ABSTRACT
Error-corrected next-generation sequencing (ecNGS) is an emerging technology for accurately measuring somatic mutations. Here, we report paired-end and complementary consensus sequencing (PECC-Seq), a high-accuracy ecNGS approach for genome-wide somatic mutation detection. We characterize a novel 2-aminoimidazolone lesion besides 7,8-dihydro-8-oxoguanine and the resulting end-repair artifacts originating from NGS library preparation that obscure the sequencing accuracy of NGS. We modify library preparation protocol for the enzymatic removal of end-repair artifacts and improve the accuracy of our previously developed duplex consensus sequencing method. Optimized PECC-Seq shows an error rate of <5 × 10-8 with consensus bases compressed from approximately 25 Gb of raw sequencing data, enabling the accurate detection of low-abundance somatic mutations. We apply PECC-Seq to the quantification of in vivo mutagenesis. Compared with the classic gpt gene mutation assay using gpt delta transgenic mice, PECC-Seq exhibits high sensitivity in quantitatively measuring dose-dependent mutagenesis induced by Aristolochic acid I (AAI). Moreover, PECC-Seq specifically characterizes the distinct genome-wide mutational signatures of AAI, Benzo[a]pyrene, N-Nitroso-N-ethylurea and N-nitrosodiethylamine and reveals the mutational signature of Quinoline in common mouse models. Overall, our findings demonstrate that high-accuracy PECC-Seq is a promising tool for genome-wide somatic mutagenesis quantification and for in vivo mutagenicity testing.
Subject(s)
High-Throughput Nucleotide Sequencing , Animals , Mice , Consensus , High-Throughput Nucleotide Sequencing/methods , Mice, Transgenic , Mutagenesis/genetics , Mutation , Sequence Analysis, DNA/methods , MaleABSTRACT
Several recent reports indicate health hazards for workers with below occupational limit exposure to benzene (BZ). Our updated review indicates that such low exposures induced traditional as well as novel toxicity/genotoxicity, e.g., increased mitochondria copy numbers, prolongation of telomeres, impairment of DNA damage repair response (DDRR), perturbations of expression in non-coding RNAs, and epigenetic changes. These abnormalities were associated with alterations of gene expression and cellular signaling pathways which affected hematopoietic cell development, expression of apoptosis, autophagy, etc. The overarching mechanisms for induction of health risk are impaired DDRR, inhibition of tumor suppressor genes, and changes of MDM2-p53 axis activities that contribute to perturbed control for cancer pathways. Evaluation of the unusual dose-responses to BZ exposure indicates cellular over-compensation and reprogramming to overcome toxicity and to promote survival. However, these abnormal mechanisms also promote the induction of leukemia. Further investigations indicate that the current exposure limits for workers to BZ are unacceptable. Based on these studies, the new exposure limits should be less than 0.07 ppm rather than the current 1 ppm. This review also emphasizes the need to conduct appropriate bioassays, and to provide more reliable decisions on health hazards as well as on exposure limits for workers. In addition, it is important to use scientific data to provide significantly improved risk assessment, i.e., shifting from a population- to an individual-based risk assessment.
Subject(s)
Benzene , Occupational Exposure , Humans , Benzene/toxicity , Occupational Exposure/adverse effects , Occupational Exposure/analysis , DNA Damage , DNA Repair , Risk AssessmentABSTRACT
To achieve carbon neutrality (i.e., net zero carbon emissions) by 2060, China must make significant changes in its socioeconomic systems, including appropriately allocating emissions responsibility. Traditional methods of delineating responsibilities (such as production-based and consumption-based accounting) can lead to double counting when applied simultaneously and therefore difficulty in determining responsibilities of different agents. An alternative approach based on economic welfare gains from environmental externalities has been refined, ensuring that the responsibilities of consumers and producers add up to the total emissions. The application of this approach to 48 countries and 31 Chinese provinces reveals that regions with less elastic supply and demand, such as Hebei in China and Russia, have higher responsibilities. Furthermore, larger externalities associated with unitary product value shift the burden of obligations from producers to consumers. Regions with high levels of wealth and carbon-intensive imports, such as Zhejiang and Guangdong in China, as well as the United States, typically have higher consumer-based accounting (CBA) emissions than production-based accounting (PBA) emissions and, as a result, redistributed responsibilities between PBA and CBA emissions. The new distribution results vary significantly from PBA or CBA emissions, indicating opportunities for more comprehensive and accessible policy goals.
Subject(s)
Carbon Dioxide , Carbon , Carbon Dioxide/analysis , China , Russia , Economic DevelopmentABSTRACT
Caenorhabditis elegans (C. elegans), an established model organism, has been widely used in environmental toxicology research. However, most of the current toxicity testing methods based on worms are time-consuming. In this study we aimed to develop an automated and highly-integrated platform for high-throughput and in situ toxicity testing. Considering the superiority of C. elegans as a neurotoxicological model, this platform mainly evaluates general toxicology and neurotoxicology endpoints, which are usually induced by metals and pesticides, the major environmental contaminants. Microplates were used as a worm culturing system, which have good compatibility with any commercial microplate applicable instruments. We developed a microfluidic-based module for worm dispensing, and an image acquisition/analysis module for monitoring worms and detecting toxicity endpoints in bright filed. These were collectively incorporated with a commercial pipetting workstation for automated food/drug delivery and a high-content analysis system for fluorescence detection. The integrated platform achieved an efficient on-demand worm dispensing, long-term maintenance, regular monitoring and imaging, survival assay and behavioral analyses, and visualized gene reporter assay. Moreover, "Lab on Web" was achieved by connecting the platform to the web for remote operation, worm monitoring, and phenotype calculation. To demonstrate the ability of the platform for automated toxicity testing assays; worms were treated with cadmium and longevity, neurotoxicity, developmental toxicity and gst-4 expression were evaluated. We determined its feasibility and proposed the potential application in high-throughput toxicity screening for environmental risk assessment in the nearest future.
Subject(s)
Caenorhabditis elegans , Pesticides , Animals , Cadmium/metabolism , Cadmium/toxicity , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , High-Throughput Screening Assays/methods , Pesticides/metabolism , Pesticides/toxicity , PhenotypeABSTRACT
As the carcinogenic risk of herbs containing aristolochic acids (AAs) is a global health issue, quantitative evaluation of toxicity is needed for the regulatory decision-making and risk assessment of AAs. In this study, we selected AA I (AAI), the most abundant and representative compound in AAs, to treat transgenic gpt delta mice at six gradient doses ranging from 0.125 to 4 mg/kg/day for 28 days. AAI-DNA adduct frequencies and gpt gene mutation frequencies (MFs) in the kidney, as well as Pig-a gene MFs and micronucleated reticulocytes (MN-RETs) frequencies in peripheral blood, were monitored. The dose-response (DR) relationship data for these in vivo genotoxicity endpoints were quantitatively evaluated using an advanced benchmark dose (BMD) approach with different critical effect sizes (CESs; i.e., BMD5, BMD10, BMD50 and BMD100). The results showed that the AAI-DNA adduct frequencies, gpt MFs and the MN-RETs presented good DR relationship to the administrated doses, and the corresponding BMDL100 (the lower 90% confidence interval of the BMD100) values were 0.017, 0.509 and 3.9 mg/kg/day, respectively. No positive responses were observed in the Pig-a MFs due to bone marrow suppression caused by AAI. Overall, we quantitatively evaluated the genotoxicity of AAI at low doses for multiple endpoints for the first time. Comparisons of BMD100 values across different endpoints provide a basis for the risk assessment and regulatory decision-making of AAs and are also valuable for understanding the genotoxicity mechanism of AAs.
Subject(s)
Aristolochic Acids/toxicity , DNA Adducts , DNA Damage , Escherichia coli Proteins/metabolism , Mutation Rate , Pentosyltransferases/metabolism , Animals , Benchmarking , Escherichia coli Proteins/genetics , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Micronucleus Tests , Mutagenicity Tests , Mutagens/toxicity , Pentosyltransferases/geneticsABSTRACT
Aristolochic acids (AAs) are a family of natural compounds with AA I and AA II being known carcinogens, whose bioactivation causes DNA adducts formation. However, other congeners have rarely been investigated. This study aimed to investigate genotoxicity of AA IVa, which differs from AA I by a hydroxyl group, abundant in Aristolochiaceae plants. AA IVa reacted with 2'-deoxyadenosine (dA) and 2'-deoxyguanosine (dG) to form three dA and five dG adducts as identified by high-resolution mass spectrometry, among which two dA and three dG adducts were detected in reactions of AA IVa with calf thymus DNA (CT DNA). However, no DNA adducts were detected in the kidney, liver, and forestomach of orally dosed mice at 40 mg/kg/day for 2 days, and bone marrow micronucleus assay also yielded negative results. Pharmacokinetic analyses of metabolites in plasma indicated that AA IVa was mainly O-demethylated to produce a metabolite with two hydroxyl groups, probably facilitating its excretion. Meanwhile, no reduced metabolites were detected. The competitive reaction of AA I and AA IVa with CT DNA, with adducts levels varying with pH of reaction revealed that AA IVa was significantly less reactive than AA I, probably by hydroxyl deprotonation of AA IVa, which was explained by theoretical calculations for reaction barriers, energy levels of the molecular orbits, and charges at the reaction sites. In brief, although it could form DNA adducts in vitro, AA IVa was non-genotoxic in vivo, which was attributed to its low reactivity and biotransformation into an easily excreted metabolite rather than bioactivation.
Subject(s)
Aristolochic Acids/toxicity , DNA Adducts/drug effects , DNA Damage/drug effects , DNA/drug effects , Animals , Aristolochic Acids/administration & dosage , Aristolochic Acids/chemistry , Carcinogens/administration & dosage , Carcinogens/chemistry , Carcinogens/toxicity , Hydrogen-Ion Concentration , Male , Mice , Mice, Inbred C57BL , Micronucleus Tests , Mutagenicity TestsABSTRACT
To improve the accuracy and the cost-efficiency of next-generation sequencing in ultralow-frequency mutation detection, we developed the Paired-End and Complementary Consensus Sequencing (PECC-Seq), a PCR-free duplex consensus sequencing approach. PECC-Seq employed shear points as endogenous barcodes to identify consensus sequences from the overlap in the shortened, complementary DNA strand-derived paired-end reads for sequencing error correction. With the high accuracy of PECC-Seq, we identified the characteristic base substitution errors introduced by the end-repair process of mechanical fragmentation-based library preparations, which were prominent at the terminal 7 bp of the library fragments in the 5'-NpCpA-3' and 5'-NpCpT-3' trinucleotide context. As demonstrated at the human genome scale (TK6 cells), after removing these potential end-repair artifacts from the terminal 7 bp, PECC-Seq could reduce the sequencing error frequency to mid-10-7 with a relatively low sequencing depth. For TA base pairs, the background error rate could be suppressed to mid-10-8. In mutagen-treated (6 µg/mL methyl methanesulfonate or 12 µg/mL N-nitroso-N-ethylurea) TK6, increases in mutagen treatment-related mutant frequencies could be detected, indicating the potential of PECC-Seq in detecting genome-wide ultra-rare mutations. In addition, our finding on the patterns of end-repair artifacts may provide new insights into further reducing technical errors not only for PECC-Seq, but also for other next-generation sequencing techniques.
Subject(s)
DNA Mutational Analysis/methods , High-Throughput Nucleotide Sequencing/methods , Mutation Rate , Cell Line , Consensus , Genome, Human , Humans , Mutation , Polymerase Chain Reaction , Sensitivity and Specificity , Sequence Analysis, DNAABSTRACT
Among neonicotinoid insecticides, the fastest growing class of insecticides worldwide over the past decade, imidacloprid (IMI) is the most widely used one. The effects of IMI on human health, especially on genetic toxicity have gradually aroused more attention. In this study, a combined in vitro approach employing the thymidine kinase (TK) gene mutation assay, the comet assay and the micronucleus test was taken to assess the genotoxicity of IMI. The mechanism behind IMI was also explored by measuring reactive oxygen species (ROS) in the human lymphoblastoid TK6 cells. The cells were treated with 0.01, 0.1, 1, 5, and 10 µg/mL IMI, and ROS generation was measured by the use of 2,7,-dichlorofluorescin diacetate (DCFH-DA) assay. IMI significantly increased the micronucleus (MN) frequency, TK mutations and DNA damage with a dose-effect relationship, and the lowest effective concentration in those tests was 0.1 µg/mL. However, no obvious change of intracellular ROS was observed for any concentrations. The results indicate that IMI has potential genotoxic effects on TK6 cells, but ROS does not seem to be involved as a mechanism of genotoxicity under the experimental conditions.
Subject(s)
Insecticides/toxicity , Lymphocytes/drug effects , Mutagens/toxicity , Neonicotinoids/toxicity , Nitro Compounds/toxicity , Cell Line , Comet Assay , Dose-Response Relationship, Drug , Humans , Insecticides/administration & dosage , Lymphocytes/pathology , Micronucleus Tests , Mutagenicity Tests , Mutagens/administration & dosage , Mutation , Neonicotinoids/administration & dosage , Nitro Compounds/administration & dosage , Reactive Oxygen Species/metabolismABSTRACT
Metabolic activation is essential in standard in vitro genotoxicity test systems. At present, there is a lack of suitable cell models that can express the major characteristics of liver function for predicting substance toxicity in humans. Human-induced hepatocytes (hiHeps), which have been generated from fibroblasts by lentiviral expression of liver transcription factors, can express hepatic gene programs and can be expanded in vitro and display functional characteristics of mature hepatocytes, including cytochrome P450 enzyme activity and biliary drug clearance. Our purpose was to investigate whether hiHeps could be used as a more suitable model for genotoxicity evaluation of chemicals. Therefore, a direct mutagen, methylmethanesulfonate (MMS), and five promutagens [2-nitrofluorene (2-NF), benzo[a]pyrene (B[a]P), aflatoxin B1, cyclophosphamide and N-nitrosodiethylamine] were tested by the cytokinesis-block micronucleus test and the comet assay. Results from genotoxicity tests showed that the micronucleus frequencies were significantly increased by all of the six clastogens tested. Moreover, MMS, 2-NF and B[a]P induced significant increases in the % Tail DNA in the comet assay. In conclusion, our findings from the preliminary study demonstrated that hiHeps could detect the genotoxicity of indirect carcinogens, suggesting their potential to be applied as an effective tool for in vitro genotoxicity assessments.
Subject(s)
DNA Damage , Hepatocytes/drug effects , Micronuclei, Chromosome-Defective , Mutagens/toxicity , Aflatoxin B1/toxicity , Benzo(a)pyrene/toxicity , Cells, Cultured , Comet Assay , Cyclophosphamide/toxicity , Cytochrome P-450 Enzyme System/metabolism , Diethylnitrosamine/toxicity , Fluorenes/toxicity , Hepatocytes/cytology , Hepatocytes/enzymology , Hepatocytes/metabolism , Humans , Karyotype , Methyl Methanesulfonate/toxicity , Micronucleus Tests , Mutagenicity TestsABSTRACT
Flame retardant polybrominated diphenyl ethers (PBDEs) are a class of persistent organic pollutants (POPs). 2,2',4,4'-Tetrabromodiphenyl ether (BDE-47) is a representative PBDE congener with widespread distribution and relatively high toxicity potential. Although it has been reported that BDE-47 can cause DNA damage in various in vitro systems, few studies have provided in vivo genotoxicity information. The aim of the present study was to investigate the genotoxicity of BDE-47 in mice. Male gpt delta mice were administered BDE-47 by gavage at 0, 0.0015, 1.5, 10 and 30 mg/kg/day, and 6 days per week for six consecutive weeks. Before the first treatment, and at 2.5 and 5 weeks after the first treatment, peripheral blood was collected from tails and the micronucleus assay and the Pig-a gene mutation assay were performed. After the last treatment, the mutant frequencies of the gpt gene in the liver and the germ cells from seminiferous tubules were determined. All these assays failed to produce positive results, suggesting that BDE-47 was neither clastogenic nor mutagenic in both target and non-target tissues in gpt delta mice.
Subject(s)
Escherichia coli Proteins/genetics , Halogenated Diphenyl Ethers/toxicity , Membrane Proteins/genetics , Mutagens/toxicity , Pentosyltransferases/genetics , Animals , DNA Damage/drug effects , Environmental Pollutants/toxicity , Liver/drug effects , Mice , Mice, Transgenic , Micronucleus Tests , Mutagenicity Tests , MutationABSTRACT
Cytochrome P450s are involved in detoxification and activation of benzo[a]pyrene (BaP) with unclear balance and unknown contribution of other oxidoreductases. Here, we investigated the BaP and BaP-induced mutagenicity in hepatic and extra-hepatic tissues using hepatic P450 reductase null (HRN) gpt mice. After 2-week treatment (50 mg/kg, i.p. 4 days), BaP in the liver and lung of HRN-gpt mice were increased. BaP promoted gpt mutant frequency (MF) in HRN-gpt mice liver. MF of gpt in the lung and Pig-a in hematopoietic cells induced by BaP in HRN-gpt mice were increased than in gpt mice. BaP-7,8-diol-9,10-epoxide (BPDE)-DNA adducts in vitro was analyzed for enzymes detection in BaP bioactivation. Specific inhibitors of 5-lipoxygenase, cyclooxygenase-1&2, and aldo-keto reductase resulted in more than 80% inhibition rate in the DNA adduct formation, further confirmed by Macaca fascicularis hepatic S9 system. Our results suggested the detoxification of BaP primarily depends on cytochrome P450, while the bioactivation involves additional oxidoreductases.
Subject(s)
Aldo-Keto Reductases/metabolism , Arachidonate 5-Lipoxygenase/metabolism , Benzo(a)pyrene/pharmacokinetics , Cytochrome P-450 Enzyme System/metabolism , Liver/enzymology , Aldo-Keto Reductases/genetics , Animals , Arachidonate 5-Lipoxygenase/genetics , Benzo(a)pyrene/pharmacology , Cytochrome P-450 Enzyme System/genetics , Hematopoietic Stem Cells/enzymology , Inactivation, Metabolic , Macaca fascicularis , Mice , Mice, KnockoutABSTRACT
The hepatitis B virus (HBV) is responsible for most of hepatocellular carcinoma (HCC). However, whether HBV plays an important role during hepatocarcinogenesis through effecting miRNAs remains unknown. Here, we reported that HBV up-regulated microRNA-181a (miR-181a) by enhancing its promoter activity. Simultaneously, we found that miR-181a inhibited apoptosis in vitro and promoted tumor cell growth in vivo. TNF receptor superfamily member 6 (Fas) was further identified as a target of miR-181a. We also found that Fas could reverse the apoptosis-inhibition effect induced by miR-181a. Moreover, HBV could inhibit cell apoptosis by down-regulating Fas expression, which could be reversed by miR-181a inhibitor. Our data demonstrated that HBV suppressed apoptosis of hepatoma cells by up-regulating miR-181a expression and down-regulating Fas expression, which may provide a new understanding of the mechanism in HBV-related HCC pathogenesis.
Subject(s)
Carcinoma, Hepatocellular/virology , Hepatitis B virus/physiology , Liver Neoplasms/virology , MicroRNAs/genetics , fas Receptor/genetics , Animals , Apoptosis/genetics , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Caspase 8/analysis , Cell Line, Tumor , Cell Proliferation/genetics , Cell Transformation, Neoplastic/genetics , DNA, Viral/genetics , Down-Regulation , Hep G2 Cells , Hepatitis B/pathology , Hepatitis B/virology , Humans , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Mice , Mice, Nude , MicroRNAs/biosynthesis , Promoter Regions, Genetic/genetics , Transcriptional Activation/genetics , Tumor Burden/genetics , Up-Regulation , fas Receptor/biosynthesisABSTRACT
AIM: Leflunomide is an immunosuppressive agent marketed as a disease-modifying antirheumatic drug. But it causes severe side effects, including fatal hepatitis and liver failure. In this study we investigated the contributions of hepatic metabolism and transport of leflunomide and its major metabolite teriflunomide to leflunomide induced hepatotoxicity in vitro and in vivo. METHODS: The metabolism and toxicity of leflunomide and teriflunomide were evaluated in primary rat hepatocytes in vitro. Hepatic cytochrome P450 reductase null (HRN) mice were used to examine the PK profiling and hepatotoxicity of leflunomide in vivo. The expression and function of sodium/bile acid cotransporter (NTCP) were assessed in rat and human hepatocytes and NTCP-transfected HEK293 cells. After Male Sprague-Dawley (SD) rats were administered teriflunomide (1,6, 12 mg · kg(-1) · d(-1), ig) for 4 weeks, their blood samples were analyzed. RESULTS: A nonspecific CYPs inhibitor aminobenzotriazole (ABT, 1 mmol/L) decreased the IC50 value of leflunomide in rat hepatocytes from 409 to 216 µmol/L, whereas another nonspecific CYPs inhibitor proadifen (SKF, 30 µmol/L) increased the cellular accumulation of leflunomide to 3.68-fold at 4 h. After oral dosing (15 mg/kg), the plasma exposure (AUC0-t) of leflunomide increased to 3-fold in HRN mice compared with wild type mice. Administration of leflunomide (25 mg·kg(-1) · d(-1)) for 7 d significantly increased serum ALT and AST levels in HRN mice; when the dose was increased to 50 mg·kg(-1) · d(-1), all HRN mice died on d 6. Teriflunomide significantly decreased the expression of NTCP in human hepatocytes, as well as the function of NTCP in rat hepatocytes and NTCP-transfected HEK293 cells. Four-week administration of teriflunomide significantly increased serum total bilirubin and direct bilirubin levels in female rats, but not in male rats. CONCLUSION: Hepatic CYPs play a critical role in detoxification process of leflunomide, whereas the major metabolite teriflunomide suppresses the expression and function of NTCP, leading to potential cholestasis.
Subject(s)
Antirheumatic Agents/toxicity , Chemical and Drug Induced Liver Injury/metabolism , Cytochrome P-450 Enzyme System/metabolism , Isoxazoles/toxicity , Liver/drug effects , Liver/pathology , Organic Anion Transporters, Sodium-Dependent/metabolism , Symporters/metabolism , Animals , Antirheumatic Agents/metabolism , Antirheumatic Agents/pharmacokinetics , Cells, Cultured , Chemical and Drug Induced Liver Injury/pathology , Crotonates/metabolism , Crotonates/pharmacokinetics , Crotonates/toxicity , Cytochrome P-450 Enzyme Inhibitors/pharmacology , Female , HEK293 Cells , Humans , Hydroxybutyrates , Isoxazoles/metabolism , Isoxazoles/pharmacokinetics , Leflunomide , Liver/metabolism , Male , Mice, Inbred C57BL , Nitriles , Organic Anion Transporters, Sodium-Dependent/antagonists & inhibitors , Rats, Sprague-Dawley , Symporters/antagonists & inhibitors , Toluidines/metabolism , Toluidines/pharmacokinetics , Toluidines/toxicityABSTRACT
Perfluorooctane sulfate (PFOS), a persistent organic pollutant, has recently been closely linked with an increased risk of tumorigenesis. However, PFOS has yielded negative results in various tests of genotoxicity. The present study aimed to investigate the mutagenic response to PFOS in the gpt delta transgenic mouse mutation system and to illustrate the contribution of hydrogen peroxide (H2O2) to PFOS genotoxicity. Mutations at the redBA/gam loci were determined by Spi(-) assay both in vitro and in vivo. DNA damage was measured by phosphorylated histone H2AX (γ-H2AX) and mouse bone marrow micronucleus (MN) testing. Our data showed that PFOS induced concentration-dependent increases in γ-H2AX foci and in mutation frequencies at redBA/gam loci in transgenic mouse embryonic fibroblast cells, which were confirmed by the formation of MNs in the bone marrow and the observations of mutation induction in the livers of gpt delta transgenic mice. Concurrent treatment with catalase, an efficient H2O2 scavenger, significantly decreased the formation of γ-H2AX foci and the mutation yields induced by PFOS. In addition, the generation of H2O2 was found to be closely related to the abnormal peroxisomal ß-oxidation caused by PFOS. These finding might provide new mechanistical information about genotoxic effects of PFOS.
Subject(s)
Alkanesulfonic Acids/toxicity , DNA Damage/drug effects , Fluorocarbons/toxicity , Hydrogen Peroxide/metabolism , Animals , Mice , Mice, Transgenic , Mutagenicity TestsABSTRACT
MicroRNA-101(miR-101) has been shown to be down-regulated in hepatocellular carcinoma (HCC). The hepatitis B virus (HBV) is a major risk factor in the development and progression of HCC. However, the correlation between HBV and miR-101 has not yet been fully elucidated. In this study, we reported that HBV could repress miR-101-3p by inhibiting its promoter activity and identified the potential effects of miR-101-3p on some important biological properties of HCC cells by targeting Rap1b. Dual-luciferase reporter assays showed that HBV down-regulated miR-101-3p by inhibiting its promoter activity. Down-regulation of miR-101-3p promoted cell proliferation, migration, and reduced apoptosis, and resulted in up-regulation of Rap1b, while overexpression of miR-101-3p inhibited these processes. Moreover, overexpression of Rap1b was able to reverse the suppressed cell proliferation and migration mediated by miR-101-3p. Our data showed that HBV down-regulated miR-101-3p expression by inhibiting its promoter activity, which resulted in up-regulation of Rap1b, and down-regulation of miR-101-3p or up-regulation of Rap1b promoted proliferation and migration of HCC cells. This provides a new understanding of the mechanism of HBV-related HCC pathogenesis and the potential application of miR-101-3p in cancer therapy.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Hepatitis B virus/physiology , Liver Neoplasms/metabolism , MicroRNAs/metabolism , rap GTP-Binding Proteins/metabolism , Apoptosis , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/virology , Cell Line, Tumor , Cell Movement , Cell Proliferation , Humans , Liver Neoplasms/pathology , Liver Neoplasms/virology , Promoter Regions, GeneticABSTRACT
BACKGROUND: Accumulating evidence showed that microRNAs are involved in development and progression of multiple tumors. Recent studies have found that miR-181a were dysregulated in several types of cancers, however, the function of miR-181a in hepatocellular carcinoma (HCC) remains unclear. In this study we assessed the potential association between miR-181a, HBV and HCC. METHODS: The expression of miR-181a in HBV-expressing cells was determined by using qRT-PCR. Dual-Luciferase reporter Assay, qRT-PCR and western blot were performed to investigate the target genes of miR-181a. The effects of miR-181a on HCC proliferation were analyzed by MTS and colony formation assay. Tumor growth assay was used to analyze the effect of miR-181a on tumor formation. RESULTS: HBV up-regulated miR-181a expression by enhancing its promoter activity. Overexpression of miR-181a in hepatoma cells promoted cell growth in vitro and tumor formation in vivo. Conversely, inhibition of miR-181a suppressed the proliferation of HBV-expressing cells. Mechanism investigation revealed that miR-181a inhibited the expression of transcription factor E2F5 by specifically targeting its mRNA 3'UTR. Moreover, E2F5 inhibition induced cell growth and rescued the suppressive effect of miR-181a inhibitor on the proliferation of SMMC-7721 cells. Interestingly, we also discovered that HBV could down-regulate E2F5 expression. CONCLUSIONS: Those results strongly suggested that HBV down-regulated E2F5 expression, in part, by up-regulating the expression of miR-181a. Up-regulation of miR-181a by HBV in hepatoma cells may contribute to the progression of HCC possibly by targeting E2F5, suggesting miR-181a plays important role in HCC development.
Subject(s)
Carcinogenesis/metabolism , Carcinoma, Hepatocellular/metabolism , E2F5 Transcription Factor/biosynthesis , Hepatitis B virus/metabolism , Liver Neoplasms/metabolism , MicroRNAs/biosynthesis , Animals , Carcinogenesis/pathology , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/virology , Disease Progression , Female , Hep G2 Cells , Humans , Liver Neoplasms/pathology , Liver Neoplasms/virology , Mice , Mice, Inbred BALB C , Mice, Nude , Up-Regulation/physiologyABSTRACT
RAB GTPase 5A (RAB5A), a member of the Rab subfamily of small GTPases, acts as an oncogene and has been associated with various key cellular functions, including cell growth, differentiation, apoptosis and angiogenesis. Recently, it has been reported that the Rab5a gene is involved in the progression of cancer. Hepatocellular carcinoma (HCC) is one of the most common and aggressive cancers, and it is usually associated with persistent hepatitis B virus (HBV) infections. Emerging evidence suggests that HBV alters microRNA (miRNA) expression profiles, but the mechanisms underlying this process have not yet been fully elucidated. Here, we examine how HBV affects the production of miR-101-1, which has been shown to be downregulated in HCC. We found that HBV could repress miR-101-3p by inhibiting its promoter activity. Downregulation of miR-101-3p promoted cancer cell growth and migration, and a specific miR-101-3p inhibitor was able to enhance proliferation and migration. Moreover, we identified Rab5a was one of the target genes of miR-101-3p in HBV-related HCC. Forced expression of miR-101-3p in liver cell lines resulted in a marked reduction of the expression of Rab5a at both the mRNA and protein level by directly targeting the 3'untranslated region of Rab5a. Overexpression of Rab5a resulted in a reversal of the suppression of proliferation and migration of SMMC-7721 cells mediated by miR-101-3p. Taken together, our data show that HBV can downregulate miR-101-3p expression by inhibiting its promoter activity and that downregulation of miR-101-3p promotes HCC cell proliferation and migration by targeting Rab5a. This provides new insights into the mechanisms of HBV-related HCC pathogenesis.
Subject(s)
Hepatitis B virus/physiology , Hepatocytes/physiology , MicroRNAs/antagonists & inhibitors , rab5 GTP-Binding Proteins/metabolism , Cell Line, Tumor , Cell Movement , Cell Proliferation , Down-Regulation , HumansABSTRACT
Aristolochic acid (AA)-containing herbs have been prescribed for thousands of years as anti-inflammatory drugs, despite the active pharmaceutical ingredients remaining unclear. However, exposure to AAI and AAII has been proven to be a significant risk factor for severe nephropathy and carcinogenicity. AAIVa, an analogue abundant in AA-containing herbs, showed neither carcinogenicity nor nephrotoxicity in our study and other reports, implying that the pharmacological effects of AAIVa on inflammation are worth studying. Herein, we employed RAW 264.7 cells, the ear edema mouse model, and the lipopolysaccharide (LPS)-induced systematic inflammation model in TNF-IRES-Luc mice (tracking TNFα luciferase activities in real-time) to evaluate the anti-inframammary effect of AAIVa. Our results showed that AAIVa could decrease pro-inflammatory cytokines (TNFα and IL-6) production in LPS-stimulated RAW 264.7 cells, indicating its anti-inflammatory effects in vitro. Furthermore, the application of AAIVa (400 and 600 µg/ear) could significantly inhibit phorbol 12-myristate 13-acetate-induced ear edema, suggesting its topical anti-inflammatory activity in vivo. Moreover, LPS-stimulated TNF-IRES-Luc mice were used to investigate the onset and duration of AAIVa on systematic inflammation. A single dosage of AAIVa (100 mg/kg, i.g.) could suppress LPS-triggered inflammation, by decreasing luciferase activities of TNFα at 3 h in TNF-IRES-Luc mice. In addition, the online pharmacological databases predicted that AAIVa might target the regulation of T cell activation-related protein (ADA, ADORA2A, ERBB2) to exhibit anti-inflammatory effect. In conclusion, we demonstrated that AAIVa had anti-inflammatory effect for the first time; our findings are constructive for further studies on pharmacological mechanism of AAIVa.
Subject(s)
Lipopolysaccharides , Tumor Necrosis Factor-alpha , Mice , Animals , Tumor Necrosis Factor-alpha/metabolism , Lipopolysaccharides/pharmacology , Macrophages , Anti-Inflammatory Agents/pharmacology , Inflammation/drug therapy , Edema/chemically induced , Edema/drug therapy , Edema/metabolism , Luciferases/metabolism , Luciferases/pharmacology , Luciferases/therapeutic useABSTRACT
The fat mass and obesity-associated protein FTO is an "eraser" of N6-methyladenosine, the most abundant mRNA modification. FTO plays important roles in tumorigenesis. However, its activities have not been fully elucidated and its possible involvement in DNA damage - the early driving event in tumorigenesis - remains poorly characterized. Here, we have investigated the role of FTO in the DNA damage response (DDR) and its underlying mechanisms. We demonstrate that FTO responds to various DNA damage stimuli. FTO is overexpressed in mice following exposure to the promutagens aristolochic acid I and benzo[a]pyrene. Knockout of the FTO gene in TK6 cells, via CRISPR/Cas9, increased genotoxicity induced by DNA damage stimuli (micronucleus and TK mutation assays). Cisplatin- and diepoxybutane-induced micronucleus frequencies and methyl methanesulfonate- and azathioprine-induced TK mutant frequencies were also higher in FTO KO cells. We investigated the potential roles of FTO in DDR. RNA sequencing and enrichment analysis revealed that FTO deletion disrupted the p38 MAPK pathway and inhibited the activation of nucleotide excision repair and cell-cycle-related pathways following cisplatin (DNA intrastrand cross-links) treatment. These effects were confirmed by western blotting and qRT-PCR. FTO deletion impaired cell-cycle arrest at the G2/M phase following cisplatin and diepoxybutane treatment (flow cytometry analysis). Our findings demonstrated that FTO is involved in several aspects of DDR, acting, at least in part, by impairing cell cycle progression.
Subject(s)
Cisplatin , DNA Damage , Mice , Animals , Cisplatin/toxicity , Mice, Knockout , Cell Division , Carcinogenesis , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/metabolismABSTRACT
Environmental pollutants, such as quinoline (QN) and 4-methylquinoline (4-MeQ), may be genotoxic and carcinogenic. Earlier studies, including in vitro genotoxicity tests, indicated that 4-MeQ is more mutagenic than QN. However, we hypothesized that the methyl group of 4-MeQ favors detoxication over bioactivation, and this factor may be overlooked in in vitro tests that do not incorporate supplementation with cofactors for enzymes that catalyze conjugation reactions. We used human induced hepatocyte cells (hiHeps), which express such enzymes, and compared the genotoxicity of 4-MeQ and QN. We also carried out an in vivo micronucleus (MN) test in rat liver, since 4-MeQ is not genotoxic in rodent bone marrow. In the Ames test and the Tk gene mutation assay, with rat S9 activation, 4-MeQ was more mutagenic than QN. However, QN induced significantly higher MN frequencies in hiHeps and rat liver than did 4-MeQ. Furthermore, QN upregulated genotoxicity marker genes much more than did 4-MeQ. We also investigated the roles of two important detoxication enzymes, UDP-glucuronosyltransferases (UGTs) and cytosolic sulfotransferases (SULTs). When hiHeps were preincubated with hesperetin (UGT inhibitor) and 2,6-dichloro-4-nitrophenol (SULT inhibitor), MN frequencies were elevated approximately 1.5-fold for 4-MeQ, whereas no significant effects were seen for QN. This study shows that QN is more genotoxic than 4-MeQ, when the roles of SULTs and UGTs in detoxication are considered and our results may improve understanding the structure-activity relationships of quinoline derivatives.