Advertisement

Toxicity and metabolism of 3-bromopyruvate in Caenorhabditis elegans

3-溴丙酮酸对秀丽隐杆线虫的毒性和代谢的影响

Abstract

In this study, we aimed to evaluate the toxic effects, changes in life span, and expression of various metabolism-related genes in Caenorhabditis elegans, using RNA interference (RNAi) and mutant strains, after 3-bromopyruvate (3-BrPA) treatment. C. elegans was treated with various concentrations of 3-BrPA on nematode growth medium (NGM) plates, and their survival was monitored every 24 h. The expression of genes related to metabolism was measured by the real-time fluorescent quantitative polymerase chain reaction (qPCR). Nematode survival in the presence of 3-BrPA was also studied after silencing three hexokinase (HK) genes. The average life span of C. elegans cultured on NGM with 3-BrPA was shortened to 5.7 d compared with 7.7 d in the control group. hxk-1, hxk-2, and hxk-3 were overexpressed after the treatment with 3-BrPA. After successfully interfering hxk-1, hxk-2, and hxk-3, the 50% lethal concentration (LC50) of all mutant nematodes decreased with 3-BrPA treatment for 24 h compared with that of the control. All the cyp35 genes tested were overexpressed, except cyp-35B3. The induction of cyp-35A1 expression was most obvious. The LC50 values of the mutant strains cyp-35A1, cyp-35A2, cyp-35A4, cyp-35B3, and cyp-35C1 were lower than that of the control. Thus, the toxicity of 3-BrPA is closely related to its effect on hexokinase metabolism in nematodes, and the cyp-35 family plays a key role in the metabolism of 3-BrPA.

概要

目 的

通过3-溴丙酮酸 (3-BrPA)处理秀丽隐杆线虫 (Caenorhabditis elegans), 观察 3-BrPA 对线虫的毒性和生存周期的影响. 通过秀丽隐杆线虫 RNA 干扰 (RNAi) 和突变株, 分析 3-BrPA 对线虫糖酵解途径己糖激酶家族和药物代谢的关键酶细胞色素 P450(cytochrome P450, CYP) 家族的影响.

创新点

首次报道了 3-BrPA 对秀丽隐杆线虫有毒性作用, 己糖激酶是 3-BrPA 对线虫作用的重要靶点, 而 CYP-35A 家族是线虫代谢 3-BrPA 的主要酶类.

方 法

用不同浓度的 3-BrPA 处理秀丽隐杆线虫, 每 24 h 监测一次存活率; 用实时荧光定量聚合酶链反应 (qPCR) 检测代谢相关基因的表达; 通过 RNAi 沉默己糖激酶家族基因 hxk-1hxk-2hxk-3; 计算 3-BrPA 处理 hxk 家族 RNAi 株和细胞色素 P450 cyp-35 家族突变株后的致死中浓度 (LC50).

结 论

3-BrPA 对线虫有明显的毒性效应 (图 1); 与对照组比较, 3-BrPA 处理组的线虫平均寿命明显缩 短 (图 2); 3-BrPA 处理线虫后 hxk-1hxk-2hxk-3 的信使 RNA (mRNA) 表达明显升高 (P < 0.05, 图 5); 3-BrPA 处理 hxk RNAi 株后的 LC50 均减小 (P<0.05, 表 5); 3-BrPA 处理 cyp-35 突变株后的 LC50 也均减小 (P<0.05, 表 6). 综上所述, 3-BrPA 的毒性与其对秀丽隐杆线虫己糖激酶代谢的影响密切相关; CYP-35 家族在线虫中对 3_BrPA 代谢中起着关键作用.

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

References

  1. Alcántar-Aguirre FC, Chagolla A, Tiessen A, et al., 2013. ATP produced by oxidative phosphorylation is channeled toward hexokinase bound to mitochondrial porin (VDAC) in beetroots (Beta vulgaris). Planta, 237(6):1571–1583. https://doi.org/10.1007/s00425-013-1866-4

  2. Cárdenas ML, Cornish-Bowden A, Ureta T, 1998. Evolution and regulatory role of the hexokinases. Biochim Biophys Acta, 1401(3):242–264. https://doi.org/10.1016/S0167-4889(97)00150-X

  3. Chen FZ, Wang H, Lai JD, et al., 2018. 3-Bromopyruvate reverses hypoxia-induced pulmonary arterial hypertension through inhibiting glycolysis: in vitro and in vivo studies. Int J Cardiol, 266:236–241. https://doi.org/10.1016/j.ijcard.2018.03.104

  4. Chen GH, Zhang YD, Liang JF, et al., 2018. Deregulation of hexokinase II is associated with glycolysis, autophagy, and the epithelial-mesenchymal transition in tongue squamous cell carcinoma under hypoxia. BioMed Res Int, 2018:8480762. https://doi.org/10.1155/2018/8480762

  5. de Meis L, Grieco MAB, Galina A, 1992. Reversal of oxidative phosphorylation in submitochondrial particles using glucose 6-phosphate and hexokinase as an ATP regenerating system. FEBS Lett, 308(2):197–201. https://doi.org/10.1016/0014-5793(92)81273-O

  6. Dewaal D, Nogueira V, Terry AR, et al., 2018. Hexokinase-2 depletion inhibits glycolysis and induces oxidative phosphorylation in hepatocellular carcinoma and sensitizes to metformin. Nat Commun, 9:446. https://doi.org/10.1038/s41467-017-02733-4

  7. Dyląg M, Lis P, Niedźwiecka K, et al., 2013. 3-Bromopyruvate: a novel antifungal agent against the human pathogen Cryptococcus neoformans. Biochem Biophys Res Commun, 434(2):322–327. https://doi.org/10.1016/j.bbrc.2013.02.125

  8. el Sayed SM, Baghdadi H, Zolaly M, et al., 2017. The promising anticancer drug 3-bromopyruvate is metabolized through glutathione conjugation which affects chemoresistance and clinical practice: an evidence-based view. Med Hypotheses, 100:67–77. https://doi.org/10.1016/j.mehy.2017.01.014

  9. García-Espiñeira MC, Tejeda-Benitez LP, Olivero-Verbel J, 2018. Toxic effects of bisphenol A, propyl paraben, and triclosan on Caenorhabditis elegans. Int J Environ Res Public Health, 15(4):684. https://doi.org/10.3390/ijerph15040684

  10. Guengerich FP, 2008. Cytochrome P450 and chemical toxicology. Chem Res Toxicol, 21(1):70–83. https://doi.org/10.1021/tx700079z

  11. Huang RT, Huang Q, Wu GL, et al., 2017. Evaluation of the antioxidant property and effects in Caenorhabditis elegans of Xiangxi flavor vinegar, a Hunan local traditional vinegar. J Zhejiang Univ-Sci B (Biomed & Biotechnol), 18(4):324–333. https://doi.org/10.1631/jzus.B1600088

  12. Kim JS, Ahn KJ, Kim JA, et al., 2008. Role of reactive oxygen species-mediated mitochondrial dysregulation in 3-bromopyruvate induced cell death in hepatoma cells: ROS-mediated cell death by 3-BrPA. J Bioenerg Biomembr, 40(6):607–618. https://doi.org/10.1007/s10863-008-9188-0

  13. Kim W, Yoon JH, Jeong JM, et al., 2007. Apoptosis-inducing antitumor efficacy of hexokinase II inhibitor in hepatocellular carcinoma. Mol Cancer Ther, 6(9):2554–2562. https://doi.org/10.1158/1535-7163.MCT-07-0115

  14. Lai CH, Chou CY, Ch’ang LY, et al., 2000. Identification of novel human genes evolutionarily conserved in Caenorhabditis elegans by comparative proteomics. Genome Res, 10(5):703–713. https://doi.org/10.1101/gr.10.5.703

  15. Menzel R, Bogaert T, Achazi R, 2001. A systematic gene expression screen of Caenorhabditis elegans cytochrome P450 genes reveals CYP35 as strongly xenobiotic inducible. Arch Biochem Biophys, 395(2):158–168. https://doi.org/10.1006/abbi.2001.2568

  16. Mycielska ME, Moser C, Wagner C, et al., 2012. Abstract 3211: inhibition of Hsp90 impairs expression of VDAC in plasma and mitochondrial membrane influencing cancer cell metabolism. Cancer Res, 72(S8):3211. https://doi.org/10.1158/1538-7445.AM2012-3211

  17. Olsen BB, Gjedde A, Vilstrup MH, et al., 2019. Linked hexokinase and glucose-6-phosphatase activities reflect grade of ovarian malignancy. Mol Imaging Biol, 21(2):375–381. https://doi.org/10.1007/s11307-018-1247-2

  18. Patra KC, Hay N, 2013. Hexokinase 2 as oncotarget. Oncotarget, 4(11):1862–1863. https://doi.org/10.18632/oncotarget.1563

  19. Patra KC, Wang Q, Bhaskar PT, et al., 2013. Hexokinase 2 is required for tumor initiation and maintenance and its systemic deletion is therapeutic in mouse models of cancer. Cancer Cell, 24(2):213–228. https://doi.org/10.1016/j.ccr.2013.06.014

  20. Qiao Y, Zhao YL, Wu QL, 2014. Full toxicity assessment of Genkwa Flos and the underlying mechanism in nematode Caenorhabditis elegans. PLoS ONE, 9(3):e91825. https://doi.org/10.1371/journal.pone.0091825

  21. Roh JY, Choi J, 2011. Cyp35a2 gene expression is involved in toxicity of fenitrothion in the soil nematode Caenorhabditis elegans. Chemosphere, 84(10):1356–1361. https://doi.org/10.1016/j.chemosphere.2011.05.010

  22. Slein MW, Cori GT, Cori CF, 1950. A comparative study of hexokinase from yeast and animal tissues. J Biol Chem, 186(2):763–780.

  23. Wilson JE, 2003. Isozymes of mammalian hexokinase: structure, subcellular localization and metabolic function. J Exp Biol, 206:2049–2057. https://doi.org/10.1242/jeb.00241

  24. Xu D, Jin JZ, Yu H, et al., 2017. Chrysin inhibited tumor glycolysis and induced apoptosis in hepatocellular carcinoma by targeting hexokinase-2. J Exp Clin Cancer Res, 36(1):44. https://doi.org/10.1186/s13046-017-0514-4

  25. Yadav S, Pandey SK, Kumar A, et al., 2017a. Antitumor and chemosensitizing action of 3-bromopyruvate: implication of deregulated metabolism. Chem Biol Interact, 270:73–89. https://doi.org/10.1016/j.cbi.2017.04.015

  26. Yadav S, Pandey SK, Singh VK, et al., 2017b. Molecular docking studies of 3-bromopyruvate and its derivatives to metabolic regulatory enzymes: implication in designing of novel anticancer therapeutic strategies. PLoS ONE, 12(5): e0176403. https://doi.org/10.1371/journal.pone.0176403

Download references

Acknowledgments

We thank technical guidance of Lihsia CHEN, PhD from the College of Biological Sciences, University of Minnesota, USA.

Author information

Conceptualization: Qiao-ling GU, Yan ZHANG, Gen CHEN; Methodology: Qiao-ling GU, Yan ZHANG, Xi-mei FU, Zhao-lian LU; Formal analysis: Qiao-ling GU, Yan ZHANG, Xi-mei FU, Zhao-lian LU; Resources: Gen CHEN, Rong MA, Wei KOU, Yong-mei LAN; Data curation: Qiao-ling GU, Yan ZHANG, Xi-mei FU, Yao YU; Writing original draft: Qiao-ling GU, Yan ZHANG, Xi-mei FU, Yao YU; Supervision: Gen CHEN; Funding acquisition: Qiao-ling GU, Yan ZHANG, Gen CHEN.

Correspondence to Gen Chen.

Ethics declarations

Qiao-ling GU, Yan ZHANG, Xi-mei FU, Zhao-lian LU, Yao YU, Gen CHEN, Rong MA, Wei KOU, and Yong-mei LAN declared that they have no conflict of interest.

All institutional and national guidelines for the care and use of laboratory animals were followed.

Additional information

Project supported by the National Natural Science Foundation of China (Nos. 31172174 and 81460677), the Fundamental Research Funds for the Central Universities of China (No. 31920170039), and the Natural Science Found of Gansu Province (No. 18JR3RA283), China

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Gu, Q., Zhang, Y., Fu, X. et al. Toxicity and metabolism of 3-bromopyruvate in Caenorhabditis elegans. J. Zhejiang Univ. Sci. B 21, 77–86 (2020). https://doi.org/10.1631/jzus.B1900370

Download citation

Key words

  • 3-Bromopyruvate
  • Caenorhabditis elegans
  • Hexokinase
  • Cytochrome P450

关键词

  • 3-溴丙酮酸
  • 秀丽隐杆线虫
  • 己糖激酶
  • 细胞色素 P450

CLC number

  • R965.3