Pub Date : 2025-08-29DOI: 10.1016/j.tiv.2025.106134
Yi-Peng Ng , Mei-Leng Tee , Shi-Ngar Pang, Chung-Lee Yee, Ursula Rho-Wan Chong, Khan-Loon Ng, Ka-Heng Lee, Mong-Sah Toh
The Forearm Controlled Application Test (FCAT) is widely used to assess skin irritancy and drying potential of rinse-off product but limited by its low throughput and long testing duration. To address these limitations, we explored the development of predictive models using New Approach Methodology (NAM) as a screening tool for evaluating skin mildness potential of rinse-off products. The skin irritancy and drying potential of standard surfactants were evaluated through multiple in vitro assays to identify relevant biomarkers, including a skin irritation bioassay by using Human Reconstituted Skin, a protein solubilization assay by using Zein test and a lipid solubilization assay by using Fatty Acid Methyl Ester (FAME) test. The in vitro test results were correlated to the clinical FCAT data, leading to the development of two multiple linear regression models in predicting changes of Transepidermal Water Loss (TEWL) and skin moisture parameters with R2 of 0.7062 and 0.8270, respectively. The models achieved prediction accuracies of 100 % and 89 % with relatively low Mean Absolute Error (MAE), outperforming single biomarker model. In summary, this proof-of-concept study demonstrated potential of integrative predictive models to predict clinical outcomes of FCAT, serving as a valuable NAM tool for evaluating the safety and skin mildness potential of rinse-off formulation.
{"title":"NAM-based development of a predictive test model for evaluating skin mildness potential of rinse-off products via integrated in vitro assays","authors":"Yi-Peng Ng , Mei-Leng Tee , Shi-Ngar Pang, Chung-Lee Yee, Ursula Rho-Wan Chong, Khan-Loon Ng, Ka-Heng Lee, Mong-Sah Toh","doi":"10.1016/j.tiv.2025.106134","DOIUrl":"10.1016/j.tiv.2025.106134","url":null,"abstract":"<div><div>The Forearm Controlled Application Test (FCAT) is widely used to assess skin irritancy and drying potential of rinse-off product but limited by its low throughput and long testing duration. To address these limitations, we explored the development of predictive models using New Approach Methodology (NAM) as a screening tool for evaluating skin mildness potential of rinse-off products. The skin irritancy and drying potential of standard surfactants were evaluated through multiple <em>in vitro</em> assays to identify relevant biomarkers, including a skin irritation bioassay by using Human Reconstituted Skin, a protein solubilization assay by using Zein test and a lipid solubilization assay by using Fatty Acid Methyl Ester (FAME) test. The <em>in vitro</em> test results were correlated to the clinical FCAT data, leading to the development of two multiple linear regression models in predicting changes of Transepidermal Water Loss (TEWL) and skin moisture parameters with R<sup>2</sup> of 0.7062 and 0.8270, respectively. The models achieved prediction accuracies of 100 % and 89 % with relatively low Mean Absolute Error (MAE), outperforming single biomarker model. In summary, this proof-of-concept study demonstrated potential of integrative predictive models to predict clinical outcomes of FCAT, serving as a valuable NAM tool for evaluating the safety and skin mildness potential of rinse-off formulation.</div></div>","PeriodicalId":54423,"journal":{"name":"Toxicology in Vitro","volume":"110 ","pages":"Article 106134"},"PeriodicalIF":2.7,"publicationDate":"2025-08-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144979084","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 : 2025-08-26DOI: 10.1016/j.tiv.2025.106132
Aleksandra K. Gładysz , Jan Stępniak , Laura López-Pingarrón , Joaquin J. Garcia , Małgorzata Karbownik-Lewińska
Chromium (Cr) is a harmful heavy metal pollutant. Cr(VI) is a group 1 carcinogen (carcinogenic to humans), whereas Cr(III) is a group 3 carcinogen (not classifiable as to its carcinogenicity to humans). Cr is also documented to be an endocrine disrupting chemical. The study aimed to check whether Cr(VI) compound (K2Cr2O7) or Cr(III) compound (CrCl3·6H2O) induce oxidative damage to membrane lipids (lipid peroxidation, LPO) in homogenates of two endocrine (the thyroid and the ovary) and two non-endocrine (the kidney and the liver) tissues, and whether antioxidants, such as melatonin, indole-3-propionic acid (IPA) and 17β-estradiol, reveal protective effects. Of note, the healthy thyroid gland is characterized by relatively high oxidative stress. Porcine tissue homogenates were incubated in the presence of Cr(VI) (0.05–10.0 mM) or Cr(III) (5.0–200.0 mM) with/without melatonin (5 mM) or IPA (5 mM) or 17β-estradiol (1 mM). The malondialdehyde+4-hydroxyalkenals (MDA + 4-HDA) concentration (LPO index) was measured spectrophotometrically. Cr(VI) (≥0.1–1.25 mM) significantly increased LPO in all tissues but these damaging effects were not prevented by any antioxidant tested. In turn, Cr(III) (≥25 mM) also significantly increased LPO in all examined tissues. All antioxidants reduced Cr(III)-induced LPO in the ovary, kidney, and liver but had no protective effects in the thyroid. Our findings highlight chromium's strong prooxidative effects, especially in the thyroid. The inability of antioxidants to prevent Cr-induced damage, especially in the thyroid, underscores the need for further research to identify effective protective strategies.
{"title":"Stronger prooxidative effects of chromium(VI) comparing to chromium(III) in endocrine and non-endocrine tissues with the thyroid being completely resistant to antioxidant protection","authors":"Aleksandra K. Gładysz , Jan Stępniak , Laura López-Pingarrón , Joaquin J. Garcia , Małgorzata Karbownik-Lewińska","doi":"10.1016/j.tiv.2025.106132","DOIUrl":"10.1016/j.tiv.2025.106132","url":null,"abstract":"<div><div>Chromium (Cr) is a harmful heavy metal pollutant. Cr(VI) is a group 1 carcinogen (carcinogenic to humans), whereas Cr(III) is a group 3 carcinogen (not classifiable as to its carcinogenicity to humans). Cr is also documented to be an endocrine disrupting chemical. The study aimed to check whether Cr(VI) compound (K<sub>2</sub>Cr<sub>2</sub>O<sub>7</sub>) or Cr(III) compound (CrCl<sub>3</sub>·6H<sub>2</sub>O) induce oxidative damage to membrane lipids (lipid peroxidation, LPO) in homogenates of two endocrine (the thyroid and the ovary) and two non-endocrine (the kidney and the liver) tissues, and whether antioxidants, such as melatonin, indole-3-propionic acid (IPA) and 17β-estradiol, reveal protective effects. Of note, the healthy thyroid gland is characterized by relatively high oxidative stress. Porcine tissue homogenates were incubated in the presence of Cr(VI) (0.05–10.0 mM) or Cr(III) (5.0–200.0 mM) with/without melatonin (5 mM) or IPA (5 mM) or 17β-estradiol (1 mM). The malondialdehyde+4-hydroxyalkenals (MDA + 4-HDA) concentration (LPO index) was measured spectrophotometrically. Cr(VI) (≥0.1–1.25 mM) significantly increased LPO in all tissues but these damaging effects were not prevented by any antioxidant tested. In turn, Cr(III) (≥25 mM) also significantly increased LPO in all examined tissues. All antioxidants reduced Cr(III)-induced LPO in the ovary, kidney, and liver but had no protective effects in the thyroid. Our findings highlight chromium's strong prooxidative effects, especially in the thyroid. The inability of antioxidants to prevent Cr-induced damage, especially in the thyroid, underscores the need for further research to identify effective protective strategies.</div></div>","PeriodicalId":54423,"journal":{"name":"Toxicology in Vitro","volume":"110 ","pages":"Article 106132"},"PeriodicalIF":2.7,"publicationDate":"2025-08-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144913329","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 : 2025-08-25DOI: 10.1016/j.tiv.2025.106133
Liyuan Wang, Ruifang Zhang, Xuelian Liu
Hyperuricemia is a recognized risk factor for cardiovascular diseases, including peripheral arterial disease (PAD), though molecular mechanisms linking uric acid to endothelial dysfunction remain unclear. This study investigates the role of sphingomyelin synthase 2 (SMS2) and endoplasmic reticulum (ER) stress in uric acid-induced endothelial impairment. Human umbilical vein endothelial cells were exposed to physiologically relevant concentrations of uric acid, with SMS2 function modulated through siRNA knockdown and ER stress inhibited using 4-phenylbutyric acid. Uric acid exhibited concentration-dependent cytotoxicity (IC50 ∼ 9 mg/dL) and significantly upregulated SMS2 expressions. At this concentration, uric acid significantly increased apoptosis, impaired migratory capacity, and diminished angiogenic potential (p < 0.01). These functional deficits coincided with marked elevation of ER stress markers and intracellular calcium disruption. Notably, both SMS2 knockdown and ER stress inhibition substantially reversed these uric acid-induced endothelial dysfunctions, restoring cell survival, migration, and angiogenic capacity while normalizing ER stress markers and calcium homeostasis (p < 0.01). These findings identify SMS2 as a potential therapeutic target for vascular complications in hyperuricemia and suggest ER stress modulators may protect against uric acid-induced endothelial damage.
{"title":"Hyperuricemia impairs endothelial function through SMS2-dependent activation of the endoplasmic reticulum stress response","authors":"Liyuan Wang, Ruifang Zhang, Xuelian Liu","doi":"10.1016/j.tiv.2025.106133","DOIUrl":"10.1016/j.tiv.2025.106133","url":null,"abstract":"<div><div>Hyperuricemia is a recognized risk factor for cardiovascular diseases, including peripheral arterial disease (PAD), though molecular mechanisms linking uric acid to endothelial dysfunction remain unclear. This study investigates the role of sphingomyelin synthase 2 (SMS2) and endoplasmic reticulum (ER) stress in uric acid-induced endothelial impairment. Human umbilical vein endothelial cells were exposed to physiologically relevant concentrations of uric acid, with SMS2 function modulated through siRNA knockdown and ER stress inhibited using 4-phenylbutyric acid. Uric acid exhibited concentration-dependent cytotoxicity (IC50 ∼ 9 mg/dL) and significantly upregulated SMS2 expressions. At this concentration, uric acid significantly increased apoptosis, impaired migratory capacity, and diminished angiogenic potential (<em>p</em> < 0.01). These functional deficits coincided with marked elevation of ER stress markers and intracellular calcium disruption. Notably, both SMS2 knockdown and ER stress inhibition substantially reversed these uric acid-induced endothelial dysfunctions, restoring cell survival, migration, and angiogenic capacity while normalizing ER stress markers and calcium homeostasis (<em>p</em> < 0.01). These findings identify SMS2 as a potential therapeutic target for vascular complications in hyperuricemia and suggest ER stress modulators may protect against uric acid-induced endothelial damage.</div></div>","PeriodicalId":54423,"journal":{"name":"Toxicology in Vitro","volume":"110 ","pages":"Article 106133"},"PeriodicalIF":2.7,"publicationDate":"2025-08-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144979070","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 : 2025-08-19DOI: 10.1016/j.tiv.2025.106131
Mairin Schott , Charles Elias Assmann , Bianca Vedoin Copês Rambo , Marcylene Vieira da Silveira , Milagros Fanny Vera Castro , Vitor Bastianello Mostardeiro , Adriel Antônio Schirmann , Robson Lourenço da Silva Santos , Pâmela de Almeida Milioni , Ana Barbosa Viana , Valderi Luiz Dressler , Daniel Lázaro Gallindo Borges , Nilda Berenice de Vargas Barbosa , Vera Maria Melchiors Morsch , Michael Aschner , João Batista Teixeira da Rocha , Maria Rosa Chitolina Schetinger
An in vitro model using human peripheral blood mononuclear cells (PBMCs) was established to investigate the cytotoxic, oxidative and inflammatory effects and changes in purinergic system parameters caused by mercuric chloride (HgCl2). Cells were exposed to concentrations of HgCl2 (0.05, 0.5, 5, and 50 μM) for 24, 48 and 72 h. Cell viability was measured by trypan blue and MTT assays, and IC50; apoptosis and cell death were assessed by flow cytometry. Oxidative stress was evaluated by reactive species (RS) generation, determination of Mercury (ICP-MS), Nitric Oxide (NO), and lipid peroxidation. Increased levels of HgCl₂ induced RS generation, NO, lipid peroxidation, apoptosis, and altered the cell cycle, reducing DNA synthesis and cell division of PBMCs. Flow cytometry confirmed decreased viability and increased late apoptosis. HgCl₂ accumulation in PBMCs was confirmed by ICP-MS. HgCl₂ altered purinergic system components, inhibiting NTPDase and increasing ADA activity. At 5 μM, it increased gene expression of purinergic receptors and both anti-inflammatory (IL-10) and pro-inflammatory markers (IL-1β, IL-6, TNF-α, NLRP3, CASP-1, NF-κB). Overexpression of the P2X7/NLRP3/CASP-1/IL-1β axis triggered cell death by pyroptosis. These findings provide compelling evidence HgCl2 induces cytotoxic and inflammatory effects in PBMCs.
{"title":"Cytotoxic and immunotoxic profile of HgCl2 involves alterations in purinergic signaling through the P2X7/NLRP3/CASP-1/IL-1β pathway: An in vitro study using human blood immune cells","authors":"Mairin Schott , Charles Elias Assmann , Bianca Vedoin Copês Rambo , Marcylene Vieira da Silveira , Milagros Fanny Vera Castro , Vitor Bastianello Mostardeiro , Adriel Antônio Schirmann , Robson Lourenço da Silva Santos , Pâmela de Almeida Milioni , Ana Barbosa Viana , Valderi Luiz Dressler , Daniel Lázaro Gallindo Borges , Nilda Berenice de Vargas Barbosa , Vera Maria Melchiors Morsch , Michael Aschner , João Batista Teixeira da Rocha , Maria Rosa Chitolina Schetinger","doi":"10.1016/j.tiv.2025.106131","DOIUrl":"10.1016/j.tiv.2025.106131","url":null,"abstract":"<div><div>An in vitro model using human peripheral blood mononuclear cells (PBMCs) was established to investigate the cytotoxic, oxidative and inflammatory effects and changes in purinergic system parameters caused by mercuric chloride (HgCl<sub>2</sub>). Cells were exposed to concentrations of HgCl<sub>2</sub> (0.05, 0.5, 5, and 50 μM) for 24, 48 and 72 h. Cell viability was measured by trypan blue and MTT assays, and IC<sub>50</sub>; apoptosis and cell death were assessed by flow cytometry. Oxidative stress was evaluated by reactive species (RS) generation, determination of Mercury (ICP-MS), Nitric Oxide (NO), and lipid peroxidation. Increased levels of HgCl₂ induced RS generation, NO, lipid peroxidation, apoptosis, and altered the cell cycle, reducing DNA synthesis and cell division of PBMCs. Flow cytometry confirmed decreased viability and increased late apoptosis. HgCl₂ accumulation in PBMCs was confirmed by ICP-MS. HgCl₂ altered purinergic system components, inhibiting NTPDase and increasing ADA activity. At 5 μM, it increased gene expression of purinergic receptors and both anti-inflammatory (IL-10) and pro-inflammatory markers (IL-1β, IL-6, TNF-α, NLRP3, CASP-1, NF-κB). Overexpression of the P2X7/NLRP3/CASP-1/IL-1β axis triggered cell death by pyroptosis. These findings provide compelling evidence HgCl<sub>2</sub> induces cytotoxic and inflammatory effects in PBMCs.</div></div>","PeriodicalId":54423,"journal":{"name":"Toxicology in Vitro","volume":"109 ","pages":"Article 106131"},"PeriodicalIF":2.7,"publicationDate":"2025-08-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144890368","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 : 2025-08-14DOI: 10.1016/j.tiv.2025.106130
Andressa Rubim Lopes , Michael González-Durruthy , Maria Natália D.S. Cordeiro , Ana S. Moura , Ramón Rial , Juan M. Ruso , Juliana Zomer Sandrini , Carlos Eduardo da Rosa , Camila de Martinez Gaspar Martins
This study explores the toxicodynamics of glyphosate in zebrafish (Danio rerio) acetylcholinesterase (zf-AChE) using a combined computational and experimental approach to reveal its potential cholinergic neurotoxic effects. Computational modeling suggested that glyphosate could block critical amino acid residues in the zf-AChE binding site, disrupting acetylcholine positioning and potentially leading to its pathological accumulation in cholinergic synapses. Additionally, glyphosate may adversely impact zf-AChE's flexibility, inducing conformational rigidity via hydrophobic van der Waals and hydrogen-bond interactions. These effects mirrored the binding behavior of physostigmine, a known specific zf-AChE inhibitor. Interestingly, the structural similarity between zf-AChE and human AChE (hs-AChE) suggests potential neurotoxicity in humans. Spectrofluorimetry confirmed binding between glyphosate and hs-AChE, resembling physostigmine binding. To sum up, our findings provide insights into glyphosate-induced cholinergic neurotoxicity in zebrafish, supporting extrapolations to humans and contributing valuable insights for ecotoxicology, new approach methodologies, and environmental risk assessment.
{"title":"Computational and spectrofluorimetric validation on glyphosate interactions with zebrafish (Danio rerio) acetylcholinesterase: Mechanistic and ecotoxicological implications","authors":"Andressa Rubim Lopes , Michael González-Durruthy , Maria Natália D.S. Cordeiro , Ana S. Moura , Ramón Rial , Juan M. Ruso , Juliana Zomer Sandrini , Carlos Eduardo da Rosa , Camila de Martinez Gaspar Martins","doi":"10.1016/j.tiv.2025.106130","DOIUrl":"10.1016/j.tiv.2025.106130","url":null,"abstract":"<div><div>This study explores the toxicodynamics of glyphosate in zebrafish (<em>Danio rerio</em>) acetylcholinesterase (zf-AChE) using a combined computational and experimental approach to reveal its potential cholinergic neurotoxic effects. Computational modeling suggested that glyphosate could block critical amino acid residues in the zf-AChE binding site, disrupting acetylcholine positioning and potentially leading to its pathological accumulation in cholinergic synapses. Additionally, glyphosate may adversely impact zf-AChE's flexibility, inducing conformational rigidity <em>via</em> hydrophobic van der Waals and hydrogen-bond interactions. These effects mirrored the binding behavior of physostigmine, a known specific zf-AChE inhibitor. Interestingly, the structural similarity between zf-AChE and human AChE (hs-AChE) suggests potential neurotoxicity in humans. Spectrofluorimetry confirmed binding between glyphosate and hs-AChE, resembling physostigmine binding. To sum up, our findings provide insights into glyphosate-induced cholinergic neurotoxicity in zebrafish, supporting extrapolations to humans and contributing valuable insights for ecotoxicology, new approach methodologies, and environmental risk assessment.</div></div>","PeriodicalId":54423,"journal":{"name":"Toxicology in Vitro","volume":"109 ","pages":"Article 106130"},"PeriodicalIF":2.7,"publicationDate":"2025-08-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144862703","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 : 2025-08-14DOI: 10.1016/j.tiv.2025.106129
Ke Xu, Krittika Mittal, Niladri Basu
N-(1,3-Dimethyl butyl)-N′-phenyl-phenylenediamine-quinone (6ppd-quinone) is of emerging concern due to its widespread presence and toxicity to aquatic species. The chemical has been detected in human biofluids though little is known about its effects on human tissues. The objective of this study was to increase understanding of 6ppd-quinone's potential human health effects by deriving transcriptomic points of departure (tPOD) values in two human cell lines using the TPD-seq workflow. An EC20 for cytotoxicity was calculated for Caco-2 (104 μg/L) but not for HepG2 cells. Even in the absence of cytotoxicity, tPOD values (20th gene, 10th percentile, mode) were calculated in Caco-2 (6.5-25 μg/L) and HepG2 (0.36-35 μg/L) cells. These ranges capture values from 16 statistical and bioinformatic tests that examined mapping methods (CLC and Deplexer), algorithms (Limma and DESeq2), and filters (log2FC and BMR). The most common and sensitive genes with calculable benchmark doses (BMDs) in Caco-2 (DPF2, CD44, PGAP1, GDF15, H4C16) and HepG2 (SLC5A3, DKK1, ARG2, PHLDA1, TM4SF1) cells are listed. Pathway BMDs were also calculated for Caco-2 (systemic lupus erythematosus, 9.7-18 μg/L; alcoholism, 9.7-20 μg/L; viral carcinogenesis, 9.3–18.1 μg/L), and HepG2 (metabolic pathways, 50-60 μg/L) cells. These findings highlight TPD-seq as an efficient workflow to yield quantitative and mechanistic data relevant for human health risk assessment.
{"title":"Transcriptomic points of departure for 6PPD-Quinone derived from human Caco-2 and HepG2 cells","authors":"Ke Xu, Krittika Mittal, Niladri Basu","doi":"10.1016/j.tiv.2025.106129","DOIUrl":"10.1016/j.tiv.2025.106129","url":null,"abstract":"<div><div>N-(1,3-Dimethyl butyl)-<em>N</em>′-phenyl-phenylenediamine-quinone (6ppd-quinone) is of emerging concern due to its widespread presence and toxicity to aquatic species. The chemical has been detected in human biofluids though little is known about its effects on human tissues. The objective of this study was to increase understanding of 6ppd-quinone's potential human health effects by deriving transcriptomic points of departure (tPOD) values in two human cell lines using the TPD-seq workflow. An EC20 for cytotoxicity was calculated for Caco-2 (104 μg/L) but not for HepG2 cells. Even in the absence of cytotoxicity, tPOD values (20th gene, 10th percentile, mode) were calculated in Caco-2 (6.5-25 μg/L) and HepG2 (0.36-35 μg/L) cells. These ranges capture values from 16 statistical and bioinformatic tests that examined mapping methods (CLC and Deplexer), algorithms (Limma and DESeq2), and filters (log2FC and BMR). The most common and sensitive genes with calculable benchmark doses (BMDs) in Caco-2 (DPF2, CD44, PGAP1, GDF15, H4C16) and HepG2 (SLC5A3, DKK1, ARG2, PHLDA1, TM4SF1) cells are listed. Pathway BMDs were also calculated for Caco-2 (systemic lupus erythematosus, 9.7-18 μg/L; alcoholism, 9.7-20 μg/L; viral carcinogenesis, 9.3–18.1 μg/L), and HepG2 (metabolic pathways, 50-60 μg/L) cells. These findings highlight TPD-seq as an efficient workflow to yield quantitative and mechanistic data relevant for human health risk assessment.</div></div>","PeriodicalId":54423,"journal":{"name":"Toxicology in Vitro","volume":"109 ","pages":"Article 106129"},"PeriodicalIF":2.7,"publicationDate":"2025-08-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144862704","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 : 2025-08-10DOI: 10.1016/j.tiv.2025.106128
Lérica le Roux-Pullen , Jeroen J.M.W. van den Heuvel , Ilse R. Dubbelboer , Frans G.M. Russel , Ronette Gehring , Jan B. Koenderink
The use of herbal alternatives in modern agriculture and healthcare offers a sustainable approach to animal and human health, but raises concerns about safety, particularly regarding phytochemical interactions at membrane transport proteins. Phytochemicals – bioactive compounds found in herbs - can influence the function of the breast cancer resistance protein (BCRP/ABCG2). BCRP is a key efflux transporter in mammals and in particularly abundant in the blood-milk-barrier of lactating animals, with the potential to affect milk quality and safety. This study investigates species-specific interactions of eight selected phytochemicals with BCRP orthologs in humans, cows, sheep and goats, using a transport assay with membrane vesicles derived from species-specific BCRP-overexpressing HEK293 cells. Five phytochemicals, namely apigenin, berberine, kaempferol, N-isobutyldodeca-2E4E8Z10E/Z-tetraenamide and quercetin inhibited BCRP-mediated transport of its prototypic substrate [3H]estrone sulfate by more than 30 % in all species. IC50 values and, assuming competitive inhibition, Ki values were calculated for these compounds. Apigenin and kaempferol were the most potent inhibitors with Ki values <0.1 μM in all species, while N-isobutyldodeca-2E4E8Z10E/Z-tetraenamide was the weakest inhibitor (Ki > 30 μM). No significant interspecies differences in inhibitory potency were observed. Understanding the cross-species effects of bioactive compounds on BCRP activity is essential for predicting their broader biological impacts. Similar inhibition trends across species facilitate interspecies extrapolation and minimize the amount of animal studies needed to further investigate the effect of these phytochemicals on milk composition.
在现代农业和医疗保健中使用草药替代品为动物和人类健康提供了一种可持续的方法，但也引起了对安全性的担忧，特别是在膜运输蛋白上的植物化学相互作用方面。植物化学物质——在草药中发现的生物活性化合物——可以影响乳腺癌抵抗蛋白（BCRP/ABCG2）的功能。BCRP是哺乳动物体内一种重要的外排转运蛋白，在哺乳期动物的血乳屏障中含量尤其丰富，具有影响乳汁质量和安全的潜力。本研究利用来自物种特异性BCRP过表达HEK293细胞的膜泡运输实验，研究了8种选定的植物化学物质与人、牛、绵羊和山羊中BCRP同源物的物种特异性相互作用。5种植物化学物质，即芹菜素、小檗碱、山奈酚、n -异丁基十二烷- 2e4e8z10e / z -四烯酰胺和槲皮素，在所有物种中抑制bcrp介导的原型底物[3H]雌酮硫酸盐的运输超过30% %。计算了这些化合物的IC50值，并假设了竞争抑制作用，计算了Ki值。芹菜素和山奈酚是最有效的抑制剂，Ki值为 30 μM)。抑菌效力在种间无显著差异。了解生物活性化合物对BCRP活性的跨物种影响对于预测其更广泛的生物学影响至关重要。跨物种的类似抑制趋势促进了物种间的外推，并减少了进一步研究这些植物化学物质对牛奶成分影响所需的动物研究数量。
{"title":"Inhibitory potential of phytochemicals on species-specific breast cancer resistance protein transport activity","authors":"Lérica le Roux-Pullen , Jeroen J.M.W. van den Heuvel , Ilse R. Dubbelboer , Frans G.M. Russel , Ronette Gehring , Jan B. Koenderink","doi":"10.1016/j.tiv.2025.106128","DOIUrl":"10.1016/j.tiv.2025.106128","url":null,"abstract":"<div><div>The use of herbal alternatives in modern agriculture and healthcare offers a sustainable approach to animal and human health, but raises concerns about safety, particularly regarding phytochemical interactions at membrane transport proteins. Phytochemicals – bioactive compounds found in herbs - can influence the function of the breast cancer resistance protein (BCRP/ABCG2). BCRP is a key efflux transporter in mammals and in particularly abundant in the blood-milk-barrier of lactating animals, with the potential to affect milk quality and safety. This study investigates species-specific interactions of eight selected phytochemicals with BCRP orthologs in humans, cows, sheep and goats, using a transport assay with membrane vesicles derived from species-specific BCRP-overexpressing HEK293 cells. Five phytochemicals, namely apigenin, berberine, kaempferol, N-isobutyldodeca-2E4E8Z10E/<em>Z</em>-tetraenamide and quercetin inhibited BCRP-mediated transport of its prototypic substrate [<sup>3</sup>H]estrone sulfate by more than 30 % in all species. IC<sub>50</sub> values and, assuming competitive inhibition, Ki values were calculated for these compounds. Apigenin and kaempferol were the most potent inhibitors with Ki values <0.1 μM in all species, while N-isobutyldodeca-2E4E8Z10E/<em>Z</em>-tetraenamide was the weakest inhibitor (Ki > 30 μM). No significant interspecies differences in inhibitory potency were observed. Understanding the cross-species effects of bioactive compounds on BCRP activity is essential for predicting their broader biological impacts. Similar inhibition trends across species facilitate interspecies extrapolation and minimize the amount of animal studies needed to further investigate the effect of these phytochemicals on milk composition.</div></div>","PeriodicalId":54423,"journal":{"name":"Toxicology in Vitro","volume":"109 ","pages":"Article 106128"},"PeriodicalIF":2.7,"publicationDate":"2025-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144838549","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 : 2025-07-30DOI: 10.1016/j.tiv.2025.106125
Martina Daniela Benedetti , Mariela Lenze , Mora Renée García , Sofía Fátima Magaña Guerrero , Silvia Wikinski , Yonathan Garfias , María Laura Gutiérrez
Ocular toxicity assessment is essential to ensure the safety of chemicals and human-use products. Although the Draize test in rabbits has historically been the regulatory standard, it presents significant ethical and scientific limitations. While validated in vitro methods are currently available, most of them do not allow the evaluation of damage reversibility—a key parameter for hazard classification under the United Nations Globally Harmonized System (UN GHS).
This study evaluates the utility of an in vitro model based on limbal mesenchymal stromal cells (LMSC) for ocular toxicity testing and the prediction of corneal damage reversibility. LMSC were obtained from slaughterhouse-derived residual tissue, cultured under specific conditions, and briefly exposed to substances classified according to UN GHS. Cell migration was then assessed as an indicator of tissue recovery.
This preliminary report presents, to our knowledge, the first application of short-term (5-min) exposure in a scratch assay to evaluate its effect on migration capacity. Unlike previous studies relying on prolonged exposure to inhibitory compounds, this approach better simulates accidental ocular exposure scenarios. While migration was not significantly disrupted under the tested conditions, these results offer valuable insight into the relationship between viability and wound healing and highlight the importance of exposure time and concentration in in vitro ocular toxicity models. Overall, this work establishes a promising foundation for future refinement of alternative methods that aim to predict damage persistence and reversibility more accurately.
{"title":"Isolation and characterization of bovine Limbal mesenchymal stromal cells for application in ocular toxicity studies","authors":"Martina Daniela Benedetti , Mariela Lenze , Mora Renée García , Sofía Fátima Magaña Guerrero , Silvia Wikinski , Yonathan Garfias , María Laura Gutiérrez","doi":"10.1016/j.tiv.2025.106125","DOIUrl":"10.1016/j.tiv.2025.106125","url":null,"abstract":"<div><div>Ocular toxicity assessment is essential to ensure the safety of chemicals and human-use products. Although the Draize test in rabbits has historically been the regulatory standard, it presents significant ethical and scientific limitations. While validated <em>in vitro</em> methods are currently available, most of them do not allow the evaluation of damage reversibility—a key parameter for hazard classification under the United Nations Globally Harmonized System (UN GHS).</div><div>This study evaluates the utility of an <em>in vitro</em> model based on limbal mesenchymal stromal cells (LMSC) for ocular toxicity testing and the prediction of corneal damage reversibility. LMSC were obtained from slaughterhouse-derived residual tissue, cultured under specific conditions, and briefly exposed to substances classified according to UN GHS. Cell migration was then assessed as an indicator of tissue recovery.</div><div>This preliminary report presents, to our knowledge, the first application of short-term (5-min) exposure in a scratch assay to evaluate its effect on migration capacity. Unlike previous studies relying on prolonged exposure to inhibitory compounds, this approach better simulates accidental ocular exposure scenarios. While migration was not significantly disrupted under the tested conditions, these results offer valuable insight into the relationship between viability and wound healing and highlight the importance of exposure time and concentration in <em>in vitro</em> ocular toxicity models. Overall, this work establishes a promising foundation for future refinement of alternative methods that aim to predict damage persistence and reversibility more accurately.</div></div>","PeriodicalId":54423,"journal":{"name":"Toxicology in Vitro","volume":"109 ","pages":"Article 106125"},"PeriodicalIF":2.7,"publicationDate":"2025-07-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144750760","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 : 2025-07-29DOI: 10.1016/j.tiv.2025.106124
Alexandra Lopes, Patrícia Pereira, Helena Oliveira
Nail polish dryers are radiation-emitting devices designed to dry and cure nail polishes. These devices primarily emit radiation within the ultraviolet A (UVA) range, with some also emitting visible light, particularly those equipped with light-emitting diodes (LEDs). The increasing use of nail polish dryers, combined with scientific concerns regarding the potential effects of UVA radiation on human skin, has raised significant safety questions. Recent studies have demonstrated cytotoxic, genotoxic and mutagenic effects in both human and murine cells. When these findings are considered alongside earlier reports linking the use of these devices to skin cancer, there is a clear need for a broader evaluation of their potential health impacts. This study aimed to evaluate the effects of realistic radiation exposures, when considering the actual gel-nail application process, on cell viability and intracellular reactive oxygen species (ROS) levels, in human keratinocytes. Cytotoxic effects were confirmed, with cell mortality rates exceeding 95 % and intracellular ROS levels increasing by over 250 % (72 h post-exposure), depending on the positioning of the cell culture within the device. These results underscore the need for a more thorough characterization of the cytotoxic effects caused by the radiation emitted by nail polish dryers.
{"title":"Cytotoxic effects of a LED/UV nail polish dryer in human keratinocytes","authors":"Alexandra Lopes, Patrícia Pereira, Helena Oliveira","doi":"10.1016/j.tiv.2025.106124","DOIUrl":"10.1016/j.tiv.2025.106124","url":null,"abstract":"<div><div>Nail polish dryers are radiation-emitting devices designed to dry and cure nail polishes. These devices primarily emit radiation within the ultraviolet A (UVA) range, with some also emitting visible light, particularly those equipped with light-emitting diodes (LEDs). The increasing use of nail polish dryers, combined with scientific concerns regarding the potential effects of UVA radiation on human skin, has raised significant safety questions. Recent studies have demonstrated cytotoxic, genotoxic and mutagenic effects in both human and murine cells. When these findings are considered alongside earlier reports linking the use of these devices to skin cancer, there is a clear need for a broader evaluation of their potential health impacts. This study aimed to evaluate the effects of realistic radiation exposures, when considering the actual gel-nail application process, on cell viability and intracellular reactive oxygen species (ROS) levels, in human keratinocytes. Cytotoxic effects were confirmed, with cell mortality rates exceeding 95 % and intracellular ROS levels increasing by over 250 % (72 h post-exposure), depending on the positioning of the cell culture within the device. These results underscore the need for a more thorough characterization of the cytotoxic effects caused by the radiation emitted by nail polish dryers.</div></div>","PeriodicalId":54423,"journal":{"name":"Toxicology in Vitro","volume":"109 ","pages":"Article 106124"},"PeriodicalIF":2.7,"publicationDate":"2025-07-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144762358","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 : 2025-07-29DOI: 10.1016/j.tiv.2025.106123
Martín Nicolás Rivero , Mariela Lenze , Martina Daniela Benedetti , Mora Renée García , Romina Martínez , Silvia Wikinski , María Laura Gutiérrez
The Bovine Corneal Opacity and Permeability (BCOP) assay is a validated ex vivo method widely used to assess the ocular irritation potential of chemical substances. It is useful for identifying both chemicals that cause serious eye damage and those that are not classified for eye irritation. This study incorporated histopathological parameters into the BCOP assay to enhance the characterization of corneal damage. Fifteen reference chemicals were tested, and IVIS scores were obtained from corneal opacity and permeability measurements. Fixed corneal tissues were then analyzed histologically. Three complementary models were applied: Depth of Injury (DOI), Stromal Thickness (ST), and Total Histopathological Damage (THD). The DOI model helped categorize damage by affected corneal layers and provided relevant information about the depth of the lesions. ST showed a strong correlation with IVIS scores and emerged as a robust indicator of edema. In contrast, the more complex THD model did not improve the classification. Overall, integrating ST and DOI into the BCOP framework provides complementary and informative endpoints that enhance the interpretation of ocular irritation potential. In particular, we highlight ST as a quantitative, and easy-to-measure endpoint that does not rely on expert interpretation.
{"title":"Employing histopathology to enhance the BCOP test: The emerging role of stromal thickness as a quantitative endpoint","authors":"Martín Nicolás Rivero , Mariela Lenze , Martina Daniela Benedetti , Mora Renée García , Romina Martínez , Silvia Wikinski , María Laura Gutiérrez","doi":"10.1016/j.tiv.2025.106123","DOIUrl":"10.1016/j.tiv.2025.106123","url":null,"abstract":"<div><div>The Bovine Corneal Opacity and Permeability (BCOP) assay is a validated <em>ex vivo</em> method widely used to assess the ocular irritation potential of chemical substances. It is useful for identifying both chemicals that cause serious eye damage and those that are not classified for eye irritation. This study incorporated histopathological parameters into the BCOP assay to enhance the characterization of corneal damage. Fifteen reference chemicals were tested, and IVIS scores were obtained from corneal opacity and permeability measurements. Fixed corneal tissues were then analyzed histologically. Three complementary models were applied: Depth of Injury (DOI), Stromal Thickness (ST), and Total Histopathological Damage (THD). The DOI model helped categorize damage by affected corneal layers and provided relevant information about the depth of the lesions. ST showed a strong correlation with IVIS scores and emerged as a robust indicator of edema. In contrast, the more complex THD model did not improve the classification. Overall, integrating ST and DOI into the BCOP framework provides complementary and informative endpoints that enhance the interpretation of ocular irritation potential. In particular, we highlight ST as a quantitative, and easy-to-measure endpoint that does not rely on expert interpretation.</div></div>","PeriodicalId":54423,"journal":{"name":"Toxicology in Vitro","volume":"109 ","pages":"Article 106123"},"PeriodicalIF":2.7,"publicationDate":"2025-07-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144750715","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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