Biomarker responses and metabolites identification in Lumbricus terrestris exposed to drugs of environmental concern, an in vivo and in vitro approach

Abstract

The earthworm Lumbricus terrestris is an anecic species living in natural soils but it is also a sentinel in pollution monitoring. Specimens of L. terrestris were exposed for 48 h though the filter paper contact test at 1 mg/mL of the chemicals: Lamotrigine (LMG), Cocaine (COC), Fipronil (FIP) and the pesticide bis-4-nitrophenyl phosphate (BNPP). After that period, the activities of Acetylcholinesterase, Glutathione S-transferase, Carboxylesterase (CE) using different substrates, and lipid peroxidation levels were evaluated in the exposed whole tissue earthworms. The results revealed differences only in CE activity, with 4-nitrophenyl butyrate (4NPB) and 1-naphthyl butyrate (1NB) the most responsive substrates to COC. The kinetic parameters of CE were characterized, for the first time, in whole tissue of this species. The chemical analysis by LC-MS/MS, confirmed the exposure to the parent compounds, identified metabolites and evidenced biotransformation pathways in earthworms. Metabolic reactions included oxidation (LMG and FIP), hydrolysis (COC and FIP) as well as glycosylation (LMG, COC and FIP). A hitherto unknown metabolite of LMG due to the conjugation with phenylalanine glutamine was formed. The in vivo results on CE activity with the specific inhibitor, BNPP, were confirmed in vitro. Moreover, in the in vitro approach, the inclusion of other contaminants of environmental concern supports the potential of CE as biomarker. This study identifies the main metabolites formed by earthworms for further in vivo exposures under more realistic conditions and the potential use of CE measures as biomarker of emerging contaminants