Characterization of S-(N,N-Dialkylaminocarbonyl)cysteine Adducts and Enzyme Inhibition Produced by Thiocarbamate Herbicides in the Rat

Abstract

Thiocarbamates are a major class of herbicides used extensively in the agricultural industry. It has been shown that thiocarbamates can form reactive sulfoxide and sulfone intermediates, which may be involved in the toxicity of thiocarbamates through covalent modification of cysteine and serine active sites of enzymes. Molinate has been shown to generate an S-hexahydro-1H-azepine-1-carbonyl adduct on the Cys-125 residue of the beta2- and beta3-chains of rat globin analogous to that reported for disulfiram and to inhibit aldehyde dehydrogenase and nonspecific esterase activity. The present study examined whether other thiocarbamate herbicides produce similar covalent protein modifications and enzyme inhibition to that reported for molinate and whether S-(N,N-dialkylaminocarbonyl)cysteine adduct levels are correlated to enzyme inhibition or the structure of thiocarbamate herbicides. Additionally, the potential of molinate to act as a peripheral demyelinating agent similar to disulfiram was evaluated. To address these aims, rats were exposed ip to molinate, vernolate, ethiolate, EPTC, or butylate for 5 days after which hemogloblin was isolated and analyzed for protein adducts using HPLC and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. In addition, brain, liver, and testes mitochondrial and microsomal fractions were assayed for nonspecific esterase, low Km ALDH, or total ALDH activities, and S-(N,N-dialkylaminocarbonyl)cysteine adducts were measured by LC/MS/MS. For the neurotoxicity assessments, rats were administered molinate parenterally for subchronic periods and morphological evaluations performed on peripheral nerves. All of the thiocarbamates except butylate produced S-(N,N-dialkylaminocarbonyl)cysteine adducts on globin and the quantity of adducts detected decreased with increasing size of the nitrogen substituents. In contrast, a clear relationship between cysteine modification in mitochondrial and microsomal samples to nitrogen substituents was not evident, and although molinate produced relatively high levels of adducts and esterase inhibition and butylate low levels of adducts and esterase inhibition for most samples, in general, the level of S-(N,N-dialkylaminocarbonyl)cysteine adducts did not appear to be related to enzyme inhibition. Molinate did not produce segmental demyelination in peripheral nerve, suggesting that molinate and possibly other thiocarbamates do not share the neurotoxic potential of dithiocarbamates.

Relative Inhibitory Potency of Molinate and Metabolites with Aldehyde Dehydrogenase 2: Implications for the Mechanism of Enzyme Inhibition

“…There have also been a couple in vivo studies assessing ALDH activity. One study found liver ALDH inhibition in mice dosed ip with molinate (13), and the other found inhibition of ALDHs from the brain, liver, and testicle in rats dosed with molinate (10). These studies all indicate molinate or a metabolite is capable of inhibiting ALDHs, but the rates of this inhibition and the metabolite responsible for it are not clear.…”

“…In mice dosed with molinate, peripheral nerve demyelination was observed in addition to eosinophilic bodies (swollen, degenerated axons) in the medulla and spinal cord, hindlimb adduction, splayed hindlimbs, and ataxia (7, 9). It should be noted that no peripheral neuropathy was observed in rats dosed with molinate (10). …”

S -Ethyl- N,N -dipropylthiocarbamate Exposure and Cancer Incidence among Male Pesticide Applicators in the Agricultural Health Study: A Prospective Cohort

“…EPA 2006). Toxicity occurs through the byproducts of EPTC metabolism, which inhibit the enzymatic activities of aldehyde dehydrogenase and cholinesterase (Smulders et al 2003, 2004; Staub et al 1995; Zimmerman et al 2004). …”

Development of PBPK model of molinate and molinate sulfoxide in rats and humans

“…A previous study has demonstrated adduct formation of molinate sulfoxide with blood proteins. (Zimmerman et al, 2004). For the oral model the fecal excretion constant (Kf) (expressed as a fraction of dose and assumed to be unmetabolized parent compound) was taken from Debaun et al, 1978.…”