Marco Binaglia

Reactive oxygen species generated by glia are responsible for neuron death induced by human immunodeficiency virus-glycoprotein 120 in vitro

Human immunodeficiency virus infection is often followed by neurodegeneration, the cause of motor and cognitive impairment in some patients affected by acquired immunodeficiency. Several in vitro data indicate glycoprotein (gp) 120 as one of the substances responsible for the neurodegenerative event that takes place only if non-neuronal cells (glial cells) are present. Our purpose was to investigate the molecular mechanisms through which glial cells could affect neuron viability after exposure to gp120 protein. We used a sandwich co-culture of primary hippocampal neurons and primary glial cells, where the two cell populations face each other but are separable. Exposure of 1-week-old rat hippocampal neurons in co-culture with glia to 600 pM gp120 protein resulted in the death of 30% of neurons after 6 days of treatment. A significant increase of intracellular calcium ([Ca2+]i), evident 72 h after gp120 exposure (control 45.8+/-7.6 nM, gp120 176.5+/-43.6 nM), preceded neuron death. The gp120 protein affected neither the viability nor the morphology or [Ca2+]i of glial cells. However, a significant amount of reactive oxygen species as well as of interleukin-1beta was produced. Treatment of the co-culture with an antibody against interleukin-1beta prevented neuron increase of [Ca2+]i and cell death but not glial production of reactive oxygen species, whereas prior incubation of glial cells with Trolox, an antioxidant analog of vitamin E, down-regulated interleukin-1beta expression and completely prevented neuron cell death. Our results indicate that reactive oxygen species produced in glial cells by gp120 exposure cause neurodegeneration by inducing the synthesis of interleukin-1beta.

Cloricromene, a semi-synthetic coumarin derivative, inhibits tumor necrosis factor-α production at a pre-transcriptional level

Cloricromene decreases myocardial infarct size after ischemic-reperfusion injury in vivo, and it has been suggested that this is due to inhibition of tumor necrosis factor-alpha (TNF-alpha). The purpose of this work was to characterize the mechanism of cloricromene-induced inhibition of TNF-alpha in rat macrophages. Cloricromene inhibited lipopolysaccharide-induced TNF-alpha release in a dose-dependent manner (IC(50)=5.9 +/- 0.8 microM). This was not due to cytotoxicity, as cloricromene was well tolerated up to 500 microM. Cloricromene inhibited lipopolysaccharide-induced expression of TNF-alpha mRNA, which suggests a pre-transcriptional effect. We then investigated the early signal transduction pathway triggered by lipopolysaccharide. The binding of lipopolysaccharide to its receptor CD14 activates protein kinase C and nuclear factor-kappaB (NF-kappaB). Cloricromene inhibited NF-kappaB activation in a dose-dependent manner, but affected protein kinase C translocation only slightly. We then established that cloricromene inhibited lipopolysaccharide-induced cellular oxidative activity, which is important for NF-kappaB activation. Our results show that cloricromene interferes with the early signal transduction pathway triggered by lipopolysaccharide.

The anti-inflammatory activity of estrogen in glial cells is regulated by the PKC-anchoring protein RACK-1

It has recently been suggested that estrogen inhibits glial activation and the release of neurotoxic mediators. The mechanisms involved in this anti‐inflammatory effect are unclear. We found that an nM concentration of 17‐β estradiol inhibits protein kinaseC‐βII translocation induced by lipopolysaccharide in primary astrocytes. Estradiol treatment did not change the total content of kinaseC‐βII or of lipopolysaccharide receptor, but dose‐dependently reduced the levels of receptors for activated C kinases‐1 (RACK‐1), the anchoring protein involved in protein kinase C (PKC) shuttling. This decrease could thus account for the defective protein kinaseC‐βII activation. Pre‐treatment with 1 nmβ‐estradiol, which reduced by ∼35% the expression of RACK‐1, prevented the lipopolysaccharide‐induced expression of tumour necrosis factor‐α mRNA and of the inducible form of nitric oxide (NO) synthase. As a consequence, the production of tumour necrosis factor‐α and NO were decreased. An antisense oligonucleotide for RACK‐1 also reduced tumour necrosis factor‐α and nitric oxide production on lipopolysaccharide stimulation. These results demonstrate that estrogen reduction of the RACK‐1 expression, leading to a defective protein kinase‐C activation counteracts the inflammatory response in astrocytes.

Risks for human health related to the presence of pyrrolizidine alkaloids in honey, tea, herbal infusions and food supplements

Abstract EFSA was asked by the European Commission to deliver a scientific opinion on the risks for human health related to the presence of pyrrolizidine alkaloids (PAs) in honey, tea, herbal infusions and food supplements and to identify the PAs of relevance in the aforementioned food commodities and in other feed and food. PAs are a large group of toxins produced by different plant species. In 2011, the EFSA Panel on Contaminants in the Food Chain (CONTAM Panel) assessed the risks related to the presence of PAs in food and feed. Based on occurrence data limited to honey, the CONTAM Panel concluded that there was a possible health concern for those toddlers and children who are high consumers of honey. A new exposure assessment including new occurrence data was published by EFSA in 2016 and was used to update the risk characterisation. The CONTAM Panel established a new Reference Point of 237 μg/kg body weight per day to assess the carcinogenic risks of PAs, and concluded that there is a possible concern for human health related to the exposure to PAs, in particular for frequent and high consumers of tea and herbal infusions. The Panel noted that consumption of food supplements based on PA‐producing plants could result in exposure levels too close (i.e. less than 100 times lower) to the range of doses known to cause severe acute/short term toxicity. From the analysis of the available occurrence data, the CONTAM Panel identified a list of 17 PAs of relevance for monitoring in food and feed. The Panel recommended continuing the efforts to monitor the presence of PAs in food and feed, including the development of more sensitive and specific analytical methods. A recommendation was also issued on the generation of data to identify the toxic and carcinogenic potency of the PAs commonly found in food.

Risks for public health related to the presence of tetrodotoxin (TTX) and TTX analogues in marine bivalves and gastropods

Abstract Tetrodotoxin (TTX) and its analogues are produced by marine bacteria and have been detected in marine bivalves and gastropods from European waters. The European Commission asked EFSA for a scientific opinion on the risks to public health related to the presence of TTX and TTX analogues in marine bivalves and gastropods. The Panel on Contaminants in the Food Chain reviewed the available literature but did not find support for the minimum lethal dose for humans of 2 mg, mentioned in various reviews. Some human case reports describe serious effects at a dose of 0.2 mg, corresponding to 4 μg/kg body weight (bw). However, the uncertainties on the actual exposure in the studies preclude their use for derivation of an acute reference dose (ARfD). Instead, a group ARfD of 0.25 μg/kg bw, applying to TTX and its analogues, was derived based on a TTX dose of 25 μg/kg bw at which no apathy was observed in an acute oral study with mice, applying a standard uncertainty factor of 100. Estimated relative potencies for analogues are lower than that of TTX but are associated with a high degree of uncertainty. Based on the occurrence data submitted to EFSA and reported consumption days only, average and P95 exposures of 0.00–0.09 and 0.00–0.03 μg/kg bw, respectively, were calculated. Using a large portion size of 400 g bivalves and P95 occurrence levels of TTX, with exception of oysters, the exposure was below the group ARfD in all consumer groups. A concentration below 44 μg TTX equivalents/kg shellfish meat, based on a large portion size of 400 g, was considered not to result in adverse effects in humans. Liquid chromatography with tandem mass spectroscopy (LC–MS/MS) methods are the most suitable for identification and quantification of TTX and its analogues, with LOQs between 1 and 25 μg/kg.

Appropriateness to set a group health based guidance value for T2 and HT2 toxin and its modified forms

Abstract The EFSA Panel on Contaminants in the Food Chain (CONTAM) established a tolerable daily intake (TDI) for T2 and HT2 of 0.02 μg/kg body weight (bw) per day based on a new in vivo subchronic toxicity study in rats that confirmed that immune‐ and haematotoxicity are the critical effects of T2 and using a reduction in total leucocyte count as the critical endpoint. An acute reference dose (ARfD) of 0.3 μg for T2 and HT2/kg bw was established based on acute emetic events in mink. Modified forms of T2 and HT2 identified are phase I metabolites mainly formed through hydrolytic cleavage of one or more of the three ester groups of T2. Less prominent hydroxylation reactions occur predominantly at the side chain. Phase II metabolism involves conjugation with glucose, modified glucose, sulfate, feruloyl and acetyl groups. The few data on occurrence of modified forms indicate that grain products are their main source. The CONTAM Panel found it appropriate to establish a group TDI and a group ARfD for T2 and HT2 and its modified forms. Potency factors relative to T2 for the modified forms were used to account for differences in acute and chronic toxic potencies. It was assumed that conjugates (phase II metabolites of T2, HT2 and their phase I metabolites), which are not toxic per se, would be cleaved releasing their aglycones. These metabolites were assigned the relative potency factors (RPFs) of their respective aglycones. The RPFs assigned to the modified forms were all either 1 or less than 1. The uncertainties associated with the present assessment are considered as high. Using the established group, ARfD and TDI would overestimate any risk of modified T2 and HT2.

Dithiocarbamate propineb induces acetylcholine release through cytoskeletal actin depolymerization in PC12 cells

Neurological complications as well as movement disorders are relevant symptoms in animals and humans chronically exposed to dithiocarbamates. Using rat pheochromocytoma cells differentiated by NGF (PC12), we investigated whether propineb affects acetylcholine (Ach) release and the molecular mechanisms involved. Propineb (0.001-100 nM) dose-dependently increased Ach release from PC12. Thus, 0.001-1 nM propineb-induced Ach release, reaching a maximal effect ( approximately 50%) at 0.1-1 nM. Higher concentrations of propineb (10-100 nM) caused a progressive disappearance of the effect. Chelation of extra- and intracellular Ca(2+) did not affect Ach release by propineb, which was prevented by the actin stabilizer jasplakinolide (500 nM). Accordingly, actin depolymerization was observed after exposure of differentiated PC12 to 0.1-1 nM propineb, a loss of effect was evident at higher concentrations (100 nM), and the effect was Ca(2+)-independent. Disulfiram, a related dithiocarbamate not coordinated with Zn(2+), also depolymerized actin, suggesting the involvement of the organic structure of dithiocarbamates rather than the leakage of Zn(2+). Nevertheless, propineb did not depolymerize actin in a cell-free system. These data suggest that dithiocarbamates, through the activation of intracellular cascade(s), impair cytoskeletal actin. This effect may contribute to affect synaptic vesicles processing resulting in an impaired cholinergic transmission.

Appropriateness to set a group health based guidance value for nivalenol and its modified forms

Abstract The EFSA Panel on Contaminants in the Food Chain (CONTAM) reviewed new studies on nivalenol since the previous opinion on nivalenol published in 2013, but as no new relevant data were identified the tolerable daily intake (TDI) for nivalenol (NIV) of 1.2 μg/kg body weight (bw) established on bases of immuno‐ and haematotoxicity in rats was retained. An acute reference dose (ARfD) of 14 μg/kg bw was established based on acute emetic events in mink. The only phase I metabolite of NIV identified is de‐epoxy‐nivalenol (DE‐NIV) and the only phase II metabolite is nivalenol‐3‐glucoside (NIV3Glc). DE‐NIV is devoid of toxic activity and was thus not further considered. NIV3Glc can occur in cereals amounting up to about 50% of NIV. There are no toxicity data on NIV3Glc, but as it can be assumed that it is hydrolysed to NIV in the intestinal tract it should be included in a group TDI and in a group ARfD with NIV. The uncertainty associated with the present assessment is considered as high and it would rather overestimate than underestimate any risk.

Update of the risk assessment on 3‐monochloropropane diol and its fatty acid esters

Abstract The CONTAM Panel updated the assessment of the risks for human health related to the presence of 3‐monochloropropane diol (3‐MCPD) and its fatty acid esters in food published in 2016 in view of the scientific divergence identified in the establishment of the tolerable daily intake (TDI) in the Joint FAO/WHO Expert Committee on Food Additives and Contaminants (FAO/WHO) report published in 2017. In this update, dose–response analysis was performed following the recent EFSA Scientific Committee guidance on the use of benchmark dose (BMD) approach in risk assessment, and a review of available data on developmental and reproduction toxicity was included. The outcome of this review indicates that in rats short‐term exposure to 3‐MCPD above 1 mg/kg body weight (bw) per day can induce reduced sperm motility associated with reduced male fecundity. Decreased sperm count and histopathological changes in the testis and epididymis were observed following longer treatment periods at higher doses. Regarding increased incidence kidney tubular hyperplasia, BMD analysis using model averaging resulted in a BMDL 10 of 0.20 mg/kg bw per day in male rats, which was selected as the new Reference Point (RP) for renal effects. For the effects on male fertility, decreased sperm motility was selected as the most sensitive relevant endpoint and a BMDL 05 of 0.44 mg/kg bw per day was calculated. The RP for renal effects was considered to derive an updated group TDI of 2 μg/kg bw per day for 3‐MCPD and its fatty acid esters and was considered protective also for effects on male fertility. The established TDI of 2 μg/kg bw per day is not exceeded in the adult population. A slight exceedance of the TDI was observed in the high consumers of the younger age groups and in particular for the scenarios on infants receiving formula only.

Dyes in aquaculture and reference points for action

Abstract The European Commission requested EFSA to evaluate whether a series of dyes are covered by the ‘Guidance on methodological principles and scientific methods to be taken into account when establishing Reference Points for Action (RPAs) for non‐allowed pharmacologically active substances present in food of animal origin’ and to which group they should be attributed according to this guidance. Although these substances are not registered for use in food‐producing animals in the European Union, they may be used illegally in aquaculture for their antimicrobial properties. It was concluded that acriflavine, 3‐aminoacridine, aminoacridine, basic blue 7, brilliant green, leucobrilliant green, C.I. basic blue 26, chloranil, crystal violet, leucocrystal violet, dichlone, ethyl violet, methylene blue, new methylene blue, Nile blue, pararosaniline base, proflavine, proflavine hydrochloride, rhodamine 6G and trypan red are covered by the guidance document and belong to group I. A toxicological screening value of 0.0025 μg/kg body weight per day is applicable. Azure blue and potassium permanganate were excluded from the evaluation due to their inorganic nature.