45 results on '"Inayat-Hussain SH"'
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2. Considerations for occupational risk management during pregnancy: A summary of a continuing education course.
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Roberts, LG, Hoberman, AM, Verpaele, S, Inayat-Hussain, SH, DeSesso, JM, and Fukumura, M
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THRESHOLD limit values (Industrial toxicology) , *CONSCIOUSNESS raising , *REPRODUCTIVE toxicology , *MEDICAL personnel , *RISK exposure - Abstract
Women comprise approximately 40% of the global workforce, and many women continue to work during pregnancy. Although occupational exposure limit values (OELVs) are intended to protect all workers, many OELVs may have been established without consideration of the unique changes in pregnant workers, and many chemicals lack OELVs altogether. A short educational course was developed to address the informational needs of health professionals who have responsibility to ensure a safe workplace for pregnant employees. The course was designed to raise awareness of the key elements in risk management and their application to the pregnant worker, such as physiological changes of pregnancy that influence susceptibility to exposures; guidance for nonclinical data interpretation; exposure assessment and control strategies; and risk management in practice in a diverse regulatory environment. This paper summarizes the course content and is intended to support informed risk management decision making to protect the health of pregnant workers and their offspring. [ABSTRACT FROM AUTHOR]
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- 2024
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3. Mapping the influence of hydrocarbons mixture on molecular mechanisms, involved in breast and lung neoplasms: in silico toxicogenomic data-mining.
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Abu-Bakar A, Ismail M, Zulkifli MZI, Zaini NAS, Shukor NIA, Harun S, and Inayat-Hussain SH
- Abstract
Background: Exposure to chemical mixtures inherent in air pollution, has been shown to be associated with the risk of breast and lung cancers. However, studies on the molecular mechanisms of exposure to a mixture of these pollutants, such as hydrocarbons, in the development of breast and lung cancers are scarce. We utilized in silico toxicogenomic analysis to elucidate the molecular pathways linked to both cancers that are influenced by exposure to a mixture of selected hydrocarbons. The Comparative Toxicogenomics Database and Cytoscape software were used for data mining and visualization., Results: Twenty-five hydrocarbons, common in air pollution with carcinogenicity classification of 1 A/B or 2 (known/presumed or suspected human carcinogen), were divided into three groups: alkanes and alkenes, halogenated hydrocarbons, and polyaromatic hydrocarbons. The in silico data-mining revealed 87 and 44 genes commonly interacted with most of the investigated hydrocarbons are linked to breast and lung cancer, respectively. The dominant interactions among the common genes are co-expression, physical interaction, genetic interaction, co-localization, and interaction in shared protein domains. Among these genes, only 16 are common in the development of both cancers. Benzo(a)pyrene and tetrachlorodibenzodioxin interacted with all 16 genes. The molecular pathways potentially affected by the investigated hydrocarbons include aryl hydrocarbon receptor, chemical carcinogenesis, ferroptosis, fluid shear stress and atherosclerosis, interleukin 17 signaling pathway, lipid and atherosclerosis, NRF2 pathway, and oxidative stress response., Conclusions: Within the inherent limitations of in silico toxicogenomics tools, we elucidated the molecular pathways associated with breast and lung cancer development potentially affected by hydrocarbons mixture. Our findings indicate adaptive responses to oxidative stress and inflammatory damages are instrumental in the development of both cancers. Additionally, ferroptosis-a non-apoptotic programmed cell death driven by lipid peroxidation and iron homeostasis-was identified as a new player in these responses. Finally, AHR potential involvement in modulating IL-8, a critical gene that mediates breast cancer invasion and metastasis to the lungs, was also highlighted. A deeper understanding of the interplay between genes associated with these pathways, and other survival signaling pathways identified in this study, will provide invaluable knowledge in assessing the risk of inhalation exposure to hydrocarbons mixture. The findings offer insights into future in vivo and in vitro laboratory investigations that focus on inhalation exposure to the hydrocarbons mixture., (© 2024. The Author(s).)
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- 2024
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4. The Malaysian Society of Toxicology: from establishment to evolution, a promising future!
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Kamaludin NF, Kamarulzaman F, Abdullah R, Chan KM, and Inayat-Hussain SH
- Abstract
The Malaysian Society of Toxicology (MySOT), founded in 2010, emerged as a response to the growing need for a collective and interdisciplinary effort to study the effects of substances on human health, and the environment. By fostering collaboration between toxicologists, researchers, regulators, industry experts, and various relevant subject matter experts, MySOT has played a vital role in generating knowledge and promoting safety to safeguard both human and environmental well-being. Within the 13 years since its establishment, MySOT has made substantial progress in the advancement of toxicology in Malaysia. Over the years, MySOT has supported many initiatives, including organizing conferences, seminars, and workshops in which experts from various fields present their research, discuss emerging trends, and propose strategies to reduce toxic substance exposure risk. The society has also been actively involved in the broader landscape of toxicology research and policy influence in Malaysia. MySOT shoulders the responsibility of conveying accurate information and educating the public about health risks associated with toxic substances. Therefore, the society aims to collaborate with governmental organizations, professional bodies, and international toxicology organizations to share ideas, resources, and expertise. MySOT seeks to gather toxicological experts in the region and significantly contribute to a safer and healthier community, therefore supporting the United Nations Sustainable Development Goals (SDGs), by being actively involved with all of its stakeholders, both local and international., (© 2023. The Author(s).)
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- 2023
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5. (E)-N-(2-(3, 5-Dimethoxystyryl) phenyl) furan-2-carboxamide (BK3C231) induces cytoprotection in CCD18-Co human colon fibroblast cells through Nrf2/ARE pathway activation.
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Tan HH, Thomas NF, Inayat-Hussain SH, and Chan KM
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- Cell Line, Colon cytology, Colon drug effects, Colon metabolism, Fibroblasts cytology, Fibroblasts metabolism, Humans, Signal Transduction drug effects, Anticarcinogenic Agents pharmacology, Antioxidant Response Elements drug effects, Cytoprotection, Fibroblasts drug effects, Furans pharmacology, NF-E2-Related Factor 2 metabolism
- Abstract
Cytoprotection involving the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway is an important preventive strategy for normal cells against carcinogenesis. In our previous study, the chemopreventive potential of (E)-N-(2-(3, 5-Dimethoxystyryl) phenyl) furan-2-carboxamide (BK3C231) has been elucidated through its cytoprotective effects against DNA and mitochondrial damages in the human colon fibroblast CCD-18Co cell model. Therefore this study aimed to investigate the molecular mechanisms underlying BK3C231-induced cytoprotection and the involvement of the Nrf2/ARE pathway. The cells were pretreated with BK3C231 before exposure to carcinogen 4-nitroquinoline N-oxide (4NQO). BK3C231 increased the protein expression and activity of cytoprotective enzymes namely NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione S-transferase (GST) and heme oxygenase-1 (HO-1), as well as restoring the expression of glutamate-cysteine ligase catalytic subunit (GCLC) back to the basal level. Furthermore, dissociation of Nrf2 from its inhibitory protein, Keap1, and ARE promoter activity were upregulated in cells pretreated with BK3C231. Taken together, our findings suggest that BK3C231 exerts cytoprotection by activating the Nrf2 signaling pathway which leads to ARE-mediated upregulation of cytoprotective proteins. This study provides new mechanistic insights into BK3C231 chemopreventive activities and highlights the importance of stilbene derivatives upon development as a potential chemopreventive agent.
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- 2021
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6. Cytoprotective effects of (E)-N-(2-(3, 5-dimethoxystyryl) phenyl) furan-2-carboxamide (BK3C231) against 4-nitroquinoline 1-oxide-induced damage in CCD-18Co human colon fibroblast cells.
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Tan HH, Thomas NF, Inayat-Hussain SH, and Chan KM
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- Cell Line, Colon cytology, DNA Repair drug effects, Fibroblasts cytology, Fibroblasts drug effects, Furans chemistry, Humans, Mitochondria drug effects, NAD(P)H Dehydrogenase (Quinone) metabolism, Stilbenes chemistry, 4-Nitroquinoline-1-oxide toxicity, Colon drug effects, Cytoprotection, DNA Damage drug effects, Furans pharmacology, Mutagens toxicity, Stilbenes pharmacology
- Abstract
Stilbenes are a group of chemicals characterized with the presence of 1,2-diphenylethylene. Previously, our group has demonstrated that synthesized (E)-N-(2-(3, 5-dimethoxystyryl) phenyl) furan-2-carboxamide (BK3C231) possesses potential chemopreventive activity specifically inducing NAD(P)H:quinone oxidoreductase 1 (NQO1) protein expression and activity. In this study, the cytoprotective effects of BK3C231 on cellular DNA and mitochondria were investigated in normal human colon fibroblast, CCD-18Co cells. The cells were pretreated with BK3C231 prior to exposure to the carcinogen 4-nitroquinoline 1-oxide (4NQO). BK3C231 was able to inhibit 4NQO-induced cytotoxicity. Cells treated with 4NQO alone caused high level of DNA and mitochondrial damages. However, pretreatment with BK3C231 protected against these damages by reducing DNA strand breaks and micronucleus formation as well as decreasing losses of mitochondrial membrane potential (ΔΨm) and cardiolipin. Interestingly, our study has demonstrated that nitrosative stress instead of oxidative stress was involved in 4NQO-induced DNA and mitochondrial damages. Inhibition of 4NQO-induced nitrosative stress by BK3C231 was observed through a decrease in nitric oxide (NO) level and an increase in glutathione (GSH) level. These new findings elucidate the cytoprotective potential of BK3C231 in human colon fibroblast CCD-18Co cell model which warrants further investigation into its chemopreventive role., Competing Interests: The authors have declared that no competing interests exist.
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- 2020
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7. Determining the endocrine disruption potential of industrial chemicals using an integrative approach: Public databases, in vitro exposure, and modeling receptor interactions.
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Alofe O, Kisanga E, Inayat-Hussain SH, Fukumura M, Garcia-Milian R, Perera L, Vasiliou V, and Whirledge S
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- Animals, Endocrine Disruptors chemistry, Environmental Pollutants chemistry, Estrogen Receptor alpha chemistry, Estrogen Receptor alpha metabolism, Female, Humans, Databases, Factual, Endocrine Disruptors pharmacology, Environmental Pollutants pharmacology, Molecular Docking Simulation
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Environmental and occupational exposure to industrial chemicals has been linked to toxic and carcinogenic effects in animal models and human studies. However, current toxicology testing does not thoroughly explore the endocrine disrupting effects of industrial chemicals, which may have low dose effects not predicted when determining the limit of toxicity. The objective of this study was to evaluate the endocrine disrupting potential of a broad range of chemicals used in the petrochemical sector. Therefore, 139 chemicals were classified for reproductive toxicity based on the United Nations Globally Harmonized System for hazard classification. These chemicals were evaluated in PubMed for reported endocrine disrupting activity, and their endocrine disrupting potential was estimated by identifying chemicals with active nuclear receptor endpoints publicly available databases. Evaluation of ToxCast data suggested that these chemicals preferentially alter the activity of the estrogen receptor (ER). Four chemicals were prioritized for in vitro testing using the ER-positive, immortalized human uterine Ishikawa cell line and a range of concentrations below the reported limit of toxicity in humans. We found that 2,6-di-tert-butyl-p-cresol (BHT) and diethanolamine (DEA) repressed the basal expression of estrogen-responsive genes PGR, NPPC, and GREB1 in Ishikawa cells, while tetrachloroethylene (PCE) and 2,2'-methyliminodiethanol (MDEA) induced the expression of these genes. Furthermore, low-dose combinations of PCE and MDEA produced additive effects. All four chemicals interfered with estradiol-mediated induction of PGR, NPPC, and GREB1. Molecular docking demonstrated that these chemicals could bind to the ligand binding site of ERα, suggesting the potential for direct stimulatory or inhibitory effects. We found that these chemicals altered rates of proliferation and regulated the expression of cell proliferation associated genes. These findings demonstrate previously unappreciated endocrine disrupting effects and underscore the importance of testing the endocrine disrupting potential of chemicals in the future to better understand their potential to impact public health., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)
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- 2019
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8. Evaluation of potential carcinogenicity of organic chemicals in synthetic turf crumb rubber.
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Perkins AN, Inayat-Hussain SH, Deziel NC, Johnson CH, Ferguson SS, Garcia-Milian R, Thompson DC, and Vasiliou V
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- Elastomers, Europe, Humans, Organic Chemicals, United States, Carcinogens, Environmental Exposure, Rubber
- Abstract
Currently, there are >11,000 synthetic turf athletic fields in the United States and >13,000 in Europe. Concerns have been raised about exposure to carcinogenic chemicals resulting from contact with synthetic turf fields, particularly the infill material ("crumb rubber"), which is commonly fabricated from recycled tires. However, exposure data are scant, and the limited existing exposure studies have focused on a small subset of crumb rubber components. Our objective was to evaluate the carcinogenic potential of a broad range of chemical components of crumb rubber infill using computational toxicology and regulatory agency classifications from the United States Environmental Protection Agency (US EPA) and European Chemicals Agency (ECHA) to inform future exposure studies and risk analyses. Through a literature review, we identified 306 chemical constituents of crumb rubber infill from 20 publications. Utilizing ADMET Predictor™, a computational program to predict carcinogenicity and genotoxicity, 197 of the identified 306 chemicals met our a priori carcinogenicity criteria. Of these, 52 chemicals were also classified as known, presumed or suspected carcinogens by the US EPA and ECHA. Of the remaining 109 chemicals which were not predicted to be carcinogenic by our computational toxicology analysis, only 6 chemicals were classified as presumed or suspected human carcinogens by US EPA or ECHA. Importantly, the majority of crumb rubber constituents were not listed in the US EPA (n = 207) and ECHA (n = 262) databases, likely due to an absence of evaluation or insufficient information for a reliable carcinogenicity classification. By employing a cancer hazard scoring system to the chemicals which were predicted and classified by the computational analysis and government databases, several high priority carcinogens were identified, including benzene, benzidine, benzo(a)pyrene, trichloroethylene and vinyl chloride. Our findings demonstrate that computational toxicology assessment in conjunction with government classifications can be used to prioritize hazardous chemicals for future exposure monitoring studies for users of synthetic turf fields. This approach could be extended to other compounds or toxicity endpoints., (Copyright © 2018 Elsevier Inc. All rights reserved.)
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- 2019
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9. Prioritization of reproductive toxicants in unconventional oil and gas operations using a multi-country regulatory data-driven hazard assessment.
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Inayat-Hussain SH, Fukumura M, Muiz Aziz A, Jin CM, Jin LW, Garcia-Milian R, Vasiliou V, and Deziel NC
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- Humans, Oil and Gas Industry, Environmental Exposure analysis, Gasoline analysis, Hazardous Substances analysis, Mutagens analysis, Risk Assessment methods
- Abstract
Background: Recent trends have witnessed the global growth of unconventional oil and gas (UOG) production. Epidemiologic studies have suggested associations between proximity to UOG operations with increased adverse birth outcomes and cancer, though specific potential etiologic agents have not yet been identified. To perform effective risk assessment of chemicals used in UOG production, the first step of hazard identification followed by prioritization specifically for reproductive toxicity, carcinogenicity and mutagenicity is crucial in an evidence-based risk assessment approach. To date, there is no single hazard classification list based on the United Nations Globally Harmonized System (GHS), with countries applying the GHS standards to generate their own chemical hazard classification lists. A current challenge for chemical prioritization, particularly for a multi-national industry, is inconsistent hazard classification which may result in misjudgment of the potential public health risks. We present a novel approach for hazard identification followed by prioritization of reproductive toxicants found in UOG operations using publicly available regulatory databases., Methods: GHS classification for reproductive toxicity of 157 UOG-related chemicals identified as potential reproductive or developmental toxicants in a previous publication was assessed using eleven governmental regulatory agency databases. If there was discordance in classifications across agencies, the most stringent classification was assigned. Chemicals in the category of known or presumed human reproductive toxicants were further evaluated for carcinogenicity and germ cell mutagenicity based on government classifications. A scoring system was utilized to assign numerical values for reproductive health, cancer and germ cell mutation hazard endpoints. Using a Cytoscape analysis, both qualitative and quantitative results were presented visually to readily identify high priority UOG chemicals with evidence of multiple adverse effects., Results: We observed substantial inconsistencies in classification among the 11 databases. By adopting the most stringent classification within and across countries, 43 chemicals were classified as known or presumed human reproductive toxicants (GHS Category 1), while 31 chemicals were classified as suspected human reproductive toxicants (GHS Category 2). The 43 reproductive toxicants were further subjected to analysis for carcinogenic and mutagenic properties. Calculated hazard scores and Cytoscape visualization yielded several high priority chemicals including potassium dichromate, cadmium, benzene and ethylene oxide., Conclusions: Our findings reveal diverging GHS classification outcomes for UOG chemicals across regulatory agencies. Adoption of the most stringent classification with application of hazard scores provides a useful approach to prioritize reproductive toxicants in UOG and other industries for exposure assessments and selection of safer alternatives., (Copyright © 2018 Elsevier Ltd. All rights reserved.)
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- 2018
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10. Lineage-related cytotoxicity and clonogenic profile of 1,4-benzoquinone-exposed hematopoietic stem and progenitor cells.
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Chow PW, Abdul Hamid Z, Chan KM, Inayat-Hussain SH, and Rajab NF
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- Animals, Antigens, Ly metabolism, Apoptosis drug effects, Biomarkers metabolism, CD11b Antigen metabolism, Cells, Cultured, Colony-Forming Units Assay, Dose-Response Relationship, Drug, Flow Cytometry, Hematopoietic Stem Cells metabolism, Hematopoietic Stem Cells pathology, Immunophenotyping, Leukocyte Common Antigens metabolism, Male, Membrane Proteins metabolism, Mice, Mice, Inbred ICR, Phenotype, Stem Cell Niche, Benzoquinones toxicity, Cell Lineage, Cell Proliferation drug effects, Hematopoietic Stem Cells drug effects
- Abstract
Hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) are sensitive targets for benzene-induced hematotoxicity and leukemogenesis. The impact of benzene exposure on the complex microenvironment of HSCs and HPCs remains elusive. This study aims to investigate the mechanism linking benzene exposure to targeting HSCs and HPCs using phenotypic and clonogenic analyses. Mouse bone marrow (BM) cells were exposed ex vivo to the benzene metabolite, 1,4-benzoquinone (1,4-BQ), for 24h. Expression of cellular surface antigens for HSC (Sca-1), myeloid (Gr-1, CD11b), and lymphoid (CD45, CD3e) populations were confirmed by flow cytometry. The clonogenicity of cells was studied using the colony-forming unit (CFU) assay for multilineage (CFU-GM and CFU-GEMM) and single-lineage (CFU-E, BFU-E, CFU-G, and CFU-M) progenitors. 1,4-BQ demonstrated concentration-dependent cytotoxicity in mouse BM cells. The percentage of apoptotic cells increased (p < 0.05) following 1,4-BQ exposure. Exposure to 1,4-BQ showed no significant effect on CD3e(+) cells but reduced the total counts of Sca-1(+), CD11b(+), Gr-1(+), and CD45(+) cells at 7 and 12 μM (p < 0.05). Furthermore, the CFU assay showed reduced (p < 0.05) clonogenicity in 1,4-BQ-treated cells. 1,4-BQ induced CFU-dependent cytotoxicity by significantly inhibiting colony growth for CFU-E, BFU-E, CFU-G, and CFU-M starting at a low concentration of exposure (5μM); whereas for the CFU-GM and CFU-GEMM, the inhibition of colony growth was remarkable only at 7 and 12μM of 1,4-BQ, respectively. Taken together, 1,4-BQ caused lineage-related cytotoxicity in mouse HPCs, demonstrating greater toxicity in single-lineage progenitors than in those of multi-lineage., (Copyright © 2014 Elsevier Inc. All rights reserved.)
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- 2015
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11. Toxicity of tetramethylammonium hydroxide to aquatic organisms and its synergistic action with potassium iodide.
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Mori IC, Arias-Barreiro CR, Koutsaftis A, Ogo A, Kawano T, Yoshizuka K, Inayat-Hussain SH, and Aoyama I
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- Aliivibrio fischeri drug effects, Animals, Chlorophyta drug effects, Daphnia drug effects, Drug Synergism, Oryzias metabolism, Aquatic Organisms drug effects, Potassium Iodide toxicity, Quaternary Ammonium Compounds toxicity, Water Pollutants, Chemical toxicity
- Abstract
The aquatic ecotoxicity of chemicals involved in the manufacturing process of thin film transistor liquid crystal displays was assessed with a battery of four selected acute toxicity bioassays. We focused on tetramethylammonium hydroxide (TMAH, CAS No. 75-59-2), a widely utilized etchant. The toxicity of TMAH was low when tested in the 72 h-algal growth inhibition test (Pseudokirchneriellia subcapitata, EC50=360 mg L(-1)) and the Microtox® test (Vibrio fischeri, IC50=6.4 g L(-1)). In contrast, the 24h-microcrustacean immobilization and the 96 h-fish mortality tests showed relatively higher toxicity (Daphnia magna, EC50=32 mg L(-1) and Oryzias latipes, LC50=154 mg L(-1)). Isobologram and mixture toxicity index analyses revealed apparent synergism of the mixture of TMAH and potassium iodide when examined with the D. magna immobilization test. The synergistic action was unique to iodide over other halide salts i.e. fluoride, chloride and bromide. Quaternary ammonium ions with longer alkyl chains such as tetraethylammonium and tetrabutylammonium were more toxic than TMAH in the D. magna immobilization test., (Copyright © 2014 Elsevier Ltd. All rights reserved.)
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- 2015
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12. Involvement of Seladin-1 in goniothalamin-induced apoptosis in urinary bladder cancer cells.
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Yen HK, Fauzi AR, Din LB, McKelvey-Martin VJ, Meng CK, Inayat-Hussain SH, and Rajab NF
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- Cell Line, Tumor, Humans, Urinary Bladder Neoplasms drug therapy, Urinary Bladder Neoplasms physiopathology, Antineoplastic Agents pharmacology, Apoptosis drug effects, Pyrones pharmacology, Urinary Bladder Neoplasms metabolism
- Abstract
Background: Selective Alzheimer Disease Indicator-1 (or Seladin-1) is a multifunctional protein first discovered by downregulation of its expression in Alzheimer's disease. Interestingly, the expression of this protein is upregulated in several cancers, including primary bladder cancer. However, its role in cancer formation has yet to be discovered. Goniothalamin is a natural product that has been demonstrated to induce apoptosis in various cancer cell lines. In this study, we have elucidated the role of Seladin-1 in goniothalamin-induced cytotoxicity towards human urinary bladder cancer cell line RT4., Methods: The cytotoxicity of goniothalamin in human urinary bladder cancer cell line RT4 was assessed using MTT assay and the mode of cell death was determined by Annexin V-FITC/PI labeling assay. Finally, the expression of Seladin-1 protein in goniothalamin-treated RT4 cells was determined by Western blot., Results: MTT assay showed that the cytotoxicity of goniothalamin on RT4 cells was concentration and time dependent with IC50 values of 61 μM (24 hr), 38 μM (48 hr) and 31 μM for 72 hr, respectively. Cell death induced was confirmed through apoptosis; as assessed using the Annexin V-FITC/PI labeling assay. Furthermore, the involvement of Seladin-1 in goniothalamin-induced apoptosis was evidenced through the cleavage of 60 kDa protein to 40 kDa and 20 kDa. This was followed by a gradual increase of 20 kDa fragment suggesting the involvement of Seladin-1 in goniothalamin-induced apoptosis on RT4 cells., Conclusion: This study demonstrates that goniothalamin induce cytotoxicity and apoptosis on RT4 cells. The involvement of Seladin-1 in goniothalamin-induced apoptosis further suggested that Seladin-1 may play a role in the formation of primary bladder cancer.
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- 2014
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13. Styryl-lactone goniothalamin inhibits TNF-α-induced NF-κB activation.
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Orlikova B, Schumacher M, Juncker T, Yan CC, Inayat-Hussain SH, Hajjouli S, Cerella C, Dicato M, and Diederich M
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- Anti-Inflammatory Agents, Non-Steroidal adverse effects, Anti-Inflammatory Agents, Non-Steroidal isolation & purification, Antineoplastic Agents, Phytogenic adverse effects, Antineoplastic Agents, Phytogenic isolation & purification, Cell Nucleus drug effects, Cell Nucleus metabolism, Cells, Cultured, Genes, Reporter drug effects, Goniothalamus chemistry, Humans, Interleukin-8 antagonists & inhibitors, Interleukin-8 metabolism, Jurkat Cells, K562 Cells, Leukocytes, Mononuclear cytology, Leukocytes, Mononuclear drug effects, Malaysia, NF-kappa B agonists, NF-kappa B antagonists & inhibitors, Neoplasm Proteins antagonists & inhibitors, Neoplasm Proteins genetics, Neoplasm Proteins metabolism, Plant Roots chemistry, Protein Transport drug effects, Pyrones adverse effects, Pyrones isolation & purification, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Response Elements drug effects, Tumor Necrosis Factor-alpha genetics, Tumor Necrosis Factor-alpha metabolism, Anti-Inflammatory Agents, Non-Steroidal pharmacology, Antineoplastic Agents, Phytogenic pharmacology, Drug Discovery, Leukemia drug therapy, NF-kappa B metabolism, Pyrones pharmacology, Tumor Necrosis Factor-alpha antagonists & inhibitors
- Abstract
(R)-(+)-Goniothalamin (GTN), a styryl-lactone isolated from the medicinal plant Goniothalamus macrophyllus, exhibits pharmacological activities including cytotoxic and anti-inflammatory effects. In this study, GTN modulated TNF-α induced NF-κB activation. GTN concentrations up to 20 μM showed low cytotoxic effects in K562 chronic myelogenous leukemia and in Jurkat T cells. Importantly, at these concentrations, no cytotoxicity was observed in healthy peripheral blood mononuclear cells. Our results confirmed that GTN inhibited tumor necrosis factor-α (TNF-α)-induced NF-κB activation in Jurkat and K562 leukemia cells at concentrations as low as 5 μM as shown by reporter gene assays and western blots. Moreover, GTN down-regulated translocation of the p50/p65 heterodimer to the nucleus, prevented binding of NF-κB to its DNA response element and reduced TNF-α-activated interleukin-8 (IL-8) expression. In conclusion, GTN inhibits TNF-α-induced NF-κB activation at non-apoptogenic concentrations in different leukemia cell models without presenting toxicity towards healthy blood cells underlining the anti-leukemic potential of this natural compound., (Copyright © 2013 Elsevier Ltd. All rights reserved.)
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- 2013
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14. Protection of hydroquinone-induced apoptosis by downregulation of Fau is mediated by NQO1.
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Siew EL, Chan KM, Williams GT, Ross D, and Inayat-Hussain SH
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- Animals, Blotting, Western, Cell Proliferation drug effects, Cells, Cultured, Comet Assay, DNA Damage drug effects, Down-Regulation, Flow Cytometry, Mice, NAD(P)H Dehydrogenase (Quinone) antagonists & inhibitors, NAD(P)H Dehydrogenase (Quinone) genetics, RNA, Small Interfering genetics, Reactive Oxygen Species metabolism, Ribosomal Proteins genetics, Thymoma drug therapy, Thymoma metabolism, Thymus Neoplasms drug therapy, Thymus Neoplasms metabolism, Apoptosis drug effects, Hydroquinones pharmacology, NAD(P)H Dehydrogenase (Quinone) metabolism, Ribosomal Proteins metabolism, Thymoma pathology, Thymus Neoplasms pathology
- Abstract
The Fau gene (Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV)-associated ubiquitously expressed gene) was identified as a potential tumor suppressor gene using a forward genetics approach. Downregulation of Fau by overexpression of its reverse sequence has been shown to inhibit apoptosis induced by DNA-damaging agents. To address a potential role of Fau in benzene toxicity, we investigated the apoptotic effects of hydroquinone (HQ), a major benzene metabolite, in W7.2 mouse thymoma cells transfected with either a plasmid construct expressing the antisense sequence of Fau (rfau) or the empty vector (pcDNA3.1) as a control. HQ induced apoptosis via increased production of reactive oxygen species and DNA damage, measured using dihydroethidine (HE) staining and alkaline Comet assay, respectively, in W7.2 pcDNA3.1 cells. In contrast, when Fau was downregulated by the antisense sequence in W7.2 rfau cells, HQ treatment did not cause DNA damage and oxidative stress and these cells were markedly more resistant to HQ-induced apoptosis. Further investigation revealed that there was an upregulation of NAD(P)H: quinone oxidoreductase 1 (NQO1), a detoxification enzyme for benzene-derived quinones, in W7.2 rfau cells. Compromising cellular NQO1 by use of a specific mechanism-based inhibitor (MAC 220) and NQO1 siRNA resensitized W7.2 rfau cells to HQ-induced apoptosis. Silencing of Fau in W7.2 wild-type cells resulted in increased levels of NQO1, confirming that downregulation of Fau results in NQO1 upregulation which protects against HQ-induced apoptosis., (Copyright © 2012 Elsevier Inc. All rights reserved.)
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- 2012
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15. The pyranoxanthone inophyllin A induces oxidative stress mediated-apoptosis in Jurkat T lymphoblastic leukemia cells.
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Chan KM, Hamzah R, Rahaman AA, Jong VY, Khong HY, Rajab NF, Ee GC, and Inayat-Hussain SH
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- Caspases metabolism, DNA Damage, Enzyme Activation, Flow Cytometry, Humans, Jurkat Cells, Leukemia, T-Cell enzymology, Leukemia, T-Cell pathology, Reactive Oxygen Species metabolism, Apoptosis drug effects, Leukemia, T-Cell metabolism, Oxidative Stress drug effects, Xanthones pharmacology
- Abstract
Inophyllin A (INO-A), a pyranoxanthone isolated from the roots of Calophyllum inophyllum represents a new xanthone with potential chemotherapeutic activity. In this study, the molecular mechanism of INO-A-induced cell death was investigated in Jurkat T lymphoblastic leukemia cells. Assessment of phosphatidylserine exposure confirmed apoptosis as the primary mode of cell death in INO-A-treated Jurkat cells. INO-A treatment for only 30 min resulted in a significant increase of tail moment which suggests that DNA damage is an early apoptotic signal. Further flow cytometric assessment of the superoxide anion level confirmed that INO-A induced DNA damage was mediated with a concomitant generation of reactive oxygen species (ROS). Investigation on the thiols revealed an early decrease of free thiols in 30 min after 50 μM INO-A treatment. Using tetramethylrhodamine ethyl ester, a potentiometric dye, the loss of mitochondrial membrane potential (MPP) was observed in INO-A-treated cells as early as 30 min. The INO-A-induced apoptosis progressed with the simultaneous activation of caspases-2 and -9 which then led to the processing of caspase-3. Taken together, these data demonstrate that INO-A induced early oxidative stress, DNA damage and loss of MMP which subsequently led to the activation of an intrinsic pathway of apoptosis in Jurkat cells., (Copyright © 2012 Elsevier Ltd. All rights reserved.)
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- 2012
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16. 3,5-dibenzyloxy-4'-hydroxystilbene induces early caspase-9 activation during apoptosis in human K562 chronic myelogenous leukemia cells.
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Roslie H, Chan KM, Rajab NF, Velu SS, Kadir SA, Bunyamin I, Weber JF, Thomas NF, Majeed AB, Myatt G, and Inayat-Hussain SH
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- Cell Survival drug effects, Humans, K562 Cells, Leukemia, Myelogenous, Chronic, BCR-ABL Positive, Membrane Potential, Mitochondrial drug effects, Stilbenes chemical synthesis, Structure-Activity Relationship, Apoptosis drug effects, Caspase 9 metabolism, Stilbenes toxicity
- Abstract
A series of 22 stilbene derivatives based on resveratrol were synthesized incorporating acetoxy-, benzyloxy-, carboxy-, chloro-, hydroxy- and methoxy functional groups. We examined the cytotoxicity of these 22 stilbenes in human K562 chronic myelogenous leukemia cells. Only four compounds were cytotoxic namely 4'-hydroxy-3-methoxystilbene (15), 3'-acetoxy-4-chlorostilbene (19), 4'-hydroxy-3,5-dimethoxystilbene or pterostilbene (3) and 3,5-dibenzyloxy-4'-hydroxystilbene (28) with IC(50)s of 78 µM, 38 µM, 67 µM and 19.5 µM respectively. Further apoptosis assessment on the most potent compound, 28, confirmed that the cells underwent apoptosis based on phosphatidylserine externalization and loss of mitochondrial membrane potential. Importantly, we observed a concentration-dependent activation of caspase-9 as early as 2 hr with resultant caspase-3 cleavage in 28-induced apoptosis. Additionally, a structure-activity relationship (SAR) study proposed a possible mechanism of action for compound 28. Taken together, our data suggests that the pro-apoptotic effects of 28 involve the intrinsic mitochondrial pathway characterized by an early activation of caspase-9.
- Published
- 2012
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17. Cytotoxic and antioxidant effects of methoxylated stilbene analogues on HepG2 hepatoma and Chang liver cells: Implications for structure activity relationship.
- Author
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Hasiah AH, Ghazali AR, Weber JF, Velu S, Thomas NF, and Inayat Hussain SH
- Subjects
- Anticarcinogenic Agents chemistry, Antioxidants chemistry, Cell Culture Techniques, Cell Survival drug effects, Dose-Response Relationship, Drug, Hep G2 Cells, Humans, Liver metabolism, Liver pathology, Molecular Structure, Stilbenes chemistry, Structure-Activity Relationship, Anticarcinogenic Agents pharmacology, Antioxidants pharmacology, Liver drug effects, Stilbenes pharmacology
- Abstract
Stilbenes possess a variety of biological activities including chemopreventive activity. This study was conducted to evaluate the structural activity relationships of six methoxylated stilbene analogues with respect to their cytotoxic effects and antioxidant activities on HepG2 hepatoma and Chang liver cells. The cytotoxic and total antioxidant activities of six stilbene analogues were determined by MTT and Ferric Reducing Antioxidant Power (FRAP) assays, respectively. We found that the cis-methoxylated stilbene: (Z)-3,4,4'-trimethoxystilbene was the most potent and selective antiproliferative agent (IC₅₀ 89 µM) in HepG2 cells. For the total antioxidant activity, compounds possessing hydroxyl groups at the 4' position namely (E)-3-methoxy-4'-hydroxystilbene, (E)-3,5-dimethoxy-4'-hydroxystilbene (pterostilbene), (E)-4-methoxy-4'-hydroxystilbene showed the highest antioxidant activity. Structure activity relationship studies of these compounds demonstrated that the cytotoxic effect and antioxidant activities of the tested compounds in this study were structurally dependent.
- Published
- 2011
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18. Goniothalamin induces coronary artery smooth muscle cells apoptosis: the p53-dependent caspase-2 activation pathway.
- Author
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Chan KM, Rajab NF, Siegel D, Din LB, Ross D, and Inayat-Hussain SH
- Subjects
- Adenosine Triphosphate analysis, Amino Acid Chloromethyl Ketones pharmacology, Cells, Cultured, Cytochromes c metabolism, Dose-Response Relationship, Drug, Enzyme Activation, Humans, Hydrogen Peroxide metabolism, Membrane Potential, Mitochondrial drug effects, NAD(P)H Dehydrogenase (Quinone) metabolism, Oxygen Consumption drug effects, Superoxides metabolism, Apoptosis drug effects, Caspase 2 metabolism, Muscle, Smooth, Vascular drug effects, Myocytes, Smooth Muscle drug effects, Pyrones toxicity, Tumor Suppressor Protein p53 physiology
- Abstract
Goniothalamin (GN), a styryl-lactone isolated from Goniothalamus andersonii, has been demonstrated to possess antirestenostic properties by inducing apoptosis on coronary artery smooth muscle cells (CASMCs). In this study, the molecular mechanisms of GN-induced CASMCs apoptosis were further elucidated. Apoptosis assessment based on the externalization of phosphatidylserine demonstrated that GN induces CASMCs apoptosis in a concentration-dependent manner. The GN-induced DNA damage occurred with concomitant elevation of p53 as early as 2 h, demonstrating an upstream signal for apoptosis. However, the p53 elevation in GN-treated CASMCs was independent of NAD(P)H: quinone oxidoreductase 1 and Mdm-2 expression. An increase in hydrogen peroxide and reduction in free thiols confirmed the role for oxidative stress in GN treatment. Pretreatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-FMK) that significantly abrogated GN-induced CASMCs apoptosis suggested the involvement of caspase(s). The role of apical caspase-2, -8, and -9 was then investigated, and sequential activation of caspase-2 and -9 but not caspase-8 leading to downstream caspase-3 cleavage was observed in GN-treated CASMCs. Reduction of ATP level and decrease in oxygen consumption further confirmed the role of mitochondria in GN-induced apoptosis in CASMCs. The mitochondrial release of cytochrome c was seen without mitochondrial membrane potential loss and was independent of cardiolipin. These data provide insight into the mechanisms of GN-induced apoptosis, which may have important implications in the development of drug-eluting stents.
- Published
- 2010
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19. Modulation of the benzene metabolite hydroquinone induced toxicity: evidence for an important role of fau.
- Author
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Inayat-Hussain SH, Ibrahim HA, Siew EL, Rajab NF, Chan KM, G T Williams, and Ross D
- Subjects
- Animals, Apoptosis drug effects, Cell Line, Tumor, Cell Survival drug effects, Gene Expression, Mice, Ribosomal Proteins genetics, Benzene metabolism, Hydroquinones adverse effects, Ribosomal Proteins metabolism
- Published
- 2010
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20. Goniothalamin-induced oxidative stress, DNA damage and apoptosis via caspase-2 independent and Bcl-2 independent pathways in Jurkat T-cells.
- Author
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Inayat-Hussain SH, Chan KM, Rajab NF, Din LB, Chow SC, Kizilors A, Farzaneh F, and Williams GT
- Subjects
- Blotting, Western, Caspase 2 metabolism, Caspase Inhibitors, Comet Assay, Cytochromes c metabolism, DNA Topoisomerases, Type II chemistry, Enzyme Inhibitors, Flow Cytometry, Glutathione metabolism, Goniothalamus chemistry, Humans, Jurkat Cells, Oligopeptides pharmacology, Proto-Oncogene Proteins c-bcl-2 metabolism, Reactive Oxygen Species, Signal Transduction drug effects, Tetrazolium Salts, Thiazoles, Antineoplastic Agents, Phytogenic toxicity, Apoptosis drug effects, Caspase 2 physiology, DNA Damage, Oxidative Stress drug effects, Proto-Oncogene Proteins c-bcl-2 physiology, Pyrones toxicity
- Abstract
Goniothalamin (GTN) isolated from Goniothalamus sp. has been demonstrated to induce apoptosis in a variety of cancer cell lines including Jurkat T leukemia cells. However, the mechanism of GTN-induced apoptosis upstream of mitochondria is still poorly defined. In this study, GTN caused a decrease in GSH with an elevation of reactive oxygen species as early as 30 min and DNA damage as assessed by Comet assay. Analysis using topoisomerase II processing of supercoiled pBR 322 DNA showed that GTN caused DNA damage via a topoisomerase II-independent pathway suggesting that cellular oxidative stress may contribute to genotoxicity. A 12-fold increase of caspase-2 activity was observed in GTN-treated Jurkat cells after 4h treatment and this was confirmed using Western blotting. Although the caspase-2 inhibitor Z-VDVAD-FMK inhibited the proteolytic activity of caspase-2, apoptosis ensued confirming that caspase-2 activity was not crucial for GTN-induced apoptosis. However, GTN-induced apoptosis was completely abrogated by N-acetylcysteine further confirming the role of oxidative stress. Since cytochrome c release was observed as early as 1h without any appreciable change in Bcl-2 protein expression, we further investigated whether overexpression of Bcl-2 confers resistance in GTN-induced cytotoxicity. Using a panel of Jurkat Bcl-2 transfectants, GTN cytotoxicity was not abrogated in these cells. In conclusion, GTN induces DNA damage and oxidative stress resulting in apoptosis which is independent of both caspase-2 and Bcl-2., (Copyright 2009 Elsevier Ireland Ltd. All rights reserved.)
- Published
- 2010
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21. A bacterial biosensor for oxidative stress using the constitutively expressed redox-sensitive protein roGFP2.
- Author
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Arias-Barreiro CR, Okazaki K, Koutsaftis A, Inayat-Hussain SH, Tani A, Katsuhara M, Kimbara K, and Mori IC
- Subjects
- Fluorescence, Oxidation-Reduction, Bacterial Physiological Phenomena, Biosensing Techniques, Green Fluorescent Proteins metabolism, Oxidative Stress
- Abstract
A highly specific, high throughput-amenable bacterial biosensor for chemically induced cellular oxidation was developed using constitutively expressed redox-sensitive green fluorescent protein roGFP2 in E. coli (E. coli-roGFP2). Disulfide formation between two key cysteine residues of roGFP2 was assessed using a double-wavelength ratiometric approach. This study demonstrates that only a few minutes were required to detect oxidation using E. coli-roGFP2, in contrast to conventional bacterial oxidative stress sensors. Cellular oxidation induced by hydrogen peroxide, menadione, sodium selenite, zinc pyrithione, triphenyltin and naphthalene became detectable after 10 seconds and reached the maxima between 80 to 210 seconds, contrary to Cd(2+), Cu(2+), Pb(2+), Zn(2+) and sodium arsenite, which induced the oxidation maximum immediately. The lowest observable effect concentrations (in ppm) were determined as 1.0 × 10(-7) (arsenite), 1.0 × 10(-4) (naphthalene), 1.0 × 10(-4) (Cu(2+)), 3.8 × 10(-4) (H(2)O(2)), 1.0 × 10(-3) (Cd(2+)), 1.0 × 10(-3) (Zn(2+)), 1.0 × 10(-2) (menadione), 1.0 (triphenyltin), 1.56 (zinc pyrithione), 3.1 (selenite) and 6.3 (Pb(2+)), respectively. Heavy metal-induced oxidation showed unclear response patterns, whereas concentration-dependent sigmoid curves were observed for other compounds. In vivo GSH content and in vitro roGFP2 oxidation assays together with E. coli-roGFP2 results suggest that roGFP2 is sensitive to redox potential change and thiol modification induced by environmental stressors. Based on redox-sensitive technology, E. coli-roGFP2 provides a fast comprehensive detection system for toxicants that induce cellular oxidation.
- Published
- 2010
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22. RACK-1 overexpression protects against goniothalamin-induced cell death.
- Author
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Inayat-Hussain SH, Wong LT, Chan KM, Rajab NF, Din LB, Harun R, Kizilors A, Saxena N, Mourtada-Maarabouni M, Farzaneh F, and Williams GT
- Subjects
- Animals, Apoptosis drug effects, Blotting, Western, Cell Death drug effects, Cell Line, Cell Survival drug effects, Clone Cells, Colony-Forming Units Assay, Coloring Agents, Comet Assay, Culture Media, DNA Damage, Humans, Jurkat Cells, Mice, Neuropeptides biosynthesis, Neuropeptides genetics, Receptors for Activated C Kinase, Tetrazolium Salts, Thiazoles, Neuropeptides physiology, Pyrones toxicity
- Abstract
Goniothalamin, a styryllactone, has been shown to induce cytotoxicity via apoptosis in several tumor cell lines. In this study, we have examined the potential role of several genes, which were stably transfected into T-cell lines and which regulate apoptosis in different ways, on goniothalamin-induced cell death. Overexpression of full-length receptor for activated protein C-kinase 1 (RACK-1) and pc3n3, which up-regulates endogenous RACK-1, in both Jurkat and W7.2 T cells resulted in inhibition of goniothalamin-induced cell death as assessed by MTT and clonogenic assays. However, overexpression of rFau (antisense sequence to Finkel-Biskis-Reilly murine sarcoma virus-associated ubiquitously expressed gene) in W7.2 cells did not confer resistance to goniothalamin-induced cell death. Etoposide, a clinically used cytotoxic agent, was equipotent in causing cytotoxicity in all the stable transfectants. Assessment of DNA damage by Comet assay revealed goniothalamin-induced DNA strand breaks as early as 1 h in vector control but this effect was inhibited in RACK-1 and pc3n3 stably transfected W7.2 cells. This data demonstrate that RACK-1 plays a crucial role in regulating cell death signalling pathways induced by goniothalamin.
- Published
- 2009
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23. Mutagenic and clastogenic characterization of poststerilized poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer biosynthesized by Delftia acidovorans.
- Author
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Siew EL, Rajab NF, Osman AB, Sudesh K, and Inayat-Hussain SH
- Subjects
- Animals, Cell Line, Cricetinae, Fibroblasts metabolism, Micronucleus Tests, Microscopy, Electron, Scanning methods, Mitomycin pharmacology, Molecular Weight, Mutagenicity Tests, Mutagens pharmacology, Mutation, Biocompatible Materials chemistry, Delftia acidovorans metabolism, Hydroxybutyrates chemistry, Polyesters chemistry, Polymers chemistry
- Abstract
Polyhydroxyalkanoates (PHA) are naturally occurring biopolyesters that have great potential in the medical field. However, the leachables resulting from sterilization process of the biomaterials may exert toxic effect including genetic damage. Here, we demonstrate that although gamma-irradiation of poly(3-hydroxybutyrate-co-50 mol % 4-hydroxybutyrate) [P(3HB-co-4HB)] did not cause any change in the morphology by scanning electron microscopy, there was a significant degradation of this copolymer where the molecular weight was reduced by 37% after sterilization indicating the generation of leachables. Therefore, further investigation on the ability of the extract of this poststerilized copolymer to induce mutagenic effect was performed using Ames test (S. typhimurium strains TA1535 and TA1537) and umu test (S. typhimurium strain TA1535/pSK1002). Additionally, the capability of the extract to induce clastogenic effect was determined using Chinese hamster lung V79 fibroblast cells. Our results showed that with and without the presence of S9 metabolic activation, no mutagenic effects were observed in both Ames and umu tests when treated with P(3HB-co-4HB) extract. Similarly, treatment of P(3HB-co-4HB) extract in V79 fibroblast cells showed no significant production of micronuclei when compared with the positive control (Mitomycin C). Together, these results indicate that leachables of poststerilized P(3HB-co-4HB) cause no mutagenic and clastogenic effects., (Copyright 2008 Wiley Periodicals, Inc.)
- Published
- 2009
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24. A comparative study of proteasomal inhibition and apoptosis induced in N27 mesencephalic cells by dopamine and MG132.
- Author
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Zafar KS, Inayat-Hussain SH, and Ross D
- Subjects
- Acetylcysteine pharmacology, Animals, Apoptosis drug effects, Caspases drug effects, Caspases metabolism, Cell Line, Transformed, Cysteine Proteinase Inhibitors pharmacology, Dopamine pharmacology, Dose-Response Relationship, Drug, Enzyme Activation drug effects, Enzyme Activation physiology, Leupeptins pharmacology, Mesencephalon cytology, Mesencephalon drug effects, Mesencephalon metabolism, Nerve Degeneration metabolism, Nerve Degeneration physiopathology, Neurons drug effects, Oxidative Stress drug effects, Oxidative Stress physiology, Parkinson Disease physiopathology, Proteasome Endopeptidase Complex drug effects, Rats, Reactive Oxygen Species metabolism, Substantia Nigra physiopathology, Apoptosis physiology, Dopamine metabolism, Neurons metabolism, Parkinson Disease metabolism, Proteasome Endopeptidase Complex metabolism, Substantia Nigra metabolism
- Abstract
Dopamine (DA) and its metabolites have been implicated in the pathogenesis of Parkinson's disease. DA can produce reactive-oxygen species and DA-derived quinones such as aminochrome can induce proteasomal inhibition. We therefore examined the ability of DA and MG132 to induce apoptosis and proteasomal inhibition in N27 rat dopaminergic cells. DA (0-500 micromol/L, 0-24 h) and MG132 (0-5 micromol/L, 0-24 h) treated N27 cells resulted in time- and concentration-dependent apoptosis. To better define DA and MG132-induced apoptosis, the activation of initiator caspases 2 and caspase 9 and the executioner caspase 3 was investigated. Activation of caspase 2, caspase 9, and caspase 3 occurred early and prior to cell death. In addition, N-acetylcysteine (NAC) blocked DA but not MG132-induced apoptosis and mitochondrial membrane potential loss. NAC can react with both reactive-oxygen and quinoid metabolites and its inhibitory activity suggests a role for reactive species in DA-induced apoptosis. Proteasomal inhibition was detected after DA treatment in N27 cells which occurred prior to cell death and was abrogated by NAC. Our results implicate DA-derived reactive species in proteasomal inhibition and caspase-dependent apoptosis in N27 cells. The ability of endogenous DA-derived metabolites to induce proteasomal inhibition and apoptosis may contribute to the selective loss of dopaminergic neurons in Parkinson's disease.
- Published
- 2007
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25. In vitro biocompatibility evaluation of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer in fibroblast cells.
- Author
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Siew EL, Rajab NF, Osman AB, Sudesh K, and Inayat-Hussain SH
- Subjects
- Animals, Apoptosis drug effects, Biocompatible Materials toxicity, Cell Line, Comet Assay, Cricetinae, Fibroblasts cytology, Fibroblasts drug effects, Fibroblasts metabolism, Hydroxybutyrates toxicity, Materials Testing, Mice, Polyesters toxicity, Tissue Engineering, Biocompatible Materials pharmacology, Hydroxybutyrates pharmacology, Polyesters pharmacology
- Abstract
Among the various biomaterials available for tissue engineering and therapeutic applications, microbial polyhydroxyalkanoates offer the most diverse range of thermal and mechanical properties. In this study, the biocompatibility of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB); containing 50 mol % of 4-hydroxybutyrate] copolymer produced by Delftia acidovorans was evaluated. The cytotoxicity, mode of cell death, and genotoxicity of P(3HB-co-4HB) extract against V79 and L929 fibroblast cells were assessed using MTT assay, acridine orange/propidium iodide staining, and alkaline comet assay, respectively. Our results demonstrate that P(3HB-co-4HB) treated on both cell lines were comparable with clinically-used Polyglactin 910, where more than 60% of viable cells were observed following 72-h treatment at 200 mg/mL. Further morphological investigation on the mode of cell death showed an increase in apoptotic cells in a time-dependent manner in both cell lines. On the other hand, P(3HB-co-4HB) at 200 mg/mL showed no genotoxic effects as determined by alkaline comet assay following 72-h treatment. In conclusion, our study indicated that P(3HB-co-4HB) compounds showed good biocompatibility in fibroblast cells suggesting that it has potential to be used for future medical applications., (Copyright 2006 Wiley Periodicals, Inc.)
- Published
- 2007
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26. Is plasma beta-glucuronidase a novel human biomarker for monitoring anticholinesterase pesticides exposure? A Malaysian experience.
- Author
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Inayat-Hussain SH, Lubis SH, Sakian NI, Ghazali AR, Ali NS, El Sersi M, Toong LM, Zainal AM, Hashim S, Ghazali MS, Saidin MN, Rahman AR, Rafaai MJ, Omar S, Rapiai R, Othman R, Chan LT, Johari A, Soon WH, Salleh AR, and Satoh T
- Subjects
- Adolescent, Adult, Aged, Aged, 80 and over, Biomarkers blood, Child, Cholinesterase Inhibitors analysis, Environmental Pollutants analysis, Female, Humans, Malaysia, Male, Middle Aged, Pesticides analysis, Cholinesterase Inhibitors toxicity, Environmental Exposure analysis, Environmental Monitoring, Environmental Pollutants toxicity, Glucuronidase blood, Pesticides toxicity
- Abstract
A cross-sectional study was conducted to investigate the effects of acute and chronic pesticide exposure on the plasma beta-glucuronidase enzyme activity among five patients of acute pesticide poisoning in Tengku Ampuan Rahimah Hospital, Klang, 230 farmers in the MADA area, Kedah and 49 fishermen in Setiu, Terengganu. The duration of pesticide exposure among the patients was unknown, but the plasma samples from patients were collected on day one in the hospital. The duration of pesticide exposure among the farmers was between 1 and 45 years. The beta-glucuronidase activity was compared with plasma cholinesterase activity in the same individual. The plasma cholinesterase activity was measured using Cholinesterase (PTC) Reagent set kit (Teco Diagnostics, UK) based on colorimetric method, while the plasma beta-glucuronidase activity was measured fluorometrically based on beta-glucuronidase assay. The plasma cholinesterase activity was significantly reduced (p<0.05) among the patients (1386.786+/-791.291 U/L/min) but the inhibition in plasma cholinesterase activity among the farmers (7346.5+/-1860.786 U/L/min) was not significant (p>0.05). The plasma beta-glucuronidase activity among the farmers was significantly elevated (p<0.05) (0.737+/-0.425 microM/h) but not significant among the patients (p>0.05). The plasma cholinesterase activity was positively correlated with the plasma beta-glucuronidase activity among the farmers (r=0.205, p<0.01) but not among the patients (r=0.79, p>0.05). Thus, plasma beta-glucuronidase enzyme activity can be measured as a biomarker for the chronic exposure of pesticide. However, further studies need to be performed to confirm whether plasma beta-glucuronidase can be a sensitive biomarker for anticholinesterase pesticide poisoning.
- Published
- 2007
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27. Overexpression of NQO1 protects human SK-N-MC neuroblastoma cells against dopamine-induced cell death.
- Author
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Zafar KS, Inayat-Hussain SH, Siegel D, Bao A, Shieh B, and Ross D
- Subjects
- Annexin A5, Apoptosis drug effects, Blotting, Western, Catalase metabolism, Cell Death drug effects, Cell Line, Tumor, Flow Cytometry, Free Radicals, Humans, Membrane Potentials drug effects, Mitochondrial Membranes drug effects, NAD(P)H Dehydrogenase (Quinone) physiology, Quinones metabolism, Superoxide Dismutase metabolism, Transfection, Brain Neoplasms pathology, Dopamine toxicity, NAD(P)H Dehydrogenase (Quinone) biosynthesis, Neuroblastoma pathology
- Abstract
NAD(P)H quinone oxidoreductase 1 (NQO1) can metabolize dopamine-derived quinones (DAQ) and absence of NQO1 due to the NQO1*2 polymorphism has been suggested to be a risk factor for Parkinson's disease. In order to define whether NQO1 plays a protective role in dopamine toxicity, we have examined the potential role of NQO1 in the SK-N-MC human neuroblastoma cell line. SK-N-MC cells were stably transfected with NQO1 to generate stable clones with NQO1 enzymatic activity of 245 nmol/mgmin while vector control and parental cells had NQO1 activities of less than 12 nmol/mgmin. Incubation of dopamine for 24 h in both parental and vector control SK-N-MC cells resulted in 85% and 72% cell death as assessed by annexin-V/propidium iodide analysis. In agreement, 88% and 84% of parental and vector control cells, respectively underwent loss of mitochondrial membrane potential (MMP) assessed by tetramethylrhodamine ethyl ester. In contrast, NQO1-transfected cells were resistant to dopamine toxicity and both cell death and loss of MMP were markedly abrogated in NQO1-transfected SK-N-MC cells. When dopamine was added to medium, oxygen uptake could be detected indicating autoxidation with concomitant formation of oxygen radicals and quinones. However, dopamine-induced cell death was not affected by the inclusion of either superoxide dismutase or catalase suggesting that superoxide and hydrogen peroxide were not involved in toxicity. Quinones formed in medium may exert toxicity extracellularly or intracellularly but the protective role of NQO1 argues for an intracellular mechanism. In summary, transfection of SK-N-MC cells with NQO1 protects against dopamine-induced toxicity.
- Published
- 2006
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28. Goniothalamin induces apoptosis in vascular smooth muscle cells.
- Author
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Chan KM, Rajab NF, Ishak MH, Ali AM, Yusoff K, Din LB, and Inayat-Hussain SH
- Subjects
- Bromodeoxyuridine, Cell Division, Cells, Cultured, Comet Assay, Humans, Muscle, Smooth, Vascular cytology, Apoptosis drug effects, Muscle, Smooth, Vascular drug effects, Pyrones pharmacology
- Abstract
Restenosis represents a major impediment to the success of coronary angioplasty. Abnormal proliferation of vascular smooth muscle cells (VSMCs) has been shown to be an important process in the pathogenesis of restenosis. A number of agents, particularly rapamycin and paclitaxel, have been shown to impact on this process. This study was carried out to determine the mechanisms of cytotoxicity of goniothalamin (GN) on VSMCs. Results from MTT cytotoxicity assay showed that the IC(50) for GN was 4.4 microg/ml (22 microM), which was lower compared to the clinically used rapamycin (IC(50) of 25 microg/ml [27.346 microM]). This was achieved primarily via apoptosis where up to 25.83 +/- 0.44% of apoptotic cells were detected after 72 h treatment with GN. In addition, GN demonstrated similar effects as rapamycin in inhibiting VSMCs proliferation using bromodeoxyuridine (BrdU) cell proliferation assay after 72 h treatment at IC(50) concentration (p > 0.05). In order to understand the mechanisms of GN, DNA damage detection using comet assay was determined at 2h post-treatment with GN. Our results showed that there was a concentration-dependent increase in DNA damage in VSMCs prior to cytotoxicity. Moreover, GN effects were comparable to rapamycin. In conclusion, our data show that GN initially induces DNA damage which subsequently leads to cytotoxicity primarily via apoptosis in VSMCs.
- Published
- 2006
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29. RH1 induces cellular damage in an NAD(P)H:quinone oxidoreductase 1-dependent manner: relationship between DNA cross-linking, cell cycle perturbations, and apoptosis.
- Author
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Dehn DL, Inayat-Hussain SH, and Ross D
- Subjects
- Apoptosis physiology, Cell Cycle physiology, Cell Line, Tumor, Humans, Apoptosis drug effects, Aziridines toxicity, Benzoquinones toxicity, Cell Cycle drug effects, Cross-Linking Reagents pharmacology, DNA metabolism, NAD(P)H Dehydrogenase (Quinone) physiology
- Abstract
Structure-based development of NAD(P)H:quinone oxidoreductase (NQO1)-directed antitumor quinones resulted in development of RH1 [2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone], a methyl-substituted diaziridinyl quinone. We conducted experiments to evaluate the mechanism of RH1-induced cytotoxicity and the inter-relationship between DNA cross-linking, cell cycle changes, and apoptosis using an isogenic cell line pair developed from the human breast cancer cell line MDA-MB-468 differing only in expression of wtNQO1 (NQ16 cells). Statistically significant DNA cross-linking was detected using a modified comet assay in cells with wtNQO1 within 1 h of dosing, whereas in parental cells, only marginal DNA cross-linking was observed and required a concentration up to 50 times higher. Cross-linking in NQ16 cells could be abrogated with 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione, a mechanism-based inhibitor of NQO1. RH1 prolonged S phase and caused a G(2)/M block. Cell cycle changes were observed up to 10-fold lower in RH1 concentrations in NQ16 cells relative to parental cells. Apoptosis was similarly observed morphologically in both cell lines after RH1 treatment but was induced preferentially in NQ16 cells at lower concentrations and earlier time points. Marked cleavage of caspase-3 was observed in NQ16 cells relative to parental cells using lower concentrations of RH1. Temporally, low doses of RH1-induced rapid DNA cross-linking in NQ16 cells followed by induction of apoptosis at times when a G(2)/M block was not observed. This suggests that cell cycle arrest is not required for RH1-induced apoptosis and that DNA damage may directly initiate apoptotic events. In summary, RH1-induced preferential DNA cross-linking, cell cycle changes, and apoptosis in an NQO1-dependent manner.
- Published
- 2005
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30. Intrinsic pathway of hydroquinone induced apoptosis occurs via both caspase-dependent and caspase-independent mechanisms.
- Author
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Inayat-Hussain SH and Ross D
- Subjects
- Amino Acid Chloromethyl Ketones pharmacology, Caspases adverse effects, Drug Combinations, Enzyme Inhibitors pharmacology, Flow Cytometry, HL-60 Cells enzymology, HL-60 Cells pathology, Humans, Jurkat Cells enzymology, Jurkat Cells pathology, Membrane Potentials drug effects, Mitochondria drug effects, Mitochondria enzymology, Phosphatidylserines metabolism, Apoptosis drug effects, Caspases biosynthesis, HL-60 Cells drug effects, Hydroquinones toxicity, Jurkat Cells drug effects
- Abstract
The role of mitochondria and apical caspases in apoptosis induced by the benzene metabolite hydroquinone (HQ) remains to be elucidated. Here, we investigated the involvement of mitochondria and activation of the apical caspases-8 and -9 in HQ induced apoptosis in myeloperoxidase (MPO)-rich HL-60 and MPO-deficient Jurkat T cells. Treatment of HL-60 and Jurkat cells with HQ resulted in apoptosis as assessed by phosphatidyl serine (PS) exposure, loss of mitochondrial transmembrane potential (MTP), release of cytochrome c, and processing of apical caspases-8 and -9 and executioner caspase-3. In HQ-treated HL-60 cells, pretreatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (ZVAD), which did not inhibit PS exposure, also failed to abrogate the loss of MTP and release of cytochrome c. However, complete processing of caspase-9 was inhibited in the presence of ZVAD. In marked contrast, in HQ-treated Jurkat cells, ZVAD significantly abrogated PS exposure, loss of MTP, and caspase-9 processing but not release of cytochrome c. Although ZVAD-sensitive caspase-8 processing occurred in both cell types, pretreatment with either fas-receptor blocking ZB4 or fas-ligand NOK1 neutralizing antibodies did not inhibit HQ-induced apoptosis. In conclusion, our results demonstrate that HQ induced apoptosis in Jurkat cells occurs via a ZVAD-inhibitable, caspase-dependent process, while in HL-60 cells, apoptosis occurs predominantly via caspase-independent mechanisms. Our results emphasize that both caspase-dependent and independent mechanisms should be considered in the intrinsic apoptotic pathway induced by HQ.
- Published
- 2005
- Full Text
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31. DNA damage evaluation of hydroxyapatite on fibroblast cell L929 using the single cell gel electrophoresis assay.
- Author
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Rajab NF, Yaakob TA, Ong BY, Hamid M, Ali AM, Annuar BO, and Inayat-Hussain SH
- Subjects
- Animals, Cell Survival drug effects, L Cells, Mice, Biocompatible Materials toxicity, Bone Substitutes toxicity, DNA Damage, Hydroxyapatites toxicity, Mutagenicity Tests, Prostheses and Implants
- Abstract
Hydroxyapatite is the main component of the bone which is a potential biomaterial substance that can be applied in orthopaedics. In this study, the biocompatibility of this biomaterial was assessed using an in vitro technique. The cytotoxicity and genotoxicity effect of HA2 and HA3 against L929 fibroblast cell was evaluated using the MTT Assay and Alkaline Comet Assay respectively. Both HA2 and HA3 compound showed low cytotoxicity effect as determined using MTT Assay. Cells viability following 72 hours incubation at maximum concentration of both HA2 and HA3 (200 mg/ml) were 75.3 +/- 8.8% and 86.7 +/- 13.1% respectively. However, the cytotoxicity effect of ZnSO4.7H2O as a positive control showed an IC50 values of 46 mg/ml (160 microM). On the other hand, both HA2 and HA3 compound showed a slight genotoxicity effect as determined using the Alkaline Comet Assay following incubation at the concentration 200 mg/ml for 72 hours. This assay has been widely used in genetic toxicology to detect DNA strand breaks and alkali-labile site. The percentage of the cells with DNA damage for both substance was 27.7 +/- 1.3% and 15.6 +/- 1.0% for HA2 and HA3 respectively. Incubation of the cells for 24 hours with 38 microg/ml (IC25) of positive control showed an increase in percentage of cells with DNA damage (67.5 +/- 0.7%). In conclusion, our study indicated that both hydroxyapatite compounds showed a good biocompatibility in fibroblast cells.
- Published
- 2004
32. Cell death induced by hydroxyapatite on L929 fibroblast cells.
- Author
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Inayat-Hussain SH, Rajab NF, Roslie H, Hussin AA, Ali AM, and Annuar BO
- Subjects
- Animals, Apoptosis drug effects, L Cells, Mice, Biocompatible Materials toxicity, Bone Substitutes toxicity, Cell Death drug effects, Durapatite toxicity, Prostheses and Implants
- Abstract
Biomaterials intended for end-use application as bone-graft substitutes have to undergo safety evaluation. In this study, we investigated the in vitro cytotoxic effects especially to determine the mode of death of two hydroxyapatite compounds (HA2, HA3) which were synthesized locally. The methods used for cytotoxicity was the standard MTT assay whereas AO/PI staining was performed to determine the mode of cell death in HA treated L929 fibroblasts. Our results demonstrated that both HA2 and HA3 were not significantly cytotoxic as more than 75% cells after 72 hours treatment were viable. Furthermore, we found that the major mode of cell death in HA treated cells was apoptosis. In conclusion, our results demonstrated that these hydroxyapatite compounds are not cytotoxic where the mode of death was primarily via apoptosis.
- Published
- 2004
33. Loss of mitochondrial transmembrane potential and caspase-9 activation during apoptosis induced by the novel styryl-lactone goniothalamin in HL-60 leukemia cells.
- Author
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Inayat-Hussain SH, Annuar BO, Din LB, Ali AM, and Ross D
- Subjects
- Caspase 3, Caspase 7, Caspase 9, Enzyme Activation drug effects, Flow Cytometry, HL-60 Cells, Humans, Indicators and Reagents, Membrane Potentials drug effects, Apoptosis physiology, Caspases metabolism, Mitochondria physiology, Pyrones pharmacology
- Abstract
Styryl-lactones such as goniothalamin represent a new class of compounds with potential anti-cancer properties. In this study, we investigated the mechanisms of goniothalamin (GTN), a plant styryl-lactone induced apoptosis in human promyelocytic leukemia HL-60 cells. This plant extract resulted in apoptosis in HL-60 cells as assessed by the externalisation of phosphatidylserine. Using the mitochondrial membrane dye (DIOC(6)) in conjunction with flow cytometry, we found that GTN treated HL-60 cells demonstrated a loss of mitochondrial transmembrane potential (Deltapsi(m)). Further immunoblotting on these cells showed activation of initiator caspase-9 and the executioner caspases-3 and -7. Pretreatment with the pharmacological caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD.FMK) abrogated apoptosis as assessed by all of the apoptotic features in this study. In summary, our results demonstrate that goniothalamin-induced apoptosis occurs via the mitochondrial pathway in a caspase dependent manner.
- Published
- 2003
- Full Text
- View/download PDF
34. Altholactone, a novel styryl-lactone induces apoptosis via oxidative stress in human HL-60 leukemia cells.
- Author
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Inayat-Hussain SH, Osman AB, Din LB, and Taniguchi N
- Subjects
- Acetylcysteine pharmacology, Annexin A5, Antioxidants pharmacology, Flow Cytometry, Fluoresceins, Furans pharmacology, HL-60 Cells, Humans, Indicators and Reagents, Pyrones pharmacology, Apoptosis drug effects, Oxidative Stress drug effects
- Abstract
Plant styryl-lactone derivatives isolated from Goniothalamus sp. are potential compounds for cancer chemotherapy. In this study, we have examined the mechanisms of apoptosis induced by altholactone, a stryl-lactone isolated from the Malaysian plant G. malayanus on human HL-60 promyelocytic leukemia cells. Flow cytometric analysis of the externalization of phosphatidylserine (PS) using the annexin V/PI method on altholactone treated HL-60 cells showed a concentration-dependent increase of apoptosis from concentrations ranging from 10.8 (2.5 microg/ml) to 172.4 microM (40 microg/ml). Pre-treatment with the antioxidant N-acetylcysteine (1 mM) completely abrogated apoptosis induced by altholactone, suggesting for the involvement of oxidative stress. Further flow cytometric assessment of the level of intracellular peroxides using the fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA) confirmed that altholactone induced an increase in cellular oxidative stress in HL-60 cells which was suppressed by N-acetylcysteine. In summary, our results demonstrate for the first time that altholactone induced apoptosis in HL-60 cells occurs via oxidative stress.
- Published
- 2002
- Full Text
- View/download PDF
35. Differential involvement of caspases in hydroquinone-induced apoptosis in human leukemic hl-60 and jurkat cells.
- Author
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Inayat-Hussain SH, Winski SL, and Ross D
- Subjects
- Caspases metabolism, Enzyme Activation drug effects, HL-60 Cells enzymology, Humans, Jurkat Cells enzymology, Amino Acid Chloromethyl Ketones pharmacology, Antioxidants pharmacology, Apoptosis drug effects, Caspase Inhibitors, Cysteine Proteinase Inhibitors pharmacology, HL-60 Cells drug effects, Hydroquinones pharmacology, Jurkat Cells drug effects, Peroxidase metabolism
- Abstract
The benzene metabolite hydroquinone (HQ) is postulated to exert its myelotoxicity by bioactivation to reactive quinone derivatives in myeloperoxidase (MPO)-containing cells. In this study, the role of caspases in hydroquinone-induced apoptosis in MPO-rich HL-60 promyelocytic leukemia and MPO-deficient Jurkat T-lymphoblastic leukemia cells was investigated. HQ-induced apoptosis in both cell types was accompanied by phosphatidylserine (PS) exposure, caspases-3/-7 activation, PARP cleavage, DNA fragmentation, and ultrastructural changes as assessed by electron microscopy. In HL-60 cells, the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD.FMK) blocked activation of caspases-3/-7, cleavage of PARP, and DNA, but PS externalization and cytoplasmic changes were not significantly affected. In marked contrast, all features of apoptosis were completely inhibited by Z-VAD.FMK in HQ-treated Jurkat cells. These data provide evidence for Z-VAD.FMK-insensitive and caspases-3/-7-independent pathway(s) in the externalization of PS and cytoplasmic changes during HQ-induced apoptosis in HL-60 cells. In contrast, in Jurkat cells, all of these changes required caspase activation. The ability of HQ to induce equivalent apoptosis in both MPO-deficient Jurkat cells and MPO-rich HL-60 cells demonstrates that MPO-catalyzed bioactivation of HQ is not a prerequisite for toxicity. The differential mechanisms of apoptosis in HL-60 and Jurkat T cells may reflect the MPO activity of these cells and, as a result, the amount of reactive BQ and other metabolites that are generated., (Copyright 2001 Academic Press.)
- Published
- 2001
- Full Text
- View/download PDF
36. Caspase-dependent and -independent mechanisms in apoptosis induced by hydroquinone and catechol metabolites of remoxipride in HL-60 cells.
- Author
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Inayat-Hussain SH, McGuinness SM, Johansson R, Lundstrom J, and Ross D
- Subjects
- Amino Acid Chloromethyl Ketones pharmacology, Antipsychotic Agents metabolism, Apoptosis physiology, Caspase Inhibitors, Caspases metabolism, Cysteine Proteinase Inhibitors pharmacology, DNA Fragmentation drug effects, Enzyme Activation, HL-60 Cells cytology, HL-60 Cells drug effects, HL-60 Cells enzymology, Humans, Isoenzymes antagonists & inhibitors, Isoenzymes metabolism, Isoenzymes physiology, Phosphatidylserines metabolism, Antipsychotic Agents toxicity, Apoptosis drug effects, Caspases physiology, Hydroquinones toxicity, Remoxipride analogs & derivatives, Remoxipride metabolism, Remoxipride toxicity
- Abstract
The hydroquinone and catechol like metabolites, NCQ344 and NCQ436 respectively, of the antipsychotic remoxipride have recently been demonstrated to induce apoptosis in myeloperoxidase (MPO)-rich human bone marrow progenitor and HL-60 cells [S.M. McGuinness, R. Johansson, J. Lundstrom, D. Ross, Induction of apoptosis by remoxipride metabolites in HL-60 and CD34+/CD19- human bone marrow progenitor cells: potential relevance to remoxipride-induced aplastic anemia, Chem. Biol. Interact. 121 (1999) 253-265]. In the present study, we determined the molecular mechanisms of apoptosis induced by these remoxipride metabolites in HL-60 cells. Our results show that apoptosis was accompanied by phosphatidylserine (PS) exposure, activation of caspases-9, -3, -7 and DNA cleavage. In HL-60 cells treated with the hydroquinone NCQ344 and catechol NCQ436, the general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp. fluoromethyl ketone (Z-VAD.FMK) blocked DNA cleavage and activation of caspases-9, -3/-7. In addition, PS exposure was significantly but not completely inhibited by Z-VAD.FMK. These results demonstrate that although Z-VAD.FMK inhibitable caspases are necessary for maximal apoptosis induced by NCQ344 and NCQ436, additional caspase-independent processes may orchestrate changes leading to PS exposure during apoptosis induced by the remoxipride polyphenolic metabolites.
- Published
- 2000
- Full Text
- View/download PDF
37. Caspases-3 and -7 are activated in goniothalamin-induced apoptosis in human Jurkat T-cells.
- Author
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Inayat-Hussain SH, Osman AB, Din LB, Ali AM, Snowden RT, MacFarlane M, and Cain K
- Subjects
- Amino Acid Chloromethyl Ketones pharmacology, Annexin A5 analysis, Annexin A5 metabolism, Caspase 3, Caspase 7, Caspase Inhibitors, Cysteine Proteinase Inhibitors pharmacology, Enzyme Activation drug effects, Humans, Jurkat Cells enzymology, Jurkat Cells pathology, Poly(ADP-ribose) Polymerases metabolism, Pyrones metabolism, Apoptosis drug effects, Caspases metabolism, Jurkat Cells drug effects, Pyrones pharmacology
- Abstract
Goniothalamin, a plant styrylpyrone derivative isolated from Goniothalamus andersonii, induced apoptosis in Jurkat T-cells as assessed by the externalisation of phosphatidylserine. Immunoblotting showed processing of caspases-3 and -7 with the appearance of their catalytically active large subunits of 17 and 19 kDa, respectively. Activation of these caspases was further evidenced by detection of poly(ADP-ribose) polymerase cleavage (PARP). Pre-treatment with the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD.FMK) blocked apoptosis and the resultant cleavage of these caspases and PARP. Our results demonstrate that activation of at least two effector caspases is a key feature of goniothalamin-induced apoptosis in Jurkat T-cells.
- Published
- 1999
- Full Text
- View/download PDF
38. A reappraisal of the role of Zn2+ in TGF-beta1-induced apoptosis in primary hepatocytes.
- Author
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Inayat-Hussain SH, Cohen GM, and Cain K
- Subjects
- Animals, Apoptosis drug effects, Cells, Cultured, Chromatin drug effects, DNA drug effects, Liver cytology, Male, Rats, Rats, Inbred F344, Apoptosis physiology, Liver drug effects, Transforming Growth Factor beta physiology, Zinc toxicity
- Abstract
There is now a wealth of information regarding the apoptotic mode of cell death and its importance in toxicological studies in many mammalian organs including the liver. In this study, we investigated the modulatory effects of the heavy metal Zn2+ on transforming growth factor-beta1 (TGF-beta1)-induced apoptosis in primary rat hepatocytes. Apoptosis induced by TGF-beta1 (1 ng/ml) in hepatocytes was accompanied by nuclear condensation as assessed morphologically by staining with Hoechst 33258 and DNA cleavage as detected biochemically by in situ end-labeling, field inversion and conventional gel electrophoresis. Pretreatment with 100 micromol/L Zn2+ abrogated the nuclear condensation, in situ end-labeling, and DNA laddering in TGF-beta1-treated hepatocytes. Surprisingly, Zn2+ did not inhibit the formation of high-molecular-weight DNA fragments (30-50 kbp to 250-300 kbp). These data provide evidence that Zn2+ exerts its effects on the endonucleases that act downstream in the execution phase of TGF-beta1-induced apoptosis in hepatocytes.
- Published
- 1999
- Full Text
- View/download PDF
39. Processing/activation of CPP32-like proteases is involved in transforming growth factor beta1-induced apoptosis in rat hepatocytes.
- Author
-
Inayat-Hussain SH, Couet C, Cohen GM, and Cain K
- Subjects
- Animals, Apoptosis drug effects, Caspase 3, Coumarins chemistry, Coumarins metabolism, Cycloheximide pharmacology, Enzyme Activation, Enzyme Induction, Kinetics, Liver cytology, Liver drug effects, Male, Oligopeptides chemistry, Oligopeptides metabolism, Poly(ADP-ribose) Polymerases chemistry, Poly(ADP-ribose) Polymerases metabolism, Protein Synthesis Inhibitors pharmacology, Rats, Rats, Inbred F344, Apoptosis physiology, Caspases, Cysteine Endopeptidases metabolism, Endopeptidases metabolism, Liver physiology, Protein Processing, Post-Translational, Transforming Growth Factor beta pharmacology
- Abstract
Apoptosis induced in rat hepatocytes by transforming growth factor beta1 (TGF-beta1) was accompanied by the activation of interleukin-1beta converting enzyme (ICE)-like proteases. Cell lysates were isolated at various times after TGF-beta1 treatment and analyzed for ICE and CPP32-like activity, using N-acetyl-Tyr-Val-Ala-Asp-7-amino-4-methylcoumarin (Ac-YVAD.AMC) and benzyloxycarbonyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin (Z-DEVD.AFC), respectively. CPP32-like but not ICE protease activity increased in a time dependent manner and preceded the onset of apoptosis. Kinetic studies in cell lysates indicated that more than one CPP32-like protease was being activated. This was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/Western blotting of TGF-beta1-treated cells, which showed limited processing of CPP32 as shown by the appearance of the catalytically active p17 subunit. Loss of pro-Mch3alpha was also observed but the catalytically active p19 subunit was not detected. Staurosporine, which induced a much greater level of hepatocyte apoptosis, produced a concomitant increase in CPP32/Mch3alpha processing as shown by the appearance of the p17/p19 subunits and the corresponding increase in CPP32-like protease activity. Apoptosis, CPP32/Mch3alpha processing and the increase in CPP32-like protease activity induced by TGF-beta1 and staurosporine were abolished in hepatocytes pretreated with Z-Asp-Glu-Val-Asp (OMe) fluoromethylketone (Z-DEVD.FMK) or Z-Val-Ala-Asp (OMe) fluoromethylketone (Z-VAD.FMK). These peptide analogues were potent inhibitors of CPP32-like protease activity in lysates. Pretreatment of hepatocytes with cycloheximide also blocked TGF-beta1-induced apoptosis and the increase in CPP32-like activity. Unlike Z-VAD.FMK and Z-DEVD.FMK, cycloheximide did not inhibit CPP32-like protease activity in cell lysates. Thus, cycloheximide may block apoptosis by inhibiting the synthesis of a protein, which is involved in the upstream events responsible for the activation of the CPP32-like protease activity. Our studies have identified two of the CPP32-like proteases, namely CPP32 and Mch3alpha, which are activated during the execution phase of hepatocyte apoptosis.
- Published
- 1997
- Full Text
- View/download PDF
40. Hepatocyte death following transforming growth factor-beta 1 addition.
- Author
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Oberhammer F, Froschl G, Tiefenbacher R, Inayat-Hussain SH, Cain K, and Stopper H
- Subjects
- Animals, Apoptosis drug effects, Cells, Cultured, Chromatin drug effects, Chromatin ultrastructure, DNA analysis, DNA genetics, DNA Damage drug effects, Female, Liver chemistry, Microscopy, Confocal, Microscopy, Ultraviolet, Rats, Rats, Wistar, Apoptosis physiology, Liver pathology, Liver physiology, Transforming Growth Factor beta pharmacology
- Abstract
Apoptosis is a morphological term which describes a sequence of events finally leading to cell death. In epithelial organs, induction of cell death is closely linked to an inhibitor of epithelial growth, transforming growth factor-beta 1 (TGF-beta 1). In this paper, we describe the morphology of TGF-beta 1-induced apoptosis in hepatocytes of the hyperplastic liver and primary cultures. Chromatin condensation, a hallmark of apoptosis, was observed in primary hepatocytes by confocal and vital UV microscopy. In addition, we have applied the morphological detection of DNA strand breaks both by in situ tailing (ISTAIL) and in situ nick translation (ISNT).
- Published
- 1996
- Full Text
- View/download PDF
41. A novel method for detecting apoptosis shows that hepatocytes undergo a time dependent increase in DNA cleavage and chromatin condensation which is augmented after TGF-beta 1 treatment.
- Author
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Cain K, Inayat-Hussain SH, Couet C, Qin HM, and Oberhammer FA
- Subjects
- Animals, Cell Nucleus, Cells, Cultured, Chromatin, Liver cytology, Male, Microscopy, Confocal, Rats, Rats, Inbred F344, Time Factors, Transforming Growth Factor beta pharmacology, Apoptosis physiology, DNA Damage physiology, Flow Cytometry methods, Liver physiology
- Abstract
This study describes a new method for quantitating apoptosis in hepatocyte monolayers in which nuclei were isolated from the cells and DNA strand breaks detected by in situ end-labeling and flow cytometry. Most (97%) nuclei from untreated hepatocytes had low end-labelling and were derived from non-apoptotic cells. Approximately 2-3% of the nuclei had high end-labelling and originated from apoptotic hepatocytes. The numbers of these nuclei increased linearly from 3 to 85% between 0 and 48 h after treatment with transforming growth factor-beta 1 (TGF-beta 1). However, a morphological assessment of apoptosis with Hoechst H33258 showed that the proportion of apoptotic nuclei plateaued at 18-19% between 24 and 48 h after TGF-beta 1 treatment. Thus, the in situ end-labeling technique also detected DNA cleavage in nuclei which did not have an obvious apoptotic morphology. Confocal microscopy of low and high end-labelled nuclei which had been separated by fluorescent cell sorting showed that nuclei with high levels of end-labeling exhibited a wide diversity of morphologies. These included nuclei with little or no chromatin condensation and nuclei with characteristic apoptotic morphology. In addition, nuclei from untreated hepatocytes contained low levels of DNA cleavage, which were localized in areas of condensed chromatin and increased according to the time in culture. Thus, hepatocytes undergo a progressive and cumulative process of DNA cleavage/chromatin condensation which is markedly enhanced by TGF-beta 1.
- Published
- 1996
- Full Text
- View/download PDF
42. A cleavage-site-directed inhibitor of interleukin-1 beta-converting enzyme-like proteases inhibits apoptosis in primary cultures of rat hepatocytes.
- Author
-
Cain K, Inayat-Hussain SH, Couet C, and Cohen GM
- Subjects
- Alkaloids pharmacology, Animals, Caspase 1, Cell Nucleus drug effects, Cells, Cultured, Chromatin drug effects, Chromatin metabolism, DNA metabolism, Electrophoresis, Agar Gel, Liver drug effects, Male, Nucleosomes drug effects, Nucleosomes metabolism, Oligopeptides pharmacology, Protease Inhibitors pharmacology, Rats, Staurosporine, Transforming Growth Factor beta pharmacology, Amino Acid Chloromethyl Ketones pharmacology, Apoptosis drug effects, Cysteine Endopeptidases metabolism, Cysteine Proteinase Inhibitors pharmacology, Liver cytology
- Abstract
Apoptosis induced in primary hepatocytes by transforming growth factor beta1 and staurosporine produced chromatin condensation, DNA cleavage is detected by in situ end-labelling, field inversion and conventional gel electrophoresis, and cell detachment. These effects were abolished by benzyloxycarbonyl-valinylalanylaspartylfluoromethyl ketone, a cleavage-site-directed inhibitor of interleukin-1beta-converting enzyme-like proteases, and this finding suggests that these enzymes are involved in liver apoptosis.
- Published
- 1996
- Full Text
- View/download PDF
43. Multi-step DNA cleavage in rat liver nuclei is inhibited by thiol reactive agents.
- Author
-
Cain K, Inayat-Hussain SH, Kokileva L, and Cohen GM
- Subjects
- Animals, Calcium metabolism, Cell Nucleus metabolism, Ethylmaleimide pharmacology, Hydrolysis, Liver metabolism, Magnesium metabolism, Protease Inhibitors pharmacology, Rats, Cell Nucleus drug effects, DNA metabolism, Liver drug effects, Sulfhydryl Compounds pharmacology
- Abstract
DNA fragmentation in isolated rat liver nuclei is a Mg(2+)-dependent, multi-step process which is potentiated by Ca2+ and cleaves the DNA into > or = 700, 200-300 and 30-50 kilobase pair (kbp) fragments, prior to internucleosomal cleavage by Ca2+/Mg(2+)-dependent endonuclease(s). We now show that Cd2+, Hg2+, dichloroisocoumarin (DCI, a serine protease inhibitor) and N-ethylmaleimide (NEM) block both Mg2+ and Ca2+/Mg(2+)-dependent processes. Inhibition of DNA cleavage produced an increase in the size of the DNA fragments, from mono-/oligonucleosomes to 30-50, 200-300, > or = 700 kbp and finally to intact DNA. NEM and DCI inhibition was blocked by dithiothreitol, and it is proposed that a critical thiol(s) is involved in the DNA cleavage reactions which are a feature of the apoptotic process.
- Published
- 1995
- Full Text
- View/download PDF
44. DNA cleavage in rat liver nuclei activated by Mg2+ or Ca2+ + Mg2+ is inhibited by a variety of structurally unrelated inhibitors.
- Author
-
Cain K, Inayat-Hussain SH, Kokileva L, and Cohen GM
- Subjects
- Animals, Apoptosis drug effects, Cadmium pharmacology, Cell Nucleus drug effects, DNA drug effects, Drug Synergism, Flow Cytometry, In Vitro Techniques, Liver drug effects, Mercury pharmacology, Rats, Serine Proteinase Inhibitors pharmacology, Sulfhydryl Reagents pharmacology, Calcium pharmacology, Cell Nucleus metabolism, DNA metabolism, Liver metabolism, Magnesium pharmacology
- Abstract
Internucleosomal DNA fragmentation is often regarded as the biochemical hallmark of apoptosis and can be reproduced in vitro in rat liver nuclei. In this study we demonstrate that DNA is initially cleaved into > or = 700, 200-250, and 30-50 kilobase pair (kbp) fragments via a Mg(2+)-dependent, multistep process which can be potentiated by Ca2+. The subsequent internucleosomal cleavage requires both Ca2+ and Mg2+. Furthermore, we show that the heavy metals Cd2+ and Hg2+, dichloroisocoumarin (a general serine protease inhibitor), and N-ethyl maleimide (NEM, a specific thiol reagent) are potent inhibitors of both the Mg(2+)- and Ca2+/Mg(2+)-stimulated DNA fragmentation. In contrast, two other serine protease inhibitors, N-alpha-tosyl-L-lysine chloromethylketone and N-tosyl-L-phenylalanine chloromethylketone are weak and ineffective, respectively, as inhibitors of DNA cleavage. Increasing inhibition of DNA cleavage is accompanied by a shift in the size of the cleaved DNA fragments, which increases from mono- + oligo-nucleosomes-->30-50 kbp-->200-300 kbp--> > or = 700 kbp-->intact DNA. Dithiothreitol, a dithiol, blocks NEM and dichloroisocoumarin inhibition, and since Cd2+ and Hg2+ are also potent--SH blocking agents it is proposed that a critical thiol is involved in the cleavage of DNA into both large kbp fragments and oligonucleosomal-sized fragments.
- Published
- 1994
- Full Text
- View/download PDF
45. DNA fragmentation into 200-250 and/or 30-50 kilobase pair fragments in rat liver nuclei is stimulated by Mg2+ alone and Ca2+/Mg2+ but not by Ca2+ alone.
- Author
-
Cain K, Inayat-Hussain SH, Wolfe JT, and Cohen GM
- Subjects
- Animals, Calcium pharmacology, Cell Nucleus drug effects, Cell Nucleus metabolism, DNA Damage drug effects, Drug Synergism, Flow Cytometry, Magnesium pharmacology, Male, Rats, Time Factors, Apoptosis physiology, Cations, Divalent pharmacology, DNA Damage physiology, Liver metabolism
- Abstract
Internucleosomal cleavage of DNA has often been regarded as the biochemical hallmark of apoptosis. We now demonstrate in isolated rat liver nuclei that DNA is initially cleaved into > or = 700, 200-250 kbp and 30-50 kbp fragments via a multi-step process, which is activated by Mg2+ and Mg2+(+)Ca2+ but not by Ca2+ alone. The subsequent internucleosomal cleavage requires both cations. These findings demonstrate that a key event in the apoptotic process is the fragmentation of DNA into large kbp fragments by either a Mg(2+)-dependent process (which can be potentiated by Ca2+) and/or by a Ca2+/Mg2+ activated endonuclease(s).
- Published
- 1994
- Full Text
- View/download PDF
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