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Prof. Dr. Angela Mally
Department of Toxicology
University of Würzburg
Versbacher Str. 9
Phone/fax: +49 931 31-81194
Point of Contact
Angela Mally (email point of contact)
- Angela Mally
|Author status||OECD status||OECD project||SAAOP status|
|Under development: Not open for comment. Do not cite|
This AOP was last modified on October 26, 2017 08:49
|Inhibition of mitochondrial DNA polymerase gamma (Pol gamma)||October 25, 2017 07:48|
|Depletion, mtDNA||October 25, 2017 07:49|
|Dysfunction, Mitochondria||October 25, 2017 07:49|
|Increase, Cytotoxicity (renal tubular cell)||September 16, 2017 10:16|
|Occurrence, Kidney toxicity||September 16, 2017 10:16|
|Inhibition, mitochondrial DNA polymerase gamma (Pol gamma) leads to Depletion, mtDNA||October 25, 2017 07:52|
|Depletion, mtDNA leads to Dysfunction, Mitochondria||October 25, 2017 07:53|
|Dysfunction, Mitochondria leads to Increase, Cytotoxicity (renal tubular cell)||October 25, 2017 07:53|
|Increase, Cytotoxicity (renal tubular cell) leads to Occurrence, Kidney toxicity||October 25, 2017 07:54|
|Tenofovir||October 25, 2017 07:45|
|Tenofovir disoproxil fumarate||October 25, 2017 07:46|
|Adefovir||October 25, 2017 07:46|
|Adefovir dipivoxil||October 25, 2017 07:46|
|Cidofovir||October 25, 2017 07:47|
This Adverse Outcome Pathway describes the sequential key events that link inhibition of mitochondrial DNA polymerase gamma (Pol gamma) to kidney toxicity. Nucleoside and nucleotide (nucleos(t)ide) analogs are widely used as antiviral drugs for the effective treatment of viral infections including HIV and chronic Hepatitis B virus infections. As structural analogs of substrate nucleotides, these drugs act as chain terminators of viral DNA synthesis via competitive inhibition of reverse transcriptase or viral DNA polymerases, thereby blocking virus replication. Besides targeting viral enzymes, nucleos(t)ide antiviral agents are also substrates for human DNA polymerases, which may lead to moderate to life-threatening adverse drug reactions, including peripheral neuropathy, myopathy, lactic acidosis, and acute and chronic kidney injury [1-4]. Toxicity of antiviral nucleos(t)ides has been linked to mitochondrial dysfunction as a consequence of inhibition of mitochondrial DNA polymerase gamma (Pol gamma), a particular sensitive target, and associated inhibition of mtDNA replication [1, 3]. In the kidney, the proximal tubule is the main target of antiviral nucleos(t)ide drug toxicity due to active uptake via basolateral organic anion transporters (e.g. OAT1 and OAT3) expressed at this site [5, 6]. Based on the current mechanistic understanding, the subsequent sequence of key events (KE) leading to kidney injury as an adverse outcome can be described as inhibition of Pol gamma as the molecular initiating event (MIE), leading to mtDNA depletion (KE1), mitochondrial dysfuntion (KE2) and proximal tubule cell toxicity (KE3).
Summary of the AOP
Events: Molecular Initiating Events (MIE)
|Sequence||Type||Event ID||Title||Short name|
|1||MIE||1481||Inhibition of mitochondrial DNA polymerase gamma (Pol gamma)||Inhibition, mitochondrial DNA polymerase gamma (Pol gamma)|
|2||KE||1482||Depletion, mtDNA||Depletion, mtDNA|
|3||KE||1483||Dysfunction, Mitochondria||Dysfunction, Mitochondria|
|4||KE||709||Increase, Cytotoxicity (renal tubular cell)||Increase, Cytotoxicity (renal tubular cell)|
|5||AO||814||Occurrence, Kidney toxicity||Occurrence, Kidney toxicity|
Relationships Between Two Key Events
(Including MIEs and AOs)
|Inhibition, mitochondrial DNA polymerase gamma (Pol gamma) leads to Depletion, mtDNA||adjacent||Moderate||Low|
|Depletion, mtDNA leads to Dysfunction, Mitochondria||adjacent||High||Low|
|Dysfunction, Mitochondria leads to Increase, Cytotoxicity (renal tubular cell)||adjacent||High||Low|
|Increase, Cytotoxicity (renal tubular cell) leads to Occurrence, Kidney toxicity||adjacent||High||Moderate|
|Tenofovir disoproxil fumarate||High|
Life Stage Applicability
|All life stages||Not Specified|
|Human, rat, mouse||Human, rat, mouse||High||NCBI|
Overall Assessment of the AOP
Mechanistic data on KEs and KERs in this AOP are derived from in vitro and in vivo studies in humans and rodents. The described AOP presents a general mechanism leading to kidney toxicity in preclinical animal species and humans. The described AOP is not limited to a specific life stage or sex.
The sequence of MIE and KEs in this AOP presents a universal mechanism by which nucleos(t)ide analogs are thought to cause toxicity not only in the kidney but also in other organs and tissues, including liver, heart, muscle and the nervous system [1, 3, 4, 7]. The tissue-specificity and severity of the response to a particular nucleos(t)ide analog is considered to be at least in part determined by toxicokinetic factors, most notably active uptake into and efflux from target cells, transport across the mitochondrial membrane and metabolic conversion into the active triphosphate form [5-8]. Nephrotoxicity presents a treatment-limiting toxicity for a number of nucleos(t)ide analogs (e.g. tenofovir, adefovir, cidofovir). Experimental evidence for inhibition of mitochondrial DNA polymerase gamma leading to kidney toxicity as an adverse outcome is comes from in vitro studies, studies in laboratory animals (rats and mice) as well as from reports of patients treated with these compounds. These studies show a strong association between mitochondrial toxicity and antiviral nucleos(t)ide induced nephrotoxicity [9-14], with some studies also demonstrating concomitant mtDNA depletion [9, 11, 12, 15].
The causal relationship between the MIE and the downstream KEs is further supported by studies investigating the mechanism of toxicity of nucleos(t)ide analogs in other cells and tissues. For instance, a significant reduction in mtDNA was observed in muscle biopsies of zidovudine-treated HIV positive patients with myopathy as compared non-HIV-patient controls . Studies with isolated human DNA polymerases demonstrate increased sensitivity of Pol gamma to inhibition by antiretroviral nucleotides as compared to nuclear polymerases. Inhibition of mtDNA synthesis and loss of cell number was observed in a T-lymphoid leukemic cell line (Molt-4) treated with several anti-HIV and anti-HBV nucleoside analogs (d4T, 3'-deoxy-2',3'-didehydrothymidine; FLT, 3'-fluoro-3'-deoxythynidine; ddC, 2',3'-dideoxycytidine), which were also identified as potent inhibition of Pol gamma. However, a number of potent Pol gamma inhibitors did not cause significant effects on mtDNA synthesis and cell viability. Based on these findings, the authors concluded that there was no clear quantitative or qualitative correlation between the inhibition of isolated Pol gamma and inhibition of mitochondrial DNA synthesis in vitro, and moreover that these data are not predictive of in vivo toxicity. It is however important to stress that toxicokinetics, most notably cellular uptake of the tested antivirals, were not considered in this assessment. Thus, it is likely that some of the most potent inhibitors of Pol gamma failed to induce mtDNA depletion and cytotoxicity in this cell model simply because of insufficient cellular uptake .
Domain of Applicability
Mechanistic data on KEs and KERs in this AOP are derived from in vitro and in vivo studies in humans and rodents. The described AOP presents a general mechanism leading to kidney toxicity in preclinical animal species (rats, mice) and humans. The described AOP is not limited to a specific life stage or sex.
Essentiality of the Key Events
MIE / KE
Inhibition, Pol gamma
Inhibition of mtDNA Pol gamma by antiviral nucleos(t)ides demonstrated using enzymatic assays [2, 18-20]
Loss of mtDNA observed in vitro, in laboratory animals and patients after treatment with antiviral nucleos(t)ides [9, 11, 12, 15, 21]
Changes in mitochondrial ultrastructure and/or function (e.g. mitochondrial enzyme activities) observed in vitro, in laboratory animals and kidney biopsies of patients after treatment with antiviral nucleos(t)ides  [10-14, 21, 22]
Cytotoxicity of antiviral nucleos(t)ides observed in a range of kidney cell models with the severity depending on cellular uptake [11-14, 21-24]
Occurrence, Kidney Toxicity
Nephrotoxicity observed in laboratory animals and patients after treatment with antiviral nucleos(t)ides  [10-14, 25-28] 
Concordance of dose-response relationships
This is still a qualitiative description of the pathway. There is at present no quantitative information on dose-response relationships. Experiments are underway to provide quantitative understanding of dose-response relationships and response-response relationships between upstream and downstream KEs. In establishing dose-response relationships, it needs to be considered that effective excision of nucleotides by proofreading exonuclease of DNA polymerase as a repair mechanism may affect downstream KEs .
Temporal concordance among the key events and adverse outcome
The individual KEs are shown to occur prior to or concomitant with the onset of nephrotoxicity.
Strength, consistency, and specificity of association of adverse outcome and initiating event
The scientific evidence on the association between inhibition of DNA Polymerase gamma (MIE) and kidney toxicity (AO) is strong and consistent. The MIE is not specific for kidney toxicity as is considered responsible for a range of adverse effects of antiviral nucleos(t)ide treatment, whereby the site of toxicity appears to be at least in part determined by the toxicokinetics of individual drugs.
Biological plausibility, coherence, and consistency of the experimental evidence
Since antiviral nucleos(t)ide analogs are specifically designed to inhibit (viral) DNA polymerases or reverse transcriptase, off-target effects via interaction of human DNA polymerases are biologically plausible and consistent with the pharmacological MoA. The described AOP is biologically plausible, coherent and supported by experimental data.
Alternative mechanism(s) that logically present themselves and the extent to which they may distract from the postulated AOP
There are no alternative mechanism(s) that logically present themselves, although a contribution of yet undefined off-target effects to the overall AO cannot be excluded.
Uncertainties, inconsistencies and data gaps
This AOP is plausible and consistent with general biological knowledge. Quantitative information on dose response-relationships as well as repsonse-response relationships for upstream and downstream KEs is needed to support its applicability for the development of alternative in vitro tests for nephrotoxicity testing.
Quantitative data on KERs between upstream and downstream KE are still lacking.
Considerations for Potential Applications of the AOP (optional)
The described AOP is intended to provide a mechanistic framework for the development of in vitro bioactivity assays capable of predicting quantitative points of departure for safety assessment with regard to nephrotoxicity. Such assays may form part of an integrated testing strategy to reduce the need for repeated dose toxicity studies (e.g. OECD Guideline 407; OECD Guideline 407) and to aid in the design of new antiviral drugs.
1. Lewis, W. and M.C. Dalakas, Mitochondrial toxicity of antiviral drugs. Nat Med, 1995. 1(5): p. 417-22.
2. Johnson, A.A., et al., Toxicity of antiviral nucleoside analogs and the human mitochondrial DNA polymerase. J Biol Chem, 2001. 276(44): p. 40847-57.
3. Fontana, R.J., Side effects of long-term oral antiviral therapy for hepatitis B. Hepatology, 2009. 49(5 Suppl): p. S185-95.
4. Fung, J., et al., Extrahepatic effects of nucleoside and nucleotide analogues in chronic hepatitis B treatment. J Gastroenterol Hepatol, 2014. 29(3): p. 428-34.
5. Izzedine, H., V. Launay-Vacher, and G. Deray, Antiviral drug-induced nephrotoxicity. American Journal of Kidney Diseases, 2005. 45(5): p. 804-817.
6. Uwai, Y., et al., Renal transport of adefovir, cidofovir, and tenofovir by SLC22A family members (hOAT1, hOAT3, and hOCT2). Pharm Res, 2007. 24(4): p. 811-5.
7. Lewis, W., B.J. Day, and W.C. Copeland, Mitochondrial toxicity of NRTI antiviral drugs: an integrated cellular perspective. Nat Rev Drug Discov, 2003. 2(10): p. 812-22.
8. Kohler, J.J., et al., Tenofovir renal proximal tubular toxicity is regulated by OAT1 and MRP4 transporters. Lab Invest, 2011. 91(6): p. 852-8.
9. Lebrecht, D., et al., Mitochondrial Tubulopathy in Tenofovir Disoproxil Fumarate-Treated Rats. Jaids-Journal of Acquired Immune Deficiency Syndromes, 2009. 51(3): p. 258-263.
10. Cote, H.C., et al., Exploring mitochondrial nephrotoxicity as a potential mechanism of kidney dysfunction among HIV-infected patients on highly active antiretroviral therapy. Antivir Ther, 2006. 11(1): p. 79-86.
11. Tanji, N., et al., Adefovir nephrotoxicity: possible role of mitochondrial DNA depletion. Hum Pathol, 2001. 32(7): p. 734-40.
12. Kohler, J.J., et al., Tenofovir renal toxicity targets mitochondria of renal proximal tubules. Lab Invest, 2009. 89(5): p. 513-9.
13. Herlitz, L.C., et al., Tenofovir nephrotoxicity: acute tubular necrosis with distinctive clinical, pathological, and mitochondrial abnormalities. Kidney Int, 2010. 78(11): p. 1171-7.
14. Ramamoorthy, H., P. Abraham, and B. Isaac, Mitochondrial dysfunction and electron transport chain complex defect in a rat model of tenofovir disoproxil fumarate nephrotoxicity. J Biochem Mol Toxicol, 2014. 28(6): p. 246-55.
15. Kohler, J.J. and S.H. Hosseini, Subcellular renal proximal tubular mitochondrial toxicity with tenofovir treatment. Methods Mol Biol, 2011. 755: p. 267-77.
16. Arnaudo, E., et al., Depletion of muscle mitochondrial DNA in AIDS patients with zidovudine-induced myopathy. Lancet, 1991. 337(8740): p. 508-10.
17. Martin, J.L., et al., Effects of antiviral nucleoside analogs on human DNA polymerases and mitochondrial DNA synthesis. Antimicrob Agents Chemother, 1994. 38(12): p. 2743-9.
18. Lee, H., J. Hanes, and K.A. Johnson, Toxicity of nucleoside analogues used to treat AIDS and the selectivity of the mitochondrial DNA polymerase. Biochemistry, 2003. 42(50): p. 14711-9.
19. Cherrington, J.M., et al., Kinetic Interaction of the Diphosphates of 9-(2-Phosphonylmethoxyethyl)Adenine and Other Anti-Hiv Active Purine Congeners with Hiv Reverse-Transcriptase and Human DNA Polymerase-Alpha, Polymerase-Beta and Polymerase-Gamma. Antiviral Chemistry & Chemotherapy, 1995. 6(4): p. 217-221.
20. Naesens, L., et al., HPMPC (cidofovir), PMEA (adefovir) and related acyclic nucleoside phosphonate analogues: A review of their pharmacology and clinical potential in the treatment of viral infections. Antiviral Chemistry & Chemotherapy, 1997. 8(1): p. 1-23.
21. Zhao, X., et al., Tenofovir and adefovir down-regulate mitochondrial chaperone TRAP1 and succinate dehydrogenase subunit B to metabolically reprogram glucose metabolism and induce nephrotoxicity. Sci Rep, 2017. 7: p. 46344.
22. Talmon, G., L.D. Cornell, and D.J. Lager, Mitochondrial changes in cidofovir therapy for BK virus nephropathy. Transplant Proc, 2010. 42(5): p. 1713-5.
23. Zhang, X., et al., Intracellular concentrations determine the cytotoxicity of adefovir, cidofovir and tenofovir. Toxicol In Vitro, 2015. 29(1): p. 251-8.
24. Nieskens, T.T., et al., A Human Renal Proximal Tubule Cell Line with Stable Organic Anion Transporter 1 and 3 Expression Predictive for Antiviral-Induced Toxicity. AAPS J, 2016. 18(2): p. 465-75.
25. Liborio, A.B., et al., Rosiglitazone reverses tenofovir-induced nephrotoxicity. Kidney Int, 2008. 74(7): p. 910-8.
26. Woodward, C.L.N., et al., Tenofovir-associated renal and bone toxicity. Hiv Medicine, 2009. 10(8): p. 482-487.
27. Gara, N., et al., Renal tubular dysfunction during long-term adefovir or tenofovir therapy in chronic hepatitis B. Aliment Pharmacol Ther, 2012. 35(11): p. 1317-25.
28. Vora, S.B., A.W. Brothers, and J.A. Englund, Renal Toxicity in Pediatric Patients Receiving Cidofovir for the Treatment of Adenovirus Infection. J Pediatric Infect Dis Soc, 2017.