DNA Damage In Colon Research Articles

Radioprotective effect of the anti-diabetic drug metformin.

Metformin is a biguanide currently used in the treatment of diabetes mellitus type 2. Besides its anti-glycemic effects, metformin has been reported to induce different cellular pleiotropic effects, depending on concentration and time of treatment. Here we report one administration of metformin (0.5 mM) has radioprotective effects in vitro on BJ human fibroblasts, increasing DNA damage repair and increasing SOD1 expression in the nucleus. Importantly, metformin (200 mg/kg) pre-administration for only 3 days in wild type 129/sv mice, decreases the formation of micronuclei in bone marrow cells and DNA damage in colon and lung tissues compared to control irradiated mice at sub-lethal and lethal doses, increasing the overall survival fraction by 37% after 10Gy total body irradiation. We next pre-treated with metformin and then exposed 129/sv mice, to a galactic cosmic rays simulation (GCRsim), at the NASA Space Radiation Laboratory (NSRL). We found metformin pre-treatment decreases the presence of bone marrow micronuclei and DNA damage in colon and lung tissues and an increase of 8-oxoguanine DNA glycosylase-1 (OGG1) expression. Our data highlight a radioprotective effect of metformin through an indirect modulation of the gene expression involved in the cellular detoxification rather than its effects on mitochondria.

New Insights into How Melatonin Ameliorates Bisphenol A-Induced Colon Damage: Inhibition of NADPH Oxidase.

Bisphenol A (BPA) is an endocrine disruptor, widely employed, and detected in many consumer products and food items. Oral intake poses a great threat to intestinal health. Melatonin (MT) stands out as an endogenous, dietary, and therapeutic molecule with potent antioxidant capacity. To explore the protective effect of MT against BPA-induced colon damage and the role of NADPH oxidase (NOX) in this process, we established mice and colonic epithelial cell (NCM460) models of BPA exposure and treated with MT. In vitro and in vivo results showed that MT ameliorated BPA-induced oxidative stress, DNA damage, and the G2/M cell cycle arrest. MT also downregulated the expression of NOX family-related genes, reversed the inhibition of the base excision repair (BER) pathway, promoted the activation of non-homologous end-joining (NHEJ) pathway, and suppressed the mRNA and protein expression of ATM, Chk1/2, and p53. Diphenyleneiodonium chloride (DPI), a NOX-specific inhibitor, also attenuated the toxic effects of BPA on NCM460 cells. Furthermore, molecular docking revealed that MT could bind to NOX. Conclusively, our finding suggested that MT can ameliorate BPA-induced colonic DNA damage by scavenging NOX-derived ROS, which further attenuates G2/M cell cycle arrest dependent on the ATM-Chk1/2-p53 axis.

Carboxymethyl Cellulose/Zn-Organic Framework Down-Regulates Proliferation and Up-Regulates Apoptosis and DNA Damage in Colon and Lung Cancer Cell Lines.

A solvothermal technique was used to prepare a Zn–benzenetricarboxylic acid (Zn@BTC) organic framework covered with a carboxymethyl cellulose (CMC/Zn@BTC). Fourier transform infrared spectroscopy (FTIR), field emission scanning electron microscope (FESEM), and Brunauer, Emmett, and Teller (BET) surface area were applied to characterize CMC/Zn@BTC. Moreover, the anticancer, anti-migrative, anti-invasive, and anti-proliferative action of CMC/Zn@BTC nanoparticles were assessed on cancer cell lines. Apoptotic markers and DNA damage were assessed to explore the cellular and biological changes induced by CMC/Zn@BTC nanoparticles. The microscopic observation revealed that CMC controls the surface morphology and surface characteristics of the Zn@BTC. The obtained BET data revealed that the Zn@BTC nanocomposite surface area lowers from 1061 m2/g to 740 m2/g, and the pore volume decreases from 0.50 cm3/g to 0.37 cm3/g when CMC is applied to Zn@BTC nanocomposites. The cellular growth of DLD1 and A549 was suppressed by CMC/Zn@BTC, with IC50 values of 19.1 and 23.1 μg/mL, respectively. P53 expression was upregulated, and Bcl-2 expression was downregulated by CMC/Zn@BTC, which promoted the apoptotic process. Furthermore, CMC/Zn@BTC caused DNA damage in both cancer cell lines with diverse impact, 66 percent (A549) and 20 percent (DLD1) compared to cisplatin’s 52 percent reduction. CMC/Zn@BTC has anti-invasive properties and significantly reduced cellular migration. Moreover, CMC/Zn@BTC aims key proteins associated with metastasis, proliferation and programmed cellular death.

Local non-pituitary growth hormone is induced with aging and facilitates epithelial damage.

SUMMARYMicroenvironmental factors modulating age-related DNA damage are unclear. Non-pituitary growth hormone (npGH) is induced in human colon, non-transformed human colon cells, and fibroblasts, and in 3-dimensional intestinal organoids with age-associated DNA damage. Autocrine/paracrine npGH suppresses p53 and attenuates DNA damage response (DDR) by inducing TRIM29 and reducing ATM phosphorylation, leading to reduced DNA repair and DNA damage accumulation. Organoids cultured up to 4 months exhibit aging markers, p16, and SA-β-galactosidase and decreased telomere length, as well as DNA damage accumulation, with increased npGH, suppressed p53, and attenuated DDR. Suppressing GH in aged organoids increases p53 and decreases DNA damage. WT mice exhibit age-dependent colon DNA damage accumulation, while in aged mice devoid of colon GH signaling, DNA damage remains low, with elevated p53. As age-associated npGH induction enables a pro-proliferative microenvironment, abrogating npGH signaling could be targeted as anti-aging therapy by impeding DNA damage and age-related pathologies.

Styrax camporum, a typical species of the Brazilian cerrado, attenuates DNA damage, preneoplastic lesions and oxidative stress in experimental rat colon carcinogenesis

ABSTRACT Styrax camporum Pohl, a typical species from the Brazilian cerrado, commonly known as “benjoeiro”, is used to treat gastroduodenal diseases. In previous studies carried out by our research group, hydroalcoholic extract of S. camporum stems (SCHE) exhibited antigenotoxic and antiproliferative effects. For a comparative analysis of the chemopreventive effect of SCHE, the aim of this study was to investigate the influence of SCHE against carcinogen 1,2-dimethylhydrazine (DMH)-induced DNA damage and pre-neoplastic lesions in Wistar rat colon. Animals were treated orally with SCHE at 250, 500 or 1000 mg/kg body weight in conjunction with a subcutaneous injection of DMH. DNA damage was assessed using the comet assay while tpre-neoplastic lesions by aberrant crypt foci (ACF) assay. The following hepatic oxidative stress markers were determined including activities of catalase (CAT) and glutathione S-transferase (GST) as well as levels of reduced glutathione (GSH) and malondialdehyde (MDA). Treatment with SCHE was not genotoxic or carcinogenic at the highest dose tested (1000 mg/kg b.w.). The extract effectively inhibited DNA damage and pre-neoplastic lesions induced by DMH administration at all concentrations tested. Measurement of CAT, and GST activities and levels of GSH showed that SCHE did not reduce oxidative processes. In contrast, treatment with SCHE (1000 mg/kg b.w.) decreased liver MDA levels. Taken together, these findings suggested the chemopreventive effect attributed to SCHE in colon carcinogenesis, may be related to its capacity to inhibit DNA damage as well as an antioxidant action associated with its chemical constituents egonol and homoegonol.

A Dietary Intervention High in Green Leafy Vegetables Reduces Oxidative DNA Damage in Adults at Increased Risk of Colorectal Cancer: Biological Outcomes of the Randomized Controlled Meat and Three Greens (M3G) Feasibility Trial.

Green leafy vegetables (GLV) may reduce the risk of red meat (RM)-induced colonic DNA damage and colorectal cancer (CRC). We previously reported the primary outcomes (feasibility) of a 12-week randomized controlled crossover trial in adults with habitual high RM and low GLV intake with body mass index (BMI) > 30 kg/m2 (NCT03582306). Herein, our objective was to report a priori secondary outcomes. Participants were recruited and enrolled in 2018, stratified by gender, and randomized to two arms: immediate intervention group (IG, n = 26) or delayed intervention group (DG, n = 24). During the 4 week intervention period, participants were provided with frozen GLV and counseled to consume 1 cooked cup equivalent daily. Participants consumed their normal diet for the remaining 8 weeks. At each of four study visits, anthropometrics, stool, and blood were taken. Overall, plasma Vitamin K1 (0.50 ± 1.18 ng/mL, p < 0.001) increased, while circulating 8OHdG (−8.52 ± 19.05 ng/mL, p < 0.001), fecal 8OHdG (−6.78 ± 34.86 ng/mL, p < 0.001), and TNFα (−16.95 ± 60.82 pg/mL, p < 0.001) decreased during the GLV intervention compared to control periods. Alpha diversity of fecal microbiota and relative abundance of major taxa did not differ systematically across study periods. Further investigation of the effects of increased GLV intake on CRC risk is warranted.

Effect of supplementing fava bean (Vicia faba L.) on ulcerative colitis and colonic mucosal DNA content in rats fed a high-sucrose diet.

Ulcerative colitis (UC) is an inflammatory bowel disease with high morbidity. Acetic acid-induced damage of colonic mucosa in rats is a commonly used experimental animal model of UC. This research aimed to explore for the first time the ameliorative effect of dietary supplementation with fava bean on the incidence of UC in rats fed with sucrose containing diet. Rats were divided into five groups as follows: G1, control healthy rats; G2, colitic rats; G3, colitic rats fed diets containing 30% sucrose, G4, healthy rats fed diets containing 30% sucrose and G5, colitic rats fed diets containing 30% sucrose supplemented with dried ground fava bean. Colonic injury and inflammation were evaluated through a disturbance of oxidative biomarkers, a significant increase in inflammatory biomarkers and inflammatory cytokines, and histological abnormalities in colonic tissues accompanied by colonic mucosal DNA damage. Colitic rats fed on sucrose containing diet demonstrated additional histological, biochemical, and DNA alterations in colonic mucosa of rats. Dietary supplementation with dried ground fava bean significantly corrected the impaired oxidative and inflammatory biomarker levels and modulated histological features and DNA alterations. Finally, fava bean attenuated the oxidative damage and colonic injury induced by acetic acid, which confirmed its high anti-oxidant and anti-incendiary properties.

Grape seed extract ameliorates PhIP-induced colonic injury by modulating gut microbiota, lipid metabolism, and NF-κB signaling pathway in rats

Abstract 2-Amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) is a common carcinogen produced in thermally processed protein-rich foods. This study explored the protective effects of grape seed extract (GSE) against colonic injury induced by short-term exposure to PhIP and the underlying mechanisms involved. Wistar rats were randomly divided into four groups: control, GSE only (GSE), PhIP (PhIP), and GSE prevention (GPhIP). In the GPhIP group, rats were fed a diet supplemented with GSE for 2 weeks before administering PhIP. GSE significantly ameliorated PhIP-induced oxidative stress and colonic DNA damage. Moreover, GSE effectively maintained homeostasis in intestinal flora, especially by preventing PhIP-mediated reduction in Lactobacillus abundance. Fecal metabolome and colonic transcriptome analyses revealed that GSE remarkably ameliorated PhIP-induced colonic injury by regulating lipid metabolic pathways. Notably, nuclear factor-κB signaling pathway involved in the process of prevention. Therefore, GSE is recommended as an effective dietary supplement to prevent the harm of PhIP in vivo.

OR16-06 Age-Associated Local GH Promotes Colon Neoplasia

Colon polyp and cancer frequency increase with age, yet little is known about age-related mechanisms underlying development of these neoplasms. Defective DNA damage response and accumulation of unrepaired DNA damage can trigger genomic instability and cellular transformation.Patients with acromegaly have a higher prevalence of colon polyps and, arguably, colon adenocarcinoma, while those with GH signaling deficiency do not develop cancer. We showed that APC+/- mice that all develop colon adenomas exhibit a significant decrease in the number and volume of colon tumors with deletion of the GH transcription factor Prop1. Further, DNA damage response triggered GH expression in colon cells, and GH, in turn, altered DNA damage repair, resulting in DNA damage accumulation and cell transformation both in vitro and in vivo. These findings prompted us to hypothesize that accumulated DNA damage in aging colon induces local GH, suppressing DNA damage repair and creating a milieu consistent with genomic instability favoring neoplastic development.In human colon tissue we now show increased expression of γH2AX, a marker of DNA breaks, associated with increased GH transcription (detected by RNA scope) and translation (assessed by immunohistochemistry) as well as GH induction in both human and murine colon after DNA damaging radiotherapy. In vitro studies support these results, showing GH induction in normal human colon epithelial cells, fibroblasts, and 3D human intestinal organoids after DNA damage. Of note, local GH was secreted in the medium, indicating a paracrine effect. Paracrine/autocrine GH expression in these cellular models resulted in suppression of p53, induction of EMT, and attenuation of DNA damage response, and accumulated unrepaired DNA damage in human colon cells and in human intestinal organoids. In vivo, colon cells infected with lentivirus expressing GH generated more metastases than did cells expressing control vector. Co-culturing of human normal colon fibroblasts expressing GH together with normal human colon cells led to increased motility and accumulation of DNA damage as well as increased proliferation of epithelial cells on a gut-on-a-chip microfluidic device, confirming paracrine GH effects. In an in vitro model of aging, culturing human intestinal organoids for up to 2 months resulted in decreased telomere length and increased GH mRNA and protein expression associated with suppressed DNA damage response evident by decreased phosphorylation of ATM and DNA-PKcs, both kinases involved in DNA repair, and DNA damage accumulation assessed by Comet assay. Suppression of GH in these aging organoids led to increased phospho-p53 and reduced DNA damage. Although somatotroph axis endocrine activity decreases with age, local GH induced in response to age-related DNA damage may trigger a “field change,” creating a milieu favorable for colon neoplastic development.

Paradoxical interaction between cancer and long-term postsepsis disorder: impairment of de novo carcinogenesis versus favoring the growth of established tumors.

BackgroundPrevious data have reported that the growth of established tumors may be facilitated by postsepsis disorder through changes in the microenvironment and immune dysfunction. However, the influence of postsepsis disorder in initial carcinogenesis remains elusive.MethodsIn the present work, the effect of postsepsis on inflammation-induced early carcinogenesis was evaluated in an experimental model of colitis-associated colorectal cancer (CAC). We also analyzed the frequency and role of intestinal T regulatory cells (Treg) in CAC carcinogenesis.ResultsThe colitis grade and the tumor development rate were evaluated postmortem or in vivo through serial colonoscopies. Sepsis-surviving mice (SSM) presented with a lower colonic DNA damage, polyp incidence, reduced tumor load, and milder colitis than their sham-operated counterparts. Ablating Treg led to restoration of the ability to develop colitis and tumor polyps in the SSM, in a similar fashion to that in the sham-operated mice. On the other hand, the growth of subcutaneously inoculated MC38luc colorectal cancer cells or previously established chemical CAC tumors was increased in SSM.ConclusionOur results provide evidence that postsepsis disorder has a dual effect in cancer development, inhibiting inflammation-induced early carcinogenesis in a Treg-dependent manner, while increasing the growth of previously established tumors.

Clonal hematopoiesis as a model for premalignant changes during aging

Over the course of the human life span, somatic DNA mutations accumulate in healthy tissues. This process has been most clearly described in blood and bone marrow, esophagus, colon, and skin, but cumulative DNA damage likely affects all tissues of the body. Although most acquired genetic variants have no discernable functional consequences, some randomly occurring somatic mutations confer a relative fitness advantage on a single stem cell and its progeny compared with surrounding cells, which may lead to progressive expansion of a clone (i.e., a genetically identical group of cells). When these mutations occur in a cell with the capacity to self-renew and expand, the mutations persist, and such clonal expansion is a risk factor for further mutation acquisition and clonal evolution. Hematopoietic stem cells are a special case of clonal expansion because both the stem cells and their blood cell progeny circulate in large numbers, and these cells are not subject to some of the anatomical restrictions that characterize other tissues in which somatic mutations conferring a fitness advantage also occur. Therefore, clonally restricted hematopoiesis can have biological and clinical consequences that are distinct from clonal expansions in other tissues. Such consequences include not only clonal progression to overt myeloid neoplasia (or, less commonly, to lymphoid neoplasia) driven by acquisition of secondary mutations in the cells of the expanded clone, but also cardiovascular events and, most likely, other diseases that are influenced by aberrant function of mutant blood cells. A more detailed understanding of how clonal hematopoiesis arises and how clonal selection and expansion occur, as well as development of strategies to avert the clinical consequences associated with clonal hematopoiesis, may both improve public health and yield more general insights into the biology of aging.

Primary Outcomes of a Randomized Controlled Crossover Trial to Explore the Effects of a High Chlorophyll Dietary Intervention to Reduce Colon Cancer Risk in Adults: The Meat and Three Greens (M3G) Feasibility Trial

Preclinical and observational research suggests green leafy vegetables (GLVs) may reduce the risk of red meat (RM)-induced colonic DNA damage and colon cancer (CC). We sought to determine the feasibility of a high GLV dietary intervention in adults with an increased risk of CC (NCT03582306) via a 12-week randomized controlled crossover trial. Participants were randomized to immediate or delayed (post-4-week washout) intervention groups. During the 4-week intervention period, participants were given frozen GLVs and counseled to consume one cooked cup equivalent daily. The primary outcomes were: accrual—recruiting 50 adults in 9 months; retention—retaining 80% of participants at completion; and adherence—meeting GLV intake goals on 90% of days. Adherence data were collected twice weekly and 24-h dietary recalls at each time point provided nutrient and food group measures. The Food Acceptability Questionnaire (FAQ) was completed to determine acceptability. On each of the four study visits, anthropometrics, stool, saliva, and blood were obtained. Fifty adults were recruited in 44 days. Participants were 48 ± 13 years of age, 62% female, and 80% Caucasian, with an average BMI at screening of 35.9 ± 5.1. Forty-eight (96%) participants were retained and completed the study. During the intervention phase, participants consumed GLVs on 88.8% of days; the adherence goal of one cup was met on 73.2% of days. Dietary recall-derived Vitamin K and GLVs significantly increased for all participants during the intervention periods. Overall satisfaction did not differ between intervention and control periods (p = 0.214). This feasibility trial achieved accrual, retention and acceptability goals, but fell slightly short of the benchmark for adherence. The analysis of biological specimens will determine the effects of GLVs on gut microbiota, oxidative DNA damage, and inflammatory cytokines.

Dietary hemin promotes colonic preneoplastic lesions and DNA damage but not tumor development in a medium-term model of colon carcinogenesis in rats

Red and processed meat consumption has been strongly related to increase the risk of colorectal cancer (CRC), although its impact is largely unknown. Hemin, an iron-containing porphyrin, is acknowledged as a putative factor of red and processed meat pro-carcinogenic effects. The aim of this study was to investigate the effects of high dietary hemin on the promotion/progression stages of 1,2-dimethylhydrazine (1,2-DMH)-induced colon carcinogenesis. Twenty-four Wistar male rats were given four subcutaneous 1,2-DMH injections and received either balanced diet or balanced diet supplemented with hemin 0.5 mmol/kg for 23 weeks. Colon specimens were analyzed for aberrant crypt foci (ACF) and tumor development. Dietary hemin significantly increased ACF number and fecal water cytotoxicity/genotoxicity in Caco-2 cells when compared to 1,2-DMH control group. However, tumor incidence, multiplicity and cell proliferation did not differ between 1,2-DMH + hemin and 1,2-DMH control group. Gene expression analysis of 91 target-genes revealed that only three genes (Figf, Pik3r5 and Tgfbr2) were down-regulated in the tumors from hemin-fed rats compared to those from 1,2-DMH control group. Therefore, the findings of this study show that high hemin intake promotes mainly DNA damage and ACF development and but does not change the number nor incidence of colon tumors induced by 1,2-DMH in male rats.

Serotonin synthesis protects the mouse colonic crypt from DNA damage and colorectal tumorigenesis.

Serotonin (5-HT) signaling pathways are thought to be involved in colorectal tumorigenesis (CRT), but the role of 5-HT synthesis in the early steps of this process is presently unknown. In this study, we used carcinogen treatment in the tryptophan hydroxylase 1 knockout (Tph1KO) and transgenic (Tph1fl/fl VillinCre ) mouse models defective in 5-HT synthesis to investigate the early mutagenic events associated with CRT. Our observations of the colonic crypt post-treatment followed a timeline designed to understand how disruption of 5-HT synthesis affects the initial steps leading to CRT. We found Tph1KO mice had decreased development of both allograft tumors and colitis-related CRT. Interestingly, carcinogenic exposure alone induced multiple colon tumors and increased cyclooxygenase-2 (Ptgs2) expression in Tph1KO mice. Deletion of interleukin 6 (Il6) in Tph1KO mice confirmed that inflammation was a part of the process. 5-HT deficiency increased colonic DNA damage but inhibited genetic repair of specific carcinogen-related damage, leading to CRT-related inflammatory reactions and dysplasia. To validate a secondary effect of 5-HT deficiency on another DNA repair pathway, we exposed Tph1KO mice to ionizing radiation and found an increase in DNA damage associated with reduced levels of ataxia telangiectasia and Rad3 related (Atr) gene expression in colonocytes. Restoring 5-HT levels with 5-hydroxytryptophan treatment decreased levels of DNA damage and increased Atr expression. Analysis of Tph1fl/fl VillinCre mice with intestine-specific loss of 5-HT synthesis confirmed that DNA repair was tissue specific. In this study, we report a novel protective role for 5-HT synthesis that promotes DNA repair activity during the early stages of colorectal carcinogenesis. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Growth Hormone Induces Colon DNA Damage Independent of IGF-1.

DNA damage occurs as a result of environmental insults and aging and, if unrepaired, may lead to chromosomal instability and tumorigenesis. Because GH suppresses ataxia-telangiectasia mutated kinase phosphorylation, decreases DNA repair, and increases DNA damage accumulation, we elucidated whether GH effects on DNA damage are mediated through induced IGF-1. In nontumorous human colon cells, GH, but not IGF-1, increased DNA damage. Stably disrupted IGF-1 receptor (IGF-1R) by lentivirus-expressing short hairpin RNA in vitro or treatment with the IGF-1R phosphorylation inhibitor picropodophyllotoxin (PPP) in vitro and in vivo led to markedly induced GH receptor (GHR) abundance, rendering cells more responsive to GH actions. Suppressing IGF-1R triggered DNA damage in both normal human colon cells and three-dimensional human intestinal organoids. DNA damage was further increased when cells with disrupted IGF-1R were treated with GH. Because GH induction of DNA damage accumulation appeared to be mediated not by IGF-1R but probably by more abundant GH receptor expression, we injected athymic mice with GH-secreting xenografts and then treated them with PPP. In these mice, high circulating GH levels were associated with increased colon DNA damage despite disrupted IGF-1R activity (P < 0.01), whereas GHR levels were also induced. Further confirming that GH effects on DNA damage are directly mediated by GHR signaling, GHR-/- mice injected with PPP did not show increased DNA damage, whereas wild-type mice with intact GHR exhibited increased colon DNA damage in the face of IGF-1 signaling suppression. The results indicate that GH directly induces DNA damage independent of IGF-1.

MON-475 Growth Hormone Induces Colon Tissue DNA Damage Independently of IGF-1

Environmental insults and aging lead to DNA damage, which, if unrepaired, results in chromosomal instability and tumorigenesis. We have shown that GH suppresses phosphorylation of ATM and its target proteins p53, Chk2, and H2AX in DNA damaged non-tumorous human colon cells. We show that GH decreased DNA repair by non-homologous end-joining and DNA damage accumulation, enhancing cell transformation as evidenced by increased growth in soft agar. Prolonged high circulating GH levels in mice bearing human GH-secreting colon cell xenografts resulted in 60% increase in unrepaired DNA damage and increased number of metastatic lesions. We sought to elucidate whether GH effects on DNA damage are mediated through induced IGF-1. In non-tumorous human colon cells treated with etoposide, GH but not IGF-1 showed enhanced DNA damage. Stable disruption of IGF-1R by lentivirus expressing shRNA in vitro, or treatment with IGF-1R inhibitor PPP in vitro and in vivo, markedly induced GH receptor expression, rendering cells more sensitive to effects of GH. Suppressing IGF-1R in normal human colon cells triggered DNA damage, while when cells with disrupted IGF-1R were treated with GH, levels of DNA damage were further increased, indicating that GH effects on DNA damage were not mediated by IGF-1R, but likely by induced GH receptor. Similar results were obtained when 3-dimensional human intestinal organoid cultures were treated with PPP. To confirm these observations in vivo, nude mice were injected with GH-secreting xenografts and 7 weeks later treated with PPP (20 mg/kg, i.p., twice daily, total 4 injections). Similar to results observed in vitro, in mice with high circulating GH colon DNA damage were significantly increased when IGF-1R was disrupted by PPP, and GH receptor levels were again induced. To further confirm that GH effects on DNA damage are directly mediated by GH signaling, GHR-/- mice were injected with PPP to block IGF-1R, resulting in increased colon DNA damage in WT mice, but animals lacking GHR did not respond to induced DNA damage in the face of IGF-1 suppression. The results imply that GH directly induces DNA damage independently of IGF-1.

Mancozeb fungicide-induced genotoxic effects, metabolic alterations, and histological changes in the colon and liver of Sprague Dawley rats.

The present study was designed to evaluate genotoxic markers of mancozeb exposure and withdrawal in colon and liver tissues together with histological changes in the gastrointestinal tract of Sprague Dawley rats. Thirty rats were divided into three equal groups; group I: treatment, 250 mg/kg mancozeb dissolved in corn oil administered twice weekly for 7 weeks; group II: withdrawal, the same treatment as group I after which animals were untreated for 5 weeks; group III: control, administered corn oil on the same schedule as group I for 7 weeks. All administrations were by oral gavage. Serum samples were analyzed for biochemical parameters. The comet assay and histopathological examinations were done on liver and colon specimens. The results demonstrated that mancozeb exposure caused significant increases in triglycerides and total cholesterol accompanied by decreases in glucose levels, with extensive DNA damage in liver and colon together with pathological changes in stomach, colon, and liver. Mancozeb withdrawal for 5 weeks improved the lipid and glucose profiles and decreased the degree of DNA damage and changes in the architecture of the stomach, colon, and liver. We concluded that discontinuing exposure to mancozeb fungicide for 5 weeks could ameliorate the adverse effects induced by 7 weeks of exposure to mancozeb. A longer withdrawal time may further reduce the observed genotoxicity.

Vitamin D Regulation of the Uridine Phosphorylase 1 Gene and Uridine-Induced DNA Damage in Colon in African Americans and European Americans

African Americans have the greatest colorectal cancer (CRC) burden in the United States; interethnic differences in protective effects of vitamin D might contribute to disparities. 1α,25(OH)2D3 vitamin D (the active form of vitamin D) induces transcription of the uridine phosphorylase gene (UPP1) in colon tissues of European Americans but to a lesser extent in colon tissues of African Americans. UPP1-knockout mice have increased intestinal concentrations of uridine and Deoxyuridine triphosphate (dUTP), have increased uridine-induced DNA damage, and develop colon tumors. We studied 1α,25(OH)2D3 regulation of UPP1 and uridine-induced DNA damage in the colon and differences in these processes between African and European Americans. We quantified expression and activity of UPP1 in response to 1α,25(OH)2D3 in young adult mouse colonic cells, human CRC cells (LS174T), and organoids (derived from rectosigmoid biopsy samples of healthy individuals undergoing colonoscopies) using quantitative polymerase chain reaction, immunoblot, and immunocytochemistry assays. Binding of the vitamin D receptor to UPP1 was tested by chromatin immunoprecipitation. Uridine-induced DNA damage was measured by fragment-length analysis in repair enzyme assays. Allele-specific 1α,25(OH)2D3 responses were tested using luciferase assays. Vitamin D increased levels of UPP1 mRNA, protein, and enzymatic activity and increased vitamin D receptor binding to the UPP1 promoter in young adult mouse colonic cells, LS174T cells, and organoids. 1α,25(OH)2D3 significantly reduced levels of uridine and uridine-induced DNA damage in these cells, which required UPP1 expression. Organoids derived from colon tissues of African Americans expressed lower levels of UPP1 after exposure to 1α,25(OH)2D3 and had increased uridine-induced DNA damage compared with organoids derived from tissues of European Americans. Luciferase assays with the T allele of single nucleotide polymorphism rs28605337 near UPP1, which is found more frequently in African Americans than European Americans, expressed lower levels of UPP1 after exposure to 1α,25(OH)2D3 than assays without this variant. We found vitamin D to increase expression of UPP1, leading to reduce uridine-induced DNA damage, in colon cells and organoids. A polymorphism in UPP1 found more frequently in African Americans than European Americans reduced UPP1 expression upon cell exposure to 1α,25(OH)2D3. Differences in expression of UPP1 in response to vitamin D could contribute to the increased risk of CRC in African Americans.

Simultaneous removal of dihydroxybenzenes and toxicity reduction by Penicillium chrysogenum var. halophenolicum under saline conditions

The dihydroxybenzenes are widely found in wastewater and usually more than one of these aromatic compounds co-exist as pollutants of water resources. The current study investigated and compared the removal efficiency of hydroquinone, catechol and resorcinol in binary substrate systems under saline conditions by Penicillium chrysogenum var. halophenolicum, to clarify the potential of this fungal strain to degrade these aromatic compounds. Since P. chrysogenum is a known penicillin producer, biosynthetic penicillin genes were examined and antibiotic was quantified in mono and binary dihydroxybenzene systems to elucidate the carbon flux of dihydroxybenzenes metabolism in the P. chrysogenum var. halophenolicum to the secondary metabolism. In binary substrate systems, the three assayed dihydroxybenzene compounds were found to be co-metabolized by fungal strain. The fungal strain preferentially degraded hydroquinone and catechol. Resorcinol was degraded slower and supports higher antibiotic titers than either catechol or hydroquinone. Dihydroxybenzenes were faster removed in mixtures compared to mono substrate systems, except for the case of hydroquinone. In this context, the expression of penicillin biosynthetic gene cluster was not related to the removal of dihydroxybenzenes. Penicillin production was triggered simultaneously or after dihydroxybenzene degradation, but penicillin yields, under these conditions, did not compromise dihydroxybenzene biological treatment. To investigate the decrease in dihydroxybenzenes toxicity due to the fungal activity, viability tests with human colon cancer cells (HCT116) and DNA damage by alkaline comet assays were performed. For all the conditions assays, a decrease in saline medium toxicity was observed, indicating its potential as detoxification agent.

Quantitative relationships between lacZ mutant frequency and DNA adduct frequency in Muta™Mouse tissues and cultured cells exposed to 3-nitrobenzanthrone.

The frequency of stable DNA adducts in a target tissue can be used to assess biologically effective dose; however, the utility of the metric in a risk assessment context depends on the likelihood that the DNA damage will be manifested as mutation. Previously, we employed the Muta™Mouse system to examine the induction of lacZ mutants and DNA adducts following exposure to the well-studied mutagenic carcinogen 3-nitrobenzanthrone (3-NBA). In this follow-up work, we examined the empirical relationships between total adduct frequency and mutant frequency (MF) in tissues and cultured cells following acute 3-NBA exposure. The results show a significant induction of DNA damage and lacZ mutants in liver, colon and bone marrow, as well as FE1 pulmonary epithelial cells. In contrast, lung and small intestine samples had low, but significantly elevated adduct levels, with no significant increases in lacZ MF. Additional analyses showed a significant relationship between the mutagenic efficiency of total adducts, measured as the slope of the relationships between MF and total adduct frequency, and tissue-specific mitotic index (MI). The lack of mutation response in lung, in contrast to the high in vitro MF in FE-1 lung cells, is likely related to the 100-fold difference in MI. The lack of small intestine mutagenic response may be related to limited metabolic capacity, differences in DNA repair, and /or chemically induced apoptosis that has been observed for other potent mutagens. The results indicate that interpretation of adduct frequency values in a risk assessment context can be improved by considering the MI of the target tissue; however, more generalised interpretation is hampered by tissue-specific variations in metabolic capacity and damage processing. The work provides a proof of principle regarding the use of the Muta™Mouse system to critically examine the health risks associated with tissue-specific adduct loads.