AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 23, Number 8, 2007, pp. 1004–1007 © Mary Ann Liebert, Inc. DOI: 10.1089/aid.2006.0286
Morphine-Mediated Deterioration of Oxidative Stress Leads to Rapid Disease Progression in SIV/SHIV-Infected Macaques ANTONIO PÉREZ-CASANOVA,1 RICHARD J. NOEL JR.,1 VANESSA RIVERA-AMILL,1 KAZIM HUSAIN,1 and ANIL KUMAR2
ABSTRACT Oxidative stress is well documented in HIV infection, but the effect of concomitant substance abuse is largely unknown. We studied oxidative stress in our macaque model of morphine abuse and AIDS. In plasma, we found an 50% decrease in catalase activity with morphine dependence that was exacerbated by infection in rapid progressors. Superoxide dismutase was decreased by a similar degree, but only in the presence of both morphine and viral infection. The loss of these antioxidant systems was coincident with significantly increased plasma malondialdehyde upon viral infection that displayed a synergistic increase in conjunction with morphine and rapid disease. INTRODUCTION
U
NDER NORMAL PHYSIOLOGICAL CONDITIONS,
a homeostatic balance exists between the formation and removal of reactive oxygen species. The pathophysiology of many disease states involves an alteration of this balance resulting in stress. Such oxidative stress is associated with HIV infection and disease progression. The balance can be disrupted very early in HIV infection resulting in decreased levels of plasma glutathione, lipid peroxidation, and altered expression of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), and others.1–5 Drug abuse is a cofactor in 30% of U.S. HIV infections and is known to contribute to increased viral replication and opportunistic infections.6–8 Morphine is a wellknown opioid analgesic used extensively, which has repercussions in patients with allergy and chronic inflammation. Morphine has been reported to release the histamine (degranulation) from the mast cells in the tissues.9 Alone, morphine use has been linked to lipid peroxidation, particularly in the liver and kidney.10 It, therefore, suggests that oxidative stress may form a common link that drives more rapid progression in the setting of HIV infection and drug abuse.
MATERIALS AND METHODS We developed a monkey model of AIDS in the setting of morphine abuse. As described in detail previously,11 a cohort 1AIDS
of six rhesus macaques was adapted to morphine dependence (5 mg/kg, TID) over a 20-week period, while three control monkeys remained drug free. After dependence was established, all animals were inoculated with a three virus cocktail (2 ml injection containing 104 TCID50 of each of SIV/17E-Fr, SHIV89.6P, and SHIVKU-1B); morphine was continued throughout the observation period. Within 20 weeks after infection, 50% of the morphine-addicted monkeys experienced very rapid progression to AIDS and death (Rapid group). Three morphine animals recovered from the acute phase (Slow group), as did all the nonmorphine macaques (Control group).11,12 Peak total viral loads measured by real time RT-PCR and CD4 loss after infection were similar in all macaques (Fig. 1); however, the Rapid group did not control replication nor recover CD4, while the Control group showed greatly reduced replication, some restoration of CD4 cells, and had evidence of an adaptive immune response.12 The Slow group showed an intermediate recovery and immune response more similar to nonmorphine controls. We examined the possible role of plasma oxidative stress in driving the more rapid progression to severe disease exclusively in the morphine-dependent monkeys. Blood samples were collected periodically during the morphine adaptation period (weeks 20 to 0) as well as regularly after infection (week 0 and following). We used a combination approach to assess oxidative stress by measuring plasma CAT activity that detoxifies hydrogen peroxide, SOD, an antioxidant enzyme against superoxide, and malondialdehyde (MDA), a marker of lipid per-
Research Program, Ponce School of Medicine, Ponce, PR 00732. of Pharmacology, School of Pharmacy, University of Missouri, Kansas City, Missouri 64108.
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FIG. 1. Total plasma viral load and CD4 T cell counts were measured at regular intervals following infection. Viral load (solid lines, left axis) was measured by real time RT-PCR using gag as a target and represents the total of all three viruses as described previously.11 CD4 T cell counts (dashed lines, right axis) were measured by immunostaining with fluorescently labeled antibodies. Data were collected using a FACSCalibur flow cytometer. Group averages are plotted for Rapid progressors (squares), Slow progressors (triangles), and nonmorphine Controls (inverted triangles).
oxidation. MDA (mol/mg protein) was assessed by detergent extraction of plasma followed by reaction with thiobarbituric acid and color development was measured at 532 nm.13 SOD activity was expressed as the amount of enzyme that inhibited the oxidation of epinephrine according to the method of Misra and Fridovich.14 CAT levels were determined using a commercially available kit (Cayman Chemical) based on the method of Johansson and Borg.15 We measured these parameters before morphine treatment, during morphine treatment before infection, and for up to 48 weeks following viral infection.
RESULTS The results shown in Fig. 2 show that oxidative imbalance was a factor in our simian AIDS/drug abuse model. At week 20, a morphine free time point, MDA (Fig. 2A), was very low (less than 10 mol/mg protein) and SOD (Fig. 2B) and CAT (Fig. 2C) activities were equal in all monkeys. At this time point, MDA, SOD, and CAT levels were highly comparable in all nine animals, indicating that the individual variation of these parameters in our macaques was small (MDA 0.2–12.5 mol/mg protein, SOD 20–30 units/ug protein, CAT 60–80 nmol/min/ ml). Over the next 20 weeks and just prior to infection (time 0 on graphs), MDA levels remained stable showing no up-regulation by morphine addiction; however, the CAT activity dropped to roughly 60% and 40% of premorphine levels in rapid
and slow progressors, respectively. In contrast to CAT, SOD showed a polymorphic response to morphine. Three animals experienced a consistent upward trend resulting in near doubling from 30 to 50 units/g protein, while the remaining 50% of morphine-addicted macaques showed stable SOD activity levels throughout the 20 weeks of morphine pretreatment. Interestingly, this phenotype directly mirrored the disease progression rate as all monkeys with SOD up-regulation became Slow progressors while the remaining monkeys comprised the Rapid group. All animals were infected with an identical triple-virus inoculum as previously described.11 The disease showed a distinctly rapid clinical course in 50% of the morphine-addicted macaques as compared to the remaining three morphine-treated animals (Slow) and the three nonmorphine-addicted monkeys (Control). The CD4 and viral load profiles for each of these three groups are presented in Fig. 1. Although all animals achieved similar peak viral loads (107, left axis) at approximately 2 weeks postinoculation, the three rapid progressors showed only a minor and transient dip in viral load by 4 weeks that quickly rose above the initial peak levels. All rapid progressors died by 20 weeks postinfection. In contrast, the Slow and Control groups maintained viral set points just above and below 105, respectively. The CD4 counts (right axis) in all animals showed a rapid, precipitous drop by 4 weeks; however, only the Rapid group failed to recover appreciably, while the Slow and Control groups showed substantial recovery.
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FIG. 2. Oxidative stress parameters were measured in morphine addicted (Rapid and Slow) and morphine free (Control) rhesus macaques infected with a three virus cocktail at multiple time points during the experimental protocol. Week 20 represents a premorphine plasma sample. Week 0 represents a noninfected plasma sample. Rapid animals (N 3) were morphine dependent and all progressed to AIDS/death between 18 and 20 weeks. Slow group animals (N 3) were morphine dependent but all survived throughout the observation period. Control group animals (N 3) were morphine free but were infected. (A) Malondialdehyde levels were determined for each animal and the average and standard error for each group at each time point are plotted. (B) SOD activity was determined for each animal and the average and standard error for each group at each time point are plotted. (C) Catalase activity was determined for each animal and then expressed as a percentage of the morphine-free/virus-free value at each time point. The group mean percentages are plotted with standard error. Statistically significant differences from Control were assessed by Student’s t-test. *p 0.05, **p 0.01.
PÉREZ-CASANOVA ET AL. The clinical course of disease after infection was also reflected in oxidative stress parameters, shown in Fig. 2 for all points after week 0. MDA increased in direct response to viral infection in all animals. While drug abuse alone (prior to week 0) did not increase plasma MDA, morphine caused a more rapid rise that by 8 weeks was significantly higher than Controls. Notably, the increase was the most dramatic in rapid progressors and paralleled the rise in viral load between weeks 8 and 16 (Figs. 1 and 2A). The slow progressors, although not as pronounced, remained higher than controls, statistically so at weeks 8, 24, and 48. This too was a reflection of the elevated mean viral load for the Slow group vs. Controls (Fig. 1). In the case of CAT (Fig. 2C), this trend of morphine-induced down-regulation continued in both Rapid and Slow groups, with the most severe decrease to approximately 30% of starting levels in the Rapid group at 8 weeks postinfection. The controls did not show decreased CAT, suggesting a direct effect of morphine that may be exaggerated by viral infection. The Slow progressors experienced a partial recovery by week 16, but this was not sustained, as by week 48 the CAT levels dropped to less than 40% of original activity. As with CAT, the SOD activity showed a marked decrease in morphine-dependent animals after infection but not in Controls (Fig. 2B). Preinfection SOD activities were 50 and 20 units/g protein in the Slow and Rapid groups, respectively, immediately prior to inoculation. By 8 weeks, each group had dropped to 50% of the week 0 level (25 and 10 units). This remained stable throughout the 48 weeks of follow-up. The predominant distinction among morphine-addicted monkeys was that the SOD set point was raised in slow progressors such that the 50% drop in the combined presence of morphine with infection produced plasma SOD activity roughly equivalent to premorphine for these animals. On the other hand, the rapid progressors did not experience SOD induction during morphine stabilization. Thus the 50% drop that was universally present in morphine-dependent, infected macaques, but uniquely absent in Control infected macaques, produced a level of SOD activity in plasma well below the basal activity of 20–30 units/g protein. We also assessed levels of plasma glutathione and nitric oxide. We found a decreasing trend in glutathione and an increasing trend in nitric oxide exclusively in morphine-dependent animals after infection, much as with SOD and MDA, respectively. However, the natural variability in baseline values for these parameters in untreated animals combined with the limited number of animals available precluded the emergence of a clearly convincing pattern (data not shown). Interestingly, the level of MDA, though dependent on viral inoculation, showed a greater rise in response to morphine dependency (compare Fig. 2A Rapid and Slow vs. Control), indicating some additive effects of drug abuse and infection on oxidative imbalance. This correlated with the drop in antioxidant capacity reflected by a 50% drop in CAT (due to morphine, week 0) and SOD (combined effect of morphine and infection, week 8 and beyond). CAT is critical for detoxification of hydrogen peroxide, a prooxidant that can directly lead to lipid peroxidation.16 SOD is a major cellular antioxidant defense system against the superoxide radical, which can cause severe membrane damage.17 Rate of progression was also a factor as the Rapid group rise in MDA was notably the sharpest with the highest peak. This was also supported by the pattern of both antioxidants: CAT, which showed the fastest
DRUG ABUSE, SIV/SHIV, AND OXIDATIVE STRESS drop in the Rapid group, while there was some recovery in the Slow group, and SOD, which dropped well below basal levels only in rapid progressors.
DISCUSSION Progression to AIDS during HIV infection has been linked to oxidative stress parameters, and although drug abuse alone can also alter oxidant balance, the impact on cellular oxidative status by a combination of drug abuse and HIV infection has not been sufficiently addressed. Morphine itself has been shown to cause oxidative stress through the release of histamine.9 The released histamine along with other inflammatory mediators induces an oxidative stress response by enhancing the reactive oxygen species generation and by depleting the antioxidant defense system.18 Hence, the aggravated MDA levels and SOD and CAT activity depression in morphine and SIV/SHIV-infected animals are most likely due to additive oxidative inactivation of the enzyme activity by both morphine and infection. To our knowledge, this is the first analysis of MDA, SOD, and CAT in AIDS progression in the setting of drug abuse in a macaque model. Our results show strong support for more severe oxidative stress in morphine-dependent infection. Furthermore, there was a notable relationship between more severe disease and the rate and extent of MDA increase, which may indicate that a combination of drug abuse and viral infection leads to more severe disease through a greater disruption in oxidant/antioxidant homeostasis. Equally interesting was the dual response of SOD activity to morphine treatment in the preinfection phase (weeks 20 to 0). While we do not know the basis for this divergent response, it may be rooted in a genetic factor as a polymorphic response to opioid drugs has been reported in humans.19 It is intriguing to consider that the up-regulation that occurred only in monkeys that showed slow progression may have had some impact on the survival of these individual animals. Although we cannot ascertain the cause and effect relationship of this phenomenon, it was clear that exclusively and universally in morphine-dependent animals SOD levels dropped by 50% upon viral infection and did not appreciably recover. This interesting relationship between drug abuse, viral infection, and oxidative stress certainly merits further study to allow better targeted strategies to treat HIV/AIDS patients, particularly among drug-abusing populations that represent a significant portion of the U.S. epidemic.
ACKNOWLEDGMENTS The authors wish to acknowledge the financial support from National Institute of Drug Abuse (DA015013) and National Institute on Alcohol and Alcoholism (AA015045).
REFERENCES 1. Marecki JC, Cota-Gomez A, Vaitaitis GM, et al.: HIV-1 Tat regulates the SOD2 basal promoter by altering Sp1/Sp3 binding activity. Free Radic Biol Med 2004;37:869–880. 2. Buhl R, Jaffe HA, Holroyd KJ, et al.: Systemic glutathione deficiency in symptom-free HIV-seropositive individuals. Lancet 1989;2:1294–1298.
1007 3. Walmsley SL, Winn LM, Harrison ML, et al.: Oxidative stress and thiol depletion in plasma and peripheral blood lymphocytes from HIV-infected patients: Toxicological and pathological implications. AIDS 1997;11:1689–1697. 4. Jareno EJ, Bosch-Morell F, Fernandez-Delgado R, et al.: Serum malondialdehyde in HIV seropositive children. Free Radic Biol Med 1998;24:503–506. 5. Sandstrom PA, Tebbey PW, Van Cleave S, and Buttke TM: Lipid hydroperoxides induce apoptosis in T cells displaying a HIV-associated glutathione peroxidase deficiency. J Biol Chem 1994;269:798–801. 6. Purcell DW, Metsch LR, Latka M, et al.: Interventions for seropositive injectors—research and evaluation: An integrated behavioral intervention with HIV-positive injection drug users to address medical care, adherence, and risk reduction. J Acquir Immune Defic Syndr 2004;37(Suppl. 2):S110–118. 7. Li Y, Merrill JD, Mooney K, et al.: Morphine enhances HIV infection of neonatal macrophages. Pediatr Res 2003;54:282–288. 8. MacFarlane AS, Peng X, Meissler JJ Jr, et al.: Morphine increases susceptibility to oral Salmonella typhimurium infection. J Infect Dis 2000;181:1350–1358. 9. Prieto-Lastra LA, Iglesias-Cadarso MM, Reano-Martos, et al.: Pharmacological stimuli in asthma/urticaria. Allergol Immunopathol (Madr) 2006;34:224–227. 10. Atici S, Cinel I, Cinel L, et al.: Liver and kidney toxicity in chronic use of opioids: An experimental long term treatment model. J Biosci 2005;30:245–252. 11. Kumar R, Torres C, Yamamura Y, et al.: Modulation by morphine of viral set point in rhesus macaques infected with simian immunodeficiency virus and simian-human immunodeficiency virus. J Virol 2004;78:11425–11428. 12. Kumar R, Orsoni S, Norman L, et al.: Chronic morphine exposure causes pronounced virus replication in cerebral compartment and accelerated onset of AIDS in SIV/SHIV-infected Indian rhesus macaques. Virology 2006;354:192–206. 13. Ohkawa H, Ohishi N, and Yagi K: Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351–358. 14. Misra HP and Fridovich I: The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 1972;247:3170–3175. 15. Johansson LH and Borg LA: A spectrophotometric method for determination of catalase activity in small tissue samples. Anal Biochem 1988;174:331–336. 16. Halliwell B and Gutteridge JM: Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem J 1984;219:1–14. 17. Halliwell B, Gutteridge JM, Blake D, et al.: Metal ions and oxygen radical reactions in human inflammatory joint disease. Philos Trans R Soc Lond B Biol Sci 1985;311:659–671. 18. Skaper SD, Facci L, and Kee WJ: Potentiation by histamine of synaptically mediated excitotoxicity in cultured hippocampal neurones: A possible role for mast cells. J Neurochem 2001; 76:47–55. 19. Mikus G, Somogyi AA, Bochner F, et al.: Polymorphic metabolism of opioid narcotic drugs: Possible clinical implications. Ann Acad Med Singapore 1991;20:9–12.
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