Pub Date : 2024-12-28DOI: 10.1016/j.tiv.2024.106004
Buket Bakan , Burak Kaptaner , Merve Tokmak , Handan Aykut , Ali Sefa Mendil , Mustafa Özkaraca
Bisphenols can enter the body, where they have potential adverse effects on human health, via different routes such as inhalation, dermally or orally. They are known as endocrine disrupting chemicals that activate signaling pathways by mimicking the estrogen actions. In this study, we aimed to investigate effects of bisphenol A (BPA), and its analogues bisphenol F (BPF) and bisphenol S (BPS) on MCF-10A cells and their impact mechanisms on autophagy, apoptosis and reduced glutathion levels. In comparison of the cytotoxic effects, while BPF and BPS showed dose-dependent high toxicity on MCF-10A cells, BPA exerted cytotoxic effects only at the highest doses. Caspase 3 and LC3B are strongly and positively correlated with BPF exposures while significant changes were not detected in the BPA and BPS applied groups. It was clearly observed that BPF and BPS displayed more toxic effects than BPA on human breast cells that are important targets for the bisphenols. These findings provide data for understanding the mechanisms for BPA, BPF and BPS-induced toxicity on human breast cells.
双酚可通过吸入、皮肤或口服等不同途径进入人体，对人体健康产生潜在的不利影响。众所周知，双酚 A 是一种干扰内分泌的化学物质，它通过模仿雌激素的作用来激活信号通路。在这项研究中，我们旨在调查双酚 A（BPA）及其类似物双酚 F（BPF）和双酚 S（BPS）对 MCF-10A 细胞的影响，以及它们对自噬、细胞凋亡和谷胱甘肽水平降低的影响机制。在细胞毒性效应比较中，双酚 F 和双酚 S 对 MCF-10A 细胞的毒性呈剂量依赖性，而双酚 A 仅在最高剂量时才产生细胞毒性效应。Caspase 3 和 LC3B 与暴露于 BPF 强烈正相关，而在暴露于 BPA 和 BPS 的组别中未检测到显著变化。可以清楚地观察到，与双酚 A 相比，BPF 和 BPS 对人类乳腺细胞的毒性作用更大，而乳腺细胞是双酚 A 的重要靶标。这些发现为了解双酚 A、双酚 F 和双酚 S 诱导人类乳腺细胞毒性的机制提供了数据。
{"title":"Toxicological investigation of bisphenol A and its derivates on human breast epithelial (MCF-10A) cells","authors":"Buket Bakan , Burak Kaptaner , Merve Tokmak , Handan Aykut , Ali Sefa Mendil , Mustafa Özkaraca","doi":"10.1016/j.tiv.2024.106004","DOIUrl":"10.1016/j.tiv.2024.106004","url":null,"abstract":"<div><div>Bisphenols can enter the body, where they have potential adverse effects on human health, <em>via</em> different routes such as inhalation, dermally or orally. They are known as endocrine disrupting chemicals that activate signaling pathways by mimicking the estrogen actions. In this study, we aimed to investigate effects of bisphenol A (BPA), and its analogues bisphenol F (BPF) and bisphenol S (BPS) on MCF-10A cells and their impact mechanisms on autophagy, apoptosis and reduced glutathion levels. In comparison of the cytotoxic effects, while BPF and BPS showed dose-dependent high toxicity on MCF-10A cells, BPA exerted cytotoxic effects only at the highest doses. Caspase 3 and LC3B are strongly and positively correlated with BPF exposures while significant changes were not detected in the BPA and BPS applied groups. It was clearly observed that BPF and BPS displayed more toxic effects than BPA on human breast cells that are important targets for the bisphenols. These findings provide data for understanding the mechanisms for BPA, BPF and BPS-induced toxicity on human breast cells.</div></div>","PeriodicalId":54423,"journal":{"name":"Toxicology in Vitro","volume":"104 ","pages":"Article 106004"},"PeriodicalIF":2.6,"publicationDate":"2024-12-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142907921","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-12-25DOI: 10.1016/j.tiv.2024.106003
Ershun Zhou , Yifei Li , Zhikai Wu , Yichun Chen , Hanpeng Wu , Yingrong Ye , Tianyu Li , Jingjing Wang , Zhengtao Yang
Bongkrekic acid (BKA), a less well-known foodborne toxin, has been implicated in numerous poisoning incidents. Recent studies suggest that BKA exerts an impact on the immune system, particularly on innate immunity. The release of neutrophil extracellular traps (NETs) is relatively a newly-discovered mechanism involving innate immunity. This study was designed to characterize and evaluate the effects of BKA on human NET formation. The co-localization of DNA, histones, and myeloperoxidase (MPO) was determined via immunostaining to confirm BKA-triggered NET formation in human neutrophils. NET quantification showed that NET formation induced by BKA was both time- and dose-dependent, and was associated with p38, ERK, PAD4 and P2X1 receptor. Moreover, immunostaining analysis observed that BKA triggered both NET formation and autophagy. Additionally, pharmacological experiments revealed that autophagy mediated BKA-triggered NET formation. Collectively, these insights offer a novel perspective on the effects of BKA exposure on host's innate immune response, and may shed new light on BKA poisoning. We call for further work to be conducted in this field to unravel the intricate mechanisms governing NET formation and autophagy in the context of BKA poisoning.
{"title":"Neutrophil extracellular traps formation and autophagy in bongkrekic acid exposed human neutrophils","authors":"Ershun Zhou , Yifei Li , Zhikai Wu , Yichun Chen , Hanpeng Wu , Yingrong Ye , Tianyu Li , Jingjing Wang , Zhengtao Yang","doi":"10.1016/j.tiv.2024.106003","DOIUrl":"10.1016/j.tiv.2024.106003","url":null,"abstract":"<div><div>Bongkrekic acid (BKA), a less well-known foodborne toxin, has been implicated in numerous poisoning incidents. Recent studies suggest that BKA exerts an impact on the immune system, particularly on innate immunity. The release of neutrophil extracellular traps (NETs) is relatively a newly-discovered mechanism involving innate immunity. This study was designed to characterize and evaluate the effects of BKA on human NET formation. The co-localization of DNA, histones, and myeloperoxidase (MPO) was determined via immunostaining to confirm BKA-triggered NET formation in human neutrophils. NET quantification showed that NET formation induced by BKA was both time- and dose-dependent, and was associated with p38, ERK, PAD4 and P2X1 receptor. Moreover, immunostaining analysis observed that BKA triggered both NET formation and autophagy. Additionally, pharmacological experiments revealed that autophagy mediated BKA-triggered NET formation. Collectively, these insights offer a novel perspective on the effects of BKA exposure on host's innate immune response, and may shed new light on BKA poisoning. We call for further work to be conducted in this field to unravel the intricate mechanisms governing NET formation and autophagy in the context of BKA poisoning.</div></div>","PeriodicalId":54423,"journal":{"name":"Toxicology in Vitro","volume":"104 ","pages":"Article 106003"},"PeriodicalIF":2.6,"publicationDate":"2024-12-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142900485","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-12-22DOI: 10.1016/j.tiv.2024.106002
Herath Mudiyanselage Udari Lakmini Herath , Musun Park , Mei Jing Piao , Kyoung Ah. Kang , Pincha Devage Sameera Madushan Fernando , Herath Mudiyanselage Maheshika Madhuwanthi Senavirathna , Hee-Sun Kim , Sungwook Chae , Young Ree Kim , Jin Won Hyun
Particulate matter 2.5 (PM2.5) exposure is responsible for skin inflammation, aging, and disruption of skin homeostasis. The objective of this investigation was to assess the potential of myricetin in protecting against skin damage caused by PM2.5. Human keratinocytes (HaCaT) were pretreated with myricetin and subsequently exposed to PM2.5. Cell viability, reactive oxygen species (ROS) generation, oxidized cellular components, mitochondrial damage, cellular apoptosis, and endoplasmic reticulum (ER) stress were assessed. A mitogen-activated protein kinase (MAPK) signaling network was constructed, and the action site of myricetin was explored through docking analysis. PM2.5 induced oxidative stress, resulting in DNA damage, lipid peroxidation, protein carbonylation, and cellular apoptosis. Myricetin counteracted these effects by reducing the PM2.5-induced ROS levels. Additionally, myricetin mitigated the PM2.5-induced cytochrome c release into the cytoplasm and caspase activation, thereby ameliorating cellular apoptosis. Myricetin reduced PM2.5-induced cytosolic Ca2+ level and ER-related signaling molecules. Furthermore, myricetin inhibited cellular cytotoxicity by downregulating the MAPK signaling pathway. Docking and network analyses identified 12 major MAPK proteins targeted by myricetin, and these proteins primarily affected the classical MAPK pathway. These findings suggest that myricetin mitigates skin impairments caused by PM2.5 exposure by reducing ROS, mitochondrial damage, ER stress, and apoptosis via downregulating the MAPK signaling pathway.
{"title":"The protective impact of myricetin against PM2.5-induced cellular apoptosis by inhibiting endoplasmic reticulum stress","authors":"Herath Mudiyanselage Udari Lakmini Herath , Musun Park , Mei Jing Piao , Kyoung Ah. Kang , Pincha Devage Sameera Madushan Fernando , Herath Mudiyanselage Maheshika Madhuwanthi Senavirathna , Hee-Sun Kim , Sungwook Chae , Young Ree Kim , Jin Won Hyun","doi":"10.1016/j.tiv.2024.106002","DOIUrl":"10.1016/j.tiv.2024.106002","url":null,"abstract":"<div><div>Particulate matter 2.5 (PM<sub>2.5</sub>) exposure is responsible for skin inflammation, aging, and disruption of skin homeostasis. The objective of this investigation was to assess the potential of myricetin in protecting against skin damage caused by PM<sub>2.5</sub>. Human keratinocytes (HaCaT) were pretreated with myricetin and subsequently exposed to PM<sub>2.5</sub>. Cell viability, reactive oxygen species (ROS) generation, oxidized cellular components, mitochondrial damage, cellular apoptosis, and endoplasmic reticulum (ER) stress were assessed. A mitogen-activated protein kinase (MAPK) signaling network was constructed, and the action site of myricetin was explored through docking analysis. PM<sub>2.5</sub> induced oxidative stress, resulting in DNA damage, lipid peroxidation, protein carbonylation, and cellular apoptosis. Myricetin counteracted these effects by reducing the PM<sub>2.5</sub>-induced ROS levels. Additionally, myricetin mitigated the PM<sub>2.5</sub>-induced cytochrome <em>c</em> release into the cytoplasm and caspase activation, thereby ameliorating cellular apoptosis. Myricetin reduced PM<sub>2.5</sub>-induced cytosolic Ca<sup>2+</sup> level and ER-related signaling molecules. Furthermore, myricetin inhibited cellular cytotoxicity by downregulating the MAPK signaling pathway. Docking and network analyses identified 12 major MAPK proteins targeted by myricetin, and these proteins primarily affected the classical MAPK pathway. These findings suggest that myricetin mitigates skin impairments caused by PM<sub>2.5</sub> exposure by reducing ROS, mitochondrial damage, ER stress, and apoptosis via downregulating the MAPK signaling pathway.</div></div>","PeriodicalId":54423,"journal":{"name":"Toxicology in Vitro","volume":"104 ","pages":"Article 106002"},"PeriodicalIF":2.6,"publicationDate":"2024-12-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142886472","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-12-19DOI: 10.1016/j.tiv.2024.106001
Krittika Mittal , Ke Xu , Samuel J. Rulli Jr , Guangyan Zhou , Jianguo Xia , Niladri Basu
There is growing scientific and regulatory interest in transcriptomic points of departure (tPOD) values from high-throughput in vitro experiments. To further help democratize tPOD research, here we outline ‘TPD-seq’ which links microplate-based exposure methods involving cell lines for human (Caco-2, Hep G2) and environmental (rainbow trout RTgill-W1) health, with a commercially available RNA-seq kit, with a cloud-based bioinformatics tool (ExpressAnalyst.ca). We applied the TPD-seq workflow to derive tPODs for solvents (dimethyl sulfoxide, DMSO; methanol) and positive controls (3,4-dichloroaniline, DCA; hydrogen peroxide, H2O2) commonly used in toxicity testing. The majority of reads mapped to protein coding genes (∼9 k for fish cells; ∼6 k for human cells), and about 50 % of differentially expressed genes were curve-fitted from which 90 % yielded gene benchmark doses. The most robust transcriptomic responses were caused by DMSO exposure, and tPOD values were 31–155 mM across the cell lines. OECD test guideline 249 (RTgill-W1 cells) recommends the use of DCA and here we calculated a tPOD of ∼5 to 76 μM. Finally, exposure of the two human cell lines to H2O2 resulted in tPOD values that ranged from 0.7 to 1.1 mM in Caco-2 cells and 5–30 μM in Hep G2 cells. The methods outlined here are designed to be performed in laboratories with basic molecular and cell culture facilities, and the performance and scalability of the TPD-seq workflow can be determined with additional case studies.
在高通量体外实验中，对转录组起始点（tPOD）值的科学和调控兴趣越来越大。为了进一步促进tPOD研究的普及，我们在这里概述了“TPD-seq”，它将涉及人类（cco -2, Hep G2）和环境（虹鳟RTgill-W1）健康的细胞系的微孔板暴露方法与市售的RNA-seq试剂盒以及基于云的生物信息学工具（expressanalyzer .ca）联系起来。我们应用TPD-seq工作流来推导溶剂的tpod(二甲基亚砜，DMSO；甲醇)和阳性对照(3,4-二氯苯胺，DCA；过氧化氢（H2O2）常用于毒性试验。大多数reads映射到蛋白质编码基因(鱼细胞~9 k；~6 k（人细胞），约50% %的差异表达基因得到曲线拟合，其中90% %得到基因基准剂量。DMSO暴露引起的转录组反应最强烈，细胞系的tPOD值为31-155 mM。OECD测试指南249 （RTgill-W1细胞）推荐使用DCA，这里我们计算出tPOD为~5至76 μM。最后，两种人类细胞系暴露于H2O2中，Caco-2细胞的tPOD值为0.7 ~ 1.1 mM， Hep G2细胞的tPOD值为5 ~ 30 μM。这里概述的方法是在具有基本分子和细胞培养设施的实验室中进行的，TPD-seq工作流程的性能和可扩展性可以通过其他案例研究来确定。
{"title":"TPD-seq: A high throughput RNA-seq method to derive transcriptomic points of departure from cell lines","authors":"Krittika Mittal , Ke Xu , Samuel J. Rulli Jr , Guangyan Zhou , Jianguo Xia , Niladri Basu","doi":"10.1016/j.tiv.2024.106001","DOIUrl":"10.1016/j.tiv.2024.106001","url":null,"abstract":"<div><div>There is growing scientific and regulatory interest in transcriptomic points of departure (tPOD) values from high-throughput <em>in vitro</em> experiments. To further help democratize tPOD research, here we outline ‘TPD-seq’ which links microplate-based exposure methods involving cell lines for human (Caco-2, Hep G2) and environmental (rainbow trout RTgill-W1) health, with a commercially available RNA-seq kit, with a cloud-based bioinformatics tool (ExpressAnalyst.ca). We applied the TPD-seq workflow to derive tPODs for solvents (dimethyl sulfoxide, DMSO; methanol) and positive controls (3,4-dichloroaniline, DCA; hydrogen peroxide, H<sub>2</sub>O<sub>2</sub>) commonly used in toxicity testing. The majority of reads mapped to protein coding genes (∼9 k for fish cells; ∼6 k for human cells), and about 50 % of differentially expressed genes were curve-fitted from which 90 % yielded gene benchmark doses. The most robust transcriptomic responses were caused by DMSO exposure, and tPOD values were 31–155 mM across the cell lines. OECD test guideline 249 (RTgill-W1 cells) recommends the use of DCA and here we calculated a tPOD of ∼5 to 76 μM. Finally, exposure of the two human cell lines to H<sub>2</sub>O<sub>2</sub> resulted in tPOD values that ranged from 0.7 to 1.1 mM in Caco-2 cells and 5–30 μM in Hep G2 cells. The methods outlined here are designed to be performed in laboratories with basic molecular and cell culture facilities, and the performance and scalability of the TPD-seq workflow can be determined with additional case studies.</div></div>","PeriodicalId":54423,"journal":{"name":"Toxicology in Vitro","volume":"104 ","pages":"Article 106001"},"PeriodicalIF":2.6,"publicationDate":"2024-12-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142873448","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-12-17DOI: 10.1016/j.tiv.2024.106000
Prabu Velumani, Naveen Palani, A. Antalin Casmie, Rajeswari Senthilvel, Vijaya Parthasarthy
The widespread use of nanoparticles raises substantial environmental, health, and safety issues. The specific mechanisms by which they impact plants and animals, as well as the entire scope of their possible impact, are still unknown. The current work investigates the impact of varying CuO NPs concentrations on phytotoxicity, cytotoxicity, genotoxicity, and antioxidant activity. Exposure of Mung bean seeds to CuO NPs results in the uptake of these particles by the roots and their subsequent transportation to various plant components, including the root, stem, and leaf. This uptake of CuO NPs triggers the production of reactive oxygen species (ROS). The CuO NPs can induce toxicological consequences due to their heightened propensity to produce excessive amounts of ROS. The absorption of CuO NPs might cause deformation and disintegration of the erythrocyte membrane, resulting in cell rupture due to osmotic pressure. The Allium cepa cytotoxicity study aimed to examine the harmful effects of CuO NPs and assess their impact on cellular structures at a microscopic scale. This work aims to analyze the cellular interaction of CuO NPs by measuring the Mitotic Index (MI) in the root cells of Allium cepa. The CuO NPs rapidly interact with plant and human cells, as well as chromosomes, leading to nano-phytotoxicity, nano-cytotoxicity, and nano-genotoxicity.
{"title":"Cellular and chromosomal interaction of bio-synthesized copper oxide nanoparticles - Induced nano-cytotoxicity and genotoxicity","authors":"Prabu Velumani, Naveen Palani, A. Antalin Casmie, Rajeswari Senthilvel, Vijaya Parthasarthy","doi":"10.1016/j.tiv.2024.106000","DOIUrl":"10.1016/j.tiv.2024.106000","url":null,"abstract":"<div><div>The widespread use of nanoparticles raises substantial environmental, health, and safety issues. The specific mechanisms by which they impact plants and animals, as well as the entire scope of their possible impact, are still unknown. The current work investigates the impact of varying CuO NPs concentrations on phytotoxicity, cytotoxicity, genotoxicity, and antioxidant activity. Exposure of <em>Mung bean</em> seeds to CuO NPs results in the uptake of these particles by the roots and their subsequent transportation to various plant components, including the root, stem, and leaf. This uptake of CuO NPs triggers the production of reactive oxygen species (ROS). The CuO NPs can induce toxicological consequences due to their heightened propensity to produce excessive amounts of ROS. The absorption of CuO NPs might cause deformation and disintegration of the erythrocyte membrane, resulting in cell rupture due to osmotic pressure. The <em>Allium cepa</em> cytotoxicity study aimed to examine the harmful effects of CuO NPs and assess their impact on cellular structures at a microscopic scale. This work aims to analyze the cellular interaction of CuO NPs by measuring the Mitotic Index (MI) in the root cells of <em>Allium cepa</em>. The CuO NPs rapidly interact with plant and human cells, as well as chromosomes, leading to nano-phytotoxicity, nano-cytotoxicity, and nano-genotoxicity.</div></div>","PeriodicalId":54423,"journal":{"name":"Toxicology in Vitro","volume":"104 ","pages":"Article 106000"},"PeriodicalIF":2.6,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142866437","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-12-17DOI: 10.1016/j.tiv.2024.105999
López-Pérez Lizardo , Roldán-Barreto Elisa , Xochiteotzin-Reyes Tania , Torres-Flores Ulises , Licea-Quintero Brandon , Monroy-Quintana Regina , Corona Juan Carlos , Zaragoza-Ojeda Montserrat , Arenas-Huertero Francisco
Benzo[ghi] perylene (b[ghi]p) is classified as non-carcinogenic to humans, and there are currently no occupational exposure models available to identify its effects. The aim of this work was to evaluate the effect of b[ghi]p on the lysosomes of NL-20 cells (a human bronchial cell line) exposed to 4.5 μM for 3 h. The effect was evaluated through an ultrastructural evaluation, morphological changes, and acridine orange staining of lysosomes. Superoxide was quantified; and SOD1, cathepsin B, LAMP1, galectin-3 and LC3α/β, and Rab7 expression was evaluated by immunocytochemistry. The expression of genes related to oxidative stress responses (NRF2, NQO1, HMOX1 and PRDX1) and genes related to autophagy (ULK1, ATG9, BCN1, VMP1, TMEM41B and p62) were quantified by RT-qPCR. The ultrastructural evaluation revealed an increase in autophagic vesicles and phagophores in cells exposed to b[ghi]p, as well as vesicles derived from mitochondria. Based on morphology, there were vesicles in the cytoplasm. B[ghi]p significantly decreased the number of lysosomes (p < 0.05), and NAC reverse this effect (p < 0.05). Superoxide production was observed from 30 min to 3 h (p < 0.05). Immunocytochemistry revealed increased galectin-3 and LC3α/β. All oxidative stress–related genes showed high expression (p < 0.05), and the expression of ATG9 gene was decreased (p < 0.05). These results demonstrate that b[ghi]p induces oxidative stress, responsible for producing the toxic effects in the lysosomes of NL-20 cells.
{"title":"Oxidative Stress, Lysosomal Permeability, and Mitochondrial-Derived Vesicles Induced in NL-20 Human Bronchial Cells Exposed to Benzo[ghi]Perylene","authors":"López-Pérez Lizardo , Roldán-Barreto Elisa , Xochiteotzin-Reyes Tania , Torres-Flores Ulises , Licea-Quintero Brandon , Monroy-Quintana Regina , Corona Juan Carlos , Zaragoza-Ojeda Montserrat , Arenas-Huertero Francisco","doi":"10.1016/j.tiv.2024.105999","DOIUrl":"10.1016/j.tiv.2024.105999","url":null,"abstract":"<div><div>Benzo[<em>ghi</em>] perylene (b[<em>ghi</em>]p) is classified as non-carcinogenic to humans, and there are currently no occupational exposure models available to identify its effects. The aim of this work was to evaluate the effect of b[<em>ghi</em>]p on the lysosomes of NL-20 cells (a human bronchial cell line) exposed to 4.5 μM for 3 h. The effect was evaluated through an ultrastructural evaluation, morphological changes, and acridine orange staining of lysosomes. Superoxide was quantified; and SOD1, cathepsin B, LAMP1, galectin-3 and LC3α/β, and Rab7 expression was evaluated by immunocytochemistry. The expression of genes related to oxidative stress responses (NRF2, NQO1, HMOX1 and PRDX1) and genes related to autophagy (ULK1, ATG9, BCN1, VMP1, TMEM41B and p62) were quantified by RT-qPCR. The ultrastructural evaluation revealed an increase in autophagic vesicles and phagophores in cells exposed to b[<em>ghi</em>]p, as well as vesicles derived from mitochondria. Based on morphology, there were vesicles in the cytoplasm. B[<em>ghi</em>]p significantly decreased the number of lysosomes (<em>p</em> < 0.05), and NAC reverse this effect (p < 0.05). Superoxide production was observed from 30 min to 3 h (p < 0.05). Immunocytochemistry revealed increased galectin-3 and LC3α/β. All oxidative stress–related genes showed high expression (p < 0.05), and the expression of ATG9 gene was decreased (p < 0.05). These results demonstrate that b[<em>ghi</em>]p induces oxidative stress, responsible for producing the toxic effects in the lysosomes of NL-20 cells.</div></div>","PeriodicalId":54423,"journal":{"name":"Toxicology in Vitro","volume":"104 ","pages":"Article 105999"},"PeriodicalIF":2.6,"publicationDate":"2024-12-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142866438","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Precise prediction of the fraction of compounds reaching the portal vein (FaFg) in humans, which could indicate the rate-limiting step of polyphenol metabolism, is particularly important for accurately evaluating the efficacy and safety of polyphenols. In this study, we aimed to develop a novel in vitro method to predict human FaFg of polyphenols using commercially available human induced pluripotent stem cell-derived small intestinal epithelial cells (hiPSC-SIECs). First, the chemicals were used at fixed test concentrations, considering their physicochemical properties and cytotoxicity. The apparent permeability coefficient (Papp) values of the six tested polyphenols in hiPSC-SIECs were considerably higher than those of the seven tested pharmaceuticals, resulting in a poor correlation between Papp in hiPSC-SIECs and human FaFg. A detailed assessment of the relationship between in vitro test concentration and metabolic activity suggested that the higher Papp value of polyphenols would be due to inadequate reflection of phase II metabolism in the human intestine. By optimizing test concentrations to reflect enzymatic metabolism in the human intestine, a good correlation was observed between the Papp values in hiPSC-SIECs and human FaFg for tested polyphenols and pharmaceuticals (R2 = 0.81). The developed method could be useful for precisely predicting human FaFg of polyphenols.
精确预测到达人体门静脉的化合物组分（FaFg）可表明多酚代谢的限速步骤，这对于准确评估多酚的功效和安全性尤为重要。在这项研究中，我们旨在开发一种新型体外方法，利用市售的人类诱导多能干细胞衍生小肠上皮细胞（hiPSC-SIECs）预测多酚的人体 FaFg。首先，考虑到化学品的理化性质和细胞毒性，以固定的试验浓度使用这些化学品。六种受测多酚在 hiPSC-SIECs 中的表观渗透系数（Papp）值远高于七种受测药物的表观渗透系数（Papp）值，导致 hiPSC-SIECs 中的 Papp 与人体 FaFg 之间的相关性较差。对体外测试浓度和代谢活性之间关系的详细评估表明，多酚的 Papp 值较高可能是由于没有充分反映人体肠道中的第二阶段代谢。通过优化测试浓度以反映人体肠道中的酶代谢，观察到受测多酚和药物在 hiPSC-SIECs 和人体 FaFg 中的 Papp 值之间存在良好的相关性（R2 = 0.81）。所开发的方法可用于精确预测人体对多酚的FaFg值。
{"title":"Development of a human FaFg prediction system for polyphenols using human induced pluripotent stem cell-derived small intestinal epithelial cells","authors":"Shunta Shigemura, Takuya Kikuchi, Yuichi Ito, Osamu Morita, Kazutoshi Saito","doi":"10.1016/j.tiv.2024.105998","DOIUrl":"10.1016/j.tiv.2024.105998","url":null,"abstract":"<div><div>Precise prediction of the fraction of compounds reaching the portal vein (<em>FaFg</em>) in humans, which could indicate the rate-limiting step of polyphenol metabolism, is particularly important for accurately evaluating the efficacy and safety of polyphenols. In this study, we aimed to develop a novel <em>in vitro</em> method to predict human <em>FaFg</em> of polyphenols using commercially available human induced pluripotent stem cell-derived small intestinal epithelial cells (hiPSC-SIECs). First, the chemicals were used at fixed test concentrations, considering their physicochemical properties and cytotoxicity. The apparent permeability coefficient (P<sub>app</sub>) values of the six tested polyphenols in hiPSC-SIECs were considerably higher than those of the seven tested pharmaceuticals, resulting in a poor correlation between P<sub>app</sub> in hiPSC-SIECs and human <em>FaFg</em>. A detailed assessment of the relationship between <em>in vitro</em> test concentration and metabolic activity suggested that the higher P<sub>app</sub> value of polyphenols would be due to inadequate reflection of phase II metabolism in the human intestine. By optimizing test concentrations to reflect enzymatic metabolism in the human intestine, a good correlation was observed between the P<sub>app</sub> values in hiPSC-SIECs and human <em>FaFg</em> for tested polyphenols and pharmaceuticals (R<sup>2</sup> = 0.81). The developed method could be useful for precisely predicting human <em>FaFg</em> of polyphenols.</div></div>","PeriodicalId":54423,"journal":{"name":"Toxicology in Vitro","volume":"104 ","pages":"Article 105998"},"PeriodicalIF":2.6,"publicationDate":"2024-12-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142824826","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-12-10DOI: 10.1016/j.tiv.2024.105995
Xianglong Wang , Yushan Tian , Huan Chen , Hongwei Hou , Qingyuan Hu
Cigarette smoking poses significant health risks, particularly to the airway, which consists predominantly of basal, club, and ciliated cells that are highly susceptible to damage from exogenous stimuli. Traditional in vitro toxicology relies on 2D cell cultures, which lack the structural complexity and functional relevance of airway architecture. As a novel category of tobacco products, the health implications of heated tobacco products (HTPs) remain largely unknown. To address this, 3D airway organoids were developed as a more physiologically relevant in vitro model for evaluating the toxicity of HTPs. Airway organoids derived from mouse lungs were induced to differentiate into various airway cell types and exposed to HTP aerosols. The exposure impaired organoid growth, reduced cell viability, and altered the proportions of secretory, basal, and ciliated cells, effectively replicating the complex cellular damage observed in vivo. Additionally, typical adverse outcomes, such as oxidative stress, inflammation, and genetic toxicity, were induced, paralleling findings from conventional 2D models. These results established the airway organoids as a viable alternative to animal testing for toxicological studies and offer critical insights into the respiratory health risks associated with HTPs.
{"title":"Airway organoids: 3D toxicology evaluation models in vitro of heated tobacco products for health risk","authors":"Xianglong Wang , Yushan Tian , Huan Chen , Hongwei Hou , Qingyuan Hu","doi":"10.1016/j.tiv.2024.105995","DOIUrl":"10.1016/j.tiv.2024.105995","url":null,"abstract":"<div><div>Cigarette smoking poses significant health risks, particularly to the airway, which consists predominantly of basal, club, and ciliated cells that are highly susceptible to damage from exogenous stimuli. Traditional <em>in vitro</em> toxicology relies on 2D cell cultures, which lack the structural complexity and functional relevance of airway architecture. As a novel category of tobacco products, the health implications of heated tobacco products (HTPs) remain largely unknown. To address this, 3D airway organoids were developed as a more physiologically relevant <em>in vitro</em> model for evaluating the toxicity of HTPs. Airway organoids derived from mouse lungs were induced to differentiate into various airway cell types and exposed to HTP aerosols. The exposure impaired organoid growth, reduced cell viability, and altered the proportions of secretory, basal, and ciliated cells, effectively replicating the complex cellular damage observed <em>in vivo</em>. Additionally, typical adverse outcomes, such as oxidative stress, inflammation, and genetic toxicity, were induced, paralleling findings from conventional 2D models. These results established the airway organoids as a viable alternative to animal testing for toxicological studies and offer critical insights into the respiratory health risks associated with HTPs.</div></div>","PeriodicalId":54423,"journal":{"name":"Toxicology in Vitro","volume":"104 ","pages":"Article 105995"},"PeriodicalIF":2.6,"publicationDate":"2024-12-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142819894","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-12-07DOI: 10.1016/j.tiv.2024.105988
Madhunika Agrawal , A.K. Saxena , Satyam Kumar Agrawal
The present study demonstrates that essential oil from Ocimum carnosum (EOC), possesses potent cytotoxic properties against human promyelocytic leukemia HL-60 cells. The results demonstrated a concentration- and time-dependent reduction in cell viability, with an IC50 value of 0.029 μl/ml after 24 h. Further mechanistic studies revealed that EOC induces apoptosis, a regulated form of cell death in HL-60 cells. This was evidenced by morphological changes characteristic of apoptosis, including cell shrinkage, membrane blebbing, and nuclear condensation. Additionally, flow cytometric analysis demonstrated a significant increase in the sub-G0 cell population, indicative of DNA fragmentation. The mitochondrial pathway of apoptosis appears to be involved in EOC-induced cell death. A loss of mitochondrial membrane potential and the subsequent release of cytochrome c into the cytosol were observed. Pronounced quantity of cytosolic cytochrome c was associated with Bcl-2 depletion. Moreover, cytochrome c, in conjunction with other apoptotic factors, activates caspases, a family of cysteine proteases that execute cell death. These findings collectively indicate that EOC possesses promising anti-cancer properties through the induction of apoptosis via a mitochondrial-dependent pathway. However, further studies are required to elucidate the precise molecular mechanisms underlying EOC's cytotoxic effects and to evaluate its therapeutic potential in vivo.
{"title":"Essential oil from Ocimum carnosum induces ROS mediated mitochondrial dysfunction and intrinsic apoptosis in HL-60 cells","authors":"Madhunika Agrawal , A.K. Saxena , Satyam Kumar Agrawal","doi":"10.1016/j.tiv.2024.105988","DOIUrl":"10.1016/j.tiv.2024.105988","url":null,"abstract":"<div><div>The present study demonstrates that essential oil from <em>Ocimum carnosum</em> (EOC), possesses potent cytotoxic properties against human promyelocytic leukemia HL-60 cells. The results demonstrated a concentration- and time-dependent reduction in cell viability, with an IC<sub>50</sub> value of 0.029 μl/ml after 24 h. Further mechanistic studies revealed that EOC induces apoptosis, a regulated form of cell death in HL-60 cells. This was evidenced by morphological changes characteristic of apoptosis, including cell shrinkage, membrane blebbing, and nuclear condensation. Additionally, flow cytometric analysis demonstrated a significant increase in the sub-G<sub>0</sub> cell population, indicative of DNA fragmentation. The mitochondrial pathway of apoptosis appears to be involved in EOC-induced cell death. A loss of mitochondrial membrane potential and the subsequent release of cytochrome <em>c</em> into the cytosol were observed. Pronounced quantity of cytosolic cytochrome <em>c</em> was associated with Bcl-2 depletion. Moreover, cytochrome <em>c</em>, in conjunction with other apoptotic factors, activates caspases, a family of cysteine proteases that execute cell death. These findings collectively indicate that EOC possesses promising anti-cancer properties through the induction of apoptosis via a mitochondrial-dependent pathway. However, further studies are required to elucidate the precise molecular mechanisms underlying EOC's cytotoxic effects and to evaluate its therapeutic potential <em>in vivo</em>.</div></div>","PeriodicalId":54423,"journal":{"name":"Toxicology in Vitro","volume":"104 ","pages":"Article 105988"},"PeriodicalIF":2.6,"publicationDate":"2024-12-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142803493","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-12-06DOI: 10.1016/j.tiv.2024.105986
Ayşe Koçak , Semih Gülle , Merih Birlik
Objectives
We evaluated potential therapeutic efficacy of LGK-974 and ETC-159 in fibrotic scleroderma cells.
Methods
Primary scleroderma dermal fibroblast cells of mouse origin (SSc fibroblasts) and primary fibrotic lung fibroblast cells of human origin (CCL-191) were used in this study. PORCN inhibitors LGK-974 (S7143, 1 μM; Selleckchem, USA) and ETC-159 (S7143, 10 μM; Selleckchem, USA) were used. The possible therapeutic effects of LGK-974 and ETC-159 on scleroderma cells and fibrosis cells were examined. Cell viability experiments were performed for each substance, and the expression levels of WNT and fibrosis marker genes were determined by qPCR. Western blotting was also used to determine collagen, fibronectin and α-SMA protein markers.
Results
This study showed that LGK-974 and ETC-159 probable protein-cysteine N-palmitoyltransferase porcupine (PORCN) inhibitors exert potent antifibrotic effects and reduce fibrosis by modulating the TGF-β signaling pathway in scleroderma cells. Using LGK-974 and ETC-159 PORCN inhibitors, either alone or in combination, can affect collagen deposition and fibrosis in patients with SSc.
Conclusions
LGK-974 and ETC-159 may be a possible long-term therapeutic target for scleroderma.
{"title":"Porcupine inhibitors LGK-974 and ETC-159 inhibit Wnt/β-catenin signaling and result in inhibition of the fibrosis","authors":"Ayşe Koçak , Semih Gülle , Merih Birlik","doi":"10.1016/j.tiv.2024.105986","DOIUrl":"10.1016/j.tiv.2024.105986","url":null,"abstract":"<div><h3>Objectives</h3><div>We evaluated potential therapeutic efficacy of LGK-974 and ETC-159 in fibrotic scleroderma cells.</div></div><div><h3>Methods</h3><div>Primary scleroderma dermal fibroblast cells of mouse origin (SSc fibroblasts) and primary fibrotic lung fibroblast cells of human origin (CCL-191) were used in this study. PORCN inhibitors LGK-974 (S7143, 1 μM; Selleckchem, USA) and ETC-159 (S7143, 10 μM; Selleckchem, USA) were used. The possible therapeutic effects of LGK-974 and ETC-159 on scleroderma cells and fibrosis cells were examined. Cell viability experiments were performed for each substance, and the expression levels of WNT and fibrosis marker genes were determined by qPCR. Western blotting was also used to determine collagen, fibronectin and α-SMA protein markers.</div></div><div><h3>Results</h3><div>This study showed that LGK-974 and ETC-159 probable protein-cysteine N-palmitoyltransferase porcupine (PORCN) inhibitors exert potent antifibrotic effects and reduce fibrosis by modulating the TGF-β signaling pathway in scleroderma cells. Using LGK-974 and ETC-159 PORCN inhibitors, either alone or in combination, can affect collagen deposition and fibrosis in patients with SSc.</div></div><div><h3>Conclusions</h3><div>LGK-974 and ETC-159 may be a possible long-term therapeutic target for scleroderma.</div></div>","PeriodicalId":54423,"journal":{"name":"Toxicology in Vitro","volume":"104 ","pages":"Article 105986"},"PeriodicalIF":2.6,"publicationDate":"2024-12-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142796423","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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