potential role of cytochrome p450s in mediating the effects of alcohol on hiv pathogenesis...
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![Page 1: Potential role of cytochrome P450s in mediating the effects of alcohol on HIV pathogenesis P.S.Shantanu Rao, Ph.D. Pharmaceutical Sciences College of Pharmacy](https://reader031.vdocuments.mx/reader031/viewer/2022020208/5697c0201a28abf838cd23b1/html5/thumbnails/1.jpg)
Potential role of cytochrome P450s in mediating the effects of alcohol on
HIV pathogenesis
P.S.Shantanu Rao, Ph.D.Pharmaceutical Sciences
College of Pharmacy
University of Tennessee Health Science Center
Memphis, TN
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RATES OF HIV DIAGNOSES PER 100,000 POPULATION
Shelby county report, 2013
~21,400 Tennesseans with HIV/AIDS.
10th in the country in the rate of new HIV infections.
5th in the country in the rate of HIV/AIDS deaths.
Memphis among the top 10 cities for HIV infection rates.
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Prevalence of mild-to-moderate alcoholic (7-14
drinks/week for Men and 4-7 drinks/week for Women): 3-fold (50-60%) higher in HIV-infected population
Alcohol
HIV-1 infection
AIDS and neuroAIDS
Neuronal damage and neuropsychological impairments
Mortality risk of AIDS and other diseases
Alcohol use amongst HIV-infected population
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Effects of alcohol on HIV-1 infection
Oxidative stress Response to antiretroviral therapy (ART) Viral replication Immune function CNS barrier permeability
CYTOCHROME P450 (CYP)?
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Alcohol induces CYP2A6, CYP2E1, CYP3A4.
Alcohol produces reactive oxygen species (ROS).
Alcohol induces anti-oxidants: SOD1, catalase.
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Hypothesis
Model:
In vitro: U937 monocytic cellsIn vitro: Primary HIV-infected macrophages In vitro: ART metabolism: Effects of ethanol Ex vivo: Human monocytes/macrophages
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Experimental Design
Day 1 Day 5 Day 9 Day 13
U937 cells(0.2*106/mL)
Collect and Replate (0.2*106/mL)
Collect and Replate (0.2*106/mL)
Collect cells
Every 12h; 0.5mL fresh media Every 12h; 0.5mL fresh media Every 12h; 0.5mL fresh media
Ethanol: 50 mMART: Darunavir (Dar) 4µM and Ritonavir (1µM)
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Results: Expression of ethanol metabolizing enzyme
β-ACTIN
CYP2E1
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Expression of major AOEs
β-ACTINSOD1
β-ACTINSOD2
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β-ACTIN
Catalase
Expression of major AOEs
β-ACTINPRDX6
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Transcription of HO-1
• Inducible heme degrading enzyme in cells
• Stress response protein induced under oxidative stress
• Reported to play a critical role in cellular protection
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Expression of major ART metabolizing enzyme
CYP3A4
β-ACTIN
• Major ART metabolizing enzyme in U937 cells.
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Transcription of major drug efflux transporter
• Major drug efflux transporter expressed in U937 cells.
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Results so far suggest---
CYP2E1
AOEs
CYP3A4
ABCC1
Increase ROS/oxidative stress/cell death
Increase drug accumulation/toxicity/cell death
ROS/Cell death?
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ROS measurement
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Cell viability (FACS)
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Cell viability (XTT)
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Day 0
Day 5
Control Ethanol+ARTART Ethanol
U937 microscopic image (10x)
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Rationalized effects of chronic ethanol+ART on HIV pathogenesis
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Hypothesis
Model:
In vitro: U937 monocytic cellsIn vitro: Primary HIV-infected macrophages In vitro: ART metabolism: Effects of ethanol Ex vivo: Human monocytes/macrophages
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Experimental design
Collect PBMCs(buffy coat)
Collect, infect, and plate (1.5-2.5*106/well)
mCSF (50 ng/ml) 7-10 days
Macrophage differentiation
HIV-Ada strain20ng/10*106 cells
Monitor p24 titer 7-10 days
Control Ethanol20 mM ethanol 14 days
0.5 ml fresh media each day
Collect RNA/Protein
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Effect of chronic ethanol on p24 production
Control Ethanol0
100
200
300
400
500
Perc
ent H
IV p
24 le
vel (
100%
con
-tr
ol)
Day 0
Day 3
Day 4
Day 7
Day 8
Day 9
Day 10
0
5
10
15
20
25
HIV replication - time course
HIV
p24
con
cent
ratio
n (n
g/m
l)
*
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Control Ethanol0.0
0.5
1.0
1.5F
old
mR
NA
exp
ress
ion
CYP2E1
β-Actin
Control Ethanol0.0
1.0
2.0
3.0
Fo
ld m
RN
A e
xpre
ssio
n
CYP3A4
β-Actin
Control Ethanol0.0
0.5
1.0
1.5
2.0
Fo
ld m
RN
A e
xpre
ssio
n
Catalase
β-Actin
Control Ethanol0.0
0.5
1.0
1.5
Fo
ld m
RN
A e
xpre
ssio
n
PRDX6
β-Actin
Control Ethanol0.0
0.5
1.0
1.5
Fo
ld m
RN
A e
xpre
ssio
n
SOD1
β-Actin
Control Ethanol0.0
0.5
1.0
1.5
2.0
Fo
ld m
RN
A e
xpre
ssio
n
SOD2
β-Actin
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Possible cellular pathways mediating the effects of chronic alcohol
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Hypothesis
Model:
In vitro: U937 monocytic cellsIn vitro: Primary HIV-infected macrophages In vitro: ART metabolism: Effects of ethanol (on-going) Ex vivo: Human monocytes/macrophages
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Experimental design
Recruited patients
Collect Plasma and Monocytes
Non-smokersNon ART/infectious diseases
Mild-to-moderate Alcohol user
Alcohol UsersN=6
HIV+alcohol UsersN=4
Collect RNA/DNA/Protein
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Oxidative stress parameters
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Relative alcohol metabolism
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Antioxidant enzymes - expression in monocytes
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Observed interactions between alcohol intake and HIV infection
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Conclusions
• In vitro studies
• Chronic ethanol and/or ART treatment significantly alter the expression of CYPs and AOEs.
• These changes were associated with enhanced production of reactive oxygen species and decreased
cell viability.
• HIV replication in primary MDM is enhanced following chronic ethanol treatment.
• Preliminary data suggests associated changes in expression of CYPs and AOEs.
• Ex vivo study
• Alcohol use amongst HIV infected patients was associated with enhanced oxidative stress compared to
non-infected alcohol users.
• Overall, chronic ethanol mediated changes in CYPs and AOEs are rationalized for the
enhancement in HIV replication.
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Acknowledgement
• Dr. Santosh Kumar (PI)
• Kumar research group
• College of pharmacy, UTHSC
• NIH - AA022063-01A1 and DA031616
Thank you for your attention!!!
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Samir Zakhari, Overview: How Is Alcohol Metabolized by the Body? NIAAA publication
Ethanol metabolism
• CYP3A4 and CYP1A2 (minor role)
• CYP2E1 (Induction by ethanol): Brain, Hearth, Lungs, Macrophages
• ADH/ALDH: primarily in Liver
• The Km of CYP2E1 for alcohol is 10 mM ,10-fold higher than the Km of ADH for ethanol
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Ethanol 50mM
Methods: “via an indwelling intragastric catheter to achieve an alcohol concentration of 50 to 60 mM for 4 consecutive days per week for the duration of the study”
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In vitro studies with primary human hepatocytes
• Alcohol (35-85 mM)
Peripheral Blood Lymphocytes
• Alcohol treatment: up to 40mM• No effect on cell viability with 80 mM ethanol
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ART concentration
• Darunavir median plasma concentration (with ritonavir): 7.2µM• Yilmaz et al., AIDS Res Hum Retroviruses. 2009 Apr; 25(4): 457–461
• Combination ratio: PI+ritonavir: 4:1 (with lopinavir as Kaletra®)
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ART affects ethanol metabolism
• Ethanol concentration in untreated HIV patients: 28mM (Dosed 1g/kg ethanol)• In patients on ART: 25 mM • DOI: 10.1097/QAI.0b013e318256625f, 2012.
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Chronic Ethanol
ROS? NFκB?
CYP2E1 AOEs
Enhanced HIV pathogenesis
Chronic Ethanol
Oxidative stress
CYP2E1 AOEs
Enhanced HIV pathogenesis?
HIV infectionEthanol+ART
ROS cell viability
CYP2E1 CYP3A4 AOEs
Enhanced HIV pathogenesis?