Fullerene C60 exposure elicits an oxidative stress response in embryonic zebrafish

Abstract

Due to its unique physicochemical and optical properties, C60 has raised interest in commercialization for a variety of products. While several reports have determined this nanomaterial to act as a powerful antioxidant, many other studies have demonstrated a strong oxidative potential through photoactivation. To directly address the oxidative potential of C60, the effects of light and chemical supplementation and depletion of glutathione (GSH) on C60-induced toxicity were evaluated. Embryonic zebrafish were used as a model organism to examine the potential of C60 to elicit oxidative stress responses. Reduced light during C60 exposure significantly decreased mortality and the incidence of fin malformations and pericardial edema at 200 and 300 ppb C60. Embryos co-exposed to the glutathione precursor, N-acetylcysteine (NAC), also showed reduced mortality and pericardial edema; however, fin malformations were not reduced. Conversely, co-exposure to the GSH synthesis inhibitors, buthionine sulfoximine (BSO) and diethyl maleate (DEM), increased the sensitivity of zebrafish to C60 exposure. Co-exposure of C60 or its hydroxylated derivative, C60(OH)(24), with H2O2 resulted in increased mortality along the concentration gradient of H2O2 for both materials. Microarrays were used to examine the effects of C60 on the global gene expression at two time points, 36 and 48 h post fertilization (hpf). At both life stages there were alterations in the expression of several key stress response genes including glutathione-S-transferase, glutamate cysteine ligase, ferritin, alpha-tocopherol transport protein and heat shock protein 70. These results support the hypothesis that C60 induces oxidative stress in this model system.

Figure 1. C₆₀ concentration-response: dark vs. light exposure

24 hpf embryos were dechorionated and exposed in the dark to graded concentrations of C₆₀. Mortality (A), fin malformations (B), and pericardial edema (C) were scored daily for 5 days post exposure. Significant difference was determined using two-way ANOVA (*p < 0.05), N = 24, compared to C₆₀ effect in the light at that concentration. Error bars represent the standard error of means (SEM).

Figure 2. NAC co-incubation with C₆₀

Embryonic zebrafish were co-incubated with 50 μM NAC and graded concentrations of C₆₀ from 24 to 120 hpf. (A) Cumulative mortality by 120 hpf for C₆₀ + NAC (●) and C₆₀ only (▽). (B) Percentage of embryos with fin malformation including those that died prior to 120 hpf. (C) Percentage of embryos with pericardial edema including those later scored for mortality. Significance was determined using two-way ANOVA, (*p < 0.05, N = 24), and error bars represent the +/− the SEM.

Figure 3. C₆₀ co-incubation with ascorbic acid

Embryos were co-exposed to 250 μM ascorbic acid (AA) and graded concentrations of C₆₀ at 24 hpf until 120 hpf. Ascorbic acid decreased (A) mortality, (B) fin malformations (FM), and (C) pericardial edema (PE) at 200 ppb C₆₀. Significance was determined by two-way ANOVA (*p < 0.05, N = 24), and error bars represent +/− the SEM.

Figure 4. DEM and BSO concentration response

DEM and BSO were used to block glutathione production. Embryos were co-exposed to 5 μM BSO or 50 nM DEM and graded concentrations of C₆₀ at 24 hpf for 24 hours. DEM shifted the LC₅₀ to 50 ppb rather than 200 ppb. BSO induced 100% mortality at 200 ppb, but no significant mortality at 100 ppb. Significance was determined using two-way ANOVA (*p < 0.05, N = 24). Error bars represent +/− the SEM.

Figure 5. Cell death in co-exposures

Cell death was measured as a result of co-incubation with either NAC or DEM and 100 ppb C₆₀. Embryos were exposed at 24 hpf and cellular death was determined at 36 hpf using acridine orange. There was not a statistically significant difference between (A) control and (C) DEM, (E) BSO, or (G) NAC only. (D,E) DEM and BSO co-incubated with C₆₀ significantly increased cell death compared to (B) C₆₀ only. (H) NAC decreased cell death compared to C₆₀ alone; however cell death was significantly higher than controls. Significance was determined using two-way ANOVA (*p < 0.05, N = 12).

Figure 6. H₂O₂ and C₆₀ co-incubation

Embryos were exposed to H₂O₂ at 24 hpf for 48 hours to determine the concentration-response. Embryos were co-exposed to H₂O₂ and either 10 ppb C₆₀ or 500 ppb C₆₀(OH)₂₄ for 48 hours. Both significantly increased mortality at 0.5 mM H₂O₂. Significance was determined using two-way ANOVA (*p < 0.05, N = 24). Error bars represent +/− the SEM.

Figure 7. Pie chart of categories of gene regulation alterations

Differentially regulated genes were put into broad categories by stage and regulation: (A) 36 hpf up, (B) 36 hpf down, (C) 48 hpf up, (D) 48 hpf down. Stress response genes were only found to be up-regulated at both stages.

Figure 8. qRT-PCR results of gene expression

Genes involved in oxidative stress responses that were identified as mis-regulated by the microarray were further validated using qRT-PCR. Significance was determined using one-way ANOVA between controls and 200 ppb C₆₀ treated (*p < 0.05, N = 3). Error bars represent the SEM.