77:103 Radiation Biology Fall 2003, The University of Iowa, D.R. Spitz, F.E. Domann, P. Goswami, and F. Ianzini. Oxidative Stress Lecture

Low Dose Radiation and Adaptive Responses to Oxidative Stress Douglas R R. Spitz Ph Ph.D. D Professor and Director Free Radical and Radiation Biology Program Department of Radiation Oncology Holden Comprehensive Cancer Center University of Iowa, Iowa City, IA

Goals: • Introduction to Free Radical Biology • Introduction to basic principles of oxidative stress responses • Presentation of recent data supporting the concept that perturbations in intracellular oxidation/reduction reactions contribute to adaptive responses to low dose radiation Spitz DR, Azzam EI, Li JJ, and Gius D: Metabolic oxidation/reduction reactions and cellular responses to ionizing radiation:a unifying concept in stress response biology. Cancer and Metastasis Reviews 2004; 23:311–322. PMID: 15197331

What is life? “An electron, given off easily, has a high energy level, while an orbital which tends to take up an electron, has a low energy level. level The transmitted electron thus goes from a high to a low level, and releases the energy which corresponds to the difference of the two levels. It is this energy which drives life.” Albert Szent-Györgyi, Electronic Biology and Cancer, Marcel Dekker Inc. 1976

• In essence, Szent-Györgyi was one of the first scientists to recognize that all of the forces necessary for the maintenance of living systems derived from the ability of higher order biological structures to extract, store, and move electrons.

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77:103 Radiation Biology Fall 2003, The University of Iowa, D.R. Spitz, F.E. Domann, P. Goswami, and F. Ianzini. Oxidative Stress Lecture

Why do we breath oxygen? The Chemiosmotic Hypothesis

Lehninger; Principles in Biochemistry 3 rd Edition

Chemiosmotic model. In this simple representation of the chemiosmotic theory applied to mitochondria, electrons from NADH and other oxidizable substrates pass through a chain of carriers arranged asymmetrically in the inner membrane. Electron flow is accompanied by proton transfer across the membrane, producing both a chemical gradient (ΔpH) and an electrical gradient (Δψ). The inner mitrochondrial membrane is impermeable to protons; protons can reenter the matrix only through proton specific channels (F0). The proton-motive force that drives protons back into the matrix provides the energy for ATP synthesis, catalyzed by the F1 complex associated with F0.

Intermembrane space Complex IV

NADH +

NAD

e-

O2

Cyt c

FMN Complex I

Cyt c1 O2

Fe S

Fe S

Q -

e

Myxothiazol Rotenone Q

QH2

Succinate Fumarate O2

O2

Cyt bL

QH2

eFAD

Cyt bH

QH2

Q

Antimycin A Q Fe S

Q

Complex III

Complex II O2

O2

Matrix Ahmad IM, Aykin-Burns N, Sim JE, Walsh SA, Higashikubo R, Buettner GR, Venkataraman S, Mackey MA, Flanagan S, Oberley LW, and Spitz DR: Mitochondrial O2•- and H2O2 mediate glucose deprivationinduced cytotoxicity and oxidative stress in human cancer cells. J. Biol. Chem. 2005; 280(6):4254-4263.

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77:103 Radiation Biology Fall 2003, The University of Iowa, D.R. Spitz, F.E. Domann, P. Goswami, and F. Ianzini. Oxidative Stress Lecture

Repair→ What is the relationship between metabolic and genetic processes necessary for life?

103-104 damaged bases per cell per day [Proc Natl Acad Sci USA 86:9697-970138; DNA Repair 9:604–616]

←Antioxidants

Prooxidants (“Bad Guys” the short list) R• HO• ROO• O2• • NO ONOORO• NO2• H2O2 ROOH -RH=O HOCL Transition metals

→ carbon centered organic radical → hydroxyl radical → organic hydroperoxyl radical → superoxide → nitric oxide → peroxynitrite → alkoxyl radical → nitrogen dioxide → hydrogen peroxide → organic hydroperoxide → aldehydes (4-hydroxyalkenals, etc.) → hypochlorous acid → Fe+2, Cu+1

Fenton Reacton and Haber-Weiss Reaction Fe+2 + H2O2 → Fe+3 + HO• + OH- and Fe+3 + O2• → Fe+2 + O2 (or ROOH)

Overall Rxn:

Fe

O2• + H2O2 → O2 + •OH + OHcatalyst

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77:103 Radiation Biology Fall 2003, The University of Iowa, D.R. Spitz, F.E. Domann, P. Goswami, and F. Ianzini. Oxidative Stress Lecture

Sources of Prooxidant Production in Mammalian Cells

Antioxidants (“Good Guys” the short list) • Small molecular weight nutritional antioxidants

vitamin E, E vitamin C, C β-carotene, β carotene selenium, selenium lipoic acid, acid etc. etc

• Small molecular weight peptides, scavengers, cofactors

glutathione, NADPH, ∝-ketoacids (i.e., pyruvate), thioredoxin, glutaredoxin, etc.

• Antioxidant Enzymes superoxide dismutases, glutathione peroxidases, catalase, thioredoxin peroxidases, etc.

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77:103 Radiation Biology Fall 2003, The University of Iowa, D.R. Spitz, F.E. Domann, P. Goswami, and F. Ianzini. Oxidative Stress Lecture

Antioxidants

• Peroxiredoxins i d i H2O2 (or ROOH) + Trx-(SH)2→H2O (or ROH) + TrxS2

• Thioredoxin Reductase Trx S2 + NADPH→ Trx-(SH) Trx-S Trx (SH)2 + NADP+

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77:103 Radiation Biology Fall 2003, The University of Iowa, D.R. Spitz, F.E. Domann, P. Goswami, and F. Ianzini. Oxidative Stress Lecture

103-104 damaged bases per cell per day [Proc Natl Acad Sci USA 86:9697-970138; DNA Repair 9:604–616]

Prooxidants

Antioxidants

ONOO ROOH ROO• •OH

H2O2 RH=O

O2•

RO•

NO• GST SOD γ-GT NADPH GPX

GR

G6PD

GCS GSH

CAT

At control steady state, the cell is maintained in a highly reducing reducing environment, so a reserve of cellular antioxidants capacity exists. exists.

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77:103 Radiation Biology Fall 2003, The University of Iowa, D.R. Spitz, F.E. Domann, P. Goswami, and F. Ianzini. Oxidative Stress Lecture

Oxidative stress occurs when prooxidant production exceeds antioxidant capacity. If oxidative stress persists, oxidative damage begins to accumulate. If oxidative damage exceeds the ability of the cellular repair processes, then cell injury and/or death results from the accumulation of damage to critical biomolecules (i.e., DNA, lipids, lipids, proteins). Antioxidants Prooxidants

GST SOD γ-GT GR NADPH

Repair Processes

G6PD

GPX GCS GSH CAT ONOO ROOH ROO• •OH

H2O2 RH=O

O2•

RO•

NO•

Oxidative Damage

Injury & Cell Death

Low dose radiation (10 cGy) induces NF-κB transcriptional activity as well as resistance to high dose radiation in JB6P+ mouse epidermal cells

Fan M, Ahmed KM, Coleman MC, Spitz DR, and Jian Jian Li JJ: Nuclear factor-κB and manganese superoxide dismutase mediate adaptive radioresistance in low-dose irradiated mouse skin epithelial cells. Cancer Res. 2007; 67(7):3220–3228.

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77:103 Radiation Biology Fall 2003, The University of Iowa, D.R. Spitz, F.E. Domann, P. Goswami, and F. Ianzini. Oxidative Stress Lecture

Low dose radiation (5-10 cGy) induces MnSOD immunoreactive protein and activity

Fan M, Ahmed KM, Coleman MC, Spitz DR, and Jian Jian Li JJ: Nuclear factor-κB and manganese superoxide dismutase mediate adaptive radioresistance in low-dose irradiated mouse skin epithelial cells. Cancer Res. 2007; 67(7):3220–3228.

• Fractionated low dose radiation (0.5 cGy x 1030 fractions) induces MnSOD immunoreactive protein as well as several other adaptive signaling pathways including p65, pERK, 14-3-3, and Cyclin B1.

Fan M, Ahmed KM, Coleman MC, Spitz DR, and Jian Jian Li JJ: Nuclear factor-κB and manganese superoxide dismutase mediate adaptive radioresistance in low-dose irradiated mouse skin epithelial cells. Cancer Res. 2007; 67(7):3220–3228.

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77:103 Radiation Biology Fall 2003, The University of Iowa, D.R. Spitz, F.E. Domann, P. Goswami, and F. Ianzini. Oxidative Stress Lecture

Inhibition of NF-κB, inhibits low dose radiation-induced (10 cGy) adaptive responses in JB6P+ mouse epidermal cells

Fan M, Ahmed KM, Coleman MC, Spitz DR, and Jian Jian Li JJ: Nuclear factor-κB and manganese superoxide dismutase mediate adaptive radioresistance in low-dose irradiated mouse skin epithelial cells. Cancer Res. 2007; 67(7):3220–3228.

Knockdown of MnSOD using siRNA inhibits Low dose radiation (10 cGy) induced adaptation in JB6P+ mouse epidermal cells

Fan M, Ahmed KM, Coleman MC, Spitz DR, and Jian Jian Li JJ: Nuclear factor-κB and manganese superoxide dismutase mediate adaptive radioresistance in low-dose irradiated mouse skin epithelial cells. Cancer Res. 2007; 67(7):3220–3228.

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77:103 Radiation Biology Fall 2003, The University of Iowa, D.R. Spitz, F.E. Domann, P. Goswami, and F. Ianzini. Oxidative Stress Lecture

Conclusions: • Low dose radiation (total doses 5-10 cGy) induces adaptive responses that render JB6P+ mouse cells resistant to higher doses of ionizing radiation • The low dose radiation-induced adaptive response in JB6P+ cells is at least in part mediated by signaling pathways leading to NF-κB and MnSOD activity

Speculation: • Similar pathways for low dose radiation-induced adaptive responses may be operational in vivo

Thanks for your attention!

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