Specific effects on the thyroid relevant for performing a dietary cumulative risk assessment of pesticide residues: 2024 update.

EFSA updated its previous work on the establishment of specific effects that are considered relevant for grouping pesticide residues targeting the thyroid and for performing the retrospective assessment of dietary cumulative risk (CRA). The two specific effects already selected in 2019 leading to the two cumulative assessment groups (CAGs) 'hypothyroidism' and 'C-cell hypertrophy, hyperplasia and neoplasia' were reconfirmed. Compared to 2019, the list of indicators that can be used to identify these specific effects was refined to only include histopathological changes. In a second phase of the work, data will be extracted on indicators of the specific effects from the dossiers on active substances (a.s.) used as plant protection products. The criteria for including a.s. into CAGs were also updated, together with the hazard characterisation methodology and the lines of evidence for assessing CAG-membership probabilities. The tasks related to the data extraction and the establishment of the CAGs on hypothyroidism and on C-cell hypertrophy, hyperplasia and neoplasia are beyond the scope of this report. This part of the CRA process has been outsourced and will be the subject of a separate report.

Re-evaluation of the risks to public health related to the presence of bisphenol A (BPA) in foodstuffs.

In 2015, EFSA established a temporary tolerable daily intake (t-TDI) for BPA of 4 µg/kg body weight (bw) per day. In 2016, the European Commission mandated EFSA to re-evaluate the risks to public health from the presence of BPA in foodstuffs and to establish a tolerable daily intake (TDI). For this re-evaluation, a pre-established protocol was used that had undergone public consultation. The CEP Panel concluded that it is Unlikely to Very Unlikely that BPA presents a genotoxic hazard through a direct mechanism. Taking into consideration the evidence from animal data and support from human observational studies, the immune system was identified as most sensitive to BPA exposure. An effect on Th17 cells in mice was identified as the critical effect; these cells are pivotal in cellular immune mechanisms and involved in the development of inflammatory conditions, including autoimmunity and lung inflammation. A reference point (RP) of 8.2 ng/kg bw per day, expressed as human equivalent dose, was identified for the critical effect. Uncertainty analysis assessed a probability of 57-73% that the lowest estimated Benchmark Dose (BMD) for other health effects was below the RP based on Th17 cells. In view of this, the CEP Panel judged that an additional uncertainty factor (UF) of 2 was needed for establishing the TDI. Applying an overall UF of 50 to the RP, a TDI of 0.2 ng BPA/kg bw per day was established. Comparison of this TDI with the dietary exposure estimates from the 2015 EFSA opinion showed that both the mean and the 95th percentile dietary exposures in all age groups exceeded the TDI by two to three orders of magnitude. Even considering the uncertainty in the exposure assessment, the exceedance being so large, the CEP Panel concluded that there is a health concern from dietary BPA exposure.

Update of the risk assessment of di-butylphthalate (DBP), butyl-benzyl-phthalate (BBP), bis(2-ethylhexyl)phthalate (DEHP), di-isononylphthalate (DINP) and di-isodecylphthalate (DIDP) for use in food contact materials.

The EFSA Panel on Food Contact Materials, Enzymes and Processing Aids (CEP Panel) was asked by the European Commission to update its 2005 risk assessments of di-butylphthalate (DBP), butyl-benzyl-phthalate (BBP), bis(2-ethylhexyl)phthalate (DEHP), di-isononylphthalate (DINP) and di-isodecylphthalate (DIDP), which are authorised for use in plastic food contact material (FCM). Dietary exposure estimates (mean and high (P95)) were obtained by combining literature occurrence data with consumption data from the EFSA Comprehensive Database. The highest exposure was found for DINP, ranging from 0.2 to 4.3 and from 0.4 to 7.0 µg/kg body weight (bw) per day for mean and high consumers, respectively. There was not enough information to draw conclusions on how much migration from plastic FCM contributes to dietary exposure to phthalates. The review of the toxicological data focused mainly on reproductive effects. The CEP Panel derived the same critical effects and individual tolerable daily intakes (TDIs) (mg/kg bw per day) as in 2005 for all the phthalates, i.e. reproductive effects for DBP (0.01), BBP (0.5), DEHP (0.05), and liver effects for DINP and DIDP (0.15 each). Based on a plausible common mechanism (i.e. reduction in fetal testosterone) underlying the reproductive effects of DEHP, DBP and BBP, the Panel considered it appropriate to establish a group-TDI for these phthalates, taking DEHP as index compound as a basis for introducing relative potency factors. The Panel noted that DINP also affected fetal testosterone levels at doses around threefold higher than liver effects and therefore considered it conservative to include it within the group-TDI which was established to be 50 µg/kg bw per day, expressed as DEHP equivalents. The aggregated dietary exposure for DBP, BBP, DEHP and DINP was estimated to be 0.9-7.2 and 1.6-11.7 µg/kg bw per day for mean and high consumers, respectively, thus contributing up to 23% of the group-TDI in the worst-case scenario. For DIDP, not included in the group-TDI, dietary exposure was estimated to be always below 0.1 µg/kg bw per day and therefore far below the TDI of 150 µg/kg bw per day. This assessment covers European consumers of any age, including the most sensitive groups. Based on the limited scope of the mandate and the uncertainties identified, the Panel considered that the current assessment of the five phthalates, individually and collectively, should be on a temporary basis.

Advancing human health risk assessment.

The current/traditional human health risk assessment paradigm is challenged by recent scientific and technical advances, and ethical demands. The current approach is considered too resource intensive, is not always reliable, can raise issues of reproducibility, is mostly animal based and does not necessarily provide an understanding of the underlying mechanisms of toxicity. From an ethical and scientific viewpoint, a paradigm shift is required to deliver testing strategies that enable reliable, animal-free hazard and risk assessments, which are based on a mechanistic understanding of chemical toxicity and make use of exposure science and epidemiological data. This shift will require a new philosophy, new data, multidisciplinary expertise and more flexible regulations. Re-engineering of available data is also deemed necessary as data should be accessible, readable, interpretable and usable. Dedicated training to build the capacity in terms of expertise is necessary, together with practical resources allocated to education. The dialogue between risk assessors, risk managers, academia and stakeholders should be promoted further to understand scientific and societal needs. Genuine interest in taking risk assessment forward should drive the change and should be supported by flexible funding. This publication builds upon presentations made and discussions held during the break-out session 'Advancing risk assessment science - Human health' at EFSA's third Scientific Conference 'Science, Food and Society' (Parma, Italy, 18-21 September 2018).

Genotoxicity testing approaches for the safety assessment of substances used in food contact materials prior to their authorization in the European Union.

Food contact materials are all materials and articles intended to come directly or indirectly into contact with food. Before being included in the positive European "Union list" of authorized substances (monomers, other starting substances and additives) for plastic food contact materials, the European Food Safety Authority (EFSA) must assess their safety "in use". If relevant for risk, the safety of the main impurities, reaction and degradation products originating from the manufacturing process is also evaluated. Information on genotoxicity is always required irrespective of the extent of migration and the resulting human exposure, in view of the theoretical lack of threshold for genotoxic events. The 2008 EFSA approach, requiring the testing of food contact materials in three in vitro mutagenicity tests, though still acceptable, is now superseded by the 2011 EFSA Scientific Committee's recommendation for only two complementary tests including a bacterial gene mutation test and an in vitro micronucleus test, to detect two main genetic endpoints (i.e., gene mutations and chromosome aberrations). Follow-up of in vitro positive results depends on the type of genetic effect and on the substance's systemic availability. In this study, we provide an analysis of the data on genotoxicity testing gathered by EFSA on food contact materials for the period 1992-2015. We also illustrate practical examples of the approaches that EFSA took when evaluating "non standard" food contact chemicals (e.g., polymeric additives, oligomer or other reaction mixtures, and nanosubstances). Additionally, EFSA's experience gained from using non testing methods and/or future possibilities in this area are discussed. Environ. Mol. Mutagen. 58:361-374, 2017. © 2017 Wiley Periodicals, Inc.

A statement on the developmental immunotoxicity of bisphenol A (BPA): answer to the question from the Dutch Ministry of Health, Welfare and Sport.

This statement addresses a request to EFSA from the Dutch Ministry of Public Health, Welfare and Sport to assess the impact of recent evidence underlying the conclusions of the 2016 RIVM report on the current temporary tolerable intake (t-TDI) for BPA of 4 µg/kg bw/day set by EFSA in 2015. The CEF Panel has then evaluated the results of two studies published by Ménard et al. in 2014, suggesting food intolerance and impaired immune response to parasitic infection in rats exposed perinatally to BPA doses in the microgram/kg bw/day range. The same appraisal criteria and weight-of-evidence analysis used for the 2015 EFSA opinion on BPA were applied to these studies. This new evidence adds to the indications of immunotoxicity of BPA in animals reported in previous reviews. For the only endpoint for which three BPA doses were tested (IgG levels), a benchmark dose analysis of the dose-response data was carried out. Due to the high inter-animal variability within the treatment groups resulting in high confidence intervals and limited dose-response, the CEF Panel concluded that these data on anti-OVA IgG antibodies are not suitable to derive a reference point for BPA on immunotoxicity. Furthermore, the limitations of both Menard et al. studies observed by the Panel confound the interpretation of the study results and prevent the assessment of the relevance to human health. The CEF Panel overall considers that the results from the two Menard et al. studies are not sufficient to call for a revision of the EFSA t-TDI for BPA. EFSA will start a review of all the scientific evidence published after 2012 and relevant for BPA hazard assessment (including immunotoxicity) in 2017. The results of immunological studies such as the two evaluated here would form a useful contribution to this evaluation provided that the limitations identified herein were addressed.

Human risk assessment of heavy metals: principles and applications.

Humans are exposed to a number of "heavy metals" such as cadmium, mercury and its organic form methylmercury, uranium, lead, and other metals as wel as metalloids, such as arsenic, in the environment, workplace, food, and water supply. Exposure to these metals may result in adverse health effects, and national and international health agencies have methodologies to set health-based guidance values with the aim to protect the human population. This chapter introduces the general principles of chemical risk assessment, the common four steps of chemical risk assessment: hazard identification, hazard characterization, exposure assessment, risk characterization, and toxicokinetic and toxicity aspects. Finally, the risk assessments performed by international health agencies such as the World Health Organisation, the Environmental Protection Agency of the United States, and the European Food Safety Authority are reviewed for cadmium, lead, mercury, uranium, and arsenic.

Developmental exposure to methylmercury and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) affects cerebral dopamine D1-like and D2-like receptors of weanling and pubertal rats.

MeHg (0.5 mg/kg/day) and/or PCB153 (5 mg/kg/day) effects, administered orally to rat dams (GD7-PND21), were explored in PND21 and PND36 offspring brain in terms of density (Bmax) and affinity (Kd) of dopamine D1-like (D1-Rs) and D2-like receptors (D2-Rs), by saturation binding studies. D1-Rs decreased density in both cortex and striatum (15-30%) by MeHg and PCB153, either alone or combined, without additivity in PND21 males. Changes disappeared by PND36. In females, only MeHg caused a 15% Bmax decrease in striatum. D2-Rs enhanced density (23-50%) and reduced affinity in cortex to a similar extent by all treatments in both weanling and pubertal males. Affinity was also decreased in females by all types of exposure at both ages, while density was enhanced by PCB153 only in a delayed manner (PND36). No changes were detected in striatum. In MeHg and MeHg + PCB153 pup cortex, Hg concentrations ranged, on PND21, between 0.25 and 0.89 and 0.94-1.40 µg/g tissue, respectively, and were 5- to sixfold lower 2 weeks later. PCB153 levels, in PCB153 ± MeHg treated rats, were about 15 µg/g tissue (PND21) and 4-8 µg/g tissue (PND36). In striatum, the Hg and PCB153 concentrations were similar to those in cortex. Brain kinetics trend also applied to blood PCB153 or Hg levels. Perinatal exposure to MeHg and/or PCB153 affects D1- and D2-Rs in a gender-, time-, and brain area-dependent manner. Combined treatment does not exacerbate the neurochemical effects of the individual compounds.

Single step determination of PCB 126 and 153 in rat tissues by using solid phase microextraction/gas chromatography-mass spectrometry: Comparison with solid phase extraction and liquid/liquid extraction.

A simple and reliable solid phase microextraction/gas chromatography-mass spectrometry (SPME/GC-MS) method was developed for the single-step determination of PCBs 126 and 153 in rat brain and serum, using liquid/liquid and solid phase extraction (SPE) as reference techniques. The multi-factor categorical experimental design used to study simultaneously the main parameters and their interactions affecting the efficiency of the method, showed that the use of an 85mum PA exposed at 100 degrees C for 40min was the optimum sampling condition for both PCBs. SPME was then validated by studying its linear dynamic (over two orders of magnitude), limits of detection (brain: 2ng/g, serum: 0.2ng/g) and analytical precision that was within 9% for SPME in both brain and serum. Finally, the method was used to determine the brain and blood target dose in mothers and pups after oral exposure of the mothers.

Neurodevelopmental toxicity of methylmercury: Laboratory animal data and their contribution to human risk assessment.

Methylmercury (MeHg) is one of the most significant public health hazards. The clinical findings in the victims of the Japanese and Iraqi outbreaks have disclosed the pronounced susceptibility of the developing brain to MeHg poisoning. This notion has triggered worldwide scientific attention toward the long-term consequences of prenatal exposure on child development in communities with chronic low level dietary exposure. MeHg neurodevelopmental effects have been extensively investigated in laboratory animals under well-controlled exposure conditions. This article provides an updated overview of the main neuromorphological and neurobehavioral changes reported in non-human primates and rodents following developmental exposure to MeHg. Different aspects of MeHg's effects on the immature organism are reported, with particular reference to the delayed onset of symptoms and the persistency of central nervous system (CNS) injury/dysfunction. Particular attention is paid to the comparative toxicity assessment across species, and to the degree of concordance/discordance between human and animal data. The contribution of animal studies to define the role of potential effect modifiers and variables on MeHg dose-response relationships is also addressed. The ultimate goal is to discuss the relevance of laboratory animal results, as a complementary tool to human data, with regard to the human risk assessment process.

Human developmental neurotoxicity of methylmercury: impact of variables and risk modifiers.

Methylmercury (MeHg) is a widespread environmental and food toxicant which has long been known to affect neurodevelopment in both humans and experimental animals. Risk assessment for MeHg is mainly based on human data coming from the massive episodes of poisoning in Japan and Iraq, as well as from large scale epidemiological studies concerning childhood development and neurotoxicity in relation to in utero exposure in various fish eating communities around the world. Despite the extensive literature and research, the threshold dose for MeHg neurotoxic effects is still unclear, in particular when it comes to subtle effects on neurobehaviour. In this article clinical and epidemiological findings concerning the neurodevelopmental toxicity of MeHg are reviewed. Much attention is focussed on the potential impact of factors, such as diet and nutrition, gender, pattern of exposure and co-exposure to other neurotoxic pollutants, which may modulate MeHg toxic effects. These factors, together with the notion that some symptoms may ensue or exacerbate with aging, contribute to the difficulties in the definition of safe levels for developmental exposure.

Perinatal co-exposure to methylmercury and PCB153 or PCB126 in rats alters the cerebral cholinergic muscarinic receptors at weaning and puberty.

In the last few decades, combined exposure to methylmercury (MeHg) and polychlorinated biphenyls (PCBs) from fish and seafood, and their potentially interactive effects on neurodevelopment, have been giving increasing cause for concern. We examined the combined effects of MeHg and either a non-dioxin PCB (PCB153) or a dioxin-like PCB (PCB126) congener on the developing brain cholinergic muscarinic receptors (MRs). These receptors are known to play a major role in many central functions including higher cognitive processes and the modulation of extrapyramidal motor activity. MRs in pup rat brains diminished following prenatal and lactational exposure, from gestational day [GD]7 to postnatal day [PND]21, to MeHg (0.5mg/kgbodyweight[bw]/day), PCB153 (5mg/kgbw/day), and PCB126 (100ng/kg/day), alone or in combination. Total MR density, as well as M1, M2, and M3 receptor subtypes of the weanling and pubertal rats, were affected in a brain-area-, gender-, time- and compound-dependent fashion. MeHg decreased (by 15-20%) the total MR density in a delayed (PND36) manner in the cerebral cortex of both genders, and early (at weaning) in the cerebellum of both genders, with the effect lasting until puberty (in males only). MeHg decreased the ACh M1- and M3-immunopositive neurons in the cerebral cortex and also increased the M2-immunopositive Bergmann glia in the cerebellum. PCB153 also induced a delayed (PND36) decrease (of 20%) in total MR number in the cerebellum of the male offspring and in the cerebral cortex of both genders. The latter effect was coupled with a decrease in ACh M1- and ACh M3-immunopositive neuron populations. PCB126 decreased (by 30-40%) total MR density in a gender-dependent manner, males being more sensitive than females. The effect was evident early (at PND21) and lasted until puberty in the cerebellum, while it was observed later (at PND36) in the cerebral cortex. The M1 and M3 receptors were similarly affected by PCB126. Co-exposure to MeHg and either PCB153 or PCB126 had the same effect on the cerebral MRs as exposure to each compound alone. The results rule out additive or synergistic interactions between MeHg and PCB153 or PCB126 on MRs in the brain areas examined. Some early-onset changes persisted until puberty, while other modifications became manifest only at the advanced time point (PND36), when the brain levels of total Hg, PCB153, and PCB126 had declined. These data support the ability of MeHg and PCBs to induce delayed neurotoxicity after developmental exposure.

Neurobehavioural and molecular changes induced by methylmercury exposure during development.

There is an increasing body of evidence on the possible environmental influence on neurodevelopmental and neurodegenerative disorders. Both experimental and epidemiological studies have demonstrated the distinctive susceptibility of the developing brain to environmental factors such as lead, mercury and polychlorinated biphenyls at levels of exposure that have no detectable effects in adults. Methylmercury (MeHg) has long been known to affect neurodevelopment in both humans and experimental animals. Neurobehavioural effects reported include altered motoric function and memory and learning disabilities. In addition, there is evidence from recent experimental neurodevelopmental studies that MeHg can induce depression-like behaviour. Several mechanisms have been suggested from in vivo- and in vitro-studies, such as effects on neurotransmitter systems, induction of oxidative stress and disruption of microtubules and intracellular calcium homeostasis. Recent in vitro data show that very low levels of MeHg can inhibit neuronal differentiation of neural stem cells. This review summarises what is currently known about the neurodevelopmental effects of MeHg and consider the strength of different experimental approaches to study the effects of environmentally relevant exposure in vivo and in vitro.

Methylmercury interaction with lymphocyte cholinergic muscarinic receptors in developing rats.

Cerebral cholinergic muscarinic receptors (MR) have been suggested as one of the sensitive biochemical endpoints of the central nervous system altered by developmental exposure to the widespread seafood contaminant methylmercury (MeHg). In adult rats, MeHg has been shown to alter MR binding both in the brain and lymphocytes, supporting the use of MR in blood cells as a surrogate marker of CNS changes. The effects of MeHg have been evaluated on rat lymphocyte MR binding (using [3H]QNB as specific muscarinic ligand) in vivo (after perinatal exposure) and in vitro. For comparison, in vitro studies were also performed on human lymphocytes. Exposure to 1 mg MeHg/kg/day during pregnancy and lactation (from GD7 to PND7) significantly enhanced lymphocyte MR density in both adult and young rats 21 days after delivery, with a more pronounced effect in the mothers (B(max) increase of 139%) than in the male offspring (+49%) and female offspring (+73%) as compared with their respective controls (33+/-4, 41+/-8, and 37+/-4 fmol/million cells), in accordance with the higher Hg levels detected in the adult blood (11.3+/-2.2 microg/mL) than in pups (1.3+/-0.4 microg/L in both genders). A lower MeHg dose (0.5 mg/kg/day) was without any effect on lymphocyte MRs. In in vitro studies, MeHg was an almost equipotent inhibitor of (3)H-QNB binding to rat and human lymphocyte MRs (IC50 values were 4.1+/-0.29, 5.2+/-0.51, and 5.0+/-0.9 microM for total rat lymphocytes, rat T lymphocytes, and total human lymphocytes, respectively). Notably, the IC50 values for MeHg to lymphocyte MRs were comparable to the Hg levels reached in blood (5-50 microM) of the PND21 rats exposed to MeHg. The finding that the MR binding is a target for the effects of MeHg in peripheral blood cells is in accordance with our previous data in brain [Coccini et al., 2006. Effects of developmental co-exposure to methylmercury and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) on cholinergic muscarinic receptors in rat brain. Neurotoxicology, in press], and supports the use of this peripheral endpoint as a biomarker of MeHg-induced cerebral muscarinic alterations. The similarity of MeHg IC50 binding data between human and rat in peripheral tissues suggests the possible application of such biomarker to humans exposed to environmental chemicals.

Brain monoaminergic neurotransmission parameters in weanling rats after perinatal exposure to methylmercury and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153).

The individual and joint effects of methylmercury (MeHg; 1 mg/kg body weight/day, GD7-PND7) and PCB153 (20 mg/kg body weight/day, GD10-GD16), administered orally to rat dams, were explored in 21-day-old rat offspring brain in terms of monoamine oxidase B (MAO-B) activity and regional content of dopamine (DA), serotonin (5-HT), 5-hydroxy-indole-3-acetic acid (5-HIAA) and homovanillic acid (HVA). Neither treatment altered MAO-B in striatum, hippocampus, cerebellum and cerebral cortex of female pups. In males the cerebellum displayed a significantly reduced enzyme activity (25-45%) following all treatments. Concerning biogenic amines, 5-HT levels were decreased by 30-50% in the cerebral cortex of males and females by PCB153 alone and combined with MeHg, without changes in 5-HIAA and dopaminergic endpoints. In cerebellum of all pups, MeHg enhanced 5-HIAA levels, whereas PCB153, either alone or combined with MeHg, did not affect this endpoint. In striatum, PCB153 reduced the content of DA, HVA and 5-HIAA (respective control values: 2-3; 60-80; 8-10 ng/mg protein) to a similar extent when administered alone or together with MeHg (20-40%). Perinatal exposure to MeHg and/or PCB153 results in regionally and/or gender-specific alterations in the central dopaminergic and serotonergic systems at weaning. The combined treatment with MeHg and PCB153 does not exacerbate the neurochemical effects of the individual compounds.

Effects of developmental co-exposure to methylmercury and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) on cholinergic muscarinic receptors in rat brain.

The developing nervous system is thought to be particularly sensitive to polychlorinated biphenyls (PCBs) present as food contaminants together with methylmercury (MeHg). Effects of perinatal co-exposure to PCB153 and MeHg on brain cholinergic muscarinic receptors (MRs) were investigated by saturation binding studies in mature and immature rats. MeHg alone (1mg/kg/day, GD7-PND7) enhanced cerebral MRs more in dams (87% and 60% in cerebellum and cerebral cortex, respectively) than in PND21 pups (0-50%) in accordance with the higher Hg levels detected in the adult brain (7-9 microg/g) than in the male and female offspring's brain (1.5-2.8 microg/g). Prenatal administration of PCB153 (20mg/kg/day, GD10-GD16), leading to higher contaminant levels in the offspring brain than in that of adults (25-66 microg/g versus 3 microg/g), induced cerebral MR changes of similar extent at both ages, namely decreased cerebellar (20-30%) and increased cortical MR density (40-50%). Co-exposure to PCB and MeHg had no more effect than exposure to either compound alone on cerebral cortex MRs, whereas, in the cerebellum, the combined treatment induced a PCB-like lowering of the MR density that masked the MeHg-induced receptor increase. None of the treatments affected the striatal and hippocampal MRs. A lower MeHg dose (0.5 mg/kg/day) was without any effect on cerebral MRs. These results show that MRs are one of the sensitive biochemical endpoints of the central nervous system altered by developmental exposure to MeHg and PCB153. Cerebral cortex and cerebellum were the most susceptible targets in the response to these neurotoxicants. MR changes were detected in both immature and adult animals and the interaction of MeHg and PCB153 at the level of these receptors occurred in a non-additive manner.

Lymphocyte cytochrome c oxidase, cyclic GMP and cholinergic muscarinic receptors as peripheral indicators of carbon monoxide neurotoxicity after acute and repeated exposure in the rat.

Changes in cerebral cytochrome oxidase (COX) activity, nitric oxide (NO)-cyclic GMP (cGMP) pathway and cholinergic muscarinic receptors (MRs) have been reported in rodents acutely exposed to carbon monoxide (CO). These endpoints measurable in lymphocytes may serve as peripheral markers of CO neurotoxicity. The early and delayed effects of repeated and acute in vivo CO inhalation were investigated on COX activity, cGMP formation and MR binding in rat brain and lymphocytes to assess whether each endpoint was similarly affected both centrally and peripherally. Male Wistar rats either inhaled 500 ppm CO, 6 h/day, 5 days/week, 4 weeks (repeated exposure) or 2,400 ppm, 1 h (single exposure). Neither treatment altered brain or lymphocyte COX activity 1 and 7 days post-treatment. Also ineffective were repeated and acute CO treatments towards (3)H-quinuclidinyl benzilate (QNB) binding to MRs in cerebral cortex, hippocampus, striatum, cerebellum (respective controls, mean+/-S.D.: 171 +/- 45, 245 +/- 53, 263 +/- 14 and 77 +/- 7 fmol/mg protein) and lymphocytes (24 +/- 10 fmol/million cells) at the same time points. In lymphocytes control cGMP levels averaged 1.98 +/- 0.99 pmol/mg protein under basal conditions, and 3.94 +/- 0.55 pmol/mg protein after NO-stimulation. One day after chronic treatment cessation, the CO-treated group displayed about a 50% decrease in both basal and NO-stimulated cGMP values, which persisted up to 7 days after, compared to air-exposed rats. Acutely, CO caused a delayed enhancement (+140%) of NO-induced activation of soluble guanylate cyclase. The finding that the NO-cGMP pathway is a target for the delayed effects of CO in peripheral blood cells is in accordance with our data in brain [Hernández-Viadel, M., Castoldi, A.F., Coccini, T., Manzo, L., Erceg, S., Felipo, V., 2004. In vivo exposure to carbon monoxide causes delayed impairment of activation of soluble guanylate cyclase by nitric oxide in rat brain cortex and cerebellum. Journal of Neurochemistry 89, 1,157-1,165], and supports the use of this peripheral endpoint as a biomarker of CO central effects.

Lymphocyte muscarinic receptors and platelet monoamine oxidase-B as biomarkers of CNS function: effects of age and gender in healthy humans.

Lymphocyte cholinergic muscarinic receptors (MRs) and platelet monoamine oxidase-B (MAO-B) activity are considered surrogate markers of the same parameters in the central nervous system. Lymphocyte MR binding and platelet MAO-B activity were measured in a consistent number of healthy human adults and analysed according to gender and age. The mean value±S.D. of MR binding neither differed between males (12.2±10.0fmol/10(6)cells, range: 0.5-37.9, n=86) and females (10.7±9.7fmol/10(6)cells, range: 0.5-39.7, n=69) nor among age groups. MAO-B activity was significantly higher in women (geometric mean: 11.3nmol/mgprotein/h, with 65% of values from 7.3 to 17.6; n=43), than in men (7.7nmol/mgprotein/h, with 65% of values from 4.5 to 13; n=95). Males aged 56-66 years displayed a higher, though not statistically significant, basal enzyme activity than younger subjects. Altogether these data indicate gender-related differences in MAO activity, but not in MR binding, and inter-individual differences in the basal values of both peripheral blood markers in healthy subjects.

In vivo exposure to carbon monoxide causes delayed impairment of activation of soluble guanylate cyclase by nitric oxide in rat brain cortex and cerebellum.

Carbon monoxide induces delayed neurological and neuropathological alterations, including memory loss and cognitive impairment. The bases for the delay remain unknown. Activation of soluble guanylate cyclase by nitric oxide modulates some forms of learning and memory. Carbon monoxide binds to soluble guanylate cyclase, activating it but interfering with its activation by nitric oxide. The aim of this work was to assess whether exposure of rats to carbon monoxide alters the activity of soluble guanylate cyclase or its modulation by nitric oxide in cerebellum or cerebral cortex. Rats exposed chronically or acutely to carbon monoxide were killed 24 h or 7 days later. Acute carbon monoxide exposure decreased cyclic guanosine monophosphate (cGMP) content and reduced activation of soluble guanylate cyclase by nitric oxide. Cortex was more sensitive than cerebellum to chronic exposure, which reduced activation of soluble guanylate cyclase by nitric oxide in cortex. In cerebellum, chronic exposure induced delayed impairment of soluble guanylate cyclase activation by nitric oxide. Acute exposure effects were also stronger at 7 days than at 24 h after exposure. This delayed impaired modulation of soluble guanylate cyclase by nitric oxide may contribute to delayed memory loss and cognitive impairment in humans exposed to carbon monoxide.

Neurotoxic and molecular effects of methylmercury in humans.

Mercurials are global environmental pollutants deriving from natural processes and anthropogenic activities. Most human exposure to mercury occurs through the intake of fish, shellfish, and sea mammals contaminated with methylmercury. Methylmercury is bioaccumulated and biomagnified in the aquatic food chain and reaches its highest levels in top predatory fish. The neurotoxic hazard posed by methylmercury to humans and the unique susceptibility of the developing brain have been well documented following the mass poisonings occurring in Japan and Iraq. Adult cases of methylmercury poisoning are characterized by the delayed onset of symptoms and by the focal degeneration of neurons in selected brain regions (for example, cerebral cortex and cerebellum). Why the fetus displays different neuropathological effects and a higher sensitivity to methylmercury relative to the adult is still unknown. Depending on the degree of in utero exposure, methylmercury may result in effects ranging from fetal death to subtle neurodevelopmental delays. On the basis of epidemiological studies performed in populations having moderate chronic methylmercury exposure, no definitive consensus has been reached to date on the safety level of maternal exposure during pregnancy. Among the multiple mechanisms believed to contribute to methylmercury neurotoxicity, methylmercury-induced microtubule alterations, oxidative damage, impairment of calcium homeostasis, and the potentiation of glutamatergic neurotransmission are presented in this review.