Tail DNA Research Articles

177 Functional Annotation of an Array of Immune Cells and Various Tissues in the Chicken

Abstract Infection and disease negatively affect performance, growth, and health in production animals and has major economic impacts on commercial poultry production. Regulatory elements control the expression of genes and consequently phenotype. Epigenetic modifications such as histone tail modifications and DNA methylation are not only key to the regulation of unique transcriptome patterns, these modifications are indispensable as genome annotators to uncover cell- and tissue-specific regulatory elements. The objectives of our current projects are to leverage transcriptomic and epigenomic methods to annotate transcripts, coding and noncoding, and cis-regulatory elements in the chicken genome, including promoters, enhancers and insulators. In addition, we are employing single-cell sequencing to characterize new or rare cell populations in tissues relevant to production and investigate underlying mechanisms of disease resistance to pathogens. The transcriptome of a number of important cells and tissues has been analyzed, including immune, intestinal and reproductive. The animal procedure was approved and conducted according to guidelines established by the Western University of Health Sciences, Pomona, California (WesternU) Institutional Animal Care and Use Committee. Cell and tissue collection, processing and bioinformatic analysis for transcriptome studies were performed as described in Overbey et al. (2021; Frontiers Genetics). Single-cell studies were performed as described in Sparling et al. (2022; Journal of Immunology) using the 10x Genomics platform. Results for the transcriptome studies show that in the immune cells, many DEGs were mostly involved in cellular processes relating to differentiation and cell metabolism as well as basic functions of immune cells such as cell adhesion and signal transduction. This was to be expected, as there was no explicit immunological stimulus involved. Nevertheless, it was notable that DEGs in the comparison between bursa and thymus that were upregulated in the thymus were related to T cell differentiation and maturation. On the other hand, genes differentially upregulated in B cell vs Bursa or Bursa vs Thymus, are mostly involved in B cell development and differentiation, or activation. Genes differentially regulated in B cells and monocytes are involved in specific functions of the cell types. In the intestinal tissues, we were able to correlate most of the differential expressed genes to metabolic processes related to nutrient digestion and absorption. Several genes in the distal part of the intestine were particularly implicated in vitamin metabolism. Genes involved in energy metabolism are also abundant in the cecum, which suggests that microbial contribution of energy production in the intestine is especially important. Finally, single cell studies on the chicken shell gland are ongoing, but we found differences in cellular populations and gene expression dependent on the chicken haplotype profiled and differences in expression of Ig-like receptors that need to be further explored.

The optimum concentration of soya-lecithin for replacing egg yolk in INRA-82-modified semen extenders improved the cryopreserved semen quality and DNA integrity of Arabian stallions

The cryopreservation of stallion spermatozoa varies greatly between animals. Different equine semen extender supplements were invented to maximize the post-thaw semen quality. Soy lecithin is one of those additives that contain plant origin phospholipids used to replace egg yolk to minimize disease transmission. This study aimed to determine which concentrations of soy lecithin can replace egg-yolk in modified INRA-82 semen extenders for the cryopreservation of horse semen. Semen was collected from six Arabian stallions and extended with modified INRA-82 semen extender supplemented with 0.0, 1.25, 2.50, and 5.0% soya lecithin. Frozen-thawed sperm motility was measured at 0, 1, 2, and 3 h. Viability index, sperm plasma membrane integrity (SPMI), and acrosome integrity in addition to the comet assay non-fragmented DNA, head DNA, tail DNA, tail length, and tail moment were statistically analyzed. The addition of 2.5% soya lecithin had the highest post-thaw sperm motility at 0, 1, 2 h (P < 0.001) and 3 h (P < 0.0001), the optimum viability index (P < 0.0001), SPMI (P < 0.05), acrosome integrity (P < 0.0001), non-fragmented DNA (P < 0.01), DNA in the comet head (P < 0.05), minimum DNA in the comet tail (P < 0.05), shortest comet tail length (P < 0.0001), and lowest comet tail moment (P < 0.01). Soy lecithin at concentrations other than 2.5% deteriorated all post-thaw semen parameters. Soy lecithin (2.5%) can replace egg yolk in the modified INRA-82 semen extender for the cryopreservation of good-quality stallion semen.

Age-related influence on DNA damage, proteomic inflammatory markers and oxidative stress in hospitalized COVID-19 patients compared to healthy controls

COVID-19 infections are accompanied by adverse changes in inflammatory pathways that are also partly influenced by increased oxidative stress and might result in elevated DNA damage. The aim of this case-control study was to examine whether COVID-19 patients show differences in oxidative stress-related markers, unconjugated bilirubin (UCB), an inflammation panel and DNA damage compared to healthy, age-and sex-matched controls.The Comet assay with and without the treatment of formamidopyrimidine DNA glycosylase (FPG) and H2O2 challenge was used to detect DNA damage in whole blood. qPCR was applied for gene expression, UCB was analyzed via HPLC, targeted proteomics were applied using Olink® inflammation panel and various oxidative stress as well as clinical biochemistry markers were analyzed in plasma.Hospitalized COVID-19 patients (n = 48) demonstrated higher serum levels of 55 inflammatory proteins (p < 0.001), including hs-C-reactive protein levels (p < 0.05), compared to healthy controls (n = 48). Interestingly, significantly increased age-related DNA damage (%-DNA in tail) after formamidopyrimidine DNA glycosylase (FPG) treatment was measured in younger (n = 24, average age 55.7 years; p < 0.05) but not in older COVID-19 patients (n = 24, average age 83.5 years; p > 0.05). Although various oxidative stress markers were not altered (e.g., FRAP, malondialdehyde, p > 0.05), a significant increased ratio of oxidized to reduced glutathione was detected in COVID-19 patients compared to healthy controls (p < 0.05). UCB levels were significantly lower in individuals with COVID-19, especially in younger COVID-19 patients (p < 0.05).These results suggest that COVID-19 infections exert effects on DNA damage related to age in hospitalized COVID-19 patients that might be driven by changes in inflammatory pathways but are not altered by oxidative stress parameters.

Comparison of the transgenic rodent mutation assay, error corrected next generation duplex sequencing, and the alkaline comet assay to detect dose-related mutations following exposure to N-nitrosodiethylamine.

N-Nitrosodiethylamine (NDEA), a well-studied N-nitrosamine, was tested in rats to compare the dose-response relationship of three genotoxicity endpoints. Mutant / mutation frequencies were determined using the transgenic rodent (TGR) gene mutation assay and error corrected next generation sequencing (ecNGS) (i.e., duplex sequencing (DS)), and genetic damage was detected by the alkaline comet assay. Big Blue® (cII Locus) animals (n=6 per dose group) were administered doses of 0.001, 0.01, 0.1, 1, 3mg/kg/day NDEA by oral gavage. Samples were collected for cII mutation and DS analyses following 28-days of exposure and 3 days recovery. In a separate study, male Sprague-Dawley (SD) rats (n=6 per dose group) were administered the same doses by oral gavage for two consecutive days and then samples collected for the alkaline comet assay. A dose-related increase in mutant / mutation frequencies of the liver but not duodenum was observed using the TGR assay and DS with DS resulting in a slightly more sensitive response, with a lower benchmark dose (BMD). In addition, a dose-related increase in percent tail DNA was observed in the liver using the alkaline comet assay. Therefore, DS and comet assays showed good utility for hazard identification and dose-response analysis of a representative N-nitrosamine comparable to the TGR gene mutation assay.

Growth inhibitory, immunosuppressive, cytotoxic, and genotoxic effects of γ-terpinene on Zeugodacus cucurbitae (Coquillett) (Diptera: Tephritidae)

γ-Terpinene, a monoterpene widely present in essential oils of many medicinal and aromatic plants with numerous biological properties, was evaluated for its insecticidal activity against melon fruit fly, Zeugodacus cucurbitae (Coquillett). Different concentrations (5, 25, 125, 625, and 3125 ppm) of γ-terpinene along with control were fed to larvae of melon fly. The number of pupae formed and adults emerged declined significantly after treatment. Morphologically deformed adults and pupae were also observed. The developmental duration too prolonged in treated larvae. Food assimilated, mean relative growth rate, larval weight gain, and pupal weight also declined. In the larvae treated with LC30 and LC50 concentrations, there was a decline in the titers of phenoloxidase and total hemocyte count, and variations were observed in the differential hemocyte count, suggesting an immunosuppressive effect of γ-terpinene on melon fly. Both concentrations also led to an increase in the apoptotic and necrotic cells as well as decrease in the viable hemocytes in the circulating hemolymph of treated larvae. Comet parameters (tail length, % tail DNA, tail moment, and olive tail moment) of γ-terpinene fed larvae increased significantly. Given the observed effects of γ-terpinene on normal developmental and nutritional physiology, its immunosuppressive properties, and its potential for genome damage, it can be considered for incorporation into integrated pest management strategies for controlling Z. cucurbitae.

Manganese nanoparticles control the gene regulations against multiple stresses in Pangasianodon hypophthalmus

Ammonia and arsenic pollution, along with the impact of climate change, represent critical factors influencing both the quantity and quality of aquaculture production. Recent developments have underscored the significance of these issues, as they not only disrupt aquatic ecosystems but also have far reaching consequences for human health. To addressed above challenges, an experiment was conducted to delineate the potential of manganese nanoparticles (Mn-NPs) to mitigate arsenic and ammonia pollution as well as high temperature stress in Pangasianodon hypophthalmus. The fish were exposed to different combination of arsenic and ammonia pollution as well as high temperature stress, while simultaneously incorporating diets enriched with Mn-NPs. The inclusion of Mn-NPs at 3 mg kg−1 in the diet led to a noteworthy downregulation of cortisol and HSP 70 gene expression, indicating their potential in mitigating stress responses. Furthermore, immune related gene expressions were markedly altered in response to the stressors but demonstrated improvement with the Mn-NPs diet. Interestingly, the expression of inducible nitric oxide synthase (iNOS), caspase (CAS), metallothionine (MT) and cytochrome P450 (CYP450) genes expression were prominently upregulated, signifying a stress response. Whereas, Mn-NPs at 3 mg kg−1 diet was significantly downregulated theses gene expression and reduces the stress. In addition to stress-related genes, we evaluated the growth-related gene expressions such as growth hormone (GH), growth hormone regulator 1 (GHR1 and GHRβ), Insulin like growth factor (IGF1 and IGF2) were significantly upregulated whereas, myostatin and somatostatin were downregulated upon the supplementation of dietary Mn-NPs with or without stressors in fish. The gene expression of DNA damage inducible protein and DNA damage in response to head DNA % and tail DNA % was protected by Mn-NPs diets. Furthermore, Mn-NPs demonstrated a capacity to enhance the detoxification of arsenic in different fish tissues, resulting in reduced bioaccumulation of arsenic in muscle and other tissues. This finding highlights Mn-NPs as a potential solution for addressing bioaccumulation associated risks. Our study aimed to comprehensively examined the role of dietary Mn-NPs in mitigating the multiple stressors using gene regulation mechanisms, with enhancing the productive performance of P. hypophthalmus.

Anti-inflammatory, antioxidant and photoprotective activity of standardised Gaultheria procumbens L. leaf, stem, and fruit extracts in UVA-irradiated human dermal fibroblasts

Ethnopharmacological relevanceGaultheria procumbens L. is a polyphenolic-rich medicinal and food plant. Its leaves, stems, and fruits are traditional anti-inflammatory, antipyretic, antioxidant, and antimicrobial herbal medicines used to treat internal and external inflammation-related ailments, including rheumatic diseases, influenza, the common cold, fever, and skin and periodontal problems. Moreover, G. procumbens leaf extract is used for skin care as an anti-ageing and anti-wrinkle ingredient. Aim of the studyVarious environmental factors, especially solar ultraviolet radiation, accelerate skin ageing by promoting oxidative stress and inflammation. Despite the dermoprotective and anti-ageing applications, the impact of G. procumbens on human dermal fibroblasts is unknown. Therefore, the study aimed to evaluate the anti-inflammatory, antioxidant, and photoprotective activity of G. procumbens standardised leaf, stem, and fruit extracts in cellular models, including human dermal fibroblasts (Hs68 cells) under UVA-irradiation, the primary pro-ageing skin stressor. Materials and methodsHs68 fibroblasts were pre-treated (24h) with G. procumbens extracts (0.5–100 μg/mL) or reference compounds followed by UVA-irradiation (8 J/cm2). Cell viability and metabolic activity were measured by CCK-8 and MTT assays in human Hs68 and mouse L929 fibroblasts, respectively. The ROS level, SOD, and GST activities were estimated by fluorescence and spectrophotometric techniques. The pro-inflammatory potential (NF-κB transcription factor activation) was checked using THP1-Blue™ NF-κB cells, and the anti-inflammatory activity was studied by measuring IL-8, ICAM-1, and NF-κB levels and phosphorylation of Erk kinase in LPS-stimulated Hs68 cells by spectrophotometry and confocal microscopy. The UVA-induced DNA damage and cell migration were evaluated by comet and scratch assays, respectively. ResultsThe extracts did not affect the metabolic activity of mouse L929 fibroblasts and the viability of unirradiated human Hs68 cells. Additionally, the extracts noticeably enhanced the viability of UVA-irradiated Hs68 cells up to 115–120% (p < 0.001) for stem and leaf extract at 25 μg/mL. All extracts in a wide concentration range (0.5–100 μg/mL) did not activate monocytes or induce the NF-κB transcription factor in LPS-stimulated Hs68 fibroblasts. On the other hand, the extracts (5–25 μg/mL) restored the activity of endogenous antioxidant enzymes, i.e., SOD and GST, up to 120–140% (p < 0.001) in the UVA-irradiated Hs68 cells. Moreover, a statistically significant reduction of ROS, IL-8, ICAM-1, and NF-κB levels by up to 48%, 88%, 43%, and 39%, respectively (p < 0.001) and strong suppression of Erk kinase activation was observed for the extracts (25–50 μg/mL) in LPS-stimulated human fibroblasts. The total DNA damage (% tail DNA) in irradiated Hs68 cells was also strongly decreased by up to 66–69% (p < 0.001) at 50 μg/mL. However, the treatment with the extracts did not relevantly enhance the cell migration of Hs68 fibroblasts. ConclusionsThe results suggest that G. procumbens may effectively protect human skin fibroblast from UVA irradiation. The leaf and stem extracts were the most potent antioxidants, while fruit and stem extracts revealed the strongest anti-inflammatory activity. The observed effects support the traditional use of aerial plant parts (leaves, stems, and fruits) in treating inflammation-related skin disorders cross-linked with oxidative stress and the topical application of Gaultheria extracts as anti-ageing agents intended for skin care.

DNA fragmentation as a bioindicator of peat fires’ smoke exposure

Introduction. The impact of combustion products of organic substances on the body is known to be accompanied by the accumulation of DNA damage, which can lead to mutations and pathological changes in the cell and the whole organism. The prevalence and scale of this phenomenon poses an important task for studying the consequences that occur in smoke-exposed organisms and their offspring. &#x0D; Materials and methods. The conditions of real peat smoke were reproduced for 40 minutes with a CO concentration of 99±2.5 mg/m3 with using of experimental bio modelling. The genotoxicity of peat fire smoke after exposure to male white rats was assessed by the occurrence of DNA damage in blood cells using the DNA comet method in the alkaline version. In the first part of the experiment, males were directly exposed to the smoke of a peat fire; in the second part – their sexually mature offspring of both sexes were examined for the occurrence of DNA damage in blood cells.&#x0D; Results. The males of the parental generation and offspring were found to be resistant to the effects of smoke components, which was confirmed by the absence of statistical significance in terms of «% DNA in the comet tail» compared with the control. At the same time, females of the received generation showed a statistically significant increase in blood cell DNA damage compared to the control group. &#x0D; Limitations. The study was limited to the study of DNA fragmentation after a single 40-minute exposure to peat smoke in male white rats and their intact offspring. &#x0D; Conclusion. The data obtained in this investigation indicate that damage to the DNA structure in the offspring of male rats exposed to peat smoke containing CO at a concentration of 99±2.5 mg/m3 can be considered as a bioindicator of genotoxic effects induced in the next generation.

Automated comet assay segmentation using combined dot enhancement filters and extended-maxima transform watershed segmentation

The comet assay, also known as single-cell gel electrophoresis, is a widely used and reliable method for assessing DNA damage and repair in individual cells. It plays a crucial role in the assessment of genetic damage potential and human biomonitoring studies in the medical and biological fields. Ensemble of comet assay individual cells and establishing accurate information on the occurrence of cellular injury followed by the process of cellular restoration is a challenging task. This paper introduces an algorithm for the detection of a distinct head, composed of undamaged DNA, and a tail, comprising damaged or fragmented DNA, in genotoxicity testing images, and provides information on the region properties of such images. The proposed approach combines a dot enhancement filter to distinguish and help in the detection of the head in each cell combined with a multilevel segmentation approach consisting of a watershed-geodesic active contour model that is capable to refine the tail estimation. The effectiveness of the suggested algorithm is quantitatively evaluated with annotation data provided by biologists, and its results are compared with those obtained from previous works. The proposed system exhibits comparable or superior performance to the existing systems while avoiding excessive computational costs.

Estimation of genomic and mitochondrial DNA integrity in the renal tissue of mice administered with acrylamide and titanium dioxide nanoparticles

The Kidneys remove toxins from the blood and move waste products into the urine. However, the accumulation of toxins and fluids in the body leads to kidney failure. For example, the overuse of acrylamide and titanium dioxide nanoparticles (TiO2NPs) in many food and consumer products increases human exposure and risks; however, there are almost no studies available on the effect of TiO2NPs coadministration with acrylamide on the integrity of genomic and mitochondrial DNA. Accordingly, this study was conducted to estimate the integrity of genomic and mitochondrial DNA in the renal tissue of mice given acrylamide and TiO2NPs. To achieve this goal, mice were administrated orally TiO2NPs or/and acrylamide at the exposure dose levels (5 mg/kg b.w) and (3 mg/kg b.w), respectively, five times per week for two consecutive weeks. Concurrent oral administration of TiO2NPs with acrylamide caused remarkable elevations in the tail length, %DNA in tail and tail moment with higher fragmentation incidence of genomic DNA compared to those detected in the renal tissue of mice given TiO2NPs alone. Simultaneous coadministration of TiO2NPs with acrylamide also caused markedly high elevations in the reactive oxygen species (ROS) production and p53 expression level along with a loss of mitochondrial membrane potential and high decreases in the number of mitochondrial DNA copies and expression level of β catenin gene. Therefore, from these findings, we concluded that concurrent coadministration of acrylamide with TiO2NPs augmented TiO2NPs induced genomic DNA damage and mitochondrial dysfunction through increasing intracellular ROS generation, decreasing mitochondrial DNA Copy, loss of mitochondrial membrane potential and altered p53 and β catenin genes expression. Therefore, further studies are recommended to understand the biological and toxic effects resulting from TiO2NPs with acrylamide coadministration.

Analyses of oxidative DNA damage among coal vendors via single cell gel electrophoresis and quantification of 8-hydroxy-2'-deoxyguanosine.

Looking at the development status of Nigeria and other developing nations, most low-income and rural households often use coal as a source of energy which necessitates its trade very close to the communities. Moreover, the effects of exposure to coal mining activities are rarely explored or yet to be studied, not to mention the numerous street coal vendors in Nigeria. This study investigated the oxidative stress levels in serum and urine through the biomarker 8-OHdG and DNA damage via single cell gel electrophoresis (alkaline comet assay). Blood and urine levels of 8-OHdG from 130 coal vendors and 130 population-based controls were determined by ELISA. Alkaline comet assay was also performed on white blood cells for DNA damage. The average values of 8-OHdG in serum and urine of coal vendors were 22.82 and 16.03ng/ml respectively, which were significantly greater than those detected in controls (p < 0.001; 15.46 and 10.40ng/ml of 8-OHdG in serum and urine respectively). The average tail length, % DNA in tail and olive tail moment were 25.06 μm, 18.71% and 4.42 respectively for coal vendors. However, for controls, the average values were 4.72 μm, 3.63% and 1.50 for tail length, % DNA in tail and olive tail moment respectively which were much lower than coal vendors (p < 0.001). Therefore, prolonged exposure to coal dusts could lead to higher serum and urinary 8-OHdG and significant DNA damage in coal vendors observed in tail length, % DNA in tail, and olive tail moment by single cell gel electrophoresis. It is therefore established that coal vendors exhibit a huge risk from oxidative stress and assessment of 8-OHdG with single cell gel electrophoresis has proven to be a feasible tool as biomarkers of DNA damage.

Genotoxicity assessment of titanium dioxide nanoparticles using a standard battery of in vivo assays

As one representative of nanometal oxides, titanium dioxide nanoparticles (TiO2-NPs) have been widely used, particularly in the food industry. The genotoxicity of TiO2-NPs has attracted great attention over the years. This study was undertaken to investigate the chromosome and DNA damage effects of TiO2-NPs (0, 50, 150, and 500 mg/kg BW) using rodent models. After a comprehensive characterization, we conducted a standard battery of in vivo genotoxicity tests, including the chromosomal aberration test (CA), micronucleus (MN) test, and the comet test. The results of all these tests were negative. There were no structural or numerical chromosomal abnormalities in mice bone marrow cells, no increase in the frequency of micronucleated polychromatic erythrocytes in mice bone marrow cells, and no elevation in % tail DNA in rat hepatocytes. This indicated that TiO2-NPs did not cause chromosomal damage or have a direct impact on DNA. These findings suggested that TiO2-NPs did not exhibit genotoxicity and provided valuable data for risk assessment purposes.

Elabela inhibits TRAF1/NF-κB induced oxidative DNA damage to promote diabetic foot ulcer wound healing

Diabetic foot ulcer (DFU) is a serious complication of diabetes. Elabela (ELA), a ligand of apelin receptor (APJ), was shown to promote angiogenesis and suppress inflammation. This study aimed to illustrate the role of ELA in DFU wound healing. A whole-skin defect model was constructed using db/m and db/db mice to observe the effects of ELA on wound healing. The function of ELA in endothelial cells cultured in high glucose medium was investigated. Administration of ELA in peri-wound area of db/db mice accelerated wound closure and reduced inflammatory infiltration. Indicators of DNA damage, elevated reactive oxygen species (ROS) levels and tail DNA amounts, were downregulated by ELA but compromised after TRAF1 overexpression. ELA-mediated inhibition of NF-κB phosphorylation improved cell migration and angiogenesis, which were blocked by APJ silencing. The findings imply that ELA suppresses TRAF1-mediated NF-κB signal activation, reducing ROS-related oxidative DNA damage and improving protection of endothelial function.

Modulatory effect of amifostine (WR-1065) against genotoxicity and oxidative stress induced by methotrexate in human umbilical vein endothelial cells (HUVECs)

Amifostine is used in chemotherapy and radiotherapy as a cytoprotective adjuvant alongside DNA-binding chemotherapeutic agents. It functions by reducing free radicals and detoxifying harmful metabolites. Methotrexate, as an antimetabolite drug has been considered for treating various cancers and autoimmune diseases. However, the cytotoxic effects of methotrexate extend beyond tumor cells to crucial organs, including the heart. This study applied the HUVEC cell line as a reference in vitro model for researching the characteristics of vascular endothelium and cardiotoxicity. The current study aimed to assess amifostine’s potential cytoprotective properties against methotrexate-induced cellular damage. Cytotoxicity was measured using the MTT assay. Apoptotic rates were evaluated by Annexin V-FITC/PI staining via flow cytometry. The genoprotective effect of amifostine was determined using the comet assay. Cells were exposed to various amifostine doses (10–200 μg/mL) and methotrexate (2.5 μM) in pretreatment culture condition. Methotrexate at 2.5 μM revealed cytotoxicity, apoptosis, oxidative stress and genotoxicity while highlighting amifostine’s cyto/geno protective properties on HUVECs. Amifostine significantly decreased the levels of ROS and LPO while preserving the status of GSH and SOD activity. Furthermore, it inhibited genotoxicity (tail length, %DNA in tail, and tail moment) in the comet assay. Amifostine markedly attenuated methotrexate-induced apoptotic cell death (early and late apoptotic rates). These findings convey that amifostine can operate as a cytoprotectant agent.

Assessment of the water quality and toxicity effects on zebrafish (Danio rerio) of a stream near a phosphorus chemical plant in Guizhou Province, southwestern China.

To reveal the influence of the phosphorus chemical industry (PCI) on regional water environmental quality and safety, the water quality and ecotoxicological effects of a stream near a phosphorus chemical plant (PCP) in Guizhou Province, southwestern China, were investigated based on water samples collected from the stream. The results showed that the average concentrations of NH3-N, TN, P, F-, Hg, Mn, and Ni were 3.14mg/L, 30.09mg/L, 3.34mg/L, 1.18mg/L, 1.06μg/L, 45.82μg/L, and 11.30μg/L, respectively. The overall water quality of the stream was in the heavily polluted category, and NH3-N, TN, P, F-, and Hg were the main pollution factors. The degree of pollution was in the order of rainy period > transitional period > dry period, and the most polluted sample site was 1100m from the PCP. After 28 days of exposure to stream water, there was no significant change in the growth parameters of zebrafish. The gills of zebrafish showed a small amount of epithelial cell detachment and a small amount of inflammatory cell infiltration, and the liver tissue displayed a large amount of hepatocyte degeneration with loose and lightly stained cytoplasm. Compared with the control group, the%DNA intail, tail length, tail moment, and olive tail moment were significantly increased (p < 0.05), indicating that the water sample caused DNA damage in the peripheral blood erythrocytes of zebrafish. The stream water in the PCI area was found to be polluted and exhibited significant toxicity to zebrafish, which could pose a threat to regional ecological security.

Gaining insight into genotoxicity with the comet assay in inhomogenoeous exposure scenarios: The effects of tritiated steel and cement particles on human lung cells in an inhalation perspective

The comet assay was recently applied for the first time to test the genotoxicity of micrometric stainless steel and cement particles, representative of those produced in the dismantling of nuclear power plants. A large dataset was obtained from in vitro exposure of BEAS-2B lung cells to different concentrations of hydrogenated (non-radiative control) and tritiated particles, to assess the impact of accidental inhalation. Starting from the distributions of the number of nuclei scored at different extent of DNA damage (% tail DNA values), we propose a new comet data treatment designed to consider the inhomogeneity of the action of such particles. Indeed, due to particle behavior in biological media and concentration, a large fraction of cells remains undamaged, and standard averaging of genotoxicity indicators leads to a misinterpretation of experimental results. The analysis we propose reaches the following goals: genotoxicity in human lung cells is assessed for stainless steel and cement microparticles; the role of radiative damage due to tritium is disentangled from particulate stress; the fraction of damaged cells and their average level of DNA damage are assessed separately, which is essential for carcinogenesis implications and sets the basis for a better-informed risk management for human exposure to radioactive particles.

Relationship between Lead Exposure and Genotoxic Effect in Paint Industry Workers

Lead-based paint is a main source of lead exposure to paint industry workers and causes an imbalance of Reactive Oxygen Species (ROS) and antioxidants, causing a genotoxic effect. Pb in the blood (PbB) level and DNA damage are frequently used as exposure and effect biomarker of lead. The purpose of this study to determine the relationship between PbB level and DNA damage due to occupational lead exposure in paint industry workers. The research design uses a cross-sectional epidemiological study involving 52 workers from three paint manufacturers in Indonesia. Blood samples were taken for PbB analysis using ICP-MS, while DNA damage was analyzed using the Comet Assay method. The PbB average obtained was 4.36±1.60 µg.dL-1, where 17 workers (32.69%) exceeded the safe limit value of PbB (5 µg.dL-1). Meanwhile, the influential factors of PbB are the working period and alcohol consumption (p=0.029). The level of DNA damage was represented as Tail DNA (%), and the average was 9.62±0.19 %. All respondents in this study were categorized as under low damage (Class 2). There was no significant relationship between PbB and Tail DNA (%) and has a negative correlation (p=0.878; r=-0.022). The study concludes that there was no difference in Tail DNA (%) between PbB ≥ 5 µg.dL-1 and PbB &lt; 5 µg.dL-1 (p=0.876). It means that lead exposure in this finding has not reached a level that can significantly cause DNA damage. However, it is necessary to monitor PbB levels in workers to minimize genotoxic or other effects.

Eco-friendly postharvest irradiation strategy with 131I isotope for environmental management of populations of migratory locust, Locusta migratoria

Purpose Irradiation of food is promising for control of pests to minimize postharvest losses of yields and thus improvement of food safety, shelf life of produce. It is a method of choice that induces a series of lethal biochemical and molecular changes culminating into the engagement of a downstream cascade to cause abnormalities in irradiated pests. In this study, the effects of iodine-131 (131I) isotope radiation on the male gonad development of the migratory locust, Locusta migratoria, were evaluated. Materials and methods Newly emerged adult male locusts, less than one-day-old, were divided into two groups, control and irradiated. Locusts in the control group (n = 20 insects) didn’t drink irradiated water and were reared under normal environmental conditions for one week. Locusts in the irradiated group (n = 20 insects) were exposed to irradiated water at a dose of 30 mCi and they were subsequently observed until they drank the whole quantity. Results At the end of the experiment, scanning and electron microscopic examination of testes obtained from irradiated locusts revealed several major abnormalities, including malformed nuclei of spermatozoa, irregular plasma membranes, shrinkage of testicular follicles, vacuolated cytoplasm, disintegrated nebenkern and agglutinations of spermatids. Flow cytometry analysis revealed that 131I radiation induced both early and late apoptosis, but not necrosis, in testicular tissues. Testes of irradiated insects also exhibited a burst in reactive oxygen species (ROS), as indicated by significant elevation in amounts of malondialdehyde (MDA), a marker for peroxidation of lipids. In contrast, irradiation coincided with significant reductions in activities of enzymatic antioxidant biomarkers. Relative to controls, a three-fold upregulation of expression of mRNA of heat shock protein, Hsp90, was observed in testicular tissue of irradiated locusts. 131I-irradiated insects exhibited genotoxicity, as indicated by significant increases in various indicators of DNA damage by the comet assay, including tail length (7.80 ± 0.80 µm; p < .01), olive tail moment (40.37 ± 8.08; p < .01) and tail DNA intensity % (5.1 ± 0.51; p < .01), in testicular cells compared to the controls. Conclusion This is the first report on elucidation of I131-irradiation-mediated histopathological, biochemical and molecular mechanisms in gonads of male L. migratoria. Herein, the findings underscore the utility of 131I radiation as an eco-friendly postharvest strategy for management of insect pests and in particular for control of populations of L. migratoria.

Evaluating the genotoxicity of salinity stress and secondary products gene manipulation in lime, Citrus aurantifolia, plants.

Salinity is a significant abiotic stress that has a profound effect on growth, the content of secondary products, and the genotoxicity of cells. Lime, Citrus aurantifolia, is a popular plant belonging to the family Rutaceae. The interest in cultivating this plant is due to the importance of its volatile oil, which is included in many pharmaceutical industries, but C. aurantifolia plants are affected by the NaCl salinity levels. In the present study, a comet assay test has been applied to evaluate the genotoxic impact of salinity at 0, 50, 100, and 200 mM of NaCl on C. aurantifolia tissue-cultured plants. Furthermore, terpene gene expression was investigated using a semi-quantitative real-time polymerase chain reaction. Results from the two analyses revealed that 200 mM of NaCl stress resulted in high levels of severe damage to the C. aurantifolia plants' DNA tail 21.8%, tail length 6.56 µm, and tail moment 3.19 Unit. The relative highest expression of RtHK and TAT genes was 2.08, and 1.693, respectively, when plants were exposed to 200 mM of NaCl, whereas pv4CL2RT expressed 1.50 in plants subjected to 100 mM of NaCl. The accumulation of transcripts for the RTMYB was 0.951 when plants were treated with NaCl at 50 mM, and RtGPPS gene was significantly decreased to 0.446 during saline exposure at 100 mM. We conclude that the comet assay test offers an appropriate tool to detect DNA damage as well as RtHK, TAT, and pv4CL2RT genes having post-transcriptional regulation in C. aurantifolia plant cells under salinity stress. Future studies are needed to assess the application of gene expression and comet assay technologies using another set of genes that show vulnerability to different stresses on lime and other plants.

Four Novel Caudoviricetes Bacteriophages Isolated from Baltic Sea Water Infect Colonizers of Aurelia aurita.

The moon jellyfish Aurelia aurita is associated with a highly diverse microbiota changing with provenance, tissue, and life stage. While the crucial relevance of bacteria to host fitness is well known, bacteriophages have often been neglected. Here, we aimed to isolate virulent phages targeting bacteria that are part of the A. aurita-associated microbiota. Four phages (Pseudomonas phage BSwM KMM1, Citrobacter phages BSwM KMM2-BSwM KMM4) were isolated from the Baltic Sea water column and characterized. Phages KMM2/3/4 infected representatives of Citrobacter, Shigella, and Escherichia (Enterobacteriaceae), whereas KMM1 showed a remarkably broad host range, infecting Gram-negative Pseudomonas as well as Gram-positive Staphylococcus. All phages showed an up to 99% adsorption to host cells within 5 min, short latent periods (around 30 min), large burst sizes (mean of 128 pfu/cell), and high efficiency of plating (EOP > 0.5), demonstrating decent virulence, efficiency, and infectivity. Transmission electron microscopy and viral genome analysis revealed that all phages are novel species and belong to the class of Caudoviricetes harboring a tail and linear double-stranded DNA (formerly known as Siphovirus-like (KMM3) and Myovirus-like (KMM1/2/4) bacteriophages) with genome sizes between 50 and 138 kbp. In the future, these isolates will allow manipulation of the A. aurita-associated microbiota and provide new insights into phage impact on the multicellular host.