Pub Date : 2026-01-24DOI: 10.1016/j.vascn.2026.108416
Isabela Nucci Galetti , Heloísa Peres Luz , Bruna Espíndola da Silva , Maitê Perin , Camila Marchioni
This study aimed to develop and validate an analytical method using dispersive solid phase extraction (d-SPE) with analysis by liquid chromatography coupled with diode array detector (HPLC-DAD). The developed technique showed detection limit of 100 ng/mL and 1300 ng/mL for cocaine, amitriptyline, and benzoylecgonine, respectively. The precision was less than 15% for 3 different analyte concentrations. The method shows no carryover, and stability was proven for cocaine and amitriptyline. When compared to other techniques, the current study presented a lower detection limit and used a smaller sample volume. The method was compared with the rapid immunoassay test on samples from patients with suspected intoxication, and was satisfactory, showing adequate sensitivity for the proposed application. The method using d-SPE/HPLC-DAD proved to be an appropriate technique to complement emergency toxicological analysis with a satisfactory total run time of 14 min for the detection of the 3 analytes of interest, which fits well into a fast-paced workflow.
{"title":"Streamlined dispersive solid phase extraction for determination of amitriptyline, cocaine, and benzoylecgonine in urine","authors":"Isabela Nucci Galetti , Heloísa Peres Luz , Bruna Espíndola da Silva , Maitê Perin , Camila Marchioni","doi":"10.1016/j.vascn.2026.108416","DOIUrl":"10.1016/j.vascn.2026.108416","url":null,"abstract":"<div><div>This study aimed to develop and validate an analytical method using dispersive solid phase extraction (d-SPE) with analysis by liquid chromatography coupled with diode array detector (HPLC-DAD). The developed technique showed detection limit of 100 ng/mL and 1300 ng/mL for cocaine, amitriptyline, and benzoylecgonine, respectively. The precision was less than 15% for 3 different analyte concentrations. The method shows no carryover, and stability was proven for cocaine and amitriptyline. When compared to other techniques, the current study presented a lower detection limit and used a smaller sample volume. The method was compared with the rapid immunoassay test on samples from patients with suspected intoxication, and was satisfactory, showing adequate sensitivity for the proposed application. The method using d-SPE/HPLC-DAD proved to be an appropriate technique to complement emergency toxicological analysis with a satisfactory total run time of 14 min for the detection of the 3 analytes of interest, which fits well into a fast-paced workflow.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"138 ","pages":"Article 108416"},"PeriodicalIF":1.8,"publicationDate":"2026-01-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146055817","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-15DOI: 10.1016/j.vascn.2026.108412
Yonghua Zhao , Dandan Liang , Yufei Guo , Ziqi Li , Zehua Yang
Objective
This study evaluated a smartphone-based system for measuring cerebrospinal fluid (CSF) total protein, verifying its compliance with clinical requirements and exploring its application in diagnosing central nervous system (CNS) diseases in remote/medically underserved areas.
Methods
The system analyzed CSF total protein standards to establish a standard curve (concentration vs. R, G, Y values). Per Clinical and Laboratory Standards Institute (CLSI) guidelines, key parameters (precision, detection capability, linear range [LR], clinical reportable range [CRR]) were assessed. Forty-two clinical samples from neurological patients were tested, with results compared to the Beckman Coulter AU5800 analyzer for consistency. Accuracy at three medical decision levels (based on CSF total protein reference ranges) was verified.
Results
The limit of blank (LOB), limit of detection (LOD), and limit of quantitation (LOQ) were 5.18 mg/dL (0.05 g/L), 11.78 mg/dL (0.12 g/L), and 12.59 mg/dL (0.13 g/L), respectively. The LR was 29.75–127.20 mg/dL (0.30–1.27 g/L), and the CRR was 12.59–508.80 mg/dL (0.13–5.09 g/L; via 1:3 dilution), both meeting clinical requirements. Total imprecision coefficients of variation (CVs) were 3.83% (high-concentration: 91.52 mg/dL [0.92 g/L]) and 3.59% (low-concentration: 31.32 mg/dL [0.31 g/L]), within acceptable ranges. The 42 clinical samples showed strong consistency with AU5800 results (R2 = 0.991). In the evaluation of the smartphone-based CSF total protein detection system, although the 95% confidence intervals of expected biases at 45 mg/dL (0.45 g/L) and 60 mg/dL (0.60 g/L) exceed 1/4 total allowable error per CLSI EP9-A2, the deviations are clinically irrelevant—with the maximum bias (5.29 mg/dL [0.0529 g/L]) fails to alter the pathological threshold classification of CSF total protein (≥45 mg/dL [0.45 g/L]), thus confirming the acceptable bias of the system for auxiliary CSF protein detection.
Conclusions
The system meets clinical standards, is user-friendly and cost-effective, and has high potential for aiding CNS disorder diagnosis in resource-constrained areas.
{"title":"Rapid quantification of CSF total protein via smartphone digital colorimetry: Enabling early diagnosis of central nervous system diseases","authors":"Yonghua Zhao , Dandan Liang , Yufei Guo , Ziqi Li , Zehua Yang","doi":"10.1016/j.vascn.2026.108412","DOIUrl":"10.1016/j.vascn.2026.108412","url":null,"abstract":"<div><h3>Objective</h3><div>This study evaluated a smartphone-based system for measuring cerebrospinal fluid (CSF) total protein, verifying its compliance with clinical requirements and exploring its application in diagnosing central nervous system (CNS) diseases in remote/medically underserved areas.</div></div><div><h3>Methods</h3><div>The system analyzed CSF total protein standards to establish a standard curve (concentration vs. R, G, Y values). Per Clinical and Laboratory Standards Institute (CLSI) guidelines, key parameters (precision, detection capability, linear range [LR], clinical reportable range [CRR]) were assessed. Forty-two clinical samples from neurological patients were tested, with results compared to the Beckman Coulter AU5800 analyzer for consistency. Accuracy at three medical decision levels (based on CSF total protein reference ranges) was verified.</div></div><div><h3>Results</h3><div>The limit of blank (LOB), limit of detection (LOD), and limit of quantitation (LOQ) were 5.18 mg/dL (0.05 g/L), 11.78 mg/dL (0.12 g/L), and 12.59 mg/dL (0.13 g/L), respectively. The LR was 29.75–127.20 mg/dL (0.30–1.27 g/L), and the CRR was 12.59–508.80 mg/dL (0.13–5.09 g/L; via 1:3 dilution), both meeting clinical requirements. Total imprecision coefficients of variation (CVs) were 3.83% (high-concentration: 91.52 mg/dL [0.92 g/L]) and 3.59% (low-concentration: 31.32 mg/dL [0.31 g/L]), within acceptable ranges. The 42 clinical samples showed strong consistency with AU5800 results (R<sup>2</sup> = 0.991). In the evaluation of the smartphone-based CSF total protein detection system, although the 95% confidence intervals of expected biases at 45 mg/dL (0.45 g/L) and 60 mg/dL (0.60 g/L) exceed 1/4 total allowable error per CLSI EP9-A2, the deviations are clinically irrelevant—with the maximum bias (5.29 mg/dL [0.0529 g/L]) fails to alter the pathological threshold classification of CSF total protein (≥45 mg/dL [0.45 g/L]), thus confirming the acceptable bias of the system for auxiliary CSF protein detection.</div></div><div><h3>Conclusions</h3><div>The system meets clinical standards, is user-friendly and cost-effective, and has high potential for aiding CNS disorder diagnosis in resource-constrained areas.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"138 ","pages":"Article 108412"},"PeriodicalIF":1.8,"publicationDate":"2026-01-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145994665","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-14DOI: 10.1016/j.vascn.2026.108411
Kate Murphy , Theo Tasoulis , Geoffrey K. Isbister
Introduction
Anticoagulant activity is an important clinical effect of human envenoming, seen in Australian black snake (Pseudechis) and cobra (Naja) envenoming. Anticoagulant toxins are not well characterised and anticoagulant assays not standardised. We aimed to develop and validate an accurate and standardised anticoagulant assay for snake venoms.
Methods
We used a turbidimetric assay previously developed to measure procoagulant activity and modified for anticoagulant activity. Recalcified fresh frozen plasma was mixed with varying concentrations of venom in a buffer for 5 s in a microplate well, and optical density was recorded for 340 nm every 10 s in a plate reader. Clotting time was defined as the time until a sharp increase occurred in optical density, which was normalised by dividing the venom clotting time by the plasma control clotting time (CT/CTPl). We investigated the effect of changing calcium concentration, the presence of a recombinant tissue factor (Innovin®), venom reconstitution, three different buffers, microplate type, and experimental timing.
Results
Optimal conditions were a calcium concentration of 0.4 M, physiological buffer solution (PBS) and a regular microplate. Freshly reconstituted venom and immediately thawed plasma modestly improved the assay. Assay sensitivity improved without a recombinant tissue factor (Innovin®) trigger, particularly for weakly anticoagulant venoms. To reduce assay variability the assays were best done by selecting 4–6 venoms, and doing these on 4–6 days, with each venom at a different time each day; median coefficient of variation for all venoms <10%.
Discussion
The anticoagulant assay had significantly reduced variability and improved sensitivity when conditions were standardised and a triggering agent was not used.
{"title":"An accurate and reproducible method to measure snake venom anticoagulant activity","authors":"Kate Murphy , Theo Tasoulis , Geoffrey K. Isbister","doi":"10.1016/j.vascn.2026.108411","DOIUrl":"10.1016/j.vascn.2026.108411","url":null,"abstract":"<div><h3>Introduction</h3><div>Anticoagulant activity is an important clinical effect of human envenoming, seen in Australian black snake (<em>Pseudechis</em>) and cobra (<em>Naja</em>) envenoming. Anticoagulant toxins are not well characterised and anticoagulant assays not standardised. We aimed to develop and validate an accurate and standardised anticoagulant assay for snake venoms.</div></div><div><h3>Methods</h3><div>We used a turbidimetric assay previously developed to measure procoagulant activity and modified for anticoagulant activity. Recalcified fresh frozen plasma was mixed with varying concentrations of venom in a buffer for 5 s in a microplate well, and optical density was recorded for 340 nm every 10 s in a plate reader. Clotting time was defined as the time until a sharp increase occurred in optical density, which was normalised by dividing the venom clotting time by the plasma control clotting time (CT/CT<sub>Pl</sub>). We investigated the effect of changing calcium concentration, the presence of a recombinant tissue factor (Innovin®), venom reconstitution, three different buffers, microplate type, and experimental timing.</div></div><div><h3>Results</h3><div>Optimal conditions were a calcium concentration of 0.4 M, physiological buffer solution (PBS) and a regular microplate. Freshly reconstituted venom and immediately thawed plasma modestly improved the assay. Assay sensitivity improved without a recombinant tissue factor (Innovin®) trigger, particularly for weakly anticoagulant venoms. To reduce assay variability the assays were best done by selecting 4–6 venoms, and doing these on 4–6 days, with each venom at a different time each day; median coefficient of variation for all venoms <10%.</div></div><div><h3>Discussion</h3><div>The anticoagulant assay had significantly reduced variability and improved sensitivity when conditions were standardised and a triggering agent was not used.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"138 ","pages":"Article 108411"},"PeriodicalIF":1.8,"publicationDate":"2026-01-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145992470","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-09DOI: 10.1016/j.vascn.2026.108410
Murillo D.L. Bernardi , Krista den Ouden , Melanie Nieuwenhuijzen-Van de Kaa , Vivian V.T. Nguyen , Frans Schutgens , Marianne C. Verhaar , Maarten B. Rookmaaker , Bas W.M. van Balkom
Nephrotoxicity remains a critical concern in drug development, with limited preclinical models that balance physiological relevance, ethical considerations, and scalability. Here, we evaluate the chicken embryo (CE) as an in vivo platform for nephrotoxicity assessment, using gentamicin-induced acute kidney injury as a proof-of-principle. Using an ex ovo culture system, we assessed renal responses in the developing CE kidney. By embryonic day 12, the CE exhibited advanced organogenesis, including a functional metanephric kidney. Immunohistochemical analyses demonstrated conservation of key structural and vascular markers between chicken and human kidneys, including α-smooth muscle actin, von Willebrand factor, and integrins. Gentamicin exposure induced dose- and time-dependent tubular injury, with significant dilation and vacuolization observed at 48 h following exposure to 0.4 mg/mL, which subsided by 96 h. As a proof-of-principle study, these findings demonstrate that the CE can detect acute tubular injury in a developing kidney. The model's primary relevance lies in developmental nephrotoxicity research, where accessible and physiologically integrated experimental systems remain limited.
{"title":"The chicken embryo model as a tool for investigating drug-induced acute kidney injury","authors":"Murillo D.L. Bernardi , Krista den Ouden , Melanie Nieuwenhuijzen-Van de Kaa , Vivian V.T. Nguyen , Frans Schutgens , Marianne C. Verhaar , Maarten B. Rookmaaker , Bas W.M. van Balkom","doi":"10.1016/j.vascn.2026.108410","DOIUrl":"10.1016/j.vascn.2026.108410","url":null,"abstract":"<div><div>Nephrotoxicity remains a critical concern in drug development, with limited preclinical models that balance physiological relevance, ethical considerations, and scalability. Here, we evaluate the chicken embryo (CE) as an in vivo platform for nephrotoxicity assessment, using gentamicin-induced acute kidney injury as a proof-of-principle. Using an ex ovo culture system, we assessed renal responses in the developing CE kidney. By embryonic day 12, the CE exhibited advanced organogenesis, including a functional metanephric kidney. Immunohistochemical analyses demonstrated conservation of key structural and vascular markers between chicken and human kidneys, including α-smooth muscle actin, von Willebrand factor, and integrins. Gentamicin exposure induced dose- and time-dependent tubular injury, with significant dilation and vacuolization observed at 48 h following exposure to 0.4 mg/mL, which subsided by 96 h. As a proof-of-principle study, these findings demonstrate that the CE can detect acute tubular injury in a developing kidney. The model's primary relevance lies in developmental nephrotoxicity research, where accessible and physiologically integrated experimental systems remain limited.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"137 ","pages":"Article 108410"},"PeriodicalIF":1.8,"publicationDate":"2026-01-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145954545","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2026-01-05DOI: 10.1016/j.vascn.2026.108409
Md Harunur Rashid , Conor O'Croinin , Tyson S. Le , Zeinab Abdi , Raimar Loebenberg , Neal M. Davies
Purpose
In this investigation, a highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was designed both to develop and to detect kratom alkaloids that are naturally occurring organic compounds present in the foliage and flowers of the Mitragyna speciosa tree.
Method
We utilize a deuterated analog of mitragynine (mitragynine-D3) as an internal standard (IS) that is spiked into and extracted from kratom extracts using liquid-liquid extraction with ethanol. Water and acetonitrile with 0.1 % formic acid constituted the mobile phase operated at a flow-rate of 0.75 mL/min through a Luna 5 μm C18(2) 100 Å, LC Column 150 × 4.6 mm analytical column and kept at 40 °C. The gradient program started at 0 min with 20 % B, then at 2 min increased to 43.7 % at 12.50 min. We used a period of 12.50 to 14.50 min, increased to 90 % B and held for 19 min, used for washing. Then, an equilibration period of the mobile phase B was maintained at 20 % for 25 min. The LC-MS/MS detection was carried out by electrospray positive ionization in the multiple-reaction monitoring (MRM) mode.
Result
We established an LC-MS/MS method to simultaneously quantify multiple kratom alkaloids. The optimized MRM transitions are mitragynine, speciogynine, speciociliatine, and mitracillatine (m/z 399.20 → 174.00); corynantheidine (369.20 → 144.05); paynantheine (397.20 → 174.00); 7-OH mitragynine (415.20 → 190.00); ajmalicine (353.00 → 144.05); mitraphylline (369.00 → 159.95); and the IS (402.20 → 177.10). Retention times ranged from 6.15 to 11.98 min. The assay (total run time 25 min) had excellent linearity over the concentration range of 5–100 ng/mL (R2 > 0.99), with a Lower Limit of Quantification of 5 ng/mL and Limit of Detection 1.5 ng/mL with a two μL injection. Analytes were well-resolved with the method, which exhibited acceptable intra- and inter-day accuracy and precision, along with confirmed stability under various conditions. Application of kratom extracts yielded reproducible and reliable quantification of alkaloid content.
Conclusion
The findings provide a standard analytical technique for testing kratom-based drug release, dissolution, comparison of different kratom products, and development of formulation. This method is also applicable for regulatory compliance and for producing validated results in fields like kratom adulteration that are unregulated and non-standardized.
{"title":"Analytical method validation with development for the detection and quantification of kratom alkaloids using LC − MS/MS","authors":"Md Harunur Rashid , Conor O'Croinin , Tyson S. Le , Zeinab Abdi , Raimar Loebenberg , Neal M. Davies","doi":"10.1016/j.vascn.2026.108409","DOIUrl":"10.1016/j.vascn.2026.108409","url":null,"abstract":"<div><h3>Purpose</h3><div>In this investigation, a highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was designed both to develop and to detect kratom alkaloids that are naturally occurring organic compounds present in the foliage and flowers of the <em>Mitragyna speciosa</em> tree.</div></div><div><h3>Method</h3><div>We utilize a deuterated analog of mitragynine (mitragynine-D3) as an internal standard (IS) that is spiked into and extracted from kratom extracts using liquid-liquid extraction with ethanol. Water and acetonitrile with 0.1 % formic acid constituted the mobile phase operated at a flow-rate of 0.75 mL/min through a Luna 5 μm C18(2) 100 Å, LC Column 150 × 4.6 mm analytical column and kept at 40 °C. The gradient program started at 0 min with 20 % B, then at 2 min increased to 43.7 % at 12.50 min. We used a period of 12.50 to 14.50 min, increased to 90 % B and held for 19 min, used for washing. Then, an equilibration period of the mobile phase B was maintained at 20 % for 25 min. The LC-MS/MS detection was carried out by electrospray positive ionization in the multiple-reaction monitoring (MRM) mode.</div></div><div><h3>Result</h3><div>We established an LC-MS/MS method to simultaneously quantify multiple kratom alkaloids. The optimized MRM transitions are mitragynine, speciogynine, speciociliatine, and mitracillatine (<em>m</em>/<em>z</em> 399.20 → 174.00); corynantheidine (369.20 → 144.05); paynantheine (397.20 → 174.00); 7-OH mitragynine (415.20 → 190.00); ajmalicine (353.00 → 144.05); mitraphylline (369.00 → 159.95); and the IS (402.20 → 177.10). Retention times ranged from 6.15 to 11.98 min. The assay (total run time 25 min) had excellent linearity over the concentration range of 5–100 ng/mL (R<sup>2</sup> > 0.99), with a Lower Limit of Quantification of 5 ng/mL and Limit of Detection 1.5 ng/mL with a two μL injection. Analytes were well-resolved with the method, which exhibited acceptable intra- and inter-day accuracy and precision, along with confirmed stability under various conditions. Application of kratom extracts yielded reproducible and reliable quantification of alkaloid content.</div></div><div><h3>Conclusion</h3><div>The findings provide a standard analytical technique for testing kratom-based drug release, dissolution, comparison of different kratom products, and development of formulation. This method is also applicable for regulatory compliance and for producing validated results in fields like kratom adulteration that are unregulated and non-standardized.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"137 ","pages":"Article 108409"},"PeriodicalIF":1.8,"publicationDate":"2026-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145919448","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Oxidative stress is a key driver of heart disease, but existing single-cell models lack the complexity of cardiac tissue. Developing advanced in vitro systems is therefore critical. This study aimed to develop a zebrafish heart oxidative stress model that can more closely mimic the in vivo effects and exploring its potential applications in in vitro research. Zebrafish hearts were isolated and cultured in optimized L15 medium. Oxidative stress was induced using hydrogen peroxide (H₂O₂), with reactive oxygen species (ROS) production monitored via 2′,7’-Dichlorodihydrofluorescein diacetate (DCFH) staining. Apoptosis was assessed by TUNEL assay, and sarcomere integrity was evaluated using F-actin staining. The protective effects of vitamin C were also examined. The optimized culture medium maintained heart viability and structural integrity. H₂O₂ treatment induced dose- and time-dependent increases in ROS levels, apoptosis, and sarcomere disorganization, closely resembling in vivo oxidative injury. Notably, vitamin C significantly mitigated these effects, demonstrating the model's utility for drug screening. This study has for the first time established an oxidative stress model using ex vivo zebrafish hearts, offering a new research platform to understand in vivo oxidative stress mechanisms and develop relevant therapies.
{"title":"An ex vivo zebrafish heart model for oxidative stress studies","authors":"Yong Wu , Huiqi Zhang , Fengnian Sun , Wantong Zhang , Ziyun Jiang","doi":"10.1016/j.vascn.2026.108408","DOIUrl":"10.1016/j.vascn.2026.108408","url":null,"abstract":"<div><div>Oxidative stress is a key driver of heart disease, but existing single-cell models lack the complexity of cardiac tissue. Developing advanced <em>in vitro</em> systems is therefore critical. This study aimed to develop a zebrafish heart oxidative stress model that can more closely mimic the <em>in vivo</em> effects and exploring its potential applications in <em>in vitro</em> research. Zebrafish hearts were isolated and cultured in optimized L15 medium. Oxidative stress was induced using hydrogen peroxide (H₂O₂), with reactive oxygen species (ROS) production monitored <em>via</em> 2′,7’-Dichlorodihydrofluorescein diacetate (DCFH) staining. Apoptosis was assessed by TUNEL assay, and sarcomere integrity was evaluated using F-actin staining. The protective effects of vitamin C were also examined. The optimized culture medium maintained heart viability and structural integrity. H₂O₂ treatment induced dose- and time-dependent increases in ROS levels, apoptosis, and sarcomere disorganization, closely resembling <em>in vivo</em> oxidative injury. Notably, vitamin C significantly mitigated these effects, demonstrating the model's utility for drug screening. This study has for the first time established an oxidative stress model using <em>ex vivo</em> zebrafish hearts, offering a new research platform to understand <em>in vivo</em> oxidative stress mechanisms and develop relevant therapies.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"137 ","pages":"Article 108408"},"PeriodicalIF":1.8,"publicationDate":"2026-01-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145919460","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-16DOI: 10.1016/j.vascn.2025.108404
Amel Hamza , David Wishart , Michael R. Doschak
<div><h3>Background</h3><div>Osteoporosis is a progressive bone disease and a significant global health issue, which places a serious economic and health burden on families and societies. Nitrogenated bisphosphonate drugs remain the first line therapy for treating osteoporosis, however their impact on bone cell metabolism and bone health after long term usage is difficult to determine for individual patients. As such, this study aimed to identify the metabolites in plasma that may serve as a diagnostic mechanism for measuring the extent of bisphosphonate drug suppression of bone loss in patients following long-term bisphosphonate drug therapy, and after active vitamin D stimulation of bisphosphonate-suppressed bone metabolism. Our approach was to evaluate combinations of alendronate bisphosphonate and active vitamin D treatment on those same plasma metabolites in an established rat model of osteoporosis, secondary to surgical ovariectomy.</div></div><div><h3>Methods</h3><div>Metabolomic analyses were performed using the commercially available Biocrates p180 metabolomics kit, which was run on a Sciex Qtrap 4000 mass spectrometer equipped with an Agilent HPLC system. Thirty 6-month old ovariectomized (OVX) rats were randomly assigned into three experimental groups, namely, control OVX rats, OVX rats dosed with alendronate, and a final group of OVX rats dosed with a combination of alendronate and active vitamin D. We used in vivo micro-Computed Tomography (μCT) imaging to confirm the developing osteoporosis phenotype in all ovariectomized rats, initially at baseline, and once more at the study endpoint of 8 weeks. Plasma from all rats was also collected at baseline and at 8 weeks and subjected to metabolomics analysis to identify potential osteoporosis biomarkers. We also determined the correlation between bone volume and specific plasma metabolites in an effort to create an osteoporosis screening tool of bisphosphonate drug metabolic suppression for potential use in clinical practice.</div></div><div><h3>Results</h3><div>Our analyses indicated that alendronate regulated several key plasma metabolites, including certain amino acids, lipids, and glucose, which are likely involved in bone resorption and formation. A distinct metabolite “fingerprint” was observed in all treatment groups compared to the control, with notable differences in metabolic changes. There was a correlation between four metabolites (proline, trans-hydroxyproline, histamine, and methionine) and micro-CT measured percent bone volume, indicating significant changes following ovariectomy surgery and with drug treatments.</div></div><div><h3>Conclusions</h3><div>For our study, metabolomic profiling served as a useful research tool for elucidating the biological activity and toxicity of bisphosphonate drugs on metabolic bone cell activity. The approach could significantly aid in gauging the impact of long-term bisphosphonate drug usage in osteoporosis patients, for the assessment of o
{"title":"Effects of alendronate and vitamin D on plasma metabolomic profiles in a rat model of osteoporosis","authors":"Amel Hamza , David Wishart , Michael R. Doschak","doi":"10.1016/j.vascn.2025.108404","DOIUrl":"10.1016/j.vascn.2025.108404","url":null,"abstract":"<div><h3>Background</h3><div>Osteoporosis is a progressive bone disease and a significant global health issue, which places a serious economic and health burden on families and societies. Nitrogenated bisphosphonate drugs remain the first line therapy for treating osteoporosis, however their impact on bone cell metabolism and bone health after long term usage is difficult to determine for individual patients. As such, this study aimed to identify the metabolites in plasma that may serve as a diagnostic mechanism for measuring the extent of bisphosphonate drug suppression of bone loss in patients following long-term bisphosphonate drug therapy, and after active vitamin D stimulation of bisphosphonate-suppressed bone metabolism. Our approach was to evaluate combinations of alendronate bisphosphonate and active vitamin D treatment on those same plasma metabolites in an established rat model of osteoporosis, secondary to surgical ovariectomy.</div></div><div><h3>Methods</h3><div>Metabolomic analyses were performed using the commercially available Biocrates p180 metabolomics kit, which was run on a Sciex Qtrap 4000 mass spectrometer equipped with an Agilent HPLC system. Thirty 6-month old ovariectomized (OVX) rats were randomly assigned into three experimental groups, namely, control OVX rats, OVX rats dosed with alendronate, and a final group of OVX rats dosed with a combination of alendronate and active vitamin D. We used in vivo micro-Computed Tomography (μCT) imaging to confirm the developing osteoporosis phenotype in all ovariectomized rats, initially at baseline, and once more at the study endpoint of 8 weeks. Plasma from all rats was also collected at baseline and at 8 weeks and subjected to metabolomics analysis to identify potential osteoporosis biomarkers. We also determined the correlation between bone volume and specific plasma metabolites in an effort to create an osteoporosis screening tool of bisphosphonate drug metabolic suppression for potential use in clinical practice.</div></div><div><h3>Results</h3><div>Our analyses indicated that alendronate regulated several key plasma metabolites, including certain amino acids, lipids, and glucose, which are likely involved in bone resorption and formation. A distinct metabolite “fingerprint” was observed in all treatment groups compared to the control, with notable differences in metabolic changes. There was a correlation between four metabolites (proline, trans-hydroxyproline, histamine, and methionine) and micro-CT measured percent bone volume, indicating significant changes following ovariectomy surgery and with drug treatments.</div></div><div><h3>Conclusions</h3><div>For our study, metabolomic profiling served as a useful research tool for elucidating the biological activity and toxicity of bisphosphonate drugs on metabolic bone cell activity. The approach could significantly aid in gauging the impact of long-term bisphosphonate drug usage in osteoporosis patients, for the assessment of o","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"137 ","pages":"Article 108404"},"PeriodicalIF":1.8,"publicationDate":"2025-12-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145784208","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-01DOI: 10.1016/j.vascn.2025.108402
Ahmad Tamim Ghafari , Yuslina Zakaria , Mizaton Hazizul Hasan , Abu Bakar Abdul Majeed , Qand Agha Nazari
{"title":"Corrigendum to ‘PolyCheck: A hybrid model for predicting polypharmacy-induced adverse drug reactions in tuberculosis treatment using heterogenous drug-target-ADR networks’ [Journal of pharmacological and toxicological methods 136 (2025) 108393]","authors":"Ahmad Tamim Ghafari , Yuslina Zakaria , Mizaton Hazizul Hasan , Abu Bakar Abdul Majeed , Qand Agha Nazari","doi":"10.1016/j.vascn.2025.108402","DOIUrl":"10.1016/j.vascn.2025.108402","url":null,"abstract":"","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"136 ","pages":"Article 108402"},"PeriodicalIF":1.8,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145277014","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-22DOI: 10.1016/j.vascn.2025.108403
Peter Hoffmann , Michael K. Pugsley
Cardiovascular adverse drug reactions remain a leading cause of drug attrition. They may emerge during non-clinical or clinical development and often remain undetected until post-marketing, prompting increased regulatory focus. Historically, non-clinical cardiovascular safety testing has centered on assessing QT interval prolongation and torsades de pointes risk, primarily via inhibition of the delayed rectifier potassium current IKr. Such focus may overlook broader cardiovascular liabilities affecting other parameters of the cardiovascular system.
This review explores the status and limitations of current non-clinical cardiovascular safety assessments, in particular the over-reliance on the core battery studies defined in ICH S7A and S7B and the insufficient use of mechanistic follow-up assessments. We highlight the gaps between the results of non-clinical cardiovascular safety testing and the emergence of cardiovascular adverse drug reactions in later clinical phases or real-world use. We conclude that to narrow the gaps, there is a need to advance non-clinical methods to detect and adequately measure adverse effects on cardiac rhythm, myocardial contraction, blood pressure, and thrombogenicity. The review further discusses emerging trends and challenges for improving translational relevance, including advanced in vitro and in vivo models, and proposes a re-evaluation of outdated regulatory frameworks to better address diverse cardiovascular risks. Emphasis is placed on functional and mechanistic endpoints over structural pathology, aligning non-clinical safety methodologies with clinical outcomes.
{"title":"Non-clinical cardiovascular safety testing: Current status, gaps and emerging trends","authors":"Peter Hoffmann , Michael K. Pugsley","doi":"10.1016/j.vascn.2025.108403","DOIUrl":"10.1016/j.vascn.2025.108403","url":null,"abstract":"<div><div>Cardiovascular adverse drug reactions remain a leading cause of drug attrition. They may emerge during non-clinical or clinical development and often remain undetected until post-marketing, prompting increased regulatory focus. Historically, non-clinical cardiovascular safety testing has centered on assessing QT interval prolongation and torsades de pointes risk, primarily <em>via</em> inhibition of the delayed rectifier potassium current IKr. Such focus may overlook broader cardiovascular liabilities affecting other parameters of the cardiovascular system.</div><div>This review explores the status and limitations of current non-clinical cardiovascular safety assessments, in particular the over-reliance on the core battery studies defined in ICH S7A and S7B and the insufficient use of mechanistic follow-up assessments. We highlight the gaps between the results of non-clinical cardiovascular safety testing and the emergence of cardiovascular adverse drug reactions in later clinical phases or real-world use. We conclude that to narrow the gaps, there is a need to advance non-clinical methods to detect and adequately measure adverse effects on cardiac rhythm, myocardial contraction, blood pressure, and thrombogenicity. The review further discusses emerging trends and challenges for improving translational relevance, including advanced <em>in vitro</em> and <em>in vivo</em> models, and proposes a re-evaluation of outdated regulatory frameworks to better address diverse cardiovascular risks. Emphasis is placed on functional and mechanistic endpoints over structural pathology, aligning non-clinical safety methodologies with clinical outcomes.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"136 ","pages":"Article 108403"},"PeriodicalIF":1.8,"publicationDate":"2025-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145363426","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-10-06DOI: 10.1016/j.vascn.2025.108401
Samantha J. Carew , Christiana M. Kennedy , Meghan L. Greenland, Matthew P. Parsons
Excitotoxicity is a key driver of neuronal death in diverse brain conditions, yet most toxicity assays rely on in vitro models that remove cells from their complex native environment within the brain parenchyma. Here, we present a novel ex vivo method to quantify N-methyl-d-aspartate (NMDA)-induced excitotoxicity using acute brain slices from male and female young adult c57bl/6 and aged B6129SF2J mice, similar to those used for conventional electrophysiological recordings. Acute hippocampal slices were recovered in an N-methyl-D-glucamine (NMDG)-based recovery solution, then treated with low-magnesium artificial cerebrospinal fluid (aCSF) containing the co-agonist glycine to promote receptor activation, with or without exogenous NMDA. Following treatment, slices were fixed, cryoprotected, and cryosectioned to 20 μm for immunohistochemistry. Apoptotic cell death was assessed by staining for cleaved caspase-3, and was combined with the percentage of dead space to calculate a toxicity index for overall excitotoxic cell death. Importantly, exposure to low-magnesium aCSF with glycine alone was sufficient to elevate active caspase-3 levels, an effect that was further enhanced by exogenous NMDA application and prevented by NMDAR antagonism. Our ex vivo method largely preserves the cytoarchitecture and local microenvironment of brain tissue, enabling the assessment of cell-specific vulnerabilities to excitotoxic damage in select brain regions at defined ages. It is particularly well-suited for use in neurodegenerative disease models, where excitotoxic susceptibility may evolve over time. In all, the approach described here provides a reliable and accessible alternative to dissociated cell cultures, bridging the gap between in vitro and in vivo systems for studying glutamate-induced cell death.
兴奋性毒性是多种脑条件下神经元死亡的关键驱动因素，然而大多数毒性试验依赖于体外模型，将细胞从脑实质内复杂的天然环境中移除。在这里，我们提出了一种新的离体方法来量化n -甲基-d-天冬氨酸（NMDA）诱导的兴奋毒性，使用雄性和雌性年轻成年c57bl/6和老年B6129SF2J小鼠的急性脑切片，类似于传统的电生理记录。急性海马切片在n -甲基- d -葡萄糖胺（NMDG）为基础的恢复液中恢复，然后用含有协同激动剂甘氨酸的低镁aCSF处理，以促进受体激活，有或没有外源性NMDA。处理后，固定切片，冷冻保护，冷冻切片至20 μm进行免疫组织化学。通过裂解caspase-3染色评估凋亡细胞死亡，并结合死亡空间百分比计算总体兴奋性毒性细胞死亡的毒性指数。重要的是，仅用甘氨酸暴露于低镁aCSF足以提高活性caspase-3水平，外源NMDA应用进一步增强了这一作用，并被NMDAR拮抗阻止。我们的离体方法在很大程度上保留了脑组织的细胞结构和局部微环境，从而能够评估特定年龄的特定大脑区域对兴奋毒性损伤的细胞特异性脆弱性。它特别适合用于神经退行性疾病模型，其中兴奋毒性易感性可能随着时间的推移而演变。总之，本文所描述的方法提供了一种可靠且可接近的解离细胞培养替代方法，弥合了体外和体内系统之间的差距，用于研究谷氨酸诱导的细胞死亡。
{"title":"A novel, flexible, and accessible method for the ex vivo induction and quantification of excitotoxicity","authors":"Samantha J. Carew , Christiana M. Kennedy , Meghan L. Greenland, Matthew P. Parsons","doi":"10.1016/j.vascn.2025.108401","DOIUrl":"10.1016/j.vascn.2025.108401","url":null,"abstract":"<div><div>Excitotoxicity is a key driver of neuronal death in diverse brain conditions, yet most toxicity assays rely on <em>in vitro</em> models that remove cells from their complex native environment within the brain parenchyma. Here, we present a novel <em>ex vivo</em> method to quantify <em>N</em>-methyl-<span>d</span>-aspartate (NMDA)-induced excitotoxicity using acute brain slices from male and female young adult c57bl/6 and aged B6129SF2J mice, similar to those used for conventional electrophysiological recordings. Acute hippocampal slices were recovered in an <em>N</em>-methyl-D-glucamine (NMDG)-based recovery solution, then treated with low-magnesium artificial cerebrospinal fluid (aCSF) containing the co-agonist glycine to promote receptor activation, with or without exogenous NMDA. Following treatment, slices were fixed, cryoprotected, and cryosectioned to 20 μm for immunohistochemistry. Apoptotic cell death was assessed by staining for cleaved caspase-3, and was combined with the percentage of dead space to calculate a toxicity index for overall excitotoxic cell death. Importantly, exposure to low-magnesium aCSF with glycine alone was sufficient to elevate active caspase-3 levels, an effect that was further enhanced by exogenous NMDA application and prevented by NMDAR antagonism. Our <em>ex vivo</em> method largely preserves the cytoarchitecture and local microenvironment of brain tissue, enabling the assessment of cell-specific vulnerabilities to excitotoxic damage in select brain regions at defined ages. It is particularly well-suited for use in neurodegenerative disease models, where excitotoxic susceptibility may evolve over time. In all, the approach described here provides a reliable and accessible alternative to dissociated cell cultures, bridging the gap between <em>in vitro</em> and <em>in vivo</em> systems for studying glutamate-induced cell death.</div></div>","PeriodicalId":16767,"journal":{"name":"Journal of pharmacological and toxicological methods","volume":"136 ","pages":"Article 108401"},"PeriodicalIF":1.8,"publicationDate":"2025-10-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145254150","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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