Archives of Toxicology最新文献

{"title":"Toxicity of ACP-105: a substance used as doping in sports: application of in silico methods for prediction of selected toxicological endpoints","authors":"Oktawia Fijałkowska,&nbsp;Kamil Jurowski","doi":"10.1007/s00204-025-03962-z","DOIUrl":"10.1007/s00204-025-03962-z","url":null,"abstract":"<div><p>ACP-105 is a novel non-steroidal Selective Androgen Receptor Modulator (SARM) used by athletes. Its action aims to increase muscle mass and is one of the options in testosterone replacement therapy. Its safety profile remains insufficiently explored, particularly regarding its toxicity in humans. The lack of information about the studied compound in the World Anti-Doping Agency (WADA) became the purpose of this study. Given the increasing use of such compounds in sports, a deeper understanding of their biological risks is crucial. This study not only fills the gap in available information but also contributes to the growing body of research on SARMs, providing insights into their potential hazards and guiding future investigations into their safety. This work aimed to use various in silico techniques to predict the toxicity of ACP-105, including acute toxicity, effects on internal organs, genotoxicity based on the Ames test, eye and skin irritation, and cardiotoxicity by testing hERG inhibitors. A preliminary safety analysis of the compound was based on its chemical structure and interactions with biological targets using various in silico techniques: qualitative (STopTox, ADMETlab, admetSAR, ProTox 3.0, and Toxtree 3.1.0) and quantitative (TEST 5.1.2, Percepta, VEGA QSAR 1.2.3, and SL-Tox) to ensure that the prediction results are as accurate as possible.</p></div>","PeriodicalId":8329,"journal":{"name":"Archives of Toxicology","volume":"99 4","pages":"1485 - 1503"},"PeriodicalIF":4.8,"publicationDate":"2025-03-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143596187","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Integrative genome-wide aberrant DNA methylation and transcriptome analysis identifies diagnostic markers for colorectal cancer.","authors":"Hengyang Shen, Zhenling Wang, Yang Chen, Changzhi Huang, Lei Xu, Ying Tong, Hongqiang Zhang, Yunfei Lu, Shuwei Li, Zan Fu","doi":"10.1007/s00204-025-03990-9","DOIUrl":"https://doi.org/10.1007/s00204-025-03990-9","url":null,"abstract":"<p><p>Colorectal cancer remains a major cause of cancer mortality, with limited sensitivity in current diagnostics. Aberrant DNA methylation in expression-regulating sites shows biomarker potential, though few studies explore such methylation-based diagnostic tools for colorectal cancer. We conducted genome-wide DNA methylation and RNA sequencing on matched colorectal cancer and normal tissues to identify expression-related differentially methylated CpG sites (DMCs). Diagnostic models were constructed with training and validation sets of 689 samples. Machine learning techniques (random forest, elastic net, support vector machine) were employed to identify optimal diagnostic markers. Methylation-specific PCR confirmed marker-host gene regulatory relationships, and targeted bisulfite sequencing validated these markers in an independent cohort of 200 samples. Host genes roles in colorectal cancer pathogenesis were further investigated through in vivo and in vitro assays and tissue microarray analysis. We identified 64,824 DMCs in colorectal cancer, with 442 associated with gene expression. These sites impact transcription factor binding, and their host genes are linked to chemotherapy resistance. Diagnostic panels showed high efficacy, with methylation changes significantly impacting RNA and protein expression of host genes. Markers cg16851417, cg19498960, and cg16302790 were validated in blood for noninvasive screening. Clustering expression-related DMCs with similar methylation patterns may facilitate diagnostic tools development. Host genes SIM2, PDX1, and TNS4 influence colorectal cancer progression and may impact therapy response. Expression-related DMCs hold strong potential as colorectal cancer biomarkers, with implications for prognosis and therapy. The specific expression patterns of these DMCs in host genes support development of non-invasive blood-based diagnostic tools.</p>","PeriodicalId":8329,"journal":{"name":"Archives of Toxicology","volume":" ","pages":""},"PeriodicalIF":4.8,"publicationDate":"2025-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143584497","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Metabolism study of two phenethylamine - derived new psychoactive substances using in silico, in vivo, and in vitro approaches.","authors":"Yiling Tang, Linhao Xu, Zhenshuo Guo, Junbo Zhao, Yue Xiao, Ping Xiang, Lili Xu, Hui Yan","doi":"10.1007/s00204-025-04010-6","DOIUrl":"https://doi.org/10.1007/s00204-025-04010-6","url":null,"abstract":"<p><p>New psychoactive substances (NPS) are substances that are not controlled by international drug control conventions but are abused and pose a threat to public health. Proscaline and methallylescaline are two phenylethylamines with psychoactive and stimulant effects and are also derivatives of the classic hallucinogen mescaline. However, limited toxicity information on proscaline and methallylescaline has hindered the identification of these two NPS. Therefore, data on the metabolic profiles of proscaline and methallylescaline are urgently needed. In this study, high-resolution mass spectrometry was used to establish three complementary metabolism models-computational prediction (in silico), zebrafish (in vivo), and human liver microsomes (in vitro)-to study the in vivo and in vitro metabolic fates of proscaline and methallylescaline. The models provided the first identification of 7 proscaline metabolites and 11 methallylescaline metabolites. In addition, hydroxylated and N-acetylated products were identified as the major metabolites of these two phenylethylamines. This enabled the selection of hydroxylated and N-acetylated metabolites as biomarkers of proscaline and methallylescaline, thereby facilitating the specific detection of the intake of these two NPSs in a relatively wide detection window.</p>","PeriodicalId":8329,"journal":{"name":"Archives of Toxicology","volume":" ","pages":""},"PeriodicalIF":4.8,"publicationDate":"2025-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143596122","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Replication stress: an early key event in ochratoxin a genotoxicity?","authors":"C Klotz, J Borchers, J Brode, P Lambeck, A Mally","doi":"10.1007/s00204-025-04004-4","DOIUrl":"https://doi.org/10.1007/s00204-025-04004-4","url":null,"abstract":"<p><p>Ochratoxin A (OTA), a widespread food contaminant and potent renal carcinogen in rodents, is weakly genotoxic in mammalian cells. The mechanisms underlying OTA-induced genetic damage are still poorly understood. In its recent risk assessment, the European food safety authority (EFSA) considered that the specific spectrum of mutations and chromosomal damage induced by OTA may derive from unresolved replication stress. The aim of the present work was to experimentally test the hypothesis that OTA interferes with DNA replication and to characterize the cellular response to OTA-mediated replication stress. Using the DNA fiber assay to study replication fork dynamics at single molecule resolution, a small but statistically significant global delay in replication fork progression was observed in human kidney (HK-2) cells exposed to OTA at ≥ 10 µM. OTA-mediated interference with DNA replication was confirmed by a concentration-related decrease in incorporation of the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) into newly replicating DNA in HK-2 cells arrested in late G₁/S via double thymidine block and treated with OTA during S phase. Western blot and immunofluorescence analyses revealed a significant concentration-related increase in γH2AX in cells exposed to OTA. Co-localization of γH2AX foci with 5-chloro-2'-deoxyuridine (CldU) incorporated into cells during S phase and increased γH2AX labeling along newly replicating chromatin fibers visualized using the extended chromatin fiber assay support a replication-coupled mechanism of OTA-induced DNA damage. Experiments with cells synchronized in late G₁/S or late G₂ demonstrated that exposure of cells to OTA during S phase, but not mitosis, leads to a significant concentration-related increase in H2AX, providing further evidence that OTA may act primarily during S phase of the cell cycle. However, OTA did not appear to efficiently activate ATR-Chk1 and ATM-Chk2 DNA damage response pathways, suggesting that cells with under-replicated DNA or unresolved DNA damage may escape checkpoint control and may continue into mitosis, with potentially deleterious consequences for genomic integrity. Overall, results from this study provide first experimental evidence for perturbation of the S phase replisome machinery by OTA and point toward replication stress as an early key event in OTA genotoxicity.</p>","PeriodicalId":8329,"journal":{"name":"Archives of Toxicology","volume":" ","pages":""},"PeriodicalIF":4.8,"publicationDate":"2025-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143596186","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Exploiting TRP channel diversity in insects: a pathway to next-generation pest management.","authors":"Amit Umesh Paschapur, Marella Sai Manoj, J S Pavan, Sabtharishi Subramanian","doi":"10.1007/s00204-025-04012-4","DOIUrl":"10.1007/s00204-025-04012-4","url":null,"abstract":"<p><p>Transient Receptor Potential (TRP) channels, a diverse family of over 30 ion channel subtypes, are pivotal in regulating sensory perception, thermoregulation, and feeding in insects. In Drosophila melanogaster, 13 TRP channels have been identified, while Aedes aegypti and Anopheles gambiae possess 11 and 10, respectively, showcasing evolutionary adaptations to their ecological niches. This review explores recent advancements in understanding the structure, classification, and physiological functions of TRP channels, emphasizing their evolutionary divergence across Diptera, Lepidoptera, and Hymenoptera. Key TRP subfamilies, such as TRPA, TRPC, TRPM, TRPV, TRPN, and TRPP, are discussed, highlighting their roles in chemo-sensation, gustation, and stress responses. Examples include TRPA1's involvement in thermal sensing and TRPV's role in osmoregulation, critical for insect survival under fluctuating environmental conditions. The review highlights the potential of TRP channels as targets for pest control, focusing on TRP-specific insecticides like pymetrozine, afidopyrifen, and flonicamid, which impact feeding and sensory pathways. RNA interference (RNAi) techniques targeting TRP genes are highlighted as promising tools for innovative pest management. TRP channels' role in mediating thermal tolerance is particularly significant in the context of climate change, where variable temperatures challenge pest dynamics and agricultural sustainability. Understanding these mechanisms is vital for developing climate-resilient pest control strategies. The review also evaluates methodologies used in TRP channel studies, including genomic, transcriptomic, and functional assays, alongside behavioural analyses. Despite progress, challenges remain in studying TRP channels in non-model insects and elucidating their regulation. Future research should integrate multidisciplinary approaches to fully harness TRP channels for sustainable pest management.</p>","PeriodicalId":8329,"journal":{"name":"Archives of Toxicology","volume":" ","pages":""},"PeriodicalIF":4.8,"publicationDate":"2025-03-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143582166","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Application of in silico methods to predict the acute toxicity of bicyclic organophosphorus compounds as potential chemical weapon.","authors":"Maciej Noga, Kamil Jurowski","doi":"10.1007/s00204-025-04000-8","DOIUrl":"https://doi.org/10.1007/s00204-025-04000-8","url":null,"abstract":"<p><p>Bicyclic organophosphorus compounds (BOPCs), including flame retardants and plasticisers, are widely used in industrial applications because of their thermal stability and resistance to degradation. However, their unique structural properties and mechanisms of toxicity raise concerns regarding their potential misuse. Unlike classical organophosphorus compounds that inhibit acetylcholinesterase, BOPCs exert toxicity by antagonising gamma-aminobutyric acid receptors, resulting in severe neurotoxic effects, including convulsions and seizures. This underscores the urgent need to prioritise predictive toxicity studies on these compounds as part of a national defence strategy. The present study represents the first extensive application of in silico toxicological approaches to investigate the acute toxicity of a BOPC dataset (n = 18) utilising advanced in silico tools, such as QSAR models and probabilistic software/platforms, to predict acute oral toxicity in rats. All the investigated BOPCs are highly acutely toxic, judging from LD₅₀ values ranging for humans between < 1 mg and > 1.000 mg/kg bw, depending on the applied model. Noticeable variation between model predictions reminds us that present in silico approaches have significant limitations, at least when addressing chemically complex compounds, such as the BOPC class. This calls for wet-laboratory experimentation. Major toxicophoric groups, such as phosphate and phosphorothione moieties, have been identified as significant contributors to their toxicity. This study considers the need for high-level computational tools, well-founded experimental validation, targeted antidotes, and regulatory measures to reduce the risks from BOPCs and improve public health protection and chemical safety.</p>","PeriodicalId":8329,"journal":{"name":"Archives of Toxicology","volume":" ","pages":""},"PeriodicalIF":4.8,"publicationDate":"2025-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143571593","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The hepatoprotective potential of ferulic acid against a spectrum of pharmaceuticals and toxic compounds.","authors":"Anandakumar Pandi, Balarko Chakraborty, Nabendu Sen, Vanitha Manickam Kalappan","doi":"10.1007/s00204-025-03996-3","DOIUrl":"https://doi.org/10.1007/s00204-025-03996-3","url":null,"abstract":"<p><p>Despite advancements in medicine, drug-induced liver injury continues to pose regulatory and clinical challenges. Ferulic acid, a naturally occurring polyphenol found in vegetables, fruits, and grains, has gained attention for its therapeutic properties. We report the hepatoprotective effects of ferulic acid against a range of pharmaceuticals and toxic compounds. Given the growing interest in natural hepatoprotective compounds, further research is essential to elucidate ferulic acid's role in liver protection.</p>","PeriodicalId":8329,"journal":{"name":"Archives of Toxicology","volume":" ","pages":""},"PeriodicalIF":4.8,"publicationDate":"2025-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143565881","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Potential role of ochratoxin A in Parkinson's disease: a systematic review of current evidence.","authors":"M Serrano-Civantos, E Beraza, L Álvarez-Erviti, A López de Cerain, A Vettorazzi","doi":"10.1007/s00204-025-03994-5","DOIUrl":"https://doi.org/10.1007/s00204-025-03994-5","url":null,"abstract":"<p><p>Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium species that contaminates various food and feed products, presenting potential risks to human health. While OTA is well-known for its nephrotoxic effects, emerging evidence highlights its neurotoxic potential. Parkinson's disease (PD) is a neurodegenerative disorder with both genetic and environmental aetiologies. Emerging lines of investigation have focused their research on the role of environmental toxins, including mycotoxins, in PD pathogenesis. However, the specific involvement of OTA in PD-related pathways still needs to be unravelled. This systematic review compiles and evaluates OTA neurotoxicity studies according to the adverse outcome pathway (AOP) for PD, established by the Organisation for Economic Cooperation and Development (OECD). The AOP framework outlines a series of key event (KEs) beginning with mitochondrial Complex I (CI) inhibition and progressing through mitochondrial dysfunction, impaired proteostasis, dopaminergic neuron degeneration, neuroinflammation, and resulting in parkinsonian motor deficits. In this systematic review, a comprehensive literature search was conducted in PubMed, to identify studies evaluating OTA neurotoxic effects. Using a search strategy of 19 terms and following a two-phased study selection, 30 relevant studies were retrieved, of which 16 dealt with in vitro adult neurotoxicity (ANT), 13 focused on in vivo ANT, and 1 gave both in vitro and in vivo approaches. Authors agree that in vitro and in vivo exposure to OTA causes mitochondrial dysfunction, impaired proteostasis, degeneration of dopaminergic (DA) neurons, and neuroinflammation. However, a notable absence of research remains on the molecular initiating event (MIE), binding to CI, and on KE1, inhibition of CI. This review identifies critical research gaps and highlights the need for further mechanistic studies on the impact of OTA on neurodegenerative pathways, particularly its binding and inhibition of CI, as well as mechanisms related to KE3: impaired proteostasis. Addressing these gaps may provide valuable insights into OTA neurotoxic potential and its relevance in PD-like neurodegeneration.</p>","PeriodicalId":8329,"journal":{"name":"Archives of Toxicology","volume":" ","pages":""},"PeriodicalIF":4.8,"publicationDate":"2025-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143565877","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"In vitro approaches to investigate the effect of chemicals on antibody production: the case study of PFASs.","authors":"Martina Iulini, Valeria Bettinsoli, Ambra Maddalon, Valentina Galbiati, Aafke W F Janssen, Karsten Beekmann, Giulia Russo, Francesco Pappalardo, Styliani Fragki, Alicia Paini, Emanuela Corsini","doi":"10.1007/s00204-025-03993-6","DOIUrl":"https://doi.org/10.1007/s00204-025-03993-6","url":null,"abstract":"<p><p>The increasing variety and quantity of new chemical substances have raised concerns about their potential immunotoxic effects, making it essential to assess their impact on human health. One key concern is the reduction of antibody production, as seen with per- and poly-fluoroalkyl substances (PFASs), commonly known as \"forever chemicals.\" Both in vivo and epidemiological data show that PFASs have immunosuppressive effects, leading to reduced antibody responses, particularly following vaccination. In animal studies, the T cell-dependent (TD) antibody response is the gold standard for assessing chemical effects on immune function. This study utilized two in vitro approaches to investigate the effects of chemicals on antibody production using human peripheral blood mononuclear cells. Initial tests used unstimulated, negative (vehicle), and positive (rapamycin) controls to confirm the robustness of the models. Subsequently, four long-chain PFASs (PFOA, PFOS, PFNA, and PFHxS) were tested. Keyhole limpet hemocyanin (KLH) was used to mimic the TD response, while a TLR9 agonist and IL-2 activated B cells for T cell-independent (TI) immunoglobulin production. The results demonstrated the ability to reproduce TD and TI responses in vitro with robust, reproducible outcomes across a cohort of 20 human donors. The data, consistent with existing literature, showed a significant reduction in anti-KLH IgM production, especially for PFOA in male donors. Similar trends were observed for all PFASs in suppressing total TI IgG and IgM production. These methods closely replicated in vivo conditions, offering a potential alternative to animal models in immunotoxicity assessments.</p>","PeriodicalId":8329,"journal":{"name":"Archives of Toxicology","volume":" ","pages":""},"PeriodicalIF":4.8,"publicationDate":"2025-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143565874","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Exploring the multifaceted impact of bisphosphonates on bone graft integration: transitioning from in vivo insights to clinical applications.","authors":"Ronnakrit Maethungkul, Apiruk Sangsin, Nipon Chattipakorn, Siriporn C Chattipakorn","doi":"10.1007/s00204-025-03991-8","DOIUrl":"https://doi.org/10.1007/s00204-025-03991-8","url":null,"abstract":"<p><p>Numerous researchers have explored the efficacy of the use of bisphosphonates, in particular alendronate and zoledronate, in bone graft integration. Mechanisms underlying the beneficial effects of bisphosphonates on bone graft integration have been previously investigated. Previous studies have demonstrated that both local and systemic applications of bisphosphonates in the integration of autogenous and allogenic bone grafts yield promising results in enhancing allograft and autograft outcomes. Several animal studies have revealed that low to moderate doses of local bisphosphonate optimize new bone formation with good mechanical properties. In addition, bisphosphonates in combination with certain growth factors, such as bone morphogenetic protein (BMP)-7, have synergistic effects on the integration of both autogenous and allogenic bone grafts. However, only some clinical parameters support these findings from in vivo studies, including increased bone volume and density in bone-grafted areas following the integration of bone grafts. Thus, the present review aims to summarize and discuss the effects of bisphosphonates and the mechanistic insights associated with the integration of autogenous and allogenic bone grafts. The contradictory findings between studies will also be presented and discussed in this review. While the promising benefits of bisphosphonates on bone grafts have been reported, further research is still needed to investigate optimal dosage. Larger clinical trials to assess the long-term outcomes of bisphosphonates are required before they can be widely adopted in orthopedic, dental, and maxillofacial bone grafting procedures.</p>","PeriodicalId":8329,"journal":{"name":"Archives of Toxicology","volume":" ","pages":""},"PeriodicalIF":4.8,"publicationDate":"2025-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143565872","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}