HIDDEN TRUTHS OF MYCOTOXINS

1. HIDDEN TRUTHS OF MYCOTOXINS Dennis Hooper, MD, PhD, CEO, Medical Director RealTime Laboratories, LLC 13016 Bee Street, Suite 203 Dallas, Texas 75234 Phone: 972-243-7754 Fax: 972-243-7759 mscmd@cox.net www.realtimelab.com 

2. Goals of this Presentation Define Mycotoxins Aflatoxins Ochratoxins Trichothecenes Others (Ergots, Etc). Show Structures of mycotoxins Discuss malignancy and mycotoxins Explain the testing of the mycotoxins and validations of the testing in a laboratory setting. Explain findings to date in specimens submitted for clinical testing (body fluids and tumors).

3. Dilemma Is there a correlation between mycotoxins and malignancies? Is there a causation between mycotoxins and malignancies?

4. Mycotoxins Low molecular weight (small molecules) Usually produced as secondary metabolites by filamentous fungi 300-400 compounds are recognized as mycotoxins 10-12 receive attention as human threats Most literature attributes mycotoxicosis to ingestion of contaminated foods but airborne contamination is a problem as well

5. MYCOTOXICITY PATIENTS ARE EXPOSED THROUGH INHALATION AS WELL AS INGESTION. IS THERE A CORRELATION/CAUSATION BETWEEN INHALATION OF FUNGAL SPORES AND MYCOTOXINS AND MYCOTOXICOSIS/MALIGNANCY?

6. Mycotoxicoses Acute Rapid onset Obvious Toxic response Chronic Low dose exposure over long period of time Results in cancers, chronic illnesses, and systemic, irreversible effects (James, 1985, Gen Prin of Toxicology)

8. BUT ??? Can we measure mycotoxins in Human Fluids and Tissues?

9. Yes, We Can ��

10. Human Health Burden Related to chronic exposure; even though the best known mycotoxin episodes are manifestations of acute effects (human ergotism, stachybotryotoxicosis). Cancer induction Kidney toxicity Immune supression

11. Aspergillus niger (Aflatoxin)

12. Aflatoxins 4 major ones: B1, B2, G1, G2 B1 is most potent natural carcinogen known Produced by Aspergillus flavus, Asp. parasiticus, Others Liver is the primary target organ; however, also found in lung and brain.

13. Aflatoxin Action Cytochrome P450 enzymes convert aflatoxins to the reactive 8,9-epoxide form Binds to DNA and proteins at the N7 position of guanines Aflatoxin B1-DNA adducts result in GC to TA transversions If there is a reactive glutathione S-transferase system found in the cytosol and microsomes- then there can be a conjugation of activated aflatoxins with reduced GSH leading to the excretion of aflatoxin

14. Exposure in diet is an important risk factor for primary Hepatocellular carcinoma. Aflatoxin B1-N7-guanine adduct represents the most reliable urinary biomarker for recent exposure Inactivation of p53 tumor suppressor gene may be important step in carcinogenesis AFLATOXIN B1 - Human Carcinogen

15. Biosynthesis and Molecular Biology During the metabolism of aflatoxins, there are many toxic metabolites formed. The mycotoxin: Sterigomatocystin is carcinogenic. Produced by Asp. versicolor Asp. nidulans Asp. parasiticus Asp. flavus Asp. oryze and Asp. sojae, both in soy sauce and sake fermentation

16. Penicillium Species

17. Ochratoxin A From Asp. ochraceus, Asp. niger Penicillium verrucosum Found in grains, coffee beans, and some wines Kidney is the primary target organ

18. Ochratoxin A Nephrotoxic Humans have the longest half-life for its elimination of any of the species studied Hepatoxic Immune suppressant Potent teratogen Carcinogen Disturbs cellular physiology in multiple ways: Inhibiting synthesis of phenylalanine-tRNA complex Inhibits mitochondrial ATP production Stimulates lipid peroxidation

19. Ochratoxin A Diseases: (Not strong evidence molecularly, but epidemiological): Endemic Balkan nephropathy with increased number of bladder tumors. May be a risk factor for testicular cancer Can be carried through the food chain (recommended levels in food intake: 5 ng/kg of body weight/day) Recommendations: pay close attention to patients with symptoms of: Renal pathology Immunosuppression THINK MYCOTOXINS!!

20. Stachybotrys (Trichothecenes) 

21. Trichothecenes Many metabolites produced by: Fusarium Stachybotrys Trichoderma Commonly found in food

22. Potent inhibitors of protein synthesis Some interfere with initiation, others elongation, and others termination stages of protein synthesis. They all inhibit peptidyl transferase by binding to the same ribosome binding site Deoxynivalenol (DON) and T-2 are best studied Cause nausea, vomiting weight loss DON is less toxic yet most prevalent Trichothecenes

23. Symptoms similar to anguidine (a simple trichothecene �diacetoxyscirpenal) Injected into humans in the 1970�s as a chemotherapeutic agent. Symptoms experienced by patients in that study are also similar to the symptoms experienced by those individuals in trichothecene infested buildings. Trichothecenes

24. Ergot Alkaloids Most fascinating of all Lysergic acid, structure common to all ergots Produced from Claviseps sp. St. Anthony�s Fire. Two clinical forms: Gangrenous Convulsive

25. ErgotaminesClinical symptoms Gangrene Abortion Convulsions Suppression of lactation Hypersensitivity Ataxia Induce Smooth muscle contractions; thus, abortion can occur and accelerates uterine contractions in labor.

26. Fumonosins Associated with human esophageal cancer

27. Case Studies

28. 38 y.o. male, exposed to Stachybotrys chartarum, Aspergillus niger, and Penicillium sp. (verified by Environmental studies) in workplace. Developed extensive skin lesions and sinusitis with asthma. Positive for Lyme Disease (By antibody titration). Pathology DX: Skin Biopsy: Pathology Bx: Psoriasiform Spongiotic Dermatitis. DNA by RT-PCR, Skin Biopsy: Stachybotrys chartarum, Aspergillus niger. Positive for Aflatoxin 4 ppb, Negative for Trichothecenes (macrocyclics). Results: Antifungal given; Itraconazole and Voraconazole. Lesions improved. Skin Lesions

29. 2 � y.o. male, with history of mold exposure in home from birth. Began having seizures at six weeks old. Also, family Labrador had seizures. Environmental studies of home showed high levels of Aspergillus, Penicillium, and Stachybotrys. Mycotoxin analysis urine: Trichothecenes: 1.5 ppb; Aflatoxins, 0 ppb,Ochratoxins, 0 ppb. Began treatment of Cholestyramine, and Glutathione. Also was believed to have Epilepsy. Began on Dilantin. One month after treatment began, urine levels of mycotoxins showed 1.5 Trichothecenes, 5 ppb Aflatoxins, and 0 ppb Ochratoxins. Results: While on reducing agents, seizure free. Medical consensus was that the patient was epileptic. Removed him off of reducing agents. Seizures increased. Now carries autistic diagnosis. Continuing to have seizures. NEUROLOGICAL DISORDERS

30. Management of Mycotoxin Positive Myoclonus in Canine Canine � 1 year old , relentless progression of myoclonus severe enough to consider euthanasia Urine (+): aflatoxin, trichothecene and ochratoxin Culture from walls: Stachybotrys chatarum, and Aspergillus Management: Moved from exposure site Resolved after ReadiSorb� Glutathione, an oral liposomal encapsulation of glutathione.

31. 6 y.o. female with astrocytoma (Grade I/II) Aflatoxin found in urine and brain biopsy 48 y.o. male with low grade glioma Aspergillus versicolor by RT-PCR Aflatoxin found at 2 ppb. In CSF and in urine Started on Voraconazole; patient showed mild improvement. Discontinued Voraconazole for chemotherapy and radiation treatments Patient expired Autopsy showed Grade 1 Astrocytoma 21 y.o. with brain inflammation by CT and by biopsy Aflatoxin and Ochratoxin A present Aspergillus and Penicillium present by RT-PCR BRAIN LESIONS (TUMORS)

32. Pulmonary Fibrosis Astrocytomas Meduloblastomas Ependymomas Muscle biopsies from ALS patients, MS patients Endometrial Tissue Breast tissue and tumors Skin CSF Types of Patients/Specimens Tested

33. Medulloblastoma Cellular medulloblastoma Aflatoxin present Trichothecenes present Patient exposed to Aspergillus sp. Penicillium sp. Stachybotrys sp.

34. Astrocytoma 13 Biopsies submitted by pathologists Aflatoxin Positive Ochratoxin Positive 7 tumors 5/7 tumors showed one or both mycotoxins 6 biopsies � gliosis or chronic inflammation 4/6 showed one or both mycotoxins

35. BREAST TUMORS 3 Breast Tumors submitted 1 Lymph node (Sentinel node) All positive for Ochratoxin A Urine Tissue Comedo Tumor Intraductal Medullary

36. Amyotrophic lateral sclerosis(ALS) 1 patient, muscle biopsy Positive for Aflatoxin in tissue and urine Lou Gehrig Note: Patient was NOT Lou Gehrig !!!!

37. LUNG TUMORS Mesothelioma Ochratoxin found in chest wall, diaphragm. No ochratoxin found in pleura Urine not sampled. Adenocarcinoma, Large Cell Aflatoxin Positive

38. Renal Cell Carcinomas All positive for Ochratoxin A 4/4 Tumors in urine and tumor

39. Uterus Tissues (Benign, atypical lesions) 5 samples received 5/5 had Aflatoxin present in tissue and urine 2/5 had Ochratoxin present in tissue and urine 1/5 had Trichothecenes present in tissue and urine Samples were atypical endometrial tissue showing different stages of endometrial atypia.

40. Esthesioneuroblastoma 1 tumor submitted Aflatoxin and Ochratoxin A in urine and tissue Various pathology labs throughout the U.S. called: Chronic inflammation Pituitary adenoma Patient is continued to be followed. Finding of mycotoxin has led to controversial discussions concerning diagnosis. Being submitted for publication.

41. SUMMARY RealTime Laboratory LLC, can measure mycotoxin by ELISA and DNA by RealTime Polymerase Chain Reaction in body fluids and tissue. There appears to be a link between toxin presence and malignancy presence. Consider using liposomal glutathione (Readisorb) WHATEVER THE CASE: ERROR ON THE SIDE OF SAFETY, BELIEVE THAT MOLDS CAN BE TOXIC, DON�T EXCLUDE CAUSATION FOR MALIGNANT LESIONS.