JAIDS Journal of Acquired Immune Deficiency Syndromes:
Clinical Science: Brief Report
Oxidant Stress and Peripheral Neuropathy During Antiretroviral Therapy: An AIDS Clinical Trials Group Study
Hulgan, Todd MD, MPH*; Hughes, Michael PhD†; Sun, Xin MS†; Smeaton, Laura M. MS†; Terry, Erin MS*; Robbins, Gregory K. MD, MPH‡; Shafer, Robert W. MD§; Clifford, David B. MD∥; McComsey, Grace A. MD¶; Canter, Jeffery A. MD, MPH*; Morrow, Jason D. MD*; Haas, David W. MD*
From the *Vanderbilt University School of Medicine, Nashville, TN; †Center for Biostatistics in AIDS Research, Harvard School of Public Health, Boston, MA; ‡Massachusetts General Hospital, Harvard University, Boston, MA; §Stanford University, Stanford, CA; ∥Washington University School of Medicine, St Louis, MO; and ¶Case Western Reserve University, Cleveland, OH.
Received for publication December 15, 2005; accepted April 20, 2006.
Supported by the National Institutes of Health/National Center for Complementary and Alternative Medicine Mentored Career Development Award AT002508 and a Developmental Core Award from the Vanderbilt-Meharry Center for AIDS Research AI54999 (TH), and in part by the Adult AIDS Clinical Trials Group (grant AI38858) funded by the National Institute of Allergy and Infectious Diseases. Additional grant support included AI46339 and AI54999 (D.W.H.); NS32228 and AI25903 (D.B.C.); and GM15431, CA77839, DK48831, and RR00095 (J.M.).
Presented at the 12th Conference on Retroviruses and Opportunistic Infections, February 2005, Boston, MA [Abstract No. 399].
Reprints: Todd Hulgan, MD, MPH, Division of Infectious Diseases, Vanderbilt University School of Medicine, 345 24th Ave N, Suite 105, Nashville, TN 37203 (e-mail: email@example.com).
Background: Peripheral neuropathy that complicates HIV nucleoside reverse transcriptase inhibitor (NRTI) therapy is likely caused by mitochondrial injury. Mitochondria play a central role in regulating oxidant stress. We explored the relationships between oxidant stress and NRTI-induced peripheral neuropathy.
Methods: The AIDS Clinical Trials Group (ACTG) studied the cases of 384 antiretroviral-naive individuals randomized to receive didanosine/stavudine or zidovudine/lamivudine, plus efavirenz, nelfinavir, or both. The participants were followed for up to 3 years. Peripheral neuropathy was ascertained by signs and symptoms. We performed a case-control study of ACTG 384 participants. Peripheral neuropathy cases and nonneuropathy control subjects were selected from didanosine/stavudine recipients. Alternate control subjects were selected from zidovudine/lamivudine recipients who developed peripheral neuropathy. Oxidant stress was assessed by quantifying F2-isoprostanes (F2-IsoPs) in cryopreserved plasma.
Results: Seventy-five cases, 71 control subjects, and 18 alternate control subjects were identified. The median baseline F2-IsoP values were 53 (interquartile range [IQR], 40-85), 57 (IQR, 41-77), and 53 (IQR, 47-101) pg/mL, respectively, and did not differ between cases and control subjects (P = 0.78) or alternate control subjects (P = 0.60). Changes in F2-IsoPs from baseline to time of peripheral neuropathy did not differ significantly between cases (median, 10 [IQR, −17 to 26] pg/mL) and control subjects (median, 4 [IQR, −11 to 17] pg/mL; P = 0.48) or alternate control subjects (median, 1 [IQR, −48 to 10] pg/mL; P = 0.21).
Conclusions: Peripheral neuropathy that complicates antiretroviral therapy with NRTIs was not associated with increased systemic oxidant stress assessed by plasma F2-IsoPs.
The availability of potent antiretroviral therapy has reduced morbidity and mortality due to AIDS.1,2 Preferred initial therapies for HIV-1 infection include 2 nucleoside reverse transcriptase inhibitors (NRTIs) plus either an HIV protease inhibitor or a nonnucleoside reverse transcriptase inhibitor (NNRTI).3 The active intracellular anabolites of approved NRTIs block viral replication by competing with endogenous cellular nucleotides for incorporation into proviral DNA and are relatively specific for HIV reverse transcriptase. However, the inhibition of human mitochondrial DNA polymerase-γ by NRTIs4 has been associated with toxicities that include peripheral neuropathy, lipoatrophy, hepatic steatosis, and lactic acidosis.5
Precise incidence rates are difficult to determine but, perhaps, as many as one third of HIV-infected persons may develop symptomatic peripheral neuropathy that is characterized by distal, symmetrical anesthesia, and/or painful dysesthesia.6,7 Although peripheral neuropathy can complicate untreated HIV infection,8 most cases since the availability of antiretroviral therapy have resulted from exposure to NRTIs, particularly the dideoxynucleosides didanosine (ddI) and stavudine (d4T).9,10 There is evidence that NRTI-associated peripheral neuropathy is the result of mitochondrial injury.11,12
AIDS Clinical Trials Group (ACTG) study 384 was a randomized, double-blind treatment strategy trial that randomized subjects using a factorial design to receive either a protease inhibitor (nelfinavir) or an NNRTI (efavirenz) or both, and to receive one of 2 dual NRTI regimens (ddI/d4T or zidovudine [ZDV]/lamivudine [3TC]) as initial therapy for HIV infection. A total of 960 participants enrolled in the United States and Italy between October 1998 and November 1999, and were followed for up to 3 years.13,14 Of the 488 subjects randomized to receive ddI/d4T, 176 (36%) developed peripheral neuropathy during the study compared with 69 (14%) of the 472 receiving ZDV/3TC. The subjects in the ddI/d4T-treated arm developed peripheral neuropathy and dose-modifying toxicities more rapidly than the subjects in the ZDV/3TC-treated arm (P < 0.001).13
Oxidant stress describes the generation of reactive free radicals that damage cells and tissues.15 Mitochondria play a central role in regulation of oxidant stress through the production and scavenging of free radicals. Oxidant stress is increased in human conditions that include Alzheimer disease, Parkinson disease, scleroderma, and hepatorenal syndrome, and exposures such as heavy cigarette smoking.16-21 A central feature of oxidant stress is lipid peroxidation. Measuring the end products of free radical-catalyzed lipid peroxidation in plasma is a noninvasive approach to quantifying this process.15 Prostaglandin F2-like products, termed F2-isoprostanes (F2-IsoPs), are accurate markers of oxidative stress in humans.22,23
Increased and/or dysregulated oxidant stress may play a role in NRTI-associated toxicities, perhaps as a consequence of mitochondrial DNA damage and dysfunction.4 In animal models, ZDV and d4T have been shown to induce mitochondrial damage in skeletal muscle that is attenuated by administration of antioxidants.24,25 Increased plasma F2-IsoP levels have been demonstrated among NRTI-treated patients with symptomatic hyperlactatemia and lipoatrophy.26 In a separate cross-sectional study, the therapeutic control of HIV replication was also associated with increased plasma F2-IsoPs.27 Neither study was designed to address associations with the use of specific antiretroviral drugs. To determine whether NRTI-associated toxicity is associated with evidence of increased systemic oxidant stress, and in an effort to identify a biomarker for predicting the development of peripheral neuropathy, we quantified plasma F2-IsoPs among ACTG 384 participants.
This study included HIV-infected individuals who had enrolled in ACTG study 384 at clinical trial sites in the United States. The eligibility criteria for ACTG 384 included plasma HIV-1 RNA greater than or equal to 500 copies per milliliter and fewer than 7 days of earlier antiretroviral therapy. The NRTIs were open-labeled: ddI (dosage, 400 mg or 250 mg once a day based on weight; enteric-coated tablets were available during the final year of study); d4T (dosage, 40 mg or 30 mg 2 times a day based on weight); and 3TC (dose, 150 mg) and ZDV (dose, 300 mg) administered as a fixed-dose combination 2 times a day. Efavirenz (dosage, 600 mg once a day) and nelfinavir (dosage, 1250 mg 2 times a day) were double-blinded with matching placebos. Clinical assessments were obtained at screening, at entry, at weeks 4, 8, 12, 16, 20, and 24, and every 8 weeks thereafter. ACTG 384 was approved by institutional review boards for each site, and the subjects gave written informed consent. The Vanderbilt Committee for the Protection of Human Subjects and the ACTG approved the use of stored plasma and data for this study.
During ACTG 384 study, signs, symptoms, or diagnoses were recorded at study entry and at each study visit using a symptom distress self-report questionnaire that included pain and neuropathy. Peripheral neuropathy was a targeted toxicity in ACTG 384 and was graded according to the Division of AIDS (National Institutes of Health, Bethesda, MD) Table for Severity of Adult Adverse Experiences.28 Grade 2 peripheral neuropathy was defined as moderate paresthesia requiring nonnarcotic analgesia, or moderate neurosensory impairment, such as decreased vibratory, pinprick, or hot/cold sensation to the ankle, or decreased position sense or mild impairment that is not asymmetrical. Subjects with peripheral neuropathy at study entry were excluded from this study, and follow-up was censored at the time that a subject changed any study drug (excluding dose adjustments) from those to which they were originally randomized. The cases were subjects who developed peripheral neuropathy of at least grade 2 while receiving ddI/d4T, and who had stored plasma available from baseline and at the time of peripheral neuropathy (or within the previous 8 weeks). The controls were subjects receiving ddI/d4T who did not develop peripheral neuropathy during follow-up. Wherever possible, for each case, a single control subject was selected at random from among the potential control subjects that had stored plasma available and at the same duration of follow-up as when the case's peripheral neuropathy occurred. To assess the effects of different NRTI combinations on oxidant stress, we also included an alternate control group comprised of subjects receiving ZDV/3TC who developed at least grade 2 peripheral neuropathy.
Plasma was from EDTA anticoagulated whole blood. The specimens were centrifuged at 400g for 10 minutes at room temperature and the plasma aliquots were stored at −70°C within 4 to 6 hours. Plasma levels of 15-F2t-isoprostane (8-iso-PGF2α) were quantified using a gas chromatographic/negative ion chemical ionization mass spectrometric approach using stable isotope dilution as described by Morrow and Roberts.29 The assay has an intraday variability of less than 10%. Normal F2-IsoP concentration in plasma of healthy volunteers is considered 35 pg/mL ± 1 SD (6 pg/mL).29
Demographics and baseline laboratory values are presented as proportions and median (interquartile range [IQR]). Comparisons of peripheral neuropathy and F2-IsoP levels were performed using Wilcoxon rank sum test.
A total of 164 ACTG 384 participants were included in this analysis: 75 cases; 71 control subjects; and 18 alternate control subjects. Most were men (84%) and non-Hispanic white (44%); the median age was 38 years (Table 1). Median baseline plasma HIV-1 RNA concentrations and CD4 lymphocyte counts were 5.2 (IQR, 4.5-5.7) log10 copies/mL and 211 (IQR, 42-405) cells/mm3, respectively. At baseline, cases (median age, 38 [IQR, 33-45] years) were older than control subjects (median age, 36 [IQR, 30-42] years; P = 0.03) and had lower CD4 lymphocyte counts (cases' median, 119 [IQR, 32-316] cells/mm3; control subjects' median, 315 [IQR, 80-455] cells/mm3; P = 0.002).
Median F2-IsoP values at baseline were 53 pg/mL (IQR, 40-85) in cases, 57 pg/mL (IQR, 41-77) in control subjects, and 53 pg/mL (IQR, 47-101) in alternate control subjects (Table 1). Median baseline values did not differ significantly between cases and control subjects (P = 0.78), or between cases and alternate control subjects (P = 0.6). F2-Median IsoPs at the time of peripheral neuropathy also did not differ from those at a corresponding time among control subjects (59 [IQR, 43-88] vs. 61 [IQR, 43-79] pg/mL, respectively; P = 0.67), or among alternate control subjects (49 [IQR, 42-72] pg/mL; P = 0.19). Median changes in F2-IsoPs from baseline were 10 pg/mL (IQR, −17 to 26) for cases, 4 pg/mL (IQR, −11 to 17) for control subjects, and 1 pg/mL (IQR, −48 to 10) for alternate control subjects; these changes were not significantly different when comparing case patients and control subjects (P = 0.48) or comparing case patients and alternate control subjects (P = 0.21).
In this case-control study of 164 individuals who received NRTI-containing regimens during a prospective, randomized clinical trial, including 146 subjects who were randomized to receive ddI/d4T-containing regimens, the development of peripheral neuropathy was not associated with increased systemic oxidant stress as assessed by quantification of F2-IsoPs in plasma.
Toxicities resulting from prolonged exposure to antiretroviral drugs can compromise the responses to multidrug antiretroviral regimens. In particular, the NRTI class has been implicated as a cause of several insidious and sometimes irreversible chronic toxicities, including peripheral neuropathy, that are thought to result from mitochondrial damage. No intervention has been shown to prevent NRTI-associated peripheral neuropathy, and there is no specific therapy for NRTI-associated peripheral neuropathy other than avoidance of further exposure to potentially neurotoxic drugs. In particular, combined use of ddI/d4T should be avoided. In an effort to identify a biomarker that might be clinically useful for identifying individuals at greatest risk for developing peripheral neuropathy, we investigated F2-IsoPs, products of cellular oxidative damage that may be increased in the setting of mitochondrial dysfunction.30
This is the first study to explore F2-IsoPs as a potential marker of oxidant injury during NRTI-associated peripheral neuropathy. We have previously reported that F2-IsoPs are increased among HIV-infected individuals with HIV-1 RNA levels less than 400 copies per milliliter while receiving various antiretroviral regimens, compared with individuals receiving no antiretrovirals.27 In another survey, McComsey and Morrow26 described increased F2-IsoP levels in individuals with symptomatic hyperlactatemia, lactic acidosis, or lipoatrophy while receiving NRTI-containing regimens, compared with similarly treated persons with asymptomatic hyperlactatemia and control subjects. We hypothesized that the increases in F2-IsoPs would be greater in subjects who developed peripheral neuropathy than in asymptomatic individuals with similar NRTI exposure, and would be greatest among persons exposed to ddI and d4T, which have greater affinity for mitochondrial DNA polymerase-γ31 and mitochondrial toxicity.32
As has been previously reported,7,33 older age at the time of drug treatment and lower baseline CD4 lymphocyte count identified individuals with the increased risk of developing symptomatic peripheral neuropathy. This could reflect underlying asymptomatic peripheral neuropathy in some subjects that progressed after NRTI therapy. Subjects that developed grade 2 or higher-graded symptomatic peripheral neuropathy while receiving ddI/d4T did not have higher F2-IsoP levels at the time of neuropathy or greater changes in F2-IsoP levels than did the control subjects receiving ddI/d4T or subjects who developed peripheral neuropathy while receiving ZDV/3TC. One explanation for the lack of association between plasma F2-IsoP and peripheral neuropathy in this study is because neuronal injury, which may involve oxidant stress, is not of sufficient magnitude to alter circulating lipid peroxidation profiles. Oxidative damage during peripheral neuropathy may be localized to tissue and may not be discernible with a plasma-based assay, in contrast with systemic toxicities (eg, lipoatrophy or lactic acidosis). Additionally, the dynamics of oxidant stress in the setting of treatment initiation and decreasing viral replication and cellular activation are likely very complex and may have limited the capacity to detect NRTI-related changes in oxidant stress in this population.
This study had several limitations. Cigarette smoking is known to increase systemic oxidant stress and plasma F2-IsoP concentrations.21 Because smoking status was not ascertained during the ACTG 384 study, we could not control for this variable. In addition, although peripheral neuropathy was characterized uniformly as a targeted toxicity during the ACTG 384 study, ascertainment was primarily based on clinical signs and symptoms, preventing us from assessing the relationships between oxidant stress and asymptomatic or subclinical peripheral neuropathy that may be identified with more detailed neurological assessments. Plasma from ACTG 384 participants was collected and cryopreserved at numerous clinical research sites. For some specimens, inconsistent processing may have spuriously elevated F2-IsoPs because of ex vivo lipid peroxidation.
In summary, symptomatic NRTI-associated peripheral neuropathy was not associated with increased plasma F2-IsoPs among ACTG 384 participants. Additional studies in this area should focus on well-characterized phenotypes while controlling for potential confounders.
The authors gratefully acknowledge the participants of ACTG study 384. We also acknowledge M. Hirsh, T. Merrigan, V. De Grutolla, R. D'Aquila, and other members of the ACTG 384 team, including M. Fischl (Miami University), M. Dube (Indiana University), C. Pettinelli, A. Martinez (National Institutes of Health), R. Delapenha (Howard University), M. Nokta (University of Texas, Galveston), V. Johnson (University of Alabama, Birmingham), G. Morse (State University of New York, Buffalo), B. Putnam (University of Colorado), M. Klebert (Washington University), M. Testa (Harvard School of Public Health), A. Chiesi, C. Tomino (Istituto Superiore de Sanita), S. Deeks (University of California, San Francisco), T. Nevin (Social & Scientific Systems), J. Levin, V. French, O. Fennell (Adult AIDS Clinical Trials Group Community Constituency Group), M. Stevens, R. Grosso, B. Dusak, S. Hodder (Bristol-Myers Squibb), J. Tolson, C. Brothers (GlaxoSmithKline), R. Leavitt (Merck), D. Manion, N. Ruiz, K. Morrissey (DuPont Pharmaceuticals), M. Becker, B. Quart (Agouron), C. Jennings (Northwestern University), L. Gedeon, S. Dascomb, M. Cooper, M. Murphy, K. Blakelock (Frontier Science and Technology Foundation), and A. Doolan (Massachusetts General Hospital).
1. Mocroft A, Ledergerber B, Katlama C, et al. Decline in the AIDS and death rates in the EuroSIDA study: an observational study. Lancet
2. Palella FJ Jr, Delaney KM, Moorman AC, et al, HIV Outpatient Study Investigators. Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. N Engl J Med
4. Lewis W, Copeland WC, Day BJ. Mitochondrial DNA depletion, oxidative stress, and mutation: mechanisms of dysfunction from nucleoside reverse transcriptase inhibitors. Lab Invest
5. Moyle G. Clinical manifestations and management of antiretroviral nucleoside analog-related mitochondrial toxicity. Clin Ther
. 2000;22(8):911-936. discussion 898.
6. Keswani SC, Pardo CA, Cherry CL, et al. HIV-associated sensory neuropathies. AIDS
7. Schifitto G, McDermott MP, McArthur JC, et al. Incidence of and risk factors for HIV-associated distal sensory polyneuropathy. Neurology
8. Cornblath DR, McArthur JC. Predominantly sensory neuropathy in patients with AIDS and AIDS-related complex. Neurology
9. Browne MJ, Mayer KH, Chafee SB, et al. 2,3-didehydro-3-deoxythymidine (d4T) in patients with AIDS or AIDS-related complex: a phase I trial. J Infect Dis
10. Kelleher T, Cross A, Dunkle L. Relation of peripheral neuropathy to HIV treatment in four randomized clinical trials including didanosine. Clin Ther
11. Cui L, Locatelli L, Xie MY, et al. Effect of nucleoside analogs on neurite regeneration and mitochondrial DNA synthesis in PC-12 cells. J Pharmacol Exp Ther
12. Dalakas MC, Semino-Mora C, Leon-Monzon M. Mitochondrial alterations with mitochondrial DNA depletion in the nerves of AIDS patients with peripheral neuropathy induced by 23-dideoxycytidine (ddC). Lab Invest
13. Robbins GK, De Gruttola V, Shafer RW, et al. Comparison of sequential three-drug regimens as initial therapy for HIV-1 infection. N Engl J Med
14. Shafer RW, Smeaton LM, Robbins GK, et al. Comparison of four-drug regimens and pairs of sequential three-drug regimens as initial therapy for HIV-1 infection. N Engl J Med
15. Halliwell B, Grootveld M. The measurement of free radical reactions in humans. Some thoughts for future experimentation. FEBS Lett
16. Montine TJ, Markesbery WR, Morrow JD, et al. Cerebrospinal fluid F2
-isoprostane levels are increased in Alzheimer's disease. Ann Neurol
17. Jenner P, Olanow CW. Oxidative stress and the pathogenesis of Parkinson's disease. Neurology
. 1996;476 (suppl 3):S161-S170.
18. Montine KS, Quinn JF, Zhang J, et al. Isoprostanes and related products of lipid peroxidation in neurodegenerative diseases. Chem Phys Lipids
19. Stein CM, Tanner SB, Awad JA, et al. Evidence of free radical-mediated injury (isoprostane overproduction) in scleroderma. Arthritis Rheum
20. Awad JA, Roberts LJ II, Burk RF, et al. Isoprostanes-prostaglandin-like compounds formed in vivo independently of cyclooxygenase: use as clinical indicators of oxidant damage. Gastroenterol Clin North Am
21. Morrow JD, Frei B, Longmire AW, et al. Increase in circulating products of lipid peroxidation (F2
-isoprostanes) in smokers. Smoking as a cause of oxidative damage. N Engl J Med
22. Morrow JD, Hill KE, Burk RF, et al. A series of prostaglandin F2
-like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalyzed mechanism. Proc Natl Acad Sci U S A
23. Morrow JD, Chen Y, Brame CJ, et al. The isoprostanes: unique prostaglandin-like products of free radical-initiated lipid peroxidation. Drug Metab Rev
24. de la Asuncion JG, del Olmo ML, Sastre J, et al. AZT treatment induces molecular and ultrastructural oxidative damage to muscle mitochondria. Prevention by antioxidant vitamins. J Clin Invest
25. Paulik M, Lancaster M, Croom D. Anti-oxidants rescue NRTI-induced metabolic changes in AKR/J mice. Antivir Ther
. 2000;5(suppl 5):6-7.
26. McComsey GA, Morrow JD. Lipid oxidative markers are significantly increased in lipoatrophy but not in sustained asymptomatic hyperlactatemia. J Acquir Immune Defic Syndr
27. Hulgan T, Morrow J, D'Aquila R, et al. Oxidant stress is increased during treatment of human immunodeficiency virus infection. Clin Infect Dis
29. Morrow JD, Roberts LJ II. Mass spectrometric quantification of F2
-isoprostanes in biological fluids and tissues as measure of oxidant stress. Methods Enzymol
30. Canter JA, Eshaghian A, Fessel J, et al. Degree of heteroplasmy reflects oxidant damage in a large family with the mitochondrial DNA A8344G mutation. Free Radic Biol Med
31. Lee H, Hanes J, Johnson KA. Toxicity of nucleoside analogues used to treat AIDS and the selectivity of the mitochondrial DNA polymerase. Biochemistry
32. Brinkman K, ter Hofstede HJ, Burger DM, et al. Adverse effects of reverse transcriptase inhibitors: mitochondrial toxicity as common pathway. AIDS
33. Lichtenstein KA, Armon C, Baron A, et al. Modification of the incidence of drug-associated symmetrical peripheral neuropathy by host and disease factors in the HIV outpatient study cohort. Clin Infect Dis
This article has been cited 4 time(s).
HIV Clinical TrialsClinical Factors Associated with Plasma F-2-Isoprostane Levels in HIV-Infected AdultsHIV Clinical Trials
Cell Biology and ToxicologyNeurotoxicity caused by didanosine on cultured dorsal root ganglion neuronsCell Biology and Toxicology
PharmacogenomicsPharmacogenetics of nucleoside reverse-transcriptase inhibitor-associated peripheral neuropathyPharmacogenomics
Antiviral TherapyOxidant stress in HIV-infected women from the Women's Interagency HIV StudyAntiviral Therapy
HIV; peripheral neuropathies; oxidative stress; reverse transcriptase inhibitors; drug toxicity
© 2006 Lippincott Williams & Wilkins, Inc.
What does "Remember me" mean?
By checking this box, you'll stay logged in until you logout. You'll get easier access to your articles, collections,
media, and all your other content, even if you close your browser or shut down your
To protect your most sensitive data and activities (like changing your password),
we'll ask you to re-enter your password when you access these services.
What if I'm on a computer that I share with others?
If you're using a public computer or you share this computer with others, we recommend
that you uncheck the "Remember me" box.
Highlight selected keywords in the article text.
Data is temporarily unavailable. Please try again soon.