P. Cowan

Toxicology of ZnO and TiO2 nanoparticles on hepatocytes: Impact on metabolism and bioenergetics

Abstract Background and aim: Zinc oxide (ZnO) and titanium dioxide (TiO2) nanomaterials (NMs) are used in many consumer products, including foodstuffs. Ingested and inhaled NM can reach the liver. Whilst their effects on inflammation, cytotoxicity, genotoxicity and mitochondrial function have been explored, no work has been reported on their impact on liver intermediary metabolism. Our aim was to assess the effects of sub-lethal doses of these materials on hepatocyte intermediary metabolism. Material and methods: After characterisation, ZnO and TiO2 NM were used to treat C3A cells for 4 hours at concentrations ranging between 0 and 10 μg/cm2, well below their EC50, before the assessment of (i) glucose production and glycolysis from endogenous glycogen and (ii) gluconeogenesis and glycolysis from lactate and pyruvate (LP). Mitochondrial membrane potential was assessed using JC-10 after 0–40 μg/cm2 ZnO. qRT-PCR was used to assess phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression. Dihydroethidium (DHE) staining and FACS were used to assess intracellular reactive oxygen species (ROS) concentration. Results: Treatment of cells with ZnO, but not TiO2, depressed mitochondrial membrane potential, leading to a dose-dependent increase in glycogen breakdown by up to 430%, with an increase of both glycolysis and glucose release. Interestingly, gluconeogenesis from LP was also increased, up to 10-fold and correlated with a 420% increase in the PEPCK mRNA expression, the enzyme controlling gluconeogenesis from LP. An intracellular increase of ROS production after ZnO treatment could explain these effects. Conclusion: At sub-lethal concentrations, ZnO nanoparticles dramatically increased both gluconeogenesis and glycogenolysis, which warrants further in vivo studies.

PMO-122 Eicosapentaenoic acid is effective at reducing hepatocyte triglyceride content of untreated C3A cells but is not effective in two models of cellular steatosis

Introduction Eicosapentaenoic acid (EPA), one of the major physiologically active constituents of Omega-3 fatty acids, has been suggested as a treatment for non-alcoholic fatty liver disease (NAFLD). The aim of these experiments was to assess the effects of EPA on intrahepatic triglyceride content in cell culture models of steatosis. Methods Human C3a hepatocytes were incubated in MEME (standard media) and two models of cellular steatosis: oleate (a model of isolated steatosis) and LPON (a model of steatosis and mitochondrial dysfunction containing the gluconeogenic substrates Lacate, Pyruvate, Octanoate and ammoNia). Test media was either unsupplemented, or supplemented with 50 μM or 250 μM EPA. Hepatocyte triglyceride accumulation was assessed both by microscopy (using oil red staining) and by quantifying the intracellular triglyceride concentration of cells incubated in culture media for 3 and 7 days. Each cell culture experiment was performed in triplicate. Results MEME When quantified by oil red staining a 73.1% (95% CI 63% to 83%) reduction in cell triglyceride content with 250 μM EPA compared with untreated cells was seen (7659 vs 28 564 pixels; p<0.001). This was confirmed in cell culture experiments as 250 μM EPA was associated with reduced intrahepatocyte triglyceride content after both 3 (74.1 vs 94.9 mmol/gTP; p<0.05) and 7 days (62.2 vs 80.9 mmol/gTP; p<0.05) incubation compared with untreated cells equating to a 21.9% (95% CI 9% to 35%) and 23.1% (95% CI 5% to 41%) reduction respectively. For both experiments a linear trend between increasing EPA concentration and reduced triglyceride content was confirmed. Oleate Here reduced triglyceride content with both 50 μM EPA (p<0.01) and 250 μM EPA (p<0.05) was seen with oil red staining and equated to reductions of 27.6% (95% CI 16% to 39%) and 22.5% (95% CI 9% to 36%) compared with untreated cells. However these results were not reproduced in cell culture experiments although on post test analysis there was a significant linear trend between increasing EPA concentration and reduced triglyceride content (p=0.04). LPON Although incubation with 250 μM EPA reduced triglyceride content in the LPON model when quantified with oil red staining (60 308 vs 79 219 pixels in 250 μM EPA vs untreated cells; p<0.05) this was not confirmed in cell culture experiments. On post hoc analysis no trend was demonstrated between EPA concentration and triglyceride content. Conclusion These results suggest that EPA is effective at reducing triglyceride accumulation in untreated hepatocytes but is not effective in either oleate or LPON models of cellular steatosis. It is therefore possible that the presence of steatosis and mitochondrial dysfunction in NAFLD may limit the efficacy of EPA as a treatment. Competing interests None declared.

Oleate upregulates lectin galactoside-binding soluble 2 (LGALS2) in in vitro model of cellular steatosis

Introduction Galectin-2 (LGALS2) has been shown to co-localise with and bind to lymphotoxin-α (LTA); a cytokine that have been associated with insulin resistance. Thus, LGALS2 has been implicated in metabolic syndrome traits. The association between a common polymorphism of LGALS2 with myocardial infarction has further supported this notion. However, a recent study also demonstrates a contrasting finding of lower fasting insulin and glucose levels with LGALS2. The association between LGALS and the hepatic manifestation of insulin resistance, non-alcoholic fatty liver disease (NAFLD), has not been examined. Here, the authors investigated in vitro whether hepatic steatosis influenced the expression of LGALS2. Methods Human hepatoblastoma HepG2/C3A cells were pretreated for 3 days with oleate (0.25 mM) or octanoate (2 mM) to induce triglyceride accumulation. The authors have previously demonstrated that the addition of gluconeogenic substrates; lactate (L), pyruvate (P) and ammonia (N) to octanoate resulted in increased cellular steatosis that manifests many of the key features associated with steatohepatitis such as impaired mitochondrial structure/function, enhanced oxidative stress, decreased PTEN expression and altered cell cycle. LGALS2 mRNA expression was measured using quantitative real time PCR. Insulin resistance was determined by measuring concentration of glucose after a 4 h incubation of rinsed pretreated cells in the presence of insulin (0–10 nM). Results As previously demonstrated, all pretreatment induced significant intracellular triglyceride accumulation. The authors found that oleate upregulated LGALS2 expression. In contrast, the expression of LGALS2 was unchanged with LPON. Despite a higher triglyceride accumulation with octanoate, LGALS2 mRNA expression was also unaltered (oleate 1.24±0.06, octanoate 1.19±0.04, LPON 1.09±0.06, untreated 0.95±0.02 fold change from β-actin, p=0.0006). Glucose concentration in oleate showed a stepwise reduction with increasing insulin concentration (insulin 0 nM: 1.23±0.21 μg/gTP/h; 10 nM: 0.92±0.15 μg/gTP/h, where TP, total protein) contrasting to the unchanged glucose with LPON (Insulin 0 nM: 1.94±0.28 μg/gTP/h; 10 nM: 2.05±0.25 μg/gTP/h). Conclusion This data demonstrate that different FFA induces different LGALS2 expression. The presence of cellular steatosis per se or triglyceride concentration does not influence LGALS2 expression. Similar to the recent study, the upregulation of LGALS2 with oleate is associated with lower glucose concentration with preserved insulin sensitivity.

P53 The contrasting effect of octanoate and oleate on phosphatase and tensin homologue expression in in vitro model of steatosis using HepG2/C3A cells

Introduction The tumour suppressor phosphatase and tensin homologue (PTEN) is mutated or deleted in several human cancers including hepatocellular carcinoma. PTEN-deficient mice demonstrated triglyceride accumulation, steatohepatitis, progressing to liver fibrosis and hepatocellular carcinoma. Similarly, reduced PTEN expression with free fatty acid (FFA) oleate has been shown to promote hepatic steatosis. In other cancer, mitochondrial respiration defect with enhanced glycolysis and NADH formation has been suggested to be a key event in PTEN downregulation. Aim Our aims were to examine whether i) medium chain FFA octanoate altered PTEN expression ii) PTEN downregulation with FFA was associated with hepatic mitochondrial dysfunction. Method Human hepatoblastoma cell line HepG2/C3A was pretreated for 3 days with oleate (0.25 mM) or octanoate (2 mM). PTEN expression was determined using quantitative real time PCR. Mitochondrial function was measured using BDTM oxygen biosensor in the presence of 2,4 dinitrophenol. Lactate and pyruvate concentrations were measured in the supernatant to determine glycolytic activity and NADH/NAD+ ratio. Intracellular lipid accumulation was confirmed with triglyceride concentrations. Experiments were done in triplicate to n=3. Results are expressed in mean±SEM. Differences between groups were analysed by one-way ANOVA. Results We have previously demonstrated that oleate and octanoate pretreatment resulted in a similar intracellular triglyceride accumulation. In this study, we have found that despite similarities in triglyceride concentration, PTEN expression was lower in octanoate pretreated cells (octanoate 0.84±0.06, oleate 1.18±0.12, untreated 1.19±0.12 fold change from b-actin, p=0.04). However, octanoate pretreatment was not associated with impaired respiration (octanoate 0.24±0.01, oleate 0.20±0.02, untreated 0.28±0.01 AFU/gTP (gram of total protein)/min). Nevertheless, reduced PTEN expression with octanoate was associated with increased glycolysis (octanoate 315.2±42.91, oleate 100.9±14.09, untreated 145.3±8.83 μmol/gTP/hr, p=0.0001) with raised NADH/NAD ratio (octanoate 17.3±1.4, oleate 13.8±2.9 untreated 17.3±1.4; p=0.007). Conclusion To our knowledge, the effect of octanoate on PTEN expression has not been previously shown. In contrast to the previous finding, our data demonstrate that octanoate, not oleate, downregulates PTEN expression. Differences in glycolysis hence redox potential may have influenced the disparity in PTEN expression between these FFA. Octanoate has recently been proposed to be beneficial in weight loss and diabetes. However, our findings suggest that it may not have a favourable effect on the progression of nonalcoholic fatty liver disease.

PWE-002 Cell growth is impaired in a cellular model of non-alcoholic fatty liver disease, but is unaffected by simple steatosis

Introduction Non-alcoholic fatty liver disease (NAFLD) is an important cause of cirrhosis. Cirrhosis can be considered as a state where disordered growth leads to inadequate repair and a risk of malignancy. We sought to study the growth characteristics of C3A cells in an in vitro model of NAFLD. Methods C3A cells (well differentiated hepatoblastoma cells) were preconditioned for 72 h in control media, media containing oleate (which leads to steatosis, but not insulin resistance) or LPON media containing lactate (L), pyruvate (P), octanoate (O) and ammonia (N) (leading to steatosis, insulin resistance and increased reactive oxygen species). The cells were passaged and re-incubated in the appropriate media. Cells were harvested daily for cell counting and total protein estimation for 5 days. Cell cycle analysis and apoptosis estimation were performed on day 3. Results Growth curves were established, showing by both cell counting and protein estimation that growth was significantly reduced in the NAFLD model (p< 0.0001) compared to control media and oleate (Abstract 002 Figure 1). Abstract PWE-002 Figure 1 Growth curve. Cell cycle analysis showed that cells grown in LPON did not accumulate in G0/G1, but appeared to accumulate in S phase (Abstract 002 Figure 2). Cells grown in oleate cycled normally. There was an increase in apoptosis in both oleate and LPON groups (26.1 and 27.7% cells undergoing apoptosis, compared to 13.5% of control cells, p<0.0001). Abstract PWE-002 Figure 2 Cell cycle analysis. Conclusion NAFLD-induced cells showed significantly impaired growth compared with both control and simple steatotic cells in this model. This was not wholly attributable to an increase in apoptosis, as there was a similar increase in apoptosis with steatosis. While simple fat accumulation increased apoptosis, there was in addition, impairment of cell growth in our NAFLD model, which may be due to formation of reactive oxygen species, causing DNA damage and cell cycle arrest.