The first exposure of intravenously (IV) administered nanomedicines in vivo is to endothelial cells (ECs) lining blood vessels. While it is known that in vitro endothelium models to assess responses to circulating nanoparticles require shear stress, there is no consensus on when and how to include it in the experimental design. Our experimental workflow integrates shear stress by featuring a flow-induced mature endothelium (14 days) and a flow-mediated nanoparticle treatment. The mature endothelium model exhibited distinct features that indicated a structurally stable and quiescent monolayer. Upon treatment with iron sucrose under dynamic conditions, there was a lower nanoparticle uptake, lower cytotoxicity, and decreased expression of activation markers compared to the static control. This response was attributed to glycocalyx expression, predominantly observed on the mature endothelium. In conclusion, our proposed in vitro endothelium model can be leveraged to understand the dynamics of IV injectable nanomedicines at the initial nano-bio interface in veins immediately post-injection.
{"title":"Go with the flow: An in vitro model of a mature endothelium for the study of the bioresponse of IV injectable nanomedicines","authors":"Niusha Nikravesh , Alexandra Rippl , Tobias Hoch , Stephanie Eitner , Amy Barton Alston , Reinaldo Digigow , Savvina Chortarea , Liliane Diener , Vanesa Ayala-Nunez , Peter Wick","doi":"10.1016/j.tiv.2024.105953","DOIUrl":"10.1016/j.tiv.2024.105953","url":null,"abstract":"<div><div>The first exposure of intravenously (IV) administered nanomedicines <em>in vivo</em> is to endothelial cells (ECs) lining blood vessels. While it is known that <em>in vitro</em> endothelium models to assess responses to circulating nanoparticles require shear stress, there is no consensus on when and how to include it in the experimental design. Our experimental workflow integrates shear stress by featuring a flow-induced mature endothelium (14 days) and a flow-mediated nanoparticle treatment. The mature endothelium model exhibited distinct features that indicated a structurally stable and quiescent monolayer. Upon treatment with iron sucrose under dynamic conditions, there was a lower nanoparticle uptake, lower cytotoxicity, and decreased expression of activation markers compared to the static control. This response was attributed to glycocalyx expression, predominantly observed on the mature endothelium. In conclusion, our proposed <em>in vitro</em> endothelium model can be leveraged to understand the dynamics of IV injectable nanomedicines at the initial nano-bio interface in veins immediately post-injection.</div></div>","PeriodicalId":54423,"journal":{"name":"Toxicology in Vitro","volume":"101 ","pages":"Article 105953"},"PeriodicalIF":2.6,"publicationDate":"2024-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142446517","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 : 2024-10-10DOI: 10.1016/j.tiv.2024.105952
Carlos Abraham García-García , Alfredo Cruz-Gregorio , José Pedraza-Chaverri , Luis F. Montaño , Erika P. Rendón-Huerta
Carcinogenic N-nitroso compounds, especially N-nitroso dimethylamine, increase the risk of gastric cancer development. Cytochrome P450-2E1 metabolizes this compound, thus generating an oxidant microenvironment. We aimed to evaluate in gastric adenocarcinoma cells if its effect on CYP2E1 and ROS affects signaling pathways associated with gastric cancer oncogenesis. The impact of N- nitroso dimethylamine upon CYP2E1 and ROS activation/secretion was evaluated by the DCFDA assay protocol, TER measurements, Stat3, pSTAT3, ERK1/2, and pERK1/2 expression, claudins-1 and -6 expression, and finally mRNA values of IL-1β IL-6, IL-8 and TNFα. Our results showed that exposure to N- N-nitroso dimethylamine disrupts the regulation of Stat3 and Erk1/2, alters the expression of claudin-1 and claudin-6 tight junction proteins, and increases the secretion of pro-inflammatory cytokines. These alterations induce a continuous local inflammatory process, an event identified as a gastric cancer promoter. In summary, N-nitroso dimethylamine can disrupt cell mechanisms associated with gastric cancer oncogenesis.
{"title":"NDMA enhances claudin-1 and -6 expression viaCYP2E1/ROS in AGS cells","authors":"Carlos Abraham García-García , Alfredo Cruz-Gregorio , José Pedraza-Chaverri , Luis F. Montaño , Erika P. Rendón-Huerta","doi":"10.1016/j.tiv.2024.105952","DOIUrl":"10.1016/j.tiv.2024.105952","url":null,"abstract":"<div><div>Carcinogenic N-nitroso compounds, especially N-nitroso dimethylamine, increase the risk of gastric cancer development. Cytochrome P450-2E1 metabolizes this compound, thus generating an oxidant microenvironment. We aimed to evaluate in gastric adenocarcinoma cells if its effect on CYP2E1 and ROS affects signaling pathways associated with gastric cancer oncogenesis. The impact of N- nitroso dimethylamine upon CYP2E1 and ROS activation/secretion was evaluated by the DCFDA assay protocol, TER measurements, Stat3, pSTAT3, ERK1/2, and pERK1/2 expression, claudins-1 and -6 expression, and finally mRNA values of IL-1β IL-6, IL-8 and TNFα. Our results showed that exposure to N- N-nitroso dimethylamine disrupts the regulation of Stat3 and Erk1/2, alters the expression of claudin-1 and claudin-6 tight junction proteins, and increases the secretion of pro-inflammatory cytokines. These alterations induce a continuous local inflammatory process, an event identified as a gastric cancer promoter. In summary, N-nitroso dimethylamine can disrupt cell mechanisms associated with gastric cancer oncogenesis.</div></div>","PeriodicalId":54423,"journal":{"name":"Toxicology in Vitro","volume":"102 ","pages":"Article 105952"},"PeriodicalIF":2.6,"publicationDate":"2024-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142481022","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 : 2024-10-09DOI: 10.1016/j.tiv.2024.105951
Anna Małkowska , Katerina Makarowa , Katarzyna Zawada , Maksymilian Grzelak , Aleksandra Zmysłowska
Curcumin, a natural polyphenol found in the turmeric plant, has been shown to have anti-inflammatory and antioxidant properties. It has been widely studied for its potential protective effect against various health conditions, including ethanol-induced malformation.
Ethanol exposure during pregnancy can lead to various developmental abnormalities, known as fetal alcohol syndrome (FAS) and fetal alcohol spectrum disorders (FASD). Due to the high prevalence of FASD and FAS and no effective treatment, it is essential to develop preventive strategies. Recent studies have investigated the potential protective effect of curcumin against ethanol-induced malformation in animal models.
This study aimed to examine whether curcumin can reduce the toxic effects of ethanol in zebrafish embryos. The present study showed that pure curcumin applied together with 1.5 % ethanol (v/v) did not lead to a protective effect on ethanol-induced malformations such as disturbances of body length and width or pericardia oedema in growing zebrafish embryos. Moreover, curcumin extract showed a pro-oxidant effect in the Fenton reaction in the presence of ethanol.
{"title":"Effect of curcumin on the embryotoxic effect of ethanol in a zebrafish model","authors":"Anna Małkowska , Katerina Makarowa , Katarzyna Zawada , Maksymilian Grzelak , Aleksandra Zmysłowska","doi":"10.1016/j.tiv.2024.105951","DOIUrl":"10.1016/j.tiv.2024.105951","url":null,"abstract":"<div><div>Curcumin, a natural polyphenol found in the turmeric plant, has been shown to have anti-inflammatory and antioxidant properties. It has been widely studied for its potential protective effect against various health conditions, including ethanol-induced malformation.</div><div>Ethanol exposure during pregnancy can lead to various developmental abnormalities, known as fetal alcohol syndrome (FAS) and fetal alcohol spectrum disorders (FASD). Due to the high prevalence of FASD and FAS and no effective treatment, it is essential to develop preventive strategies. Recent studies have investigated the potential protective effect of curcumin against ethanol-induced malformation in animal models.</div><div>This study aimed to examine whether curcumin can reduce the toxic effects of ethanol in zebrafish embryos. The present study showed that pure curcumin applied together with 1.5 % ethanol (<em>v</em>/v) did not lead to a protective effect on ethanol-induced malformations such as disturbances of body length and width or pericardia oedema in growing zebrafish embryos. Moreover, curcumin extract showed a pro-oxidant effect in the Fenton reaction in the presence of ethanol.</div></div>","PeriodicalId":54423,"journal":{"name":"Toxicology in Vitro","volume":"101 ","pages":"Article 105951"},"PeriodicalIF":2.6,"publicationDate":"2024-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142401993","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 : 2024-09-30DOI: 10.1016/j.tiv.2024.105950
Yan-Zhu Quan , Jing-He Wang , Si-Hui Zhang , Guang-Nan Jin , Jing-Mei Lu , Yi-Ming Liu , Hong-Yan Gao , Jin-Yi Zhou , Bing-Zhe Wang , Yan Xin , Yue-Xian Cui , Xiang Xu , Lian-Xun Piao
Tanshinone IIA (Tan IIA), a neuroprotective natural compound extracted from Salvia miltiorrhiza, is used in stroke treatment. However, elucidating Tan IIA's neuroprotective mechanisms remains challenging due to limitations in assessing drug efficacy and biochemical parameters in clinical studies. This study investigated Tan IIA's impact on neuroinflammatory responses and its neuroprotective mechanisms using HMGB1- or TNF-α-stimulated BV2 microglia in a co-culture system with primary neuron cells. The results indicated that Tan IIA significantly reduced microglial activation induced by TNF-α or HMGB1. Concurrently, Tan IIA disrupted the interactions between HMGB1 and toll-like receptor 4 (TLR4), and between TNF-α and TNF receptor 1 (TNFR1), modulating the HMGB1/TLR4/nuclear factor-kappa B (NF-κB) and TNF-α/TNFR1/NF-κB signaling pathways and related protein expressions. Moreover, co-culture experiments showed that neuronal apoptosis induced by microglial activation was reversed by Tan IIA. In conclusion, Tan IIA provides neuroprotection by modulating signaling pathways in microglia, thus preventing neuronal apoptosis. This study offers new insights into therapeutic targets for ischemic stroke.
{"title":"The intervention mechanism of Tanshinone IIA in alleviating neuronal injury induced by HMGB1 or TNF-α-mediated microglial activation","authors":"Yan-Zhu Quan , Jing-He Wang , Si-Hui Zhang , Guang-Nan Jin , Jing-Mei Lu , Yi-Ming Liu , Hong-Yan Gao , Jin-Yi Zhou , Bing-Zhe Wang , Yan Xin , Yue-Xian Cui , Xiang Xu , Lian-Xun Piao","doi":"10.1016/j.tiv.2024.105950","DOIUrl":"10.1016/j.tiv.2024.105950","url":null,"abstract":"<div><div>Tanshinone IIA (Tan IIA), a neuroprotective natural compound extracted from <em>Salvia miltiorrhiza</em>, is used in stroke treatment. However, elucidating Tan IIA's neuroprotective mechanisms remains challenging due to limitations in assessing drug efficacy and biochemical parameters in clinical studies. This study investigated Tan IIA's impact on neuroinflammatory responses and its neuroprotective mechanisms using HMGB1- or TNF-α-stimulated BV2 microglia in a co-culture system with primary neuron cells. The results indicated that Tan IIA significantly reduced microglial activation induced by TNF-α or HMGB1. Concurrently, Tan IIA disrupted the interactions between HMGB1 and toll-like receptor 4 (TLR4), and between TNF-α and TNF receptor 1 (TNFR1), modulating the HMGB1/TLR4/nuclear factor-kappa B (NF-κB) and TNF-α/TNFR1/NF-κB signaling pathways and related protein expressions. Moreover, co-culture experiments showed that neuronal apoptosis induced by microglial activation was reversed by Tan IIA. In conclusion, Tan IIA provides neuroprotection by modulating signaling pathways in microglia, thus preventing neuronal apoptosis. This study offers new insights into therapeutic targets for ischemic stroke.</div></div>","PeriodicalId":54423,"journal":{"name":"Toxicology in Vitro","volume":"101 ","pages":"Article 105950"},"PeriodicalIF":2.6,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142367363","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 : 2024-09-28DOI: 10.1016/j.tiv.2024.105946
Hongyan Dong , Katie Paul Friedman , Alain Filiatreault , Errol M. Thomson , Michael G. Wade
Rapid, human relevant assays are needed to assess potential hazards of the many chemicals in commerce. An assay of thyroid peroxidase (TPO) inhibition, using the substrate Amplex Ultra Red, was recently adapted for human TPO (AUR-hTPO). We tested a large number (788) of chemicals through this AUR-hTPO assay and compared performance with published results from an assay using enzyme from rat thyroid microsomes (AUR-rTPO). Coded chemicals, from the US EPA ToxCast Inventory, were tested in a tiered approach: 1) Initial screening at a single concentration; 2) Potency estimation for active chemicals with multiple concentrations; 3) Screening active chemicals for the non-specific activity. The assay gave consistent results for positive chemical methimazole and several positive and negative reference chemicals. hTPO inhibition was observed for 190 chemicals reported as positive in rTPO. Of these, 158 showed no confounding activity (interference due to fluorescence or non-specific protein inhibition). Comparison of all result with rTPO data and with evidence of TPO inhibition found in the literature suggest that the current assay has a higher rate of false negative but a much lower rate of false positive compared with the rTPO screen. These findings underscore the effectiveness of the AUR assay, using hTPO enzyme from engineered cell lines, to identify moderate to strong inhibitors but some improvements may be needed to detect weak TPO inhibitors.
{"title":"A high throughput screening assay for human Thyroperoxidase inhibitors","authors":"Hongyan Dong , Katie Paul Friedman , Alain Filiatreault , Errol M. Thomson , Michael G. Wade","doi":"10.1016/j.tiv.2024.105946","DOIUrl":"10.1016/j.tiv.2024.105946","url":null,"abstract":"<div><div>Rapid, human relevant assays are needed to assess potential hazards of the many chemicals in commerce. An assay of thyroid peroxidase (TPO) inhibition, using the substrate Amplex Ultra Red, was recently adapted for human TPO (AUR-hTPO). We tested a large number (788) of chemicals through this AUR-hTPO assay and compared performance with published results from an assay using enzyme from rat thyroid microsomes (AUR-rTPO). Coded chemicals, from the US EPA ToxCast Inventory, were tested in a tiered approach: 1) Initial screening at a single concentration; 2) Potency estimation for active chemicals with multiple concentrations; 3) Screening active chemicals for the non-specific activity. The assay gave consistent results for positive chemical methimazole and several positive and negative reference chemicals. hTPO inhibition was observed for 190 chemicals reported as positive in rTPO. Of these, 158 showed no confounding activity (interference due to fluorescence or non-specific protein inhibition). Comparison of all result with rTPO data and with evidence of TPO inhibition found in the literature suggest that the current assay has a higher rate of false negative but a much lower rate of false positive compared with the rTPO screen. These findings underscore the effectiveness of the AUR assay, using hTPO enzyme from engineered cell lines, to identify moderate to strong inhibitors but some improvements may be needed to detect weak TPO inhibitors.</div></div>","PeriodicalId":54423,"journal":{"name":"Toxicology in Vitro","volume":"101 ","pages":"Article 105946"},"PeriodicalIF":2.6,"publicationDate":"2024-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142332392","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 : 2024-09-27DOI: 10.1016/j.tiv.2024.105945
Isabella de Souza Mota , Miguel Cardoso , João Bueno , Ingrid Gracielle Martins da Silva , João Gonçalves , Sonia N. Bao , Brenno A.D. Neto , Guilherme Brand , José Raimundo Corrêa , José Roberto S.A. Leite , Felipe Saldanha-Araujo
The anticancer potential of some antimicrobial peptides has been reported. Hs02 is a recently characterized Intragenic Antimicrobial Peptide (IAP), which was able to exhibit potent antimicrobial and anti-inflammatory action. In this study, we evaluate for the first time the antineoplastic potential of the Hs02 IAP using cell lines representing the main types of leukemia as cancer models. Interestingly, this peptide decreased the viability of several leukemic cell lines, without compromising the viability of PBMCs in the same concentration. In the HL-60 line, treatment with Hs02 controlled cell division, leading to cell arrest in the G1 phase of the cell cycle. More importantly, HL-60 cells treated with Hs02 undergo cell death, with the formation of pores in the plasma membrane and the release of LDH. Accordingly, Hs02 treatment stimulated the expression of components involved in pyroptosis, such as NLRP1, CASP-1, GSDME, and IL-1β. Taken together, our data characterize the antineoplastic potential of Hs02 and open an opportunity for both evaluating the peptide's antineoplastic potential in other cancer models and using this molecule as a template for new peptides with therapeutic potential against cancer.
{"title":"Intragenic antimicrobial peptide Hs02 toxicity against leukemia cell lines is associated with increased expression of select pyroptotic components","authors":"Isabella de Souza Mota , Miguel Cardoso , João Bueno , Ingrid Gracielle Martins da Silva , João Gonçalves , Sonia N. Bao , Brenno A.D. Neto , Guilherme Brand , José Raimundo Corrêa , José Roberto S.A. Leite , Felipe Saldanha-Araujo","doi":"10.1016/j.tiv.2024.105945","DOIUrl":"10.1016/j.tiv.2024.105945","url":null,"abstract":"<div><div>The anticancer potential of some antimicrobial peptides has been reported. Hs02 is a recently characterized Intragenic Antimicrobial Peptide (IAP), which was able to exhibit potent antimicrobial and anti-inflammatory action. In this study, we evaluate for the first time the antineoplastic potential of the Hs02 IAP using cell lines representing the main types of leukemia as cancer models. Interestingly, this peptide decreased the viability of several leukemic cell lines, without compromising the viability of PBMCs in the same concentration. In the HL-60 line, treatment with Hs02 controlled cell division, leading to cell arrest in the G1 phase of the cell cycle. More importantly, HL-60 cells treated with Hs02 undergo cell death, with the formation of pores in the plasma membrane and the release of LDH. Accordingly, Hs02 treatment stimulated the expression of components involved in pyroptosis, such as <em>NLRP1</em>, <em>CASP-1</em>, <em>GSDME</em>, and <em>IL-1β</em>. Taken together, our data characterize the antineoplastic potential of Hs02 and open an opportunity for both evaluating the peptide's antineoplastic potential in other cancer models and using this molecule as a template for new peptides with therapeutic potential against cancer.</div></div>","PeriodicalId":54423,"journal":{"name":"Toxicology in Vitro","volume":"101 ","pages":"Article 105945"},"PeriodicalIF":2.6,"publicationDate":"2024-09-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142332398","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 : 2024-09-27DOI: 10.1016/j.tiv.2024.105947
Suvarna Mini Vijayan , Moritz Baierl , Thomas Göen , Raymund E. Horch , Ingo Ludolph , Hans Drexler , Sonja Kilo
Technical products containing N-Phenyl-beta-naphthylamine (PBNA) are contaminated with beta-naphthylamine (BNA), a known carcinogen. Both amines penetrate the skin to different degrees, but little is known about their dermal-depot formation. This study investigated the dermal penetration of PBNA and its degradation product BNA using a viable human-skin model. PBNA (259 μg) or BNA (0.52 μg) in n-hexane and industrial grease were applied to freshly excised human skin (n = 6, 0.64 cm2) for 2-72 h. After temporary/continuous and single/repeated exposure, samples were taken (stratum corneum, epidermis/dermis, receptor fluid) and analyzed for their amine content by GC–MS. Continuous exposure led to a PBNA dermal depot of ∼47 μg/cm2 over 72 h. Temporary applications also resulted in lower but consistent PBNA dermal depots. A single 2-h application resulted in a dermal depot of ∼16 μg/cm2 after 72 h, while this was ∼25 μg/0.64 cm2 with repeated applications. BNA behaved differently; with repeated 2-h applications, intradermally retained BNA initially increased 3–6 fold, then dropped to ∼200–250 ng/cm2. This incomplete decline upon repeated short-term exposure to PBNA suggests that a BNA dermal depot is formed either due to contamination of PBNA with BNA or to enzymatic conversion of PBNA to BNA. Additionally, PBNA dermal depots were saturable under the given conditions. These findings highlight the importance of understanding the dermal-exposure dynamics of potential carcinogenic compounds in industrial settings.
{"title":"Intradermal and transdermal absorption of beta-naphthylamine and N-Phenyl-beta-naphthylamine in a viable human skin model","authors":"Suvarna Mini Vijayan , Moritz Baierl , Thomas Göen , Raymund E. Horch , Ingo Ludolph , Hans Drexler , Sonja Kilo","doi":"10.1016/j.tiv.2024.105947","DOIUrl":"10.1016/j.tiv.2024.105947","url":null,"abstract":"<div><div>Technical products containing N-Phenyl-beta-naphthylamine (PBNA) are contaminated with beta-naphthylamine (BNA), a known carcinogen. Both amines penetrate the skin to different degrees, but little is known about their dermal-depot formation. This study investigated the dermal penetration of PBNA and its degradation product BNA using a viable human-skin model. PBNA (259 μg) or BNA (0.52 μg) in <em>n</em>-hexane and industrial grease were applied to freshly excised human skin (<em>n</em> = 6, 0.64 cm<sup>2</sup>) for 2-72 h. After temporary/continuous and single/repeated exposure, samples were taken (stratum corneum, epidermis/dermis, receptor fluid) and analyzed for their amine content by GC–MS. Continuous exposure led to a PBNA dermal depot of ∼47 μg/cm<sup>2</sup> over 72 h. Temporary applications also resulted in lower but consistent PBNA dermal depots. A single 2-h application resulted in a dermal depot of ∼16 μg/cm<sup>2</sup> after 72 h, while this was ∼25 μg/0.64 cm<sup>2</sup> with repeated applications. BNA behaved differently; with repeated 2-h applications, intradermally retained BNA initially increased 3–6 fold, then dropped to ∼200–250 ng/cm<sup>2</sup>. This incomplete decline upon repeated short-term exposure to PBNA suggests that a BNA dermal depot is formed either due to contamination of PBNA with BNA or to enzymatic conversion of PBNA to BNA. Additionally, PBNA dermal depots were saturable under the given conditions. These findings highlight the importance of understanding the dermal-exposure dynamics of potential carcinogenic compounds in industrial settings.</div></div>","PeriodicalId":54423,"journal":{"name":"Toxicology in Vitro","volume":"101 ","pages":"Article 105947"},"PeriodicalIF":2.6,"publicationDate":"2024-09-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142332397","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 : 2024-09-27DOI: 10.1016/j.tiv.2024.105949
Janne Heikkinen , Sanna Palosaari , Petri Lehenkari
Background
Smoking and nicotine impose detrimental health effects including adipose tissue dysfunction. Despite extensive physiological evidence, the cellular mechanisms remain poorly understood, with few studies examining the effects of cigarette smoke extract (CSE) or nicotine on adipocyte differentiation.
Methods
Primary human bone marrow-derived mesenchymal stromal cells (MSCs) were exposed to CSE or nicotine (50–500 ng/ml) during adipogenic differentiation. Cell viability and metabolic activity were assessed via MTT assay. Lipid droplet accumulation was evaluated using Sudan III staining and quantitative image analysis. Adiponectin, IL6, and IL8 concentrations were measured after 35 days using ELISA.
Results
At these doses, CSE and nicotine do not immediately affect cell viability but inhibit undifferentiated cell proliferation. Notably, both agents at 50 ng/ml significantly increased lipid accumulation during adipogenesis, while higher CSE doses nearly completely inhibited this process. Additionally, CSE dose-dependently decreased adiponectin secretion and increased IL6 and IL8, indicating a shift towards an inflammatory state. Nicotine alone primarily increased IL6 secretion with less pronounced effects.
Conclusion
The study highlights the complex impact of CSE and nicotine on adipocyte function during early differentiation from MSCs. Dose-dependent changes in lipid accumulation, cytokine, and adiponectin secretion induced by CSE and nicotine can partly explain smoking-related adipose tissue dysfunction.
{"title":"Cigarette smoke extract decreases human bone marrow mesenchymal stromal cell adipogenic differentiation","authors":"Janne Heikkinen , Sanna Palosaari , Petri Lehenkari","doi":"10.1016/j.tiv.2024.105949","DOIUrl":"10.1016/j.tiv.2024.105949","url":null,"abstract":"<div><h3>Background</h3><div>Smoking and nicotine impose detrimental health effects including adipose tissue dysfunction. Despite extensive physiological evidence, the cellular mechanisms remain poorly understood, with few studies examining the effects of cigarette smoke extract (CSE) or nicotine on adipocyte differentiation.</div></div><div><h3>Methods</h3><div>Primary human bone marrow-derived mesenchymal stromal cells (MSCs) were exposed to CSE or nicotine (50–500 ng/ml) during adipogenic differentiation. Cell viability and metabolic activity were assessed <em>via</em> MTT assay. Lipid droplet accumulation was evaluated using Sudan III staining and quantitative image analysis. Adiponectin, IL6, and IL8 concentrations were measured after 35 days using ELISA.</div></div><div><h3>Results</h3><div>At these doses, CSE and nicotine do not immediately affect cell viability but inhibit undifferentiated cell proliferation. Notably, both agents at 50 ng/ml significantly increased lipid accumulation during adipogenesis, while higher CSE doses nearly completely inhibited this process. Additionally, CSE dose-dependently decreased adiponectin secretion and increased IL6 and IL8, indicating a shift towards an inflammatory state. Nicotine alone primarily increased IL6 secretion with less pronounced effects.</div></div><div><h3>Conclusion</h3><div>The study highlights the complex impact of CSE and nicotine on adipocyte function during early differentiation from MSCs. Dose-dependent changes in lipid accumulation, cytokine, and adiponectin secretion induced by CSE and nicotine can partly explain smoking-related adipose tissue dysfunction.</div></div>","PeriodicalId":54423,"journal":{"name":"Toxicology in Vitro","volume":"101 ","pages":"Article 105949"},"PeriodicalIF":2.6,"publicationDate":"2024-09-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142332393","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 : 2024-09-27DOI: 10.1016/j.tiv.2024.105948
Patricia Böttcher , Laura Steinmeyer , Holger Stark , Jörg Breitkreutz , Karsten R. Mewes
The MUTZ-3 cell line is a surrogate for Langerhans cells (LCs) employed in New Approach Methodologies for assessing the skin sensitizing potential of chemicals. However, MUTZ-3 cells must first be differentiated to achieve the LC-typical phenotype. As all protocols use high fetal calf serum (FCS) concentrations, we aimed at reducing, or even replacing FCS, while maintaining MUTZ-LC characteristics. Additionally, we assessed the impact of the poorly defined 5637-conditioned medium (5637CM) on MUTZ-LC differentiation.
With reducing the FCS content by 75 %, the desired differentiation status was achieved after 7 instead of 14 days, identified by elevated CD207 and CD1a expression. Culture with Ultroser G, a synthetic surrogate for FCS, resulted in an insufficient number of MUTZ-LCs. 5 % FCS-differentiated MUTZ-LCs could be activated with DNCB, an extreme sensitizer, as demonstrated by increased CD83 expression. 5637CM did not affect MUTZ-LC differentiation and is therefore not needed as a supplement.
For their intended role in an immunocompetent skin model to assess the sensitizing potential of chemicals, MUTZ-LCs were successfully integrated into the Phenion® Full-Thickness skin model, as demonstrated by CD1a expression. These results are important steps towards medium standardization and the generation of an immunocompetent skin model according to the 3R principles.
{"title":"Integration of MUTZ-Langerhans cells into a 3D full-thickness skin equivalent and influences of serum reduction and undefined medium supplements on differentiation","authors":"Patricia Böttcher , Laura Steinmeyer , Holger Stark , Jörg Breitkreutz , Karsten R. Mewes","doi":"10.1016/j.tiv.2024.105948","DOIUrl":"10.1016/j.tiv.2024.105948","url":null,"abstract":"<div><div>The MUTZ-3 cell line is a surrogate for Langerhans cells (LCs) employed in New Approach Methodologies for assessing the skin sensitizing potential of chemicals. However, MUTZ-3 cells must first be differentiated to achieve the LC-typical phenotype. As all protocols use high fetal calf serum (FCS) concentrations, we aimed at reducing, or even replacing FCS, while maintaining MUTZ-LC characteristics. Additionally, we assessed the impact of the poorly defined 5637-conditioned medium (5637CM) on MUTZ-LC differentiation.</div><div>With reducing the FCS content by 75 %, the desired differentiation status was achieved after 7 instead of 14 days, identified by elevated CD207 and CD1a expression. Culture with Ultroser G, a synthetic surrogate for FCS, resulted in an insufficient number of MUTZ-LCs. 5 % FCS-differentiated MUTZ-LCs could be activated with DNCB, an extreme sensitizer, as demonstrated by increased CD83 expression. 5637CM did not affect MUTZ-LC differentiation and is therefore not needed as a supplement.</div><div>For their intended role in an immunocompetent skin model to assess the sensitizing potential of chemicals, MUTZ-LCs were successfully integrated into the Phenion® Full-Thickness skin model, as demonstrated by CD1a expression. These results are important steps towards medium standardization and the generation of an immunocompetent skin model according to the 3R principles.</div></div>","PeriodicalId":54423,"journal":{"name":"Toxicology in Vitro","volume":"102 ","pages":"Article 105948"},"PeriodicalIF":2.6,"publicationDate":"2024-09-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142332396","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}
Di-(2-ethylhexyl) phthalate (DEHP) is widely used as a plasticizer in polyvinyl chloride products. DEHP exposure in humans is of great concern due to its endocrine-disrupting properties. In this study, we characterized the agonistic activities of DEHP and its five metabolites, mono-(2-ethylhexyl) phthalate (MEHP), 5OH-MEHP, 5oxo-MEHP, 5cx-MEPP and 2cx-MMHP against human nuclear receptors, peroxisome proliferator-activated receptor α (PPARα), pregnane X receptor (PXR), and constitutive androstane receptor (CAR) using transactivation assays. In the PPARα assay, the order of the agonistic activity was MEHP >> 5cx-MEPP >5OH-MEHP, 5oxo-MEHP >2cx-MMHP > DEHP, with DEHP significantly inhibiting MEHP-induced PPARα agonistic activity. This finding was compared to the results from in silico docking simulation. In the PXR assay, DEHP showed PXR agonistic activity more potent than that of MEHP, whereas the other metabolites showed little activity. In the CAR assay, none of the tested compounds showed agonistic activity. Moreover, the expression levels of PPARα-, PXR-, and CAR-target genes in HepaRG cells exposed to DEHP or MEHP were investigated using qRT-PCR analysis. As a result, exposure to these compounds significantly upregulated PXR/CAR target genes (CYP3A4 and CYP2B6), but not PPARα target genes (CYP4A11, etc.) in HepaRG cells. Taken together, these results suggest that direct PXR and/or indirect CAR activation by several DEHP metabolites may be involved in the endocrine disruption by altering hormone metabolism.
{"title":"Effects of di-(2-ethylhexyl) phthalate and its metabolites on transcriptional activity via human nuclear receptors and gene expression in HepaRG cells","authors":"Ayaka Yasuda , Wataru Murase , Atsuhito Kubota , Naoto Uramaru , Katsuhiro Okuda , Ryo Hakota , Atsuko Ikeda , Hiroyuki Kojima","doi":"10.1016/j.tiv.2024.105943","DOIUrl":"10.1016/j.tiv.2024.105943","url":null,"abstract":"<div><div>Di-(2-ethylhexyl) phthalate (DEHP) is widely used as a plasticizer in polyvinyl chloride products. DEHP exposure in humans is of great concern due to its endocrine-disrupting properties. In this study, we characterized the agonistic activities of DEHP and its five metabolites, mono-(2-ethylhexyl) phthalate (MEHP), 5OH-MEHP, 5oxo-MEHP, 5cx-MEPP and 2cx-MMHP against human nuclear receptors, peroxisome proliferator-activated receptor α (PPARα), pregnane X receptor (PXR), and constitutive androstane receptor (CAR) using transactivation assays. In the PPARα assay, the order of the agonistic activity was MEHP >> 5cx-MEPP >5OH-MEHP, 5oxo-MEHP >2cx-MMHP > DEHP, with DEHP significantly inhibiting MEHP-induced PPARα agonistic activity. This finding was compared to the results from in silico docking simulation. In the PXR assay, DEHP showed PXR agonistic activity more potent than that of MEHP, whereas the other metabolites showed little activity. In the CAR assay, none of the tested compounds showed agonistic activity. Moreover, the expression levels of PPARα-, PXR-, and CAR-target genes in HepaRG cells exposed to DEHP or MEHP were investigated using qRT-PCR analysis. As a result, exposure to these compounds significantly upregulated PXR/CAR target genes (<em>CYP3A4</em> and <em>CYP2B6</em>), but not PPARα target genes (<em>CYP4A11</em>, etc.) in HepaRG cells. Taken together, these results suggest that direct PXR and/or indirect CAR activation by several DEHP metabolites may be involved in the endocrine disruption by altering hormone metabolism.</div></div>","PeriodicalId":54423,"journal":{"name":"Toxicology in Vitro","volume":"101 ","pages":"Article 105943"},"PeriodicalIF":2.6,"publicationDate":"2024-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142332394","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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