Environmental and Molecular Mutagenesis最新文献

{"title":"A Survey of DNA Damage in American Alligators (Alligator mississippiensis) in Florida.","authors":"Haiyan Lu, Idoia Meaza, Rachel M Wise, James T F Wise, Sandra S Diven, Tayler J Croom-Pérez, Jennifer H Toyoda, John Pierce Wise","doi":"10.1002/em.70047","DOIUrl":"10.1002/em.70047","url":null,"abstract":"<p><p>The scientific concept of One Environmental Health is a research strategy focused on the study of toxicants, aiming to incorporate human, wildlife, and ecosystem health to establish a more comprehensive understanding of health. A One Environmental Health approach, studying the responses of American alligators (Alligator mississippiensis) that are considered apex sentinel organisms to environmental toxicants, is crucial. Due to their long-lived lifestyle on land and water, including a wide range of prey items in their diet and their ability to bioaccumulate metals, alligators serve as effective indicators of heavy metal pollution, which poses risks to aquatic ecosystems and human health. In this study, we examined DNA damage in American alligators from three locations in Florida using comet assay. We found alligators in Merritt Island National Wildlife Refuge had significantly higher DNA damage levels compared to those in Lake Apopka and Lake Woodruff. This trend was consistent across both sexes, with no observed sex differences. Similarly, DNA damage levels were significantly higher in both adult and juvenile alligators from Merritt Island compared to the other locations. Notably, juvenile alligators exhibited higher DNA damage than adults, with animals from Merritt Island exhibiting particularly elevated levels.</p>","PeriodicalId":11791,"journal":{"name":"Environmental and Molecular Mutagenesis","volume":"67 3","pages":"e70047"},"PeriodicalIF":2.7,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13109689/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147766169","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Alizarin Dye: Toxicity, Genotoxicity, and Histopathological Alterations in Model Organisms.","authors":"Amanda Rocha Rodrigues, Gabriela Cristina Fonseca Almeida, Natália Oliveira de Farias, Anjaina Fernandes de Albuquerque, Adria Caloto de Oliveira, Jessica Camila Miranda Cardoso, Gabriely Fernanda Groto Militão, Inês Moutinho Cabral, Catarina A Faustino, João D Vitorino, Marina Tenório Botelho, Pedro M Costa, Gisela de Aragão Umbuzeiro","doi":"10.1002/em.70046","DOIUrl":"10.1002/em.70046","url":null,"abstract":"<p><p>Alizarin is an anthraquinone red dye from natural or synthetic sources, widely used in textiles. Effluents of this activity can contain residual dyes, which may contaminate the aquatic environment. Studies report alizarin's aquatic toxicity, mutagenic, and carcinogenic effects. This study aimed to complement the aquatic toxicity evaluation and confirm its ability to cause genotoxicity in alternative models. Acute toxicity was performed with crustaceans, mussels, and fish embryos, while chronic toxicity was assessed in algae. Light effects on toxicity were evaluated using Daphnia similis. Histopathological effects on the gonads of Mytilus galloprovincialis and somatic mutations and sperm genotoxicity in Parhyale hawaiensis were investigated. Mutagenicity was confirmed using a miniaturized Ames test. The effect concentration 50% (EC₅₀) for D. similis was 90.3, 105, and 68.6 μg L^-1 for photoperiod (16 h light:8 h dark), light and dark, respectively. For Danio rerio embryos, the lethal concentration 50% (LC₅₀) was 45.8 μg L^-1, and an EC₁₀ of 20.8 μg L^-1 was calculated for sublethal effects. In vivo exposures caused alterations in the digestive gland and gonads of M. galloprovincialis, even in a short-term exposure, and increased the frequency of micronuclei and DNA damage in hemocytes and spermatozoids, respectively, of P. hawaiensis. It was mutagenic in the miniaturized Ames test using strain TA1537 (10% and 30% S9). Alizarin can be classified as a Category 1 acute aquatic toxicity according to the globally harmonized system (GHS). Due to adverse histopathological and DNA effects on reproductive systems in model organisms, it is considered a potential germ cell mutagen.</p>","PeriodicalId":11791,"journal":{"name":"Environmental and Molecular Mutagenesis","volume":"67 3","pages":"e70046"},"PeriodicalIF":2.7,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13109681/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147766227","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Longitudinal Assessment of DNA Damage in PBMCs From Hospitalized COVID-19 Patients via Alkaline Comet Assay.","authors":"Lenin Rueda-Torres, Dulce E Alarcón-Yaquetto, Beatriz Ayala-Quintanilla, Germán Málaga, Jaime Rosales-Rimache","doi":"10.1002/em.70055","DOIUrl":"10.1002/em.70055","url":null,"abstract":"<p><p>Growing evidence suggests an association between SARS-CoV-2 infection and oxidative stress-related genomic damage, primarily from cross-sectional studies. However, longitudinal evaluations remain limited. Here, we assessed DNA damage in peripheral blood mononuclear cells (PBMCs) from hospitalized COVID-19 patients during the first days of hospitalization, using the alkaline comet assay. A longitudinal descriptive study was conducted at a modular COVID-19 hospital in Lima, Peru, throughout October 2020. Blood samples were collected at admission (day 0), day 3, and day 6. DNA damage was assessed by measuring tail length, tail moment, and tail intensity, which were modeled using mixed-effects linear models. Concurrent hematological parameters and acute-phase proteins were also analyzed to characterize their time-course patterns in conjunction with DNA damage. Results from the adjusted mixed-effects model showed a significant temporal increase in DNA damage parameters, particularly tail intensity (β = 0.25 per day, p = 0.003), relative to baseline measurements, with a peak observed on day 3 of hospitalization. These changes aligned with variations in platelet counts and transferrin levels, while lymphocyte and monocyte counts, along with C-reactive protein, showed an inverse trend, reflecting the dynamic interplay of the inflammatory response and DNA damage. No significant differences in comet assay parameters were observed between survivors and non-survivors, likely due to the limited and unbalanced sample. The findings indicate the presence of a measurable DNA damage burden in PBMCs of hospitalized COVID-19 patients, highlighting the need to further studies to clarify the mechanisms underlying DNA damage and its potential long-term biological implications.</p>","PeriodicalId":11791,"journal":{"name":"Environmental and Molecular Mutagenesis","volume":"67 3","pages":"e70055"},"PeriodicalIF":2.7,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147766158","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Polymorphisms in CLAUDIN1 and SPINK5 Influence Skin Absorption of Pyrene, Pyrimethanil, and Oxybenzone in Human Volunteers.","authors":"Emmy Keysendal, Gunnar Johanson, Lina Hagvall, Nanna Fyhrqvist, Christian Lindh, Karin Broberg, Emelie Rietz Liljedahl","doi":"10.1002/em.70050","DOIUrl":"10.1002/em.70050","url":null,"abstract":"<p><p>Absorption of chemicals through the skin affects occupational and environmental exposure to diverse compounds. We previously showed that loss-of-function (null) mutations and low-copy number variants (CNV) of Filaggrin (FLG), which encodes a key skin barrier protein, increased dermal chemical absorption; however, the FLG genotype did not explain all the observed variation. Here, we explore the effects of variation in genes encoding skin proteins that could affect chemical uptake. In a dermal exposure test, 23 null-FLG and 31 wild-type carriers were exposed to three common organic compounds: the polycyclic aromatic hydrocarbon pyrene, the fungicide pyrimethanil, and the ultraviolet-light absorber oxybenzone. Liquid chromatography-mass spectrometry was used to measure the concentrations of these chemicals or their metabolites in the subjects' urine collected over a 40-h period following exposure. We genotyped the participants for 14 polymorphisms in seven skin function-related genes (Filaggrin 2 [FLG2], including a new method for assessing FLG2 CNV, claudin 1 [CLDN1], serine peptidase inhibitor kazal type 5 [SPINK5], S100 calcium binding protein A7 [S100A7], transmembrane protein 79 [TMEM79], laminin subunit alpha 3 [LAMA3], and involucrin [IVL]) and performed a population toxicokinetic analysis. While controlling for FLG genotype, the CLDN1 rs893051 minor allele was associated with increased absorption, faster absorption rate, and longer lag time, while the SPINK5 rs2303067 minor allele was associated with shorter lag time. However, the differences in total systemic absorption were minor compared with FLG variants. Thus, FLG remains the predominant genetic determinant of chemical uptake through the skin.</p>","PeriodicalId":11791,"journal":{"name":"Environmental and Molecular Mutagenesis","volume":"67 3","pages":"e70050"},"PeriodicalIF":2.7,"publicationDate":"2026-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13109691/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147766222","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The In Vitro Transgenic Rodent Assay in Primary MutaMouse Hepatocytes Compared to the Mammalian Cell Gene Mutation Assay Using the HPRT Gene.","authors":"Alina Göpfert, Kylee Kendra Marie Ronnenberg, Claudia Ruelker, Silke Spang, Naveed Honarvar, Robert Landsiedel","doi":"10.1002/em.70040","DOIUrl":"10.1002/em.70040","url":null,"abstract":"<p><p>Gene mutations can be detected in mammalian cells in vitro using indicator genes such as the hypoxanthine-guanine-phosphoribosyltransferase (HPRT) gene. These assays have been adopted as OECD test guidelines (TG, e.g., OECD TG no. 476) and are used for regulatory purposes. The in vitro transgenic rodent assay (TGRA) in primary MutaMouse hepatocytes is a novel approach for the detection and quantification of gene mutations. Its methodology follows the same principles as the in vivo TGRA, an in vivo gene mutation assay with regulatory adoption (OECD TG no. 488). Although the potential of the in vitro TGRA to identify mutagens has been reported, its performance compared to an established in vitro gene mutation assay has not been reported. This study compared the in vitro TGRA with the HPRT assay using 10 known in vivo mutagens. The in vitro TGRA correctly identified all 10 mutagens, whereas the HPRT assay identified only nine. Benchmark concentration (BMC) modeling for the nine substances detected by both assays revealed overlapping confidence intervals for six compounds, indicating comparable sensitivity. For three mutagens, the HPRT assay yielded lower BMC intervals. Additionally, eight substances known to be non-mutagenic in vivo tested negative in the in vitro TGRA. While increased cytotoxicity did not induce increased mutant frequencies, it reduced DNA yield, thereby impairing mutagenicity assessment. The results of this study contribute to the understanding of the sensitivity and robustness of the in vitro TGRA and provide essential information for the validation of the assay.</p>","PeriodicalId":11791,"journal":{"name":"Environmental and Molecular Mutagenesis","volume":" ","pages":"e70040"},"PeriodicalIF":2.7,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13063353/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145387923","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Meta-Analysis of CYP1A1 MspI and Ile462Val Polymorphisms in Cancer Susceptibility Among Different Ethnic Populations.","authors":"Amjad Yousuf, Najeeb Ullah Khan, Ahsanullah Unar","doi":"10.1002/em.70042","DOIUrl":"10.1002/em.70042","url":null,"abstract":"<p><p>The Cytochrome P450 1A1 (CYP1A1) gene plays a crucial role in the production of enzymes involved in the metabolic activation and detoxification of harmful carcinogens, which are essential for genetic susceptibility to cancer. Due to the inconsistent findings obtained from population-based studies, it is crucial to systematically investigate the association between CYP1A1 polymorphisms and diverse ethnic groups. To assess the link between CYP1A1 polymorphisms and cancer risk across different ethnic populations. The studies published in the last decade were searched through PubMed, Cochrane Library, and Embase, based on PRISMA guidelines and eligibility criteria. Meta-analysis includes subgroup analysis based on ethnicity with odds ratio (OR) and 95% confidence intervals through R Studio. Genotypic and allelic data were analyzed under genetic models (allelic, dominant, and recessive) using a random-effects model. The quality of the included case-control studies was assessed using the Newcastle-Ottawa scale. Twenty case-control studies containing various ethnic populations, of which eleven contain the MspI polymorphism, and the other nine contain the Ile462Val polymorphism of the CYP1A1, while none explained both SNPs. The research studies involved 3976 cases and 4891 controls in this meta-analysis. For MspI polymorphisms, the overall pooled analysis revealed a significant association with cancer risk in the Brazilian ethnic group (2.46 [95% CI: 0.00; 305699178.1]) with moderate heterogeneity observed within the genetic models of CYP1A1 polymorphisms. For Ile462Val polymorphisms, the overall pooled effect size was significant among the Asian group (2.11 [95% CI: 1.45; 3.06]). Meanwhile, the subgroup analysis provides some evidence of cancer risk association with polymorphisms among different ethnicities. The results of this meta-analysis indicate that the understanding of CYP1A1 polymorphisms is necessary to determine the etiology of cancer. The significant association among CYP1A1 polymorphisms and cancer can further be studied by selecting studies focused on a particular cancer type and containing a large sample size within a specific ethnic population.</p>","PeriodicalId":11791,"journal":{"name":"Environmental and Molecular Mutagenesis","volume":" ","pages":"e70042"},"PeriodicalIF":2.7,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13063359/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145713358","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Identification of Most Stably Expressed Internal Control Genes for Normalization of Target Gene Expression in Buffalo Skin Tissue.","authors":"Ankita Gurao, Anurag Kumar, Namita Kumari, Rashi Vasisth, Manishi Mukesh, Vikas Vohra, Sanjay Kumar, Naresh Kumar, Ranjit Singh Kataria","doi":"10.1002/em.70038","DOIUrl":"10.1002/em.70038","url":null,"abstract":"<p><p>Water buffalo (Bubalus bubalis) skin exhibits unique morphological and physiological adaptations compared to other domestic bovines to enhance thermoregulation facilitated by abundant dermal melanin. Melanin synthesis and distribution, governed by the melanogenesis pathway and associated genes underpin coat color phenotypes and determine thermotolerance. The molecular mechanisms regulating pigmentation in buffalo skin remain underexplored as well as the reliable internal control genes (ICGs) for qPCR normalization. This study addresses the critical gap by systematically evaluating a panel of nine candidate reference genes for their expression stability in skin tissues from contrastingly pigmented buffaloes, jet-black Murrah and Nili Ravi (CC1) versus extensively white-spotted Nili Ravi (CC2). From the application of comprehensive statistical algorithms (deltaC_q, NormFinder, geNorm, BestKeeper), the combination of GAPDH, EEF1A1, and RPS23 emerged as the most stable reference gene set for normalization. Validation using key melanogenesis genes (KIT and TYR) confirmed that normalization with this trio yields consistent and biologically relevant expression profiles, with KIT robustly downregulated in white versus black skin. The recommended reference gene combination ensures robust normalization in gene expression profiling of melanogenesis-related genes in buffalo skin tissue.</p>","PeriodicalId":11791,"journal":{"name":"Environmental and Molecular Mutagenesis","volume":" ","pages":"e70038"},"PeriodicalIF":2.3,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145451330","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"","authors":"","doi":"","DOIUrl":"","url":null,"abstract":"","PeriodicalId":11791,"journal":{"name":"Environmental and Molecular Mutagenesis","volume":"67 1-2","pages":""},"PeriodicalIF":2.3,"publicationDate":"2025-12-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148070763","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Outcome of IWGT workshop on transcriptomic biomarkers for genotoxicity: Key considerations for bioinformatics.","authors":"Matthew J Meier, Florian Caiment, J Christopher Corton, Roland Frötschl, Yurika Fujita, Danyel Jennen, Roman Mezencev, Carole Yauk","doi":"10.1002/em.22644","DOIUrl":"10.1002/em.22644","url":null,"abstract":"<p><p>As a part of the International Workshop on Genotoxicity Testing (IWGT) in 2022, a workgroup was formed to evaluate the level of validation and regulatory acceptance of transcriptomic biomarkers that identify genotoxic substances. Several such biomarkers have been developed using various molecular techniques and computational approaches. Within the IWGT workgroup on transcriptomic biomarkers, bioinformatics was a central topic of discussion, focusing on the current approaches used to process the underlying molecular data to distill a reliable predictive signal; that is, a gene set that is indicative of genotoxicity and can then be used in later studies to predict potential DNA damaging properties for uncharacterized chemicals. While early studies used microarray data, a technological shift occurred in the past decade to incorporate modern transcriptome measuring techniques such as high-throughput transcriptomics, which in turn is based on high-throughput sequencing. Herein, we present the workgroup's review of the current bioinformatic approaches to identify genes comprising transcriptomic biomarkers. Within the context of regulatory toxicology, the reproducibility of a given analysis is critical. Therefore, the workgroup provides consensus recommendations on how to facilitate sufficient reporting of experimental parameters for the analytical procedures used in a transcriptomic biomarker study, including the recommendation to develop a biomarker-specific reporting module within the OECD Omics Reporting Framework.</p>","PeriodicalId":11791,"journal":{"name":"Environmental and Molecular Mutagenesis","volume":" ","pages":"71-80"},"PeriodicalIF":2.7,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12988029/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142827734","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Interpretation of in vitro concentration-response data for risk assessment and regulatory decision-making: Report from the 2022 IWGT quantitative analysis expert working group meeting.","authors":"Marc A Beal, Guangchao Chen, Kerry L Dearfield, Min Gi, Bhaskar Gollapudi, Robert H Heflich, Katsuyoshi Horibata, Alexandra S Long, David P Lovell, Barbara L Parsons, Stefan Pfuhler, John Wills, Andreas Zeller, George Johnson, Paul A White","doi":"10.1002/em.22582","DOIUrl":"10.1002/em.22582","url":null,"abstract":"<p><p>Quantitative risk assessments of chemicals are routinely performed using in vivo data from rodents; however, there is growing recognition that non-animal approaches can be human-relevant alternatives. There is an urgent need to build confidence in non-animal alternatives given the international support to reduce the use of animals in toxicity testing where possible. In order for scientists and risk assessors to prepare for this paradigm shift in toxicity assessment, standardization and consensus on in vitro testing strategies and data interpretation will need to be established. To address this issue, an Expert Working Group (EWG) of the 8th International Workshop on Genotoxicity Testing (IWGT) evaluated the utility of quantitative in vitro genotoxicity concentration-response data for risk assessment. The EWG first evaluated available in vitro methodologies and then examined the variability and maximal response of in vitro tests to estimate biologically relevant values for the critical effect sizes considered adverse or unacceptable. Next, the EWG reviewed the approaches and computational models employed to provide human-relevant dose context to in vitro data. Lastly, the EWG evaluated risk assessment applications for which in vitro data are ready for use and applications where further work is required. The EWG concluded that in vitro genotoxicity concentration-response data can be interpreted in a risk assessment context. However, prior to routine use in regulatory settings, further research will be required to address the remaining uncertainties and limitations.</p>","PeriodicalId":11791,"journal":{"name":"Environmental and Molecular Mutagenesis","volume":" ","pages":"101-120"},"PeriodicalIF":2.7,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12988033/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138797526","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}