Lipid Profile Altered in Phenanthrene Exposed Zebrafish Embryos with Implications for Neurological Development and Early Life Nutritional Status

Abstract

Lecithotrophic fish embryos rely on finite maternally deposited yolk resources for early development. Toxicant exposure can disrupt the uptake of yolk resources with consequences for development. In this study, we investigate the impacts of altered yolk utilization on fish embryos using the cardiotoxic compound phenanthrene. Zebrafish embryos were exposed to a cardiotoxic concentration of phenanthrene beginning at 6 hpf (hours post-fertilization) until a maximum of 72 hpf. Embryos were stained with Oil Red O to visualize neutral lipids. We then used a nontargeted approach to profile lipids in 24 and 72 hpf embryos after phenanthrene treatment. To assess changes in lipid movement within the embryo, the yolk sac was dissected from the body at 24 and 72 hpf and analyzed separately from the body at 72 hpf. Overall, total metabolites were significantly reduced in the yolk sac, and staining for neutral lipids was reduced in the embryo body at 72 hpf. This result is consistent with significant reductions in triglycerides in both the embryo body and yolk, indicating a limited contribution of impaired cardiac function to lipid mobilization at the dose tested. Additionally, lysophosphatidylcholines and lysophosphatidylethanolamines were significantly increased in the 72 hpf embryo body. Bioinformatic pathway analysis indicated that changes to these lysophospholipids could be linked to a disease model associated with inflammation and neuron demyelination consistent with previously observed injuries to neuronal and eye development in fish embryos and larvae.

Figure 1

Pericardial area (mm²) in 72 hpf zebrafish embryos exposed to 0.06% DMSO control (n = 32) or 12 μM phenanthrene (n = 28). Data were analyzed by Wilcoxon rank sum test; *p < 0.05.

Figure 2

(A) Representative images of 72 hpf zebrafish embryos stained with Oil Red O. (B) Mean Oil Red O staining measured in the body of the embryo (head and trunk) excluding the yolk at 24, 27, 30, 48, and 72 hpf (n = 8–23). Errors bars represent the bootstrapped 95% confidence intervals surrounding the mean; *p < 0.05.

Figure 3

(A) Principal component analysis (PCA) considering all metabolites, both known and unknown. Highlighted areas represent 95% confidence regions. (B) Total metabolites, both known and unknown, within each treatment and time point. Errors bars represent the bootstrapped 95% confidence intervals surrounding the mean. Each group contains n = 5; *p < 0.05.

Figure 4

Number of lipids identified in each lipid class: phosphatidylcholine (PC), triacylglycerides (TG), phosphatidylethanolamine (PE), phosphatidylinositol (PI), lysophosphatidylcholine (LPC), diacylglyceride (DG), sphingomyelin (SM), ceramide (Cer), sterol (ST), lysophosphatidylethanolamine (LPE), phosphatidylserine (PS), fatty acid (FA), hexosylceramide (HexCer), N-acyl phosphatidylethanolamine (LNPE), phosphatidylglycerol (PG), acyl carnitine (CAR), coenzyme Q (CoQ), monogalactosyldiacylgylcerol (MGDG), saccharolipids (SL).

Figure 5

Relative abundance of metabolites by lipid class. Errors bars represent the bootstrapped 95% confidence intervals surrounding the mean. Data are grouped by treatment, developmental stage, and sample type. Each group contains n = 5: Ceramide (Cer), diacylglyceride (DG), fatty acid (FA), hexosylceramide (HexCer), N-acyl phosphatidylethanolamine (LNPE), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin (SM), sterol (ST), triacylglyceride (TG); *p < 0.05.

Figure 6

(A) Relative abundance of LPC and LPE species highlighted by Ingenuity Pathway Analysis in the 72 hpf zebrafish embryo body samples. Gray bars represent the DMSO control, and blue bars represent the 12 μM phenanthrene exposures. Points represent individual replicates (n = 5). Error bars represent mean ± SEM; *p < 0.05; **p < 0.01. (B) Lipids linked to the development of the disease model chronic phase experimental autoimmune encephalomyelitis by Ingenuity Pathway Analysis. Red indicates increased abundance, and white indicates predicted upstream regulators, or downstream events. The orange line predicts activation.