Table of Contents

1. Toxic Effects of Mercury on Central Nervous System Nucleotide Binding Proteins: Potential Role in Alzheimer’s Disease

2. I. Sources and Fate of Absorbed Elemental Mercury Vapor (Hg⁰)

3. Estimated Average Daily Intake of Mercury from Environmental Sources

4. Metabolism and Transport of Elemental Mercury Vapor (Hg⁰)

5. Oxidation of Elemental Mercury Vapor (Hg⁰) by the Enzyme Catalase

6. Oxidation of Mercury Vapor in the Brain and Trapping of Hg²⁺ by Binding to Brain Proteins

7. Membrane Associated Targets of Mercuric Cation and Methylmercury

8. Partial List of Nucleotide Binding Proteins Inhibited by Hg²⁺

9. II. Toxic Effects of Mercury on Brain Nucleotide Binding Proteins (NBPs)

10. Many Nucleotide Binding Proteins Contain Cysteine Residues At Their Active Sites

11. These Active Site Cysteine Sulfhydryl (-SH) Groups Are Critical for Proper Enzyme Function

12. Mercury Can Covalently Bind to Active Site Sulfhydryls (-SH) and Inhibit Enzyme Activity

13. Schematic of Nucleotide Photoaffinity Labeling

14. Inhibitory Effects of Mercury on a Mixture of Nucleotide Binding Proteins can be Detected and Quantified by Photoaffinity Labeling

15. Neuronal Tubulin, the Most Abundant Brain Protein, Is Especially Vulnerable to Mercury

16. Reported Effects of Mercury and Other Sulfhydryl Reactive Heavy Metals on the In Vitro Polymerization of Purified Brain Tubulin

17. Reported Effects of Mercury and Other Sulfhydryl Reactive Heavy Metals on Microtubules (MTs) in Cell Culture

18. Photoaffinity Labeling With [³²P]8N₃GTP Has Been Used Extensively to Study Tubulin Biochemistry

19. Biochemical Properties of Brain Tubulin

20. Structure of Neuronal Microtubules

21. Morphological Arrangement of the Neuronal Cytoskeleton

22. Microtubules Form the Structural Framework for Axonal Transport - A Process Essential for the Survival of Neurons

23. Disruption of Axonal Transport

24. III. Possible Role of Mercury and Sulfhydryl Reactive Heavy Metals in the Etiology of Alzheimer’s Disease (AD)

25. Diagnosis of Alzheimer’s Disease

26. Pathological Hallmarks of AD

27. Proteins Associated with Senile Plaques

28. Possible Relationship Between Microtubule Disruption and Plaque and Tangle Formation

29. Genes Linked to Alzheimer’s Disease

30. Apolipoprotein E4 Genotype Increases the Susceptibility to the Development of AD

31. Apolipoprotein E (Apo E)

32. Substitution of Arginine for Cysteine in Apo E3 and Apo E4 at Positions 112 and 158 Results in Loss of Potential Binding Sites for Sulfhydryl Reactive Heavy Metals such as Mercury

33. Mercury is Significantly Elevated in the Brains of Alzheimer’s Disease Subjects Relative to Controls

34. Hg²⁺ Induces Aberrant [³²P]8N₃GTP-b-Tubulin Interactions Indicative of Alzheimer’s Disease

35. SDS-PAGE Separation of Control and AD Brain Hippocampus Homogenates After Photolabeling with [³²P]8N₃GTP

36. Autoradiogram of the Photolabeled Control & AD Brain Hippocampus Homogenates Showing Decreased [³²P]8N₃GTP-b-Tubulin Interactions

37. Western Blotting of the Hippocampus Homogenates with Anti-b-Tubulin Antibodies Shows the Amount of b-Tubulin Protein is Not Reduced in the AD Brain Relative to Controls Despite a Significant Decrease in Photolabeling

38. Illustration of Western Blotting

39. [³²P]8N₃GTP-b-Tubulin Interactions are Aberrant in Both the Hippocampus and Frontal Pole of the Majority of AD Brain Homogenates Despite Normal Levels of Total b-Tubulin

40. Decreased [³²P]8N₃GTP-b-Tubulin Interactions in Hg⁰ Vapor Exposed Rats Correlates with Elevated Brain Hg

41. Decreased [³²P]8N₃GTP-b-Tubulin Interactions in Hg⁰ Vapor Exposed Rats Correlates with Elevated Brain Hg

42. Decreased [³²P]8N₃GTP-b-Tubulin Interactions in Hg⁰ Exposed Rats & AD Brain Homogenates is Not Due to Decreased Levels of b-Tubulin Protein

43. Treatment of Human Control Brain Homogenate with Sulfhydryl Reactive Heavy Metals Results in a Concentration Dependent Decrease [³²P]8N₃GTP Photolabeling of b-Tubulin

44. EDTA Prevents Cd, Cu & Zn But Potentiates Hg Inhibition of [³²P]8N₃GTP Photolabeling of Brain b-Tubulin

45. Partial List of Studies Demonstrating the Cytotoxic Effects of Mercury Containing Amalgams

46. Cytotoxicity of Endodontic Materials

47. Study Design

48. Table 2. Root-End Filling Materials Tested

49. Osorio et al., (1998). J. Endodon. 24,91-96.

50. Extraction and In Vitro Toxicity Testing of a Mercury Amalgam

51. Sequential Extracts of a Mercury Containing Amalgam Significantly Inhibit [³²P]8N₃GTP Interactions with b-Tubulin in Human Control Brain Homogenate

52. Inhibition of [³²P]8N₃GTP Photolabeling of Brain b-Tubulin Was Greater Than 65% for All Amalgam Extracts Tested While the 45 kDa Protein Band was Not Significantly Effected

53. Sequential Extracts of a Mercury Containing Amalgam Inhibit [³²P]N₃ATP Interactions with Purified ATP Binding Enzymes

54. The Extract of a Mercury Containing Amalgam Inhibits [³²P]N₃ATP Interactions with Purified ATP Binding Enzymes

55. Phosphorylase a (Phos a) Catalyzes the Sequential Removal of Glycosyl Residues from Glycogen

56. Phosphoglycerate Kinase (PGK) and Pyruvate Kinase (PK) Function in the Breakdown of Glucose to Pyruvate in Glycolysis and in the Substrate Level Production of ATP

57. Creatine Kinase (CK) and Adenylate Kinase (AK) Maintain ATP Levels in Tissues With High, Fluctuating Energy Demands Such as Brain and Muscle