Comparing the effects of developmental exposure to alpha lipoic acid (ALA) and perfluorooctanesulfonic acid (PFOS) in zebrafish (Danio rerio)

Abstract

Alpha lipoic acid (ALA) is a dietary supplement that has been used to treat a wide range of diseases, including obesity and diabetes, and have lipid-lowering effects, making it a potential candidate for mitigating dyslipidemia resulting from exposures to the per- and polyfluoroalkyl substance (PFAS) family member perfluorooctanesulfonic acid (PFOS). ALA can be considered a non-fluorinated structural analog to PFOS due to their similar 8-carbon chain and amphipathic structure, but, unlike PFOS, is rapidly metabolized. PFOS has been shown to reduce pancreatic islet area and induce β-cell lipotoxicity, indicating that changes in β-cell lipid microenvironment is a mechanism contributing to hypomorphic islets. Due to structural similarities, we hypothesized that ALA may compete with PFOS for binding to proteins and distribution throughout the body to mitigate the effects of PFOS exposure. However, ALA alone reduced islet area and fish length, with several morphological endpoints indicating additive toxicity in the co-exposures. Individually, ALA and PFOS increased fatty acid uptake from the yolk. ALA alone increased liver lipid accumulation, altered fatty acid profiling and modulated PPARɣ pathway signaling. Together, this work demonstrates that ALA and PFOS have similar effects on lipid uptake and metabolism during embryonic development in zebrafish.

Figure 1.. ALA is a structural analog of PFOS.

When the disulfide bond in (A) ALA is reduced, the ring structure opens forming (B) DHLA. Both DHLA and (C) PFOS have an 8-carbon chain and amphipathic structure. Figure created with Biorender.com.

Figure 2.. Developmental exposure to ALA, PFOS and co-exposure affects zebrafish morphology at 4 dpf. Zebrafish were exposed to ALA (0, 8, 16 μM), PFOS (16 μM) or a combination of ALA+PFOS from 3–96 hpf.

(A) Fish length, n = 39–44 fish per treatment group. Letters determined by Kruskal-Wallis with Dunn’s post hoc test. (B) Islet area, n = 29–42 fish per treatment group. Letters determined by One-way ANOVA with Tukey’s post hoc test. Letters in A and B represent significant differences between groups (p < 0.05). (C) Swim bladder inflation, n = 39–44 fish per treatment group, ****p < 0.0001 determined by Fisher’s exact test comparing each treatment group to control (0.01% DMSO). Representative images of exposed fish (ruler = 500 μm) and islets (ruler = 25 μm) for each treatment. Error bars represent SEM. Data compiled from 3 independent experimental replicates.

Figure 3.. Exposure to PFOS or ALA increases lipid uptake from the yolk.

Yolk fluorescence measurements were corrected for background fluorescence and autofluorescence, then normalized to the 0 μM (0.01% DMSO) group mean yolk fluorescence for each replicate. (A) Timeline of yolk fluorescence in zebrafish and representative images of fish at 4 dpf following PFOS exposure from 6 hpf-4 dpf. n = 29–32 fish per treatment group. ****p < 0.0001 determined by a two-tailed Mann-Whitney test. (B) Relative yolk fluorescence and representative images of fish at 4 dpf in zebrafish following ALA exposure from 6 hpf-4 dpf. n = 27–40 fish per treatment group. ***p = 0.0006 determined by Kruskal-Wallis with Dunn’s post hoc test. Error bars represent SEM. Data compiled from 3–4 independent experimental replicates.

Figure 4.. ALA exposure alters fatty acid profiles.

(A) Palmitic acid (*p = 0.047), (B) Stearic acid, (C) Palmitoleic acid (*p = 0.0495) and (D) Linoleic acid (*p = 0.02) as a percent of total fatty acid content. (E) Desaturation index calculated from fatty acid profiling. n = 3 groups of 10 zebrafish at 96 hpf. p = 0.057 determined by One-way ANOVA with Dunnett’s post hoc test. Error bars represent SEM. Data compiled from 3 independent experimental replicates.

Figure 5.. ALA exposure increases liver lipid accumulation.

Oil Red O staining for neutral lipid accumulation in the liver and skeletal muscle (4^th somite, internal control) at 96 hpf. Measurements for each treatment group are relative to the 0 μM (0.01% DMSO) group. Representative images of Oil Red O-stained fish with small images focus on the liver (outlined in yellow, avoiding pigment and overlap with intestines). n = 3 groups of 4–9 zebrafish per treatment. **p = 0.0014 determined by One-way ANOVA with Dunnett’s post hoc test. Error bars represent SEM. Data compiled from 3 independent experimental replicates.

Figure 6.. Developmental exposure to ALA, PFOS and co-exposures affect pparɣ pathway gene expression.

Gene expression of (A) pparɣ and pparɣ targets, (B) fabp1b1 and (C) apoa1a. Fold change relative to DMSO was calculated using β-actin as the housekeeping gene. Letters indicate significant differences between treatments (p < 0.05) determined by One-way ANOVA with Fisher’s LSD post hoc test (pparɣ and fabp1b1) or Kruskal-Wallis with Uncorrected Dunn’s post hoc test (apoa1a). n = 3–6 pools of 14–20 zebrafish at 96 hpf. Error bars represent SEM. Data compiled from 4–6 independent experimental replicates.