University of Tasmania
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Towards understanding the mechanism of clioquinol neurotoxicity

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posted on 2023-05-27, 10:39 authored by Chhetri, J
The quinoline derivative clioquinol (CQ) was widely used as a topical disinfectant for skin conditions and as an oral antibiotic against diarrhoea. However, in 1970 the oral formulation was taken off the market in many countries after it was linked to about 10 000 cases of subacute myelo-optic neuropathy (SMON) in Japan. SMON was characterised by optic neuritis and axonopathy of the spinal cord. Although a clear mechanistic connection is still disputed, a clear reduction in SMON cases was witnessed following drug withdrawal from the market. Furthermore, the common pathological features of SMON-associated neurotoxicity have been successfully recapitulated in CQ-treated animals, which support a direct connection. Several hypotheses have tried to explain CQ-dependent toxicity such as CQ-induced DNA damage and oxidative stress. However, none of these studies has attempted to explain the restriction of CQ-induced neurotoxicity to the Japanese population. Despite this toxicity, CQ and its structural analogues are currently under investigation as a disease modifying treatment for the neurodegenerative disorders such as Alzheimer's and Huntington's Disease, with some encouraging preclinical and clinical results. In light of the re-emergence of CQ and its structural analogues, it is crucial to understand the underlying mechanism of CQ-induced toxicity beyond the scope of SMON to prevent any potential CQ-associated risks to future patients. From a previous research project that screened marketed drugs and drug-like compounds against their potential to cause mitochondrial dysfunction in vitro, CQ and 8-HQ were identified as hits (unpublished). The mitochondrial liability of these compounds (0.5-10) ˜í¬¿M was subsequently confirmed in RGC5 cells by measuring galactose-hypersensitive ATP levels, lipid peroxidation and cellular viability. However, the confirmation of CQ-induced mitochondrial toxicity in another cell line, HepG2, failed. Prior unrelated observations (N. Gueven, unpublished) showed that these cell lines differ in the response to oxidants, which is associated with different cellular levels of the antioxidant enzyme NADPH Quinone Oxidoreductase 1 (NQO1). NQO1-dependent CQ associated-mitochondrial-dysfunction was confirmed in isogenic cell lines that differ only in the expression levels of NQO1. NQO1 expression was inversely correlated to CQ-induced mitochondrial-dysfunction. While cells with low NQO1 were highly sensitive to CQ-induced mitochondrial-dysfunction, recombinant NQO1 expression in these cells provided protection against CQ-toxicity. CQ-induced reduction of cellular ATP levels, increased lipid peroxidation and elevated cell death could be attenuated by concomitant treatment with different antioxidants, implicating oxidative stress as the core mechanism of CQ-induced toxicity. Furthermore, biochemical studies also revealed that CQ and 8-HQ directly inhibited NQO1 enzyme activity at a concentration range of 10-100 ˜í¬¿M. In order to translate the in-vitro findings of NQO1-dependent CQ toxicity into an in-vivo situation, zebrafish were selected as a suitable animal model. Zebrafish are characterised by variable NQO1 expression in the retina during different stages of their life. While larval zebrafish are reported to lack retinal NQO1 expression, the protein is highly expressed in the adult zebrafish retina. The observed general CQ-induced toxicity in zebrafish was strain-dependent. Petshop (PET) strain zebrafish larvae were less sensitive compared to a commonly used laboratory strain (AB). The maximum tolerated concentration of CQ that did not lead to systemic toxicity was 10 ˜í¬¿M in PET and 3 ˜í¬¿M in AB. To demonstrate retinal CQ effects in this model, visual CQ toxicity in PET zebrafish larvae was determined by measuring eye velocity as a function of different stimulus velocities. Comparable to the human situation, CQ inhibited visual function in a dose-dependent manner between 3-10 ˜í¬¿M, which is surprisingly consistent with the concentration range that caused mitochondrial-dysfunction in-vitro. In contrast to larval responses, even the highest CQ concentration (10 ˜í¬¿M) did not affect visual function in the adult zebrafish. This is in line with the in-vitro results where high NQO1-expressing HepG2 cells were resistant to CQ-induced mitochondrial-dysfunction. NQO1-dependent protection against CQ-induced visual impairment was confirmed by the presence of NQO1 enzyme activity in the adult zebrafish retina. Consistent with this, pharmacological inactivation of NQO1 resulted in CQ-induced oxidative stress in the retina and acute systemic toxicity in the adult fish. Overall, these results highlight the importance of NQO1 expression in mitigating CQ toxicity both in-vitro and in-vivo and that CQ induces mitochondrial-dysfunction through the generation of oxidative stress. Strikingly, nearly 50 years after the drug was retracted from the market, this connection between NQO1 and CQ-toxicity might explain the geographic restriction of SMON cases. It is important to note that a much higher prevalence of the inactivating C609T NQO1 polymorphism has been reported for the Japanese population compared to the European population. Based on the results of this study, this population group should, therefore, be highly susceptible to the toxicity of CQ. The results of the present study could for the first time explain the geographic restriction of CQ-induced neurotoxicity to Japan. Currently, this hypothesis is tested by assessing the presence of the C609T NQO1 polymorphism in Japanese SMON survivors. Furthermore, if CQ or its derivatives are to be employed for the treatment of neurodegenerative diseases, it appears imperative that NQO1 status of the patients should be ascertained before the start of the treatment. Therefore, in addition to potentially solving a long-standing medical mystery, this project offers a significant benefit towards personalised medicine to minimise drug treatment-associated risk to a specific group of patients.


Publication status

  • Unpublished

Rights statement

Copyright 2016 the author Chapter 1 appears to be, in part, the equivalent of a post-print version of an article published as: Chhetri, J., Jacobson, G., Gueven, N., 2014. Zebrafish-on the move towards ophthalmological research, Eye, 7(10), 367-380 Chapter 1 appears to be, in part, the equivalent of an Accepted Manuscript of an article published by Taylor & Francis Group in Expert opinion on therapeutic targets on 13/1/2016, available online: (Chhetri, J., Gueven, N. 2016. Targeting mitochondrial function to protect against vision loss, Expert opin ther targets, 20(6), 721-736 Chapters 1 and 2 appear to be, in part, the equivalent of a post-print version of an article published as: Chhetri, J., King, A. E., Gueven, N., 2018. Alzheimer's disease and NQO1: is there a link?, Current Alzheimer research, 15(1), 56-66

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