Christopher W. Shade, Ph.D. chris@quicksilverscientific Quicksilver Scientific, LLC

1. Looking Inside the Black Box: Understanding Analytical Approaches, Diagnostic Tools, and Detoxification Strategies for Mercury Intoxication Heretical Content Christopher W. Shade, Ph.D. chris@quicksilverscientific.com Quicksilver Scientific, LLC Lafayette, CO 80026 (303)263-6903

2. Mercury and The Human Detoxification System • What the key forms of Hg are • How the Natural Detoxification System Works • How the Detox System gets subverted leading to biochemical stress • Mercury Analysis (Blood is not a dirty word) • Intestinal Metals Detox System to repair and amplify the bodies Detoxification System and safely remove mercury

3. Forms of mercury • Hg0 – Elemental Mercury • The metal form; both liquid and gas forms • HgII – Inorganic Mercury • The salt, formed by oxidation of Hg0 • MeHg - Methylmercury • Organomercurial, formed by bacterial synthesis • EtHg - Ethylmercury • Synthetic organomercurial; antimicrobial

4. Transport of mercury • Hg0 • 80% uptake in lungs, crossed BBB, diffuses in to tissues; moderate uptake from intestines • HgII • Very poor uptakes in intestines; poor mobility; does not cross BBB • MeHg • 95% uptake from intestines, good mobility, crosses BBB • EtHg • 100% absorbtion (inj), good mobility, crosses BBB

5. Targets of mercury forms • Brain/CNS • Kidney • Liver • Heart • Pituitary/Thyroid • And thus all glandular

6. The Heart of the Toxicity • Inappropriate Binding – enzymes, redox proteins, membranes • Oxidative Damage and related Inflammation, including membrane damage (Parinanada) and apoptosis (cell death)

7. The Heart of the Toxicity • Thiol Binding and Redox Reaction • Reduced sulfur groups, R-SH, • Hg replaces proton and binds to sulfur • R-SH + Hg2+ = R-SHg+ + H+ • Enzymes use thiols to anchor functional metals (Zn, Ni, Cu, Fe) • Bind and alter membrane or trigger membrane reorganization and consequent auto-oxidation • Oxidize Thioredoxin (protein repair molecule) • Deplete Glutathione system

8. Inorganic Hg and Poisoning of the Extracellular Matrix

9. Mercury and The Human Detoxification System • What the key forms of Hg are • How the Natural Detoxification System Works • How the Detox System gets subverted leading to biochemical stress • Mercury Analysis (Blood is not a dirty word) • Intestinal Metals Detox System to repair and amplify the bodies Detoxification System and safely remove mercury

10. Defense – Glutathione SystemAntioxidant, Detoxification, Protein Repair • Glutathione (GSH) - A thiolic tripeptide composed of glutamate, cysteine, and glycine

11. Defense – Glutathione SystemAntioxidant, Detoxification, Protein Repair • Synthetases (synthesize GSH from precursors) • Transpeptidases (take apart and reassemble) • Transferases (Phase II conjugation) • Peroxidases (radical quenching) • Reductases (repair after quenching) • Redoxins (using GSH as reducing equivalent for protein repair) • Glutathionylation – protection of Proteins • What to do? Quench the fire or protect the children?

12. The Human Detoxification System • Detoxification Phases I, II, III • Phase I is an activation, • Phase IIis conjugation • Phase IIIis transport (recently delineated; control point)

13. The Human Detoxification System • Phase I - an oxidative activation, usually the Cytochrome P450 system • Prepares toxin for conjugation in Phase II with GSH, Glucuronic acid, Sulfate, Gycine or other amino acid, Taurine, Methyl group • Not needed for metals, but very important to have coupled to Phase II • Creates Essentially Free-Radicals

14. The Human Detoxification System • Phase II – conjugation makes toxin more water soluble and recognizable by transporters • Glutathione S-Transferases (GST) responsible for GSH conjugation • Low expression in people with high MeHg and with sensitivity (allergy) to Thimerosal (EthylHg)

15. The Human Detoxification System • Phase III is the transport out! • Several transport proteins (cMOAT, OAT, MRP1, MRP2, GS-X) • Organic Anion Transporters • Same transporters for many pathways (glucuronide, sulfate, glycinate, GSH) • In cells, liver, intestines, kidneys – biggest in liver then intestines

16. Mercury and The Human Detoxification System • What the key forms of Hg are • How the Natural Detoxification System Works • How the Detox System gets subverted leading to biochemical stress • Mercury Analysis (Blood is not a dirty word) • Intestinal Metals Detox System to repair and amplify the bodies Detoxification System and safely remove mercury

17. Breakdown of the defense system • GSH deficiency – • Genetic (GCS polymorphisms, epigenetic dysfunction) • Environmental (oxidative consumption or inflammation) • GST problems – • Genetic (GST polymorphisms, epigenetic dysfunction) • Environmental (Inflammatory cascade/ARE dysfunction)

18. Disease and Gen Polymorphisms of GSH genes • Hemolytic Anemia – GST (Beutler et al 1988) • Sensitivity to DDT – GCS and GST (Hung et al. 2004) • Bladder Cancer (Hung et al. 2004) • O.R. GSTM1 = 1.69 • O.R. GSTT1 = 1.74 • O.R. GSTM1 + Env Exposure = 2.77 • Acute leukemia – GST

19. Breakdown of the defense system • GSH deficiency – • Genetic (GCS polymorphisms, epigenetic dysfunction) • Environmental (oxidative consumption or inflammation) • GST problems – • Genetic (GST polymorphisms, epigenetic dysfunction) • Environmental (Inflammatory cascade/ARE dysfunction) • Phase III can get blocked and then downregulates Phase II enzymes • Can stop multiple detoxification pathways!

20. Biggest Reason for Phase III Dysfunction Inflammation! Especially in Gut! -Hallmark of Autism cases -Easily caused by heavy metal induced oxidative damage

24. Coordinated Expression of Phase II and III MRP2 and GSTπ coregulated

25. Oxidative Activation Phase I Glutathione Conjugation Sulfation Glucuronidation Phase II Cellular MRP1 Blood Phase III OATP LIVER MRP2 Normal Small Intestine

26. Oxidative Activation Phase I Glutathione Conjugation Sulfation Glucuronidation Phase II Cellular MRP1 Blood Phase III OATP LIVER MRP2 Inflamed Small Intestine

27. Oxidative Activation Phase I Glutathione Conjugation Sulfation Glucuronidation Phase II Cellular MRP1 Negative Feedback – Inhibition of Phase II Blood Phase III Inflammation causes Downregulation of MRP2 OATP LIVER MRP2 Inflamed Small Intestine

28. Oxidative Activation Oxidative Activation Oxidative Stress From Phase I/Phase II mismatch Phase I Glutathione Conjugation Sulfation Glucuronidation Phase II Build-up of both cellular and blood-borne toxins Cellular MRP1 Negative Feedback – Inhibition of Phase II Blood Phase III Inflammation causes Downregulation of MRP2 OATP LIVER MRP2 Inflamed Small Intestine

29. Amalgam as Cause for Inflammation Cysteine Mouth Rinse HgII MeHg MeHg MeHg HgII HgII No Amalgam 25ppt MeHg, 5ppt HgII Amalgam – 10ppb Amalgam – 10ppb Amalgam (2nd Rinse) – 16ppb

30. MeHg Mitochondrial Inferno – Breakdown of the Thiolic Protection System Mitochondrial Stress – The MeHg-induced Blaze • Covering Exposed Thiols • Mopping up ROS • Binding Hg Leaking ROS and cyt c Decay in GSH Insufficient Protection from GSH TrX releases ASK1 ROS and Hg Oxidize TrX Apoptosis – Cell Death

31. Mercury and The Human Detoxification System • What the key forms of Hg are • How the Natural Detoxification System Works • How the Detox System gets subverted leading to biochemical stress • Mercury Analysis (Blood is not a dirty word) • Intestinal Metals Detox System to repair and amplify the bodies Detoxification System and safely remove mercury

32. Testing for Hg • Ambient Measures • Blood • Hair • Urine • Stool • Provoked Measure • Urine

33. H H H g H Testing for Hg • Ambient • Blood – MeHg + HgII (MeHg larger) • Hair – MeHg only • Urine – HgII (little bit of MeHg) • Stool – MeHg + HgII • Provoked • Urine – MeHg + HgII

34. Challenge Tests – What do they mean? Are They Necessary?

35. Mercury Industry Workers Dentists Controls (Amalgam) Amalgam-free Referents

36. Mercury Industry Workers Dentists Controls (Amalgam) Amalgam-free Referents

39. Chelation therapy – Dump from blood followed by reloading from cellular burden

40. Mercury Speciation Testing • Separates the two main forms of mercury in the human body • Once separately measured, ambient measurements reveal A LOT without challenge tests

41. Whole Blood vs. RBC • Partitioning between RBC and plasma different for MeHg and HgII • MeHg 90% RBC/10% Plasma • Access to brain, intracellular, metabolic-heme • HgII 50%/50% • Lymph an extension of plasma: ~5X the volume of blood • Extracellular Matrix, also concept of terrain • Look at both forms with whole blood

42. Inorganic Hg and Poisoning of the Extracellular Matrix…Uhhh, What Matrix?

43. H H H g H MethylMercuryThe Unsuspected Factor • Direct toxicity not as high as Hg(II) • One binding site vs. 2 major and 2 minor • BUT…has many insidious properties • Is slow to excrete bank of mercury • Constant source of inorganic mercury from breakdown • Can continue symptoms of hypersensitivity from amalgam after amalgams removed • Has “all-access pass” • Intracellular, BBB, placental barrier • Can form allergy that will persist till all gone • If phase II enzymes weak, can stay for long, long time

44. H H H g H Accumulation Enterohepatic Circulation of MeHg

45. H H H g H Blood Testing • Old Dictum – blood is only recent exposure, 3-days • Reality • 3-day residence only the quick decay after a large dose • For MeHg, Steady state develops after initial decay; Then blood reflects body burden! • Real Problem • Most labs detection limits too high to see dynamics • Need sensitive equipment!

46. Blood Testing: The Great Tuna Experiment • 2 cans of Albacore Tuna in one sitting 24 hours later 24 hours later 24 hours later 2 hours later Pre-Tuna 2 hours later 2 hours later Initial Decay – Redistribution to Tissues

47. The Great(er) Walleye Experiment*Nothing is new under the sun* Clarkson et al., Arch. Env. Health, 1980

48. Walleye Experiment – Decay and Body Burden After initial peak and decay, Blood reflects Body Burden!

49. H H H g H Mercury Speciation Testing

50. H H H g H Testing for MeHg MeHg HgII MeHg HgII