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

Carboxylesterases (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 (

Human variability in polymorphic CYP2D6 metabolism: Implications for the risk assessment of chemicals in food and emerging designer drugs.

The major human cytochrome P450 CYP2D6 isoform enzyme plays important roles in the liver and in the brain with regards to xenobiotic metabolism. Xenobiotics as CYP2D6 substrates include a whole range of pharmaceuticals, pesticides and plant alkaloids to cite but a few. In addition, a number of endogenous compounds have been shown to be substrates of CYP2D6 including trace amines in the brain such as tyramine and 5-methoxytryptamine as well as anandamide and progesterone. Because of the polymorphic nature of CYP2D6, considerable inter-phenotypic and inter-ethnic differences in the pharmaco/toxicokinetics (PK/TK) and metabolism of CYP2D6 substrates exist with potential consequences on the pharmacology and toxicity of chemicals. Here, large extensive literature searches have been performed to collect PK data from published human studies for a wide range of pharmaceutical probe substrates and investigate human variability in CYP2D6 metabolism. The computed kinetic parameters resulted in the largest open source database, quantifying inter-phenotypic differences for the kinetics of CYP2D6 probe substrates in Caucasian and Asian populations, to date. The database is available in supplementary material (CYPD6 DB) and EFSA knowledge junction (DOI to added). Subsequently, meta-analyses using a hierarchical Bayesian model for markers of chronic oral exposure (oral clearance, area under the plasma concentration time curve) and acute oral exposure (maximum plasma concentration (Cmax) provided estimates of inter-phenotypic differences and CYP2D6-related uncertainty factors (UFs) for chemical risk assessment in Caucasian and Asian populations classified as ultra-rapid (UM), extensive (EMs), intermediate (IMs) and poor metabolisers (PMs). The model allowed the integration of inter-individual (i.e. inter-phenotypic and inter-ethnic), inter-compound and inter-study variability together with uncertainty in each PK parameter. Key findings include 1. Higher frequencies of PMs in Caucasian populations compared to Asian populations (>8% vs 1-2%) for which EM and IM were the most frequent phenotype. 2. Large inter-phenotypic differences in PK parameters for Caucasian EMs (coefficients of variation (CV) > 50%) compared with Caucasian PMs and Asian EMs and IMs (i.e CV < 40%). 3. Inter-phenotypic PK differences between EMs and PMs in Caucasian populations increase with the quantitative contribution of CYP2D6 for the metabolism (fm) for a range of substrates (fmCYP2D6 range: 20-95% of dose) (range: 1-54) to a much larger extent than those for Asian populations (range: 1-4). 4. Exponential meta-regressions between FmCYP2D6 in EMs and inter-phenotypic differences were also shown to differ between Caucasian and Asian populations as well as CYP2D6-related UFs. Finally, implications of these results for the risk assessment of food chemicals and emerging designer drugs of public health concern, as CYP2D6 substrates, are highlighted and include the integration of in vitro metabolism data and CYP2D6-variability distributions for the development of quantitative in vitro in vivo extrapolation models.

Impact of the environment on the health: From theory to practice.

The Erice 56 Charter titled "Impact of the environment on the health: from theory to practice" was unanimously approved at the end of the 56th course of the "International School of Epidemiology and Preventive Medicine G. D'Alessandro" held from 3rd to November 7, 2019 in Erice - Sicily (Italy) and promoted by the Study Group of "Environment and Health" of the Italian Society of Hygiene, Preventive Medicine and Public Health. The course, that included lectures, open discussions and guided working groups, was aimed to provide a general training on epidemiological and toxicological aspects of the environmental health impact, to be used by public health professionals for risk assessment, without forgetting the risk communications. At the end of the course 12 key points were agreed among teachers and students: they underlined the need of specific training and research, in the perspective of "One Health" and "Global Health", also facing emerging scientific and methodological issues and focusing on communication towards stakeholders. This Discussion highlight the need to improve knowledge of Health and Environment topic in all sectors of health and environmental prevention and management.

Water quality and human health: A simple monitoring model of toxic cyanobacteria growth in highly variable Mediterranean hot dry environments.

Due to population growth, urbanization and economic development, demand for freshwater in urban areas is increasing throughout Europe. At the same time, climate change, eutrophication and pollution are affecting the availability of water supplies. Sicily, a big island in southern Italy, suffers from an increasing drought and consequently water shortage. In the last decades, in Sicilian freshwater reservoirs several Microcystis aeruginosa and more recently Planktothrix rubescens blooms were reported. The aims of the study were: (1) identify and quantify the occurring species of cyanobacteria (CB), (2) identify which parameters, among those investigated in the waters, could favor their growth, (3) set up a model to identify reservoirs that need continuous monitoring due to the presences, current or prospected, of cyanobacterial blooms and of microcystins, relevant for environmental and, consequentially, for human health. Fifteen artificial reservoirs among the large set of Sicilian artificial water bodies were selected and examined for physicochemical and microbiological characterization. Additional parameters were assessed, including the presence, identification and count of the cyanobacterial occurring species, the measurement of microcystins (MCs) levels and the search for the genes responsible for the toxins production. Principal Component Analysis (PCA) was used to relate environmental condition to cyanobacterial growth. Water quality was poor for very few parameters, suggesting common anthropic pressures, and PCA highlighted clusters of reservoirs vulnerable to hydrological conditions, related to semi-arid Mediterranean climate and to the use of the reservoir. In summer, bloom was detected in only one reservoir and different species was highlighted among the Cyanobacteria community. The only toxins detected were microcystins, although always well below the WHO reference value for drinking waters (1.0 µg/L). However, molecular analysis could not show the presence of potential cyanotoxins producers since a few numbers of cells among total could be sufficient to produce these low MCs levels but not enough high to be proved by the traditional molecular method applied. A simple environmental risk-based model, which accounts for the high variability of both cyanobacteria growth and cyanotoxins producing, is proposed as a cost-effective tool to evaluate the need for monitoring activities in reservoirs aimed to guarantee supplying waters safety.

Human variability in influx and efflux transporters in relation to uncertainty factors for chemical risk assessment.

Transporters are divided into the ABC and SLC super-families, mediating the cellular efflux and influx of various xenobiotic and endogenous substrates. Here, an extensive literature search was performed to identify in vivo probe substrates for P-gp, BCRP and OAT1/3. For other transporters (e.g. OCT, OATP), no in vivo probe substrates could be identified from the available literature. Human kinetic data (Cmax, clearance, AUC) were extracted from 142 publications and Bayesian meta-analyses were performed using a hierarchical model to derive variability distributions and related uncertainty factors (UFs). For P-gp, human variability indicated that the kinetic default UF (3.16) would cover over 97.5% of healthy individuals, when considering the median value, while the upper confidence interval is exceeded. For BCRP and OAT1/3 human variability indicated that the default kinetic UF would not be exceeded while considering the upper confidence interval. Although limited kinetic data on transporter polymorphisms were available, inter-phenotypic variability for probe substrates was reported, which may indicate that the current default kinetic UF may be insufficient to cover such polymorphisms. Overall, it is recommended to investigate human genetic polymorphisms across geographical ancestry since they provide more robust surrogate measures of genetic differences compared to geographical ancestry alone. This analysis is based on pharmaceutical probe substrates which are often eliminated relatively fast from the human body. The transport of environmental contaminants and food-relevant chemicals should be investigated to broaden the chemical space of this analysis and assess the likelihood of potential interactions with transporters at environmental concentrations.

Bayesian meta-analysis of inter-phenotypic differences in human serum paraoxonase-1 activity for chemical risk assessment.

Human variability in paraoxonase-1 (PON1) activities is driven by genetic polymorphisms that affect the internal dose of active oxons of organophosphorus (OP) insecticides. Here, an extensive literature search has been performed to collect human genotypic frequencies (i.e. L55M, Q192R, and C-108T) in subgroups from a range of geographical ancestry and PON1 activities in three probe substrates (paraoxon, diazoxon and phenyl acetate). Bayesian meta-analyses were performed to estimate variability distributions for PON1 activities and PON1-related uncertainty factors (UFs), while integrating quantifiable sources of inter-study, inter-phenotypic and inter-individual differences. Inter-phenotypic differences were quantified using the population with high PON1 activity as the reference group. Results from the meta-analyses provided PON1 variability distributions and these can be implemented in generic physiologically based kinetic models to develop quantitative in vitro in vivo extrapolation models. PON1-related UFs in the Caucasian population were above the default toxicokinetic UF of 3.16 for two specific genotypes namely -108CC using diazoxon as probe substrate and, -108CT, -108TT, 55MM and 192QQ using paraoxon as probe substrate. However, integration of PON1 genotypic frequencies and activity distributions showed that all UFs were within the default toxicokinetic UF. Quantitative inter-individual differences in PON1 activity are important for chemical risk assessment particularly with regards to the potential sensitivity to organophosphates' toxicity.

The importance of protein binding for the in vitro-in vivo extrapolation (IVIVE)-example of ibuprofen, a highly protein-bound substance.

A physiologically based human kinetic model (PBHKM) was used to predict the in vivo ibuprofen dose leading to the same concentration-time profile as measured in cultured human hepatic cells (Truisi et al. in Toxicol Lett 233(2):172-186, 2015). We parameterized the PBHKM with data from an in vivo study. Tissue partition coefficients were calculated by an algorithm and also derived from the experimental in vitro data for the liver. The predicted concentration-time profile in plasma was in excellent agreement with human experimental data when the liver partition coefficient was calculated by the algorithm (3.01) demonstrating values in line with findings obtained from human postmortem tissues. The results were less adequate when the liver partition coefficient was based on the experimental in vitro data (11.1). The in vivo doses necessary to reach the in vitro concentrations in the liver cells were 3610 mg using the best fitting model with a liver partition coefficient of 3.01 compared to 2840 mg with the in vitro liver partition coefficient of 11.1. We found that this difference is possibly attributable to the difference between protein binding in vivo (99.9 %) and in vitro (nearly zero) as the partition coefficient is highly dependent on protein binding. Hence, the fraction freely diffusible in the liver tissue is several times higher in vitro than in vivo. In consequence, when extrapolating from in vitro to in vivo liver toxicity, it is important to consider non-intended in vitro/in vivo differences in the tissue concentration which may occur due to a low protein content of the medium.

Kinetics and dynamics of cyclosporine A in three hepatic cell culture systems.

In vitro experiments have a high potential to improve current chemical safety assessment and reduce the number of animals used. However, most studies conduct hazard assessment alone, largely ignoring exposure and kinetic parameters. Therefore, in this study the kinetics of cyclosporine A (CsA) and the dynamics of CsA-induced cyclophilin B (Cyp-B) secretion were investigated in three widely used hepatic in vitro models: primary rat hepatocytes (PRH), primary human hepatocytes (PHH) and HepaRG cells. Cells were exposed daily to CsA for up to 14 days. CsA in cells and culture media was quantified by LC-MS/MS and used for pharmacokinetic modeling. Cyp-B was quantified by western blot analysis in cells and media. All cell systems took up CsA rapidly from the medium after initial exposure and all showed a time- and concentration-dependent Cyp-B cellular depletion and extracellular secretion. Only in PRH an accumulation of CsA over 14 days repeated exposure was observed. Donor-specific effects in CsA clearance were observed in the PHH model and both PHH and HepaRG cells significantly metabolized CsA, with no bioaccumulation being observed after repeated exposure. The developed kinetic models are described in detail and show that all models under-predict the in vivo hepatic clearance of CsA, but to different extents with 27-, 24- and 2-fold for PRH, PHH and HepaRG cells, respectively. This study highlights the need for more attention to kinetics in in vitro studies.

Cell type-specific expression and localization of cytochrome P450 isoforms in tridimensional aggregating rat brain cell cultures.

Within the Predict-IV FP7 project a strategy for measurement of in vitro biokinetics was developed, requiring the characterization of the cellular model used, especially regarding biotransformation, which frequently depends on cytochrome P450 (CYP) activity. The extrahepatic in situ CYP-mediated metabolism is especially relevant in target organ toxicity. In this study, the constitutive mRNA levels and protein localization of different CYP isoforms were investigated in 3D aggregating brain cell cultures. CYP1A1, CYP2B1/B2, CYP2D2/4, CYP2E1 and CYP3A were expressed; CYP1A1 and 2B1 represented almost 80% of the total mRNA content. Double-immunolabeling revealed their presence in astrocytes, in neurons, and to a minor extent in oligodendrocytes, confirming the cell-specific localization of CYPs in the brain. These results together with the recently reported formation of an amiodarone metabolite following repeated exposure suggest that this cell culture system possesses some metabolic potential, most likely contributing to its high performance in neurotoxicological studies and support the use of this model in studying brain neurotoxicity involving mechanisms of toxication/detoxication.

Cyclosporine A kinetics in brain cell cultures and its potential of crossing the blood-brain barrier.

There is an increasing need to develop improved systems for predicting the safety of xenobiotics. However, to move beyond hazard identification the available concentration of the test compounds needs to be incorporated. In this study cyclosporine A (CsA) was used as a model compound to assess the kinetic profiles in two rodent brain cell cultures after single and repeated exposures. CsA induced-cyclophilin B (Cyp-B) secretion was also determined as CsA-specific pharmacodynamic endpoint. Since CsA is a potent p-glycoprotein substrate, the ability of this compound to cross the blood-brain barrier (BBB) was also investigated using an in vitro bovine model with repeated exposures up to 14 days. Finally, CsA uptake mechanisms were studied using a parallel artificial membrane assay (PAMPA) in combination with a Caco-2 model. Kinetic results indicate a low intracellular CsA uptake, with no marked bioaccumulation or biotransformation. In addition, only low CsA amounts crossed the BBB. PAMPA and Caco-2 experiments revealed that CsA is mostly trapped to lipophilic compartments and exits the cell apically via active transport. Thus, although CsA is unlikely to enter the brain at cytotoxic concentrations, it may cause alterations in electrical activity and is likely to increase the CNS concentration of other compounds by occupying the BBBs extrusion capacity. Such an integrated testing system, incorporating BBB, brain culture models and kinetics could be applied for assessing neurotoxicity potential of compounds.

Interleukin-1 gene polymorphisms and gastric cancer risk in a high-risk Italian population.

OBJECTIVES: Host genetic factors, including the IL1 gene cluster, play a key role in determining the long-term outcome of Helicobacter pylori infection. The aim of the study was to investigate the relationship between selected IL1 loci polymorphisms and gastric cancer risk in an Italian population. METHODS: In a case-control study we compared the IL1B-31 and IL1B+3954 biallelic and IL1RN pentaallelic variable number of tandem repeats (VNTR) polymorphisms in 185 gastric cancer patients and 546 controls randomly sampled from the general population of an area at high gastric cancer risk (Tuscany, Central Italy). RESULTS: Genotype frequencies of the IL1B-31 T/C, IL1B+3954 C/T, and IL1RN polymorphisms among our population controls were in Hardy-Weinberg equilibrium. In multivariate analyses, no increase in gastric cancer risk was observed for the IL1B-31*C- and IL1B+3954*T- carriers; a significant 50% increase emerged for IL1RN*2 allele carriers (OR = 1.49; 95% CI: 1.01-2.21). Analyses based on combined genotypes showed also that the association with IL1RN*2 allele was limited to two-variant allele carriers who were also homozygous for the IL1B-31*T allele (OR = 2.23; 95% CI: 1.18-4.23) with a statistically significant interaction between these two genotypes (p= 0.043). Haplotype analysis showed an increased risk for the haplotype IL1RN*2/IL1B-31*T. CONCLUSIONS: Our results suggest that host genetic factors (such as the IL1RN and the IL1B-31 polymorphisms) interact in the complex process of gastric carcinogenesis in this high-risk Italian population. Overall, this effect appears more modest than previously reported in other populations, supporting the hypothesis that other still-to-be-defined factors are important in gastric carcinogenesis. These findings might be due to a haplotype effect.

Toxicology investigations with cell culture systems: 20 years after.

From almost 20 years the "in vitro" model has gained a wide ground in toxicological investigation, providing advanced tools, reliable protocols, mechanistic information. These advancements have been done thanks to different approaches, addressed at improving chemical testing and validating procedures, at exploring the cellular and molecular basis of toxicity, at studying the modifications that xenobiotics undergo in the cellular environment. In this review the most advanced cellular models, the mechanisms of cell death, the techniques to monitor gene activation, following chemical exposure, is highlighted. Moreover the more recent in vitro models to approach the biotransformation issue will be presented.

Mechanistic aspects of organophosphorothionate toxicity in fish and humans.

Metabolic transformation plays a major role in the mechanism of toxicity of organophosphorous (OP) pesticides. The modulation of their toxicity by oxonases and monooxygenases, alone or in combination, has been shown in mammals and fish. Very limited information exists for the identification of the metabolic factors relevant in the human toxicology of such chemicals. In this paper, we develop a simple algorithm, based on in vitro data, for the identification of fish species more susceptible to diazinon (D). Similar algorithms are likely to be applicable to other organophosphothionate (OPT) pesticides. We also report on preliminary studies on the OPT substrate specificity of human liver cytochromes P450 (CYPs): such information may be useful to understand the role of sulphoxidation in OPT toxicity to humans and to identify individuals with increased susceptibility to OPT toxicity. Studies of the mechanism of OPT toxicity may provide useful tools for a more detailed characterisation of these chemicals, with reference to the risk for the human population and to the impact on the fish species present in specific environments.

Correlation of a specific mitochondrial phospholipid-phosgene adduct with chloroform acute toxicity.

The dose and time dependence of formation of a specific adduct between mitochondrial phospholipid and phosgene have been determined in the liver of Sprague-Dawley (SD) rats as well as in the liver and kidney of B6C3F1 mice after dosing with chloroform. Rats were induced with phenobarbital or non-induced. Determination of tissue glutathione (GSH) and of serum markers of hepatotoxicity and nephrotoxicity was also carried out. With dose-dependence experiments, a strong correlation between the formation of the specific phospholipid adduct, GSH depletion and organ toxicity could be evidenced in all the organs studied. With non-induced SD rats, no such effects could be induced up to a dose of 740 mg/kg. Time-course studies with B6C3F1 mice indicated that the specific adduct formation took place at very early times after chloroform dosing and was concurrent with GSH depletion. The adduct formed during even transient GSH depletion (residual level: 30% of control) and persisted after restoration of GSH levels. Following a chloroform dose at the hepatotoxicity threshold (150 mg/kg), the elimination of the adduct in the liver occurred within 24 h and correlated with the recovery of ALT, which was slightly increased (12 times) after treatment. Following a moderately nephrotoxic dose (60 mg/kg), the renal adduct persisted longer than 48 h, when a 100% increase in blood urea nitrogen and a 40% increase in serum creatinine indicated the onset of organ damage. The formation of the adduct in the liver mitochondria of B6C3F1 mice was associated with the decrease of phosphatidyl-ethanolamine (PE), in line with previous results in rat liver indicating that the adduct results from the reaction of phosgene with PE. The adduct levels implicated the reaction of phosgene with about 50% PE molecules in the liver mitochondrial membrane of phenobarbital-induced SD rats and of about 10% PE molecules of the inner mitochondrial membrane of the liver of B6C3F1 mice. The association of this adduct with the toxic effects of chloroform makes it a very good candidate as the primary critical alteration in the sequence of events leading to cell death caused by chloroform.

The relevance of xenobiotic metabolism in the interindividual susceptibility to chemicals.

Biotransformation enzymes may catalyze either detoxication or bioactivation reactions; indeed, many xenobiotics exert their toxic effects after metabolic activation to electrophilic chemicals, interacting with nucleophilic sites on cellular macromolecules. On the other hand, by increasing xenobiotic hydrophilicity, the drug-metabolizing enzymes favors excretion of lipophilic chemicals, not allowing their bioaccumulation up to toxic levels. The expression of the enzymes of the drug-metabolizing system is modulated by genetic, pathological, developmental, environmental and dietary factors. Genetic polymorphism resulting in interindividual and interethnic variation in xenobiotic metabolism is responsible for differences in the susceptibility to chemical-induced toxicity and carcinogenicity, allowing the identification of people at increased risk. Moreover, differences in drug metabolism may correspond to variability in drug response during pharmacological therapy, which can be manifest either as adverse reactions or as a lack of benefit.

Comparative characterization of CHCl(3) metabolism and toxicokinetics in rodent strains differently susceptible to chloroform-induced carcinogenicity.

A comparative kinetic study in B6C3F1 mice, Osborne-Mendel (OM) and Sprague-Dawley (SD) rats has been undertaken with the major aim to determine the extent of covalent binding of chloroform reactive metabolites produced in vivo through oxidative and/or reductive metabolism in the target organs of chloroform carcinogenicity. Some additional kinetic observations of chloroform biotransformation were also collected comparatively. Expiration of [14C]-CO(2) showed that chloroform metabolism went to saturation in all tested rodent strains. In the B6C3F1 mouse maximal rates of approximately 135 µmol [14C]-CO(2)/kg b.w./h were reached at a dose of approximately 150 mg/kg, while in the two rat strains saturation occurred at a dose of approximately 60 mg/kg, with a maximal rate of approximately 40 µmol [14C]-CO(2)/kg b.w./h. At doses of 150-180 mg/kg b.w., limited differences were found in the distribution and elimination of [14C]-chloroform in the liver and kidney. Species differences have been found in the kinetics of alkali-extractable radioactivity in the blood. The levels of adducts of electrophilic intermediates with the polar heads (PH) of phospholipids (PL) showed a limited variability accross the rodents tested and did not correlate with the species and organ susceptibility to chloroform carcinogenicity. The levels of adducts of radical intermediates with the fatty acyl chains (FC) of PL were much lower than the PH adducts in all the samples analyzed; at the carcinogenicity bioassay doses, statistically significant levels of hepatic FC adducts were present only in the B6C3F1 mouse, where chloroform is hepatocarcinogenic. The observations in the rat kidney were suggestive of the formation of electrophilic reactive metabolites, presumably different from phosgene and associated with an initial chloroform reduction.

Time dependence of chloroform-induced metabolic alterations in the liver and kidney of B6C3F1 mice.

The time course of some biochemical changes in the liver and in the kidney was studied in B6C3F1 male mice dosed with a single i.p. injection of 150 mg/kg body weight (b.w.) CHCl(3). Hepatic and renal microsomal cytochrome P450 (P450) content and some related monooxygenase activities, CHCl(3) oxidative and reductive metabolism, cytosolic reduced glutathione (GSH) content and serum markers of nephrotoxicity were measured. In the liver no biochemical changes were produced up to a week after chloroform treatment. On the contrary, the drug-metabolizing enzyme system in the kidney was dramatically and rapidly inactivated by chloroform treatment. Maximum loss of GSH (50%), P450 (80%) and of different enzymatic activities, including CHCl(3) bioactivation, occurred during the first 5 h. These biochemical alterations are early effects, not secondary to morphological tissue changes. Kidney parameters, altered by chloroform treatment, returned to control values at different times: renal function markers became normal in 48 h; GSH levels were recovered at 96 h and the drug-metabolizing enzyme activities at longer times. The present results clearly show that repeated daily doses of chloroform, as those used in carcinogenicity tests, find renal tubular cells not at their physiological status, due to the changes produced by the first chloroform dose. Therefore the similarity in P450-dependent chloroform metabolism shown in vitro by hepatic and renal microsomes from untreated B6C3F1 male mice or in vivo in animals treated once, is lost during repeated treatments. These features should be considered in understanding the different susceptibility of the liver and the kidney to chloroform-induced tumours.