Prevention of Genomic Instability and Cancer by Dietary

Antioxidant N-acetyl Cysteine

Ramune Reliene, Ph.D.Cancer Research Center

Department of Environmental Health Sciences

STUDY OUTLINE

•

Antioxidant: N-acetyl cysteine

•

Cancer: Lymphoma

•

Cancer Cause: ATM deficiency

•

Biological Model: Atm-/-

•

Free radical scavenger•

Glutathione precursor

•

Prevents acetaminophen toxicity & liver failure

•

Mucolytic agent •

Effective against flu

•

Non-toxic•

Available as over-the-counter supplement

N-Acetyl Cysteine (NAC)

HS CH2

O

CH3HN

OH

O

•

DSS-induced colon cancer in mice (Seril et al. Carcinogenesis 23, 2002)

•

NF-B and inflammatory cytokine production:

Flu-infected lung A549 cells (Geiler et al. Biochem Pharmacol 79,2010)

H.pylori-infected gastric AGS cells

(Seo et al. Ann NY Acad Sci 973, 2002)

•

IL-1-induced COX-2 expression in osteoblasts MG63

(Origuchi et al. J Lab Clin Med

136, 2000)

NAC and Inflammation

Ataxia Telangiectasia (AT)•

“mysterious from the start-

an entity easily diagnosed on

purely clinical grounds, often by inspection-

but elusive for a long time”, Boder E, Kroc. Found. Ser 19: 1-63, 1985

• Autosomal recessive disorder

• Occurs in 1 of ~300,000 birth

•

Arises from a mutation of the ATM gene

Clinical Phenotype of AT•

Uncoordinated or ataxic

movements•

Ocular telangiectasia

(dilated blood vessels of the eye)

• Radiosensitivity• Sterility• Endocrine abnormalities• Immunodeficiency (from EMBO Reports v. 5, 2004)

Clinical Phenotype of AT (cont.)

There is no treatment to prevent AT

• High cancer incidence (30 -

40%): 40% of all tumors are non-Hodgkin’s lymphoma 20% are acute lymphocytic leukemia 5% are Hodgkin’s lymphoma

• The median survival 19 -

25 years

Cellular Phenotype of AT

• Genetic instability: chromosomal breaks translocations aneuploidy telomere shortening

• Hypersensitivity to ionizing radiation

• Chronic oxidative stress

ATM is a Protein Kinase that Responds to DNA Double-stranded Breaks

Shiloh et al. Seminars in Cancer Biology 2004 v.14

Atm Deficient Mice Recapitulate AT

• Thymic lymphoma• Immunodeficiency• Mild ataxia • Cells display genetic

instability• Oxidative stress: catalase activity superoxide dismutase activity thioredoxin levels ROS levels

(from Nature Genetics 32, 2002)

Oxidative Stress → Cancer

ROS

CANCERGENOMIC INSTABILITY

Can NAC suppress genetic instability and/or cancer in Atm-/-

mice?

HYPOTHESIS

Oxidative stress is a cause of genomic instability and cancer in Atm-/-

mice

STUDY DESIGN

•

Atm-/-

and Atm+/+ mice were chronically exposed to NAC-drinking water throughout life

40 mM NAC

short-term study• Oxidative stress: 8-OHdG• Genomic instability: DNA deletions

long-term study • Survival • Cancer

STUDY DESIGN

8-OHdG Determination by HPLC

min0 5 10 15 20 25

mAu

0

100

200

300

400

ADC1 A, ADC1 (07-10-07\DNA00028.D)

0.0

15 0

.060

1.4

94 1

.737

2.1

56 2

.293

2.7

23 2

.913

3.0

18 3

.554

4.1

87 5.1

76

6.6

38

8.8

92

9.9

83

14.

044

23.

547

min0 5 10 15 20 25

mAU

0

200

400

600

800

1000

1200

1400

1600

VWD1 A, Wavelength=245 nm (07-10-07\DNA00028.D)

1.6

82 2

.043

2.2

88 2

.536 3.0

36 3.3

23

4.1

02

7.6

07 8

.113

9.7

10

11.

244

20.

309

24.

581

8-OHdG

dG

Mouse DNA Deletion Assay

Exons 1-5 6-18 6-18 19-23

70 kbpun locus

p genehomologous deletion/ pun

reversion

Exons 1-5 6-18 19-23

pun

mouse (C57BL/6J pun/pun)

Reliene et al. Methods Mol Biol 262, 2003

retinal pigment epithelium

optical nerve head

neural retina

choroid

optical nerve

Eye-spot

70 kb DNA Deletions Result in Black Spots on the Retinal Pigment Epithelium

Dissection of the retinal pigment epithelium(eye-spot assay)

1 Eye-spot = 1 DEL event

Oxi

dativ

e D

NA

dam

age,

8-

OH

dG/1

06 dG

NAC Suppressed Oxidative DNA Damage

0

10

20

30

40

WT AT M AT M +NAC WT +NAC

ATM+ NAC

WT+ NAC

WT ATM

p = 0.0003 p = 0.004

Reliene et al. Cancer Res 2004, v.64

0

2

4

6

8

WT A T M AT M +NAC WT +NAC

ATM+ NAC

WT+ NAC

WT ATM

p = 0.001 p = 0.003Fr

eque

ncy

of D

NA

del

etio

ns,

No

of e

ye-s

pots

/RP

E

NAC Suppressed DNA Deletions in Atm-/-

Mice

Reliene et al. Cancer Res 2004, v.64

10

15

20

25

30

35

0 2 4 6 8 10

ATM

ATM-NAC

Num

ber o

f 8-O

HdG

/106

dG

Number of eye-spots/RPE

The Level of Oxidative DNA Damage is Associated with the Frequency of Deletions

single-strand DNA break

double-strand DNA break

Stalling & re-initiation of DNA replication

DNA deletion

Oxidative DNA base damage

DNA Deletions in Atm-/- Mice may Occur through Oxidative Stress

(S-phase)

0

0.5

1

0 20 40 60 80 100

NAC Prolonged Survival of Atm-/- Mice

Survival time, weeks

Pro

porti

on s

urvi

ving

p = 0.03Log rank test

Reliene et al. DNA Repair 2006, v.5

Atm-/- NAC

Atm-/-

Atm+/+ NAC

Atm+/+

68w50w

NAC Suppressed Lymphoma in Atm-/- Mice

0

20

40

60

80

0 20 40 60 80 100

Survival time, weeks

Inci

denc

e of

lym

phom

a, %

p = 0.02

Reliene et al. DNA Repair 2006, v.5

Atm-/- NAC

Atm-/-

Kidney 40x

Liver 40x

Lung10x

Control mouse Mouse with lymphoma

NAC Reduced Tumor MultiplicityLy

mph

oma

tissu

e di

strib

utio

n, %

0

20

40

60

80

100

T hymus Spleen Liver Lymph n odes Lun g Hear t K idn ey Pan cr eas St omach Duoden um Adr en al glan d

AtmAtm+NAC

Thym

us

Spl

een

Lym

ph

node

s

Lung

Live

r

Hea

rt

Kid

ney

Pan

crea

s

Sto

mac

h

Adr

enal

gl

ands

Duo

denu

m

Inci

denc

e of

lym

phom

a, %

NAC Reduced Tumor Incidence & Multiplicity in Atm-/- Mice

0

20

40

60

80

100

1 2

Atm-/- NACAtm-/-

p = 0.02

Num

ber o

f tu

mor

s/ m

ouse

0

1

2

3

4

5

1 2

Atm-/- NACAtm-/-

p = 0.038

SUMMARY

• Oxidative stress • Genomic instability • Longevity • Lymphoma • Metastasis

CONCLUSION

Antioxidant therapy may be beneficial in AT patients or other disorders associated

with oxidative stress

Our Findings were Translated to the Clinics

ACKNOWLEDGMENTS

Robert H. SchiestlElvira FischerGregory LawsonRichard Gatti

Lymphoma Research Foundation

UCLA School of Medicine

70 kb DNA Deletions Result in Black Spots on the Fur

Fur-spot assay

Fur-spot

Fur-spot

Chemical Dose % mice withfur-spots

Chemical Dose % mice withfur-spots

control - 5 -

10% BaP(benzo[a]pyrene)

150 mg/kg 63%

X-rays 1 Gy 23% BEN(benzene)

100 mg/kg 27%

EMS(ethylmethane

sulfonate)

100 mg/kg 29% TCE(trichloroethylene)

200 mg/kg 32%

MMS(methylmethane

sulfonate)

100 mg/kg 25% Aroclor1221

500 mg/kg 20%

ENU(ethyl nitrosourea)

25 mg/kg 53% Aroclor1260

500 mg/kg 26%

SOA(sodium arsenate)

20 mg/kg 29% TCDD(dioxin)

2.25 g/kg 25%

Carcinogens Induce DNA Deletions in the Exposed in utero Mice

reviewed in Reliene

& Schiestl Oncogene 2003 v. 22

Air Pollutants Induce DNA Deletions in the Exposed in utero Mice

Agent Dose Eye spots,% above control

Diesel exhaust particles

500 mg/kg/d125 mg/kg/d62 mg/kg/d

58%50%30%

Environmentaltobacco smoke 0.5 mg/m3 TPM 27%

Reliene

et al. Mutation Research 2005 v. 570