DNA Damage In Epithelial Cells Research Articles

Disordered Expression of Tight Junction Proteins Is Involved in the Mo-induced Intestinal Microenvironment Dysbiosis in Sheep.

To evaluate the molybdenum (Mo)-induced changes of intestinal morphology and the relationship of intestinal tight junction (TJ) proteins expression and intestinal barrier function, a total of 20 healthy sheep were randomly divided into five groups of four: 0, 5, 10, 20, and 50mg/kg BW/day Na2MoO4·2H2O were administrated in five groups named control group, Mo 5 group, Mo 10 group, Mo 20 group, and Mo 50 group, respectively. After 28days of Mo treatment, the duodenum, the jejunum, and the ileum tissue were collected. The histopathology and the developmental parameters were evaluated by hematoxylin-eosin staining. The intestinal epithelial cell DNA damage was detected by TdT-mediated dUTP nick end labeling assay. The intestinal glycoprotein and the goblet cells were analyzed by Alcian Blue-Periodic Acid-Schiff (AB-PAS) staining and PAS staining, respectively. TJ proteins were determined by immunofluorescence technology. Results showed that excessive Mo significantly decreased the small intestinal villus height (VH), crypt depth (CD), VH/CD, and mucosal thickness (P < 0.05 or P < 0.01) while induced the damage of DNA in small intestinal epithelial cells. Moreover, excessive Mo injured intestinal barrier function by decreasing the percent of glycoprotein distribution area (P < 0.05) and the relative density of intestinal goblet cells (P < 0.05). Mo treatment induced decreased (P < 0.01) expression of Zonula Occludens-1, Occludin, and Claudin-1. In conclusion, excessive Mo interfered with the expression of TJ proteins, inhibited intestinal epithelial development, and further aggravated the intestinal barrier function damage, leading to disturbing the small intestinal microenvironment balance.

Abstract P2-06-03: Obesity is associated with DNA damage in the breast epithelium of BRCA1 and BRCA2 mutation carriers: A role for estrogens &amp; strategies for prevention

Abstract Background: Elevated bodyweight is a risk factor for breast cancer development in women who carry a mutation in the DNA repair enzymes BRCA1 and BRCA2. However, the mechanistic basis for this association is unknown. Breast adipose tissue undergoes significant changes in the setting of weight gain and obesity, including elevation in aromatase expression which leads to the increased biosynthesis of estrogens. Given that estrogens and estrogen metabolites have known pro-proliferative and genotoxic effects, we hypothesized that in BRCA1/2 mutation carriers, obesity may be positively associated with breast epithelial cell DNA damage, thereby increasing the risk of tumorigenesis. Furthermore, we examined the impact of inhibiting estrogen signaling or production on breast epithelium DNA damage in BRCA1/2 mutation carriers. Methods: Tissue microarrays were generated from non-cancerous breast tissue derived from 72 women carrying a mutation in BRCA1 or BRCA2 with known body mass index (BMI, kg/m2). Breast epithelium DNA damage was quantified by immunofluorescence (IF) staining of the DNA damage marker γH2AX. RNA-Seq was performed on breast organoids to assess differences in gene expression in relation to BMI. Associations between DNA damage and biomarkers of estrogen biosynthesis and bioavailability, including aromatase expression in the breast and circulating steroid hormone binding globulin (SHBG), were also evaluated. To explore the effect of blocking estrogen signaling or production on DNA damage, non-tumorous breast tissue explants from BRCA1/2 mutation carriers were cultured with fulvestrant, an estrogen receptor degrader, or metformin, an anti-diabetic drug that also reduces aromatase expression in the breast. Breast epithelial cell DNA damage was measured in control vs treated explants by γH2AX IF staining after 24 hours of treatment. Results: BMI was positively correlated with DNA damage in the breast epithelium of BRCA1/2 mutation carriers. Upstream analysis of gene expression in organoids derived from women with a BMI ≥ 30 compared to &amp;lt;25, revealed activation of estrogen signaling. Further supporting a contribution of locally-derived and circulating estrogens to obesity-related DNA damage, breast aromatase expression was found to be positively correlated with DNA damage while circulating SHBG levels showed a negative correlation. Targeting estrogen signaling with fulvestrant significantly reduced breast epithelium DNA damage in breast explants from women carrying a mutation in either BRCA1 or BRCA2. Interestingly, metformin, also caused a significant reduction in DNA damage in breast explants. Conclusion: These data provide mechanistic evidence for the link between obesity and breast cancer in BRCA1 and BRCA2 mutation carriers through identification of a positive association between BMI and breast epithelial cell DNA damage. Importantly, these studies demonstrate that fulvestrant and metformin, drugs already approved for clinical use, decrease breast epithelial cell DNA damage. Further studies are warranted to determine whether targeting estrogens or use of metformin may be effective risk reduction strategies in BRCA1/2 mutation carriers with excess bodyweight who are at high risk for breast cancer development and currently have limited options for prevention beyond surgical intervention. Support: NIH R01CA215797, NIH F31CA236306, Anne Moore Breast Cancer Research Fund Citation Format: Priya Bhardwaj, Neil M. Iyengar, Sofya Oshchepkova, Phoebe Piloco, Rohan Bareja, Olivier Elemento, Dilip D. Giri, Michael Pollak, Monica Morrow, Jason A. Spector, Kristy A. Brown. Obesity is associated with DNA damage in the breast epithelium of BRCA1 and BRCA2 mutation carriers: A role for estrogens &amp; strategies for prevention [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-06-03.

Bcl-2 Interacts with Beclin 1 and Regulates Autophagy in 7, 12-Dimethylbenz[a]anthracene-Induced Hamster Buccal-Pouch Squamous-Cell Tumorigenesis.

Autophagy is a programmed cell death procedure, which has essential functions in tumorigenesis. However, its temporal expression and function under different status are yet to be determined. This study aims to investigate the temporal expression of autophagy and its possible function in 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal-pouch cancer model (HBPCM). A total of 50 hamster buccal-pouch tumorigenesis models were established by painting DMBA for 4, 8, 10 and 13 weeks. The expression and subcellular localization of LC3, Beclin 1 and Bcl-2 in buccal lesions were evaluated by immunohistochemical staining and Western blotting. DNA damage was observed by immunohistochemical staining of 8-oHdG. The relationship between Beclin 1 and Bcl-2 was analyzed by immunofluorescence colocalization. The expression levels of LC3 and Beclin 1 associated with autophagy in the experimental buccal pouch of HBPCM were significantly upregulated after 4 weeks (P<0.05), but gradually downregulated after 13 weeks of HBPCM induction. By contrast, the expression level of Bcl-2 was significantly upregulated after 13 weeks. The co-localized regions of Bcl-2 and Beclin 1 peaked after 4 weeks and then decreased gradually. The DNA damage in epithelial cells increased slightly after 4 weeks, and then rapidly decreased over the next 2 months. Autophagy is motivated by a tumor suppressor that diminishes carcinogen-induced DNA damage. However, autophagy is gradually suppressed, which may be attributed to the interaction between Bcl-2 and Beclin 1. This result indicates that the promotion of autophagy may suppress malignant transformation and provide new insights on future potential treatments of HBPCM.

Lactobacillus rhamnosus GG Colonization in Early Life Ameliorates Inflammaging of Offspring by Activating SIRT1/AMPK/PGC-1α Pathway.

Inflammaging refers to chronic, low-grade inflammation during aging, which contributes to the pathogenesis of age-related diseases. Studies have shown that probiotic intervention in the aging stage could delay aging-related disorders. However, whether the application of probiotics in early life could have antiaging effects on offspring was unknown. Here, we investigated the effects of Lactobacillus rhamnosus GG (LGG) colonization in early life on inflammaging of offspring. Pregnant mice with the same conception time were given LGG live bacteria (LC group) or LGG fixed bacteria (NC group) from the 18th day after pregnancy until natural birth. The progeny mice were treated with 107 cfu of live or fixed LGG for 0-5 days after birth, respectively. LGG colonization could be detected in the feces of 3-week offspring. The 16S rRNA sequencing analysis of 3-week-old offspring showed that colonization of LGG in early life could alter the composition and diversity of gut microbiota. Interestingly, the beneficial effects of LGG colonization in early life on the microbiota lasted to 8 months old. The abundance of longevity-related bacteria (Lactobacillus, Bifidobacterium, and Akkermansia muciniphila) increased significantly in the LGG colonization group. In addition, LGG colonization increased the abundance of short-chain fatty acid- (SCFA-) producing bacteria and the production of cecal SCFAs. LGG colonization in early life protected the intestinal barrier, enhanced antioxidant defense, attenuated epithelial cell DNA damage, and inhibited intestinal low-grade inflammation in 8-month-old progeny mice. Mechanically, LGG could upregulate Sirtuin1 (SIRT1)/Adenosine 5′-monophosphate-activated protein kinase (AMPK)/Peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) pathway and repress activation of nuclear factor-kappa B (NF-κB), while the protective effect of LGG was blunted after SIRT1 gene silencing. Together, LGG colonization in early life could ameliorate inflammaging of offspring, which would provide a new strategy for the prevention of age-related diseases.

Abstract IA06: Colon cancer: Microbes and communities in microbiome translation

Abstract Intensive efforts are under way to understand how the colon microbiome contributes to the pathogenesis of colorectal cancer (CRC) and to determine if and how the microbiome can be utilized in the prevention and/or therapy of CRC. Both single microbes and microbial communities are proposed as contributors to human CRC. Among single microbes, attention to date has focused on enterotoxigenic Bacteroides fragilis (ETBF), (pks+) Escherichia coli, and Fusobacterium spp., although other candidates are emerging. Among communities, one aspect has been the potential contribution of mucus-invasive, dense mucosal microbial communities termed biofilms to CRC disease pathogenesis. This talk will review select concepts within ETBF’s and biofilm contribution to colon carcinogenesis. While ETBF exposure is common, the risk of colon carcinogenesis in an individual is unknown but potentially influenced by colonization state, B. fragilis toxin isotype secreted by ETBF, colon site, and the underlying genomic state of the colonic epithelial cells. With respect to colon biofilms, about 50% of human sporadic colon cancer, particularly in the right colon, display polymicrobial biofilms with marked tumor infiltration with bacteria whereas ~15% of normal colonoscopy biopsies also reveal polymicrobial biofilms. These polymicrobial biofilms are typically composed of Bacteroidetes and Lachnospiraceae with a subset of tumors, but not biopsies, also displaying Fusobacterium. Further, samples from biofilm-positive human colon cancers induce assembly of biofilms in the distal germ-free mouse colon within a week following inoculation and are procarcinogenic (JCI 129:1699, 2019). One of the common hereditary human colon cancers (familial adenomatous polyposis; APC+/−) also displays biofilms but dominated by two bacteria, (pks+) E. coli and ETBF. A mouse model of (pks+) E. coli and ETBF cocolonization exhibited accelerated tumor formation and mortality when cocolonized with the cancer-inducing bacteria, potentially by fostering increased microbial adherence, IL-17, changes in gene expression, and enhanced colonic epithelial cell DNA damage (Science 2018;359:592-97). Current directions of translational research will be presented. Citation Format: Cynthia L. Sears. Colon cancer: Microbes and communities in microbiome translation [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr IA06.

Ocular Cell Lines and Genotoxicity Assessment.

Genotoxicity screening tests aim to evaluate if and to what extent a compound in contact with the human body (e.g., a drug molecule, a compound from the environment) interacts with DNA. The comet assay is a sensitive method used to predict the risk of DNA damage in individual cells, as it quantifies the tape breaks, being the alkaline version (pH > 13) the most commonly used in the laboratory. Epithelial cells serve as biomatrices in genotoxicity assessments. As ca. 80% of solid cancers are of epithelial origin, the quantification of the DNA damage upon exposure of epithelial cells to a drug or drug formulation becomes relevant. Comet assays run in epithelial cells also have clinical applications in human biomonitoring, which assesses whether and to what extent is the human body exposed to environmental genotoxic compounds and how such exposure changes over time. Ocular mucosa is particularly exposed to environmental assaults. This review summarizes the published data on the genotoxicity assessment in estimating DNA damage in epithelial cells with a special focus on ocular cell lines. General comet assay procedures for ex vivo and in vivo epithelium samples are also described.

Association between Mobile Phone Using and DNA Damage of Epithelial Cells of the Oral Mucosa

Exposure to high levels of radiofrequency radiation can potentially cause tissue damage characteristic of many diseases including cancer. This study explored relationship between use of mobile phone and DNA damage in oral mucosal cells. One hundred individuals completed a questionnaire were grouped according to frequency and duration of mobile usage. Comet and TUNEL assays were used to determine DNA damage and rate of apoptosis, respectively. Number of damaged cells in right cheek was significantly higher than in left cheek in right ear phone dominant users, but not in left ear dominant people. Years of phone use was not positively correlated to degree of DNA damage, however, damage increased with increased frequency of phone use. Apoptotic cells were highest in medium (30-60 min/day) phone users. Although no significant correlation was observed between degree of apoptosis and cumulative year of mobile phone use, phone use must be minimized to reduce health effects.

ZEB1 promotes inflammation and progression towards inflammation-driven carcinoma through repression of the DNA repair glycosylase MPG in epithelial cells

ObjectiveChronic inflammation is a risk factor in colorectal cancer (CRC) and reactive oxygen species (ROS) released by the inflamed stroma elicit DNA damage in epithelial cells. We sought to identify...

FTIR SPECTROSCOPIC TRENDS AND DNA DAMAGE IN RABBIT LENS DUE TO LONG RUN OF TAMOXIFEN TREATMENT

Objective: The aim of the present work is to evaluate the molecular structure changes of the lens of rabbits and DNA damage of epithelial cells due to tamoxifen administration.&#x0D; Methods: Twenty four healthy New Zealand white rabbits were divided into 2 main groups. The first group is served as control (n=12) kept untreated, second one is Tamoxifen administrative group (n=12) received orally daily dose of 15 mg/kg. Rabbits were decapitated after 2, 4, 6 and 8 mo, respectively. Using fourior transform infrared (FTIR) to study the molecular structure changes due to tamoxifen and comet assay analysis for discovering DNA damage.&#x0D; Results: FTIR data indicated that tamoxifen affects structural components in NHOH and fingerprint region. Increases of β-turns of the protein secondary structure while, reducing the content of both α-helix after 8 mo and Turns appeared for all periods of administrative tamoxifen were observed. On the other hand tamoxifen induced a statistically significant increase in comet assay parameters as tail moment compared to control animals that indicated DNA damage due to single or double strand break.&#x0D; Conclusion: Tamoxifen uses for more than 6 mo may lead to changes in the molecular structure of the lens and damage of DNA cells. An ophthalmic baseline examination prior to anti-cancer treatment may help detect any pre-existing ocular condition and lead to reduction of ocular side effects when predisposed patients are screened and examined regularly during and after chemotherapeutic therapy.

SAT-339 Cross-Talk with Breast Adipose Tissue Contributes to Obesity-induced DNA Damage in BRCA Mutant Breast Epithelial Cells

Background: Obese women who carry a mutation in the DNA repair enzymes BRCA1 and/or BRCA2 are at a greater risk of developing breast cancer compared with lean BRCA1/2 mutation carriers. Causative factors that drive the increased penetrance of breast cancer in obese women are poorly characterized. Previously, we showed that BMI was positively correlated with DNA damage in breast epithelium of BRCA mutation carriers. We hypothesized that obesity-associated dysfunctional adipose tissue may cause DNA damage in neighboring breast epithelial cells. If true, this would provide evidence for the initial insults associated with increased risk of tumor formation in obese women. Methods: To test the effect of locally secreted factors on DNA damage induction, conditioned media was collected over 24 hours from primary breast adipose explants obtained from lean and obese women. Conditioned media was used to treat normal breast epithelial cells (MCF10A) harboring a heterozygous BRCA1 mutation or wildtype for BRCA. Immunofluorescence (IF) staining of the DNA damage marker ƴH2AX was carried out after treatment. Cell line findings were validated in primary breast epithelial organoids isolated by collagenase digestion of breast tissue obtained from the clinic. Additionally, RNA-seq was conducted on primary breast epithelial organoids isolated from lean and obese women to identify potential drivers of gene expression differences associated with obesity and DNA damage. Results: Conditioned media from obese breast adipose tissue stimulated DNA damage in BRCA mutant breast epithelial cells while lean conditioned media did not have this effect. Similar findings were made in primary breast epithelial organoids, suggesting a role for locally secreted factors in mediating DNA damage. Ingenuity Pathway Analysis of RNA-seq data from lean and obese primary breast organoids identified several upstream regulators of gene expression differences, including signaling by estradiol and pro-inflammatory cytokines, both of which are elevated in obese breast tissue and are known inducers of DNA damage. Conclusions: These data show for the first time that local factors produced by obese breast adipose tissue induce breast epithelial cell DNA damage. Our results suggest that therapies targeting weight or dysfunctional breast adipose tissue may reduce the increased DNA damage observed in obese BRCA mutation carriers and thereby decrease tumor formation in this high-risk population of women.

Distinct differences in the mechanisms of mucosal damage and γ-H2AX formation in the rat urinary bladder treated with o-toluidine and o-anisidine.

Although aromatic amines are widely used as raw materials for dyes, some of them have been concerned about carcinogenicity in the urinary bladder. We examined early changes in histopathology and the formation of γ-H2AX, a biomarker of DNA damage, in the urinary bladder of rats to investigate the mechanisms of mucosal damage induced by monocyclic aromatic amines. 6-week-old male F344 rats were administered 0.4% or 0.8% o-toluidine, 0.3% or 1.0% o-anisidine, 0.4% 2,4-xylidine, 0.2% p-toluidine, or 0.6% aniline in the diet for 4 weeks. Animals were sequentially killed from day 2 to after 2 weeks of recovery, and histopathological and immunohistochemical analyses were performed. In the 0.8% o-toluidine group, there was sequential progression of bladder lesions, characterized by edematous changes and intramucosal hemorrhage at day 2 and formation of granulation tissue with mononuclear cell infiltration at week 1, followed by diffuse hyperplasia at weeks 2 and 4. In the 1.0% o-anisidine group, simple hyperplasia only with slight inflammation was detected from week 1. Whereas γ-H2AX-positive bladder epithelial cells in the 1.0% o-anisidine group were significantly increased in a time-dependent manner, transient increases in γ-H2AX-positive cells were detected at day 2 and week 1 in the 0.8% o-toluidine group. No apparent bladder lesions or increases in γ-H2AX formation were observed in any other groups. These results revealed different mechanisms of bladder mucosal damage associated with o-toluidine and o-anisidine. Moreover, immunohistochemical analysis for γ-H2AX suggested that both compounds may induce DNA damage in epithelial cells, mainly basal cells, of the bladder mucosa.

5-Aminosalicylic acid downregulates the growth and virulence of Escherichia coli associated with IBD and colorectal cancer, and upregulates host anti-inflammatory activity.

5-aminosalicylate (5-ASA) is widely prescribed for the treatment of inflammatory bowel disease (IBD) and prevention of inflammation-associated colorectal cancer (CRC). Its clinical effect is widely attributed to modulation of host inflammatory responses. However, the recent association of intestinal dysbiosis and selective enrichment in Escherichia coli in patients with IBD and CRC raises the possibility that 5-ASA might also affect the enteric microflora. The aim of this study was to investigate the effect of 5-ASA on the growth and virulence of E. coli associated with IBD and CRC, and its impact on host cell inflammatory responses. Our results show that 5-ASA inhibited E. coli growth in a dose-dependent manner and downregulated the expression of bacterial virulence genes associated with IBD (fliC, fimH, ompC, yfgL, nlpL, lpfA, htrA, dsbA, fyuA, and chuA) and CRC (pks). 5-ASA inhibited E. coli motility (30-70%), epithelial adherence and invasion, and IL-8 secretion (p < 0.05). 5-ASA reduced E. coli survival in J774A.1 macrophages by 20 to 50% (p < 0.01) and TNF-α secretion by infected macrophages up to 30% (p < 0.05). In addition, 5-ASA reduced DNA damage in epithelial cells (Caco-2) induced by pks-positive E. coli. Our results reveal a multifaceted and previously unrecognized effect of 5-ASA on the growth and virulence of IBD- and CRC-associated E. coli, in addition to its inhibitory effect on host cell inflammatory responses. These results suggest that 5-ASA may abrogate the proinflammatory and oncogenic effects of E. coli in patients with IBD and CRC.

Abstract 5317: JAK2 regulates oxidative damage-induced epigenetic alterations

Abstract Elevated levels of reactive oxygen species at sites of chronic inflammation cause oxidative DNA damage in epithelial cells, contributing to carcinogenesis. Aberrant silencing of key genes by promoter CpG island DNA hypermethylation occurs in many cancers that form at sites of chronic inflammation. Yet, it is not completely understood how these aberrant DNA methylation alterations are initiated. We hypothesize that the recruitment of epigenetic silencing proteins to sites of oxidative DNA damage is important for initiating DNA methylation. Here we demonstrate that after hydrogen peroxide (H2O2) treatment, mismatch repair proteins MSH2 and MSH6 become localized to sites of oxidative DNA damage, resulting in the recruitment of DNA methyltransferase 1 (DNMT1) and Polycomb repressive complex 2 (PRC2) members to chromatin. This recruitment results in the formation of repressive chromatin in the promoters of tumor-suppressor genes and the concomitant transient reduction in their expression. We sought to further understand the mechanism that drives H2O2-induced MSH2 and MSH6 chromatin binding. Janus kinase 2 (JAK2) is a kinase that is canonically known to be activated by cytokines and growth hormones and phosphorylate the transcription factor signal transducer and activator of transcription (STATs). Here, we demonstrate that in response to treatment with H2O2, but not interleukin 6 (IL-6), JAK2 translocates to the nucleus and interacts with MSH2 and MSH6. Nuclear JAK2 is important for H2O2-induced chromatin interaction of MSH2, MSH6, DNMT1 and PRC2 members. Interestingly, H2O2-induces a global and site-specific increase in trimethylation of histone H3 at lysine 27 (H3K27me3) that is also dependent on JAK2. Furthermore, by analyzing TCGA datasets, we demonstrate high JAK2 mRNA expression correlates with CpG island methylator phenotype (CIMP) status in gastric and colorectal cancer. All together, these findings suggest JAK2 plays a key role in initiating oxidative damage-induced epigenetic alterations during inflammation-driven tumorigenesis. Ultimately, understanding the mechanism of initiation of cancer-specific promoter DNA hypermethylation may lead to treatment strategies to reduce aberrant epigenetic changes induced by chronic inflammation and therefore reduce tumorigenesis. Citation Format: Ning Ding, Sam Miller, Heather O'Hagan. JAK2 regulates oxidative damage-induced epigenetic alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5317.

Differences between co-cultures and monocultures in testing the toxicity of particulate matter derived from log wood and pellet combustion.

BackgroundIn vitro studies with monocultures of human alveolar cells shed deeper knowledge on the cellular mechanisms by which particulate matter (PM) causes toxicity, but cannot account for mitigating or aggravating effects of cell-cell interactions on PM toxicity.MethodsWe assessed inflammation, oxidative stress as well as cytotoxic and genotoxic effects induced by PM from the combustion of different types of wood logs and softwood pellets in three cell culture setups: two monocultures of either human macrophage-like cells or human alveolar epithelial cells, and a co-culture of these two cell lines. The adverse effects of the PM samples were compared between these setups.ResultsWe detected clear differences in the endpoints between the mono- and co-cultures. Inflammatory responses were more diverse in the macrophage monoculture and the co-culture compared to the epithelial cells where only an increase of IL-8 was detected. The production of reactive oxygen species was the highest in epithelial cells and macrophages seemed to have protective effects against oxidative stress from the PM samples. With no metabolically active cells at the highest doses, the cytotoxic effects of the PM samples from the wood log combustion were far more pronounced in the macrophages and the co-culture than in the epithelial cells. All samples caused DNA damage in macrophages, whereas only beech and spruce log combustion samples caused DNA damage in epithelial cells. The organic content of the samples was mainly associated with cytotoxicity and DNA damage, while the metal content of the samples correlated with the induction of inflammatory responses.ConclusionsAll of the tested PM samples induce adverse effects and the chemical composition of the samples determines which pathway of toxicity is induced. In vitro testing of the toxicity of combustion-derived PM in monocultures of one cell line, however, is inadequate to account for all the possible pathways of toxicity.

The interactive effect of dietary n-6:n-3 fatty acid ratio and vitamin E level on tissue lipid peroxidation, DNA damage in intestinal epithelial cells, and gut morphology in chickens of different ages.

Feeding chickens diets high in n-3 fatty acids (FA) increases their incorporation into tissue lipids, but leads to oxidative stress in cells. This study investigated the effect of the dietary polyunsaturated FA ratio (PUFA n-6: n-3) and vitamin E (vE) level on DNA damage and morphological changes in the gut epithelium of chickens. One-day-old female broiler chicks (n = 176) were divided into 4 groups fed for 43 d diets with a high (HR) or low (LR) PUFA n-6: n-3 ratio and supplemented with 50 or 300 mg vE kg−1. Performance was calculated for periods of d 1 to 9, d 9 to 16, d 9 to 35, and d 9 to 42, while organs were sampled at d 9, d 17, d 36, and d 43. At d 17 and d 43, DNA damage of epithelial cells in the duodenum and jejunum was measured and duodenal and jejunal morphology was analyzed. HR diets improved FCR for the periods of d 1 to 9, d 9 to 16 and d 9 to 42, whereas the increased vE level improved FCR for the period of d 9 to 16. In the jejunum DNA damage was greater in chickens fed LR than HR diets at d 17 (P < 0.001) and the increased vE level promoted DNA damage in both intestinal segments (P < 0.02) in younger birds. The morphology of the duodenum was marginally affected by the diets, whereas LR diets in the jejunum reduced villus surface area at d 17 (P = 0.022), and mucosa thickness (P = 0.029) and villus height (P = 0.035) at d 43. The results indicated that feeding birds LR diets and vE levels significantly exceeding the recommendation induced DNA damage in epithelial cells, but this effect varied depending on the intestinal segment and the age of birds.

Status of Salivary Nitric Oxide Levels and Buccal Epithelial Cell DNA Damage in Potentially Malignant Disorders – A Biochemical Study

Oral cancer is a progressive, multistage disease in which changes in genetic structure and cellular morphology occur from the normal to the premalignant state and then to the malignant state. Nitric oxide (NO.) is an uncharged molecule with an unpaired electron. It is highly reactive and interacts with DNA molecules, resulting in DNA damage. Objective: To evaluate the salivary nitric oxide levels and buccal epithelial cell DNA damage in patients with potentially malignant oral disorders. Methods: The salivary nitric oxide levels and buccal epithelial cell DNA damage were estimated in 20 healthy individuals without oral lesions, in 20 subjects having smoking and/or tobacco chewing habits without oral lesions, and 20 patients with a potentially malignant oral disorder. Results: The salivary nitric oxide levels were significantly greater in the subjects with tobacco chewing and/or smoking habits without oral lesions than in the healthy controls. Similarly, the extent of DNA damage was higher in the subjects with potentially malignant disorders and in the subjects with tobacco chewing and/or smoking habits without oral lesions than in the healthy controls. Conclusion: The encouraging results of the present study indicated the potential involvement of nitric oxide in the pathogenesis of potentially malignant oral disorders.

Personal samplers of bioavailable pesticides integrated with a hair follicle assay of DNA damage to assess environmental exposures and their associated risks in children

Agriculture in the United States employs youth ages ten and older in work environments with high pesticide levels. Younger children in rural areas may also be affected by indirect pesticide exposures. The long-term effects of pesticides on health and development are difficult to assess and poorly understood. Yet, epidemiologic studies suggest associations with cancer as well as cognitive deficits. We report a practical and cost-effective approach to assess environmental pesticide exposures and their biological consequences in children. Our approach combines silicone wristband personal samplers and DNA damage quantification from hair follicles, and was tested as part of a community-based participatory research (CBPR) project involving ten Latino children from farmworker households in North Carolina. Our study documents high acceptance among Latino children and their caregivers of these noninvasive sampling methods. The personal samplers detected organophosphates, organochlorines, and pyrethroids in the majority of the participants (70%, 90%, 80%, respectively). Pesticides were detected in all participant samplers, with an average of 6.2±2.4 detections/participant sampler. DNA damage in epithelial cells from the sheath and bulb of plucked hairs follicles was quantified by immunostaining 53BP1-labled DNA repair foci. This method is sensitive, as shown by dose response analyses to γ radiations where the lowest dose tested (0.1Gy) led to significant increased 53BP1 foci density. Immunolabeling of DNA repair foci has significant advantages over the comet assay in that specific regions of the follicles can be analyzed. In this cohort of child participants, significant association was found between the number of pesticide detections and DNA damage in the papilla region of the hairs. We anticipate that this monitoring approach of bioavailable pesticides and genotoxicity will enhance our knowledge of the biological effects of pesticides to guide education programs and safety policies.

Salivary Nitric Oxide Levels and Buccal Epithelial Cell DNA Damage in Oral Cancer - A Biochemical Study

Abstract Background: Cancer is the one of the common cause of mortality in developed and developing countries. Tobacco in any form is found to induce cancer formation. Oral cancer is a multistage process with progressive changes occurring in genetic and cellular morphology as the disease progresses from the normal to premalignant state and then to malignant state. Recent studies have emphasised that nitric oxide is a highly reactive molecule that interacts with DNA resulting in DNA damage.The objective of the present study was to evaluate the nitric oxide levels in the saliva and buccal epithelial cell DNA damage in patients with oral cancer. Material and methods: In this study nitric oxide levels in saliva and buccal epithelial cell DNA damage were estimated in 20 healthy individuals without oral lesions and 20 patients with oral cancer. Results: The mean values of salivary nitric oxide levels were significantly greater in oral cancer patients (126.2155+/- 2.7587) when compared to that of controls (76.111+/-μM/L). Similarly the extent of DNA damage was higher in subjects with oral cancer (28.099+/- 1.2696) than in controls (9.15+/- 0.9131). Pearson's correlation between nitric oxide levels in saliva and extent of DNA damage were found to be moderately correlated in controlsr=0.491 (p&lt;0.01) and in oral cancer patientsr=0.428 (p&lt;0.01). Conclusion: The results of present study illustrate an active involvement of nitric oxide which is derived from tobacco and its related products in the initiation of DNA damage and subsequent formation of oral cancer.

Areca nut exposure increases secretion of tumor-promoting cytokines in gingival fibroblasts that trigger DNA damage in oral keratinocytes.

Molecular crosstalk between cancer cells and fibroblasts has been an emerging hot issue in understanding carcinogenesis. As oral submucous fibrosis (OSF) is an inflammatory fibrotic disease that can potentially transform into squamous cell carcinoma, OSF has been considered to be an appropriate model for studying the role of fibroblasts during early stage carcinogenesis. In this sense, this study aims at investigating whether areca nut (AN)‐exposed fibroblasts cause DNA damage of epithelial cells. For this study, immortalized hNOF (hTERT‐hNOF) was used. We found that the levels of GRO‐α, IL‐6 and IL‐8 increased in AN‐exposed fibroblasts. Cytokine secretion was reduced by antioxidants in AN‐exposed fibroblasts. Increase in DNA double strand breaks (DSB) and 8‐oxoG FITC‐conjugate was observed in immortalized human oral keratinocytes (IHOK) after the treatment of cytokines or a conditioned medium derived from AN‐exposed fibroblasts. Cytokine expression and DNA damage were also detected in OSF tissues. The DNA damage was reduced by neutralizing cytokines or antioxidant treatment. Generation of reactive oxygen species (ROS) and DNA damage response, triggered by cytokines, were abolished when NADPH oxidase (NOX) 1 and 4 were silenced in IHOK, indicating that cytokine‐triggered DNA damage was caused by ROS generation through NOX1 and NOX4. Taken together, this study provided strong evidence that blocking ROS generation might be a rewarding approach for cancer prevention and intervention in OSF.

The use of genetically modified Roundup Ready soyabean meal and genetically modified MON 810 maize in broiler chicken diets. Part 2. Functional status of the small intestine

ABSTRACT. The effects of genetically modified (GM) soyabean meal (SBM) and GM maize on the functional status of the small intestine of broiler chickens was studied. Eight diets were prepared, based on conventional or GM SBM (GTS 40-3-2) combined with non-modified maize cvs. Clarica and PR39 F58 or their isogenic GM (MON 810) counterparts cvs. Bacilla or PR39 F56. Diets were fed in mash form from days 1 to 30 of age to 192 Ross 308 broiler fe-males, assigned to 8 groups and kept in individual cages from day 8 of life. The functional status of the small intestine was studied in 8 birds per group after slaughter. The investigation included morphological evaluation of jejunum and ileum sections, calculation of apoptotic and mitotic indices, and measurement of DNA damage in jejunal epithelial cells. There were no statistically significant differences in morphological parameters of the jejunum and ileum except the width of the tunica mucosa in the jejunum, which was greater in birds fed GM than in those given conventional maize. GM SBM had no effect on the apoptotic and mitotic indices in the crypt area or in villi tips, while GM maize did not affect these indices in the crypt area, but increased the apoptotic index in the villi tips of the jejunum. Neither substituting conventional SBM by GM SBM, nor con-ventional maize by GM maize induced significant DNA damage in the epithelial cells of the jejunum. Our results indicate that the functional development and maturation of the small intestinal epithelium is not greatly modified by the use of GM SBM and GM maize in broiler diets.