Research Studies
Study Tracker
Oxidative Stress in Zebrafish Gut Induced by Individual and Combined Exposure to Amoxicillin, Arsenic and Fluoride: Engagement of Nrf2-Keap1-ARE Pathway.Abstract
This study investigates the effects of oxidative stress on zebrafish gut as a consequence of exposure to amoxicillin (AMX, 100 mg/L) alone or in combination with arsenic (As2O3, 50 ug/L, equivalent to 37.87 ug/L of As), and fluoride (NaF, 15 mg/L, equivalent to 6.8 mg/L of F) for 15 days. While the toxic ramifications of antibiotics and heavy metals have been extensively studied individually, their co-toxicity on aquatic piscine models and the associated cellular stress responses remain poorly understood. The research focuses on understanding the mechanisms of stress and simultaneous toxicity in this specific scenario. The study revealed elevated levels of cellular ROS, MDA, and GSH, along with increased CAT enzyme activity in all treated groups, indicating oxidative stress. Histological damages, including increased numbers of goblet cells and necrotic spots, further confirmed oxidative injury. Gene expression analysis showed enhancement of stress-responsive genes such as nrf2, GPx-1, hsp70, keap1, nqo1, Cyp 1a, ucp2, Cu/Zn-SOD, and Mn-SOD, aligning with the observed biochemical changes. The translocation and heightened expression of Nrf2 in the nucleus were notable across all treatments. Furthermore, the combined effects of AMX, As, and F were more pronounced compared to other combinations, while exposure to AMX alone had the least impact. These findings underscore the role of the Nrf2-Keap1-ARE pathway in mediating the observed responses and highlight the synergistic toxicity of AMX, As, and F in the environment, posing a significant threat to both ecosystems and human health.
Conflicts of Interest
The authors declare no conflicts of interest.
Data Availability Statement
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.
Supporting Information
jat4842-sup-0001-TableS1.docxWord 2007 document , 15.3 KB
- 1984. “ [13] Catalase In Vitro.” In Methods in Enzymology, vol. 105, 121–126. Academic Press. https://doi.org/10.1016/S0076-6879(84)05016-3.
- 2003. “Interaction of Human NAD (P) H: Quinone Oxidoreductase 1 (NQO1) With the Tumor Suppressor Protein p53 in Cells and Cell-Free Systems.” Journal of Biological Chemistry 278, no. 12: 10368–10373. https://doi.org/10.1074/jbc.M211981200. , , , et al.
- 2018. “Sodium Fluoride Induces Hepato-Renal Oxidative Stress and Pathophysiological Changes in Experimental Animals.” Open Journal of Apoptosis 7, no. 1: 1–23. https://doi.org/10.4236/ojapo.2018.71001. , , , and .
- 1963. “Improved Method for Determination of Blood Glutathione.” Journal of Laboratory and Clinical Medicine 61: 882–888. , , and .
- 2017. “Assessment of Potential Health Risk of Fluoride Consumption Through Rice, Pulses, and Vegetables in Addition to Consumption of Fluoride-Contaminated Drinking Water of West Bengal, India.” Environmental Science and Pollution Research 24: 20300–20314. https://doi.org/10.1007/s11356-017-9649-2. , , , , and .
- 1978. “ Microsomal Lipid Peroxidation.” In Methods in Enzymology, vol. 52, 302–310. Academic Press. https://doi.org/10.1016/S0076-6879(78)52032-6. , and .
- 2017. “The Gastrointestinal Tract as a Key Target Organ for the Health-Promoting Effects of Dietary Proanthocyanidins.” Frontiers in Nutrition 3: 57. https://doi.org/10.3389/fnut.2016.00057. , , , and .
- 2020. “Occurrence, Seasonal Variation and Human Exposure to Pharmaceuticals and Personal Care Products in Surface Water, Groundwater and Drinking Water in Lagos State, Nigeria.” Emerging Contaminants 6: 124–132. https://doi.org/10.1016/j.emcon.2020.02.004. , , , , and .
- 2017. “Effect of Amoxicillin Exposure on Brain, Gill, Liver, and Kidney of Common Carp (Cyprinus?carpio): The Role of Amoxicilloic Acid.” Environmental Toxicology 32, no. 4: 1102–1120. https://doi.org/10.1002/tox.22307. , , , et al.
- 2020. “Exposure to a High Dose of Amoxicillin Causes Behavioral Changes and Oxidative Stress in Young Zebrafish.” Metabolic Brain Disease 35: 1407–1416. https://doi.org/10.1007/s11011-020-00610-6. , , , et al.
- 2015. “A Concurrent Exposure to Arsenic and Fluoride From Drinking Water in Chihuahua, Mexico.” International Journal of Environmental Research and Public Health 12, no. 5: 4587–4601. https://doi.org/10.3390/ijerph120504587. , , , et al.
- 2022. “Daidzein Normalized Gentamicin-Induced Nephrotoxicity and Associated Pro-Inflammatory Cytokines in MDCK and Zebrafish: Possible Mechanism of Nephroprotection.” Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 258: 109364. https://doi.org/10.1016/j.cbpc.2022.109364. , , , et al.
- 2022. “The Role of Glutathione Peroxidase-1 in Health and Disease.” Free Radical Biology and Medicine 188: 146–161. https://doi.org/10.1016/j.freeradbiomed.2022.06.004. , and .
- 2022. “Effects of Plastic Particles on Aquatic Invertebrates and Fish–A Review.” Environmental Toxicology and Pharmacology 96: 104013. https://doi.org/10.1016/j.etap.2022.104013. , , , , and .
- 2013. “The Zebrafish Reference Genome Sequence and Its Relationship to the Human Genome.” Nature 496, no. 7446: 498–503. https://doi.org/10.1038/nature12111. , , , et al.
- 2004. “Nrf2 Signaling in Coordinated Activation of Antioxidant Gene Expression.” Free Radical Biology and Medicine 36, no. 10: 1199–1207. https://doi.org/10.1016/j.freeradbiomed.2004.02.074.
- 2016. “Immunological Aspects of Intestinal Mucus and Mucins.” Nature Reviews Immunology 16, no. 10: 639–649. , and .
- 2011. “Protective Effects of Nigella?sativa Extract Against Chromium (VI)-Induced Genotoxicity in Nile Tilapia (Oreochromis?niloticus) and Zebrafish (Danio?rerio).” Global Veterinaria 7, no. 3: 283–293. , , , , and .
- 2018. “Global Increase and Geographic Convergence in Antibiotic Consumption Between 2000 and 2015.” Proceedings of the National Academy of Sciences of the United States of America 115, no. 15: E3463–E3470. https://doi.org/10.1073/pnas.1717295115. , , , et al.
- 2015. “Increased Activity of Mitochondrial Uncoupling Protein 2 Improves Stress Resistance in Cultured Endothelial Cells Exposed In Vitro to High Glucose Levels.” American Journal of Physiology. Heart and Circulatory Physiology 309, no. 1: H147–H156. https://doi.org/10.1152/ajpheart.00759.2014. , , and .
- 2003. “Significance of Antibiotics in the Environment.” Journal of Antimicrobial Chemotherapy 52, no. 1: 5–7. https://doi.org/10.1093/jac/dkg293.
- 2021. “Ecological Risk Assessment of Amoxicillin, Enrofloxacin, and Neomycin: Are Their Current Levels in the Freshwater Environment Safe?” Toxics 9, no. 8: 196. https://doi.org/10.3390/toxics9080196. , , , et al.
- 2024. “Interplay Between Fluorine and Cadmium on Intestinal Accumulation, Oxidative Stress, Permeability and Inflammatory Response in Rats.” Ecotoxicology and Environmental Safety 284: 117030. https://doi.org/10.1016/j.ecoenv.2024.117030. , , , et al.
- 2015. “Cellular and Transcriptional Responses in Microcystis Aeruginosa Exposed to Two Antibiotic Contaminants.” Microbial Ecology 69: 535–543. https://doi.org/10.1007/s00248-014-0515-1. , , and .
- 2001. “Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2???CT Method.” Methods 25, no. 4: 402–408. https://doi.org/10.1006/meth.2001.1262. , and .
- 2015. “Insight Into the Oxidative Stress Induced by Lead and/or Cadmium in Blood, Liver and Kidneys.” Food and Chemical Toxicology 78: 130–140. https://doi.org/10.1016/j.fct.2015.02.011. , , , and .
- 2019. “Mixture Effect of Arsenic and Fluoride at Environmentally Relevant Concentrations in Zebrafish (Danio?rerio) Liver: Expression Pattern of Nrf2 and Related Xenobiotic Metabolizing Enzymes.” Aquatic Toxicology 213: 105219. https://doi.org/10.1016/j.aquatox.2019.06.002. , , , et al.
- 2021. “Combined Effect of Arsenic and Fluoride at Environmentally Relevant Concentrations in Zebrafish (Danio?rerio) Brain: Alterations in Stress Marker and Apoptotic Gene Expression.” Chemosphere 269: 128678. https://doi.org/10.1016/j.chemosphere.2020.128678. , , , et al.
- 2024. “Gut Microbiota Perturbation and Subsequent Oxidative Stress in Gut and Kidney Tissues of Zebrafish After Individual and Combined Exposure to Inorganic Arsenic and Fluoride.” Science of the Total Environment 957: 177519. https://doi.org/10.1016/j.scitotenv.2024.177519. , , , , , and .
- 2025. “Oxidative Stress in Kidney of Zebrafish Due to Individual and Combined Exposure to Amoxicillin, Arsenic, and Fluoride: Involving Nrf2-Keap1-ARE Pathway.” Journal of Applied Toxicology 45: 964–975. https://doi.org/10.1002/jat.4763. , , , and .
- 2014. “Induction of Oxidative Stress and Related Transcriptional Effects of Sodium Fluoride in Female Zebrafish Liver.” Bulletin of Environmental Contamination and Toxicology 93: 64–70. https://doi.org/10.1007/s00128-014-1271-0. , and .
- 2015. “Sodium Fluoride Affects Zebrafish Behaviour and Alters mRNA Expressions of Biomarker Genes in the Brain: Role of Nrf2/Keap1.” Environmental Toxicology and Pharmacology 40, no. 2: 352–359. https://doi.org/10.1016/j.etap.2015.07.003. , , and .
- 2015. “Sodium Fluoride Generates ROS and Alters Transcription of Genes for Xenobiotic Metabolizing Enzymes in Adult Zebrafish (Danio?rerio) Liver: Expression Pattern of Nrf2/Keap1 (INrf2).” Toxicological Mechanisms and Methods 25: 364–373. https://doi.org/10.3109/15376516.2015.1025348. , , and .
- 2012. “Amoxycillin/Clavulanic Acid Combinations (Augmentin® 375 and 625® Tablets) Induce-Oxidative Stress, and Renal and Hepatic Damage in Rats.” African Journal of Pharmacy and Pharmacology 6, no. 33: 2441–2449. https://doi.org/10.5897/AJPP10.165. , , and .
- 2019. “Genotoxic and Cytotoxic Alterations Induced by Environmentally-Relevant Concentrations of Amoxicillin in Blood Cells of Cyprinus?carpio.” Chemosphere 236: 124323. https://doi.org/10.1016/j.chemosphere.2019.07.054. , , , et al.
- 2020. “Impact of Neonicotinoids to Aquatic Invertebrates—In Vitro Studies on Mytilus?galloprovincialis: A Review.” Journal of Marine Science and Engineering 8, no. 10: 801. https://doi.org/10.3390/jmse8100801. , , , and .
- 2023. “Research Progress of Glutathione Peroxidase Family (GPX) in Redoxidation.” Frontiers in Pharmacology 14: 1147414. https://doi.org/10.3389/fphar.2023.1147414. , , , and .
- 2001. “A New Mathematical Model for Relative Quantification in Real-Time RT–PCR.” Nucleic Acids Research 29, no. 9: e45. https://doi.org/10.1093/nar/29.9.e45.
- 2019. “Toxic Effect of Arsenic Trioxide on Biochemical Response in Catfish, Clarias?batrachus.” International Journal of Recent Scientific Research 10, no. 08: 34033–34036. https://doi.org/10.24327/ijrsr.2019.1008.3804. , and .
- 2020. “Maternal Exposure to Environmental Antibiotic Mixture During Gravid Period Predicts Gastrointestinal Effects in Zebrafish Offspring.” Journal of Hazardous Materials 399: 123009. https://doi.org/10.1016/j.jhazmat.2020.123009. , , , et al.
- 2019. “The Hsp70 Chaperone Network.” Nature Reviews Molecular Cell Biology 20: 665–680. https://doi.org/10.1038/s41580-019-0133-3. , , , and .
- 2007. “Mechanisms of Cell Death in Oxidative Stress.” Antioxidants & Redox Signaling 9, no. 1: 49–89. https://doi.org/10.1089/ars.2007.9.49. , , , et al.
- 2014. “Low Dose of Arsenic Trioxide Triggers Oxidative Stress in Zebrafish Brain: Expression of Antioxidant Genes.” Ecotoxicology and Environmental Safety 107: 1–8. https://doi.org/10.1016/j.ecoenv.2014.05.012. , , , and .
- 2017. “Differential Modulation of Cellular Antioxidant Status in Zebrafish Liver and Kidney Exposed to Low Dose Arsenic Trioxide.” Ecotoxicology and Environmental Safety 135: 173–182. https://doi.org/10.1016/j.ecoenv.2016.09.025. , , , and .
- 2019. “Environmentally Relevant Concentration of Chromium Activates Nrf2 and Alters Transcription of Related XME Genes in Liver of Zebrafish.” Chemosphere 214: 35–46. https://doi.org/10.1016/j.chemosphere.2018.09.104. , , , and .
- 2020. “Environmentally Relevant Concentration of Chromium Induces Nuclear Deformities in Erythrocytes and Alters the Expression of Stress-Responsive and Apoptotic Genes in Brain of Adult Zebrafish.” Science of the Total Environment 703: 135622. https://doi.org/10.1016/j.scitotenv.2019.135622. , , , and .
- 2023. “Effect of Arsenic Stress on the Intestinal Structural Integrity and Intestinal Flora Abundance of Cyprinus?carpio.” Frontiers in Microbiology 14: 1179397. https://doi.org/10.3389/fmicb.2023.1179397. , , , et al.
- 2021. “The Role of NRF2/KEAP1 Signaling Pathway in Cancer Metabolism.” International Journal of Molecular Sciences 22: 4376. https://doi.org/10.3390/ijms22094376. , , , and .
- 2013. “In Vivo Effect of Arsenic Trioxide on Keap1-p62-Nrf2 Signaling Pathway in Mouse Liver: Expression of Antioxidant Responsive Element-Driven Genes Related to Glutathione Metabolism.” International Scholarly Research Notices 2013, no. 1: 817693. https://doi.org/10.1155/2013/817693. , , , et al.
- 1997. “Functional Antioxidant Responsive Elements.” Proceedings of the National Academy of Sciences of the United States of America 94, no. 10: 5361–5366. https://doi.org/10.1073/pnas.94.10.5361. , and .
- World Health Organization. 2011. Guidelines for Drinking-Water Quality. 4th ed. WHO.
- 2023. “Immobilization, Oxidative Stress and Antioxidant Response of Daphnia?magna to Amoxicillin and Ciprofloxacin.” Environmental Toxicology and Pharmacology 98: 104078. https://doi.org/10.1016/j.etap.2023.104078. , , , , , and .
- 2013. “Antibiotics in the Offshore Waters of the Bohai Sea and the Yellow Sea in China: Occurrence, Distribution and Ecological Risks.” Environmental Pollution 174: 71–77. https://doi.org/10.1016/j.envpol.2012.11.008. , , , et al.
- 2018. “Environmental Concentrations of Antibiotics Impair Zebrafish Gut Health.” Environmental Pollution 235: 245–254. https://doi.org/10.1016/j.envpol.2017.12.073. , , , et al.
- 2015. “Dietary Squid Ink Polysaccharide Induces Goblet Cells to Protect Small Intestine From Chemotherapy Induced Injury.” Food & Function 6, no. 3: 981–986. , , , and .
ABSTRACT ONLINE AT
https://analyticalsciencejournals.onlinelibrary.wiley.com/doi/10.1002/jat.4842