a new antioxidant prevents toxicity of hiv proteins with methamphetamine

A New Antioxidant Prevents Toxicity of HIV Proteins with Methamphetamine

Nuran Ercal MD PhDDepartment of Chemistry,

Missouri University of Science and Technology, Rolla, MO

&

Department of Internal Medicine, St. Louis University, St. Louis, MO

Our Hypothesis:

Toxic HIV ProteinsAddictive Drugs

(METH)

OXIDATIVESTRESS

BBB

BBB integrity disrupted

NEURONs are exposed to TOXINS!

What is Oxidative Stress?

Increased generation of ROS and/or a decrease in the antioxidant capacity of cells result in “Oxidative Stress”.

Oxidative stress compromises crucial cellular functions.

Sources of Reactive Oxygen Species

1) Non mitochondrial: NADPH OxidasesMicrosomal cytochrome P-450CyclooxygenasesMonoamine oxidases

Peroxisomal oxidation of fatty acids

Phagocytes

2) >90% is mitochondrial electron transport chain contains several redox centers

that may leak electrons to oxygen----- Superoxide radical!

ROS Superoxide (O2

¯• ) – No direct effects on targets Penetrates important sites Subsequently converted to other ROI

Hydrogen Peroxide (H2O2) – Dismutation of superoxide radical

2H + + 2O2¯• H2O2 + O2

Reacts with thiols Bacteriocidal only at higher concentrations Secondary oxidants from H2O2 responsible for

killing

SOD

Hydroxyl Radicals (OH•) – Fenton Reaction

Fe 2+ +H2O2 Fe 3+ + OH¯ + OH•

OH• as a major component of neutrophil bacteriocidal arsenal is controversial

Limited radius of action

Consequences of oxidative stress

Membrane peroxidationDecreased membrane fluidity

Oxidative Damage to Lipids

8-iso-PGF2 F2-Isoprostanes

Oxidative Damage

to DNA

MutationsDeletions O

H

N

NH

NH

N

NH2

Sugar

oxo8dG

Oxidative Damage

to Proteins

Oxidation of sulfhydryl groupsReactions with aldehydesProtein aggregation

Protein carbonylsPROTEIN

Marker

Outline

Introduction (background) HIV Associated Dementia

Methamphetamine

Blood Brain Barrier (BBB)

Proposed Research Goals

Experimental Methods Previous Results: RBE4

Recent Results: HBMVEC

Future Studies: immortal HBMVEC and transgenic animals

Conclusion

What is the problem?

Neurological disorders are serious complications of human immunodeficency virus type 1 (HIV-1).

What are these neurological complications? HIV-associated dementia (HAD) HIV-related encephalitis (HIVE) More commonly minor neurocognitive

problems DEMENTIA is the most challenging

complications of HIV infection.

Avindra Nath et al International Review of Psychiatry, Feb 2008, 20 (1), 25-31.

What is HAD?

A neurological syndrome characterized by cognitive deficits and motor and behavioral dysfunction.

Symptoms include:Sluggish mental capabilities and motor controlMarked apathyLoss of interest in previously enjoyable activitiesTremors and impaired balance, slow eye

movement, abnormal reflexes..The disorder continues to escalate, eventually resulting in severe dementiaSome experience mania or even psychosis

What are the symptoms of HAD?

How often is HAD seen?

One third of adults and half of children with HIV have dementia in the western countries

HAD is the most common case of dementia among people aged 40 or less and a significant independent risk factor for death due to AIDS!

Steiner J et al, Antioxidants &Redox Signaling, 2006; McArthur JC et al, Neurology, 1993; Bouwman FH et al, Neurology, 1998

HAART (Highly Active AntiRetroviral Therapy) therapy has reduced HAD in infected population some but it does not seem to be effective.

In the era of HAART, the course of HIV dementia appears to have changed.

Avindra Nath et al International Review of Psychiatry, Feb 2008, 20 (1), 25-31.

Treatment of AIDS/HIV

Highly active antiretroviral therapy (HAART) is introduced in the mid-90s.

HAART consists of combination of nucleoside reverse transcriptase inhibitors non-nucleoside RT inhibitors and protease inhibitors

There are more than 20 (zidovudine, lamivudine, stavudine, emtriva, crixivan, kaletra and more) approved antiretroviral drugs.

HAART therapy has been shown to prolong survival in AIDS patients.

Current Pharmaceutical Design, 2006, 12,2031-2055

How about: World Wide AIDS Statistics

Over 22 million people have died from AIDS Over 42 million people are living with HIV/AIDS Over 19 million women are living with HIV/AIDS There are over 14,000 new infections every day

(95% are in developing countries)

http://www.until.org/statistics.shtml

World Wide Future Estimates

The UN estimates that, currently, there are 14 million AIDS orphans and that by 2010 there will be 25 million

By the year 2010, five countries (Ethiopia, Nigeria, China, India, and Russia) with 40% of the world’s population will add 50 to 75 million infected people to the worldwide pool of HIV disease if nothing is done to help stop the spread of HIV/AIDS


United States AIDS Statistics

One million people are currently living with HIV in the U.S., with approximately 40,000 new infections occurring each year

70% of these new infections occur in men and 30% occur in women

75% of the new infections in women are heterosexually transmitted

Half of all new infections occur in people 25 years old or younger


What is the possible mechanism of HIV dementia?

HIV envelope protein (gp120) and transregulatory protein (Tat) may play a role in the development of HAD by increasing the production of reactive oxygen species (ROS) in blood brain barrier (BBB).

Oxidative stress may be involved in HIV neuropathogenesis.

Oxidative Stress Related Disorders

Parkinson’s Disease Alzheimer’s Disease Stroke Rheumatoid arthritis Atherosclerosis Vascular dysfunction Multiple sclerosis Inflammatory bowel disease H. pylori-associated gastritis Systemic inflammatory

response syndrome

And the list is growing…

Autoimmune thyroid disease Cystic fibrosis Diabetes Aging Macular degeneration HIV/AIDS Cancer Septic shock Heavy metal toxicity Nanoparticle toxicity EtOH abuse Meth ALS

Is there “Oxidative Stress” in HIV Dementia? Analyses of brain tissue and CSF of patients with

HIV-1 dementia shows evidence for oxidative stress correlated with disease pathogenesis and cognitive impairment.

Evidences are: 4-HNE is high Protein carbonyls are high Nitrated tyrosine residues are increased in HIV

dementia brains

Another important concern: Methamphetamine (METH)

It is widely known that many people with HIV-1 use addictive drugs. Among them: Alcohol Methamphetamine Cocaine Nitrite Inhalants Hallucinogens

Nearly 50% of HIV positive women in the US contract the infection via drug use!! Methamphetamine abuse is common

9.4 million people in the US Midwest – 90% of all drug cases Methamphetamine (METH) induces ROS.

Crystal

PowderPill

Missouri – Highest Rate of METH Lab Activity in the Country

Picture source: US Department of Justice (www.usdoj.gov)

More on Methamphetamine

Street names of methamphetamine (METH) Speed Ice Crystal Meth Chalk

Consumption Smoke Snort Inject Oral Ingestion

National Drug Intelligence Center, National Drug Threat Assessment 2008, October 2007; Picture Source: http://cerhr.niehs.nih.gov/chemicals/stimulants/amphetamines/methamphetamine.gif

Surge in catecholamine levels: Dopamine (DA) Serotonin Norepinephrine (NE)

Meth and Oxidative Stress

Oxidative stress has been shown to play an important role in the toxic effects of METH.

METH increases levels of dopamine.

Dopamine can react to form ROS through several different pathways.

Josephine W.S et al., Annals of the New York Academy of Sciences (2000)Zecca, L., et al., Nature Reviews (2004)

Dopamine autoxidation

DA + O2 SQ· + O2· - + H+

DA + O2· - + 2 H+ SQ· + H2O2

Dopamine enzymatic oxidation MAO

DA + O2 + H2O-------------------

3,4,dihydroxyphenylacetic acid + NH3 +

H2O2

What are toxic HIV proteins?

HIV envelope protein (gp120) and transregulatory protein (Tat) may play a role in the development of HAD by increasing the production of reactive oxygen species (ROS) in blood brain barrier (BBB).

What exactly is happening?

The BBB has been implicated in the development of HIV Dementia.

The BBB is a barrier between the blood and the fluid that surrounds the cells of the brain.

The BBB is selectively permeable, allowing some substances to cross and not others. It is generally more permeable to lipophilic substances.

The cells that line the capillaries of the rest of the body usually have small gaps between them, that allow substance exchange. The BBB lacks these small gaps, prohibiting much exchange.

Blood Brain Barrier (BBB)

What is the function of the BBB?

The BBB maintains that delicate balance by regulating the entry and exit of substances.

The BBB also provides protection by preventing toxic chemicals from entering the brain.

The structural integrity of the BBB is compromised in HIV/AIDs demented patients.

What is the cause of this destruction?

Studies have found that HIV-1 envelope protein gp120 and/or Tat may have a role in the structural damage that the BBB experiences.

What’s causing the blood-brain barrier to fail?

Our Hypothesis:

Toxic HIV ProteinsGp120 and tat

Addictive Drugs(METH)

OXIDATIVESTRESS

BBB


NEURONs are exposed to TOXINS..

Let’s study HIV-1 and its proteins

gp120 gp120 is a protein on the outer envelope of the HIV-1 virus.

It binds to a receptor on CD4 cells and aides in the injection of viral nucleic acid into the host cell.

The amino acids responsible for this binding interaction are highly conserved amongst strains of the virus.

Gp120 (red) in complex with a CD4 surface protein (yellow)

gp120 is composed of an outer and inner domain made of α helices and β sheets.

The negatively charged central cavity contains the binding site for a positively charged region of a surface protein on CD4 (electrostatic interactions).

More on gp120

Molecular Structure of gp120

gp120 changes confirmation when bound to different proteins

Picture Source: Tongqing Zhou et al. Nature 2007

Transregulatory Protein (Tat)

It is a viral protein released from HIV-1-infected T cells and monocytes/macrophages

Transactivator of Transcription (Tat) protein consists of between 86 and 101 amino acids depending on the subtype

Jeang, K. T. (1996) In: Human Retroviruses and AIDS: A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences. Los Alamos National Laboratory (Ed.) pp. III-3–III-18; Picture Source: Grant R. Campbell et al. J. Biol. Chem. 2004

Tat and gp120-Induced Oxidative Stress

Although the mechanism is not explicitly known, it has been shown that both gp120 and tat increase oxidative stress levels in cells.

Tat Decreases GSH by inhibiting GSH synthetase Increases NO secretion which induces apoptosis Increases calcium uptake resulting in production of ROS

gp120 Decreases GSH by inhibiting GSH synthetase Increases cell permeability Promotes calcium overload Oxyradical production Mitochondrial dysfunction by altering ion channels

Choi J et al, J Biol Chem 2000; Toneatto S et al, AIDS 1999; Visalli, Valeria, Neuroscience 2007

METH

Gp120TAT

Oxidative Stress

Microglialactivation

NOO¯

Dopamine

Glutamate mobilization by NMDARactivation

Autooxidation

Oxidation by MAO

iNOSeNOS

•NO

Ca2+

overload

nNOS

ROS

O2•¯

DNA oxidationProtein oxidationLipid Peroxidation

H2O2

QuinonesO2

•¯, OH•

Cytokines & Chemokines IL-1B,IL-8,TNF-a CCL2, CCL4,CXCL8

ERK1/2

MAPK

RNS

Antioxidants

Oxidative Stress: Possible Pathways

Oxidative Stress in HIV Dementia

Analyses of brain tissue and CSF of patients with HIV-1 dementia shows evidence for oxidative stress correlated with disease pathogenesis and cognitive impairment.

Evidences are: 4-HNE is high Protein carbonyls are high Nitrated tyrosine residues are increased in HIV

dementia brains

HIV Associated Dementia and METH

Overlap of pathways leading to neurodegeneration in HAD

METH use enhances gp120 and Tat mediated neurotoxicity

Oxidative stress common to viral protein and METH mediated toxicity

Therapeutic approaches Block rise in intracellular calcium Antagonize NMDA receptors Target oxidative damage

Disruption of Blood Brain Barrier

Disruption of the BBB is seen more in AIDS/HIV dementia patients then in non-demented AIDS/HIV patients.

The disruption of the BBB may be due to an increase in ROS by viral proteins

Tat and gp120 induce oxidative stress Stimulation of iNOS and production of NO Glutamate mediated excitotoxicity Disruption of calcium homeostasis

METH may potentiate Tat and gp120 induced oxidative stress on BBB.

Antioxidants in Neurodegeneration

Highly active antiretroviral therapy (HAART) does not prevent BBB disruption nor does it decrease ROS production.

Therefore, ANTIOXIDANTS should be included in the treatment to prevent HAD.

Dealing with BBB Most antioxidants (AO) unable to cross BBB Design AO capable of crossing BBB Low MW thiol antioxidants with ability to cross BBB

Thiols as Antioxidants

The most important biological thiol, Glutathione (GSH), protects cells against ROS.

GSH deficiency has been associated with various neurodegenerative diseases.

A significant decrease in GSH levels in patients with AIDS/HIV in various biological samples (blood, liver, brain).

Thiol antioxidants in HAD: NAC (decreased the mortality rate of HIV-infected patients) NAC analogs (N-acetyl-L-cysteinyl)-S-acetylcysteamine (NACA, possible candidate)

Pocernich CB et al., Brain Research Reviews, 2005 .

Structures of NAC and NACA

NAC NACA

NACA related projects

NACA in radiation damage NACA in neurological complications of

HIV Dementia Lead poisoning (possibly via Glu)

NACA in medicinal and abusive drug complications Acetaminophen poisoning METH neurotoxicity

NACA in macular degeneration

30-day percentage survival rates of SD-rats after irradiation with pre-treatment of NAC or NACA and post-treatment of NAC or NACA.

A: XRT only; B: XRT + NAC (pre-treated); C: XRT + NACA (pre-treated);D: Control (no XRT and any treatment); E:NAC only; F: NACA only; G:XRT + NAC (post-treated); H: XRT + NACA (post-treated).

0%

25%

50%

75%

100%%

30-

day

su

rviv

al r

ate

A B C D E F G H

Animal groups

Control NACA only (750 μM)

Glutamate only (10 mM)Glutamate (10 mM) + NACA (750 μM)

PC12 cells were plated at a density 25 x 103 cells/well in a 24 well plate and grown for 24 h in culture medium; then they were treated or not (control) with 10 mM Glu with or without NACA.. Twenty four h later, cells were examined and photographed.

Immortalized HBMVEC

Control METH 1mM METH +NACA NACA0.5mM0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

***

###

HCMVEC/D3 Cells were pretreated with 1mM NACA for 2 h, followed by24 h incubation with METH 1mM. NACA protected HCMVEC/D3 cells fromcytotoxicity caused by METH. Values were the means +SD of threesamples.Statistically different values of ***p <0.0001 were determined,compared to control. ###p < 0.0001 compared to METH 1mM group.

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Leading cause of blindness in people over 55

Degeneration of macular region of the eye

Macula – oval yellow spot near the center of the retina of the eye

Near its center is the fovea, a small pit containing the largest concentration of cone cells of the eye. Responsible for central vision

Disease begins when transport of nutrients and waste via retinal pigment epithelial cells begins to slow and down, leading to accumulation of waste products

Age Related Macular degeneration

Changes in Macula in ARMD

Image Source: www.sfn.org (Society for Neuroscience)

Retinal Pigment Epithelium

RPE affected in AMD

Undergoes oxidative stress: Metabolically active cells Large oxygen fluxes across its boundary Exposure to sunlight RPE phagocytose photoreceptor outer segments

containing PUFAS which can undergo lipid peroxidation

Age decreases antioxidants in RPE, increasing effect of oxidative insults

Experimental design Cell model – Adult retinal pigment epithelial cells

(ARPE-19)

Oxidative stress induction – tert butylhydroperoxide (tBHP), a cell permeant oxidant that causes lipid peroxidation and loss of GSH

Antioxidant – Various concentrations of N-acetycysteine amide

Pretreat cells for 24 h with antioxidant, followed by treatment with tBHP (0.4 mM) for 4 h

Experimental design Cell model – Adult retinal pigment epithelial cells

Oxidative stress induction – tert butylhydroperoxide (tBHP), a cell permeant oxidant that causes lipid peroxidation and loss of GSH

Antioxidant – Various concentrations of N-acetycysteine amide

Pretreat cells for 24 h with antioxidant, followed by treatment with tBHP (0.4 mM) for 4 h

Cell Viability

tBHP induced cell death and protection by NACA pretreatment

n=3, *p<0.0001 as compared to control

0

20

40

60

80

100

*

%

Cell

Su

rviv

al

Reactive oxygen species (ROS) production

tBHP induced production of ROS (incubation with tBHP for 45 min)

0

100

200

300

400

*

****

Concentration of tBHP(M)

Re

lati

ve

Flu

ore

sc

en

ce

(%

Co

ntr

ol)

n=3, *p<0.00 as compared to control, **p<0.005 as compared to control

Reactive oxygen species production

Protection by NACA pretreatment

0

50

100

150

200*

#

Rela

tive F

luo

rescen

ce (

% C

on

tro

l)

n=3, *p<0.0001 as compared to control, #p<0.005 as compared to tBHP treated group

GSH levels

0

20

40

60

80

*

#

GS

H (

nm

ole

s/m

g p

rote

in)

n=3, * p<0.005 as compared to control, # p<0.0001 as compared to tBHP treated group.

Restoration of GSH levels by NACA pretreatment

Conclusions NACA is capable of supplying GSH to cells

undergoing tBHP induced oxidative stress

NACA has a protective effect against tBHP induced cell death and reactive oxygen species production

NACA protected cells against

XRT-induced oxidative stress Glutamate-toxicity HIV-proteins (gp120and Tat)-METH-induced

oxidative stress Acetaminophen-toxicity Macular degeneration

HPLC Analysis of Thiol Antioxidants

A reversed-phase HPLC method developed to separate and quantify NAC and NACA by fluorescence detection by using N-(1-pyrenyl) maleimide (NPM) as the derivatizing agent.

Biological thiols such as glutathione (GSH), cysteine (CYS), and homocysteine (HCYS) can be determined simultaneously.

NPM-thiol derivative

N OO

HS R

N OO

SHH H

R

Wu W et al., Biomedical Chromatography, 20: 415-422 (2006)

NPM

thiol

Chromatogram of a Plasma Sample from an Animal Sacrificed 30 min After Administration of 500 mg/kg Body Weight NACA.

Minutes0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

mV

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

mV

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

NA

CN

AC

A Hyd

roly

sis

peaks

GS

H

CY

S

HC

YS

Antioxidants: N-acetylcysteine (NAC)

Replenishes GSH through deacetylation to cysteine

Low oral bioavailability (30%) due to negative charge

Low solubility and tissue distribution May cause a severe, anaphylaxis-like allergic

reaction when given intravenously

http://www.medicinenet.com/acetylcysteine-injection/article.htm

Antioxidants: NACA

Neutral charge: Lipophilic Easily crosses the cell membranes Crosses blood brain barrier, scavenges free-radicals,

chelates copper, and protects red blood cells from oxidative stress.

Current research areas using NACA include: Parkinson’s treatment cerebral ischemia other neurodegenerative diseases

Penugonda S et.al., Brain Research, September 21,

2005

HIV Dementia and Antioxidants

A few antioxidants have been used in clinical trials in HIV dementia.

OPC-14117 (lipophilic compound structurally similar to Vit E), Selegiline (L-Deprenyl, MAO inhibitor), and CPI-1189 (lipophilic antioxidant scavenging superoxide radicals).

Unfortunately, the findings are very disappointing with side effects of cataract formation, elevation of hepatic enzymes and decrease in mean corpuscular volume.

Steiner J et al, Antioxidants & Redox Signaling, 2006

Our Hypothesis:

Toxic HIV ProteinsAddictive Drugs

(METH)

OXIDATIVESTRESS

BBB


NEURONs are exposed to TOXINS

AIMS

Aim I: To determine whether oxidative stress induced by gp120 and Tat at the BBB is potentiated by METH.

Aim II: To determine whether the potent antioxidant NACA protects the BBB from gp120, Tat, or METH alone and in combination.

Cell Models

RBE4-rat blood brain barrier cell model (Banks Lab)

HBMEC-primary human brain microvascular endothelial cells (Banks Lab)

Immortilized HBMEC (Banks Lab)

Oxidative Stress Parameters

Reduced glutathione (GSH) and oxidized glutathione (GSSG) assay by HPLC method

ROS measurement by DCF fluorescence-DCFH2DA converted by intracellular ROS into fluorescent DCF

Lipid peroxidation by-product (MDA) by HPLC Caspase-3 for apoptosis Antioxidant enzyme levels

GSH (glutathione) -glutamyl-cyteinyl-glycine

HOOC CH CH2 CH2 CHC N C N CH2 COOH

NH3+ O O

HH CH2

SH

Importance of glutathione GSH is the most abundant non-protein thiol Critical in maintaining the redox environment Cellular GSH is increased in times of stress, and

down-regulated after a challenge has been faced. GSH is crucial antioxidant in the BRAIN because:

20% oxygen is used in the brain (2% of the BW) Result: large quantities of ROS in the brain

In addition, There is high levels of iron in some parts of the brain Brain is rich in pufas Brain has very low amounts of SOD, CAT and GPx

So, defense against ROS by GSH becomes crucial in the brain.

Treatment of RBE4 Cells RBE4 cells divided into groups:

Control METH only (100M) Tat only (40nM) gp120 only (3nM) NACA only (1mM) Tat (40nM)+ METH (100M) gp120 (3nM) + METH (100M) Tat (40nM)+ METH (100M) + NACA (1mM) gp120 (3nM) + METH (100M) + NACA (1mM)

Incubation times – 3 h METH + 12 h for gp120 and Tat groups

Serine-Borate buffer used to prevent artifactual oxidation

Effects of increasing concentrations of METH on GSH levels in RBE4 cells.

Results (RBE4 Cells)

N=4; *Significantly different when compared with control p<0.005, **p<0.0005, ***p<0.00003. (n=3/group).

Effect of METH on GSH Levels

Gp120+Meth+NACA treated cells have similar GSH levels as controls.

N=4; *p<0.05, **p<0.05, ***p<0.05, #p<0.05, ##p<0.003, ###p<0.001.

Effect of NACA on GSH Levels in gp120 & METH Treated Cells

GSH Levels (% control) ***###*

#******

***

0

20

40

60

80

100

120

140

NACA significantly increased the GSH levels in Tat+METH-incubated RBE4 cells.

N=4; *p<0.05 compared with control, **p<0.05, ***p<0.02, #p<0.03, ##p<0.02.

Effect of NACA on GSH Levels in Tat and METH Treated Cells

Measurement of Intracellular ROS by DCF Method

Intracellular ROS production was measured using the dye 2,7-dichlorofulrescin diacetate (DCFH2-DA) method.

This measurement of cell oxidation is based on ROS-mediated conversion of nonfluorescent compound (DCFH2-DA) into a highly fluorescent compound (DCF).

Effects of NACA on the Generation of ROS in gp120 Treated Cells

N=4; *p<0.05, **p<0.05

1 nM gp120 treated cells have significantly higher intracellular ROS as compared to

controls. The increase of ROS was inhibited in the presence of ROS scavenger NACA (1 mM)

N=4; *p<0.05, **p<0.05

Effects of NACA on the Generation of ROS in Tat Treated Cells

10nm Tat treated cells have significantly higher intracellular ROS as compared to controls. The increase of ROS was inhibited in the presence of ROS scavenger

NACA (1mM).

Oxidative Stress Parameter-MDA

Malondialdehyde (MDA) levels: Polyunsaturated fatty acids, exposed to free radicals, can be oxidized to hydroperoxides which decompose (in the presence of metals) to hydrocarbons and aldehydes such as malondialdehyde (MDA).

N=4; *p<0.05, **p<0.05

NACA (1mM) attenuated lipid peroxidation in gp120 treated cells.1nM gp120 induced significant increase in MDA levels

Lipid Peroxidation Levels in gp120 Treated Cells

N=4; ***p<0.05, **p<0.05, #p<0.01

Lipid Peroxidation Levels in Tat Treated Cells

NACA (1mM) attenuated lipid peroxidation in Tat treated cells. 10 nM Tat induced significant increases in MDA levels.

Caspase-3 apoptotic Activity

Caspase-3 activity was measured by a spectrophotometric assay kit. The protease activity was measured by the addition of a specific peptide substrate for caspsase-3. The cleavage of peptide by the caspase releases the chromophore, which can be quantitated spectrophotometrically at a wavelength of 405nm.

N=4; *p<0.05, **p<0.05

Effect of NACA on Caspase 3 Activity in gp120 Treated Cells

NACA (1mM) treated cells have significantly lower caspase-3 activity as compared to the gp120 alone treated cells.

N=4; *p<0.05

Effect of NACA on caspase 3 activity in Tat treated cells

NACA (1mM) treated cells have significantly lower caspase-3 activity as compared to the Tat alone treated cells.

Effect of NACA on Antioxidant Enzyme Levels in RBE4 Cells

Groups Catalase(mU/mg protein)

GPx(mU/mg protein)

GR(mU/mg protein)

Control 6.5 ± 0.7 6.25 ± 0.73 13.94 ± 1.4

gp120 3.38 ± 0.14* 3.62 ± 0.25* 9.82 ± 0.09*

Tat 5.02 ± 0.16 3.96 ± 0.52* 9.73 ± 0.33*

gp120+NACA 8.45 ± 0.68** 8.87 ± 0.47** 11.82 ± 0.23**

Tat+NACA 9.2 ± 0.8# 10.12 ± 0.51# 11.71 ± 1.44#

N=4*p<0.005-0.01, Compared with control group **p<0.005-0.01, Compared with gp120 group#p<0.005-0.01, Compared with Tat group

Further Studies

Determine whether the potent antioxidant NACA protects the BBB from gp120, Tat, or METH (alone and/or in combination) In vitro: Use Human brain microvascular

endothelial cells (HBMVECs) New cell line :immortilized Human brain

microvascular endothelial cells (HBMVECs) In vivo: Use transgenic mice over expressing

gp120 or Tat. Permeability experiments in BBB models both in

HBMVECs and cells isolated from the transgenic mice

Treatment of HBMVEC Cells

HBMVEC cells divided into groups: Control METH only (100M) Tat only (40nM) gp120 only (3nM) NACA only (1mM) Tat (40nM)+ METH (100M) gp120 (3nM) + METH (100M) Tat (40nM)+ METH (100M) + NACA (1mM) gp120 (3nM) + METH (100M) + NACA (1mM)

Incubation times – 3 h METH + 12 h for gp120 and Tat groups

Serine-Borate buffer used to prevent artifactual oxidation

Cytotoxicity Assays

MTS tetrazolium compound

NADH or NADPH (in living cells)

NAD+ or NADP+

Colored MTS Formazan Product

(The absorbance at 490nm is proportional to the number of living cells in culture.)

Toxicological Profile of NAC and NACA in HBMVEC (MTS Assay)

0

20

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60

80

100

120

140

0.5 5 10

Concentration (mM)

Cel

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bili

ty (

% u

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d c

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tro

l)NACA

NAC

*

* p<0.05 as compared to NACA

NACA treated cells have significantly higher cell viability as compared to NAC treated cells.

N=4

NACA + gp120 treated cells have significantly higher cell viability as compared to gp120 alone treated cells. (Cells were treated with gp120 for 12 hours, followed by NACA

treatment for 12 hours)

Recent Results in HBMVEC Cells

N=4; *P<0.05 Control vs. 5 nM gp120, **P<0.05 5 nM gp120 vs. 5 nM gp120+ 1 mM NACA

Effect of gp120 on cell viability

Protective Effect of NACA on Tat and METH Treated cells

0

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*

#

* p<0.005 as compared to METH only group# p < 0.05 as compared to METH + Tat group

Tat (100nM) +METH (4nM)+NACA treated cells have significantly higher cell viability as compared to Tat+METH alone treated group. Cell viability was measured using MTS assay. Cells were exposed to Tat and/or METH for 24 hours.

Immortalized HBMVEC

Control METH 1mM METH +NACA NACA0.5mM0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

***

###

HCMVEC/D3 Cells were pretreated with 1mM NACA for 2 h, followed by24 h incubation with METH 1mM. NACA protected HCMVEC/D3 cells fromcytotoxicity caused by METH. Values were the means +SD of threesamples.Statistically different values of ***p <0.0001 were determined,compared to control. ###p < 0.0001 compared to METH 1mM group.

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ol)

Therefore

The BBB cells (rat and human) do show oxidative stress when exposed to HIV proteins (gp120 and tat) and Meth.

NACA reverses all the oxidative stress parameters to their control levels.

How does it affect the functionality of BBB? Meaning, what happens to its permeability?

Permeability assay: Transepithelial Electric Resistance (TEER)

TEER was measured using Millicell- electric resistance system. The cells were placed in the upper chamber of 24-well tissue inserts and were cultured for 4 days before use. Change in TEER during experimental conditions was calculated as a percentage of the corresponding baseline values. Unit: ohm/cm2 (Ohm: resistance, cm2: surface area of the monolayer).

N=3

Transepithelial Electrical Resistance

NACA treated cells have higher electric resistance as compared to Tat and gp120 alone treated groups.

Permeability results

TEER decreases with gp120, tat and both NACA again returns it to its control level. Meaning:

BBB permeability increases in the presence of gp120, tat or both.

BBB integrity DISRUPTED. NACA helps keep BBB integrity.

Glutathione Peroxidase (GPx) Increases in brains of gp120 Transgenic mice.

Gp120 transgenic (SJL/(C57BL/6/SV129) and non-transgenic (C57BL/6) mice were used. Genotyping was done by PCR using DNA isolated from tail cuts. Brain samples were removed and GPx activities were measured.

Groups GPx (mU/mg protein)

Non-transgenic 17.22 ± 1.83

gp120-transgenic 20.49 ± 1.64*

*p<0.05 compared to the control group. (N = 4/group)

Review

HIV proteins, gp120 and tat, along with many addictive drugs, specifically methamphetamine, can induce oxidative stress in the blood-brain barrier.

Oxidative stress disturbs the integrity of BBB. New thiol antioxidants may protect the BBB

against oxidative stress and help reduce the risk of neurodegenerative diseases.

Publications on NACA1. Ates B, Abraham LS, Ercal N. In vitro Antioxidant and Free-Radical

Scavenging Properties of N-Acetylcysteine Amide: A Novel Thiol Antioxidant. (Accepted, Free Radical Research).

2. Wu W, Abraham LS, Ogony J, Matthews R, Ercal N. Effects of N-Acetylcysteine Amide, a Novel Thiol Antioxidant on Radiation Induced Cytotoxicity in Chinese Hamster Ovary cells. (Accepted, Life Sciences).

3. Yan M, Shen J, Person MD, Kuang X, Lynn WS, Atlas D, Wong PK. Endoplasmic reticulum stress and unfolded protein response in Atm-deficient thymocytes and thymic lymphoma cells are attributable to oxidative stress. Neoplasia. 2008;10(2):160-7.

4. Lee KS, Kim SR, Park HS, Park SJ, Min KH, Lee KY, Choe YH, Hong SH, Han HJ, Lee YR, Kim JS, Atlas D, Lee YC. A novel thiol compound, N-acetylcysteine amide, attenuates allergic airway disease by regulating activation of NF-kappaB and hypoxia-inducible factor-1alpha. Exp Mol Med. 200;39(6):756-68.

5. Amer J, Atlas D, Fibach E. N-acetylcysteine amide (AD4) attenuates oxidative stress in beta-thalassemia blood cells. Biochim Biophys Acta. 2008;1780(2):249-55.

6. Penugonda S, Mare S, Lutz P, Banks WA, Ercal N. Potentiation of lead-induced cell death in PC12 cells by glutamate: protection by N-acetylcysteine amide (NACA), a novel thiol antioxidant. Toxicol Appl Pharmacol. 2006;216(2):197-205.

7. Price TO., Uras F, Banks WA, Ercal N. A novel antioxidant N-acetylcysteine amide prevents gp120- and Tat-induced oxidative stress in brain endothelial cells. Exp Neurol. 2006;201(1):193-202.

8. Bartov O, Sultana R, Butterfield DA, Atlas D. Low molecular weight thiol amides attenuate MAPK activity and protect primary neurons from Abeta(1-42) toxicity. Brain Res. 2006;1069(1):198-206.

9. Wu W, Goldstein G, Adams C, Matthews RH, Ercal N. Separation and quantification of N-acetyl-l-cysteine and N-acetyl-cysteine-amide by HPLC with fluorescence detection. Biomed Chromatogr. 2006; 20(5):415-22.

10. Sadan O, Bahat-Stromza M, Gilgun-Sherki Y, Atlas D, Melamed E, Offen D. A novel brain-targeted antioxidant (AD4) attenuates haloperidol-induced abnormal movement in rats: implications for tardive dyskinesia. Clin Neuropharmacol. 2005;28(6):285-8.

Publications on NACA

Publications on NACA11. Penugonda S, Mare S, Goldstein G, Banks WA, Ercal N. Effects of

N-acetylcysteine amide (NACA), a novel thiol antioxidant against glutamate-induced cytotoxicity in neuronal cell line PC12. Brain Res. 2005; 1056(2): 132-8.

12. Gilgun-Sherki Y, Barhum Y, Atlas D, Melamed E, Offen D. Analysis of gene expression in MOG-induced experimental autoimmune encephalomyelitis after treatment with a novel brain-penetrating antioxidant. J Mol Neurosci. 2005;27(1):125-35.

13. Grinberg L, Fibach E, Amer J, Atlas D. N-acetylcysteine amide, a novel cell-permeating thiol, restores cellular glutathione and protects human red blood cells from oxidative stress. Free Radic Biol Med. 2005;38(1):136-45.

14. Offen D, Gilgun-Sherki Y, Barhum Y, Benhar M, Grinberg L, Reich R, Melamed E, Atlas D. A low molecular weight copper chelator crosses the blood-brain barrier and attenuates experimental autoimmune encephalomyelitis. J Neurochem. 2004;89(5):1241-51.

Acknowledgements

NIH R15 AIDS Reagent Program

Tulin Price-Otamis William A. Banks Glenn Goldstein Linu Abraham Xinsheng Zhang Karissa Braaten Atrayee Banerjee Shinya Dohgu Burhan Ates Wei Wu

Thank You

Thank You !

a new antioxidant prevents toxicity of hiv proteins with methamphetamine

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