Chemoproteomic Profiling of Acetanilide Herbicides Reveals Their Role in Inhibiting Fatty Acid Oxidation

Abstract

Acetanilide herbicides are among the most widely used pesticides in the United States, but their toxicological potential and mechanisms remain poorly understood. Here, we have used chemoproteomic platforms to map proteome-wide cysteine reactivity of acetochlor (AC), the most widely used acetanilide herbicide, in vivo in mice. We show that AC directly reacts with >20 protein targets in vivo in mouse liver, including the catalytic cysteines of several thiolase enzymes involved in mitochondrial and peroxisomal fatty acid oxidation. We show that the fatty acids that are not oxidized, due to impaired fatty acid oxidation, are instead diverted into other lipid pathways, resulting in heightened free fatty acids, triglycerides, cholesteryl esters, and other lipid species in the liver. Our findings show the utility of chemoproteomic approaches for identifying novel mechanisms of toxicity associated with environmental chemicals like acetanilide herbicides.

Metabolome disruption of pregnant rats and their offspring resulting from repeated exposure to a pesticide mixture representative of environmental contamination in Brittany

“…This is supported by recent findings published by Counihan et al . who showed that acetochlor could inhibit FA oxidation in mice, resulting in heightened levels of free FAs, triglycerides, cholesteryl esters, and other lipid species in the liver [ 49 ]. The alteration in FA profiles may be related to the inhibition of FA oxidation.…”

Proving the Mode of Action of Phytotoxic Phytochemicals

“…Protein binding interactions (not enzymatic) with potential inhibitors when many proteins are examined at the same time have been termed chemoproteomics [ 66 , 67 ]. This method was used by Campe et al [ 13 ] to identify the molecular target of cinmethylin and by Counihan et al [ 68 ] to identify the thiolase enzymes involved in fatty acid oxidation that are targeted by the herbicide acetochlor in mice livers. This method is especially useful to identify targets for which in vitro enzyme assays are not available or are very difficult.…”

A novel missense variant in ACAA1 contributes to early-onset Alzheimer’s disease, impairs lysosomal function, and facilitates amyloid-β pathology and cognitive decline

“…As ACAA1 is a member of the acetyl-CoA acyltransferase family and plays an important role in fatty acid β-oxidation of the very-long-chain fatty acid (VLCFA), 34 we compared the enzymatic activities of wild-type (WT) ACAA1 and mutant p.N299S by an in vitro enzymatic activity assay. 35 The enzymatic activity of ACAA1 p.N299S is lower than that of ACAA1 WT protein (Fig. 1b ), indicating that ACAA1 p.N299S is a loss-of-function variant causing a reduction in enzymatic activity.…”

“…The enzymatic activities of ACAA1 WT and ACAA1 p.N299S were measured using the Fluorometric Acetyltransferase Activity Assay Kit (Abcam, ab204536), as previously described. 35 Briefly, the assay was performed with 5 μg of pure protein and 100 nM acetoacetyl coenzyme A sodium salt hydrate (Sigma, A1625), and fluorescence was detected at Ex/Em of 380/520 nm. Both ACAA1 WT and ACAA1 p.N299S proteins were extracted using TnT® Quick Coupled Transcription/Translation Systems (Promega, L1170) and purified using the His-tag Protein Purification Kit (Beyotime, P2226).…”