Metabolomics reveals target and off-target toxicities of a model organophosphate pesticide to roach (Rutilus rutilus): implications for biomonitoring

Abstract

The ability of targeted and nontargeted metabolomics to discover chronic ecotoxicological effects is largely unexplored. Fenitrothion, an organophosphate pesticide, is categorized as a "red list" pollutant, being particularly hazardous to aquatic life. It acts primarily as a cholinesterase inhibitor, but evidence suggests it can also act as an androgen receptor antagonist. Whole-organism fenitrothion-induced toxicity is well-established, but information regarding target and off-target molecular toxicities is limited. Here we study the molecular responses of male roach ( Rutilus rutilus ) exposed to fenitrothion, including environmentally realistic concentrations, for 28 days. Acetylcholine was assessed in brain; steroid metabolism was measured in testes and plasma; and NMR and mass spectrometry-based metabolomics were conducted on testes and liver to discover off-target toxicity. O-demethylation was confirmed as a major route of pesticide degradation. Fenitrothion significantly depleted acetylcholine, confirming its primary mode of action, and 11-ketotestosterone in plasma and cortisone in testes, showing disruption of steroid metabolism. Metabolomics revealed significant perturbations to the hepatic phosphagen system and previously undocumented effects on phenylalanine metabolism in liver and testes. On the basis of several unexpected molecular responses that were opposite to the anticipated acute toxicity, we propose that chronic pesticide exposure induces an adapting phenotype in roach, which may have considerable implications for interpreting molecular biomarker responses in field-sampled fish.

Figure 1

Correlations between the ranked intensities of S-methylglutathione [M − H]⁻ and desmethylfenitrothion [M − H]⁻ measured in (A) liver and (B) testes of roach exposed to fenitrothion, confirming that O-demethylation is a major route of pesticide degradation.

Figure 2

Mean concentrations (±SEM) of (A) cortisone and (B) 11-hydroxyandrostenedione in roach testes and (C) 11-ketotestosterone in plasma. Key: water control (WC), solvent control (SC), and 2, 20, and 200 μg/L fenitrothion exposures. Lowercase letters indicate significance, such that bars with the same letter are not significantly different.

Figure 3

Representative spectra of tissue extracts from an untreated roach: 1D projections of J-resolved ¹H NMR spectra of (A) testes and (B) liver, and negative-ion direct infusion mass spectra of (C) testes and (D) liver. Selected metabolites that changed significantly in response to fenitrothion exposure are indicated: (i) creatine, (ii) phosphocreatine, (iii) isoleucine, (iv) valine, (v) N-acetylphenylalanine, and (vi) tyrosine.

Figure 4

PCA score plots for (A) NMR liver data, (B) DIMS liver data, (C) NMR testes data, and (D) DIMS testes data. Key: (●) water control, (○) solvent control, and (▲, green) 2 μg/L, (×, blue) 20 μg/L, and (◆, red) 200 μg/L fenitrothion exposures. The PC axes were selected (up to PC5) based upon which axes showed a significant difference between groups (FDR < 10%; see Table S7 in Supporting Information), except for panel D, in which no PCs were significant so the axes capturing the highest variance were plotted.

Figure 5

Overview of metabolic changes discovered in (A) steroidogenesis and (B) phenylalanine metabolism in roach. Observed metabolites are in boldface type, with arrows (broad = significant, narrow = near-significant) indicating the intensity change from solvent control to 200 μg/L fenitrothion exposure. Biological compartments of the observations: black arrow = testes; gray arrow = liver; open arrow = plasma. ^†Data from Sancho et al.(12) (i) Phenylalanine N-acetyltransferase; (ii) phenylalanine hydroxylase.