Mitochondria as a target of organophosphate and carbamate pesticides: Revisiting common mechanisms of action with new approach methodologies

Abstract

Mitochondrial toxicity has been proposed as a potential cause of developmental defects in humans. We evaluated 51 organophosphate and carbamate pesticides using the U.S. EPA ToxCast and Tox21 databases. Only a small number of them bind directly to cholinesterases in the parent form. The hydrophobicity of organophosphate pesticides is correlated significantly to TSPO binding affinity, mitochondrial membrane potential reduction in HepG2 cells, and developmental toxicity in Caenorhabditis elegans and Danio rerio (p < 0.05). Structural analysis suggests that in some cases the Krebs cycle is a potential target of organophosphate and carbamate exposure at early life stages. The results support the hypothesis that mitochondrial effects of some organophosphate pesticides-particularly those that require enzymatic activation to the oxon form-may augment the documented effects of disruption of acetylcholine signaling. This study provides a proof of concept for applying new approach methodologies to interrogate mechanisms of action for cumulative risk assessment.

Keywords: Carbamate; Developmental toxicity; Mitochondria; Mixture toxicity; Organophosphate; Risk assessment; ToxCast.

Figure 1.. Molecular weight and hydrophobicity of organophosphates and carbamates in ToxCast Phase I and II libraries in comparison with endogenous ligands.

The molecular weight and logarithm of P_{octanol-water} (log P_ow) of acetylcholine, cholesterol, 14 organophosphate oxons, 18 organophosphate thions, 8 N-methyl carbamates, and 11 other carbamates were visualized in a scatter plot. Acetylcholine is the third smallest and most hydrophilic compound and cholesterol is the second largest and most hydrophobic.

Figure 2A and 2B.. Unsupervised two-directional heat map from hierarchical clustering of 32 organophosphates and 19 carbamates across 110 molecular targets.

The heat map was generated with the “gplot” package in R version 3.5.1, using Euclidean distance for measure and Ward’s method for linkage analysis. The intensity of blue color indicates the value of affinity score. Organophosphate (OP) thions are enriched in Cluster A (in bold), which bind to mitochondrial translocator proteins (TSPOs) and cytochrome P450 (CYP) 1A1, 1A2, 2B6, 2C9, 2C19, and 2D6; but not acetylcholinesterases (AChEs) and butyrylcholinesterase (BChE). Another group of 2 OP oxons and 6 N-methyl carbamates bind to the ChEs, but not TSPOs and CYPs (i.e. Cluster B; in italic). For improved visualization of these two groups, Cluster A and B were re-clustered with their 84 molecular targets in Figure 2B. Animal species of these targets are specified by a first-letter initial (b: bovines; g: guinea pigs; h: humans; m: mouse; r: rats; and rab: rabbits) preceding the gene symbol.

Figure 3.. Quantitative bipartite network that shows in vitro binding affinity of 30 organophosphates and 18 carbamates to 110 molecular targets.

The network was constructed with ForceAtlas layout using Gephi version 0.8.2 beta, with 5 target classifications in different colors. The locality of the pesticides and their targets was determined by affinity score and connectivity, as visualized in edge thickness and node size. Cluster A (see Figure 2B) was connected to mitochondrial translocator proteins (TSPOs), nuclear receptors, and cytochrome P450 (CYP) enzymes. Cluster B was connected to acetylcholinesterases (AChEs) and butyrylcholinesterase (BChE) on the opposite side. Animal species of these targets were specified by a first-letter initial (b: bovines; g: guinea pigs; h: humans; m: mouse; r: rats; and rab: rabbits) preceding the gene symbol.

Figure 4A and 4B.. Correlation matrices of molecular weight and octanol-water partition coefficients (MW and Log P_ow); toxicity scores of developmental effects in Caenorhabditis elegans and Danio rerio and mitochondrial membrane potential (MMP) reduction in HepG2 cells; and affinity scores of acetylcholinesterase in humans and rats (hAChE and rAChE), butyrylcholinesterase in humans (hBChE), and mitochondrial translocator proteins in humans and rats (hTSPO and rTSPO) of 32 organophosphates and 18 carbamates.

The affinity scores, toxicity scores, molecular weight, and log P_ow of OPs, carbamates were examined with Spearman's rank correlation [44], followed by multiple testing correction with Holm’s method in R version 3.5.1 [33, 45]. The hydrophobicity of organophosphate pesticides was correlated significantly to TSPO binding affinity, MMP reduction, and developmental toxicity in C. elegans and D. rerio. * p < 0.05; ** p < 0.01

Figure 5.. Unsupervised two-directional heat map from hierarchical clustering of 32 organophosphates and 19 carbamates with acetylcholine, cholesterol, sarin, PK-11195, and Krebs cycle intermediates by structural similarity.

The heat map was generated with the “gplot” package in R version 3.5.1, using Euclidean distance for measure and Ward’s method for linkage analysis. The chemicals were clustered into three groups based on two-dimensional structural similarity: (1) 28 pesticides with acetylcholine and sarin; (2) 21 pesticides with cholesterol, PK-11195, and succinyl coenzyme A; and (3) malathion and mevinphos with fumarate and other Krebs cycle intermediates. Sarin is a chemical warfare agent that targets acetylcholinesterase. PK-11195 is a pharmaceutical ligand of mitochondrial translocator proteins The intensity of blue color indicates the value of similarity score, as computed with the PubChem score matrix (https://pubchem.ncbi.nlm.nih.gov/score_matrix).