The Role Of Cytochrome P450 2E1

On Ethanol-Mediated Oxidative Stress

And HIV Replication
In Human Monocyte-Derived Macrophages

Biochemistry Journal Report

Arciaga, Frances Geline R.

1MD - 1
 The Role Of Cytochrome P450 2E1 On Ethanol-Mediated Oxidative Stress And
HIV Replication In Human Monocyte-Derived Macrophages

Abstract

One of the major health problems among people with HIV/AIDS is alcohol
consumption. Previous reports have shown that there is a decrease in the intracellular
concentrations of antiretroviral drugs elvitegravir and darunavir in the HIV-1-infected U1
cell line due to ethanol. Despite the presence of elvitegravir, ethanol is still able to
increase HIV-1 replication. Other previous findings has also shown that the levels of
cytochrome P450 enzyme 2E1 (CYP2E1) and oxidative stress in blood monocytes were
induced, while the concentration of alcohol in the plasma was reduced in HIV-1-infected
alcohol users compared to uninfected alcohol users. However, the role of CYP2E1 in
ethanol-enhanced oxidative stress and HIV-1 replication is still unclear.

Introduction

Alcohol consumption is considered to be a major health problem among people

living with HIV/AIDS. It is known to raise the risk of acquiring HIV-1 infection and the
rate of HIV-1 disease progression. Chronic alcohol consumption has been accounted to
impact HIV-1 replication and response to antiretroviral therapy. Despite this, much is still
unknown about the cellular pathways involved with alcohol consumption and HIV-1
replication.

Macrophage lineage cells or monocytes serve as a major viral reservoir for HIV-1
and play an initial role in HIV-1 infection. HIV-1-infected monocytes / macrophages can
penetrate the brain and spread the virus into the central nervous system and cause
neurocognitive disorders. MDM express CYP2E1, which plays a central role in ethanol
metabolism. CYP2E1 or Cytochrome P450 2E1 can metabolize alcohol and generate
acetaldehyde and reactive oxygen species, which can cause oxidative stress.
Objective:
To examine the cellular pathway associated with alcohol metabolism and HIV-1
replication in monocyte-derived macrophages.

Materials and Methods

2.1. Cell Culture and Treatment

Primary MDM obtained yielded 95% purity in CD14+ monocytic cells with less
than 5% contamination from B and T cells.

To evaluate the role of CYP2E1 in HIV-1 replication after ethanol exposure, two
treatment protocols were set up in differentiated U1 cells.
1) U1 cells were transfected with scramble siRNA or CYP2E1 siRNA for 24 hours
following manufacturer's protocol. Cells were then washed and treated with control
(media only) or ethanol (20 mM) every day for 3 days.
 2) U1 cells were treated with 20mM ethanol
ethanol then treated with CYP2E1 inhibitor
diethyl ether every day for 3 days. Cell culture media was collected to measure HIV p24
level.

2.2. Quantitation of oxidative DNA damage

A common marker for DNA damage is the concentration of 8-hydroxy-2’

8 -
deoxyguanosine (8-OHdG)
OHdG). Two test kits were used: the Oxiselect oxidative DNA
damage ELISA kit and EpiQuik 8-OHdG
8 DNA Damage kit. Absorbance
bsorbance was measured at
450
0 nm using a microplate reader, with the amount of 8-OHdG
OHdG calculated using the
standards provided with the kit.

Oxiselect oxidative DNA damage ELISA kit EpiQuik 8-OHdG

OHdG DNA Damage kit

 Used for MDM samples.  Uses the colorimetric method

 Has a low DNA yield of < 300 ng/sample  Used for U1 cell line samples
 Has a high DNA of ≥300
ng/sample
2.3. Reactive Oxygen Species
pecies Quantitation

MDM from Cells were

Cells were
chronic washed with
incubated Data were
ethanol PBS and the
with the 5 μM analyzed by
treatment (MFI) was
H2DCFDA dye using the BD
were measured at
in PBS for 30 FACS software
collected and 525 ± 20 nm
min at room version 8
washed with using flow
temperature
PBS. cytometer

• MDM - Monocyte-derived
derived macrophages
• MFI - Median
edian fluorescent intensity

2.4. Western Blot Analysis

nalysis

Equal amount of proteins (20 μg) from 14-days

14 treated
experiments of MDM were loaded on 10% acrylamide gel.

After SDS-PAGE,
PAGE, the proteins were transferred to a PVDF
membrane.

The membrane was blocked in LiLi-Cor blocking buffer for 1

h and incubated with primary antibodies against the ff.
AOEs at 4 °C
C overnight:
CYP2E1, Catalase, PRDX6, SOD1 , SOD2, β β-actin

The membrane was then washed and incubated with

secondary antibodies: goat anti-mouse
anti Mab, goat anti-
rabbit Mab

The blots were scanned with a Li

Li-Cor Scanner (LI--COR
Biosciences)

• AOEs: Antioxidant
ntioxidant enzymes
2.5. Reverse Transcription PCR

Real-time reverse transcription polymerase chain reaction was performed to

measure the relative mRNA fold expression of CYP2E1 and beta-actin in MDM after
ethanol exposure. Three test kits were used to perform the PCR reaction:
1) TaqMan Gene Expression kit
2) SimpliAmp Thermal Cycler
3) Step-One Plus Real-Time PCR system
The relative fold expression was calculated by using 2-ΔΔCt method by using beta-
actin as the housekeeping gene.

TaqMan Gene Expression kit

2.6. HIV-1 p24 ELISA

The level of HIV-1 viral load was determined from cell culture media by using
HIV-1 p24 antigen ELISA kit following the manufacturer's protocol.
1) The p24 was detected by measuring optical density at 450 nm using a
microplate reader.
2) The concentration of HIV-1 p24 antigen was calculated using standard
calibration curve.
3) p24 level were normalized reported as a percentage of the control group.

2.7. Safety precautions

All experiments related with HIV-1 were performed in a biocontainment

laboratory and conducted according to biosafety level BSL-3 practices under the
protection of biosafety cabinets, personal protective equipment (PPE), and N95
respirators. 10% (v/v) bleach was used to disinfect liquid waste while solid waste was
double bagged, autoclaved then removed from containment areas.
2.8. Statistical analysis

All statistical analysis were performed using RStudio while graphs were
performed using GraphPad Prism 6. Student's t-test analysis was used for comparisons
between two groups. The statistical significance among the various methods was
determined by using one-way ANOVA post-hoc Tukey HSD Test. A p-value ≤0.05
among the treatment groups was considered significant.

Results

3.1. Chronic ethanol exposure in non-infected MDM

A 14-day ethanol treatment in human primary MDM was performed to examine

the effect of chronic ethanol exposure on oxidative DNA damage, ROS production, and
CYP2E1 expression. A ~1.5-fold increase on oxidative DNA damage after 14-day
ethanol treatment in non-infected MDM was observed (Fig. 1A) while there was also an
increase in the ROS production in ethanol-treated cells (Fig. 1B). A pattern of increased
CYP2E1 protein expression in the ethanol-treated non-infected MDM (Fig. 1C) was also
observed.
To confirm the induction of CYP2E1,
an examination was done on the mRNA level
of CYP2E1 in non-infected MDM after chronic
ethanol exposure. An increase in the CYP2E1
mRNA expression was observed. (Fig. 1D)
3.2. Effect of chronic ethanol exposure on oxidative DNA damage and HIV-1
replication in HIV-1-infected MDM

A ~2-fold increase in DNA damage level was observed in ethanol-treated MDM

compared to control MDM (Fig. 2A) and was associated with enhanced viral replication
while a significant ~2.5-fold increase in viral load was observed in chronic ethanol
exposure. (Fig. 2B).

3.3. Effect of chronic ethanol exposure on the expression of CYP2E1 and

antioxidant enzymes in HIV-1-infected MDM

An increase of ~20% in the protein

expression of CYP2E1 (Fig. 3A) was observed
when compared with the non-ethanol treated
group, as well as a pattern of increase in the
mRNA expression of CYP2E1 (Fig. 3B) in
chronic ethanol-treated HIV-1-infected MDM
among 3 different donors. However, there was
a decrease in PRDX6 protein expression but no
significant change in the expression of other
AOEs upon ethanol treatment (Fig. 3C).
3.4. Role of CYP2E1 on ethanol-mediated oxidative DNA damage and HIV-1
replication in U1 cells

After ethanol exposure, there was an increased oxidative DNA damage in

ethanol-treated group rather than in the non-ethanol treated group. This increase was
rescued by CYP2E1 siRNA, which significantly decreased the DNA damage to the
control level compared with the treatment of scrambled siRNA.
HIV-1 replication after CYP2E1 siRNA transfection was also examined and an
increased HIV-1 replication was observed in the ethanol-treated scramble group. The
viral replication in both control and ethanol-treated groups was reduced by CYP2E1
siRNA. On the other hand, the CYP2E1 siRNA effect appears to be more evident in the
ethanol-treated group, having a significant decrease in HIV-1 replication.
Selective CYP2E1 inhibitor diethyl ether greatly reduced the level of HIV-1
replication upon ethanol exposure (Fig. 4C).
Discussion

Alcohol-induced liver disease is associated with a state of “oxidative stress” as

well as among many other conditions. Oxidative stress has been recognized as a potent
inducer of HIV-1 replication and uses oxidative DNA damage as a biomarker. In this
study, we observed increased oxidative DNA damage in non-infected and HIV-1-
infected human primary MDM, suggesting increased oxidative stress associated with
chronic ethanol exposure. Oxidative stress also occurs when the generation of reactive
oxygen species increase to an degree that cellular antioxidants cannot counteract.
Reactive oxygen species play a role in activating nuclear factor kappalight- chain-
enhancer of activated B cells (NF-κB), inducing HIV-1 replication, which results from a
build-up of ROS in ethanol-treated macrophages. Observations also show that acute
ethanol treatment caused significant up regulation of CYP2E1 protein in U937
monocytic cell, as well as in SVGA astrocyte, whereas in chronic ethanol treatment,
there is an increase in protein expression of CYP2E1 in HIV-1-infected primary MDM.
There is a higher expression of CYP2E1 in blood monocytes and reduced alcohol
concentration in the plasma seen in HIV-1-infected alcohol users compared to
uninfected alcohol users. It has also been recommended that alcohol consumption can
decrease the expression of antioxidant enzymes and can further attenuate cellular
antioxidant capacity. Also, changes in some AOE expression during HIV-1 infection put
PLWHA under chronic oxidative stress. An antioxidant enzyme, PRDX6, is able to
reduce hydrogen peroxide, fatty-acid hydroperoxides, and phospholipid hydroperoxides.
It plays an important role in ethanol-induced oxidative stress, showing a significant
decrease in PRDX6 expression after ethanol exposure in U937 cells. This decrease
while there is no change in other AOEs but having an increase in CYP2E1 expression,
propose an increase in oxidative stress which results to an increase in HIV-1 replication.
Aside from the liver being a primary location of CYPs, they are also expressed in
extrahepatic cells, together with monocytes and astrocytes but CYPs levels in
monocytes or astrocytes ultimately cannot cause ethanol-mediated oxidative stress.
The effect of alcohol consumption on antiretroviral drugs have also been studied.
Darunavir, an important ARV drug, is influenced by ethanol, and has faster hepatic
intrinsic clearance in the presence of a ritonavirboosted darunavir combination. Studies
suggest a potential role of ethanol in causing a reduced response to antiretroviral drugs,
as it has the ability to interact with microsomal CYP3A4 and alter elvitegravir
metabolism and HIV-1 replication in monocytic cells. Overall, ethanol has the capability
of increasing HIV-1 replication, not only through CYP2E1-mediated oxidative stress but
also through a decreased efficacy on antiretroviral drugs. Results signify the next step in
discovering new and improved treatment strategies for people living with HIV/AIDS.

Summary

Alcohol consumption is considered to be one of the major health problems

among people living with HIV/ AIDS. Previous reports have shown that ethanol can
reduce intracellular concentrations of antiretroviral drugs in the HIV-1-infected U1 cell
line, but it can also increase HIV-1 replication in the presence of elvitegravir.
Cytochrome P450 enzyme 2E1 (CYP2E1) levels and oxidative stress in blood
monocytes are induced, whereas alcohol concentration in the plasma was significantly
decreased in HIV-1-infected alcohol users compared to uninfected alcohol users.
Nevertheless, given the studies shown, the role of CYP2E1 in ethanol-enhanced
oxidative stress and HIV-1 replication is still unclear.
The chronic effects of ethanol on HIV viral load, oxidative DNA damage,
CYP2E1, antioxidant enzymes and reactive oxygen species expression in human
monocyte-derived macrophages were examined for 2 weeks. Following ethanol
treatment, CYP2E1 siRNA and CYP2E1 selective inhibitor were used in the HIV-1-
infected U1 cell line to evaluate the role of CYP2E1 in mediating ethanol-induced viral
replication. Chronic ethanol exposure showed a 3-fold significant increase in oxidative
DNA damage and CYP2E1 expression in both non-infected and HIV-1-infected MDM.
This ethanol-enhanced HIV-1 replication was associated with an increased CYP2E1
expression and oxidative DNA damage, with a decreased PRDX6 expression. A
decrease in viral replication and DNA damage after repression of CYP2E1 by siRNA,
was observed upon ethanol exposure. Thus, suggesting that chronic ethanol exposure
increases HIV-1 replication in MDM, through CYP2E1-mediated oxidative stress.

Conclusion

The study shows an enhanced HIV-1 replication in human primary monocyte-

derived macrophages following chronic ethanol treatment. It includes the involvement of
CYP2E1 and oxidative stress in ethanol-induced HIV-1 replication in monocyte-derived
macrophages, by the use of CYP2E1 siRNA to remove CYP2E1 expression and a
CYP2E1 novel inhibitor to reduce CYP2E1activity.
The results of the study suggest that chronic ethanol exposure increases HIV-1
replication and oxidative stress in human primary monocyte-derived macrophages. To
maintain homeostasis, the enhanced oxidative stress is accompanied by CYP2E1
induction and failure of cellular antioxidant mechanisms.
These results are clinically relevant to potentially find effective treatment
strategies for HIV-1-infected alcohol users.

Recommendation

Use of other analytical biochemistry assay for studying about the role of
cytochrome p450 in alcohol-mediated stress and potentially discover more strategies for
treatment in HIV-1 infected alcohol users.
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