EUROPLAN: a project to support the development of national plans on rare diseases in Europe.

BACKGROUND/AIMS: National Plans for Rare Diseases (RDs) are the common denominator of current public health policy concerns on RDs across the EU. With the aim of a better distribution of the available resources, they conjugate the European objective that aims at ensuring that patients with RDs have access to high-quality care - including diagnostics, treatment and rehabilitation - with the national priorities of selecting specific measures for adoption and implementation. METHODS: The European Project for Rare Diseases National Plans Development (EUROPLAN, www.europlanproject.eu) is cofunded by the EU Commission (DG-SANCO) and is coordinated by the Italian National Center for Rare Diseases of the Istituto Superiore di Sanità (ISS). The EUROPLAN goal is to promote the implementation of National Plans or Strategies to tackle RDs and share relevant experiences within countries, linking national efforts, through a common strategy at a European level. In order to fulfill these objectives, EUROPLAN involved health authorities, clinicians, scientists, the European Organisation for Rare Diseases (EURORDIS), and many other patient groups as associated and collaborating partners from several European countries. RESULTS: The project was launched in 2008 and foresaw 2 implementation phases: phase 1 (2008-2011) to build the consensus definition of operational tools (recommendations and indicators), and the ongoing phase 2 (2012-2015), mainly aimed at capacity building with the proactive involvement of multilevel stakeholders. EUROPLAN is facilitating and accelerating the implementation of National Plans in almost all EU and several non-EU Countries. CONCLUSIONS: EUROPLAN is a European and an international process more than a project, and it could be defined as a 'litmus test' demonstrating how the collaboration between institutions and patients' associations can accelerate the process of awareness and development of policies and actions.

The current situation and needs of rare disease registries in Europe.

BACKGROUND: Registries are considered key instruments for developing rare disease (RD) clinical research, enhancing patient care and health planning, and improving social, economic and quality-of-life outcomes. Indeed, it is usually the case that no single institution, and in many cases no single country, has sufficient data to provide results that can be applied broadly to clinical and translational research. However, the fragmentation and heterogeneity of the registries, which are often the result of spontaneous initiatives, limit the general applicability of their observations. METHODS: An inquiry has been carried out by the EPIRARE, a European Union (EU)-funded project ('Building Consensus and Synergies for the EU Registration of Rare Disease Patients') aiming at paving the way to the creation of a European Platform for RD Registries, by means of an on-line questionnaire among European RD registries on their main activities and needs, the way they deal with methodological, technical and regulatory issues and the way they find resources to carry on their activities. RESULTS: In spite of the heterogeneity of the European registries, some elements of relevance for an action to improve the situation of patient registries in the EU are apparent. The needs more frequently indicated by registry holders were financial support, motivation of data providers, data quality assessment, improvement of communication and visibility, and extension of collaborations. Moreover, the registry holders were in favor of a common EU platform providing services for RD registries. CONCLUSION: It appears that the current situation of the European registries provides the transition towards a more uniform, higher quality and better coordinated approach.

A model for the European platform for rare disease registries.

BACKGROUND: The current situation of rare disease (RD) registries is rather heterogeneous, and new ways to support the registration of RD patients are being sought in the European Union (EU) and the US. The project 'Building Consensus and Synergies for the EU Registration of RD Patients', funded by the EU, aimed to define a model platform for EU RD registries. METHODS: A number of surveys and extensive consultations among registry stakeholders have been carried out to study how the platform can best fulfill their needs. RESULTS: This web-based, multidisease and multipurpose platform is intended to provide a number of functions: a metadata and data repository function supporting the planning of research studies and the production of predefined outputs for the funding organizations and the public, provision of tools and resources of use to registries, promotion of registration and networking among patients and professionals. CONCLUSION: Its main impact is expected to be on data and procedures standardization, on the establishment of new registries, on the sustainability of the smaller ones, and on the registration of those RDs for which a dedicated registry is not sustainable, e.g. ultra-rare diseases or diseases for which there is no special research, clinical or economic interest. It will also impact on the production of sounder information on RD and RD-dedicated health systems, by promoting registry data comparability and quality.

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.

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.

Identification of the cytochrome P450 isoenzymes involved in the metabolism of diazinon in the rat liver.

The metabolism of diazinon, an organo-phosphorothionate pesticide, to diazoxon and pyrimidinol has been studied in incubations with hepatic microsomes from control Sprague-Dawley (SD) rats or SD rats treated with different P450-specific inducers (phenobarbital, dexamethasone, beta-napthoflavone, and pyrazole). Results obtained indicate an involvement of CYP2C11, CYP3A2, and CYP2B1/2, whereas CYP2E1 and CYP1A1 do not contribute to the pesticide oxidative metabolism. Indeed, diazinon was metabolized by microsomes from control rats; among the inducers, phenobarbital and dexamethasone only increased the production of either metabolites, although to different extents. The production of the two metabolites is self-limiting, due to P450 inactivation; therefore, the inhibition of CYP-specific monooxygenase activities after diazinon preincubation has been used to selectively identify the competent CYPs in diazinon metabolism. Results indicate that, after diazinon preincubation, CYP3A2-catalyzed reactions (2beta- and 6beta-testosterone hydroxylation) are very efficiently inhibited; CYP2C11- and CYP2B1/2-catalyzed reactions (2alpha- and 16beta-testosterone hydroxylation, respectively) are weakly inhibited, while CYP2E1-, CYP2A1/2-, and CYP1A1/2-related activities were unaffected. Results obtained by using chemical inhibitors or antibodies selectively active against specific CYPs provide a direct evidence for the involvement of CYP2C11, CYP3A2, and CYP2B1/2, indicating that each of them contributed about 40-50% of the diazinon metabolism, in hepatic microsomes from untreated, phenobarbital-, and dexamethasone-treated rats, respectively. The higher diazoxon/pyrimidinol ratio observed after phenobarbital-treatment together with the significantly more effective inhibition toward diazoxon production exerted by metyrapone in microsomes from phenobarbital-treated rats supports the conclusion that CYP2B1/2 catalyze preferentially the production of diazoxon.

Characterization of a phospholipid adduct formed in Sprague Dawley rats by chloroform metabolism: NMR studies.

The formation of a covalent adduct to a single phospholipid by the oxidative chloroform metabolite, phosgene, is demonstrated in liver mitochondria of phenobarbital-pretreated Sprague Dawley (SD) rats treated with CHCl3. The densitometric analysis of the phosphorus stained extracted phospholipids showed that the formation of this adduct in liver mitochondria is accompanied by a decrease of phosphatidylethanolamine and cardiolipin. The characterization of this adduct was performed with a multinuclear NMR approach by comparison with the decreased phospholipids. Treatment of rats with [13C]chloroform resulted in an intense 13C NMR peak from either an esteric or amidic carbonyl. Very strong similarities in fatty acid composition were found between phosphatidylethanolamine and the phosgene-modified PL, using 13C and 1H NMR spectroscopy. A multiplet at 3.91 ppm coupled to a signal at 3.41 ppm was shown by two-dimensional 1H NMR in the adduct spectrum. This cross peak was interpreted as arising from the shifted resonances of the two PE head group methylene groups, due to the binding with phosgene. 31P spectrum of the adduct was identical to that of phosphatidylethanolamine. We concluded that the chloroform adduct is a modified phosphatidylethanolamine, with the phosgene-derived carbonyl bound to the amine of the head group.

Food plant toxicants and safety Risk assessment and regulation of inherent toxicants in plant foods.

The ADI as a tool for risk management and regulation of food additives and pesticide residues is not readily applicable to inherent food plant toxicants: The margin between actual intake and potentially toxic levels is often small; application of the default uncertainty factors used to derive ADI values, particularly when extrapolating from animal data, would prohibit the utilisation of the food, which may have an overall beneficial health effect. Levels of inherent toxicants are difficult to control; their complete removal is not always wanted, due to their function for the plant or for human health. The health impact of the inherent toxicant is often modified by factors in the food, e.g. the bioavailability from the matrix and interaction with other inherent constituents. Risk-benefit analysis should be made for different consumption scenarios, without the use of uncertainty factors. Crucial in this approach is analysis of the toxicity of the whole foodstuff. The relationship between the whole foodstuff and the pure toxicant is expressed in the `product correction factor' (PCF). Investigations in humans are essential so that biomarkers of exposure and for effect can be used to analyse the difference between animals and humans and between the food and the pure toxicant. A grid of the variables characterising toxicity is proposed, showing their inter-relationships. A flow diagram for risk estimate is provided, using both toxicological and epidemiological studies.

In vivo CHCl3 bioactivation, toxicokinetics, toxicity, and induced compensatory cell proliferation in B6C3F1 male mice.

Chloroform carcinogenicity has often been associated with acute tissue damage and consequent compensatory cell proliferation. However, available data do not fully support this hypothesis, and other biological factors may play a role in the tumor induction by chloroform. The purpose of this study was to characterize the in vivo CHCl3 metabolism and the time course of toxic effects and of cell proliferation in the liver and kidney of B6C3F1 male mice dosed i.p. or by gavage with 150 mg CHCl3/kg body wt. Microsomal phospholipid adducts attributed to (14)CHCl3 metabolism by both oxidative and reductive pathways were detected in both liver and kidney. The levels and composition of the adducts were similar in the liver and kidney of treated animals. In the liver, although no necrosis was histologically detectable, a transient cell proliferation was found starting at 24 and peaking at 48 hr post-treatment. Kidney toxicity was evident by biochemical and cytochemical methods at 5 hr after dosing and progressed to severe necrosis at 48 and 96 hr. An intense kidney cell regeneration began 48 hr after CHCl3 treatment, became maximal at 96 hr, and was sustained for at least the following 3 days. These observations raise questions about the purely epigenetic action of chloroform in tumor induction since bioassays have found tumors in liver but not kidneys of CHCl3-treated B6C3F1 mice.

The role of different cytochrome P450 isoforms in in vitro chloroform metabolism.

The two CHCl3 activation pathways have been studied in incubations at different oxygenation conditions with hepatic microsomes from control Sprague Dawley (SD) rats or SD rats treated with different cytochrome P450 inducers (acetone, phenobarbital, pyrazole, dexamethasone, and beta-naphthoflavone). The present results provide direct evidence that CHCl3 concentration is critical in determining the role of different cytochrome P450 isoforms (CYP) and the related effects of metabolic inducers. At 0.1 mM CHCl3 concentration, the only major contribution to its oxidative biotransformation in liver microsomes from untreated rats was due to CYP2E1, as shown by metabolic inhibition due to 4-methylpyrazole or by anti-CYP2E1 antibodies. Moreover, animal treatments with acetone and pyrazole increased the production of adducts of phosgene to microsomal phospholipid by about 10-15 times. At 5 mM chloroform, in control rat liver microsomes, CYP2B1/2 was the major participant responsible for chloroform activation, while CYP2E1 and CYP2C11 were also significantly involved. Consistently, at this chloroform concentration, the effect of phenobarbital (CYP2B1/2 inducer) was maximal, producing very high levels of adducts. The reductive pathway was expressed at 5 mM CHCl3 only and was not significantly increased by any of the inducers used. Moreover, it was not inhibited by metyrapone and 4-methylpyrazole or by anti CYP2C11 antibodies. Therefore, it may be concluded that, in the range of chloroform concentrations tested, those CYPs involved in CHCl3 oxidative bioactivation do not participate in CHCl3 reduction. Chloroform oxidative metabolism in PB-microsomes could achieve very high absolute rates, much higher than those in C-microsomes; in contrast, the metabolic rates in AC- and PYR-microsomes remained within the activity levels observable in C-microsomes at high chloroform concentration. Therefore, it can be argued that the CYP2B1/2-mediated induction of CHCl3 activation is the basis for the effect of PB in potentiating chloroform hepatotoxicity. Moreover, processes other than CYP2E1-mediated metabolic induction may be more relevant in the ketones potentiation of chloroform-induced acute toxicity.

In vitro quantitative determination of phospholipid adducts of chloroform intermediates in hepatic and renal microsomes from different rodent strains.

We have comparatively studied in vitro the oxidative and reductive pathways of chloroform metabolism in hepatic and renal microsomes of rodent strains used for carcinogenicity testing (B6C3F1 mice, Osborne Mendel and Sprague Dawley rats). To this aim we exploited the regioselective binding of phosgene to phospholipid (PL) polar heads and of dichloromethyl radical to PL fatty acyl chains, using a method based on the chemical transmethylation of PL adducts, followed by phase partitioning of the resulting products (De Biasi et al., 1992). The analysis of results let us to conclude at first that a (14)C label partitioning by 89.2 (±6.5)% or 13.7 (±5.0)% in the aqueous phase is typical of the PL adduct with phosgene (PL-PHOS) or with dichloromethyl radical (PL-RAD), respectively. Metabolism of 0.1 mM CHCl(3) was mainly oxidative in all the samples, being hepatic microsomes more active than renal ones by about one order of magnitude and levels of CHCl(3)-derived PL adducts in B6C3F1 mouse liver microsomes higher than in rat samples. At 5 mM CHCl(3), total levels of PL adducts in renal microsomes reached levels almost similar to those found in liver microsomes. However, while B6C3F1 mouse kidney microsomes produced both reactive metabolites, similarly as the hepatic samples, Osborne Mendel rat kidney microsomes bioactivated CHCl(3) only reductiveiy, producing the radical. The relevance of this finding depends on the fact that phosgene is known to be the major cause of CHCl(3) toxicity, based on data with the rat liver and mouse liver and kidney, while nephrotoxicity in rats occurs with minimal production of COCl(2). Chloroform reductive bioactivation may therefore provide a reasonable explanation for the toxicity of chloroform to the rat kidney. The same finding may be of interest in elucidating the metabolic reasons of the chloroform-induced kidney tumors in Osborne Mendel rats.

Enzymological differences of AChE and diazinon hepatic metabolism: correlation of in vitro data with the selective toxicity of diazinon to fish species.

The in vitro hepatic metabolism of diazinon, as well as the sensitivity of the brain acetylcholine esterase, to diazoxon inhibitory action have been studied in order to explain the different toxicity of diazinon to Oncorhynchus mykiss (rainbow trout), Poecilia reticulata (guppy), Brachydanio rerio (zebra fish) and Cyprinus carpio (carp). In spite of a very sensitive acetylcholine esterase the carp is very resistant to diazinon toxicity because of its very low rate of bioactivation and relatively high activity of detoxicating enzymes. The trout is very sensitive towards diazinon in spite of its low activity of bioactivation, because of its lack of detoxicating enzymes and a very sensitive acetylcholine esterase. Diazinon is very toxic for the guppy, because this fish combines a relatively sensitive acetylcholine esterase with a high rate of bioactivation. The zebra fish has the most insensitive acetylcholine esterase, associated with a limited activation rate, thus resulting a rather resistant species. The results obtained indicate that diazinon toxicity differences among the fish species studied can largely be explained in relation to metabolic balances in the liver and with the features of the target enzyme.

An in vitro investigation of the reductive metabolism of chloroform.

In hypoxic (1% pO2) and anoxic (0% pO2) incubations of CHCl3 with rat liver microsomes from PB-induced animals, no evidence of formation of monochloromethyl carbene could be found. Dichloromethane was detected as a volatile metabolite of CHCl3 in incubations with rat liver microsomes from PB-induced animals, under different oxygenation conditions (from 0% to 20% pO2). With uninduced microsomes, significant levels of dichloromethane were formed only in hypoxic (1% pO2) or anoxic incubations. The amount of dichloromethane measured was 2-6 times lower than the levels of adducts to the fatty acyl chains (FC) of microsomal phospholipid. The very low rate of dichloromethane formation suggests that the assay of expired dichloromethane is not suitable to detect the reductive metabolism of CHCl3 in vivo.

Multiple activation of chloroform in kidney microsomes from male and female DBA/2J mice.

Microsomes from the renal cortex of DBA/2J mice can metabolize chloroform through oxidative and reductive pathways, similar to hepatic microsomes. The oxidative or reductive nature of CHCl3 activation is strictly dependent on the oxygenation of the incubation mixture, as indicated by the formation of qualitatively different adducts to phospholipids (PLs). The protein and lipid binding levels measured in kidney microsomes from control females differed significantly from the binding levels observed with kidney microsomes from male and testosterone-treated female DBA/2J mice in aerobic conditions only. Therefore, the sex-dependent CHCl3-induced acute nephrotoxicity seems related only with the oxidative CHCl3 activation. The levels of adducts to PL polar heads and to protein showed a strict correlation with each other. Therefore, the assay of adducts to PL polar heads may be used as a substitute for the assay of adducts to protein. This might be especially convenient when studying the effects of both phosgene and the trichloromethyl radicals.

The contribution of electrophilic and radicalic intermediates to phospholipid adducts formed by halomethanes in vivo.

The different production of phosgene and free-radicals from CHCl3 and CCl4 was determined in vitro and in vivo, by measuring the regioselective binding to the two intermediates to phospholipid (PL) molecules. Results clearly indicated that this assay can be successfully used to selectively detect electrophilic and radicalic metabolites produced in vivo and selectively quantitate their adducts. The in vivo biotransformation of CCl4, similarly to the in vitro situation, resulted in the formation of radicals only, the contribution of phosgene to the structural damage of PL being negligible. These findings allowed us to rule out the hypothesis of substantial formation of radicalic intermediates from CHCl3 in phenobarbital (PB)-pretreated Sprague-Dawley (SD) rats, derived from in vitro data. While the role of reduced glutathione (GSH) in preventing COCl2-derived damages seems to be less important in vivo than in vitro, it is not possible to rule out the action of radical scavenging systems in decreasing the level of adducts with fatty acyl chains (FC) of PL measured in vivo.

Effect of ethanol on CHCl3 metabolism in hepatic microsomes from Osborne-Mendel rats.

The treatment of Osborne-Mendel rats with ethanol in drinking water for 2 weeks resulted in a 3-fold increase of hepatic microsomal hydroxylation of both p-nitrophenol and aniline, two substrates considered highly selective for P4502E1. No other forms of P450 seemed to be affected. These results, confirmed by the immunoblot analysis of microsomal protein, showed an induction of P4502E1. The levels of total covalent binding to microsomal phospholipid due to 14CHCl3 reactive intermediates in ethanol-pretreated microsomes were identical to those measured in microsomes from untreated rats at any pO2. The distribution of radioactivity obtained after transmethylation of the adducts of 14CHCl3 intermediates with microsomal phospholipids (PL) indicated that binding to fatty acyl chains (due to .CHCl2 radicals) increased with decreasing pO2. On the contrary, the binding to polar heads due to phosgene decreased. The ethanol treatment did not affect binding to either PL moieties. These results indicated that, in our experimental conditions, the in vitro production of both oxidative and reductive intermediates of CHCl3 in the liver of Osborne-Mendel rats were not influenced by ethanol consumption.