Pasquale Mosesso

Furan carcinogenicity: DNA binding and genotoxicity of furan in rats in vivo

SCOPE Furan is a potent hepatotoxicant and liver carcinogen in rodents. However, short-term tests for genotoxicity of furan are inconclusive. The aim of this study was to assess the potential of furan to covalently bind to DNA, and to assess furan genotoxicity in rats in vivo. MATERIALS AND METHODS Accelerator mass spectrometry was used to determine the (14) C-content in DNA following administration of [3,4-(14) C]-furan (0.1 and 2.0 mg/kg bw) to F344 rats. DNA damage, micronuclei, chromosomal aberrations, and sister chromatid exchanges were analyzed in F344 rats treated with furan for up to 28 days. CONCLUSION The (14) C-content in liver DNA was significantly increased in a dose-dependent manner, with mean concentrations of 7.9 ± 3.5 amol (14) C/μg DNA and 153.3 ± 100.2 amol (14) C/μg DNA, corresponding to 16.5 ± 7.4 and 325.2 ± 212.7 adducts/10(9) nucleotides at 0.1 and 2.0 mg/kg bw, respectively. There was no evidence for genotoxicity of furan in peripheral blood and bone marrow cells. However, a dose-related increase in the incidence of chromosomal aberrations in rat splenocytes and some indication of DNA damage in liver were observed. Collectively, results from this study indicate that furan may operate-at least in part-by a genotoxic mode of action.

Relationship between chromatin structure, DNA damage and repair following X-irradiation of human lymphocytes

Earlier studies using the technique of premature chromosome condensation (PCC) have shown that in human lymphocytes, exchange type of aberrations are formed immediately following low doses (<2 Gy) of X-rays, whereas at higher doses these aberrations increase with the duration of recovery. This reflects the relative roles of slow and fast repair in the formation of exchange aberrations. The underlying basis for slow and fast repairing components of the DNA repair may be related to differential localization of the initial damage in the genome, i.e., between relaxed and condensed chromatin. We have tried to gain some insight into this problem by (a) X-irradiating lymphocytes in the presence of dimethyl sulfoxide (DMSO) a potent scavenger of radiation-induced .OH radicals followed by PCC and (b) probing the damage and repair in two specific chromosomes, 18 and 19, which are relatively poor and rich in transcribing genes by COMET-FISH, a combination of Comet assay and fluorescence in situ hybridization (FISH) techniques. Results obtained show (a) that both fast appearing and slowly formed exchange aberrations seem to take place in relaxed chromatin, since they are affected to a similar extent by DMSO, (b) significant differential DNA breakage of chromosome 18 compared to chromosome 19 in both G0 and G1 phases of the cell cycle as detected by Comet assay, indicating that relaxed chromatin containing high densities of transcriptionally active genes shows less fragmentation due to fast repair (chromosome 19) compared to chromosome 18, and (c) that relaxed chromatin is repaired or mis-repaired faster than more compact chromatin.

A selective de-O-methylation of guaiacyl lignans to corresponding catechol derivatives by 2-iodoxybenzoic acid (IBX). The role of the catechol moiety on the toxicity of lignans

We report here the first selective de-O-methylation of a large panel of guaiacyl lignans to the corresponding catechol derivatives by using IBX as primary oxidant under green conditions (dimethyl carbonate-H(2)O solvent) through an in situ reduction procedure. The influence of the catechol moiety on the cytotoxicity and genotoxicity of new lignan derivatives has been investigated. The results obtained indicated that the presence of the catechol moiety sharply enhances the clastogenic potential (e.g. induction of chromosomal aberrations), the cytotoxicity and the modulation of cell cycle progression with respect to the parent compounds. Thus, despite the in vitro antioxidant activity usually described for catechol derivatives, our results show for the first time the generation of a clastogenic potential, highly indicative of a long-term genetic and cancer risk.

Terrestrial gastropods (Helix spp) as sentinels of primary DNA damage for biomonitoring purposes: A validation study

We validated the alkaline comet assay in two species of land snail (Helix aspersa and Helix vermiculata) to test their suitability as sentinels for primary DNA damage in polluted environments. The study was conducted under the framework of a biomonitoring program for a power station in Central Italy that had recently been converted from oil to coal‐fired plant. After optimizing test conditions, the comet assay was used to measure the % Tail DNA induced by in vitro exposure of hemocytes to different concentrations of a reactive oxygen species (H2O2). The treatment induced significant increases in this parameter with a concentration effect, indicating the effectiveness of the assay in snail hemocytes. After evaluating possible differences between the two species, we sampled them in three field sites at different distances from the power station, and in two reference sites assumed to have low or no levels of pollution. No species differences emerged. Percent Tail DNA values in snails from the sites near the power station were higher than those from control sites. An inverse correlation emerged between % Tail DNA and distance from the power station, suggesting that the primary DNA damage decreased as distance increased away from the pollution source. Detection of a gradient of heavy metal concentration in snail tissues suggests that these pollutants are a potential cause of the observed pattern. The comet assay appears to be a suitable assay and Helix spp. populations suitable sentinels to detect the genotoxic impact of pollutants. Environ. Mol. Mutagen. 54:204–212, 2013.

In Vitro Cytogenetic Assays: Chromosomal Aberrations and Micronucleus Tests

Chromosome damage is a very important indicator of genetic damage relevant to environmental and clinical studies. Detailed descriptions of the protocols used for detection of chromosomal aberrations induced by unknown agents in vitro both in the presence or the absence of rat liver-derived metabolizing systems are given. Structural chromosomal aberrations that can be observed and quantified at metaphases are described here. For the detection of chromosomal damage (fragments or whole chromosome) in interphase, the micronucleus test can be used and a description of this test is also presented. Criteria for determining a positive result using appropriate statistical methods are described.

A comparative study of the anticlastogenic effects of chlorophyllin on N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or 7,12-dimethylbenz (α) anthracene (DMBA) induced micronuclei in mammalian cells in vitro and in vivo.

Chlorophyllin (CHL), a water soluble derivative of chlorophyll has been shown to have both anticarcinogenic and antigenotoxic properties. We evaluated the protective effects of CHL (25μM in vitro, 4 and 100mg/kg. b.w.) on the clastogenic action of two model carcinogens, MNNG and DMBA (25μM and 2μM respectively) in vitro on human hepatoma cells (HepG2) and (40mg and 25mg/Kg/b.w. respectively) in vivo on bone marrow of mice, using the frequencies of induced micronuclei as the end point. Pre-, post- and simultaneous treatments with CHL and the carcinogen were carried out in vitro. With MNNG, only simultaneous treatment with CHL was effective in reducing the frequencies of MN, suggesting a direct interaction between CHL and MNNG. A statistically significant reduction in of DMBA induced MN was found by pre-or post treatment with CHL while a reduction (not significant) was observed by simultaneous treatment. In in vivo experiments, CHL pre-treatment did not affect the frequencies of MN in PCEs of bone marrow induced by MNNG or DMBA. However, increased the toxic effect of DMBA (reduction in percent of PCEs) was accompanied by a reduction in the induced frequencies of MN. CHL was not clastogenic in both in vitro and in vivo tests. It can be concluded that (a) CHL has a protective effect against MNNG and DMBA. This effect is dependent upon the protocol employed in in vitro experiments. In vivo, CHL did not have a protective effect against MNNG and DMBA. A protective effect of CHL against DMBA was evident only at high toxic levels.

Re‐evaluation of alginic acid and its sodium, potassium, ammonium and calcium salts (E 400–E 404) as food additives

Abstract The present opinion deals with the re‐evaluation of alginic acid and its sodium, potassium, ammonium and calcium salts (E 400–E 404) when used as food additives. Alginic acid and its salts (E 400–E 404) are authorised food additives in the EU in accordance with Annex II and Annex III to Regulation (EC) No 1333/2008. Following the conceptual framework for the risk assessment of certain food additives re‐evaluated under Commission Regulation (EU) No 257/2010, the Panel concluded that there was no need for a numerical Acceptable Daily Intake (ADI) for alginic acid and its salts (E 400, E 401, E 402, E 403 and E 404), and that there was no safety concern at the level of the refined exposure assessment for the reported uses of alginic acid and its salts (E 400, E 401, E 402, E 403 and E 404) as food additives. The Panel further concluded that exposure of infants and young children to alginic acid and its salts (E 400, E 401, E 402, E 403 and E 404) by the use of these food additives should stay below therapeutic dosages for these population groups at which side‐effects could occur. Concerning the use of alginic acid and its salts (E 400, E 401, E 402, E 403 and E 404) in ‘dietary foods for special medical purposes and special formulae for infants’ (Food category 13.1.5.1) and ‘in dietary foods for babies and young children for special medical purposes as defined in Directive 1999/21/EC’ (Food category 13.1.5.2), the Panel further concluded that the available data did not allow an adequate assessment of the safety of alginic acid and its salts (E 400, E 401, E 402, E 403 and E 404) in infants and young children consuming the food belonging to the categories 13.1.5.1 and 13.1.5.2.

Re‐evaluation of guar gum (E 412) as a food additive

Abstract The Panel on Food Additives and Nutrient Sources added to Food (ANS) provides a scientific opinion re‐evaluating the safety of guar gum (E 412) as a food additive. In the EU, guar gum was evaluated by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 1970, 1974 and 1975, who allocated an acceptable daily intake (ADI) ‘not specified’. Guar gum has been also evaluated by the Scientific Committee for Food (SCF) in 1977 who endorsed the ADI ‘not specified’ allocated by JECFA. Following the conceptual framework for the risk assessment of certain food additives re‐evaluated under Commission Regulation (EU) No 257/2010, the Panel considered that adequate exposure and toxicity data were available. Guar gum is practically undigested, not absorbed intact, but significantly fermented by enteric bacteria in humans. No adverse effects were reported in subchronic and carcinogenicity studies at the highest dose tested; no concern with respect to the genotoxicity. Oral intake of guar gum was well tolerated in adults. The Panel concluded that there is no need for a numerical ADI for guar gum (E 412), and there is no safety concern for the general population at the refined exposure assessment of guar gum (E 412) as a food additive. The Panel considered that for uses of guar gum in foods intended for infants and young children the occurrence of abdominal discomfort should be monitored and if this effect is observed doses should be identified as a basis for further risk assessment. The Panel considered that no adequate specific studies addressing the safety of use of guar gum (E 412) in food categories 13.1.5.1 and 13.1.5.2 were available. Therefore, the Panel concluded that the available data do not allow an adequate assessment of the safety of guar gum (E 412) in infants and young children consuming these foods for special medical purposes.

Re‐evaluation of glycerol (E 422) as a food additive

Abstract The ANS Panel provides a scientific opinion re‐evaluating the safety of glycerol (E 422) used as a food additive. In 1981, the Scientific Committee on Food (SCF) endorsed the conclusion from the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 1976 of ‘acceptable daily intake (ADI) for man not specified’. The Panel concluded that glycerol has low acute toxicity and that local irritating effects of glycerol in the gastrointestinal tract reported in some gavage studies was likely due to hygroscopic and osmotic effects of glycerol. Glycerol did not raise concern with respect to genotoxicity and was of no concern with regard to carcinogenicity. Reproductive and prenatal developmental studies were limited to conclude on reproductive toxicity but no dose‐related adverse effects were reported. None of the animal studies available identified an adverse effect for glycerol. The Panel conservatively estimated the lowest oral dose of glycerol required for therapeutic effect to be 125 mg/kg bw per hour and noted that infants and toddlers can be exposed to that dose by drinking less than the volume of one can (330 mL) of a flavoured drink. The Panel concluded that there is no need for a numerical ADI and no safety concern regarding the use of glycerol (E 422) as a food additive at the refined exposure assessment for the reported uses. The Panel also concluded that the manufacturing process of glycerol should not allow the production of a food additive, which contains genotoxic and carcinogenic residuals at a level which would result in a margin of exposure below 10,000. The Panel recommended modification of the EU specifications for E 422. The Panel also recommended that more information on uses and use levels and analytical data should be made available to the Panel.

Synthesis and antioxidant activity of DOPA peptidomimetics by a novel IBX mediated aromatic oxidative functionalization

DOPA peptidomimetics with stable O–C and N–C covalent bonds between amino acid residues have been prepared by aromatic oxidative functionalization of tyrosine with 2-iodoxybenzoic acid (IBX). The reaction involves the Michael-like nucleophilic addition of different oxygen and nitrogen protected amino acids on a reactive DOPA quinone intermediate. Similar results were obtained in heterogeneous conditions using supported IBX-amide for more runs. Among the novel derivatives, compounds containing glycine residues showed a more pronounced antioxidant activity in the 2,2-diphenyl picrylhydrazyl (DPPH) radical scavenging cell free assay. Instead, valine derivatives showed the highest biological effect in L5178Y mouse lymphoma cells, by assessing the ability to reduce H2O2 induced DNA breakage in the alkaline comet assay.