PLoS One. 2015 Apr 16;10(4):e0122402. doi: 10.1371/journal.pone.0122402.  

Effect of mild-to-moderate smoking on viral load, cytokines, oxidative stress, and cytochrome P450 enzymes in HIV-infected individuals.

 

Ande A1, McArthur C2, Ayuk L3, Awasom C3, Achu PN4, Njinda A4, Sinha N5, Rao PS5, Agudelo M6, Nookala AR1, Simon S7, Kumar A1, Kumar S5.

Author information

1Division of Pharmacology and Toxicology, School of Pharmacy, University of Missouri-Kansas City, Kansas City, Missouri, United States of America.

2Department of Oral and Craniofacial Science, School of Dentistry, University of Missouri-Kansas City, Kansas City, Missouri, United States of America.

3Regional Hospital, Box 818, Bamenda, North West Province, Cameroon.

4Mezam Polyclinic HIV/AIDS Treatment Center, Bamenda, Cameroon.

5Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America.

6Department of Immunology, Herbert Wertheim College of Medicine, Florida International University, Miami, Florida, United States of America.

7Department of Medical Informatics, School of Medicine, University of Missouri-Kansas City, Kansas City, Missouri, United States of America.

 

Abstract

Mild-to-moderate tobacco smoking is highly prevalent in HIV-infected individuals, and is known to exacerbate HIV pathogenesis. The objective of this study was to determine the specific effects of mild-to-moderate smoking on viral load, cytokine production, and oxidative stress and cytochrome P450 (CYP) pathways in HIV-infected individuals who have not yet received antiretroviral therapy (ART). Thirty-two human subjects were recruited and assigned to four different cohorts as follows: a) HIV negative non-smokers, b) HIV positive non-smokers, c) HIV negative mild-to-moderate smokers, and d) HIV positive mild-to-moderate smokers. Patients were recruited in Cameroon, Africa using strict selection criteria to exclude patients not yet eligible for ART and not receiving conventional or traditional medications. Those with active tuberculosis, hepatitis B or with a history of substance abuse were also excluded. Our results showed an increase in the viral load in the plasma of HIV positive patients who were mild-to-moderate smokers compared to individuals who did not smoke. Furthermore, although we did not observe significant changes in the levels of most pro-inflammatory cytokines, the cytokine IL-8 and MCP-1 showed a significant decrease in the plasma of HIV-infected patients and smokers compared with HIV negative non-smokers. Importantly, HIV-infected individuals and smokers showed a significant increase in oxidative stress compared with HIV negative non-smoker subjects in both plasma and monocytes. To examine the possible pathways involved in increased oxidative stress and viral load, we determined the mRNA levels of several antioxidant and cytochrome P450 enzymes in monocytes. The results showed that the levels of most antioxidants are unaltered, suggesting their inability to counter oxidative stress. While CYP2A6 was induced in smokers, CYP3A4 was induced in HIV and HIV positive smokers compared with HIV negative non-smokers. Overall, the findings suggest a possible association of oxidative stress and perhaps CYP pathway with smoking-mediated increased viral load in HIV positive individuals.

PMID: 25879453

 

Supplement

Over the past several decades, cigarette smoking has been identified as a serious health hazard and the impact of cigarette smoking has been evaluated on several disease models. In addition to its significant impact on neurochemistry and the inherent addictive properties of nicotine, cigarette constituents are well-known carcinogens and leading cause of lung cancer. Importantly, compared to patients with other chronic ailments and the general population, a higher prevalence of cigarette smoking has been reported in HIV-infected individuals. Some studies have reported as high as 50-70 % of recruited HIV patients to be current smokers. Such observations have resulted in critical analysis of the impact of cigarette smoking on HIV disease progression and adherence to anti-HIV medication in HIV positive smokers. However, the direct impact of cigarette smoking on HIV pathogenesis remains unknown.

Since the discovery of antiretroviral drugs, HIV infection has transformed from a lethal infective disease to a chronic ailment. The increased lifespan for HIV positive individuals has resulted in an enhanced risk for developing substance abuse problems. Given the ease of access to cigarette and other tobacco products, cigarette smoking has emerged as one of the predominantly abused drugs amongst HIV individuals. Since little is known about the cellular mechanisms by which cigarette smoking might have an impact on HIV replication, we designed the current study to evaluate the effects of cigarette smoking in HIV-infected subjects. We recruited HIV-infected and uninfected patients who were either mild-to-moderate smokers or non-smokers, and evaluated the status on HIV replication and other parameters. In particular, we examined the changes in monocytic cells since these cells are known to harbor HIV and serve as viral reservoirs in HIV-infected patients.

Mainstream cigarette smoke is known to be a complex mixture of chemicals which includes nicotine, heavy metals, polycyclic aromatic hydrocarbons (PAHs), aromatic amines, N-nitrosamines etc. Upon entering the systemic circulations, cigarette constituents are extensively metabolized by specific isoforms of cytochrome P450 (CYP) enzymes. For instance, about 70-80 % of systemic nicotine is metabolized to cotinine by CYP2A6 enzyme. Similarly, PAHs are metabolized/activated by CYP1A/CYP1B enzymes to reactive intermediates that can form carcinogenic DNA adducts. Therefore, CYP enzymes play a critical role in determining the bioavailability and activation of various cigarette constituents. The involvement and significance of CYP enzymes in context of governing cigarette smoke mediated harmful effects in HIV positive individuals has been reviewed by us previously [1, 2]. In addition, the harmful effects of cigarette constituents are known to be mediated, in part, through the generation of free radicals and reactive oxygen species (ROS). Importantly, an intricate link between CYP-mediated metabolism and generation of ROS was confirmed by our group and others. Considering the fact that HIV replication and pathogenesis are enhanced by oxidative stress, we hypothesized a direct involvement of cigarette smoking-induced changes in CYP and oxidative stress pathways in enhanced HIV replication in current smokers.

Our hypothesis was further supported by preliminary study, wherein, we have demonstrated enhanced expression of CYP2A6 and CYP1A1 enzymes in nicotine treated astrocytes [3]. This nicotine-mediated change in CYP2A6 expression was found to be associated with an enhanced ROS production and was inhibited in the presence of selective CYP2A6 inhibitor, tryptamine. A follow up study reported an enhanced nicotine metabolism in HIV-positive smokers, compared to HIV-negative smokers, suggesting a direct impact of HIV infection on nicotine metabolism [4]. These in vitro and ex vivo studies provided substantial evidence for a significant change in metabolism of nicotine, and possibly other cigarette constituents, in HIV-infected smokers. Taking into consideration the impact of cigarette smoking on CYP and oxidative stress pathways, these results warranted a closer examination of changes in CYP expression and antioxidant activity in HIV-infected smokers.

Therefore, to validate our previous findings, in this study, upon stringent inclusion and exclusion criteria, we recruited uninfected nonsmokers, infected nonsmokers, uninfected smokers, and infected smokers, and collected blood samples. To confirm our hypothesis, we first tested the effect of smoking status on plasma viral load of HIV patients. As rationalized, mild-to-moderate smoking in HIV patients was associated with a significant increase in plasma viral load compared to non-smokers. To replicate and confirm the observed clinical effects of smoking, in a different set of experiments, we treated HIV-infected primary macrophages with cigarette smoke condensate (CSC) and analyzed p24 levels in cell-free supernatant. As reported in the paper, CSC treatment of HIV-infected primary macrophages was also found to be associated with an increase in HIV replication. In addition, several aberrations in key antioxidant defense parameters were observed in HIV-infected smokers, indicating an elevated oxidative stress in these individuals. However, an absence of induction in the levels of antioxidant enzymes (AOEs) to counter the elevated oxidative stress was seen in HIV infected smokers. Lastly, significant changes were also observed in expression of CYPs in monocytes collected from recruited subjects. Importantly, the expression of major antiretroviral drugs metabolizing enzyme, CYP3A4, was significantly upregulated in monocytes collected from both HIV-infected nonsmokers and HIV-infected smokers. Thus, an enhanced metabolism and reduced bioavailability of antiretroviral drugs is rationalized in monocytic cells of HIV positive individuals, promoting the development of monocytes as HIV reservoir.

 

KS fig1

Figure 1: The effects of cigarette smoking in HIV-infected individuals includes significant changes in the expression of cytochrome P450 and antioxidant enzymes. These changes are rationalized to decrease the bioavailability of commonly used antiretroviral drugs and increase the oxidative stress in monocytic cells. In combination, these changes are associated with an enhanced HIV replication.

 

Overall, the observed changes in this study support our hypothesis that CYP and oxidative stress pathways play a critical role in smoking-mediated enhanced HIV replication (Figure 1). Changes in expression of AOEs and CYPs can potentially lead to an enhanced oxidative stress while reducing the bioavailability of antiretroviral drugs leading to enhanced viral replication. On-going studies are aimed at delineating the cellular mechanisms involved in the observed smoking-induced effects on HIV replication. We expect the current efforts to help us identify novel targets for treating the smoking-mediated adverse effects on HIV pathogenesis in HIV positive cigarette smokers.

 

References:

  1. Rao, P.S. and S. Kumar, Polycyclic aromatic hydrocarbons and cytochrome P450 in HIV pathogenesis. Front Microbiol, 2015. 6: p. 550.
  2. Ande, A., et al., Tobacco smoking effect on HIV-1 pathogenesis: role of cytochrome P450 isozymes. Expert Opin Drug Metab Toxicol, 2013. 9(11): p. 1453-64.
  3. Ande, A., et al., An LC-MS/MS method for concurrent determination of nicotine metabolites and the role of CYP2A6 in nicotine metabolite-mediated oxidative stress in SVGA astrocytes. Drug Alcohol Depend, 2012. 125(1-2): p. 49-59.
  4. Earla, R., et al., Enhanced nicotine metabolism in HIV-1-positive smokers compared with HIV-negative smokers: simultaneous determination of nicotine and its four metabolites in their plasma using a simple and sensitive electrospray ionization liquid chromatography-tandem mass spectrometry technique. Drug Metab Dispos, 2014. 42(2): p. 282-93.

 

Acknowledgements: The work was supported by the NIH grants DA031616 (SK), AA022063 (SK), and AA020806 (AK).

 

KS fig2Contact:

Santosh Kumar, Ph.D.

Associate Professor

881 Madison Ave, Rm 456

Memphis, TN 38163

Phone: 901-448-7157

Fax: 901-448-2062

Email: ksantosh@uthsc.edu

 

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