The effect of cruciferous and leguminous sprouts on genotoxicity, in vitro and in vivo

Vegetable consumption is associated with a reduced risk of colorectal cancer, which is the second most common cancer after lung/breast cancer within Europe. Some putative protective phytochemicals are found in higher amounts in young sprouts than in mature plants. The effect of an extract of mixed cruciferous and legume sprouts on DNA damage induced by H(2)O(2) was measured in HT29 cells using single cell microgelelectrophoresis (comet). Significant antigenotoxic effect (P < or = 0.05) was observed when HT29 cells were pre-incubated with the extract (100 and 200 microL/mL) for 24 hours and then challenged with H(2)O(2). A parallel design intervention study was carried out on 10 male and 10 female healthy adult volunteers (mean age = 25.5 years) fed 113 g of cruciferous and legume sprouts daily for 14 days. The effect of the supplementation was measured on a range of parameters, including DNA damage in lymphocytes (comet), the activity of various detoxifying enzymes (glutathione S-transferase, glutathione peroxidase, and superoxide dismutase), antioxidant status using the ferric reducing ability of plasma assay, plasma antioxidants (uric acid, ascorbic acid, and alpha-tocopherol), blood lipids, plasma levels of lutein, and lycopene. A significant antigenotoxic effect against H(2)O(2)-induced DNA damage was shown in peripheral blood lymphocytes of volunteers who consumed the supplemented diet when compared with the control diet (P = 0.04). No significant induction of detoxifying enzymes was observed during the study, neither were plasma antioxidant levels or activity altered. The results support the theory that consumption of cruciferous vegetables is linked to a reduced risk of cancer via decreased damage to DNA.

Do variations in substitution rates and male mutation bias correlate with life-history traits? A study of 32 mammalian genomes

Life-history traits vary substantially across species, and have been demonstrated to affect substitution rates. We compute genomewide, branch-specific estimates of male mutation bias (the ratio of male-to-female mutation rates) across 32 mammalian genomes and study how these vary with life-history traits (generation time, metabolic rate, and sperm competition). We also investigate the influence of life-history traits on substitution rates at unconstrained sites across a wide phylogenetic range. We observe that increased generation time is the strongest predictor of variation in both substitution rates (for which it is a negative predictor) and male mutation bias (for which it is a positive predictor). Although less significant, we also observe that estimates of metabolic rate, reflecting replication-independent DNA damage and repair mechanisms, correlate negatively with autosomal substitution rates, and positively with male mutation bias. Finally, in contrast to expectations, we find no significant correlation between sperm competition and either autosomal substitution rates or male mutation bias. Our results support the important but frequently opposite effects of some, but not all, life history traits on substitution rates. KEY WORDS: Generation time, genome evolution, metabolic rate, sperm competition.

A comparison of the anticancer properties of isoxanthohumol and 8-prenylnaringenin using in vitro models of colon cancer

The hops plant (Humulus lupulus L.) is an essential ingredient in beer and contains a number of potentially bioactive prenylflavonoids, the predominant being the weakly estrogenic isoxanthohumol (Ix), which can be converted to the more strongly estrogenic 8-PN by the colonic microbiota. The aim of this study was to investigate the biological activity of 8-PN and Ix using in vitro models representing key stages of colorectal carcinogenesis, namely cell growth and viability (MTT assay), cell-cycle progression (DNA content assay), DNA damage (Comet assay), and invasion (Matrigel assay). A significant decrease in Caco-2 cell viability was noted after both 8-PN and Ix treatments at the higher doses (40 and 50 μM, respectively) although the impact on cell cycle differed between the two compounds. The decreased cell viability observed after Ix treatment was associated with a concentration-dependent increase in G2/M and an increased sub-G1 cell-cycle fraction, whereas treatment with 8-PN was associated with an elevated G0/G1 and an increased sub-G1 cell-cycle fraction. Significant antigenotoxic activity was noted at all 8-PN concentrations tested (5-40 μM). Although significant antigenotoxic activity was also noted with Ix treatment at ≤25 μM, at a higher dose, Ix itself exerted genotoxic activity. In a dose-dependent manner, both compounds inhibited HT115 cell invasion with reductions up to 52 and 46% for Ix and 8-PN, respectively, in comparison to untreated cells. This study demonstrated that both Ix and its gut microbial metabolite 8-PN exert anticancer effects on models of key stages of colon tumourigenesis.

Effect of aluminium on migratory and invasive properties of MCF-7 human breast cancer cells in culture

Aluminium (Al) has been measured in human breast tissue, nipple aspirate fluid and breast cyst fluid, and recent studies have shown that at tissue concentrations, aluminium can induce DNA damage and suspension growth in human breast epithelial cells. This paper demonstrates for the first time that exposure to aluminium can also increase migratory and invasive properties of MCF-7 human breast cancer cells. Long-term (32 weeks) but not short-term (1 week) exposure of MCF-7 cells to 10-4M aluminium chloride or 10-4M aluminium chlorohydrate increased motility of the cells as measured by live cell imaging (cumulative length moved by individual cells), by a wound healing assay and by migration in real time through 8m pores of a membrane using xCELLigence technology. Long-term exposure (37weeks) to 10-4M aluminium chloride or 10-4M aluminium chlorohydrate also increased the ability of MCF-7 cells to invade through a matrigel layer as measured in real time using the xCELLigence system. Although molecular mechanisms remain to be characterized, the ability of aluminium salts to increase migratory and invasive properties of MCF-7 cells suggests that the presence of aluminium in the human breast could influence metastatic processes. This is important because mortality from breast cancer arises mainly from tumour spread rather than from the presence of a primary tumour in the breast.

Parabens can enable hallmarks and characteristics of cancer in human breast epithelial cells: a review of the literature with reference to new exposure data and regulatory status

A framework for understanding the complexity of cancer development was established by Hanahan and Weinberg in their definition of the hallmarks of cancer. In this review, we consider the evidence that parabens can enable development in human breast epithelial cells of 4/6 of the basic hallmarks, 1/2 of the emerging hallmarks and 1/2 of the enabling characteristics. Hallmark 1: parabens have been measured as present in 99% of human breast tissue samples, possess oestrogenic activity and can stimulate sustained proliferation of human breast cancer cells at concentrations measurable in the breast. Hallmark 2: parabens can inhibit the suppression of breast cancer cell growth by hydroxytamoxifen, and through binding to the oestrogen-related receptor gamma (ERR) may prevent its deactivation by growth inhibitors. Hallmark 3: in the 10nM to 1M range, parabens give a dose-dependent evasion of apoptosis in high-risk donor breast epithelial cells. Hallmark 4: long-term exposure (>20weeks) to parabens leads to increased migratory and invasive activity in human breast cancer cells, properties which are linked to the metastatic process. Emerging hallmark: methylparaben has been shown in human breast epithelial cells to increase mTOR, a key regulator of energy metabolism. Enabling characteristic: parabens can cause DNA damage at high concentrations in the short term but more work is needed to investigate long-term low-doses of mixtures. The ability of parabens to enable multiple cancer hallmarks in human breast epithelial cells provides grounds for regulatory review of the implications of the presence of parabens in human breast tissue.

MiR-375 is essential for human spinal motor neuron development and may be involved in motor neuron degeneration

The transcription factor REST is a key suppressor of neuronal genes in non-neuronal tissues. REST has been shown to suppress pro-neuronal microRNAs in neural progenitors indicating that REST-mediated neurogenic suppression may act in part via microRNAs. We used neural differentiation of Rest-null mouse ESC to identify dozens of microRNAs regulated by REST during neural development. One of the identified microRNAs, miR-375, was upregulated during human spinal motor neuron development. We found that miR-375 facilitates spinal motor neurogenesis by targeting the cyclin kinase CCND2 and the transcription factor PAX6. Additionally, miR-375 inhibits the tumor suppressor p53 and protects neurons from apoptosis in response to DNA damage. Interestingly, motor neurons derived from a spinal muscular atrophy patient displayed depressed miR-375 expression and elevated p53 protein levels. Importantly, SMA motor neurons were significantly more susceptible to DNA damage induced apoptosis suggesting that miR-375 may play a protective role in motor neurons.

Iridaea cordata (Gigartinales, Rhodophyta): responses to artificial UVB radiation

The effects of UVB radiation on the different developmental stages of the carrageenan-producing red alga Iridaea cordata were evaluated considering: (1) carpospore and discoid germling mortality; (2) growth rates and morphology of young tetrasporophytes; and (3) growth rates and pigment content of field-collected plant fragments. Unialgal cultures were submitted to 0.17, 0.5, or 0.83 W m(-2) of UVB radiation for 3 h per day. The general culture conditions were as follows: 12 h light/12 h dark cycles; irradiance of 55 mu mol photon. per square meter per second; temperature of 9 +/- 1 degrees C; and seawater enriched with Provasoli solution. All UVB irradiation treatments were harmful to carpospores (0.17 W m(-2) = 40.9 +/- 6.9%, 0.5 W m(-2) = 59.8 +/- 13.4%, 0.83 W m(-2) = 49 +/- 17.4% mortality in 3 days). Even though the mortality of all discoid germlings exposed to UVB radiation was unchanged when compared to the control, those germlings exposed to 0.5 and 0.83 W m(-2) treatments became paler and had smaller diameters than those cultivated under control treatment. Decreases in growth rates were observed in young tetrasporophytes, mainly in 0.5 and 0.83 W m(-2) treatments. Similar effects were only observed in fragments of adult plants cultivated at 0.83 W m(-2). Additionally, UVB radiation caused morphological changes in fragments of adult plants in the first week, while the young individuals only displayed this pattern during the third week. The verified morphological alterations in I. cordata could be interpreted as a defense against UVB by reducing the area exposed to radiation. However, a high level of radiation appears to produce irreparable damage, especially under long-term exposure. Our results suggest that the sensitivity to ultraviolet radiation decreases with increased algal age and that the various developmental stages have different responses when exposed to the same doses of UVB radiation.

Genotoxic effects of X-rays on keratinized mucosa cells during panoramic dental radiography

Objectives: The aim of this study was to evaluate the genotoxic effects of X-rays on epithelial gingival cells during panoramic dental radiography using a differentiated protocol for the micronucleus test. Methods: 40 healthy individuals who underwent this procedure for diagnostic purposes on request from their dentists agreed to participate in this study. All of them answered a questionnaire before the examination. Epithelial gingival cells were obtained from the keratinized mucosa of the upper dental arcade by gentle scraping with a cervical brush immediately before exposure and 10 days later. Cytological preparations were stained according to the Feulgen-Rossenbeck reaction, counterstained with fast green 1% for 1 min and analysed under a light microscope. Micronuclei, nuclear projections (broken eggs) and degenerative nuclear alterations (pyknosis, karyolysis, karyorrhexis and condensed chromatin) were scored. Results: The frequency of micronuclei was significantly higher after exposure (P < 0.05), as were frequencies of nuclear alterations indicate of apoptosis (P < 0.001). Conclusions: These results indicate that X-ray radiation emitted during panoramic dental radiography induces a genotoxic effect on epithelial gingival cells that increases the frequency of chromosomal damage and nuclear alterations indicative of apoptosis.

CPDs and 6-4PPs play different roles in UV-induced cell death in normal and NER-deficient human cells

Ultraviolet (UV) light generates two major DNA lesions: cyclobutane pyrimidine dimers (CPDs) and pyrimidine-(6-4)-pyrimidone photoproducts (6-4PPs), but the specific participation of these two lesions in the deleterious effects of UV is a longstanding question. In order to discriminate the precise role of unrepaired CPDs and 6-4PPs in UV-induced responses triggering cell death, human fibroblasts were transduced by recombinant adenoviruses carrying the CPD-photolyase or 6-4PP-photolyase cDNAs. Both photolyases were able to prevent UV-induced apoptosis in cells deficient for nucleotide excision repair (NER) to a similar extent, while in NER-proficient cells UV-induced apoptosis was prevented only by CPD-photolyase, with no effects observed when 6-4PPs were removed by the specific photolyase. These results strongly suggest that both CPDs and 6-4PPs contribute to UV-induced apoptosis in NER-deficient cells, while in NER-proficient cells, CPDs are the only lesions responsible for UV-killing, probably due to the rapid repair of 6-4PPs by NER. As a consequence, the difference in skin photosensitivity, including carcinogenesis, of most of the xeroderma pigmentosum patients and of normal people is probably not only a quantitative aspect, but depends on the type of DNA damage induced by sunlight and its rate of repair. (c) 2007 Elsevier B.V. All rights reserved.

Sustained activation of p53 in confluent nucleotide excision repair-deficient cells resistant to ultraviolet-induced apoptosis

p53 activation is one of the main signals after DNA damage, controlling cell cycle arrest, DNA repair and apoptosis. We have previously shown that confluent nucleotide excision repair (NER)-deficient cells are more resistant to apoptosis induced by ultraviolet irradiation (UV). Here, we further investigated the effect of cell confluence on UV-induced apoptosis in normal and NER-deficient (XP-A and XP-C) cells, as well as the effects of treatments with the ATWATR inhibitor caffeine, and the patterns of p53 activation. Strong p53 activation was observed in either proliferating or confluent cells. Caffeine increased apoptosis levels and inhibited p53 activation in proliferating cells, suggesting a protective role for p53. However, in confluent NER-deficient cells no effect of caffeine was observed. Transcription recovery measurements showed decreased recovery in proliferating XPA-deficient cells, but no recovery was observed in confluent cells. The levels of the cyclin/Cdk inhibitor, p21(Waf1/Cip1), correlated well with p53 activation in proliferating cells. Surprisingly, confluent cells also showed similar activation of p21(Waf1/Cip1). These results indicate that reduced apoptosis in confluent cells is associated with the deficiency in DNA damage removal, since this effect is not clearly observed in NER-proficient cells. Moreover, the strong activation of p53 in confluent cells, which barely respond to apoptosis, suggests that this protein, under these conditions, is not linked to UV-induced cell death signaling. (c) 2008 Elsevier B.V. All rights reserved.

Defective transcription/repair factor IN recruitment to specific UV lesions in trichothiodystrophy syndrome

Most trichothiodystrophy (TTD) patients present mutations in the xeroderma pigmentosum D (XPD) gene, coding for a subunit of the transcription/repair factor IIH (TFHH) complex involved in nucleotide excision repair (NER) and transcription. After UV irradiation, most TTD/XPD patients are more severely affected in the NER of cyclobutane pyrimidine dimers (CPD) than of 6-4-photoproducts (6-4PP). The reasons for this differential DNA repair defect are unknown. Here we report the first study of NER in response to CPDs or 6-4PPs separately analyzed in primary fibroblasts. This was done by using heterologous photorepair; recombinant adenovirus vectors carrying photolyases enzymes that repair CPD or 64PP specifically by using the energy of light were introduced in different cell lines. The data presented here reveal that some mutations affect the recruitment of TFHH specifically to CPDs, but not to 6-4PPs. This deficiency is further confirmed by the inability of TTD/XPD cells to recruit, specifically for CPDs, NER factors that arrive in a TFIIH-dependent manner later in the NER pathway. For 6-4PPs, we show that TFHH complexes carrying an NH2-terminal XPD mutated protein are also deficient in recruitment of NER proteins downstream of TFUH. Treatment with the histone deacetylase inhibitor trichostatin A allows the recovery of TFHH recruitment to CPDs in the studied TTD cells and, for COOH-terminal XPD mutations, increases the repair synthesis and survival after UV, suggesting that this defect can be partially related with accessibility of DNA damage in closed chromatin regions.

p53 Mutant Human Glioma Cells Are Sensitive to UV-C-Induced Apoptosis Due to Impaired Cyclobutane Pyrimidine Dimer Removal

The p53 protein is a key regulator of cell responses to DNA damage, and it has been shown that It sensitizes glioma cells to the alkylating agent temozolomide by up-regulating the extrinsic apoptotic pathway, whereas it increases the resistance to chloroethylating agents, such as ACNU and BCNU, probably by enhancing the efficiency of DNA repair. However, because these agents induce a wide variety of distinct DNA lesions, the direct Importance of DNA repair is hard to access. Here, it is shown that the Induction of photoproducts by UV light (UV-C) significantly Induces apoptosis In a p53-mutated glioma background. This Is caused by a reduced level of photoproduct repair, resulting In the persistence of DNA lesions in p53-mutated glioma cells. UV-C-Induced apoptosis in p53 mutant glioma cells Is preceded by strong transcription and replication inhibition due to blockage by unrepaired photolesions. Moreover, the results Indicate that UV-C-induced apoptosis of p53 mutant glioma cells Is executed through the intrinsic apoptotic pathway, with Bcl-2 degradation and sustained Bax and Bak up-regulation. Collectively, the data Indicate that unrepaired DNA lesions Induce apoptosis In p53 mutant gliomas despite the resistance of these gliomas to temozolomide, suggesting that efficiency of treatment of p53 mutant gliomas might be higher with agents that Induce the formation of DNA lesions whose global genomic repair is dependent on p53. (Mol Cancer Res 2009;7(2):237-46)

Characterization of the SOS regulon of Caulobacter crescentus

The SOS regulon is a paradigm of bacterial responses to DNA damage. A wide variety of bacterial species possess homologs of lex,4 and recA, the central players in the regulation of the SOS circuit. Nevertheless, the genes actually regulated by the SOS have been determined only experimentally in a few bacterial species. In this work, we describe 37 genes regulated in a LexA-dependent manner in the alphaproteobacterium Caulobacter crescentus. In agreement with previous results, we have found that the direct repeat GTTCN(7)GTTC is the SOS operator of C. crescentus, which was confirmed by site-directed mutagenesis studies of the imuA promoter. Several potential promoter regions containing the SOS operator were identified in the genome, and the expression of the corresponding genes was analyzed for both the wild type and the lex,4 strain, demonstrating that the vast majority of these genes are indeed SOS regulated. Interestingly, many of these genes encode proteins with unknown functions, revealing the potential of this approach for the discovery of novel genes involved in cellular responses to DNA damage in prokaryotes, and illustrating the diversity of SOS-regulated genes among different bacterial species.

Gamma-linolenic Acid Alters Ku80, E2F1, and Bax Expression and Induces Micronucleus Formation in C6 Glioma Cells In Vitro

Gamma-linolenic acid (GLA) is an inhibitor of tumor cell proliferation in both in vitro and in vivo conditions. The aim of this study was to investigate the effects of 150 mu M GLA on the expression of E2F1, cyclin D1, bax, bcl2, Ku70, and Ku80 in C6 rat glioma cells. The Ku proteins were chosen as previous studies have shown that loss or reduction in their expression causes increased DNA damage and micronucleus formation in the presence of radiation. The fact that GLA exposure is known to enhance the efficacy of radiation treatment raised the question whether the Ku proteins could be involved in this effect as seen for other molecules such as roscovitine and flavopiridol. GLA altered the mRNA expression of E2F1, cyclin D1, and bax, but no changes were found for bcl2, Ku70, and Ku80. Alterations in protein expression were observed for bax, Ku80, and E2F1. The 45% decrease in E2F1 expression was proportional to decreased cell proliferation (44%). Morphological analysis found a 25% decrease in mitotic activity in the GLA-treated cells, which was accompanied by a 49% decrease in S-phase by FACS analysis. A 39% increase in the number of micronuclei detected by Hoechst fluorescence points to GLA`s effects on cell division even at concentrations that do not produce significant increases in apoptosis. Most important was the finding that Ku80 expression, a critical protein involved in DNA repair as a heterodimer with Ku70, was decreased by 71%. It is probable that reduced Ku80 is responsible for the increase in micronucleus formation in GLA-treated cells in a similar manner to that found in Ku80 null cells exposed to radiation. The decreased expression of Ku80 and E2F1 could make cells more susceptible to radiotherapy and chemotherapy. (C) 2009 IUBMB

Establishment of the comet assay in the freshwater snail Biomphalaria glabrata (Say, 1818)

The single cell gel eletrophoresis or the comet assay was established in the freshwater snail Biomphalaria glabrata. For detecting DNA damage in circulating hemocytes, adult snails were irradiated with single doses of 2.5. 5, 10 and 20 Gy of Co-60 gamma radiation. Genotoxic effect of ionizing radiation was detected at all doses as a dose-related increase in DNA migration. Comet assay in B. glabrata demonstrated to be a simple, fast and reliable tool in the evaluation of genotoxic effects of environmental mutagens. (c) 2008 Elsevier B.V. All rights reserved.