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Combined incubation of cadmium, docosahexaenoic and eicosapentaenoic acid results in increased uptake of cadmium and elevated docosapentaenoic acid content in hepatocytes in vitro.

Linhartova P, Sampels S - Lipids Health Dis (2015)

Bottom Line: The resarzurin assay, evaluating cell viability, showed a significant decrease in cell viability between Cd(2+) incubation time and, further, the pre-incubation with BSA-FA complex resulted in significantly increased cell viability.The most important finding is that DHA and EPA reduced the detrimental effect of Cd(2+) on cell viability.The exact effects and kinetics behind our observations still need further evaluation.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Institute of Aquaculture and Protection of Waters, University of South Bohemia in Ceske Budejovice, Husova Tř. 458/102, 370 05, České Budějovice, Czech Republic. linhap01@frov.jcu.cz.

ABSTRACT

Background: Human hepatocellular cells Hep G2 were used to mimic and investigate the effect of the intake of cadmium (Cd(2+)) contaminated fish on cytotoxicity, fatty acid (FA) and phospholipid class composition.

Methods: Cells were incubated with a combination of Cd(2+) and eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) fish specific FA.

Results: We measured a significant increased proportion of EPA and DHA in the treated cells compared to the control line confirming the uptake. While doses of 25 μM DHA showed to be toxic to the cells, repeated short term incubations (2 h) at lower doses resulted in an increased uptake of DHA. The resarzurin assay, evaluating cell viability, showed a significant decrease in cell viability between Cd(2+) incubation time and, further, the pre-incubation with BSA-FA complex resulted in significantly increased cell viability. On the metabolic level, increased concentrations of EPA and DHA resulted in an increased proportion of docosapentaenoic acid (DPA) which indicated an increased metabolism. Also 24-h combined incubations of 5 μM Cd(2+) and EPA and DHA showed a significant increase DPA in the total lipid fraction of the cells. In addition, incubation with 5 μM Cd(2+) for 24 h also decreased the total cardiolipin (CL) fraction from the identified phospholipids.

Conclusions: We confirmed that the applied FA were taken up by the cells. A combination of EPA, DHA and Cd(2+) influenced lysosomal integrity, cell viability and lipid metabolism in the cells. The most important finding is that DHA and EPA reduced the detrimental effect of Cd(2+) on cell viability. The exact effects and kinetics behind our observations still need further evaluation.

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Lysosomal integrity after in vitro pre-incubations of Hep G2 for 24 h with EPA (a) at nominal concentrations of 10, 25, 40 and 50 μM and DHA (b) at nominal concentrations of 25, 50, 75 and 100 μM compared to control cells. Data are presented as means ± SD, n = 3. Different letters denote significant difference between treatments (ANOVA, p < 0.05)
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Fig1: Lysosomal integrity after in vitro pre-incubations of Hep G2 for 24 h with EPA (a) at nominal concentrations of 10, 25, 40 and 50 μM and DHA (b) at nominal concentrations of 25, 50, 75 and 100 μM compared to control cells. Data are presented as means ± SD, n = 3. Different letters denote significant difference between treatments (ANOVA, p < 0.05)

Mentions: In order to establish suitable incubation concentrations of FA, we first incubated the cells with the individual FA ranging for EPA from 1 μM to 50 μM (Fig. 1a) and for DHA from 2 μM to 100 μM (Fig. 1b), respectively. For EPA, the highest 50 μM concentration was above toxic effects for the cells (Fig. 1a). Neither the level of IC50, nor level of the IC70 was reached. No significant change on cell growth measured by lysosomal integrity was found for this FA. For DHA, significant changes were found only between control line (Hep G2) and cells treated with 25 μM DHA and higher DHA concentrations showed significant toxic effects on the cells (Fig. 1b). A significantly negative effect of DHA on Hep G2 cell growth was observed after in vitro for 24 h at concentrations 50 μM DHA (IC70) and the IC50 was reached at the level of 76 μM DHA (Fig. 1b). Furthermore, the cells showed only 29.3 % vitality at a level of 100 μM DHA (Fig 1b). In a second step we evaluated the effects of the incubation with a combination of EPA and DHA. The combination of 40 μM EPA+ 75 μM DHA resulted in a cell viability significantly below 50 % (18.9 % viable cells), while the combination of 10 μM EPA+ 20 μM DHA, was above the level IC70 (85.3 % vital cells) (Fig. 2), but was significantly lower compared to the control cells without added FA. As the level of DHA was still too high, we decreased the concentration to a combination of 5 μM EPA+ 10 μM DHA (Fig. 2). With these concentrations a cell viability of 96.9 % (EPA5 + DHA10) was reached. No cytotoxic effects of FA on cell growth in this combination with control line were found.Fig. 1


Combined incubation of cadmium, docosahexaenoic and eicosapentaenoic acid results in increased uptake of cadmium and elevated docosapentaenoic acid content in hepatocytes in vitro.

Linhartova P, Sampels S - Lipids Health Dis (2015)

Lysosomal integrity after in vitro pre-incubations of Hep G2 for 24 h with EPA (a) at nominal concentrations of 10, 25, 40 and 50 μM and DHA (b) at nominal concentrations of 25, 50, 75 and 100 μM compared to control cells. Data are presented as means ± SD, n = 3. Different letters denote significant difference between treatments (ANOVA, p < 0.05)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4666081&req=5

Fig1: Lysosomal integrity after in vitro pre-incubations of Hep G2 for 24 h with EPA (a) at nominal concentrations of 10, 25, 40 and 50 μM and DHA (b) at nominal concentrations of 25, 50, 75 and 100 μM compared to control cells. Data are presented as means ± SD, n = 3. Different letters denote significant difference between treatments (ANOVA, p < 0.05)
Mentions: In order to establish suitable incubation concentrations of FA, we first incubated the cells with the individual FA ranging for EPA from 1 μM to 50 μM (Fig. 1a) and for DHA from 2 μM to 100 μM (Fig. 1b), respectively. For EPA, the highest 50 μM concentration was above toxic effects for the cells (Fig. 1a). Neither the level of IC50, nor level of the IC70 was reached. No significant change on cell growth measured by lysosomal integrity was found for this FA. For DHA, significant changes were found only between control line (Hep G2) and cells treated with 25 μM DHA and higher DHA concentrations showed significant toxic effects on the cells (Fig. 1b). A significantly negative effect of DHA on Hep G2 cell growth was observed after in vitro for 24 h at concentrations 50 μM DHA (IC70) and the IC50 was reached at the level of 76 μM DHA (Fig. 1b). Furthermore, the cells showed only 29.3 % vitality at a level of 100 μM DHA (Fig 1b). In a second step we evaluated the effects of the incubation with a combination of EPA and DHA. The combination of 40 μM EPA+ 75 μM DHA resulted in a cell viability significantly below 50 % (18.9 % viable cells), while the combination of 10 μM EPA+ 20 μM DHA, was above the level IC70 (85.3 % vital cells) (Fig. 2), but was significantly lower compared to the control cells without added FA. As the level of DHA was still too high, we decreased the concentration to a combination of 5 μM EPA+ 10 μM DHA (Fig. 2). With these concentrations a cell viability of 96.9 % (EPA5 + DHA10) was reached. No cytotoxic effects of FA on cell growth in this combination with control line were found.Fig. 1

Bottom Line: The resarzurin assay, evaluating cell viability, showed a significant decrease in cell viability between Cd(2+) incubation time and, further, the pre-incubation with BSA-FA complex resulted in significantly increased cell viability.The most important finding is that DHA and EPA reduced the detrimental effect of Cd(2+) on cell viability.The exact effects and kinetics behind our observations still need further evaluation.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Fisheries and Protection of Waters, South Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses, Institute of Aquaculture and Protection of Waters, University of South Bohemia in Ceske Budejovice, Husova Tř. 458/102, 370 05, České Budějovice, Czech Republic. linhap01@frov.jcu.cz.

ABSTRACT

Background: Human hepatocellular cells Hep G2 were used to mimic and investigate the effect of the intake of cadmium (Cd(2+)) contaminated fish on cytotoxicity, fatty acid (FA) and phospholipid class composition.

Methods: Cells were incubated with a combination of Cd(2+) and eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) fish specific FA.

Results: We measured a significant increased proportion of EPA and DHA in the treated cells compared to the control line confirming the uptake. While doses of 25 μM DHA showed to be toxic to the cells, repeated short term incubations (2 h) at lower doses resulted in an increased uptake of DHA. The resarzurin assay, evaluating cell viability, showed a significant decrease in cell viability between Cd(2+) incubation time and, further, the pre-incubation with BSA-FA complex resulted in significantly increased cell viability. On the metabolic level, increased concentrations of EPA and DHA resulted in an increased proportion of docosapentaenoic acid (DPA) which indicated an increased metabolism. Also 24-h combined incubations of 5 μM Cd(2+) and EPA and DHA showed a significant increase DPA in the total lipid fraction of the cells. In addition, incubation with 5 μM Cd(2+) for 24 h also decreased the total cardiolipin (CL) fraction from the identified phospholipids.

Conclusions: We confirmed that the applied FA were taken up by the cells. A combination of EPA, DHA and Cd(2+) influenced lysosomal integrity, cell viability and lipid metabolism in the cells. The most important finding is that DHA and EPA reduced the detrimental effect of Cd(2+) on cell viability. The exact effects and kinetics behind our observations still need further evaluation.

Show MeSH
Related in: MedlinePlus