“Disturbances of Tryptophan Metabolism and Human Health”

King-Thom Chung Department of Biology

The University of MemphisMemphis, TN 39152

Do Tryptophan Metabolites Really Cause Cancer ?

Tryptophan (W) Cancer

Tryptophan (73-22-3) Essential amino acidAromatic amino acidCoded by only a single codon UGGSusceptible 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

Sources of TryptophanRed meat, Turkey and chicken,Cheese, cheddar, other milk productsChocolate

Produced industrially by using microorganisms with Indole as precursor

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:

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

Tryptophan Metabolism

• 1. Nicotinamide Pathway

• 2. Serotonin pathway

• 2. Indole pathway

Nicotinamide pathway

NAD Pathway

source : Metacyc database

serSeratonin pathway

Indole pathway

Nicotinamide pathway

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

4. 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?

Decongugation of Glucouronides and Sulfate Esters

Liver turns tryptophan metabolites into glucouronides and sulphate estersThese metabolites are excreted to urine and accumulate in bladderBladder has sulphurylases and β-glucouronidase which cleave these conjugatesand revert them to original metabolites which may cause cancer

Glucouronidation and sulphate esterisation

Glucuronides and sulphate esters are

deconjugatedBy action of

Glucuronidase and sulphurylaseIn Bladder

Bladder CancerThe uncontrolled proliferations of cells in the urinary bladder

Especially in the transitional cells that line the inner membrane of thebladder

Transitional cell lining of the bladder

Bladder with transitional carcinoma

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?)

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.

Implication of Tryptophan as a Cause for Bladder CancerA. Suggesting Evidences1. 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 Information1. 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.

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.

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.

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

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.

Formation of Reactive Oxygen Species through formation of radical

Trp-metabolite + O2 Trp-metabolite * + O2-

H2O2 + Fe 2+ H2O2 + O2-

HO. + OH- + Fe3+H2O2 + Fe 2+

Try-Metabolite*+ Fe 2+Fe3+ + Trp metabolite

SOD

Fenton reaction

Tryptophan metabolites like kynurenine, 3-hydroxylkynurenine, anthranilic acid, 3-hydroxy anthranilic acid are excited to form these free radicals and mediate molecules

Tryptophan and Reactive Oxygen Species

O2

Tryptophan metabolite(3-HAA)

Tryptophan radical(anthranilyl radical)

Tryptophan adduct

O2.-

2H+H2O2

Transition metalsFe(II), Cu(II), Mn(III)

Hydroxyl radicalOH.

ResultsEffect of 3-hydroxyanthranilinic acid on DNA damage

in response to change in Cu2+ P+

DMSO

P+ 1

0μm

Fe2

+

P+10

0 μM

3O

HAA

P+

100

μM 3

OHA

A+2.

5 μC

u2+

P+

100

μM 3

OHA

A+5

μCu2

+

P+10

0 μM

3O

HAA+

10 μ

Cu2+

P+

100

μM 3

OHA

A+15

μCu

2+

P+10

0 μM

3O

HAA+

20 μ

Cu2+

ResultsEffect of 3-hydroxyanthranilinic acid on DNA damage

in response to change in Cu2+ P+

DMSO

P+ 1

0μm

Fe2

+

P+10

0 μM

3O

HAA

P+

100

μM 3

OHA

A+2.

5 μC

u2+

P+

100

μM 3

OHA

A+5

μCu2

+

P+10

0 μM

3O

HAA+

10 μ

Cu2+

P+

100

μM 3

OHA

A+15

μCu

2+

P+10

0 μM

3O

HAA+

20 μ

Cu2+

Effect of Cu2+ on DNA damage in response to change in 3-hydroxyanthranilinic acid

P+40

0 μM

3O

HAA+

10 μ

Cu2+

P+20

0 μM

3O

HAA+

10 μ

Cu2+

P+15

0 μM

3O

HAA+

10 μ

Cu2+

P+10

0 μM

3O

HAA+

10 μ

Cu2+

P+50

μM 3

OHA

A+10

μCu

2+

P+10

μCu

2

P+DM

SO

P+ 1

0μm

Fe2

+

Results

S No Metabolite Transition metal

Concentration (μM)

Result

1 3-hydroxyanthranilinic acid

_ 0-1000 _

2 3-hydroxyanthranilinic acid

Cu2+ 0-1000 +

3 Anthranilic acid _ 0-1000 _

4 Anthranilic acid Cu2+, Fe2+, Mn2+

0-1000 _

5 Kynurenine Cu2+, Fe2+, Mn2+

0-1000 _

6 kynurenine Cu2+, Fe2+, Mn2+

0-1000 _

7 3-hydroxykynurenine 0-1000 _

Results

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

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

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

Carcinogen Co-carcinogen Effect Specimen source

2-AAF Tryptophan Bladder tumors Rat Dunning et al. 1950

2-AAF Tryptophan Abnormal excretion of tryptophan metabolites

rat Dyer and Morris, 1961

Tryptophan No No effect Wistar rats Boyland et al. 1958

Tryptophan 2-AAF Bladder tumors Wistar rats Boyland et al. 1958

*3-KN, 3-HKN, AA, 3-HAA, XA

No Bladder tumors mice Bryan et al. 1971

Tryptophan No Bladder hyperplasia beagle dogs Oliver et al. 1978

Tryptophan Exposure to 4-aminobiphenyl or 2-naphthylamine

Bladder tumors beagle dogs Oliver et al. 1978

Indole 2-AAF Neoplasms hamster Oyasu et al. 1970Tryptophan CCl4 or aflatoxin B1 or

bothLiver cirrhosis, megalocytosis, hepatoma

Wistar rats Roschlau et al. 1962

Tryptophan B6 deficiency Bladder tumors Wistar rats Yoshida et al. 1970; Birt et al. 1987

Tryptophan metabolites No Hyperplasia and eventually tumors

mice Sims and Renwick, 1982

Tryptophan Saccharin Additive effect on carcinogenesis

Sims and Renwick, 1982

Carcinogen Co-carcinogen Effect Specimen source

2-AAF Tryptophan Bladder tumors Rat Dunning et al. 1950

2-AAF Tryptophan Abnormal excretion of tryptophan metabolites

rat Dyer and Morris, 1961

Tryptophan No No effect Wistar rats Boyland et al. 1958

Tryptophan 2-AAF Bladder tumors Wistar rats Boyland et al. 1958

*3-KN, 3-HKN, AA, 3-HAA, XA

No Bladder tumors mice Bryan et al. 1971

Tryptophan No Bladder hyperplasia beagle dogs Oliver et al. 1978

Tryptophan Exposure to 4-aminobiphenyl or 2-naphthylamine

Bladder tumors beagle dogs Oliver et al. 1978

Indole 2-AAF Neoplasms hamster Oyasu et al. 1970Tryptophan CCl4 or aflatoxin B1 or

bothLiver cirrhosis, megalocytosis, hepatoma

Wistar rats Roschlau et al. 1962

Tryptophan B6 deficiency Bladder tumors Wistar rats Yoshida et al. 1970; Birt et al. 1987

Tryptophan metabolites No Hyperplasia and eventually tumors

mice Sims and Renwick, 1982

Tryptophan Saccharin Additive effect on carcinogenesis

Sims and Renwick, 1982

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 (?)

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

Tryptophan metabolite like tryptamine or indoleacetic acid

Induction of XMEs by tryptophan metabolites

Mimura et al. 2003

Ah receptor dependent binding to DNA

Competitive displacement of TCDDby IAA and TA

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

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.

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.