A New Antioxidant Prevents Toxicity of HIV Proteins with Methamphetamine

1
A New Antioxidant Prevents Toxicity of HIV
Proteins with Methamphetamine

• Nuran Ercal MD PhD
• Department of Chemistry,
• Missouri University of Science and Technology,
  Rolla, MO
• Department of Internal Medicine, St. Louis
  University, St. Louis, MO

2
Our Hypothesis
Toxic HIV Proteins
Addictive Drugs (METH)
OXIDATIVE STRESS BBB
BBB integrity disrupted
NEURONs are exposed to TOXINS!
3
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.
•      

4
Sources of Reactive Oxygen Species 1) Non
mitochondrial NADPH Oxidases Microsomal
cytochrome P-450 Cyclooxygenases Monoamine
oxidases Peroxisomal b oxidation of fatty
acids Phagocytes
2) gt90 is mitochondrial electron transport
chain contains several redox centers that may
leak electrons to oxygen-----? Superoxide
radical!
5
(No Transcript)
6
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
7

• 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

8
Consequences of oxidative stress
Marker
F2-Isoprostanes
8-iso-PGF2a
9
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

10
What is the problem?

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

11
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.
12
What is HAD?

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

13
What are the symptoms of HAD?

• Symptoms include
• Sluggish mental capabilities and motor control
• Marked apathy
• Loss of interest in previously enjoyable
  activities
• Tremors and impaired balance, slow eye movement,
  abnormal reflexes..
• The disorder continues to escalate, eventually
  resulting in severe dementia
• Some experience mania or even psychosis

14
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!

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

• 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.

Steiner J et al, Antioxidants Redox Signaling,
2006 McArthur JC et al, Neurology, 1993
Bouwman FH et al, Neurology, 1998
15
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

16
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

17
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 worlds 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
• http//www.until.org/statistics.shtml

18
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
• http//www.until.org/statistics.shtml

19
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.

20
Oxidative Stress Related Disorders

• Parkinsons Disease
• Alzheimers 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

21
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

22
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.

23
Crystal
Powder
Pill
24

• Missouri Highest Rate of METH Lab Activity in
  the Country

Picture source US Department of Justice
(www.usdoj.gov)
25
More on Methamphetamine

• Street names of methamphetamine (METH)
• Speed
• Ice
• Crystal Meth
• Chalk
• Consumption
• Smoke
• Snort
• Inject
• Oral Ingestion

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

National Drug Intelligence Center, National Drug
Threat Assessment 2008, October 2007 Picture
Source http//cerhr.niehs.nih.gov/chemicals/stim
ulants/amphetamines/methamphetamine.gif
26
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.

Dopamine enzymatic oxidation
MAO DA O2 H2O-------------------? 3,4,dih
ydroxyphenylacetic acid NH3 H2O2
Dopamine autoxidation DA O2 ? SQ O2 -
H DA O2 - 2 H ? SQ H2O2

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

27
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).

28
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.

29
Blood Brain Barrier (BBB)
30
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?

31
Whats causing the blood-brain barrier to fail?

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

32
Our Hypothesis
Toxic HIV Proteins Gp120 and tat
Addictive Drugs (METH)
OXIDATIVE STRESS BBB
BBB integrity disrupted
NEURONs are exposed to TOXINS..
33
Lets study HIV-1 and its proteins
34

• 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.

35
Gp120 (red) in complex with a CD4 surface protein
(yellow)
36
More on gp120

• gp120 is composed of an outer and inner domain
  made of a helices and ß sheets.
• The negatively charged central cavity contains
  the binding site for a positively charged
  region of a surface protein on CD4 (electrostatic
  interactions).

37
Molecular Structure of gp120

• gp120 changes confirmation when bound to
  different proteins

Picture Source Tongqing Zhou et al. Nature 2007
38
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-3III-18 Picture
Source Grant R. Campbell et al. J. Biol. Chem.
2004
39
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
40
Oxidative Stress Possible Pathways
Antioxidants
41
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

42
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

43
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.

44
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

45
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-acetylcysteam
  ine
• (NACA, possible candidate)

Pocernich CB et al., Brain Research Reviews, 2005
.
46
Structures of NAC and NACA
NAC
NACA
47
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

48
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) ENAC only F NACA only GXRT NAC
  (post-treated) H XRT NACA (post-treated).

49
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.
50
Immortalized HBMVEC
51
Age Related Macular degeneration

• 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

52
Changes in Macula in ARMD
Image Source www.sfn.org (Society for
Neuroscience)
53
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

54
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

55
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

56
Cell Viability

• tBHP induced cell death and protection by NACA
  pretreatment

n3, plt0.0001 as compared to control
57
Reactive oxygen species (ROS) production

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

n3, plt0.00 as compared to control, plt0.005 as
compared to control
58
Reactive oxygen species production

• Protection by NACA pretreatment

n3, plt0.0001 as compared to control, plt0.005
as compared to tBHP treated group
59
GSH levels

• Restoration of GSH levels by NACA pretreatment

n3, plt0.005 as compared to control, plt0.0001
as compared to tBHP treated group.
60
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

61
NACA protected cells against

• XRT-induced oxidative stress
• Glutamate-toxicity
• HIV-proteins (gp120and Tat)-METH-induced
  oxidative stress
• Acetaminophen-toxicity
• Macular degeneration

62
NACA protected cells against

• XRT-induced oxidative stress
• Glutamate-toxicity
• HIV-proteins (gp120and Tat)-METH-induced
  oxidative stress
• Acetaminophen-toxicity
• Macular degeneration

63
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.

thiol
NPM

• NPM-thiol derivative

Wu W et al., Biomedical Chromatography, 20
415-422 (2006)
64
Chromatogram of a Plasma Sample from an Animal
Sacrificed 30 min After Administration of 500
mg/kg Body Weight NACA.
65
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-injectio
n/article.htm
66
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
• Parkinsons treatment
• cerebral ischemia
• other neurodegenerative diseases

Penugonda S et.al., Brain Research, September 21,
2005
67
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
68
Our Hypothesis
Toxic HIV Proteins
Addictive Drugs (METH)
OXIDATIVE STRESS BBB
BBB integrity disrupted
NEURONs are exposed to TOXINS
69
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.

70
Cell Models

• RBE4-rat blood brain barrier cell model (Banks
  Lab)
• HBMEC-primary human brain microvascular
  endothelial cells (Banks Lab)
• Immortilized HBMEC (Banks Lab)

71
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

72

• GSH (glutathione)
• ?-glutamyl-cyteinyl-glycine

73
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.

74
Treatment of RBE4 Cells

• RBE4 cells divided into groups
• Control
• METH only (100mM)
• Tat only (40nM)
• gp120 only (3nM)
• NACA only (1mM)
• Tat (40nM) METH (100mM)
• gp120 (3nM) METH (100mM)
• Tat (40nM) METH (100mM) NACA (1mM)
• gp120 (3nM) METH (100mM) NACA (1mM)
• Incubation times 3 h METH 12 h for gp120 and
  Tat groups
• Serine-Borate buffer used to prevent artifactual
  oxidation

75
Results (RBE4 Cells)
Effect of METH on GSH Levels

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

N4 Significantly different when compared with
control plt0.005, plt0.0005, plt0.00003.
(n3/group).
76
Effect of NACA on GSH Levels in gp120 METH
Treated Cells

• Gp120MethNACA treated cells have similar GSH
  levels as controls.

N4 plt0.05, plt0.05, plt0.05, plt0.05,
plt0.003, plt0.001.
77
Effect of NACA on GSH Levels in Tat and METH
Treated Cells

• NACA significantly increased the GSH levels in
  TatMETH-incubated RBE4 cells.

N4 plt0.05 compared with control, plt0.05,
plt0.02, plt0.03, plt0.02.
78
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).

79
Effects of NACA on the Generation of ROS in gp120
Treated Cells
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)
N4 plt0.05, plt0.05
80
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).
N4 plt0.05, plt0.05
81
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).

82
Lipid Peroxidation Levels in gp120 Treated Cells
NACA (1mM) attenuated lipid peroxidation in gp120
treated cells.1nM gp120 induced significant
increase in MDA levels
N4 plt0.05, plt0.05
83
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.
N4 plt0.05, plt0.05, plt0.01
84
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.

85
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.
N4 plt0.05, plt0.05
86
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.
N4 plt0.05
87
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
gp120NACA 8.45 0.68 8.87 0.47 11.82 0.23
TatNACA 9.2 0.8 10.12 0.51 11.71 1.44
N4 plt0.005-0.01, Compared with control group
plt0.005-0.01, Compared with gp120
group plt0.005-0.01, Compared with Tat group
88
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

89
Treatment of HBMVEC Cells

• HBMVEC cells divided into groups
• Control
• METH only (100mM)
• Tat only (40nM)
• gp120 only (3nM)
• NACA only (1mM)
• Tat (40nM) METH (100mM)
• gp120 (3nM) METH (100mM)
• Tat (40nM) METH (100mM) NACA (1mM)
• gp120 (3nM) METH (100mM) NACA (1mM)
• Incubation times 3 h METH 12 h for gp120 and
  Tat groups
• Serine-Borate buffer used to prevent artifactual
  oxidation

90
Cytotoxicity Assays
91
Toxicological Profile of NAC and NACA in HBMVEC
(MTS Assay)
N4
NACA treated cells have significantly higher cell
viability as compared to NAC treated cells.
92
Recent Results in HBMVEC Cells
Effect of gp120 on cell viability
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)
N4 Plt0.05 Control vs. 5 nM gp120, Plt0.05 5
nM gp120 vs. 5 nM gp120 1 mM NACA
93
Protective Effect of NACA on Tat and METH Treated
cells
Tat (100nM) METH (4nM)NACA treated cells have
significantly higher cell viability as compared
to TatMETH alone treated group. Cell viability
was measured using MTS assay. Cells were exposed
to Tat and/or METH for 24 hours.
94
Immortalized HBMVEC
95
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?

96
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).

97
Transepithelial Electrical Resistance
N3
NACA treated cells have higher electric
resistance as compared to Tat and gp120 alone
treated groups.
98
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.

99
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
• plt0.05 compared to the control group. (N
  4/group)

100
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.

101
Publications on NACA

• 1. 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.
  200810(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. 20039(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. 20081780(2)249-55.

102
Publications on NACA

• 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.
  2006216(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. 2006201(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.
  20061069(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. 200528(6)285-8.

103
Publications on NACA

• 11. 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.
  200527(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. 200538(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.
  200489(5)1241-51.

104
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

105
Thank You
106
Thank You !