Dose In Cells Research Articles

The organotin triphenyltin disrupts cholesterol signaling in mammalian ovarian steroidogenic cells through a combination of LXR and RXR modulation

Organotins, a chemical family with over 30 congeners to which humans are directly exposed to through food consumption, are a chemical class widely used as stabilizers in polyvinyl chloride, and biocides in antifouling products. Aside from tributyltin (TBT), toxicological information on other organotin congeners, such as triphenyltin (TPT), remains scarce. Our previous work has demonstrated that TBT can interfere with cholesterol trafficking in steroidogenic cells. Given their structural similarities, we hypothesized that TPT, similar to TBT, disrupts intracellular cholesterol transport and impairs steroidogenesis in ovarian theca cells. To test this, human and ovine primary ovarian theca cells were isolated, purified and exposed to TPT at environmentally relevant doses (1 or 10 ng/ml) in pre-luteinized (48 h exposure) or luteinizing cells (72 h exposure). Intracellular cholesterol levels, progesterone, and testosterone secretion and gene expression of nuclear receptors, cholesterol transporters, and steroidogenic enzymes were evaluated. In ovine cells, TPT upregulated StAR, ABCA1, and SREBF1 mRNA and ABCA1 protein in both pre-luteinized and luteinized stages. TPT did not alter intracellular cholesterol or testosterone synthesis, but upregulated progesterone production. Inhibitor and shRNA knockdown approaches were then used to evaluate the role of retinoid X receptor (RXR) and liver X receptor (LXR) on TPT's effects. TPT upregulated ABCA1 and StAR expression was blocked by both LXR and RXR antagonists. TPT's effect on ABCA1 expression was reduced in LXRβ and RXRβ knockdown theca cells. Similar findings were obtained with primary human theca cells. No synergistic effect of TBT and TPT was observed. In conclusion, at an environmentally relevant dose, TPT upregulates theca cell cholesterol transporter ABCA1 expression via RXR and LXR pathways. Similar effects of TPT on human and sheep theca cells supports its conserved mechanism across mammalian theca cells.

Albumin-Based Nanocarriers for the Simultaneous Delivery of Antioxidant Gene and Phytochemical to Combat Oxidative Stress.

Human serum albumin (HSA) nanoparticles are promising biocompatible, nontoxic, and non-immunogenic platforms for biomedical applications such as bioimaging and drug and gene delivery. The development of nonviral gene delivery vectors is a great challenge for efficient and safe gene therapy. Sulforaphane (SF) can stimulate the expression of antioxidant genes via activation of a nuclear transcription factor, the erythroid-2 related factor 2 (Nrf-2). Here, we use polyethyleneimine (PEI)-stabilized HSA nanoparticles to stimulate endogenous antioxidant defense mechanisms in lung epithelial cells L-132 through the combinatorial effect of SF drug and antioxidant superoxide dismutase 1 gene (pSOD1 plasmid) delivered by HSA-PEI-SF-pSOD1 nanocomposites (NCs). The developed NCs demonstrated high biocompatibility (L-132 viability, >95%, MTT assay) and high antioxidant activity because of efficient entry of the SOD1 gene and SF-loaded NCs at a very low (3 μg) dose in L-132 cells. A high transfection efficiency of L-132 cells (∼66%, fluorescent microscopy) was obtained with the GFP-tagged transgene SOD1-GFP. We speculate that the antioxidant activity of HSA-PEI-SF-pSOD1 NCs in L-132 cells is due to the initial release of SF followed by subsequent SOD1 gene expression after three to four days of incubation. Hence, the developed HSA-based NCs can be efficient biocompatible nanocarriers for safe and effective drug and gene delivery applications to treat diseases with high oxidative stress due to combinatorial SF and SOD1 gene mechanisms.

INVESTIGATION OF CIRCULAR RNAs AS BIOMARKERS IN HEAD AND NECK CANCER CELLS AND THE EFFECTS OF RADIOTHERAPY ON CIRCULAR RNAs

Objective: The head and neck squamous cell carcinoma (HNSCC) is the sixth most common type of cancer worldwide and the 5-year survival rate of patients is by 40-50 %. Radiotherapy is one of the important treatments for HNSCC. circRNAs, which are much more stabil than other RNAs because of their circular structure, have the potential to be a biomarker for all types of cancer. Therefore, the alterations of circRNAs expression in HNSCC cells and after irradiation was investigated. Methods: Pharyngeal cancer (Detroit-562) and healthy fibroblast (WI-38) cell lines were used. After appropriate irradiation dose for Detroit-562 cells was determined by colony formation assay, Detroit-562 cells were irradiated with 4 grays. The expression levels of 122 circRNA molecules in all groups were examined by RT-qPCR. The results were evaluated using CircNet and TCGA platforms. Results: In this study, 60 circRNAs were not expressed in any cell line, whereas 62 were found to be expressed in at least one of the groups, including irradiated Detroit-562, Detroit-562, and WI-38 cell lines. Among them, seven circRNAs were detected in all groups and expressed higher in Detroit-562 than two groups. Interestingly, these circRNAs were significantly decreased in irradiated group, exhibiting similar expression levels with healthy group. Bioinformatics analyses showed that the host genes of these circRNAs was also higher expressed in HNSCC tissue. Conclusions: Differential expression of circRNAs in cancers and changes with treatment revealed that they could be potential diagnostic and monitoring biomarkers in cancer. However, further studies need in this regard.

Puerarin attenuates isoproterenol‑induced myocardial hypertrophy via inhibition of the Wnt/β‑catenin signaling pathway.

Myocardial hypertrophy (MH) is an independent risk factor for cardiovascular disease, which in turn lead to arrhythmia or heart failure. Therefore, attention must be paid to formulation of therapeutic strategies for MH. Puerarin is a key bioactive ingredient isolated from Pueraria genera of plants that is beneficial for the treatment of MH. However, its molecular mechanism of action has not been fully determined. In the present study, 40 µM puerarin was demonstrated to be a safe dose for human AC16 cells using Cell Counting Kit-8 assay. The protective effects of puerarin against MH were demonstrated in AC16 cells stimulated with isoproterenol (ISO). These effects were characterized by a significant decrease in surface area of cells (assessed using fluorescence staining) and mRNA and protein expression levels of MH-associated biomarkers, including atrial and brain natriuretic peptide, assessed using reverse transcription-quantitative PCR and western blotting, as well as β-myosin heavy chain mRNA expression levels. Mechanistically, western blotting demonstrated that puerarin inhibited activation of the Wnt signaling pathway. Puerarin also significantly decreased phosphorylation of p65; this was mediated via crosstalk between the Wnt and NF-κB signaling pathways. An inhibitor (Dickkopf-1) and activator (IM-12) of the Wnt signaling pathway were used to demonstrate that puerarin-mediated effects alleviated ISO-induced MH via the Wnt signaling pathway. The results of the present study demonstrated that puerarin pre-treatment may be a potential therapeutic strategy for preventing ISO-induced MH and managing MH in the future.

Low-Dose Rifabutin Increases Cytotoxicity in Antimitotic-Drug-Treated Resistant Cancer Cells by Exhibiting Strong P-gp-Inhibitory Activity

The cytotoxicity of various antibiotics at low doses in drug-resistant cancer cells was evaluated. Low doses of rifabutin were found to markedly increase the cytotoxicity of various antimitotic drugs, such as vincristine (VIC), to P-glycoprotein (P-gp)-overexpressing antimitotic-drug-resistant KBV20C cells. Rifabutin was also found to exert high levels of P-gp-inhibitory activity at 4 and 24 h posttreatment, suggesting that the cytotoxicity of VIC + rifabutin was mainly due to the direct binding of rifabutin to P-gp and the reduction of VIC efflux by P-gp. The combination of VIC + rifabutin also increased early apoptosis, G2 arrest, and the DNA damaging marker, pH2AX protein. Interestingly, only the combination of VIC + rifabutin induced remarkable levels of cytotoxicity in resistant KBV20C cells, whereas other combinations (VIC + rifampin, VIC + rifapentine, and VIC + rifaximin) induced less cytotoxicity. Such finding suggests that rifabutin specifically increases the cytotoxicity of VIC in KBV20C cells, independent of the toxic effect of the ansamycin antibiotic. Only rifabutin had high P-gp-inhibitory activity, which suggests that its high P-gp-inhibitory activity led to the increased cytotoxicity of VIC + rifabutin. As rifabutin has long been used in the clinic, repositioning this drug for P-gp-overexpressing resistant cancer could increase the availability of treatments for patients with drug-resistant cancer.

Novel antigens for targeted radioimmunotherapy in hepatocellular carcinoma.

Liver cancer is the sixth common cancer and forth cause of cancer-related death worldwide. Based on usually advanced stages of hepatocellular carcinoma (HCC) at the time of diagnosis, therapeutic options are limited and, in many cases, not effective, and typically result in the tumor recurrence with a poor prognosis. Radioimmunotherapy (RIT) offers a selective internal radiation therapy approach using beta or alpha emitting radionuclides conjugated with tumor-specific monoclonal antibodies (mAbs), or specific selective peptides. When compared to chemotherapy or radiotherapy, radiolabeled mAbs against cancer-associated antigens could provide a high therapeutic and exclusive radiation dose for cancerous cells while decreasing the exposure-induced side effects to healthy tissues. The recent advances in cancer immunotherapy, such as blockade of immune-checkpoint inhibitors (ICIs), has changed the landscape of cancer therapy, and the efficacy of different classes of immunotherapy has been tested in many clinical trials. Taking into account the use of ICIs in the liver tumor microenvironment, combined therapies with different approaches may enhance the outcome in the future clinical studies. With the development of novel immunotherapy treatment options in the recent years, there has been a great deal of information about combining the diverse treatment modalities to boost the effectiveness of immunomodulatory drugs. In this opinion review, we will discuss the recent advancements in RIT. The current status of immunotherapy and internal radiotherapy will be updated, and we will propose novel approaches for the combination of both techniques. Potential target antigens for radioimmunotherapy in Hepatocellular carcinoma (HCC). HCC radioimmunotherapy target antigens are the most specific and commonly accessible antigens on the surface of HCC cells. CTLA-4 ligand and receptor, TAMs, PD-1/PD-L, TIM-3, specific IEXs/TEXs, ROBO1, and cluster of differentiation antigens CD105, CD147 could all be used in HCC radioimmunotherapy. Abbreviations: TAMs, tumor-associated macrophages; CTLA-4, cytotoxic T-lymphocyte associated antigen-4; PD-1, Programmed cell death protein 1; PD-L, programmed death-ligand1; TIM-3, T-cell immunoglobulin (Ig) and mucin-domain containing protein-3; IEXs, immune cell-derived exosomes; TEXs, tumor-derived exosomes.

Integrating Sex Chromosome and Endocrine Theories to Improve Teaching of Sexual Differentiation.

Major sex differences in mammalian tissues are functionally tied to reproduction and evolved as adaptations to meet different reproductive needs of females and males. They were thus directly controlled by gonadal hormones. Factors encoded on the sex chromosomes also cause many sex differences in diverse tissues because they are present in different doses in XX and XY cells. The sex chromosome effects likely evolved not because of demands of reproduction, but as side effects of genomic forces that adaptively reduced sexual inequality. Sex-specific effects of particular factors, including gonadal hormones, therefore, are not necessarily explained as adaptations for reproduction, but also as potential factors offsetting, rather than producing, sex differences. The incorporation of these concepts would improve future teaching about sexual differentiation.

СРАВНИТЕЛЬНЫЙ АНАЛИЗ СТОЙКОСТИ РЕЗИСТИВНОЙ И ДРУГИХ ВИДОВ ЭНЕРГОНЕЗАВИСИМОЙ ПАМЯТИ К ВОЗДЕЙСТВИЮ ИОНИЗИРУЮЩЕГО ИЗЛУЧЕНИЯ КОСМИЧЕСКОГО ПРОСТРАНСТВА

The aim of the study is to consider the prospects of using resistive memory microcircuits (RRAM, CBRAM) for space applications. Total dose and single event effects in resistive memory test cells and finished CMOS RRAM microcircuits were investigated. A comparative assessment of typical radiative hardness levels for other types of non-volatile memories such as flash, ferroelectric (FRAM), magnetoresistive (MRAM) memory was provided in this study. The main advantages and disadvantages of using various technologies for the manufacture of non-volatile memory microcircuits with increased resistance to ionizing effects are described.

Co-treatment of Low Dose Pacritinib, a Phase III Jak2 Inhibitor, Greatly Increases Apoptosis of P-gp Over-expressing Cancer Cells With Multidrug Resistance.

The over-expression of P-glycoprotein (P-gp) is a major mechanism underlying multidrug resistance (MDR). Co-treatment with Janus kinase 2 (Jak2) inhibitors sensitizes P-gp-over-expressing drug-resistant cancer cells. In this study, we evaluated pacritinib, a Jak2 inhibitor currently in phase III clinical trials. Microscopic observation, cell viability assay, colony forming assay, rhodamine uptake tests, annexin V analyses, fluorescence-activated cell sorting (FACS), and western-blot analysis were performed to further investigate the mechanism of action. We found that pacritinib reduced cell viability, induced G2 arrest, and upregulated early apoptosis when administered to P-gp-over-expressing resistant KBV20C cells with vincristine (VIC). Moreover, apoptosis and G2 arrest in VIC-pacritinib-treated cells were involved in the upregulation of pH2AX expression. Pacritinib had an approximately 2-fold higher P-gp-inhibitory activity than the dimethyl sulfoxide (DMSO)-treated control, indicating that VIC-pacritinib sensitization involves the P-gp-inhibitory effects of pacritinib. Similar to VIC, other antimitotic drugs (vinorelbine, vinblastine, and eribulin) could also sensitize against KBV20C cells by co-treatment with pacritinib. Furthermore, comparison of pacritinib with previously characterized Jak2 inhibitors revealed that the VIC-pacritinib combination had sensitization effects similar to those of VIC- CEP-33779 or VIC-NVP-BSK805 combinations at lower doses in KBV20C cells. Generally, Jak2 inhibitor and VIC co-treatment sensitized P-gp-over-expressing resistant cancer cells by inducing early apoptosis. Collectively, pacritinib, induced G2 arrest, reduced cell viability, had high P-gp inhibitory activity, and upregulated the expression of pH2AX when used in combination with VIC. As pacritinib is a Jak2 inhibitor currently in phase III clinical trials, our findings may facilitate the application of this co-treatment in patients with MDR cancer.

Analysis of dose distribution in proton therapy for lung cancer with MCNP code

Proton beam characteristics for proton therapy have been obtained by simulation method using MCNP6 software. The proton beam is modelled as a monodirectional disk with a diameter of 3 cm is 23 cm from the left lung cancer, a sphere with a diameter of 3 cm. The variation of proton beam energy that produces the best isodose in cancer cells is 94 MeV, 104 MeV, and 112 MeV fired alternately at the target from the left. The result is a total equivalent dose in cancer cells of (0.858 ± 0.003) Sv. The scattered amount received by healthy cells in the left lung was (1.39 ± 0.01) mSv, rib (0.12 ± 0.01) mSv, and skin (0.11 ± 0.01) mSv. According to the organ at risk (OAR) provisions, the scattered dose is declared safe. The simulation results prove that proton therapy is a cell targeting therapy. The dose used to kill lung cancer cells is 60 Gy, so with a proton beam of 1 µA, the total exposure time for therapy is (2.31 ± 0.01) minutes.

Effect of Malvidin on Induction of Apoptosis and Inhibition of Cell Proliferation on Myeloid and Lymphoid Leukemia

Malvidin is one of 6 most common anthocyanidins found in fruits and vegtables. studies suggest that anthocyanidins are bioactive chemicals have potential for treatment of cancer. Malvidin have been demonstrated to induce apoptosis and inhibition of cell proliferation in a number of cancer cell lines. However, to date, few studies have tested the potential of malvidin in the treatment of leukemia. In the present study, we investigated the antiproliferative and proapoptotic properties of malvidin in two human leukemia cell lines lymphoid cell line: SUP-B15 (acute lymphoblastic leukemia) and myeloid cell line: KG-1a (acute myelogenous leukemia) in addition to non-tumour control cells (CD133+ HSC). We found that treatment of cells with malvidin at concentration 0, 25, 50 and 100 µM for 24 h resulted in significant inhibition of cell proliferation in both leukemia cell lines with obvious less cytotoxic effect on non-tumour control cells. In addition, malvidin induce apoptosis in a dose and dependent manner in both leukemia cells and confirmed that by activation of caspas-3 following 24 h treatment. Cell arrest at S phase has been indicated in both cell lines using DNA analysis by flow cytometry. These finding indicated that malvidin could inhibit leukemic cell proliferation and induce their apoptosis and might be used as one kind of functional food component or a novel nutraceutical beneficial for blood health.

Shift in G1-Checkpoint from ATM-Alone to a Cooperative ATM Plus ATR Regulation with Increasing Dose of Radiation.

The current view of the involvement of PI3-kinases in checkpoint responses after DNA damage is that ATM is the key regulator of G1-, S- or G2-phase checkpoints, that ATR is only partly involved in the regulation of S- and G2-phase checkpoints and that DNA-PKcs is not involved in checkpoint regulation. However, further analysis of the contributions of these kinases to checkpoint responses in cells exposed to ionizing radiation (IR) recently uncovered striking integrations and interplays among ATM, ATR and DNA-PKcs that adapt not only to the phase of the cell cycle in which cells are irradiated, but also to the load of DNA double-strand breaks (DSBs), presumably to optimize their processing. Specifically, we found that low IR doses in G2-phase cells activate a G2-checkpoint that is regulated by epistatically coupled ATM and ATR. Thus, inhibition of either kinase suppresses almost fully its activation. At high IR doses, the epistatic ATM/ATR coupling relaxes, yielding to a cooperative regulation. Thus, single-kinase inhibition suppresses partly, and only combined inhibition suppresses fully G2-checkpoint activation. Interestingly, DNA-PKcs integrates with ATM/ATR in G2-checkpoint control, but functions in its recovery in a dose-independent manner. Strikingly, irradiation during S-phase activates, independently of dose, an exclusively ATR-dependent G2 checkpoint. Here, ATM couples with DNA-PKcs to regulate checkpoint recovery. In the present work, we extend these studies and investigate organization and functions of these PI3-kinases in the activation of the G1 checkpoint in cells irradiated either in the G0 or G1 phase. We report that ATM is the sole regulator of the G1 checkpoint after exposure to low IR doses. At high IR doses, ATM remains dominant, but contributions from ATR also become detectable and are associated with limited ATM/ATR-dependent end resection at DSBs. Under these conditions, only combined ATM + ATR inhibition fully abrogates checkpoint and resection. Contributions of DNA-PKcs and CHK2 to the regulation of the G1 checkpoint are not obvious in these experiments and may be masked by the endpoint employed for checkpoint analysis and perturbations in normal progression through the cell cycle of cells exposed to DNA-PKcs inhibitors. The results broaden our understanding of organization throughout the cell cycle and adaptation with increasing IR dose of the ATM/ATR/DNA-PKcs module to regulate checkpoint responses. They emphasize notable similarities and distinct differences between G1-, G2- and S-phase checkpoint regulation that may guide DSB processing decisions.

Extracts of Tagetes erecta exhibit potential cytotoxic and antitumor activity that could be employed as a promising therapeutic agent against cancer: A study involving in vitro and in vivo approach

Background and purposeGlobally, the burden of cancer is increasing consistently. Modern cancer therapies include lots of toxicity in the non-targeted organs reducing the life expectancy of the patients. Therefore, the development of safer alternative medicines with less toxicity and high efficacy is of immense importance. The present study was designed to evaluate the anticancer activity of a medicinal plant, “Tagetes erecta L.” (TE), in established cancer cell lines in vitro and in animal models in vivo. MethodsGC–MS analysis was performed to reveal the bioactive compounds present in the leaves of TE. To evaluate the antitumor potential of aqueous extract of TE (AETE), EAC (Ehrlich ascites carcinoma) induced solid tumor model was prepared in Swiss albino mice. The effect of AETE on body weight (BW), tumor weight (TW), and tumor volume (TV) was assessed in control and tumor-bearing animals. Further, the survival of the experimental animals was also monitored to assess the effect of AETE on life span. To determine the cytotoxicity induced by AETE, trypan blue dye exclusion assay, MTT assay, and LDH release assay was performed in EAC and HEp-2 cell lines by in vitro methods. Further, cell cycle assay and apoptosis assay was employed to ascertain the AETE-induced cytotoxicity is due to apoptosis or not. In addition, we have done a mitochondrial membrane potential assay using a flow cytometer to understand the mechanism of apoptosis. ResultsGC–MS analysis revealed the presence of hexadecanoic acid, Linolenic acid, Quinic acid, 2,3- dihydro benzofuran (Coumaran), and β-stigmasterol as major bioactive compounds in TE leaves. Results in animal studies showed that AETE treatment potentially reduced the TW and TV and increased the life span in EAC-induced tumor-bearing animals. Side effect analysis was also done in normal Swiss albino mice that confirmed the lack of toxicity of AETE. Studies in cancer cell lines indicated dose and time-dependent cytotoxicity in HEp-2 and EAC cells. Flow cytometric analysis established significant induction of apoptosis in EAC cells without arresting the cell cycle. In addition, AETE treatment led to a significant increase in cells with depolarised mitochondrial membrane potential. ConclusionThe present study indicated that AETE potentially inhibits tumor progression without disturbing normal body physiology. Thus, we conclude that AETE can be used as a potential therapeutic agent against cancer.

Preparation of Liposomal Formulations for Ocular Delivery of Thymoquinone: In Vitro Evaluation in HCEC-2 e HConEC Cells.

Thymoquinone (TQ) is the main constituent of Nigella sativa L. essential oil. In vitro studies have shown its protective effect against H2O2-induced oxidative stress in human retinal pigment epithelium cells, and in vivo experiments have demonstrated its effect in decreasing corneal neovascularization and reducing the inflammation in an experimental dry eye model in mice. Its therapeutic use is limited by poor bioavailability, low solubility, and scarce permeability. In this study, two liposomal formulations have been developed, both of which consist of phosphatidylcholine and Plurol Oleique, a liquid lipid, and one of which is coated with 0.1% w/v hyaluronic acid (HA) to increase both TQ solubility and its ocular therapeutic potential. Each formulation has a size <200 nm and an EE% around 70%, determined by scattering techniques and the HPLC-DAD analytical method, respectively, and they result in a 2-fold increase in TQ solubility. HA-coated liposomes are stable over 2 months at +4 °C, and coated and uncoated liposomes present a gradual and prolonged release of TQ. Two cell lines, human corneal epithelial cells (HCEC-2) and human conjunctival epithelial cells (HConEC) were used to investigate the safety of the liposomal formulations. Uptake studies were also performed using fluorescent liposomes. Both liposomes and, in particular, HA-coated liposomes reduce the TQ toxicity observed at high doses in both HCEC-2 and HConEC cells, and both formulations increase the absorption at the cellular level and especially at the nucleus level, with a more pronounced effect for HA-coated liposomes.

High-dose vitamin D metabolite delivery inhibits breast cancer metastasis.

Besides its well‐known benefits on human health, calcitriol, the hormonally active form of vitamin D3, has been being evaluated in clinical trials as an anticancer agent. However, currently available results are contradictory and not fundamentally deciphered. To the best of our knowledge, hypercalcemia caused by high‐dose calcitriol administration and its low bioavailability limit its anticancer investigations and translations. Here, we show that the one‐step self‐assembly of calcitriol and amphiphilic cholesterol‐based conjugates leads to the formation of a stable minimalist micellar nanosystem. When administered to mice, this nanosystem demonstrates high calcitriol doses in breast tumor cells, significant tumor growth inhibition and antimetastasis capability, as well as good biocompatibility. We further reveal that the underlying molecular antimetastatic mechanisms involve downregulation of proteins facilitating metastasis and upregulation of paxillin, the key protein of focal adhesion, in primary tumors.

Monte Carlo Simulation-Based Calculations of Complex DNA Damage for Incidents of Environmental Ionizing Radiation Exposure

In this paper, we present a useful Monte Carlo (MC)-based methodology that can be utilized to calculate the absorbed dose and the initial levels of complex DNA damage (such as double strand breaks-DSBs) in the case of an environmental ionizing radiation (IR) exposure incident (REI) i.e., a nuclear accident. Our objective is to assess the doses and complex DNA damage by isolating only one component of the total radiation released in the environment after a REI that will affect the health of the exposed individual. More specifically, the radiation emitted by radionuclide 137Cs in the ground (under the individual’s feet). We use a merging of the Monte Carlo N-Particle Transport code (MCNP) with the Monte Carlo Damage Simulation (MCDS) code. The DNA lesions have been estimated through simulations for different surface activities of a 137Cs ground-based γ radiation source. The energy spectrum of the emitted secondary electrons and the absorbed dose in typical mammalian cells have been calculated using the MCNP code, and then these data are used as an input in the MCDS code for the estimation of critical DNA damage levels and types. As a realistic application, the calculated dose is also used to assess the Excess Lifetime Cancer Risk (ELCR) for eight hypothetical individuals, living in different zones around the Chernobyl Nuclear Power Plant, exposed to different time periods at the days of the accident in 1986. We conclude that any exposition of an individual in the near zone of Chernobyl increases the risk of cancer at a moderate to high grade, connected also with the induction of complex DNA damage by radiation. Generally, our methodology has proven to be useful for assessing γ rays-induced complex DNA damage levels of the exposed population, in the case of a REI and for better understanding the long-term health effects of exposure of the population to IR.

From single-molecule to genome-wide mapping of DNA lesions: repair-assisted damage detection sequencing

Mapping DNA damage and its repair has immense potential in understanding environmental exposures, their genotoxicity, and their impact on human health. Monitoring changes in genomic stability also aids in the diagnosis of numerous DNA-related diseases, such as cancer, and assists in monitoring their progression and prognosis. Developments in recent years have enabled unprecedented sensitivity in quantifying the global DNA damage dose in cells via fluorescence-based analysis down to the single-molecule level. However, genome-wide maps of DNA damage distribution are challenging to produce. Here, we describe the localization of DNA damage and repair loci by repair-assisted damage detection sequencing (RADD-seq). Based on the enrichment of damage lesions coupled with a pull-down assay and followed by next-generation sequencing, this method is easy to perform and can produce compelling results with minimal coverage. RADD-seq enables the localization of both DNA damage and repair sites for a wide range of single-strand damage types. Using this technique, we created a genome-wide map of the oxidation DNA damage lesion 8-oxo-7,8-dihydroguanine before and after repair. Oxidation lesions were heterogeneously distributed along the human genome, with less damage occurring in tight chromatin regions. Furthermore, we showed repair is prioritized for highly expressed, essential genes and in open chromatin regions. RADD-seq sheds light on cellular repair mechanisms and is capable of identifying genomic hotspots prone to mutation.

Regulations of expressions of rat/human sulfotransferases by anticancer drug, nolatrexed, and micronutrients.

Cancer is related to the cellular proliferative state. Increase in cell-cycle regulatory function augments cellular folate pool. This pathway is therapeutically targeted. A number of drugs influences this metabolism, that is, folic acid, folinic acid, nolatrexed, and methotrexate. Our previous study showed methotrexate influences on rat/human sulfotransferases. Present study explains the effect of nolatrexed (widely used in different cancers) and some micronutrients on the expressions of rat/human sulfotransferases. Female Sprague-Dawley rats were treated with nolatrexed (01-100 mg/kg) and rats of both sexes were treated to folic acid (100, 200, or 400 mg/kg) for 2-weeks and their aryl sulfotransferase-IV (AST-IV; β-napthol sulfation) and sulfotransferase (STa; DHEA sulfation) activities, protein expression (western blot) and mRNA expression (RT-PCR) were tested. In human-cultured hepatocarcinoma (HepG2) cells nolatrexed (1 nM-1.2 mM) or folinic acid (10 nM-10 μM) were applied for 10 days. Folic acid (0-10 μM) was treated to HepG2 cells. PPST (phenol catalyzing), MPST (dopamine and monoamine), DHEAST (dehydroepiandrosterone and DHEA), and EST (estradiol sulfating) protein expressions (western-blot) were tested in HepG2 cells. Present results suggest that nolatrexed significantly increased sulfotransferases expressions in rat (protein, STa, F = 4.87, P < 0.05/mRNA, AST-IV, F = 6.702, P < 0.014; Student's t test, P < 0.01-0.05) and HepG2 cells. Folic acid increased sulfotransferases activity/protein in gender-dependant manner. Both folic and folinic acid increased several human sulfotransferases isoforms with varied level of significance (least or no increase at highest dose) in HepG2 cells pointing its dose-dependent multiphasic responses. The clinical importance of this study may be furthered in the verification of sulfation metabolism of several exogenous/endogenous molecules, drug-drug interaction and their influences on cancer pathophysiological processes. Further studies are necessary.

Anti-inflammatory and prolonged protective effects of Artemisia herba-alba extracts via glutathione metabolism reinforcement

Artemisia herba-alba (AHA) is a medicinal plant known for its preventive capacities against chronic diseases. Inflammation is a naturally occurring cellular response to noxious stimuli, and both acute and chronic inflammation are involved in human disorders. Preventing and limiting the negative impact of inflammation by using phytocompounds has attracted a lot of attention. Here, we show that AHA extracts are potent anti-inflammatory products at low doses in white blood cells, treated with phorbol myristate acetate (PMA), a pro-inflammatory trigger. Regardless of the mode of extraction, i.e. maceration or microwave, all extracts reduced on dose-dependent manner reactive oxygen species (ROS) production induced by the pro-inflammatory compound PMA in white blood cells and leukemic Jurkat cell line. The induction of the anti-inflammatory effect was evaluated by the measurement of glutathione metabolism (glutathione reductase, transferase, and peroxidase), and protein-free thiols in Jurkat cells. Jurkat cells incubated with low to high concentrated AHA extracts, ranged from 10 to 100 g/mL, induced a fast cell response measured at one and six hours. Interestingly, extracts from the microwave showed a stronger induction, 5 to 10 fold changes at early time points, and maintained over 72 h. On the other hand, AHA extracts showed a moderate anti-proliferative effect in the leukemic Jurkat line. We concluded that AHA extracts enhance directly anti-inflammatory cell response, earlier via ROS induction. Later, however, vigorously maintained, independently from ROS, suggesting the intervention of the cellular metabolism in enhancing these effects. Considering the preventive and anti-inflammatory potential of AHA, regular consumption is advisable, and phytotherapeutic applications are encouraged to treat local inflammations.

Comparison of biological and transcriptomic effects of conventional cigarette and electronic cigarette smoke exposure at toxicological dose in BEAS-2B cells

Cigarette seriously affects human health, and electronic cigarette (e-cigarette), considered as cigarette substitutes, become popular as its contribution to quit smoking. But scientific evidence about the absolute safety of e-cigarette is insufficient. Previous studies also have indicated that different dosages of cigarette can lead to different biological effects. Thus, the impact of cigarette at toxicological dose such as IC50 compared with that of e-cigarette are highly needed. In this study, we investigated the effects of cigarette smoke condensate (CSC) at toxicological dose compared with e-cigarette smoke condensate (ECSC) in equivalent nicotine level. Nicotine content of CSC and ECSC were determined by UPLC. Human lung epithelial cells (BEAS-2B) were exposed to 0–32 μg/ml of CSC and ECSC for 24 h to determine IC50 of cell viability and morphological assessment. Inflammation, apoptosis, cell cycle analysis and RNA-Seq transcriptome analysis were performed to characterize the differences between CSC and ECSC. We found that acute exposure of BEAS-2B cells to CSC at IC50 leaded to morphological change, inflammatory cytokines production and cell apoptosis, while ECSC did not exert such cell effects in equivalent nicotine level. The transcriptome analysis showed that differentially expressed genes in CSC were far more than that in ECSC, and mainly enriched in the category of cell cycle, DNA repair, cancer, and metabolic related pathways. Such cell cycle arrest was further experimentally confirmed. These results suggested that toxicological dose of ECSC might be much higher than that of CSC. Based on equivalent nicotine content, an acute exposure to CSC had significant impacts on cell effects and gene expression profile compared to ECSC. Our results provided a reference for the safety studies of conventional cigarette and e-cigarette.