Inhibitor Report for: Phenmedipham

Enchytraeus albidus are important organisms of the soil biocenosis, used as standard test species in environmental risk assessment. The inhibition of cholinesterases (ChE) activity of several species has been widely used to assess the exposure and effects of anti-cholinesterase environmental contaminants. Several studies have shown the association between ChE activity inhibition and adverse effects on behaviour and survival. Extensive studies addressing survival and behavioural endpoints, as well as other biomarkers, have been done in E. albidus with different types of soil contaminants. The main objectives of this study were: (1) to characterize biochemically the ChE present in the soluble post-mitochondrial fraction of E. albidus whole body homogenates, using different substrates and selective inhibitors; (2) to assess the in vivo effects of copper, phenmedipham and different soil properties (pH, organic matter, clay) on the ChE activity; (3) to assess the in vitro effects of copper and phenmedipham on the ChE activity. The results suggest the presence of one ChE in the soluble post-mitochondrial fraction of E. albidus whole body homogenates, which displays properties of both acetylcholinesterase and pseudocholinesterase considering the typical mammalian enzymes. It is also shown that ChE activity is not inhibited by exposure to different soil properties and that copper and phenmedipham inhibited ChE activity both in in vivo and in in vitro conditions and therefore ChE inhibition seems to be a robust biomarker for this herbicide and this heavy metal. This study showed that ChE activity in E. albidus might be correlated to previously determined higher level effects like survival and reproduction, as well as avoidance behaviour.

Arthrobacter oxydans P52 isolated from soil samples was found to degrade the phenylcarbamate herbicides phenmedipham and desmedipham cometabolically by hydrolyzing their central carbamate linkages. The phenylcarbamate hydrolase (phenmedipham hydrolase) responsible for the degradative reaction was purified to homogeneity. The enzyme was shown to be a monomer with a molecular weight of 55,000. A 41-kb wild-type plasmid (pHP52) was identified in A. oxydans P52, but not in a derivative of this strain that had spontaneously lost the ability to hydrolyze phenylcarbamates, indicating that the gene for phenylcarbamate degradation (pcd) is plasmid encoded. Determination of two partial amino acid sequences allowed the localization of the coding sequence of the pcd gene on a 3.3-kb PstI restriction fragment within pHP52 DNA by hybridization with synthetic oligonucleotides. The phenylcarbamate hydrolase was functionally expressed in Escherichia coli under control of the lacZ promoter after the 3.3-kb PstI fragment was subcloned into the vector pUC19. A stretch of 1,864 bases within the cloned Pst fragment was sequenced. Sequence analysis revealed an open reading frame of 1,479 bases containing the amino acid partial sequences determined for the purified enzyme. Sequence comparisons revealed significant homology between the pcd gene product and the amino acid sequences of esterases of eukaryotic origin. Subsequently, it was demonstrated that the esterase substrate p-nitrophenylbutyrate is hydrolyzed by phenmedipham hydrolase.

This method for determining carbamates is based on the inhibiting action of these substances on acetylcholinesterase activity. The use of radioactively labelled acetylcholine as a substrate, the ensuing extractive separation of the radioactive acetic acid (formed by hydrolysis) and its radiometric determination permit to detect very small amounts of carbamates. The limit of detection for aldicarb, baygon, benomyl, bux, carbaryl, CIPC, matacil, phenmedipham and promecarb lies in the picogram range; that for barban and methomyl, in the nanogram range. The lower, linear parts of the curves for the different carbamates fall within the range 0.001-10 ng. The sensitivity (expressed as delta% inhibition/delta lg ng carbamate) ranges from 1.0 to 9.7.

Enchytraeus albidus are important organisms of the soil biocenosis, used as standard test species in environmental risk assessment. The inhibition of cholinesterases (ChE) activity of several species has been widely used to assess the exposure and effects of anti-cholinesterase environmental contaminants. Several studies have shown the association between ChE activity inhibition and adverse effects on behaviour and survival. Extensive studies addressing survival and behavioural endpoints, as well as other biomarkers, have been done in E. albidus with different types of soil contaminants. The main objectives of this study were: (1) to characterize biochemically the ChE present in the soluble post-mitochondrial fraction of E. albidus whole body homogenates, using different substrates and selective inhibitors; (2) to assess the in vivo effects of copper, phenmedipham and different soil properties (pH, organic matter, clay) on the ChE activity; (3) to assess the in vitro effects of copper and phenmedipham on the ChE activity. The results suggest the presence of one ChE in the soluble post-mitochondrial fraction of E. albidus whole body homogenates, which displays properties of both acetylcholinesterase and pseudocholinesterase considering the typical mammalian enzymes. It is also shown that ChE activity is not inhibited by exposure to different soil properties and that copper and phenmedipham inhibited ChE activity both in in vivo and in in vitro conditions and therefore ChE inhibition seems to be a robust biomarker for this herbicide and this heavy metal. This study showed that ChE activity in E. albidus might be correlated to previously determined higher level effects like survival and reproduction, as well as avoidance behaviour.

Phenmedipham [methyl-3-(3-methylphenylcarbamoyloxy)carbamate] is used as a herbicide, especially in the growing of sugar beet and strawberries. During metabolism of the substance in rats, the two carbamate moieties of phenmedipham are cleaved and the metabolites methyl-N-(3-hydroxyphenyl)-carbamate, m-aminophenol and hydroxyacetanilide are formed. These compounds and their conjugates are excreted in urine. Additionally, it has been suggested that m-toluidine is formed during metabolism. For the first time it has been possible to detect this metabolite in the urine of workers after agricultural use of phenmedipham. The concentrations of m-toluidine in urine were significantly higher in persons occupationally exposed than in controls. The median values for each group were 0.36 microg/l and 0.16 microg/l, respectively. This means that persons not exposed to phenmedipham also excrete m-toluidine, possibly as a result of the uptake of pesticides like phenmedipham from the diet.

Arthrobacter oxydans P52 isolated from soil samples was found to degrade the phenylcarbamate herbicides phenmedipham and desmedipham cometabolically by hydrolyzing their central carbamate linkages. The phenylcarbamate hydrolase (phenmedipham hydrolase) responsible for the degradative reaction was purified to homogeneity. The enzyme was shown to be a monomer with a molecular weight of 55,000. A 41-kb wild-type plasmid (pHP52) was identified in A. oxydans P52, but not in a derivative of this strain that had spontaneously lost the ability to hydrolyze phenylcarbamates, indicating that the gene for phenylcarbamate degradation (pcd) is plasmid encoded. Determination of two partial amino acid sequences allowed the localization of the coding sequence of the pcd gene on a 3.3-kb PstI restriction fragment within pHP52 DNA by hybridization with synthetic oligonucleotides. The phenylcarbamate hydrolase was functionally expressed in Escherichia coli under control of the lacZ promoter after the 3.3-kb PstI fragment was subcloned into the vector pUC19. A stretch of 1,864 bases within the cloned Pst fragment was sequenced. Sequence analysis revealed an open reading frame of 1,479 bases containing the amino acid partial sequences determined for the purified enzyme. Sequence comparisons revealed significant homology between the pcd gene product and the amino acid sequences of esterases of eukaryotic origin. Subsequently, it was demonstrated that the esterase substrate p-nitrophenylbutyrate is hydrolyzed by phenmedipham hydrolase.

This method for determining carbamates is based on the inhibiting action of these substances on acetylcholinesterase activity. The use of radioactively labelled acetylcholine as a substrate, the ensuing extractive separation of the radioactive acetic acid (formed by hydrolysis) and its radiometric determination permit to detect very small amounts of carbamates. The limit of detection for aldicarb, baygon, benomyl, bux, carbaryl, CIPC, matacil, phenmedipham and promecarb lies in the picogram range; that for barban and methomyl, in the nanogram range. The lower, linear parts of the curves for the different carbamates fall within the range 0.001-10 ng. The sensitivity (expressed as delta% inhibition/delta lg ng carbamate) ranges from 1.0 to 9.7.