Histone acetylation and arachidonic acid cytotoxicity in HepG2 cells overexpressing CYP2E1

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.

Fig. 1

Acetylated histone H3 (AcH3) and histone H4 (AcH4) in HepG2 cells and HepG2 cells overexpressing CYP2E1 grown for 1 week in culture medium supplemented with 1 mM acetate or 100 mM ethanol. Figures represent mean results ± SD. Data are relative values (digitized relative density) expressed as percentages over untreated cells taken as 100 %. Representative Western blot pictures are included. Statistically significant differences from controls are indicated as: *P < 0.05 or **P < 0.01

Fig. 2

Histone deacetylase 2 (HDAC2) expression in HepG2 cells and HepG2 cells overexpressing CYP2E1 grown for 1 week in culture medium supplemented with 1 mM acetate or 100 mM ethanol. Figures represent mean results ± SD of five experiments. Data are shown as relative values (digitized relative density) expressed as percentages over untreated cells taken as 100 %. Representative Western blot picture is also included. Statistically significant difference from control is indicated as: **P < 0.01

Fig. 3

The effect of arachidonic acid (AA; 60 μM) on viability of HepG2 and HepG2 cells overexpressing CYP2E1. Cell viability was assessed 1, 3, 6, 12, and 24 h after AA treatment using lactate dehydrogenase (LDH) assay. The same AA treatment was also done in cells grown prior to AA treatment for 1 week in culture medium supplemented with 1 mM acetate (Acetate + AA) or 100 mM ethanol (Ethanol + AA). Values of maximal LDH release reflecting total cell destruction were obtained after cell sonication. Each plotted point is a mean value from six experiments. Cytotoxicity data after 24 h of AA treatment are shown also in Table 1

Fig. 4

The effect of arachidonic acid (AA; 60 μM; 24 h) on viability of HepG2 and HepG2 cells overexpressing CYP2E1. Cell viability was assessed with MTT test. The same treatment was done in cells grown for 1 week with medium supplemented with 1 mM acetate or 100 mM ethanol. Results from six independent experiments are expressed as mean percentages of viable cells ± SD and control was taken as 100 %. Statistically significant difference from the control is indicated as *P < 0.05 and ^## P < 0.01—from ethanol-treated cells

Fig. 5

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

Fig. 6

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

Fig. 7

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