Epoxides Derived from Dietary Dihomo-Gamma-Linolenic Acid Induce Germ Cell Death in C. elegans

Abstract

Dietary fats are not created equally, slight differences in structure lead to crucial differences in function. Muticellular organisms use polyunsaturated fatty acid as substrates to produce potent signaling molecules crucial for many physiological processes, including reproduction. Here we explored the mechanism responsible for germ cell loss induced by dietary supplementation of dihomo-gamma-linolenic acid (DGLA, 20:3n-6) in the roundworm Caenorhabditis elegans. In this study we found that C. elegans CYP-33E2 activity produces a range of epoxy and hydroxy metabolites from dietary DGLA. Knockdown of cyp-33E2 suppressed the DGLA-induced sterility phenotype. Additionally, direct exposure of two specific DGLA-derived epoxy products, 8,9- and 14,15-epoxyeicosadienoic acids, produced germ cell abnormalities in the C. elegans gonad. We propose that sterility is mediated by the production of toxic DGLA-derived epoxides that trigger germ cell destruction. These studies are the first to establish a biological activity for a CYP-produced metabolite of DGLA.

Figure 1. Schematic diagram of C. elegans DGLA cascade.

Figure 2. Genetic manipulation of DGLA cascade alters sterility from DGLA supplementation.

Potential DGLA metabolism pathways were inhibited with RNAi or mutant alleles and assayed for sterility induced by DGLA supplementation. (A) RNAi knock-down of cyp-33E2 suppresses DGLA-induced sterility. (B) ceeh-1 and ceeh-2 mutations do not significantly alter sterility. (C) Knockdown of alpha-keto reductase and PGE synthase homologs increases sensitivity to DGLA supplementation. (D) Knock-down of cyp-33E2 decreases C. elegans reproduction success. For all panels, EV = empty vector control; ND = no detectable sterility on 0 mM DGLA; error bars, S.E.M, **P < 0.0005 compared to empty vector control.

Figure 3. C. elegans DGLA metabolism is mediated by CYP activity.

(A) Microsomes from a CYP-33E2/hCPR expressing baculovirus system were assayed with radiolabeled DGLA and separated by HPLC. The resulting products were identified and quantified by LC-MS/MS. (B–D) Endogenous DGLA metabolites of C. elegans fed 0 mM or 0.3 mM DGLA, assayed by LC-MS/MS. (B) Monohydroxy-metabolites (HETrE) increase in the presence of 0.3 mM DGLA. (C) Epoxy-metabolites (EED) and (D) Dihydroxy-metabolites (DHED) also increase in the presence of 0.3 mM DGLA. For all panels, error bars are S.E.M., *P < 0.05, **P < 0.001 compared to 0 mM DGLA.

Figure 4. Specific epoxides derived from DGLA trigger germ cell abnormalities.

Confocal microscopy of injected gonads indicates the defects produced by direct injection of specific DGLA metabolites. (A) Full gonad image of mock injection. White box indicates area of gonad enlarged for B–E. (B) Mock injected, (C) 10 μM DGLA injection, (D) 2.5 μM 14,15-EED, (E) 2.5 μM 8,9-EED. (F) The resulting defects are summarized as the mean number of defective germ cells per injection. White bars at the top right of A and B indicate 50 μm length. White arrows indicate multinucleated germ cells. Error bars, S.E.M, *P < 0.01 compared to mock injection. Injection of other DGLA metabolites did not result in increased germ cell defects compared to mock injections.