Anthocyanins protect against DNA damage induced by tert-butyl-hydroperoxide in rat smooth muscle and hepatoma cells.

Anthocyanins are flavonoids present in a variety of pigmented food and, like other flavonoids, seem to play a role in preventing human pathologies related to oxidative stress. In fact, anthocyanins have been shown to exert antiproliferative effects in cell cultures and exhibit antiinflammatory and vasoprotective activities in animal models. Although these biological activities have been related to their antioxidant properties, little is known on the molecular mechanism of action of anthocyanins. The effects of pretreatment with the anthocyanins delphinidin, cyanidin, and their glycoside and rutinoside derivatives against induction of DNA damage induced by tert-butyl-hydroperoxide (TBHP) were evaluated in rat smooth muscle and in rat hepatoma cell lines using alkaline single cell gel electrophoresis (Comet test). In addition, a possible protection exerted by anthocyanins on cell killing, lipid peroxidation, and redox state alterations induced by TBHP was also investigated. It was found that the treatment with TBHP induces the formation of DNA single strand breaks (SSB) and oxidised bases, along with cell killing, lipid peroxidation and redox state alteration. Our data demonstrate that anthocyanins are effective against cytotoxicity, DNA SSB formation and lipid peroxidation induced by TBHP, but they do not have any detectable effect against impairment by TBHP of cellular redox state and on protection against DNA bases oxidation. The presence of a sugar moiety in anthocyanin derivatives reduced this protective effect, mainly in rat hepatoma cells. The different activity of anthocyanins and their derivatives may be explained taking into account a structure/function relationship that could also influence anthocyanin intracellular localisation.

The antiproliferative effect of beta-carotene requires p21waf1/cip1 in normal human fibroblasts.

In normal human fibroblasts, beta-carotene induces a cell-cycle delay in the G1 phase independent of its provitamin A activity via a mechanism not yet elucidated. In this study we provide biochemical evidence showing that delayed progression through the G1 phase occurs concomitantly with: an increase in both nuclear-bound and total p21waf1/cip1 protein levels; an increase in the amount of p21waf1/cip1 associated with cdk4; the inhibition of cyclin D1-associated cdk4 kinase activity; and a reduction in the levels of hyperphosphorylated forms of retinoblastoma protein, and particularly, in phosphorylated Ser780. The role of p21waf1/cip1 in the antiproliferative effect of the carotenoid was further supported by genetic evidence that neither changes in cell-cycle progression nor in the phosphorylation status of retinoblastoma protein were observed in p21waf1/cip1-deficient human fibroblasts treated with beta-carotene. These results clearly demonstrate that p21waf1/cip1 is involved directly in the molecular pathway by which beta-carotene inhibits cell-cycle progression.

The effect of selective low-density lipoprotein apheresis on plasma lipoperoxides and antioxidant vitamins in familial hypercholesterolemic patients.

Familial hypercholesterolemia (FH) is an autosomal dominant genetic disorder characterized by a lifelong elevation in the concentration of low-density lipoprotein (LDL) bound cholesterol in blood by cholesterol deposits and by early coronary artery disease. The LDL apheresis technique has been introduced with the goal of reducing LDL cholesterol levels, thereby preventing the development of atherosclerosis. The literature on LDL apheresis reports 2 different facets, the therapeutic aspect associated with the lessening of LDL concentration and the initiation of a peroxidation process associated with the biocompatibility of the artificial membrane. Lipid and protein peroxidation gives rise to toxic and atherogenic hydroperoxide, mostly lipid hydroperoxides, and derivative compounds, which may offset the benefit of the procedure. In this paper, plasma hydroperoxide levels are determined along with the elevation of the serum and LDL antioxidant status in hypercholesterolemic patients before and following repeated LDL apheresis sessions. Hydroperoxide concentration has been expressed both in terms of plasma volume and LDL concentration. A highly significant increase in LDL lipid hydroperoxides is demonstrated when expressed in terms of LDL concentration and is associated with the LDL apheresis procedure. The usefulness of antioxidant supplementation in LDL apheresis is discussed.

Effect of beta-carotene on cell cycle progression of human fibroblasts.

The uptake of beta-carotene (BC) and its effect on the cell cycle progression of normal human fibroblasts in primary culture were investigated by using two different delivery methods: exposure to BC solubilized in the organic solvent tetrahydrofuran (THF) or to BC incorporated into dipalmitoylphosphatidylcholine (DPPC) liposomes. Cell cycle progression was evaluated by immunofluorescence detection and flow cytometric analysis of the proliferating cell nuclear antigen (PCNA). In contrast to THF, which induced a marked reduction in the number of cells in S phase and in the extent of PCNA immunolabeling, DPPC liposomes proved to be an effective delivery system that does not interfere with cell proliferation. Cellular uptake of 0.23 nmol/10(6) cells was found after 24 h incubation in BC-containing DPPC liposomes. This value increased to 1.2 nmol/10(6) cells after 72 h. After the first day of incubation, the number of cells in S phase was reduced by approximately 50%, with a consequent accumulation of cells in G1 phase. This effect was maintained up to 3 days incubation, with no detectable effects on cell viability. This cell cycle delay was found to be reversible, returning the percentage of cells in S phase to the control value 24 h after removal of BC from the medium. In order to determine whether the activity of BC could be attributed to the molecule itself or to its conversion into retinoids, the production of BC metabolites was assessed. Analysis of cellular levels of retinoids failed to demonstrate the presence of retinal, retinol, retinoic acid or retinyl esters during an incubation period of 6 days. These results suggest that in normal human fibroblasts, BC induces a cell cycle delay in the G1 phase and that this effect is independent of conversion to known retinoids.

Effects of beta-carotene and alpha-tocopherol on photogenotoxicity induced by 8-methoxypsoralen: the role of oxygen.

The protective effect of beta-carotene (beta-C) and alpha-tocopherol (alpha-T), singularly and in equimolar mixtures, toward the photomutagenicity induced by 8-methoxypsoralen (8-MOP), at different oxygen partial pressure (pO2), was evaluated in two different experimental models: Salmonella typhimurium TA102 and Saccharomyces cerevisiae D7. After phototreatment with 8-MOP, the results show a lethal effect under hypoxic conditions in both experimental model systems, an increase in revertants associated to the pO2 increase in S. typhimurium TA102, and a decrease in revertants and convertants associated to the pO2 increase in S. cerevisiae D7. In S. typhimurium TA102, in atmospheric condition, beta-C and alpha-T (1.86 or 18.6 microM) show a protective effect only at the higher dosage. Alpha-T was more protective than beta-C. The equimolar mixtures show an antimutagenic effect at both dosage used with a synergistic effect at lower dosage and an additive antimutagenic activity at higher dosage. An inhibition of the spontaneous mutagenicity by mixtures at higher dosage was also observed. The results obtained in S. typhimurium TA102 show an antimutagenic effects of beta-C, alpha-T and their mixture at 190 mmHg pO2, confirming the data obtained in air condition. At 380 mmHg pO2, alpha-T and the mixture show a significant antimutagenic activity; at 570 mmHg pO2, only alpha-T is protective. At 760 mmHg pO2, no protective effect was observed by the two antioxidants, and beta-C increases the photomutagenicity induced by 8-MOP. In S. cerevisiae D7 a protective effect was only observed at 380 mmHg pO2 with the mixture. No antigenotoxic effect was found in the other experimental conditions, even if the uptake of the two antioxidants was confirmed by HPLC. Our results underline the role of oxygen in the photomutagenicity induced by 8-MOP and in the antimutagenic activity of beta-C and alpha-T. This is the first report confirming in a cellular experimental model the data obtained in some chemical systems: the protective effect of beta-C only at low pO2 and the synergistic effect of mixture of beta-C and alpha-T.

Photoinduction of micronuclei by 4,4',6-trimethylangelicin and 8-methoxypsoralen in different experimental models.

The frequencies of micronuclei induced by treatment with 4,4',6-trimethylangelicin (TMA) and 8-methoxypsoralen (8-MOP) have been compared in the following experimental models: (1) peripheral normochromatic erythrocytes (NCE) during 10 days after single p.o. administration of TMA or 8-MOP in male and female mice; (2) peripheral NCE during photocarcinogenesis by TMA or 8-MOP topically administered to female mice; (3) primary cultures of human skin fibroblasts treated with TMA or 8-MOP. The frequency of micronuclei in peripheral NCE of mice (both sexes) was significantly enhanced after p.o. administration of TMA or 8-MOP. This latter was more active than TMA in inducing chromosomal damage. No increased frequencies of micronuclei in peripheral NCE were detected in mice subjected to TMA or 8-MOP photocarcinogenic treatment, even when malignancies developed. In human fibroblast cultures, at equimolar concentrations, the induction of lethal effects by TMA in the presence of 365-nm radiation was higher than that exerted by 8-MOP. At equal survival, however, TMA showed practically the same activity as 8-MOP in the induction of micronuclei. Our findings provide evidence of genotoxicity by TMA administered p.o. without irradiation and give further information about photogenotoxicity of these substances.

Genotoxicity assay of five pesticides and their mixtures in Saccharomyces cerevisiae D7.

Four organophosphorus pesticides (azinphos-methyl, diazinone, dimethoate, and pirimiphos-methyl), and one carbamate (benomyl) were tested for cytotoxicity, reverse mutation and gene conversion in Saccharomyces cerevisiae D7, with and without the S9 metabolic system. Furthermore, two mixtures of the above compounds, namely benomyl + pirimiphos-methyl (6/1 ratio) and dimethoate + diazinone + azinphos-methyl (10/4/6 ratio) were tested in the same experimental model. Azinphos-methyl, benomyl, and pirimiphos-methyl alone did not induce any genotoxic effect, whereas azinphos-methyl and diazinone were active in inducing reversion and gene conversion. The benomyl + pirimiphos-methyl mixture did not show any genotoxic activity. The dimethoate + diazinone + azimphos-methyl mixture was genotoxic, although an antagonistic effect between the components was observed. The addition of S9 post-mitochondrial liver fraction decreased the activity of both single and mixed genotoxic agents.

Carotenoids reduce the chromosomal damage induced by bleomycin in human cultured lymphocytes.

The supplementation with beta-carotene and canthaxanthin, two carotenoids with and without pro-vitamin A activity, reduced in vitro the number of micronuclei induced in human cultured lymphocytes by the chemotherapeutic radiomimetic drug bleomycin. The genotoxic activity of this substance is supposed to be mediated by a free radical mechanism. The reduction of micronucleated cells was in correlation with donors' carotenoid blood levels. It has been observed that 20 weeks are needed, following the end of carotenoid supplementation, in order to return to the high levels of micronuclei induced by bleomycin. It is suggested that this protective effect is due to the antioxidant properties of carotenoids acting against free radical-mediated genotoxic damage.

Assisted evaluation of antibiotic resistance development in Pseudomonas aeruginosa from intensive care units.

421 strains of Pseudomonas aeruginosa were isolated from patients admitted to intensive care units and tested with automated systems for sensitivity to 21 antimicrobial agents. Data were collected in a database for evaluation and monitoring of resistance development. Results showed that assisted monitoring of antimicrobial resistance gives continuously updated information, with particular attention to the different local therapeutical schedules. It is therefore advisable that clinicians constantly exchange information with the microbiology laboratory through a hospital information system in which data from different laboratories are pooled in real time.

Use of serum markers in the diagnosis and management of laryngeal cancer.

Many neoplastic diseases are reported to be accompanied by the presence or associated with an increase in biological substances identified as tumour markers. The most common markers implicated in head and neck cancers are CEA, TPA, LASA, SCC, CA 19-9, and ferritin. These markers (except SCC) were evaluated in 50 patients with a laryngeal carcinoma, in 20 patients with benign lesions, and in 20 healthy subjects. The results show for each marker assayed the following sensitivity values (true positives): CEA, 10%; CA 19-9, 30%; TPA, 30%; LASA, 90%; ferritin, 60%. Specificity (true negatives) was as follows: CEA, 85%; CA 19-9, 99.4%; TPA, 98%; LASA, 99.8%; ferritin, 97%. LASA and ferritin seem to be the most suitable markers for patient monitoring because of their higher sensitivity in all phases of cancer disease.

Reduction of chromosomal damage by bleomycin in lymphocytes from subjects supplemented with carotenoids. Relevance in bleomycin tumour chemotherapy. Preliminary results.

In a one year-study, 9 healthy human donors were being supplemented with beta-carotene (BC) plus canthaxanthin (CX), to determine the effect of carotenoids on chromosomal damage (micronuclei) induced in the donors' lymphocyte cell cultures by exposure to bleomycin (BLM), an antineoplastic drug that has been shown to produce chromosomal aberrations through the production of free radicals. The first four months monitoring data, including determination of carotenoid blood levels, are here reported. These data show that carotenoid supplementation significantly decrease (up to 50%) the formation of micronuclei induced by BLM in human lymphocyte cell cultures. This decrease is in correlation with carotenoid blood levels.

Chemoprevention of indirect and direct chemical carcinogenesis by carotenoids as oxygen radical quenchers.

Beta-carotene (BC) and canthaxanthine (CX), two carotenoids with and without pro-vitamin A activity, respectively, were found to help to prevent benzo[a]pyrene (BP)-induced skin carcinogenesis in the dark and BP photocarcinogenesis (UV 300-400 nm) when given as an oral supplement to female Swiss albino mice. The same experimental procedure was adapted to 8-methoxypsoralen (8-MOP) photoinduction of mammary carcinomas in mice. Here also, the two carotenoids were strongly antitumorigenic. Indeed, 8-MOP photomutagenesis, tested in S. typhimurium TA 102, appeared to depend on a two-step reaction, namely an oxygen-independent DNA-8-MOP photoadduct, followed by an oxygen-dependent second step, sensitive to carotenoids. This result suggests that dietary carotenoids (powerful antioxidant molecules) might prevent the carcinogenic risk caused by substances that are transformed into ultimate carcinogens by oxidative processes which are indirectly carcinogenic. Finally, to verify whether supplemental carotenoids can affect carcinogenesis where neither light excitation nor oxidative metabolic processes are involved, an experimental attempt was made on gastric carcinogenesis induced in rats by the direct carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The results demonstrate that supplemental carotenoids do not affect initiation and progression stages, but do prevent the progression stage of dysplasias to infiltrating gastric carcinomas. Thus, this provides strong presumptive evidence for oxygen radical involvement in the later stages of this neoplastic development, as recently reported in the literature. As far as mutagenicity in S. typhimurium is concerned, carotenoids do not exert, as expected, any protective effect on MNNG mutagenic activity. The above experimental data suggest that supplemental carotenoids, instead of sunscreen preparations, can be adopted by outdoor workers to prevent skin cancer. Accordingly, such natural antioxidants may be useful in human chemoprevention against neoplasias of the lung, breast, urinary bladder, and colon and rectum even after radical surgery.