4NQO Research Articles

An Ultra-Sensitive Electrochemical Sensor for the Detection of Carcinogen Oxidative Stress 4-Nitroquinoline N-Oxide in Biologic Matrices Based on Hierarchical Spinel Structured NiCo2O4 and NiCo2S4; A Comparative Study.

Various factors leads to cancer; among them oxidative damage is believed to play an important role. Moreover, it is important to identify a method to detect the oxidative damage. Recently, electrochemical sensors have been considered as the one of the most important techniques to detect DNA damage, owing to its rapid detection. However, electrode materials play an important role in the properties of electrochemical sensor. Currently, researchers have aimed to develop novel electrode materials for low-level detection of biomarkers. Herein, we report the facile hydrothermal synthesis of NiCo2O4 micro flowers (MFs) and NiCo2S4 micro spheres (Ms) and evaluate their electrochemical properties for the detection of carcinogen-causing biomarker 4-nitroquinoline n-oxide (4-NQO) in human blood serum and saliva samples. Moreover, as-prepared composites were fabricated on a glass carbon electrode (GCE), and its electrochemical activities for the determination of 4-NQO were investigated by using various electrochemical techniques. Fascinatingly, the NiCo2S4-Ms showed a very low detection limit of 2.29 nM and a wider range of 0.005 to 596.64 µM for detecting 4-NQO. Finally, the practical applicability of NiCo2S4-Ms in the 4-NQO spiked human blood serum and saliva samples were also investigated.

Cytoprotective effects of (E)-N-(2-(3, 5-dimethoxystyryl) phenyl) furan-2-carboxamide (BK3C231) against 4-nitroquinoline 1-oxide-induced damage in CCD-18Co human colon fibroblast cells.

Stilbenes are a group of chemicals characterized with the presence of 1,2-diphenylethylene. Previously, our group has demonstrated that synthesized (E)-N-(2-(3, 5-dimethoxystyryl) phenyl) furan-2-carboxamide (BK3C231) possesses potential chemopreventive activity specifically inducing NAD(P)H:quinone oxidoreductase 1 (NQO1) protein expression and activity. In this study, the cytoprotective effects of BK3C231 on cellular DNA and mitochondria were investigated in normal human colon fibroblast, CCD-18Co cells. The cells were pretreated with BK3C231 prior to exposure to the carcinogen 4-nitroquinoline 1-oxide (4NQO). BK3C231 was able to inhibit 4NQO-induced cytotoxicity. Cells treated with 4NQO alone caused high level of DNA and mitochondrial damages. However, pretreatment with BK3C231 protected against these damages by reducing DNA strand breaks and micronucleus formation as well as decreasing losses of mitochondrial membrane potential (ΔΨm) and cardiolipin. Interestingly, our study has demonstrated that nitrosative stress instead of oxidative stress was involved in 4NQO-induced DNA and mitochondrial damages. Inhibition of 4NQO-induced nitrosative stress by BK3C231 was observed through a decrease in nitric oxide (NO) level and an increase in glutathione (GSH) level. These new findings elucidate the cytoprotective potential of BK3C231 in human colon fibroblast CCD-18Co cell model which warrants further investigation into its chemopreventive role.

Novel Genes Involved in Resistance to Both Ultraviolet Radiation and Perchlorate From the Metagenomes of Hypersaline Environments.

Microorganisms that thrive in hypersaline environments on the surface of our planet are exposed to the harmful effects of ultraviolet radiation. Therefore, for their protection, they have sunscreen pigments and highly efficient DNA repair and protection systems. The present study aimed to identify new genes involved in UV radiation resistance from these microorganisms, many of which cannot be cultured in the laboratory. Thus, a functional metagenomic approach was used and for this, small-insert libraries were constructed with DNA isolated from microorganisms of high-altitude Andean hypersaline lakes in Argentina (Diamante and Ojo Seco lakes, 4,589 and 3,200 m, respectively) and from the Es Trenc solar saltern in Spain. The libraries were hosted in a UV radiation-sensitive strain of Escherichia coli (recA mutant) and they were exposed to UVB. The resistant colonies were analyzed and as a result, four clones were identified with environmental DNA fragments containing five genes that conferred resistance to UV radiation in E. coli. One gene encoded a RecA-like protein, complementing the mutation in recA that makes the E. coli host strain more sensitive to UV radiation. Two other genes from the same DNA fragment encoded a TATA-box binding protein and an unknown protein, both responsible for UV resistance. Interestingly, two other genes from different and remote environments, the Ojo Seco Andean lake and the Es Trenc saltern, encoded two hypothetical proteins that can be considered homologous based on their significant amino acid similarity (49%). All of these genes also conferred resistance to 4-nitroquinoline 1-oxide (4-NQO), a compound that mimics the effect of UV radiation on DNA, and also to perchlorate, a powerful oxidant that can induce DNA damage. Furthermore, the hypothetical protein from the Es Trenc salterns was localized as discrete foci possibly associated with damaged sites in the DNA in cells treated with 4-NQO, so it could be involved in the repair of damaged DNA. In summary, novel genes involved in resistance to UV radiation, 4-NQO and perchlorate have been identified in this work and two of them encoding hypothetical proteins that could be involved in DNA damage repair activities not previously described.

An Ultra-Sensitive Electrochemical Sensor for the Detection of Carcinogen Oxidative Stress 4-nitroquinoline N-oxide in Biological matrices Based on Hierarchical Spinel Structured NiCo2O4 and NiCo2S4; A Comparative Study

An Ultra-Sensitive Electrochemical Sensor for the Detection of Carcinogen Oxidative Stress 4-nitroquinoline N-oxide in Biological matrices Based on Hierarchical Spinel Structured NiCo2O4 and NiCo2S4; A Comparative Study

An Ultra-Sensitive Electrochemical Sensor for the Detection of Carcinogen Oxidative Stress 4-nitroquinoline N-oxide in Biological matrices Based on Hierarchical Spinel Structured NiCo2O4 and NiCo2S4; A Comparative Study

An Ultra-Sensitive Electrochemical Sensor for the Detection of Carcinogen Oxidative Stress 4-nitroquinoline N-oxide in Biological matrices Based on Hierarchical Spinel Structured NiCo2O4 and NiCo2S4; A Comparative Study

An Ultra-Sensitive Electrochemical Sensor for the Detection of Carcinogen Oxidative Stress 4-nitroquinoline N-oxide in Biological matrices Based on Hierarchical Spinel Structured NiCo2O4 and NiCo2S4; A Comparative Study

An Ultra-Sensitive Electrochemical Sensor for the Detection of Carcinogen Oxidative Stress 4-nitroquinoline N-oxide in Biological matrices Based on Hierarchical Spinel Structured NiCo2O4 and NiCo2S4; A Comparative Study

Specific Deletion of p16INK4a with Retention of p19ARF Enhances the Development of Invasive Oral Squamous Cell Carcinoma

The cyclin-dependent kinase inhibitor 2A (CDKN2A)/alternate reading frame (ARF) locus consists of two overlapping tumor suppressor genes, p16INK4a and p14ARF (p19ARF in mice), encoding two unrelated proteins in alternative reading frames. Previous reports suggest that p16INK4a and p14ARF alterations independently exhibit differential roles, and p16INK4a is more closely associated with a poor prognosis in oral cancer. However, the role of p16INK4a-specific loss in oral squamous cell carcinogenesis remains unclear. The authors assessed chemical carcinogen 4-nitroquinoline 1-oxide (4NQO)-induced multistep oral squamous cell carcinogenesis in mice carrying p16INK4a-specific loss with retention of the p19ARF gene (p16INK4a-/-). 4NQO-treated p16-/- mice exhibited a higher incidence and multiplicity of oral squamous cell carcinoma (OSCC) development relative to 4NQO-treated wild-type mice. 4NQO-treated p16INK4a-/- OSCC cells exhibited higher proliferation and up-regulation of Arf, transcription factor E2f1, tumor protein p63 (tp63), and oncogenic ΔNp63, an isoform p63, compared with observations in 4NQO-treated wild-type OSCC cells. Furthermore, the overexpression of oncogenic ΔNp63 was associated with human OSCC. In conclusion, these results in mice indicate the biological significance of p16INK4a-specific loss with retention of p19ARF in oral squamous cell carcinogenesis, and ΔNp63 may be a potential target for OSCC.

Chemopreventive activity of Tualang honey against oral squamous cell carcinoma-in vivo.

The aim of this study was to evaluate the chemopreventive activity of Malaysian jungle Tualang honey (TH) after oral carcinogenesis induced with 4-nitroquinoline 1-oxide (4 NQO). A total of 28 male Sprague-Dawley (SD) rats were distributed into 4 groups as follows: group 1 (nontreated group); group 2 (control), which received 4 NQO during 8 weeks in drinking water only; and groups 3 and 4, which received 4 NQO for 8 weeks in drinking water and treated with TH 1000 mg/kg and 2000 mg/kg by oral gavage for 10 weeks. All rats from all experiments were sacrificed after 22 weeks, and the incidence of oral neoplasms and histopathologic changes were microscopically evaluated. Moreover, immunohistochemical expression was analyzed in tongue specimens by using image analysis software. The expression of particular genes associated with oral cancer were assessed by using RT2 Profiler PCR Array (Qiagen, Germantown, MD). TH significantly reduced the incidence of oral squamous cell carcinoma (OSCC) and suppressed cancer cell proliferation via diminishing the expression of CCND1, EGFR, and COX-2. Furthermore, TH preserved cellular adhesion (epithelial polarity) through overexpression of β-catenin and e-cadherin and inhibited the OSCC aggressiveness by downregulating TWIST1 and RAC1. Our data suggest that TH exerts chemopreventive activity in an animal model in which oral cancer was induced by using 4 NQO.

Mutations in long-lived epithelial stem cells and their clonal progeny in pre-malignant lesions and in oral squamous cell carcinoma.

Oral squamous cell carcinomas (OSCCs) are the most common cancers of the oral cavity, but the molecular mechanisms driving OSCC carcinogenesis remain unclear. Our group previously established a murine OSCC model based on a 10-week carcinogen [4-nitroquinoline 1-oxide (4-NQO)] treatment. Here we used K14CreERTAM;Rosa26LacZ mice to perform lineage tracing to delineate the mutational profiles in clonal cell populations resulting from single, long-lived epithelial stem cells, here called LacZ+ stem cell clones (LSCCs). Using laser-capture microdissection, we examined mutational changes in LSCCs immediately after the 10-week 4-NQO treatment and >17 weeks after 4-NQO treatment. We found a 1.8-fold ±0.4 (P = 0.009) increase in single-nucleotide variants and insertions/deletions (indels) in tumor compared with pre-neoplastic LSCCs. The percentages of indels and of loss of heterozygosity events were 1.3-fold±0.3 (P = 0.02) and 2.2-fold±0.7 (P = 0.08) higher in pre-neoplastic compared with tumor LSCCs. Mutations in cell adhesion- and development-associated genes occurred in 83% of the tumor LSCCs. Frequently mutated genes in tumor LSCCs were involved in planar cell polarity (Celsr1, Fat4) or development (Notch1). Chromosomal amplifications in 50% of the tumor LSCCs occurred in epidermal growth factor receptor, phosphoinositide 3-kinase and cell adhesion pathways. All pre-neoplastic and tumor LSCCs were characterized by key smoking-associated changes also observed in human OSCC, C>A and G>T. DeconstructSigs analysis identified smoking and head and neck cancer as the most frequent mutational signatures in pre-neoplastic and tumor LSCCs. Thus, this model recapitulates a smoking-associated mutational profile also observed in humans and illustrates the role of LSCCs in early carcinogenesis and OSCCs.

Doxycycline-induced exogenous Bmi-1 expression enhances tumor formation in a murine model of oral squamous cell carcinoma

ABSTRACTB Cell-Specific Moloney Murine Leukemia Virus Integration Site 1 (Bmi-1, Bmi1), an epigenetic protein, is necessary for normal stem cell self-renewal in adult animals and for cancer stem cell (CSC) functions in adult animals. To elucidate the functions of Bmi-1 in the oral cavity we created a transgenic mouse line (KrTBmi-1) that expresses ectopic, Flag-tagged Bmi-1 in tongue basal epithelial stem cells only upon doxycycline (DOX) treatment. Genome wide transcriptomics and Ingenuity Pathway Analysis identified several pathways altered by exogenous Bmi-1 expression in the normal tongue epithelium, including EIF2 signaling (P value = 1.58 x 10−49), mTOR signaling (P value = 2.45 x 10−12), oxidative phosphorylation (P = 6.61 x 10−3) and glutathione redox reactions I (P = 1.74 x 10−2). Overall, our data indicate that ectopic Bmi-1 expression has an impact on normal tongue epithelial homeostasis. We then assessed the KrTBmi-1 mice in the 4-nitroquinoline 1-oxide (4-NQO) model of oral carcinogenesis. We found that 80% of mice expressing exogenous Bmi-1 (+DOX, +4-NQO KrTBmi-1; N = 10) developed tumors classified as grade 3 or higher, compared to 60% and 40% of mice expressing just endogenous Bmi-1 (+DOX, +4-NQO Kr and -DOX, +4-NQO KrTBmi-1 groups, respectively; N = 10/group; P value = <0.0001); and 30% of mice expressing ectopic Bmi-1 mice developed 20 or more lesions compared to 10% of mice expressing only endogenous Bmi-1 (P = .009). This demonstrates that exogenous Bmi-1 expression increases the susceptibility of mice to 4-NQO-induced oral carcinogenesis, strengthening the evidence for Bmi-1 as a therapeutic target in human oral squamous cell carcinoma.

A PI3K/AKT Scaffolding Protein, IQ Motif-Containing GTPase Associating Protein 1 (IQGAP1), Promotes Head and Neck Carcinogenesis.

Head and neck cancer (HNC) is the sixth most common cancer worldwide with a 5-year survival rate of less than 50%. The PI3K/AKT/mTOR signaling pathway is frequently implicated in HNC. Recently, IQ motif-containing GTPase-activating protein 1 (IQGAP1) was discovered to scaffold the PI3K/AKT signaling pathway. IQGAP1 gene expression is increased in HNC, raising the hypothesis that IQGAP1 contributes to HNC. We performed a combination of in vitro studies using human cancer cell lines treated with a cell-permeable peptide that interferes with IQGAP1's ability to bind to PI3K, and in vivo studies utilizing mice genetically knocked out for the Iqgap1 (Iqgap1 -/-). In vivo EGF stimulation assays were used to evaluate PI3K signaling. To study the role of IQGAP1 in HNC, we used a well-validated mouse model that drives HNC via a synthetic oral carcinogen, 4-nitroquinoline 1-oxide (4NQO). IQGAP1 is necessary for efficient PI3K signaling in vitro and in vivo. Disruption of IQGAP1-scaffolded PI3K/AKT signaling reduced HNC cell survival. Iqgap1 -/- mice had significantly lower cancer incidences, lesser disease severity, and fewer cancer foci. IQGAP1 protein levels were increased in HNC arising in Iqgap1+/+ mice. The level of PI3K signaling in 4NQO-induced HNC arising in Iqgap1 -/- mice was significantly reduced, consistent with the hypothesis that IQGAP1 contributes to HNC at least partly through PI3K signaling. High IQGAP1 expression correlated with reduced survival, and high pS6 levels correlated with high IQGAP1 levels in patients with HNC. These data demonstrate that IQGAP1 contributes to head and neck carcinogenesis.

The efficiency of propranolol on occurrence and development of 4-nitroquinoline 1-oxide-induced squamous cell carcinoma of the tongue in rats.

Aims:The aim of this study to investigate the efficiency of propranolol on occurrence and development of 4-nitroquinoline 1-oxide (4NQO)-induced squamous cell carcinogenesis of the tongue in rats.Subjects and Methods:The sample was composed of 27 male Sprague Dawley rats that received 50 ppm 4NQO for 20 weeks in drinking water. Group 1 (n = 9) was treated with 50 mg/kg/day propranolol for 20 weeks, Group 2 (n = 9), after carcinogenesis inducement for 20 weeks, received propranolol (50 mg/kg/day) for 2 weeks and Group 3 (n = 9) received no treatment. At the end of the experimental stage, the tongue specimens were evaluated under a light microscope and categorized as low- or high-risk lesions according to a binary system.Statistical Analysis Used:The statistical comparison was performed with a likelihood ratio test.Results:Histopathological analysis revealed the risk of malignant transformation rates as 33.3% in Group 1, 55.5% in Group 2 and 77.8% in Group 3; however, the difference between the groups was not statistically significant (P > 0.05).Conclusion:The results of the study suggest that propranolol has a tendency to preventive effect against carcinogenesis.

Development of an integrated assay in human TK6 cells to permit comprehensive genotoxicity analysis in vitro

In vitro genetic toxicology assays are used to assess the genotoxic potential of chemicals or mixtures. They measure chromosome damage (e.g., micronucleus [MN] formation) or gene mutation, and different combinations of data generated from such assays are evaluated in concert in order to identify genotoxic hazards. Mode-of-action (MoA) information is also fundamental to understanding any apparent genotoxic response. In view of the importance of these types of data for full characterization of genotoxic potential, we leveraged relevant endpoints already established in the human TK6 cell line to develop a single integrated assay that measures MN formation, gene mutation (at the thymidine kinase locus), and MoA (DNA damage response biomarkers). Several prototypical direct-acting genotoxins (methyl methanesulfonate, mitomycin C, and 4-nitroquinoline 1-oxide), pro-genotoxins (benzo[a]pyrene and cyclophosphamide monohydrate), and one non-DNA reactive genotoxin (vinblastine sulfate) were assessed in the approach and found to elicit genotoxic profiles that were generally consistent with their MoA. In contrast, the non-genotoxic agents D-mannitol and (2-chloroethyl) trimethyl-ammonium chloride induced negligible effects on all endpoints up to a top concentration of 10 mM. Sodium diclofenac, presumed to be non-genotoxic, provoked an induction in the phosphoserine10-H3-positive cell population within a small window of concentrations (0.157–0.314 mM), as well as increases in γH2AX, nuclear p53, and MN at higher concentrations, although it had no effect on the mutation frequency endpoint. G2M cell cycle arrest was also largely observed in cells that exhibited genotoxicity in the in vitro MN assay. The TK6 cell-based integrated assay represents an in vitro approach that permits comprehensive genotoxicity analysis in a human-relevant test system. Moreover, its vis-à-vis nature may facilitate further comprehension of the range of effects that can manifest in human cells in response to DNA-damaging agents.

ERK Activation Modulates Cancer Stemness and Motility of a Novel Mouse Oral Squamous Cell Carcinoma Cell Line.

We established the NHRI-HN1 cell line from a mouse tongue tumor induced by 4-nitroquinoline 1-oxide (4-NQO)/arecoline, with further selection for cell stemness via in vitro sphere culture, to evaluate potential immunotherapies for oral squamous cell carcinoma (OSCC) in East and Southeast Asia. In vivo and in vitro phenotypic characterization, including tumor growth, immune modulator administration, gene expression, morphology, migration, invasion, and sphere formation assays, were conducted. NHRI-HN1 cells are capable of generating orthotopic tumors in syngeneic mice. Interestingly, immune stimulation via CpG oligodeoxynucleotide (CpG-ODN) dramatically reduced the tumor growth in NHRI-HN1 cell-injected syngeneic mice. The pathways enriched in genes that were differentially expressed in NHRI-HN1 cells when compared to non-tumorigenic cells were similar to those that were identified when comparing human OSCC and non-tumorous tissues. NHRI-HN1 cells have characteristics of epithelial–mesenchymal transition (EMT), including enhanced migration and invasion. NHRI-HN1 cells showed aggressive cell growth and sphere formation. The blockage of extracellular signal-regulated kinase (ERK) activation suppressed cell migration and reduced stemness characteristics in NHRI-HN1 cells, similar to human OSCC cell lines. Our data suggest that NHRI-HN1 cells, showing tumorigenic characteristics of EMT, cancer stemness, and ERK activation, are sufficient in modeling human OSCC and also competent for use in investigating oral cancer immunotherapies.

MTA1 promotes tumorigenesis and development of esophageal squamous cell carcinoma via activating the MEK/ERK/p90RSK signaling pathway.

Metastasis-associated protein 1 (MTA1) is upregulated in multiple malignancies and promotes cancer proliferation and metastasis, but whether and how MTA1 promotes esophageal squamous cell carcinoma (ESCC) tumorigenesis remain unanswered. Here, we established an ESCC model in MTA1 transgenic mice induced by the chemical carcinogen 4-nitroquinoline 1-oxide (4-NQO) and found that MTA1 promotes ESCC tumorigenesis in mice. MTA1 overexpression was observed in ESCC cells and clinical ESCC samples. Overexpressed MTA1 increased colony formation and the invasiveness and migration of ESCC cells, whereas knock down of MTA1 in ESCC cells significantly decreased colony formation, invasion and migration in vitro and inhibited the growth of xenograft tumors in vivo. RNA sequencing (RNA-seq) analysis combined with western blot assays revealed that MTA1 promotes carcinogenesis by enhancing MEK/ERK/p90RSK signaling. The phosphorylation of MEK, ERK and their downstream target p90RSK was significantly decreased after MTA1 knockdown in ESCC cells and was increased in MTA1-overexpressing cells. Moreover, colony formation, invasion and migration potential were dramatically suppressed when cells overexpressing MTA1 were treated with MEK (PD0325901) or ERK (SCH772948) inhibitors. In conclusion, MTA1 plays a pivotal oncogenic role in ESCC tumorigenesis and development through activating the MEK/ERK/p90RSK pathway.

Isolation, characterization and identification of antigenotoxic and anticancerous indigenous probiotics and their prophylactic potential in experimental colon carcinogenesis

Colorectal cancer, the third most commonly diagnosed cancer, is a lifestyle disease where diet and gut microbiome contribute intricately in its initiation and progression. Prophylactic bio-interventions mainly probiotics offer an alternate approach towards reducing or delaying its progression. Therefore, the present study was designed wherein a robust protocol for the isolation, characterization, and identification of indigenous probiotics having antigenotoxic and anticancerous activity was followed along with their prophylactic potential assessment in early experimental colorectal carcinogenesis. Among forty-six isolated lactic acid bacterial strains, only three were selected on the basis of antigenotoxicity against N,N-Dimethyl dihydrazine dihydrochloride and 4-Nitroquinoline 1-oxide and probiotic attributes. All three selected probiotic strains exhibited anticancerous potential as is evident by the reduced Aberrant Crypt Foci, reduced fecal pH, enhanced fecal lactic acid bacteria and altered fecal enzymes (β-glucuronidase, nitroreductase, β-glucosidase) that modulated gut microbiota and microenvironment resulting into restored histoarchitecture of the colon. The results are a clear indicator of the prophylactic potential of selected indigenous probiotics which may be used as an alternative prophylactic biological therapy against colon carcinogenesis particularly in highly susceptible individuals.

Cancer susceptibility of beta HPV49 E6 and E7 transgenic mice to 4-nitroquinoline 1-oxide treatment correlates with mutational signatures of tobacco exposure

We have previously showed that a transgenic (Tg) mouse model with cytokeratin 14 promoter (K14)-driven expression of E6 and E7 from beta-3 HPV49 in the basal layer of the epidermis and of the mucosal epithelia of the digestive tract (K14 HPV49 E6/E7 Tg mice) are highly susceptible to upper digestive tract carcinogenesis upon exposure to 4-nitroquinoline 1-oxide (4NQO). Using whole-exome sequencing, we show that in K14 HPV49 E6/E7 Tg mice, development of 4NQO-induced cancers tightly correlates with the accumulation of somatic mutations in cancer-related genes. The mutational signature in 4NQO-treated mice was similar to the signature observed in humans exposed to tobacco smoking and tobacco chewing. Similar results were obtained with K14 Tg animals expressing mucosal high-risk HPV16 E6 and E7 oncogenes. Thus, beta-3 HPV49 share some functional similarities with HPV16 in Tg animals.

Feasibility and safety of papermaking wastewater in using as ecological water supplement after advanced treatment by fluidized-bed Fenton coupled with large-scale constructed wetland

Reuse of pulp-and-paper industry wastewater as reclaimed water is an effective way to mitigate water resource shortage. In this study, the feasibility and safety of papermaking wastewater for the use as ecological water supplement after the treatment by fluidized-bed Fenton (FBF) coupled with constructed wetland (CW), were investigated from laboratory-scale to large-scale field. The optimum pH, H2O2, H2O2/Fe2+ ratio and hydraulic retention time (HRT) of FBF were 3.5, 0.93 mL/L, 4 and 60 min, respectively, based on reduction of both total organic carbon (TOC) and genotoxicity. Furthermore, the safety of effluent was evaluated using SOS/umu assay and 8-hydroxy-2-deoxyguanosine (8-OHdG) in zebrafish. Results showed FBF followed by CW improved the conventional water quality indicators and reduced the toxicity. Average removal rates of chemical oxygen demand (COD), ammonia nitrogen (NH3-N), total nitrogen (TN), total phosphorus (TP) and colority were 87.3%, 93.59%, 51.73%, 84.75% and 95.86%, respectively. The equivalent concentration of 4-nitroquinoline 1-oxide (4-NQO-EQ) decreased from 30.6 ± 1.6 μg/L in influent to 12.4 ± 1.0 μg/L after treated by FBF, then decreased to 5.9 ± 0.4 μg/L after treated by CW and to 3.2 ± 0.3 μg/L after 12-km downstream self-purification. The chronic survival rates of 21-d zebrafish significantly increased from 0.0% in influent to 58.8 ± 4.0% in effluent of CW and gradually increased to 68.8 ± 2.6% after 12-km downstream self-purification. Similarly, 8-OHdG level in zebrafish decreased from 120.0 ± 19.3 ng/L in effluent of ecological oxidation pond to 94.0 ± 7.5 ng/L in effluent of CW and gradually decreased to 42.0 ± 3.0 ng/L after 12-km downstream self-purification. The study concluded that FBF-CW is an efficient detoxication and water quality improvement technology for papermaking wastewater to be used as an ecological water supplement.

Investigation of comet assays under conditions mimicking ocular instillation administration in a three-dimensional reconstructed human corneal epithelial model

Purpose: A comet assay is one of the genotoxicity methods for evaluating the potential of chemicals to induce DNA strand breaks. To investigate the usefulness of comet assays for evaluating the genotoxic potential of ophthalmic solutions, a three-dimensional (3D) reconstructed human corneal epithelial model (3D corneal model) was exposed to conditions mimicking topical ocular instillation administration.Methods: The 3D corneal model was exposed to acridine orange, ethidium bromide, hydrogen peroxide, 1,1′-dimethyl-4,4′-bipyridinium dichloride (paraquat), 4-nitroquinoline 1-oxide (4-NQO), acrylamide and methyl methanesulfonate (MMS). To mimic the ocular surface condition to which ophthalmic solutions are administered, the exposure time was set to 1 minute. Likewise, human corneal epithelial (HCE-T) cells, as monolayer cultured cells, were exposed to the same chemicals, for comparison.Results: In the 3D corneal model, the amount of DNA fragments was statistically significantly increased in cells treated with each of the test chemicals except acrylamide. In HCE-T cells, the amount of DNA fragments was statistically significantly increased in acridine orange-, ethidium bromide-, hydrogen peroxide-, 4-NQO- and MMS-treated cells but not in paraquat- or acrylamide-treated cells. In the 3D corneal model, the lowest concentrations at which we observed DNA damage were about 100 times higher than the concentrations in HCE-T cells. Since the 3D corneal model is morphologically similar to human corneal tissue, form a multilayer and having tight junctions, it may be that the test chemicals only permeated about 1% into the 3D corneal model.Conclusion: These results suggest that the comet assay using 3D cell culture models may reflect in vivo conditions better than do monolayer cultured cells, and that the comet assay may be useful for the evaluation of genotoxic potential of topical ophthalmic solution.

Abstract 4297: MYC HIF-2 alpha embryonic stemness genes mediate a transcriptional network to maintain asymmetric self-renewal in oral cancer

Abstract Cancer Stem Cells (CSCs) explains therapeutic failure across several tumor types. Despite being evolutionarily successful, the extreme ability to self-renew is asymmetric and limited to CSCs in heterogeneous tumor population. Thus, understanding molecular mechanisms maintaining CSCs asymmetric self-renewal would be therapeutically critical.Here, we identified putative molecular transcriptional network that may regulate CSCs asymmetric self-renewal. We developed 4-NQO (4-Nitroquinoline 1-oxide) induced mouse model of oral carcinogenesis that exhibited similar phenotype as human oral cancer. ABCG2+ CSCs enriched from 4-NQO derived mouse tumors and human oral cancer (n=7) patients indicated high expression of embryonic stemness genes such as Nanog, Sox-2, and Oct-4 in addition to MYC and HIF-2a. Higher engraftment potential was observed when ABCG2+ cells were injected to NOD/SCID mice, while HIF-2a silencing markedly increased mice survival during engraftments. ChIP assay revealed preferential binding of MYC to HIF-2a promoter regions in ABCG2+ vs ABCG2- cells. Nanog and Oct4 facilitated MYC binding to HIF-2a while gene silencing of Nanog and Sox2 led to loss of MYC and HIF-2a expression, indicating the possible regulation by MYC and HIF-2a. Further, we utilized clinical data from The Cancer Genome Atlas (TCGA) and demonstrated significant correlation between HIF-2a/MYC and embryonic stemness genes on Structural Equation Modeling Analysis (SEMA) platform, thereby confirming our in vitro/in vivofindings. Thus,our results may provide evidence of a biologically robust stemness transcriptional network regulating the asymmetric self-renewal of ABCG2+ CSCs. Note: This abstract was not presented at the meeting. Citation Format: Bidisha Pal, Wael Tasabehji, Sandhya Sorra, Joyeeta Talukdar, Bikul Das. MYC HIF-2 alpha embryonic stemness genes mediate a transcriptional network to maintain asymmetric self-renewal in oral cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4297.