“Disturbances of Tryptophan Metabolism and Human Health” King-Thom Chung Department of Biology

1. “Disturbances of Tryptophan Metabolism and Human Health” King-Thom Chung Department of Biology The University of Memphis Memphis, TN 39152

2. Do Tryptophan Metabolites Really Cause Cancer ? Cancer Tryptophan (W)

3. Tryptophan (73-22-3) Essential amino acid Aromatic amino acid Coded by only a single codon UGG Susceptible to Silent Mutation Indole IUPAC: (D)-α-Amino-3-indolepropionic acid (R)-2-Amino-3-(3-indolyl)propionic acid (S)-2-Amino-3-(3-indolyl)propionic acid

4. Sources of Tryptophan Red meat, Turkey and chicken,Cheese, cheddar, other milk products Chocolate Produced industrially by using microorganisms with Indole as precursor

5. Functions of Tryptophan A. Synthesizes Serotonin and Melatonin: Serotonin: neurotransmitter, vasoconstrictor: depression, schizophrenia, obesity, autoimmune disorder and immune deficiency, and probably in cancer. Melatonin: Sleep patterns, Zn uptake, Appetite patterns, mood, etc. B. Synthesizes Niacin (Vitamin B3 ): In the Liver C. Synthesizes Protein:

6. Tryptophan Deficiency Related Human Diseases 1. Cataracts • Totter. J. R. and Day, P. L. Cataract and other ocular changes resulting from • tryptophan deficiency. J. Nutri., 24:159, 1942. • Ferraro, A. and Roizin, L. Ocular involvement in rats on diet • deficient in amino acids. I. tryptophan. Arch. Ophthalmol., 38,331, • 1947. 2. Corneal vascularization • Sydenstricker, V. P., Hall, W. K. Bowels, L. L. And Schmidt, H. L., Jr., The • corneal vascularization resulting from deficiencies of amino acids in the • rat. J. Nutr., 34, 481, 1947. 3. Hematological manifestation of anemia, reduction in plasma proteins, fatty liver, and pancreatic atrophy. 4. Pellagra

7. Tryptophan Metabolism • 1. Nicotinamide Pathway • 2. Serotonin pathway • 2. Indole pathway

8. Nicotinamide pathway

9. NAD Pathway source : Metacyc database

10. ser Seratonin pathway Indole pathway Nicotinamide pathway

11. Tryptophan Metabolism A. Nicotinamide Pathway: 1. Mainly occurs in the liver through various intermediate compounds: Xanthurenic acid , Kynurenine, 3-hydroxykynurenine , anthranlic acid, 3-hydroxyanthranilic acid, xanthurenine tryptamine, etc. 2. Conjugation (Glucuronidation, Sulphate esterification) in the liver. 3. Filtered into Kidney • Deconjugation in the bladder by glucuronidase and sulphurylase and interact with the bladder tissue or excreted through urine------- Bladder Cancer ? B. Serotonin Pathway 5-hydroxytryptophan- Serotonin--Melatonin Occur in the serotonergic cells in the brain and intestine and by the platelets. C. Indole Pathway Occur in the GI tract and excreted or reabsorbed into the blood- Colorectal cancer?

13. Decongugation of Glucouronides and Sulfate Esters Liver turns tryptophan metabolites into glucouronides and sulphate esters These metabolites are excreted to urine and accumulate in bladder Bladder has sulphurylases and β-glucouronidase which cleave these conjugates and revert them to original metabolites which may cause cancer Glucouronidation and sulphate esterisation Glucuronides and sulphate esters are deconjugated By action of Glucuronidase and sulphurylase In Bladder

14. Bladder Cancer The uncontrolled proliferations of cells in the urinary bladder Especially in the transitional cells that line the inner membrane of the bladder Transitional cell lining of the bladder Bladder with transitional carcinoma

15. Statistics of Bladder Cancer 4th most prevalent among men and 8th most prevalent among women 38, 000 males 14, 000 females (USA, 2008) Chemotherapy is offered. (references?)

16. Factors in Etiology of Bladder Cancer 1. Exposure to industrial carcinogens like 4-Aminobiphenyl, 2-Aminonaphthol, Benzidine, β-Naphythlamine 2. Cigarette smoking 3. Disturbance of tryptohan metabolism when intake of excessive amount of tryptophan 4. Nutritional inbalanace : Deficiency of vitamin B6. Combination of excessive intake of tryptohan and deficiency of vitamin B, resulting in accumulation of tryptohan metabolites such as: Xanthurenic acid (XA), Kynurenine and 3-hydroxy Kynurenine, anthranilic acid, and 3-hydroxyanthranilic acid, etc. in the urine. 5. Infections of bladder : protozoan parasites of Schistosoma Cystitis, etc.

17. Implication of Tryptophan as a Cause for Bladder Cancer A. Suggesting Evidences 1. Smokers excreted more tryptophan metabolites in the urine than that of non smokers. (Kerr et al. 1965) 2. Hill, M. J. (1974) suggested that tryptohan might be converted to carcinogenic metabolites by intestinal bacteria. (Hill, M. J. 1974. Cancer 34:816. ) 3. Tryptohan concentration were higher in the feces of rat fed with all-meat diet than that of rats fed with normal chow diet (Chung et al. 1975. J. Natl Cancer Inst., 54: 1073-1078). 4. Co-carcinogenicity of tryptophan: No carcinogenic effect was observed when the hamsters were treated with high tryptophan diet, however these animals developed high incidences of tumors in bladder when their diets were supplemented with both tryptophan and acetyl 2-aminoflourerene. (Oyasu et al. 1972) 5. Most of the tryptophan metabolites were positive in Ames test when treated with nitrite. (Hashizume et al. 1991) B. Not supportive Information 1. Patients treated with the bladder cancer also excreted same level of the tryptophan metabolites as before. (Wolf et al. 1975) 2. Patients with schistomiasis were more susceptible to bladder cancer due to the rupture caused by the eggs of the parasite. (Badawi et al. 1994) 3. Industrial workers had high risk of cancer than that of those in rural area although they excreted comparable levels of tryptophan metabolites.

18. Comparison of fecal tryptophan contents of rats on all-meat and on normal chow diet • Tryptophan concentration (µmole/100 mg /wet weight) • ___________________________________________________ • All-Meat diet Norma diet • __________________________________________________ 0.051* 0.023* __________________________________________________ Average of 9 day’s sampling. *p< 0.001 From: Chung et al., J. Natl Cancer Inst., 54:1073-1078, 1975.

19. Proposed Molecular Mechanisms of Carcinogenesis caused by Tryptophan Metabolites Because of the chemical structure similar with industrial carcinogens, it is possible to have the similar molecular mechanisms 1. Induction of the formation of Reactive Oxygen Species (ROS) Hydroxyl radical (OH_.), Superoxide radical (O2.-) Hydrogen Peroxide (H2O2) 2. Formation of DNA adducts through the generation of cinnabaric acid. 3. Formation of DNA adducts through lipid peroxidation. 4. Metal ion mediated oxidation. 5. Receptor mediated mis-regulation of genes involved in carcinogenesis. 6. Inhibition of cellular apoptosis due to deficiency of niacin.

20. Structural similarities of anthranilic acid, kynurenine, naphthylamine and their carcinogenic hydroxy metabolites

21. Formation of cinnabarinate by autoxidation of the anthranilic acid 1.O2 mediated. 2. Cinnabarinate may react with the DNA to form an adduct, and cause mutations.

22. Formation of Reactive Oxygen Species through formation of radical Trp-metabolite + O2 Trp-metabolite * + O2- SOD H2O2 + Fe 2+ H2O2 + O2- Fenton reaction HO. + OH- + Fe3+ H2O2 + Fe 2+ Try-Metabolite*+ Fe 2+ Fe3+ + Trp metabolite Tryptophan metabolites like kynurenine, 3-hydroxylkynurenine, anthranilic acid, 3-hydroxy anthranilic acid are excited to form these free radicals and mediate molecules

23. Tryptophan and Reactive Oxygen Species Tryptophan metabolite (3-HAA) Tryptophan radical (anthranilyl radical) Tryptophan adduct O2.- O2 H2O2 2H+ Hydroxyl radical OH. Transition metals Fe(II), Cu(II), Mn(III)

24. Results Effect of 3-hydroxyanthranilinic acid on DNA damage in response to change in Cu2+ P+100 μM 3OHAA+20 μCu2+ P+100 μM 3OHAA+2.5 μCu2+ P+100 μM 3OHAA+5 μCu2+ P+100 μM 3OHAA+10 μCu2+ P+100 μM 3OHAA+15 μCu2+ P+100 μM 3OHAA P+ 10μm Fe2+ P+DMSO

25. Results Effect of 3-hydroxyanthranilinic acid on DNA damage in response to change in Cu2+ P+100 μM 3OHAA+20 μCu2+ P+100 μM 3OHAA+2.5 μCu2+ P+100 μM 3OHAA+5 μCu2+ P+100 μM 3OHAA+10 μCu2+ P+100 μM 3OHAA+15 μCu2+ P+100 μM 3OHAA P+ 10μm Fe2+ P+DMSO

26. Results Effect of Cu2+ on DNA damage in response to change in 3-hydroxyanthranilinic acid P+10 μCu2 P+50μM 3OHAA+10 μCu2+ P+150 μM 3OHAA+10 μCu2+ P+400 μM 3OHAA+10 μCu2+ P+DMSO P+100 μM 3OHAA+10 μCu2+ P+200 μM 3OHAA+10 μCu2+ P+ 10μm Fe2+

27. Results

28. Lipid peroxidation caused by Tryptophan metabolites Hydroxyl radicals transfer electron by attacking lipid membranes leading to formation of lipid radicals generating malondialdehyde that forms DNA adducts malondialdehyde

29. Plasmid relaxation assay Production of hydroxyl radical or singlet oxygen radicals would cause damage to the plasmid DNA thus relaxing the supercoiled plasmid to relaxed form. Relative mobilities of various plasmids on gel electrophoresis

30. Lipid peroxidation caused by Tryptophan metabolites Hydroxyl radicals transfer electron by attacking lipid membranes leading to formation of lipid radicals generating malondialdehyde that forms DNA adducts malondialdehyde

33. Receptor mediated Carcinogenesis Ah receptor (aryl hydro carbon receptor) mediates many toxicological reactions. Strong affinity to tetra dihlorodibenzodioxin(dioxin) that mediates carcinogenesis. Tryptamine and Indole acetic acid had good affinity to the Ah receptor, Induce receptor mediated binding to the DNA (gene) and regulate high expression of the gene leading to carcinogenesis. Cytochrome P4501A1 is a well characterized Ah-TCDD inducible Gene. TA competitively displaces the strong ligand TCDD resulting in a hint for the underlying mechanism for carcinogenesis (?)

34. Modification of the tryptophan metabolites by the Xenobiotic metabolizing enzymes (XMEs) Trp Trp Trp The tryptophan metabolite generated induces the generation of P450 and phase II enzymes that alter the compound Mechanism similar to the classic dioxin toxicity. Mimura et al. 2003

35. Induction of XMEs by tryptophan metabolites Tryptophan metabolite like tryptamine or indoleacetic acid Mimura et al. 2003

36. Ah receptor dependent binding to DNA Competitive displacement of TCDD by IAA and TA

37. Metal mediated carcinogenic effect (Hypothesis) 1. Mitochondria plays a role in apoptosis of cell. 2. Three forms of SOD: Mn-SOD, CuZn-SOD, Fe-SOD. Mn-SOD in mitochondria is chiefly responsible for cell apoptosis. 3. The displacement of Mn in SOD by Fe-SOD may prevent it from going to apoptosis, which promotes Cancer

38. Deficiency of Niacin (Tryptophan metabolism) leads to propagation of Cancer • Niacin cofactor in tryptophan catabolism • T cell recognize tryptophan metabolites as a signal to destroy pathogens or • carcinogenic cells • Low Niacin leads low Tryptophan metabolites down the path (?) • T cells do not recognize the carcinogenic cells leading to propagation of the cancer • cell.

39. Conclusion? • Tryptophan metabolites may generate ROS and thus cause mutations. • Affinity of tryptophan-ligands to various receptors, which lead to carcinogenesis • Factors that determine requirement of a co-metabolite to promote mutagenesis • Role of tryptophan metabolites in the Immune system.