Chemoprevention in BRCA1 Mutation Carriers—A Proof-of-Concept Study

Background: Women with germline BRCA1 mutations have a high lifetime risk of breast cancer, with the only available risk-reduction strategies being risk-reducing surgery or chemoprevention. These women predominantly develop triple-negative breast cancers; hence, it is unlikely that selective estrogen receptor modulators (serms) will reduce the risk of developing cancer, as these have not been shown to reduce the incidence of estrogen receptor–negative breast cancers. Preclinical data from our laboratory suggest that exposure to estrogen and its metabolites is capable of causing dna double-strand breaks (dsbs) and thus driving genomic instability, an early hallmark of BRCA1-related breast cancer. Therefore, an approach that lowers circulating estrogen levels and reduces estrogen metabolite exposure may prove a successful chemopreventive strategy.

Aims: To provide proof of concept of the hypothesis that the combination of luteinizing-hormone releasing-hormone agonists (lhrha) and aromatase inhibitors (ais) can suppress circulating levels of estrogen and its metabolites in BRCA1 mutation carriers, thus reducing estrogen metabolite levels in breast cells, reducing dna dsbs, and potentially reducing the incidence of breast cancer.

Methods: 12 Premenopausal BRCA1 mutation carriers will undergo baseline ultrasound-guided breast core biopsy and plasma and urine sampling. Half the women will be treated for 3 months with combination goserelin (lhrha) plus anastrazole (ai), and the remainder with tamoxifen (serm) before repeat tissue, plasma, and urine sampling. After a 1-month washout period, groups will cross over for a further 3 months treatment before final biologic sample collection. Tissue, plasma, and urine samples will be examined using a combination of immunohistochemistry, comet assays, and ultrahigh performance liquid chromatography tandem mass spectrometry to assess the impact of lhrha plus ai compared with serm on levels of dna damage, estrogens, and genotoxic estrogen metabolites. Quality of life will also be assessed during the study.

Results: This trial is currently ongoing.

Importância do polimorfismo OGG1 Ser326Cys no estudo dos efeitos para a saúde que resultam da exposição ocupacional a genotóxicos: o caso da exposição a antioneoplásicos

O ensaio cometa é um dos ensaios de avaliação de genotoxicidade mais promissores de avaliação do risco humano, sendo recomendado para a monitorização de populações com exposição crónica a agentes genotóxicos. Os laboratórios são ambientes profissionais em que os trabalhadores podem encontrar-se expostos a agentes químicos. As drogas antineoplásicas são agentes químicos que são considerados carcinogénicos e, desta forma, medidas de protecção especiais devem ser adoptadas. O objectivo desta comunicação é contribuir para o desenvolvimento de um programa de biomonitorização que inclui a avaliação de genotoxicidade relacionada com biomarcadores de susceptibilidade genética.

Avaliação do potencial cancerigénico de microcistinas (cianotoxinas)

Tese de doutoramento em Farmácia (Toxicologia), apresentada à Faculdade de Farmácia da Universidade de Lisboa, 2009.

Cyto- and Genotoxicity assessment of manufactured nano cerium dioxide in the A549 cell line

In the past decades the growing application of nanomaterials (NMs) in diverse consumer products has raised various concerns in the field of toxicology. They have been extensively used in a broad range of applications and cover most of the industrial sectors as well as the medicine and the environmental areas. The most common scenarios for human exposure to NMs are occupational, environmental and as consumers and inhalation is the most frequent route of exposure, especially in occupational settings. Cerium dioxide NMs (nano-CeO2) are widely used in a number of applications such as in cosmetics, outdoor paints, wood care products as well as fuel catalysts. For such reason, nano-CeO2 is one of the selected NMs for priority testing within the sponsorship program of the Working Party of Manufactured Nanomaterials of the OECD. In this context, the aim of this study is to assess the safety of nano-CeO2 (NM-212, Joint Research Center Repository) through the characterization of its cytotoxicity and genotoxicity in a human alveolar epithelial cell line. A dispersion of the NM in water plus 0.05% BSA was prepared and sonicated during 16 minutes, according to a standardized protocol. DLS analysis was used to characterize the quality of the NM dispersion in the culture medium. To evaluate the cytotoxicity of nano-CeO2 in the A549 cell line, the colorimetric MTT assay was performed; the capacity of cells to proliferate when exposed to CeO2 was also assessed with the Clonogenic assay. The genotoxicity of this NM was evaluated by the Comet Assay (3 and 24h of exposure) to quantify DNA breaks and the FPG-modified comet assay to assess oxidative DNA damage. The Cytokinesis-Block Micronucleus (CBMN) assay was used to further detect chromosome breaks or loss. The nano-CeO2 particles are spherical, displaying a diameter of 33 nm and 28 m2/g of surface area. The results of the MTT assay did not show any decreased in cells viability following treatment with a dose-range of nano-CeO2 during 24h. Nevertheless, the highest concentrations of this NM were able to significantly reduce the colony forming ability of A549 cells, suggesting that a prolonged exposure may be cytotoxic to these cells. Data from both genotoxicity assays revealed that nano-CeO2 was neither able to induce DNA breaks nor oxidative DNA damage. Likewise, no significant micronucleus induction was observed. Taken together, the present results indicate that this nano-CeO2 is not genotoxic in this alveolar cell line under the tested conditions, although further studies should be performed, e.g., gene mutation in somatic cells and in vivo chromosome damage (rodent micronucleus assay) to ensure its safety to human health.

Parental exposure to the herbicide diuron results in oxidative DNA damage to germinal cells of the Pacific oyster Crassostrea gigas

Chemical pollution by pesticides has been identified as a possible contributing factor to the massive mortality outbreaks observed in Crassostrea gigas for several years. A previous study demonstrated the vertical transmission of DNA damage by subjecting oyster genitors to the herbicide diuron at environmental concentrations during gametogenesis. This trans-generational effect occurs through damage to genitor-exposed gametes, as measured by the comet-assay. The presence of DNA damage in gametes could be linked to the formation of DNA damage in other germ cells. In order to explore this question, the levels and cell distribution of the oxidized base lesion 8-oxodGuo were studied in the gonads of exposed genitors. High-performance liquid chromatography coupled with UV and electrochemical detection analysis showed an increase in 8-oxodGuo levels in both male and female gonads after exposure to diuron. Immunohistochemistry analysis showed the presence of 8-oxodGuo at all stages of male germ cells, from early to mature stages. Conversely, the oxidized base was only present in early germ cell stages in female gonads. These results indicate that male and female genitors underwent oxidative stress following exposure to diuron, resulting in DNA oxidation in both early germ cells and gametes, such as spermatozoa, which could explain the transmission of diuron-induced DNA damage to offspring. Furthermore, immunostaining of early germ cells seems indicates that damages caused by exposure to diuron on germ line not only affect the current sexual cycle but also could affect future gametogenesis.

DNA Damage and Oxidative DNA Damage in Inflammatory Bowel Disease

Background and aims: Inflammation has long been regarded as a major contributor to cellular oxidative damage and to be involved in the promotion of carcinogenesis. Methods: We aimed to investigate the oxidative damage in inflammatory bowel disease [IBD] patients through a case–control and prospective study involving 344 IBD patients and 294 healthy controls. DNA damage and oxidative DNA damage were measured by comet assay techniques, and oxidative stress by plasmatic lipid peroxidation, protein carbonyls, and total antioxidant capacity. Results: Higher DNA damage [p < 0.001] was found both in Crohn’s disease [CD] (9.7 arbitrary units [AU]; interquartile range [IQR]: 6.2–14.0) and ulcerative colitis [UC] [7.1 AU; IQR: 4.4–11.7], when compared with controls [5.4 AU; IQR: 3.8–6.8], and this was also the case with oxidative DNA damage [p < 0.001] [CD: 3.6 AU; IQR: 1.8–6.8; UC: 4.6 AU; IQR: 2.4–8.1], when compared with controls: 2.3 AU; IQR: 1.2–4.2]. Stratifying patients into groups according to therapy (5-aminosalicylic acid [5-ASA], azathioprine, anti-TNF, and combined therapy [azathioprine and anti-TNF]) revealed significant between-group differences in the level of DNA damage, both in CD and UC, with the combined therapy exhibiting the highest DNA damage levels [11.6 AU; IQR: 9.5–14.3, and 12.4 AU; IQR: 10.6–15.0, respectively]. Among CD patients, disease behaviour [B1 and B2], and age at diagnosis over 40 years [A3] stand as risk factors for DNA damage. For UC patients, the risk factors found for DNA damage were disease activity, treatment, age at diagnosis under 40 years [A1 + A2] and disease locations [E2 and E3]. Conclusions: In IBD there is an increase in DNA damage, and treatment, age at diagnosis and inflammatory burden seem to be risk factors.

Effects of physical exercise training in DNA damage and repair activity in humans with different genetic polymorphisms ofhOGG1(Ser326Cys)

The main purpose of this pilot study was to investigate the possible influence of genetic polymorphisms of the hOGG1 (Ser326Cys) gene in DNA damage and repair activity by 8-oxoguanine DNA glycosylase 1 (OGG1 enzyme) in response to 16 weeks of combined physical exercise training. Thirty-two healthy Caucasian men (40-74 years old) were enrolled in this study. All the subjects were submitted to a training of 16 weeks of combined physical exercise. The subjects with Ser/Ser genotype were considered as wild-type group (WTG), and Ser/Cys and Cys/Cys genotype were analysed together as mutant group (MG). We used comet assay in conjunction with formamidopyrimidine DNA glycoslyase (FPG) to analyse both strand breaks and FPG-sensitive sites. DNA repair activity were also analysed with the comet assay technique. Our results showed no differences between DNA damage (both strand breaks and FPG-sensitive sites) and repair activity (OGG1) between genotype groups (in the pre-training condition). Regarding the possible influence of genotype in the response to 16 weeks of physical exercise training, the results revealed a decrease in DNA strand breaks in both groups, a decrease in FPG-sensitive sites and an increase in total antioxidant capacity in the WTG, but no changes were found in MG. No significant changes in DNA repair activity was observed in both genotype groups with physical exercise training. This preliminary study suggests the possibility of different responses in DNA damage to the physical exercise training, considering the hOGG1 Ser326Cys polymorphism.

Chemical characterization and in vitro cyto- and genotoxicity of ‘legal high’ products containing Kratom (Mitragyna speciosa)

Kratom is a popular ‘legal high’ mainly constituted by alkaloids extracted from the Mitragyna speciosa plant with mitragynine (MG) as the dominant active substance. The increasing use of Kratom for recreational purposes has alerted risk assessment bodies of the lack of information on the real composition and its potential health risks. The present study aimed to determine and compare the MG composition of 13 commercial products of Kratom sold online and in “smartshops”, by gas chromatography–mass spectrometry. For the first time, the cytotoxicity induced by pure MG and Kratom, extracts was evaluated in in vitro models of human intestinal (Caco-2) and neuronal (SH-SY5Y) cells after 6 and 24 h. Genotoxicity was also evaluated in intestinal Caco-2 cells following 24 h of exposure to subtoxic concentrations using the comet assay. The obtained results revealed an inconsistency between the information (‘power’) provided in labels and the MG content. Cytotoxicity tests revealed a concentration-dependent decrease in cell viability in both cellular models, with the SH-SY5Y cells being more sensitive to the Kratom extracts. The resin and the ‘powered extracts’ were the most cytotoxic samples, with IC50 values significantly lower than the leaf extracts and pure MG (P < 0.0001 vs. leaf extracts and MG). In addition, significant DNA damage was observed in Caco-2 cells exposed to these extracts but not to pure MG, which suggests that other substances present in the extracts or interactions involving Kratom components might be responsible for the observed effects.

Análise toxicológica in vitro e in vivo de uma fucana antitrombótica da alga marrom Spatoglossum schdöederi

Fucan is a term used to denominate a family of sulfated polysaccharides rich in L-fucose. They are extracted mainly from the extracellular matrix of brown algae and echinoderms. The brown alga Spatoglossum schröederi (Dictyotaceae) has three heterofucans named A, B and C. Our research group have been extracted non anticoagulant heterofucan from S. schröederi which possess antithrombotic activity in vivo. However, their toxicity in vitro and in vivo has not yet been determined. For the results in toxicity in vitro, we observed that the fucan A at 20, 500 and 1000 μg/plate showed no mutagenic activity in Kado test (Microsuspension), when the bacterial strains TA97a, TA98, TA100 and TA102, with and without S9 were used. The comet assay showed that fucan A (from 20 to 1000 μg/mL) did not cause any genotoxic effect on CHO cells. There was no damage to the DNA of these cells, as evidenced by the tail length and tail moment, which were similar to that found for the negative control. The fucan A from S. schröederi was administered at 20 μg/g of rat (dose which it showed high antithrombotic activity) during two months. After that, the animals were killed and examined. The data showed that fucan A did not cause any change in biochemistry and hematological parameters, as well as, in the morphology and size of the rat s organs analyzed. In conclusion, this study indicates that fucan is a compound with potential pharmacological that has no toxicity

Effect of temperature on genomic and some biological indices during embryonic and larval development of Caspian trout Salmo trutta caspius Kessler, 1877

Since 1966 especially recent decade, Caspian trout (Salmo trutta caspius Kessler, 1877) considered as a strategic endemic species for Caspian Sea fisheries resources also coldwater aquaculture in Iran. Nowadays habitat condition effects on this subspecies during life stages, artificial breeding and incubation period noticed by research and execution sessions of fisheries in Iran. Incubation duration of Caspian trout from artificial fertilization followed by green egg and eyed egg, hatching and yolk sac absorption identified as most sensitive stages for fish and any pollution, stress and deviation by natural life conditions of embryo up to larvae could provide possible mortalities and observable or hidden alterations. Among all vital factors for Caspian trout welfare even in conservation plans and stocks rehabilitation programs or recent attempts for domestication of this fish for introduction to cold water aquaculture industry, water temperature as the most important physical factor which might conserve or induce stress to rearing environment condition is not considered yet. In hatcheries activities, the temperature for incubation and rearing Caspian trout eggs is determining by available water temperature and wide range of temperatures in governmental or private farms is using depend on the water resources availability. Also global climate change consideration and increase temperature trend accompany with group of physical and chemical factors provided by fish farm discharges and other source points entered to the migration pathway of Caspian trout in spawning season were not investigated before. Natural spawning migration pathway is upstream of Caspian tout south and south west rivers especially in Cheshmehkileh upstream in Tonekabon, Iran directed this research focus on the mentioned location. For simulation of natural spawning bed for Caspian trout, water supplied from the upstream of Daryasar branch as headwater of Cheshmehkileh River which provided REDD water condition for in vitro incubation. Green eggs treatments of wild and F1 cultured brooders both 3+ were incubated. Incubation implemented in dark, constant temperature (4, 8, 12 degree centigrade) and DO–pH–temperature digital monitoring in 3 recycling incubators ended to yolk sac absorption and entering larval stage. Hatching success, possible genome alterations by HSP70 gene expression and comet assay implemented as diagnostic tools in 3 life stages of eyed egg– Alevin and Larvae. Numbers and diameters of larvae white fiber muscles measured by histology experiment and Hematoxylin–eosine staining. Results stated significant effect of incubation temperature on hatching success, genome and white fiber muscles of wild and F1 samples. Hatching success measured as 31% and 38% for cultured and wild cold treatments, 79% and 91% for normal and 64% and 73% for warm cultured and wild treatments respectively. Considerable mortality occurred for cold treatment and 8 degree centigrade stated the best thermal condition in normal incubator according to hatching success in wild Caspian trout samples.

Evaluation of the cytotoxic and genotoxic effects of benchmark multi-walled carbon nanotubes in relation to their physicochemical properties

To contribute with scientific evidence to the grouping strategy for the safety assessment of multi-walled carbon nanotubes (MWCNTs), this work describes the investigation of the cytotoxic and genotoxic effects of four benchmark MWCNTs in relation to their physicochemical characteristics, using two types of human respiratory cells. The cytotoxic effects were analysed using the clonogenic assay and replication index determination. A 48h-exposure of cells revealed that NM-401 was the only cytotoxic MWCNT in both cell lines, but after 8-days exposure, the clonogenic assay in A549 cells showed cytotoxic effects for all the tested MWCNTs. Correlation analysis suggested an association between the MWCNTs size in cell culture medium and cytotoxicity. No induction of DNA damage was observed after any MWCNTs in any cell line by the comet assay, while the micronucleus assay revealed that both NM-401 and NM-402 were genotoxic in A549 cells. NM-401 and NM-402 are the two longest MWCNTs analyzed in this work, suggesting that length may be determinant for genotoxicity. No induction of micronuclei was observed in Beas-2B cell line and the different effect in both cell lines is explained in view of the size-distribution of MWCNTs in the cell culture medium, rather than cell's specificities.

Células de anêmonas Bunodosoma cangicum expostas ao cobre: citotoxidade, defesa e dano de DNA

Anêmonas-do-mar são pólipos solitários, bentônicos, de pouca mobilidade, que habitam regiões entre-marés. Devido a estas características, são organismos que podem ser atingidos diretamente pela poluição aquática, no entanto, são pouco utilizados como modelo ecotoxicológico. O cobre é um metal essencial, que em altas concentrações pode ser tóxico, sendo bastante comum em ecossistemas marinhos. Um dos mecanismos de toxicidade do cobre envolve a produção de espécies reativas de oxigênio (ERO), podendo levar as células ao estresse oxidativo, que tem como característica danos celulares, inclusive no DNA. Muitos organismos possuem um mecanismo que bombeia os xenobióticos para fora da célula – multixenobiotic resistance (MXR) – que visa prevenir as células dos danos tóxicos causados pelo contaminante. Com isso, o presente trabalho estudou a capacidade de defesa e dano ao DNA à toxicidade causada pelo cobre em células de anêmonas Bunodosoma cangicum. Para isto, células de anêmonas, mantidas em cultura primária através de explante do disco podal, foram expostas ao cobre a duas concentrações (7,8 µg.L-1 Cu e 15,6 µg.L-1 Cu), além do grupo controle, por 6 e 24 h. Antes e após as exposições as células tiveram sua viabilidade avaliada através do método de exclusão por azul de tripan (0,08%) para analisar a citotoxicidade. Parâmetros como a indução do mecanismo MXR através do método de acúmulo de rodamina-B, espécies reativas de oxigênio e ensaio cometa, também foram avaliados. Os resultados obtidos mostram que o cobre é citotóxico, sendo constatada uma queda na viabilidade e no número de células, principalmente após 24 h de exposição, sendo que na concentração de cobre de 15,6 µg.L-1 , foi possível observar uma diminuição de 40% na viabilidade e uma redução em 36% no número de células (p < 0,05, n = 6). Em relação ao fenótipo MXR, foi observada uma ativação do mecanismo apenas naquelas células expostas ao cobre 7,8 µg.L-1 (53%) no tempo de 24 h (p < 0,05, n = 5). Na análise da geração de ERO foi observado um aumento de 11,5% naquelas células expostas por 6 h na concentração mais alta de cobre 15,6 µg.L-1 . Nas células que foram expostas por 24 h, o aumento de espécies reativas pode ser percebido já na concentração de 7,8 µg.L-1 , elevando-se para cerca de 20% quando exposto a 15,6 µg.L-1 (p < 0,05, n = 4-5). Quanto ao dano de DNA, foram vistas quebras na molécula desde 7,8 µg.L-1 Cu em 6 h, com danos ainda mais salientes naquelas células expostas por 24 h, na concentração de 7,8 µg.L -1 Cu (p < 0,05, n = 3-4), e para 15,6 µg.L-1 Cu a viabilidade celular (número de células) não permitiu a análise. Com base nestes dados, pode-se dizer que o cobre, mesmo em baixas concentrações causa estresse em células de B. cangicum, sendo citotóxico. Este metal causa estresse oxidativo com dano à molécula de DNA mesmo com a ativação do mecanismo de defesa.

Evaluation of the genotoxicity of cellulose nanofibers

Background: Agricultural products and by products provide the primary materials for a variety of technological applications in diverse industrial sectors. Agro-industrial wastes, such as cotton and curaua fibers, are used to prepare nanofibers for use in thermoplastic films, where they are combined with polymeric matrices, and in biomedical applications such as tissue engineering, amongst other applications. The development of products containing nanofibers offers a promising alternative for the use of agricultural products, adding value to the chains of production. However, the emergence of new nanotechnological products demands that their risks to human health and the environment be evaluated. This has resulted in the creation of the new area of nanotoxicology, which addresses the toxicological aspects of these materials.Purpose and methods: Contributing to these developments, the present work involved a genotoxicological study of different nanofibers, employing chromosomal aberration and comet assays, as well as cytogenetic and molecular analyses, to obtain preliminary information concerning nanofiber safety. The methodology consisted of exposure of Allium cepa roots, and animal cell cultures (lymphocytes and fibroblasts), to different types of nanofibers. Negative controls, without nanofibers present in the medium, were used for comparison.Results: The nanofibers induced different responses according to the cell type used. In plant cells, the most genotoxic nanofibers were those derived from green, white, and brown cotton, and curaua, while genotoxicity in animal cells was observed using nanofibers from brown cotton and curaua. An important finding was that ruby cotton nanofibers did not cause any significant DNA breaks in the cell types employed.Conclusion: This work demonstrates the feasibility of determining the genotoxic potential of nanofibers derived from plant cellulose to obtain information vital both for the future usage of these materials in agribusiness and for an understanding of their environmental impacts.

Looking at the night sky

There are two series of images in this exhibition: Series # 1: Images 4 – 9 Astronomical observatories in different countries and telescopes which have made some important discoveries in science of astronomy. These images were taken with simple primitive camera including a homemade toy camera and a Holga pinhole camera. The toy camera use a simple plastic lens and film. It produces a very softly focused and blurred image. The Holga Pinhole camera has a panoramic format and also uses film. (The pinhole camera has no lens and only a very small pinhole to lets the light into the camera to produce a simple image) For me the limited resolution of these primitive cameras invoke a sense of wonder, mystery and imagination which the ancient observers must have experienced when looking at the night sky. Series # 2: Images 1 – 3 and 9 - 14 presents individual celestial objects including, the planet Saturn, the Moon, the Sun, a comet and a Star Cluster. All these images have been re-photographed through a number of large primitive lenses. Some are hand made glass lenses and others are hollow and filled with water. These primitive lenses distort and stretch the images and represent the way in which the lens and (the telescope) have changed our vision of the cosmos. They also represent the subjectivity of the lens, something that all photographers know about - just because we see something through a lens, does not mean that all has been revealed and that what finally perceive is both a combination of what we see and what we feel inside and our imagination. The toy and pinhole camera images were made during a 5 year period starting in 2010 and up to 2015. The second series have all been made during 2015.

Associations between DNA damage, DNA base excision repair gene variability and Alzheimer's disease risk

BACKGROUND: Increased oxidative damage to DNA is one of the pathways involved in Alzheimer's disease (AD). Insufficient base excision repair (BER) is in part responsible for increased oxidative DNA damage. The aim of the present study was to assess the effect of polymorphic variants of BER-involved genes and the peripheral markers of DNA damage and repair in patients with AD. MATERIAL AND METHODS: Comet assays and TaqMan probes were used to assess DNA damage, BER efxFB01;ciency and polymorphic variants of 12 BER genes in blood samples from 105 AD patients and 130 controls. The DNA repair efficacy (DRE) was calculated according to a specific equation. RESULTS: The levels of endogenous and oxidative DNA damages were higher in AD patients than controls. The polymorphic variants of XRCC1 c.580C>T XRCC1 c.1196A>G and OGG1 c.977C>G are associated with increased DNA damage in AD. CONCLUSION: Our results show that oxidative stress and disturbances in DRE are particularly responsible for the elevated DNA lesions in AD. The results suggest that oxidative stress and disruption in DNA repair may contribute to increased DNA damage in AD patients and risk of this disease. In addition, disturbances in DRE may be associated with polymorphisms of OGG1 and XRCC1.