Phosmet bioactivation by isoform-specific cytochrome P450s in human hepatic and gut samples and metabolic interaction with chlorpyrifos

Data on the bioactivation of Phosmet (Pho), a phthalimide-derived organophosphate pesticide (OPT), to the neurotoxic metabolite Phosmet-oxon (PhOx) in human are not available. The characterization of the reaction in single human recombinant CYPs evidenced that the ranking of the intrinsic clearances was: 2C18 > 2C19 > 2B6 > 2C9 > 1A1 > 1A2 > 2D6 > 3A4 > 2A6. Considering the average human hepatic content, CYP2C19 contributed for the great majority (60%) at relevant exposure concentrations, while CYP2C9 (33%) and CYP3A4 (31%) were relevant at high substrate concentration. The dose-dependent role of the active isoforms was confirmed in human liver microsomes by using selective CYP inhibitors. This prominent role of CYP2C in oxon formation was not shared by other OPTs. The pre-systemic Pho bioactivation measured in human intestinal microsomes was relevant accounting for ¼ of that measured in the liver showing two reaction phases catalysed by CYP2C and CYP3A4. Phosmet efficiently inhibited CPF bioactivation and detoxication, with Ki values (≈30 μM) relevant to pesticide concentrations achievable in the human liver, while the opposite is unlikely (Ki ≈ 160 μM) at the actual exposure levels, depending on the peculiar isoform-specific Pho bioactivation. Kinetic information in humans can support the development of quantitative in vitro/in vivo extrapolation and in silico models for risk assessment refinement for single and multiple pesticides

emerging health issues of cyanobacterial blooms

This paper describes emerging issue related to cyanobacterial dynamics and toxicity and human health risks. Data show an increasing cyanobacteria expansion and dominance in many environments. However there are still few information on the toxic species fitness, or on the effects of specific drivers on toxin production. Open research fields are related to new exposure scenario (cyanotoxins in water used for haemodialysis and in food supplements); to new patterns of co-exposure between cyanotoxins and algal toxins and/or anthropogenic chemicals; to dynamics affecting toxicity and production of different cyanotoxin variants under environmental stress; to the accumulation of cyanotoxins in the food web. In addition, many data gaps exist in the characterization of the toxicological profiles, especially about long term effects

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

L’USO DI UN SISTEMA 3D DI EPITELIO INTESTINALE UMANO PER STUDIARE IL RUOLO DEI TRASPORTATORI NELL’ASSORBIMENTO INTESTINALE DELLE MICROCISTINE

Le microcistine (MCs) sono un gruppo di circa 200 eptapeptidi, prodotti secondari del metabolismo dei cianobatteri le cui fioriture nei corpi idrici stanno aumentando in frequenza ed estensione a causa di eutrofizzazione, attività antropiche e cambiamenti climatici. L’esposizione orale è la più frequente per ingestione di cibo (es prodotti ittici), acqua potabile ed integratori alimentari (BGAS) contaminati. L’organo bersaglio principale è il fegato, e la tossicità è congenere-specifica. Poiché le varianti sono equipotenti nell’inibizione delle fosfatasi 1 e 2A (evento molecolare iniziale), tali differenze sono state attribuite a una diversa cinetica. Le MCs sono detossificate attraverso coniugazione con il GSH, reazione sia spontanea che catalizzata delle Glutatione-S-trasferasi. L’uptake negli epatociti di queste tossine è mediato dai trasportatori OATP; anche l’assorbimento gastrointestinale è dovuto a trasporto attivo, ma ad oggi non sono disponibili studi specifici sul sistema di trasporto intestinale che comprende proteine di influsso ed estrusione. Nel presente lavoro abbiamo studiato l’assorbimento di cinque MCs (MC-LR, RR, YR, LW e LF), utilizzando un sistema in vitro 3D di epitelio intestinale umano (EpiIntestinalTM), costituito da vari tipi cellulari, differenziato e polarizzato, che riproduce sia l’anatomia (lume intestinale/ comparto basolaterale per il flusso sanguigno) che le funzionalità intestinali. Mediante il calcolo della velocità di trasporto espressa in termini di coefficiente di permeabilità Papp, si è misurata la capacità di queste tossine di attraversare l’epitelio intestinale. Papp= (dQ/dt)/C0*A, dove dQ/dt è il flusso (quantità di sostanza/ unità di tempo), C0 è la concentrazione iniziale e A è l’area dell’epitelio. L’uso di un sistema substrato/inibitore specifico ha confermato la presenza di trasportatori di efflusso (PgP, MRP2 e BCRP) e di assorbimento (OATP 2B1 e 1A2), ulteriormente supportata da un’analisi western blotting del tessuto e ha permesso di identificare il coinvolgimento relativo nel trasporto delle MCs. La quantificazione dei vari substrati, delle MCs e del coniugato nei vari comparti è stata fatta mediante metodi HPLC ad hoc. Non è mai stata evidenziata la presenza del coniugato indicando una bassa capacità metabolica del sistema o la sua degradazione successiva. I risultati hanno mostrano la partecipazione sia di trasportatori di efflusso che di assorbimento delle varie MCs (40 μM) che portano a valori netti di Papp simili (meno di 4 x 10-7 cm2/sec). L’assorbimento della MCLW sembra legato solo all’OATP 2B1, la forma principalmente espressa nell’intestino. I dati di uptake congenere-specifico ottenuti solo rispetto all’attività degli OATP, dovrebbero essere rivisti alla luce di un'azione combinata con le proteine di efflusso

In vitro detoxication of microcystins in human samples: variability among variants with different hydrophilicity and structure

Cyanotoxins, among which>200 variants of Microcystins (MC), constitute an emerging issue in food safety. Microcystins (MC) toxicity is congener-specific; however, the in vitro inhibition of PP1/PP2A (the key molecular event of MC toxicity) by single MC variants is comparable and MC toxicokinetics seems to be the critical point. Here, the variability in GSH conjugation catalysed by human recombinant enzymes and human hepatic cytosol has been compared between hydrophilic (MC-LR and MC-RR) and hydrophobic (MC-LW, MC-YR and MC-LF) variants, according to measured logPow. In vitro detoxication reaction (spontaneous plus enzymatic) is favored by the variant hydrophilicity, with MC-LF very poorly detoxified. With MC-YR and -LW the spontaneous reaction always gave the major contribution, whereas with MC-LR and -RR the enzymatic reaction became by far predominant when GSH was depleted. Consequently, the well-known GST polymorphisms seems not to be the major driver for potential human variability in susceptibility towards the MC-toxicity, except for MC-RR and –LR when GSH is depleted. Looking at these results and literature data, MC-RR (the least cytotoxic and acutely toxic in rodents) is the more hydrophilic, has the lowest OATP-mediated hepatic uptake and the highest detoxication efficiency. The opposite is true for the most lipophilic MC-LF: once entered in the cells with the highest uptake, it is very poorly detoxified, and resulted as the most toxic in various cell types. MC-dependent TK should be considered in order to estimate the variability in toxicity and to support the use of quantitative in vitro-in vivo extrapolation models of single toxins and their mixtures co-occurring in the environment

Emerging health issues of cyanobacterial blooms

This paper describes emerging issue related to cyanobacterial dynamics and toxicity and human health risks. Data show an increasing cyanobacteria expansion and dominance in many environments. However there are still few information on the toxic species fitness, or on the effects of specific drivers on toxin production. Open research fields are related to new exposure scenario (cyanotoxins in water used for haemodialysis and in food supplements); to new patterns of co-exposure between cyanotoxins and algal toxins and/or anthropogenic chemicals; to dynamics affecting toxicity and production of different cyanotoxin variants under environmental stress; to the accumulation of cyanotoxins in the food web. In addition, many data gaps exist in the characterization of the toxicological profiles, especially about long term effects

Human Variability in Carboxylesterases and carboxylesterase-related Uncertainty Factors for Chemical Risk Assessment

International audienceCarboxylesterases (CES) are an important class of enzymes involved in the hydrolysis of a range of chemicals and show large inter-individual variability in vitro. An extensive literature search was performed to identify in vivo probe substrates for CES1 and CES2 together with their protein content and enzymatic activity. Human pharmacokinetic (PK) data on Cmax, clearance, and AUC were extracted from 89 publications and Bayesian meta-analysis was performed using a hierarchical model to derive CES-related variability distributions and related uncertainty factors (UF). The CES-related variability indicated that 97.5% of healthy adults are covered by the kinetic default UF (3.16), except for clopidogrel and dabigatran etexilate. Clopidogrel is metabolised for a small amount by the polymorphic CYP2C19, which can have an impact on the overall pharmacokinetics, while the variability seen for dabigatran etexilate might be due to differences in the absorption, since this can be influenced by food intake. The overall CES-related variability was moderate to high in vivo (<CV 50%), which might be due to possible polymorphism in the enzyme but also to the small sample size available per chemical. The presented CES-related variability can be used in combination with in vitro data to derive pathway-specific distributions

Remediation Strategies to Control Toxic Cyanobacterial Blooms: Effects of Macrophyte Aqueous Extracts on Microcystis aeruginosa (Growth, Toxin Production and Oxidative Stress Response) and on Bacterial Ectoenzymatic Activities

Increasing toxic cyanobacterial blooms in freshwater demand environmentally friendly solutions to control their growth and toxicity, especially in arid countries, where most drinking water is produced from surface reservoirs. We tested the effects of macrophyte allelochemicals on Microcystis aeruginosa and on the fundamental role of bacteria in nutrient recycling. The effects of Ranunculus aquatilis aqueous extract, the most bioactive of four Moroccan macrophyte extracts, were tested in batch systems on M. aeruginosa growth, toxin production and oxidative stress response and on the ectoenzymatic activity associated with the bacterial community. M. aeruginosa density was reduced by 82.18%, and a significant increase in oxidative stress markers was evidenced in cyanobacterial cells. Microcystin concentration significantly decreased, and they were detected only intracellularly, an important aspect in managing toxic blooms. R. aquatilis extract had no negative effects on associated bacteria. These results confirm a promising use of macrophyte extracts, but they cannot be generalized. The use of the extract on other toxic strains, such as Planktothrix rubescens, Raphidiopsis raciborskii and Chrysosporum ovalisporum, caused a reduction in growth rate but not in cyanotoxin content, increasing toxicity. The need to assess species-specific cyanobacteria responses to verify the efficacy and safety of the extracts for human health and the environment is highlighted

OpenCYP: An open source database exploring human variability in activities and frequencies of polymophisms for major cytochrome P-450 isoforms across world populations

International audienceThe open source database "OpenCYP database" has been developed based on the results of extensive literature searches from the peer-reviewed literature. OpenCYP provides data on human variability on baseline of activities and polymophism frequencies for selected cytochrome P-450 isoforms (CYP1A2, CYP2A6, CYP2D6, CYP3A4/3A5 and CYP3A7) in healthy adult populations from world populations. CYP enzymatic activities were generally expressed as the metabolic ratio (MR) between an unchanged probe drug and its metabolite(s) in urine or plasma measured in healthy adults. Data on other age groups were very limited and fragmented, constituting an important data gap. Quantitative comparisons were often hampered by the different experimental conditions used. However, variability was quite limited for CYP1A2, using caffeine as a probe substrate, with a symmetrical distribution of metabolic activity values. For CYP3A4, human variability was dependent on the probe substrate itself with low variability when data considering the dextromethorphan/demethilathed metabolite MR were used and large variability when the urinary 6β-hydroxycortisol/cortisol ratio was used. The largest variability in CYP activity was shown for CYP2D6 activity, after oral dosing of dextromethorphan, for which genetic polymorphisms are well characterised and constitute a significant source of variability. It is foreseen that the OpenCYP database can contribute to promising tools to support the further development of QIVIVE and PBK models for human risk assessment of chemicals particularly when combined with information on isoform-specific content in cells using proteomic approaches