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

The 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 6ß-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.

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.

Human variability in glutathione-S-transferase activities, tissue distribution and major polymorphic variants: Meta-analysis and implication for chemical risk assessment.

The input into the QIVIVE and Physiologically-Based kinetic and dynamic models of drug metabolising enzymes performance and their inter-individual differences significantly improve the modelling performance, supporting the development and integration of alternative approaches to animal testing. Bayesian meta-analyses allow generating and integrating statistical distributions with human in vitro metabolism data for quantitative in vitro-in vivo extrapolation. Such data are lacking on glutathione-S-transferases (GSTs). This paper reports for the first time results on the human variability of GST activities in healthy individuals, their tissue localisation and the frequencies of their major polymorphic variants by means of extensive literature search, data collection, data base creation and meta-analysis. A limited number of papers focussed on in vivo GST inter-individual differences in humans. Ex-vivo total GST activity without discriminating amongst isozymes is generally reported, resulting in a high inter-individual variability. The highest levels of cytosolic GSTs in humans are measured in the kidney, liver, adrenal glands and blood. The frequencies of GST polymorphisms for cytosolic isozymes in populations of different geographical ancestry were also presented. Bayesian meta-analyses to derive GST-related uncertainty factors provided uncertain estimates, due to the limited database. Considering the relevance of GST activities and their pivotal role in cellular adaptive response mechanisms to chemical stressors, further studies are needed to identify GST probe substrates for specific isozymes and quantify inter-individual differences.

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 » 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.

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.

The contribution of human small intestine to chlorpyrifos biotransformation.

Despite the oral intake is the major route of exposure to chlorpyrifos for the general population, few data are available on human intestine biotransformation. In this study the contribution of chlorpyrifos (CPF) metabolism in human small intestine was investigated in microsomes from duodenum (HDM) and ileum/jejunum (HS2M) from 11 individual donors. Samples were characterized for testosterone hydroxylated metabolite formation and CYP content quantification by means of Western blotting. The two methods gave consistent results, evidencing the presence of CY3A4 and its-related activity in 10/11 samples, among which one showed also the presence of CYP2C9. Analogously, although with high interindividual variability (about 10 fold), CPF bioactivation to chlorpyrifos-oxon (CPFO) was observed in 10/11 HDM: intrinsic clearance highest value was 0.75 pmolCPFO/(mgproteinminµM). Detoxication to 3,5,6-trichloropyrin-2-ol formation was negligible. The comparison between HDM and HS2M indicates that most CPF bioactivation was confined in the duodenum, declining toward the distal ileum. Results suggest that following oral exposure, the small intestine CPF bioactivation, although much lower when compared to the total hepatic metabolism, could play a role in the pre-systemic CPF clearance, with CPFO transported into the lumen by the efflux P-glycoprotein and further metabolized by esterases.

Foetal and neonatal exposure to chlorpyrifos: biochemical and metabolic alterations in the mouse liver at different developmental stages.

The mechanisms implicated in the age-related toxicity, including its neurobehavioral effects after subtoxic developmental exposure to chlorpyrifos (CPF), a widely used insecticide, have not been fully elucidated yet. With the aim of investigating whether metabolic differences during ontogeny could account for the age-related susceptibility to CPF, we examined the developmental time-course of hepatic metabolizing enzymes and CPF metabolism in a cohort of mice exposed either prenatally (gestational day 15-18) and/or postnatally (postnatal day (PND) 11-14) to CPF at doses which were previously reported to induce neurobehavioural alterations, in the absence of brain acetyl-cholinesterase inhibition. Testosterone hydroxylase activity, CPF ex vivo biotransformation, glutathione content, as well as aromatase activity were determined in the liver of control and treated male and female mice at PND0, 9, 15 and 150. In control mice most Cyp activities were detectable and progressively increased up to PND15. In newborn control mice CPF bioactivation was much higher than the Cyp-catalysed detoxication, negligible at birth, indicating a possible increased susceptibility to CPF-induced effects in newborn mice. Detoxication rapidly increased with age, so that Cyp-related metabolic features cannot explain the higher susceptibility of juvenile mice. The observed age-dependent metabolic picture was partially altered by CPF prenatal treatment. Following in utero exposure CPF detoxifying capability was enhanced at birth and reduced at PND15, when CPF-oxon formation was slightly increased. No effects were evident at adulthood. Prenatal dosing was more effective in causing metabolic alterations than CPF postnatal treatment; no potentiation was observed in mice experiencing pre- plus post-natal CPF administration. Both in utero and postnatal CPF exposure decreased aromatase activity by 50% at PND9 and 15; this effect together with the presence of higher levels of the sex-specific Cyp2c activity at adulthood in male mice may suggest the occurrence of long-lasting impairment in the expression of hepatic Cyps under hormonal regulation. Altogether, the alterations in CPF Cyp-mediated biotransformation caused by perinatal CPF exposure seem not sufficient per se to explain the reported vulnerability of developing central nervous system to this insecticide, which can be due also to the parent compound itself or to the activation of different toxicological pathways. The hypothesis that observed effects on aromatase and sex-specific Cyp activity may be associated with a possible interference with the long-term alterations in sex-specific behavioural pattern deserves further investigation.

Cholinesterase inhibition and alterations of hepatic metabolism by oral acute and repeated chlorpyrifos administration to mice.

Chlorpyrifos (CPF) is a broad spectrum organophosphorus insecticide bioactivated in vivo to chlorpyrifos-oxon (CPFO), a very potent anticholinesterase. A great majority of available animal studies on CPF and CPFO toxicity are performed in rats. The use of mice in developmental neurobehavioural studies and the availability of transgenic mice warrant a better characterization of CPF-induced toxicity in this species. CD1 mice were exposed to a broad range of acute (12.5-100.0mg/kg) and subacute (1.56-25mg/kg/day from 5 to 30 days) CPF oral doses. Functional and biochemical parameters such as brain and serum cholinesterase (ChE) and liver xenobiotic metabolizing system, including the biotransformation of CPF itself, have been studied and the no observed effect levels (NOELs) identified. Mice seem to be more susceptible than rats at least to acute CPF treatment (oral LD(50) 4.5-fold lower). The species-related differences were not so evident after repeated exposures. In mice a good correlation was observed between brain ChE inhibition and classical cholinergic signs of toxicity. After CPF-repeated treatment, mice seemed to develop some tolerance to CPF-induced effects, which could not be attributed to an alteration of P450-mediated CPF hepatic metabolism. CPF-induced effects on hepatic microsomal carboxylesterase (CE) activity and reduced glutathione (GSH) levels observed at an early stage of treatment and then recovered after 30 days, suggest that the detoxifying mechanisms are actively involved in the protection of CPF-induced effects and possibly in the induction of tolerance in long term exposure. The mouse could be considered a suitable experimental model for future studies on the toxic action of organophosphorus pesticides focused on mechanisms, long term and age-related effects.