Study of the biotransformation kinetics of pesticides and natural toxins for the identification of markers of exposure and susceptibility and possible groups of population at greatest risk.

Background: We are daily exposed to many xenobiotics, including natural toxins or pesticides, via the environment, water and food. They can be toxic, depending on the exposure dose. These substances are generally lipophilic and tend to accumulate in adipose tissue and membranes. As a defence mechanism, organisms promote their excretion, biotransforming them to more hydrophilic compounds through reactions catalysed by specific enzymes as Glutathione-S-Transferases (GSTs) or cytochromes P450 (CYPs) characterized by different polymorphic isoforms. Objectives: i) Characterization of the metabolic pathway of natural toxins, as Microcystins (MCs) and organophosphorothionate pesticides (OPs); ii) Identification of the human GSTs or CYPs involved in metabolism suggestive of group of population more susceptible to the toxic effects due to genetic polymorphism; iii) Characterization of new biomarkers of exposure. This study will be carried out applying an integrated approach using human recombinant enzymes and human liver microsomes and cytosols and advanced analytical techniques. Expected (or Preliminary) results: The study started with the characterization of the detoxification reaction of three variants of MCs (MC-LW, YR and LF), natural hepatotoxic and tumor promoters compounds, produced as secondary substances by cyanobacteria. They are >200 congeners with different in vivo toxicity. The in vitro inhibition potency of protein phosphatase by single MC congener, the key event in their mechanism of toxicity is comparable; therefore, the toxicokinetic of detoxification, mediated by the conjugation with GSH, seems to be the critical point to explain the MC congener-dependent toxicity. The variants, such as MC-LW, MC-YR and MC-LF, having hydrophobic amino acids (e.g. tyrosine, tryptophan) may be more cell permeable than MC-LR and also the detoxification reaction could be dependent on lipophilicity. At first, the logPow of 5 MC congeners has been determined using the OECD guideline 117 and ranking compound from the most lipophilic at the least lipophilic is: MC-LF>MC-LW>MC-LR>MC-YR>MC-RR. Moreover we have calculate the kinetics parameters (Vmax, Km and Cli) of MC-LW and MC-YR and results indicate that the efficiencies of recombinant GSTs used are quite similar (0.022-0.066 pmolGSMC-LW/(μgprot*min*μM) and 0.048-0.09 pmolGSMC-YR/(μgprot*min*μM); the highest Cli were shown by GSTP1 and A1 (the most abundant in the liver) for MC-LW and P1=O1>A1 for MC-YR. Future perspectives: This work is a part of an EU project financed by EFSA aimed to modeling the kinetics variability to predict the dynamic variability, our data will be used as an input for PBPK models. As next step OPs will be studied as well as other xenobiotics of toxicological interest.