Wei Wu and Nuran Ercal Department of Chemistry University of Missouri-Rolla

1. Effects of N-acetylcysteine amide (NACA), a novel thiol antioxidant, on radiation-induced oxidative stress Wei Wu and Nuran Ercal Department of Chemistry University of Missouri-Rolla

2. Objectives • Explore the radio-protective effects of a newly synthesized thiol antioxidant, N-acetylcysteine amide (NACA). • Explain its protective mechanism against radiation-induced oxidative stress.

3. Radiation Exposure • Clinic setting (cancer patient treatments) • Unexpected event (terrorist attack, nuclear accident, etc.)

4. Types of Ionizing Radiations • A) Electromagnetic: 1. X-ray (produced extra-nuclearly) 2. Gamma-ray (produced intra-nuclearly) • B) Particulate: Beta particles (electrons), alpha particles, protons, neutrons, heavy charged ions, and others.

5. Actions of Radiation • Direct action • A radiation absorbed by biological molecules interacts directly with critical targets. • Indirect action • Radiation interacts with atoms or molecules within a cell (particularly water) to produce reactive free radicals that damage critical targets (DNA, protein, lipid., etc.).

6. Radiation Damage Depending on radiation dosage (1Gy=100 rads): • Cerebrovascular syndrome: death within 24-48 h after 100Gy exposure (very high). • Gastrointestinal syndrome: within 4-10 d after whole-body exposure at 5-12 Gy. • Hematopoietic (bone marrow) syndrome: after several weeks of 3-8 Gy exposure. Current treatment of radiation-induced syndromes is only supportive therapy.

7. Radiation-Induced Oxidative Stress • Oxidative stress is an imbalance in the pro-oxidant/antioxidant ratio in favor of pro-oxidants. • Reactive oxygen species (ROS), produced by indirect action of radiation, disrupts the pro-oxidant/antioxidant balance of tissues, leading to protein, lipid, and DNA oxidation.

8. Mechanism of Radiation Effect and Damage • Systemic damage (following irradiation), partially due to overproduction of free radicals. • Biological thiols and antioxidant enzymes can remove free radicals.

9. Thiols as Radioprotectors • Research on thiol supplementation to maintain tissue redox balance has demonstrated that thiol radioprotectants (such as WR-2721) prevent radiation-induced damage. • Cysteine and glutathione delivery compounds protect normal cells from anti-tumor agents and radiation.

10. Thiols as Radioprotectors (cont) • Most thiols have side effects or are not available orally. • A strong, non-toxic, and orally-available antioxidant is needed to prevent radiation damage.

11. Exposure to radiation Oxidative stress Overproduction of O2·-, OH-, H2O2 Direct-Acting Antioxidants Antioxidant Enzymes -GSH/CYS -α-Tocopherol -ascorbic acid -carotenoids -retinoids -superoxide dismutase -catalase -glutathione peroxidase -glutathione reductase Oxidative Damages -Lipid Peroxidation -DNA Oxidation -Protein Oxidation

12. Objective • To investigate the role of antioxidant N-acetylcysteine amide (NACA) as a radioprotector.

13. Information about NAC N-acetyl-L-cysteine (NAC) • A well-known antioxidant that facilitates glutathione (GSH) biosynthesis. • Replenishes intracellular GSH under oxidatively challenging circumstances. • Shows radio-protective abilities.

14. Information about NACA N-acetyl-cysteine-amide (NACA) • The amide form of NAC. • Lipophilic, able to easily cross cell membranes. • Crosses blood brain barrier, scavenges free-radicals, chelates copper, and protects red blood cells from oxidative stress.

15. Structures of NAC and NACA NAC NACA

16. 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 NPM-thiol derivative Analytical Technique to Analyze Thiol Antioxidants Wu W, Goldstein G, Adams C, Matthews RH, Ercal N. Separation and Quantification of N-acetyl-L-cysteine (NAC) and N-acetylcysteine-amide (NACA) by HPLC with fluorescence detection.Biomedical Chromatography, 2005 (in press)

17. Chromatogram of a plasma sample from an animal sacrificed 30 min after administration of 500 mg/kg body weight NACA.

18. Experimental Design • Thiol-providing studies • Oxidative stress studies • Animal survival studies

19. Experimental • All experiments used adult Sprague Dawley rats. • Irradiation of the rats conducted at Department of Radiation Oncology, Phelps County Regional Medical Center in Rolla, Missouri, using a 16 MeV beam generated by a Varian linear accelerator, model 21 EX. • Selective oxidative stress parameters determined in plasma and various tissues (liver, lung, brain, and kidney).

20. I. Thiol-Providing Studies Q: Do NAC and NACA provide biological thiols for tissue? • To determine if NACA and NAC differ in providing thiols (GSH or CYS) to cells, animals(n=3) were given 500 mg/kg of NACA or NAC. • After 30 min, the rats were anesthetized and heparinized blood was collected via cardiopuncture. Following sacrifice, tissue samples were removed and analyzed. • GSH and CYS levels were measured in plasma and liver samples.

21. Thiol levels in plasma after NACA or NAC oral administration *p<0.05 compared to the control group

22. Thiol levels in liver after NACA or NAC oral administration *p<0.05 compared to the control group

23. Q: Do NAC and NACA Provide Biothiols? A: Yes. NACA is more effective than NAC.

24. II. Oxidative Stress Study Q1: Does radiation cause oxidative stress? Q2: If it does, can NAC and NACA protect animals against radiation-induced oxidative stress?

25. II. Oxidative Stress Study (cont) • 12 female Sprague-Dawley rats ( approximately 250 g) were divided into groups: 1) Control (n=3) 2) Radiation only (XRT) (n=3): rats received total- body x-ray radiation (6Gy, 16Mev). 3) XRT + NAC (n=3): received total-body x-ray radiation (6Gy, 16Mev) and 500mg/kg body weight of NAC. 4) XRT +NACA (n=3): received total-body x-ray radiation (6Gy, 16Mev) and 500mg/kg body weight of NACA. • Treatments given 30 min orally before radiation exposure, and on 4 consecutive days following the radiation exposure.

26. II. Oxidative Stress Study (cont) • Rats anesthetized and heparinized blood collected via cardiopuncture. Following sacrifice, liver, lung, brain and kidney removed. • Tissue samples analyzed. • Parameters measured: • GSH for thiol status • CYS for thiol status • Malondialdehyde (MDA) to detect lipid peroxidation • Antioxidant enzyme activities (catalase, glutathione peroxidase, glutathione reductase)

27. Oxidative stress parameters-GSH/CYS • Glutathione (GSH), principal intracellular thiol responsible for scavenging free radicals. • Cysteine (CYS), necessary for GSH biosynthesis.

28. -glutamylcysteinylglycine -glutamyl-amino acid cys GGT cysteinylglycine amino acid gly extracellular dipeptidase GSH transporter Amino acid transporters intracellular -glu-amino acid amino acid 5-oxoproline ATP ADP -glutamylcysteinylglycine cysteine glycine -glutamylcysteine glutamate GSH synthase GCL ADP ATP ADP ATP Dale A. Dickinson1, Shelly C. Lu and Henry Jay Forman. Glutathione – Synthesis Oxygen Society Education Program

29. GSH (nmol/mg protein) Levels in Liver *p<0.05 compared to the control group ** p<0.05 compared to the XRT only group

30. GSH (µM) Levels in Plasma *p<0.05 compared to the control group ** p<0.05 compared to the XRT only group *** p< 0.05 compared to the XRT+NACA-treated group

31. CYS (µM) Levels in Plasma *p<0.05 compared to the control group ** p<0.05 compared to the XRT only group *** p< 0.05 compared to the XRT+NACA-treated group

32. 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).

33. MDA (nmol/100mg protein)Levels in Liver *p<0.05 compared to the control group ** p<0.05 compared to the XRT only group *** p< 0.05 compared to the XRT+NACA-treated group

34. MDA (nmol/100mg protein)Levels in Lung *p<0.05 compared to the control group ** p<0.05 compared to the XRT only group *** p< 0.05 compared to the XRT+NACA-treated group

35. Oxidative Stress Parameter-Catalase (CAT) Catalase (CAT): in the peroxisomes of nearly all aerobic cells, a tetramer; each subunit contains a heme group and a NADP group. CAT 2H2O2 O2 + 2H2O

36. Catalase Activities (mU/mg protein) in Kidney *p<0.05 compared to the control group ** p<0.05 compared to the XRT only group

37. Catalase Activities (mU/mg protein) in Lung *p<0.05 compared to the control group ** p<0.05 compared to the XRT only group

38. Catalase Activities (mU/mg protein) in Liver *p<0.05 compared to the control group ** p<0.05 compared to the XRT only group *** p< 0.05 compared to the XRT+NACA-treated group

39. Oxidative Parameter-Glutathione Reductase (GR) Glutathione reductase (GR): a flavoprotein homodimer; each subunit has one mole of FAD. It has cysteine on it active site. GR GSSG + NADPH + H+ 2GSH + NADP+

40. Glutathione Reductase Activities (mU/mg protein) in Liver *p<0.05 compared to the control group ** p<0.05 compared to the XRT only group *** p< 0.05 compared to the XRT+NACA-treated group

41. Glutathione Reductase activities (mU/mg protein) in Kidney *p<0.05 compared to the control group ** p<0.05 compared to the XRT only group

42. Oxidative Stress Parameter-Glutathione Peroxidase (GPx) Glutathione peroxidase (GPx): a tetramer; each subunit has a selenocysteine residue in its active site. Protects mammals against oxidative damage by catalyzing the reduction of a variety of ROOH or H2O2 using GSH as the reducing substance. GPx ROOH + 2GSH ROH + GSSG + H2O

43. Glutathione Peroxidase Activities (mU/mg protein) in Liver *p<0.05 compared to the control group ** p<0.05 compared to the XRT only group *** p< 0.05 compared to the XRT+NACA-treated group

44. Glutathione Peroxidase Activities (mU/mg protein) in Kidney *p<0.05 compared to the control group ** p<0.05 compared to the XRT only group

45. Glutathione Peroxidase Activities (mU/mg protein) in Brain *p<0.05 compared to the control group ** p<0.05 compared to the XRT only group

46. Q: Does radiation induce oxidative stress? A: Yes. 6 Gy radiation: • Decreases levels of GSH (an important cellular antioxidant). • Increases levels of MDA (a lipid peroxidation indicator). • Increases catalase activities in the kidney, lung, and liver indicating that they are overwhelmed by hydrogen peroxides. • Decreases glutathione reductase and glutathione peroxidase activities in the liver and kidney, indicating that those two enzymes are suppressed by radiation.

47. Q: Does NACA provide antioxidant defense? A: Yes. • MDA levels were statistically lowered by NACA in the liver and lung. Although NAC also decreased MDA levels in the liver (but not in lung), NACA was more effective than NAC. • Levels of catalase, glutathione reductase, and glutathione peroxidase in various tissues were returned close to control levels by NACA. • More importantly, thiol levels (GSH and CYS) were returned to control levels by NACA.

48. III. Animal Survival Study • 18 rats were divided into the following groups. A high radiation dose (9Gy) was used for this study. 1). Radiation only (XRT) (n=6): rats received total-body x-ray radiation (9Gy, 16Mev). 2). XRT + NAC (n=6): received total-body x-ray radiation (9Gy, 16Mev) and 500mg/kg body weight of NAC. 3). XRT +NACA (n=6): received total-body x-ray radiation (9Gy, 16Mev) and 500mg/kg body weight of NACA. • Treatments given 30 min orally before radiation exposure, and on 4 consecutive days following the radiation exposure. • Rats then received a normal diet. Survival status of rats in each group recorded until death in 30 d.

49. Results of Animal Survival Study

50. NACA Prolonged Survival of Rats after Radiation • Rats in the NACA-treated group survived 30 d after irradiation, compared to lower survival rates in radiation-only groups and NAC-treated groups.