title: In vitro and in vivo studies of cholinesterases and carboxylesterases in Planorbarius corneus exposed to a phosphorodithioate insecticide: Finding the most sensitive combination of enzymes, substrates, tissues and recovery capacity.
abstract: Organophosphate insecticides (OPs) continue to be an important class of agrochemicals used in modern agriculture worldwide. Even though these pesticides persist in the environment for a relatively short time, they show a high acute toxicity that may represent a serious hazard for wildlife. Sub-lethal effects on non-target species are a focus in pest management programs and should be used as biomarkers. Cholinesterases (ChEs) are the most used biomarker of OP exposure in vertebrate and invertebrate species. However, the combined monitoring of ChE and carboxylesterase (CE) activities may provide a more useful indication of exposure and effect of the organisms. The objective of the present work was to find the most sensitive combination of enzyme, substrate, tissue and capacity to recovery of B-esterases in the freshwater gastropod Planorbarius corneus exposed to the OP azinphos-methyl. For this purpose, ChE and CE activities in different tissues of P. corneus (head-foot, pulmonary region, digestive gland, gonads and whole organism soft tissue) were studied. Measurements of ChE activity were performed using three substrates: acetylthiocholine, propionylthiocholine and butyrylthiocholine and CE activity using four different substrates: p-nitrophenyl acetate, p-nitrophenyl butyrate, 1-naphthyl acetate, and 2-naphthyl acetate in control and exposed organisms. Finally, the recovery rates of ChE and CE activities following 48h exposure to azinphos-methyl were analyzed. Our results show a preference for acetylthiocholine as substrate, a high inhibition with eserine (a selective ChE inhibitor) and inhibition with excess of substrate in all the analyzed tissues. The highest ChE and CE activity was found in the pulmonary region and in the digestive gland, respectively. The highest CE Vmaxwas obtained with 1 and 2-naphthyl acetate in all the tissues. CEs were more sensitive than ChE to azinphos-methyl exposure. The highest sensitivity was found using p-nitrophenyl acetate and butyrate as substrates. On the other hand, CEs of the digestive gland and the pulmonary region were more sensitive than CEs of the whole organism soft tissue. Regarding the recovery of enzyme activities after 48h exposure, ChE and CEs with p-nitrophenyl butyrate reached control values after 14days in the digestive gland and after 21days in the pulmonary region. Our results show marked differences in P. corneus basal ChE and CE activities depending on substrates and the tissue. Also, both tissue-dependent and substrate-dependent variations in sensitivity to azinphos-methyl exposure and recovery were obtained. CEs measured with p-nitrophenyl butyrate in the pulmonary region were the best combination to be used as biomarker of exposure to azinphos-methyl due to their sensitivity and low recovery capacity. Environmental concentrations of azinphos-methyl inhibited CE activity so they could be used as effective biomarkers of aquatic contamination.
authors.affiliation: Laboratorio de Ecotoxicología Acuática: Invertebrados Nativos (EAIN), Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Nuñez, 1428, Buenos Aires, Argentina.
authors.forename: Sofía
authors.initials: S
authors.lastname: Otero
authors.affiliation: Laboratorio de Ecotoxicología Acuática: Invertebrados Nativos (EAIN), Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Nuñez, 1428, Buenos Aires, Argentina; Universidad de Buenos Aires-Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN), Facultad de Ciencias Exactas y Naturales, Buenos Aires, Argentina. Electronic address: giselakristoff@hotmail.com.
authors.forename: Gisela
authors.initials: G
authors.lastname: Kristoff
mesh_terms: D000818:Animals; D001387:Azinphosmethyl / Q000633:toxicity; D015415:Biomarkers / Q000378:metabolism; D002087:Butyrates / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002802:Cholinesterases / Q000737:chemistry / Q000378:metabolism*; D020128:Inhibitory Concentration 50; D007306:Insecticides / Q000633:toxicity*; D007700:Kinetics; D009596:Nitrophenols / Q000378:metabolism; D009943:Organophosphorus Compounds / Q000633:toxicity; D012908:Snails / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D013379:Substrate Specificity; D014874:Water Pollutants, Chemical / Q000633:toxicity*
chemical_list: D015415:Biomarkers; D002087:Butyrates; D007306:Insecticides; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D014874:Water Pollutants, Chemical; C033592:4-nitrophenyl butyrate; D001387:Azinphosmethyl; C008642:4-nitrophenyl acetate; D002265:Carboxylic Ester Hydrolases; D002802:Cholinesterases
keywords: Azinphos-methyl; B-esterases; Biomarkers; Freshwater invertebrates