Clara Della Croce

Cell cycle and morphological alterations as indicative of apoptosis promoted by UV irradiation in S. cerevisiae

An apoptotic phenotype induced by oxygen radicals or Bax expression has been observed in Saccharomyces cerevisiae yeast cells by electron and fluorescence microscopy. In this work, we analyzed DNA content and cellular morphology of S. cerevisiae after H(2)O(2) or UV treatment by TdT-mediated dUTP nick end labeling (TUNEL)-test and flow cytofluorimetry. A TUNEL-positive phenotype was observed in both cases, on the same samples a dose-dependent increase in the sub-G(1) population was pointed out by flow cytometry. Sub-G(1) cells were isolated by flow sorting and analyzed by electron microscopy. This population showed condensed chromatin in the nucleus and cell shrinking. This paper reports the first evidence of apoptosis in yeast cells induced by DNA damage after UV irradiation.

Protective effects of vitamins and selenium compounds in yeast.

Antimutagens and anticarcinogens are known to play an important role in decreasing damages induced by oxidants. In this study, we investigated the genotoxic and antimutagenic potential of two selenium compounds (sodium selenite: Na(2)SeO(3); seleno-DL-methionine: C(5)H(11)NO(2)Se) and Vitamins A and E in yeast cells of Saccharomyces cerevisiae. An oxidative mutagen (hydrogen peroxide (H(2)O(2)), HP) was chosen as positive control. We determined the enzymatic activities involved in the protection against oxidative damages (catalase: CAT; superoxide dismutase: SOD; glutathione peroxidase: GPx) in the cytosolic extract of yeast cells. The results demonstrated that selenium compounds exerted both mutagenic and antimutagenic effect at different concentrations. Antimutagenesis was evident both in stationary and in logarithmic phase cells. Catalase, SOD, and GPx were significantly increased in the presence of all the compounds assayed. Vitamins A (retinol) and E (alpha-tocopherol) did not have toxic or mutagenic action.

Antimutagenic and antioxidant activity of Lisosan G in Saccharomyces cerevisiae.

In the present study the antimutagenic and antioxidant effects of a powder of grain (Lisosan G) in yeast Saccharomyces cerevisiae were studied. Results showed that Lisosan G treatment decreased significantly the intracellular ROS concentration and mutagenesis induced by hydrogen peroxide in S. cerevisiae D7 strain. The effect of Lisosan G was then evaluated by using superoxide dismutase (SOD) proficient and deficient strains of S. cerevisiae. Lisosan G showed protective activity in sod1Δ and sod2Δ mutant strains, indicating an in vivo antioxidant effect. A high radical scavenging activity of Lisosan G was also demonstrated in vitro using the oxygen radical absorbance capacity (ORAC) assay. The obtained results showed a protective effect of Lisosan G in yeast cells, indicating that its antioxidant capacity contributes to its antimutagenic action.

Isolation of a novel metabolizing system enriched in phase-II enzymes for short-term genotoxicity bioassays

Murine S9 liver fractions isolated from mice fed 7.5 g kg-1 2(3)-tert-Butyl-4-hydroxyanisole (BHA) for 3 weeks were tested to determine: (a) the profile of both phase-I and phase-II xenobiotic metabolizing enzymes; (b) their ability to induce in vitro covalent binding of some precarcinogens to calf thymus DNA; and (c) their activation in a standard genetic toxicology assay. With regard to phase-I pathway, the S9 fraction expressed various cytochrome P-450-(CYP) (classes 1A1, 1A2, 2B1, 2E1, and 3A)-dependent biotransformation enzymes at levels comparable with those present in murine control liver. For post-oxidative enzymes, the S9 expressed high levels of glutathione S-transferases (up to 12-fold increase), glutathione S-epoxide-transferase (up to 2.6-fold), UDP-glucuronosyl transferase (up to 5.3-fold) and epoxide hydrolase (up to 2.6-fold) activities, as compared to untreated mice. The in vitro DNA binding of the precarcinogenic agents [14C]-1,4-dichlorobenzene, [14C]-1,2-dichlorobenzene and [14C]-1,4-dibromobenzene, mediated by BHA-induced cytosol and/or microsomal preparation, showed an increase in specific activity comparable to that observed with phase-I (PB/beta NF) induced S9. In some instances, covalent binding was even more elevated using the BHA-induced systems as compared with traditional S9 fractions. For example, cytosol derived from BHA-administered mice was able to induce a significant binding to calf thymus DNA up to 26.2-fold increase for [14C]-1,4-dichlorobenzene, while cytosol from PB/beta NF was not. A high mutagenic response on diploid D7 strain of Saccharomyces cerevisiae as exemplified by a marked induction of mitotic gene conversion and point (reverse) mutation confirmed that BHA-derived S9 fractions activate precarcinogens to final genotoxins. Because a number of chemicals are activated by either oxidative or post-oxidative enzymes, the use of metabolizing biosystems, with an enhanced phase-II pathway, together with classical S9 fractions, can improve the sensitivity of the assay in detecting unknown genotoxins.

E-cigarettes induce toxicological effects that can raise the cancer risk

Electronic cigarettes (e-cigs) are devices designed to deliver nicotine in a vaping solution rather than smoke and without tobacco combustion. Perceived as a safer alternative to conventional cigarettes, e-cigs are aggressively marketed as lifestyle-choice consumables, thanks to few restrictions and a lack of regulatory guidelines. E-cigs have also gained popularity among never-smokers and teenagers, becoming an emergent public health issue. Despite the burgeoning worldwide consumption of e-cigs, their safety remains largely unproven and it is unknown whether these devices cause in vivo toxicological effects that could contribute to cancer. Here we demonstrate the co-mutagenic and cancer-initiating effects of e-cig vapour in a rat lung model. We found that e-cigs have a powerful booster effect on phase-I carcinogen-bioactivating enzymes, including activators of polycyclic aromatic hydrocarbons (PAHs), and increase oxygen free radical production and DNA oxidation to 8-hydroxy-2′-deoxyguanosine. Furthermore, we found that e-cigs damage DNA not only at chromosomal level in peripheral blood, such as strand breaks in leucocytes and micronuclei formation in reticulocytes, but also at gene level such as point mutations in urine. Our results demonstrate that exposure to e-cigs could endanger human health, particularly among younger more vulnerable consumers.

Antimutagenicity of sodium selenite in Chinese hamster V79 cells exposed to azoxymethane, methylmethansulphonate and hydrogen peroxide.

In the last 10 years, there is an increasing interest in selenium (Se) because of its environmental, biological, and toxicological importance, and in particular, because of its antioxidant properties. However, inspite of extensive studies, the optimal concentration of Se to be used for its beneficial effects in not yet known. In addition, the mechanisms of Se antioxidant property require further study. We report on the effects of various mutagens/carcinogens such as azoxymethane, methylmethanesulphonate, and hydrogen peroxide on Chinese V79 hamster cells, in presence of sodium selenite in the concentration of 0.5 microM. We found that Se reduced the genotoxic effect of these mutagens/carcinogens. We also investigated enzymatic activities of glutathione peroxidase, catalase, superoxide dismutase, and glutathione S-transferase, in order to understand the Se involvement in the detoxification of free radicals. Sodium selenite increased the activities of glutathione peroxidase and catalase.

Antimutagenicity in yeast

In recent years there has been increasing interest in antimutagenesis, and studies have been done using both prokaryotic and eukaryotic systems. In eukaryotic systems the first studies were performed with different strains of Schizosaccharomyces pombe. In particular, caffeine and L-methionine were investigated. Different strains of Saccharomyces cerevisiae were employed in studies of a wide variety of compounds, including acridine, saccharin, salts, tumor promoters and co-carcinogens. Strain D7 was widely employed and antimutagenic activity of spermine, chlorophyllin, cobaltous chloride and fermented milk is reported.

Effect of HFD/STZ on expression of genes involved in lipid, cholesterol and glucose metabolism in rats

AIMS The aim of the study was to evaluate lipid, cholesterol and glucose metabolism in a novel rat model of non-alcoholic fatty liver disease (NAFLD). MAIN METHODS Rats (Wistar) were fed high fat/cholesterol diet (HFD) and a single low dose (35mg/kg) of streptozotocin (STZ). Collagen and glycogen content, oxidative stress and glucokinase activity were measured using biochemical assays. Other metabolic pathways were assessed by qRT-PCR. KEY FINDINGS HFD/STZ treated rats, compared to control ones, showed an increase in expression of biomarkers of inflammation (TNFα, IL6), fibrosis (TGFβ), mitochondrial stress (UCP2) and oxidative stress (GSH and carbonylated proteins) but not of ER stress (CHOP, XBP1). Additionally, HFD/STZ treatment caused a reduction in glycogen content, glucokinase activity (a limiting step in glycolysis) and expression of ChREBP gene (a de novo lipogenesis regulator), suggesting a modified glycolytic pathway. The cholesterol biosynthesis in HFD/STZ treated rats was inhibited (reduced expression of SREBP-2-regulated HMGCoA red and LDLr), instead the cholesterol catabolism was increased, as shown by the mRNA induction of the CYP7A1 and CYP8B1 (key genes for BA acid). A reduced gene expression of FXR-dependent SHP (a key gene for feedback inhibition of CYP7A1 and CYP8B1) and of bile acids (NTCP, OATP1A1, BSEP) and cholesterol (ABCA1) transporters was found. SIGNIFICANCE These results widely extend the characterization of HFD/STZ rat model, which might mimic the NAFLD/NASH in diabetic humans.

Disruption of redox homeostasis and carcinogen metabolizing enzymes changes by administration of vitamin E to rats.

AIMS A large meta-analysis of randomized clinical trials has seriously questioned chemoprevention based on vitamins including vitamin E (VE), and an increased risk for cancer among long-term users was actually seen. However, the mechanism underlying these findings still remain unknown. To clarify the mechanism, in an in vivo model we studied the putative disruption of redox homeostasis and the perturbation of carcinogen metabolizing enzymes determined by VE. MAIN METHODS Male Sprague-Dawley rats were treated ip with either 100 or 200mg/kg b.w. daily for 7 or 14 consecutive days. Controls received vehicle only. Cytochrome P450 (CYP) content, CYP-reductase, CYP-linked monooxygenases, as well as phase-II and the antioxidant enzymes catalase and NAD(P)H quinone reductase were investigated in both liver and kidney. Free radical species in tissue subcellular preparations were measured by electronic paramagnetic resonance (EPR) spectroscopy coupled to a radical probe technique. KEY FINDINGS No substantial changes of hepatic xenobiotic metabolism enzymes were determined by VE. Conversely, a powerful booster effect of various renal phase-I carcinogen bioactivating enzymes at both dosages and observational times was recorded. While no relevant changes of post-oxidative phase-II reactions were found in the liver, a significant inactivating effect was caused by VE in renal tissues. Antioxidant enzymes were found mainly downregulated by the treatment. In the kidney, a marked free radical over-generation linked to CYP induction was observed. SIGNIFICANCE This study proved that VE acts as a co-carcinogen and pro-oxidant agent. Such epigenetic mechanisms may contribute to explain the harmful outcomes observed in humans.

Genotoxic and mono-oxygenase system effects of the fungicide maneb

Abstract. The in vivo effects of a commercial preparation of maneb on mono-oxygenase activities of hepatic microsomes of basal and induced rats were examined. In vitro experiments with the D7 strain of yeast Saccharomyces cerevisiae were also performed. In both basal and induced rats maneb caused a decrease in cytochrome P-450 content and aniline hydroxylase. Immunoblotting analysis using anti-P-450 IIE1 antibodies confirmed the data obtained for aniline hydroxylase activity. Maneb was toxic in cells of S. cerevisiae. On the basis of in vivo and in vitro experiments it can be concluded that maneb possesses a toxic activity attributable to its main metabolite ethylene thiourea. Immunoblotting analysis indicates that maneb biotransformation influences the IIE1 P-450 isoform.