulted in cytotoxicity in both cell types, but significantly higher toxicity was again observed in cells overexpressing CYP2E1. In HepG2 cells, AA treatment produced about 11 % of dead cells while in cells overexpressing CYP2E1 approximately 35 % of cells were damaged. AA cytotoxicity was lower in CYP2E1-expressing cells, which prior to AA treatment had grown with ethanol (20 % of dead cells; P < 0.01) or with acetate (18 % of dead cells, P < 0.01), while in HepG2 cells, such protective effects were not observed. 4MP decreased AA cytotoxicity by about 35 % (P < 0.05) in transduced cells only and similar effect was observed when transduced cells were pretreated with TSA (P < 0.05).Fig. 5

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