Antonella Di Sotto

Multi-walled carbon nanotubes: Lack of mutagenic activity in the bacterial reverse mutation assay

The mutagenic effect of multi-walled carbon nanotubes (MWCNTs) characterised by small surface/volume ratio, high diameter and less than 0.1% of metal contaminants was evaluated by the bacterial reverse mutation assay (Ames test) on Salmonella typhimurium TA 98 and TA 100 strains, and on Escherichia coli WP2uvrA strain, in presence and in absence of the metabolic activation system S9. A preliminary cytotoxicity assay was carried out to ensure that cytotoxicity did not interfere with response. MWCNTs resulted devoid of mutagenic effect in the bacterial cellular systems tested in that they did not significantly increase the number of revertant colonies. The mutagenic activity did not even appear in presence of the metabolic activator, so we can exclude that MWCNTs metabolites, produced via cytochrome-based P450 metabolic oxidation system, may act as mutagens. Carbon nanomaterials seem to exhibit different biological activities and different toxicities in relation to their physico-chemical characteristics, size, shape, crystallinity and presence of metal traces, so it is difficult to establish their health risk. Due to the limited background of genotoxicity studies and the increased occupational and public exposure to nanomaterials, present results appear useful to extend the knowledge on the safety of carbon nanotubes in view of their possible applications.

Inhibition by β-caryophyllene of ethyl methanesulfonate-induced clastogenicity in cultured human lymphocytes

beta-Caryophyllene is a bi-cyclic sesquiterpene that occurs in essential oils from several plants. A variety of biological activities have been ascribed to this compound. In particular, it seems to possess anti-carcinogenic properties, due to its capability to induce detoxifying enzymes or to enhance, in vitro and in vivo, the natural killer cell-induced cytotoxicity against tumours. Conversely, the knowledge on the DNA-damaging activity of the substance and its modulation is scanty. Therefore, in this study, we aimed at evaluating the capability of beta-caryophyllene to protect cultured human lymphocytes from the genotoxic damage induced by ethyl methanesulfonate (EMS) and colcemid (COL) in the micronucleus assay. To investigate the mechanisms of action of this sesquiterpene, the cultures were treated with the compound before (pre-treatment), during (co-treatment) and after (post-treatment) treatment with the mutagens. Up to 100 microg/ml, beta-caryophyllene by itself did not produce any cytotoxic and genotoxic effect, as shown by the value of the nuclear division index (NDI) and the frequency of micronuclei (MN). The test compound (0.1-100 microg/ml) significantly reduced the MN frequency induced by EMS in pre- and co-treatment protocols (up to 64.4% and 87% reduction, respectively). In the post-treatment protocol, beta-caryophyllene was not effective as an antimutagen. No significant reduction of COL-induced MN frequency was found. The antigenotoxic activity of beta-caryophyllene observed here suggests that this compound could act by chemical interaction with the mutagen in the growth medium (co-treatment) or in the cytoplasm of lymphocytes (pre-treatment), so acting as a desmutagen. These data encourage further studies to investigate the mode of action and the potential use of this compound as a chemopreventive agent.

Antimutagenic and mutagenic activities of some terpenes in the bacterial reverse mutation assay.

The mutagenic and antimutagenic effects of linalool, linalyl acetate and beta-caryophyllene were evaluated by the bacterial reverse mutation assay on Salmonella typhimurium TA 98 and TA 100, and on Escherichia coli WP2uvrA strains. Neither linalool nor beta-caryophyllene showed mutagenicity, but linalyl acetate induced a statistically significant increase in the number of revertant colonies in WP2uvrA, both with and without S9 mixture. Linalool was devoid of antimutagenic activity against 2-nitrofluorene (2NF), sodium azide (SA), methyl methane sulfonate (MMS) and 2-aminoanthracene (2AA). In contrast, beta-caryophyllene showed a strong antimutagenic activity against 2NF: at the maximum concentration tested (6.40mg/plate) the number of 2NF-induced revertant colonies was reduced by 83.9%. beta-Caryophyllene also showed to counteract the mutagenicity of SA (in TA 100), MMS and 2AA (in WP2uvrA): the effect was weak against SA (inhibition lower than 25%) and moderate against MMS and 2AA (up to 30.5%). The antimutagenic activity of beta-caryophyllene observed here suggests further studies to evaluate its possible chemopreventive properties.

Hepatotoxicity of green tea: an update

Green tea (GT), obtained from the leaves of Camellia sinensis (L.) Kuntze (Fam. Theaceae), is largely used for its potential health benefits such as reduction in risk of cardiovascular diseases and weight loss. Nevertheless, it is suspected to induce liver damage. Present work reviews the hepatic adverse reactions associated with GT-based herbal supplements, published by the end of 2008 to March 2015. A systematic research was carried out on PubMed, MedlinePlus, Scopus and Google Scholar databases, without any language restriction. Moreover, some accessible databases on pharmacovigilance or phytovigilance were consulted. The causality assessment was performed using the CIOMS/RUCAM score. Nineteen cases of hepatotoxicity related to the consumption of herbal products containing GT were identified. The hepatic reactions involved mostly women (16/19); the kind of liver damage was generally classified as hepatocellular (16/19). The causality assessment between consumption of herbal preparation and hepatic reaction resulted as probable in eight cases and as possible in eleven cases. In seven cases, patients used preparations containing only GT, while twelve reactions involved patients who took multicomponent preparations (MC). The reactions induced by GT had a generally long latency (179.1xa0±xa058.95xa0days), and the outcome was always resolution, with recovery time of 64.6xa0±xa017.78xa0days. On the contrary, liver injury associated with MC had a shorter latency (44.7xa0±xa013.85xa0days) and was more serious in four cases that required liver transplantation and, when resolution occurred, the recovery time was longer (118.9xa0±xa038.79). MC preparations contained numerous other components, many of which are suspected to induce liver damage, so it is difficult to ascribe the toxicity to one specific component, e.g., GT. Present data confirm a certain safety concern with GT, even if the number of hepatic reactions reported is low considering the great extent of use of this supplement. The mechanism of GT hepatotoxicity remains unclear, but factors related to the patient are becoming predominant. A major safety concern exists when GT is associated with other ingredients that can interact between them and with GT, enhancing the risk of liver damage. Patients should be discouraged from using herbal or dietary supplements containing complex mixtures and should be encouraged to use herbal and dietary supplement possibly under supervision of healthcare professionals.

Chelidonium majus is not hepatotoxic in Wistar rats, in a 4 weeks feeding experiment.

AIM OF THE STUDYnAerial parts of Chelidonium majus L. (Papaveraceae family) are traditionally used in the treatment of gallstones and dyspepsia, however several cases of hepatotoxicity are reported. In this work we evaluated the effects on liver function of a C. majus extract, obtained from the herbal material responsible for one case of hepatotoxicity.nnnMATERIALS AND METHODSnExperiments were performed in Wistar rats, after oral administration of doses corresponding to 1.5 and 3g/(kg day) of herbal drug, for 2 or 4 weeks. Blood samples were collected to perform biochemical analysis, whereas liver samples were used for histomorphological and immunohistochemical examination along with the determination of oxidative stress parameters.nnnRESULTSnNo significant modification in animal body weight, food consumption, enzyme activities, hepatic histomorphology and MDA formation, at either time or dosage level. Conversely, C. majus induced a slight but significant decrease of GSH levels and SOD activity, especially at the high dose.nnnCONCLUSIONSnOur study suggests that C. majus, at doses about 50 and 100 times higher than those generally used in humans, does not alter hepatic function. However, the reduction in GSH levels and SOD activity suggests particular attention in use of C. majus or its preparations in situations (pharmacological treatments, physio-pathological conditions, etc.) that can compromise liver function.

Genotoxicity of lavender oil, linalyl acetate, and linalool on human lymphocytes in vitro.

The potential genotoxicity of lavender essential oil and its major components, linalool, and linalyl acetate, was evaluated in vitro by the micronucleus test on peripheral human lymphocytes. In the range of non‐toxic concentrations (0.5–100 μg/ml), linalyl acetate increased the frequency of micronuclei significantly and in concentration‐dependent manner; lavender oil did so only at the highest concentration tested, whereas linalool was devoid of genotoxicity. None of the tested substances led to an increase in nucleoplasmic bridges or nuclear buds frequency. These findings suggest that the mutagenic activity of lavender oil can be related to the presence of linalyl acetate, which seems to have a profile of an aneugenic agent. Environ. Mol. Mutagen. 52:69–71, 2011.

Genotoxicity assessment of some cosmetic and food additives

α-Hexylcinnamaldehyde (HCA) and p-tert-butyl-alpha-methylhydrocinnamic aldehyde (BMHCA) are synthetic aldehydes, characterized by a typical floral scent, which makes them suitable to be used as fragrances in personal care (perfumes, creams, shampoos, etc.) and household products, and as flavouring additives in food and pharmaceutical industry. The aldehydic structure suggests the need for a safety assessment for these compounds. Here, HCA and BMHCA were evaluated for their potential genotoxic risk, both at gene level (frameshift or base-substitution mutations) by the bacterial reverse mutation assay (Ames test), and at chromosomal level (clastogenicity and aneuploidy) by the micronucleus test. In order to evaluate a primary and repairable DNA damage, the comet assay has been also included. In spite of their potential hazardous chemical structure, a lack of mutagenicity was observed for both compounds in all bacterial strains tested, also in presence of the exogenous metabolic activator, showing that no genotoxic derivatives were produced by CYP450-mediated biotransformations. Neither genotoxicity at chromosomal level (i.e. clastogenicity or aneuploidy) nor single-strand breaks were observed. These findings will be useful in further assessing the safety of HCA and BMHCA as either flavour or fragrance chemicals.

Genotoxicity assessment of β-caryophyllene oxide

β-caryophyllene oxide is a biciclic sesquiterpene, occurring naturally in essential oils from various medicinal and edible plants and used as a flavouring agent. Due to its potential hazardous chemical structure, the European Food Safety Authority reported to be pending a safety assessment for this compound. Here, this flavouring agent was tested for its mutagenic effect in the Ames test and micronucleus assay. Furthermore, considering that the penetration of a substance through phospholipid bilayers is determinant for its activity, the ability of β-caryophyllene oxide to be absorbed into cells was evaluated by differential scanning calorimetry (DSC) using multilamellar vesicles of dimyristoylphosphatidylcholine as a biomembrane model. β-caryophyllene oxide was found to be devoid of mutagenic effect, both at gene level (frameshift or base-substitution mutations), and on chromosome (clastogenicity and aneuploidogenicity). Results of DSC analysis highlighted that the substance was strongly absorbed through the membrane bilayer. Present results show that β-caryophyllene oxide, although absorbed through cell membranes and in spite of its potentially reactive chemical structure, is devoid of genotoxic effects, inducing neither point mutations nor chromosomal damages. These negative genotoxic findings will be critical to the safety assessment of β-caryophyllene oxide as used as a flavouring/fragrance ingredient.

Multidisciplinary Approach to Determine the Optimal Time and Period for Extracting the Essential Oil from Mentha suaveolens Ehrh

A comprehensive study on essential oils (EOs) extracted from some Mentha suaveolens L. samples, collected in the countryside of Tarquinia, is reported. In this study, the procedure for essential oil preparation, in terms of harvesting and extraction time, was analyzed in detail for the first time. The GC/MS analysis, carried out on 18 samples, revealed that piperitenone oxide (PO), the main essential oils’ chemical constituent, is primarily responsible for the related antifungal activity. Nevertheless, EOs with lower PO content indicate that other chemicals, such as para-cymenene, may participate in exerting the EOs’ antifungal effect. Furthermore, the bacterial reverse mutation assay highlighted lack of mutagenic effect in all tested samples. Analysis of the results indicated that for higher activity, the essential oils should be produced with 3 h maximum hydrodistillation, regardless of the harvesting time. Differently, the maximum essential oil yield can be obtained in August and the highest piperitenone oxide percentage is obtainable in July.

Antimutagenic and antioxidant activities of some bioflavours from wine.

Monoterpenes limonene and its metabolic derivatives, α-terpineol and 1,8-cineol, commonly found as aroma wine components, were studied for their antimutagenicity by the bacterial reverse mutation assay on different strains. Substances were also tested for their antioxidant activity, i.e. radical scavenger, chelation, reduction, and lipid peroxidation inhibition. Limonene and its metabolites, α-terpineol and 1,8-cineol, resulted able to inhibit the chemically-induced mutagenesis, although with a different specificity. The antimutagenicity of limonene has been generally retained by its metabolites and sometimes increased. In particular, α-terpineol exhibited the strongest inhibition, moreover it showed to be a remarkable ferrous ions chelating agent. Limonene and 1,8-cineol were devoid of antioxidant activity. Present results are a starting point in evaluating the potential of α-terpineol as a chemopreventive agent and suggest potential functional dietary benefits of wine.