Mitochondrial dysfunction and oxidative stress contribute to cross-generational toxicity of benzo(a)pyrene in Danio rerio

Abstract

The potential for polycyclic aromatic hydrocarbons (PAHs) to have adverse effects that persist across generations is an emerging concern for human and wildlife health. This study evaluated the role of mitochondria, which are maternally inherited, in the cross-generational toxicity of benzo(a)pyrene (BaP), a model PAH and known mitochondrial toxicant. Mature female zebrafish (F0) were fed diets containing 0, 12.5, 125, or 1250 μg BaP/g at a feed rate of 1% body weight twice/day for 21 days. These females were bred with unexposed males, and the embryos (F1) were collected for subsequent analyses. Maternally-exposed embryos exhibited altered mitochondrial function and metabolic partitioning (i.e. the portion of respiration attributable to different cellular processes), as evidenced by in vivo oxygen consumption rates (OCRs). F1 embryos had lower basal and mitochondrial respiration and ATP turnover-mediated OCR, and increased proton leak and reserve capacity. Reductions in mitochondrial DNA (mtDNA) copy number, increases in mtDNA damage, and alterations in biomarkers of oxidative stress were also found in maternally-exposed embryos. Notably, the mitochondrial effects in offspring occurred largely in the absence of effects in maternal ovaries, suggesting that PAH-induced mitochondrial dysfunction may manifest in subsequent generations. Maternally-exposed larvae also displayed swimming hypoactivity. The lowest observed effect level (LOEL) for maternal BaP exposure causing mitochondrial effects in offspring was 12.5 µg BaP/g diet (nominally equivalent to 250 ng BaP/g fish). It was concluded that maternal BaP exposure can cause significant mitochondrial impairments in offspring.

Keywords: Benzo(a)pyrene; Cross-generational toxicity; Mitochondria; Oxidative stress; Polycyclic aromatic hydrocarbons; Zebrafish.

Figure 1.

Metabolic partitioning of basal respiration. (A) F0 ovaries (1 ovary/female, 4–5 females/tank, 3 tanks/treatment). (B) 24 hpf F1 embryos (2 embryos/well, 5–6 wells/tank, 3 tanks/treatment) (data from oligomycin plates). Different letters denote statistical differences across treatments within a given parameter (Two-way ANOVA with maternal-treatment and tank factors; Fisher’s LSD for treatment effects; P<0.05). For Figures 1A–B, total basal respiration is represented by the entire bar (red+blue), and statistical significance is denoted by a and b. Non-mitochondrial respiration is represented in red, and significance values (when significant) are displayed within this segment, denoted by c and d. Mitochondrial respiration is represented in blue, and significance values (when significant) are displayed within this segment, denoted by e and f.

Figure 2.

Metabolic partitioning of maximal respiration. (A) F0 ovaries (1 ovary/female, 4–5 females/tank, 3 tanks/treatment group). (B) 24 hpf F1 embryos (2 embryos/well, 5–6 wells/tank, 3 tanks/treatment group) (data from FCCP plates). Different letters denote statistical differences across treatments within a given parameter (Two-way ANOVA with treatment and tank factors; Fisher’s LSD treatment effects; P<0.05). For Figures 2A and 2B, total maximal respiration is represented by the entire bar (red+green+green/pink hashed sections), and statistical significance (when significant) is denoted by n and o. Maximal mitochondrial respiration is represented by the entire green section, including the portion of the green bar which has pink crossed hashes. The portion of maximal mitochondrial respiration that reflects reserve capacity is cross hashed in pink, and significance values (when significant) are displayed within this segment, denoted by p and r. Non-mitochondrial respiration is represented in red, and significance values (when significant) are displayed within this segment, denoted by c and d.

Figure 3.

Mitochondrial function and efficiency in 24 hpf F1 embryos (2 embryos/well, 5–6 wells/tank, 3 tanks/treatment) (data from oligomycin plates). (A) Mitochondrial respiratory partitioning. (B) Mitochondrial coupling efficiency. Different letters denote statistical differences across treatments within a given parameter (Two-way ANOVA with maternal treatment and tank factors; Fisher’s LSD for treatment effects; P<0.05). For both proton leak and coupling efficiency, standard deviations were significantly different across treatments (Bartlett’s Test; P<0.0001). For Figure 3A, mitochondrial respiration is represented by the entire bar (orange+purple), and statistical significance is denoted by letters e and f. ATP turnover is represented in orange, and significance values are displayed within this segment, denoted by g and h. Proton leak is represented in purple, and significance values are displayed within this segment, denoted j and k. For Figure 3B, mitochondrial coupling efficiency is represented by the bar, individual values are presented to showcase variability, and significance values are denoted by l and m.

Figure 4.

(A) Relative metabolic state of 24 hpf F1 embryos (2 embryos/well, 5–6 wells/tank, 3 tanks/treatment). The X-axis represents total basal extracellular acidification rate (ECAR), a proxy of glycolysis. The Y-axis represents ATP-linked mitochondrial respiration, a proxy of oxidative phosphorylation. The OCR:ECAR ratio was used for statistical analyses. (B) Total basal ECAR. Data are presented as means ± SEM. Different letters denote statistical differences across treatments (Two-way ANOVA with maternal treatment and tank factors; Fisher’s LSD for treatment effects; P<0.05) (data from oligomycin plates).

Figure 5.

Mitochondrial DNA damage and copy number. (A) F0 ovaries (1 ovary sample/female, 4–5 females/treatment) mtDNA damage expressed as lesions/10 kb. (B) F0 ovaries (1 ovary sample/female, 4–5 females/treatment) mtDNA copy number/nuclear genome. (C) 36 hpf F1 embryo (12-pooled embryos/sample, 4–6 samples/tank, 3 tanks/treatment) mtDNA damage expressed as lesions/10 kb. (D) 36 hpf F1 embryo (12-pooled embryos/sample, 4–6 samples/tank, 3 tanks/treatment) mtDNA copy number/nuclear genome. All data are presented as means ± SEM. For Figures 5A and 5B, no significant effects of treatment were detected (One-way ANOVA; Fisher’s LSD for treatment effects; P<0.05). For Figures 5C and 5D, different letters denote statistical differences across treatments (Two-way ANOVA with maternal treatment and tank factors; Fisher’s LSD for treatment effects; P<0.05). For both F1 embryo mtDNA damage and copy number, standard deviations were significantly different across treatment groups (Bartlett’s Test; P<0.0002 and 0.003, respectively), thus individual values are presented to showcase variability.

Figure 6.

Activities of selected antioxidant enzymes in F0 ovaries (1 ovary/female, 2–5 females/tank, 3 tanks/treatment). (A) GST. (B) GR. (C) GPx. (D) SOD. Data are presented as a boxplot (25%–75%) with whiskers from minimum to maximum. Different letters denote statistical differences across treatments. (Two-way ANOVA with treatment and tank factors; Fisher’s LSD for treatment effects; P<0.05). Statisticss were conducted on both untransformed and log transformed values for GR, GPx, and SOD, because standard deviations were significantly different across treatments (Bartlett’s Test; P<0.05), but significant differences were only detected for GR with log transformation. Untransformed values are plotted to showcase unequal variances.

Figure 7.

Activities of selected antioxidant enzymes in pooled samples of 96 hpf F1 larvae (15-pooled larvae/sample, 3 samples/tank, 3 tanks/treatment). (A) GST. (B) GR. (C) GPx. (D) SOD. Data are presented as a boxplot (25%–75%) with whiskers from minimum to maximum. Different letters denote statistical differences across treatments. (Two-way ANOVA with maternal treatment and tank factors;Fisher’s LSD for treatment effects; P<0.05). Statisticss were conducted on both untransformed and log transformed values for SOD because standard deviations were significantly different across treatments (Bartlett’s Test; P<0.05), but no significant differences in SOD activities were detected. Untransformed values are plotted to showcase unequal variances.

Figure 8.

Glutathione concentrations in F0 ovaries (1 ovary/female, 2–5 females/tank, 3 tanks/treatment). (A) TGSH. (B) GSH = TGSH - 2GSSG. (C) GSSG. (D) GSH:GSSG. Data are presented as a boxplot (25%–75%) with whiskers from minimum to maximum. Different letters denote statistical differences across treatments. (Two-way ANOVA with treatment and tank factors; Fisher’s LSD for treatment effects; P<0.05). Statisticss were conducted on log transformed values for GSH:GSSG because standard deviations were significantly different across treatments(Bartlett’s Test; P<0.05), but untransformed values are plotted to showcase unequal variances.

Figure 9.

Glutathione concentrations in 96 hpf F1 larvae (15-pooled larvae/sample, 3 samples/tank, 3 tanks/treatment). (A) TGSH. (B) GSH = TGSH - 2GSSG. (C) GSSG. (D) GSH:GSSG. Data are presented as a boxplot (25%–75%) with whiskers from minimum to maximum. Different letters denote statistical differences across treatments. (Two-way ANOVA with maternal treatment and tank factors;Fisher’s LSD for treatment effects; P<0.05). Statistics were conducted on log transformed values for GSH:GSSG because standard deviations were significantly different across treatment groups (Bartlett’s Test; P<0.05), but untransformed values are plotted to showcase unequal variances.

Figure 10.

Locomotor activity of 6 dpf F1 larvae (24 larvae/tank, 3 tanks/treatment). (A) Total distance traveled (mm) as a function of trial minute. (B) Total distance traveled (mm/min) as a function of light condition. Data are presented as means ± SEM. * indicates significant difference compared to control (RMANOVA; Dunnett’s or Fisher’s LSD; P<0.05).