hairy research: can hair tell the story about your health?

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Hairy Research Firhan A. Malik

(BSc. Biochemistry)

January 24, 2005 Laurentian University

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Outline

Ø  Role of metals in the body Ø  Zinc’s role in the immune response Ø  Toxic effects of arsenic and nickel Ø  The chemistry and structure of hair Ø  The use of hair to measure metal exposure Ø  A brief lesson on ICP-MS Ø  No Liposomes Ø  Proposed Thesis

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Iron (Fe) Ø  Synergistic with Vitamin C and nickel Ø  Increase in concentration leads to ↓pH in stomach Ø  Primarily involved in RBC production by forming

hemoglobin Ca

↕ Zn ↔ Fe ↔ Mg

↑ P

http://www.acu-cell.com

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RBC Production

Fe

Bone Marrow

•  Fe (iron) is bound to transferrin protein in the blood.

•  Fe + transferrin are transported to the bone marrow via blood

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RBC Production

Martini FH. 2004. Fundamentals of Anatomy and Physiology. Sixth Edition. Pearson/Benjamin Cummings.

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Hemoglobin

Martini FH. 2004. Fundamentals of Anatomy and Physiology. Sixth Edition. Pearson/Benjamin Cummings.

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Too Low or Too High?

Deficiency -  fatigue -  anemia -  depression -  asthma -  weak immune system -  learning difficulties -  ovarian cysts -  gastrointestinal disorders -  amenorrhea (failure to

menstruate)

Toxicity -  arthritis -  heart disease -  high blood pressure -  liver disease -  fibroid tumors -  edema -  constipation (seen during high

supplementation) -  hemochromatosis


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Zinc (Zn) Ø  Component of more than 100 enzymes Ø  Involved in metabolism (thyroid hormone) and DNA/RNA

synthesis Ø  Part of superoxide dismutase (SOD) to help protect cells

from free radical damage Ø  Increases proliferation of T-lymphocytes Ø  Maintains levels of testosterone Ø  In animals, related to insulin metabolism


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Too Low or Too High?

Deficiency -  decreased growth -  loss of taste and smell -  muscle weakness -  low sperm count or sterility -  heart disease -  hair loss -  impotence -  kidney stones -  liver and kidney disease

Toxicity -  vomiting -  dehydration -  stomach ulcers -  loss of libido -  anemia -  ovarian cysts -  joint or back pains -  muscle spasms or cramps -  irritability


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Zn and Melatonin Ø  In animals that had their pineal

glands removed, there was reduction of Zn levels

Ø  Zn deficiency leads to reduction in plasma melatonin.

Ø  Supplementation with Zn leads to increase in plasma melatonin

Ø  Melatonin has been found to decrease the risk of breast cancer by an immunologic response

Low Zn Low Melatonin Increased risk of breast cancer

Baltaci AK et al. 2004. Opposite effects of zinc and melatonin on thyroid hormones in rats. Toxicol. 195:69-75

Baltaci AK et al. 2003. Effect of zinc and melatonin supplementation on cellular immunity in rats with toxoplasmosis. Biol. Trace. Elem. Res. 96(1-3):237-45.

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Zn and Melatonin

Mocchegiani E et al. 1998. Presence of links between zinc and melatonin during the circadian cycle in old mice: effects on thymic endocrine activity and on the survival. J. Neuroimmunol. 86:111-122

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Zn and the Immune System

Zn

Pro to Pre-Lymphocyte

Maturation of Lymphocytes

Inhibit apoptosis Erythropoiesis

Regulation of Delayed-type hypersensitivity

Fraker PJ and King LE. 2004. Reprogamming of the immune system during zinc deficiency. Annu. Rev. Nutr. 24:277-298

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Low Zn = High Death


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Low Zn = High Death Ø  Deficiency leads to increased

apoptosis of lymphocytes Ø  The glucocorticoids may act in

synergy with low Zn to initiate the death pathway

Ø  This causes accelerated death among Pro and Pre-T-lymphocytes


ZA = zinc adequate diet

ZD = zinc deficient diet

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Arsenic (As) Ø  Negative effect on immune

system (reduces survival of lymphocytes)

Ø  Increases cancer risk Ø  Stimulates neoplastic growth

by inducing cell proliferation through AP-1 activation

Ø  Decreases expression of genes that down-regulate cell proliferation (possible tumor growth)

Beyersmann D. 2002. Effects of carcinogenic metals on gene expression. Toxicol. Letters. 127:63-68

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As and the Immune System

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As and Cell Death Ø  Increased levels of As

leads to lower numbers of viable cells

Ø  As does cause apoptosis

of lymphoma cells


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Nickel (Ni): The Good News Ø  Synergistic relationship with

Vitamin C Ø  Aids in blood clotting Ø  Even found to prevent

gallstones Ø  Can decrease oxidative

damage when present with Vitamin C

Salacinski HJ and O’Brien P. 2000. Evidence that the reactions of nickel in the presence of vitamin C do not produce toxic oxygen intermediates such as hydroxyl but ascorbate and carbon radicals. Arch. Toxicol. 74:5-12

Denkaus E and Salnikow K. 2002. Nickel essentiality, toxicity and carcinogenicity. 42:35-56.

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Ni: The Bad News Ø  A carcinogen Ø  Can form DNA-protein

crosslinks leading to chromosomal aberrations when present as a sulfide

Ø  Has high affinity for chromatin proteins

Ø  When inhaled as nickel carbonyl it causes epithelial damage in the lungs

Ø  Inhibits tumor suppressors (FHIT)

Chakarabarti SK et al. 2001. DNA-protein crosslinks induced by nicekl compounds in isolated rat lymphocytes: role of reactive oxygen species and specific amino acids. Toxicol. App. Pharmacol. 170:153-165

Kowara R et al. 2004. Reduced FHIT protein expression in nickel transformed mouse cells and in nickel-induced murine sarcomas. Mol. And Cell. Biochemistry. 255:195-202.

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Ni: The Bad News


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Hypoxia Ø  When oxygen level drops

it activates hypoxia-inducible factor 1 (HIF-1)

Ø  This leads to secretion of vascular endothelial growth factor (VEGF)

Ø  Stimulation of angiogenesis

Alberts B et al. 2002. Molecular Biology of the Cell. Fourth Edition. Garland Science

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What are you trying to say? Ø  In cancer cells, HIF-1 is

over-expressed Ø  Ni can activate HIF-1 and

Cap43

Li J et al. 2004. Nickel compounds act through phosphatidylinositol-3-kinase/Akt-Dependant, p70s6k-Independent pathway to induce hypoxia inducible factor transactivation and Cap43 expression in mouse epidermal c131 cells. Canc. Res. 64:94-101.

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HIF-1 expressed in cancer cells

Cangul H. et al. 2002. Enhanced overexpression of an HIF-1/Hypoxia-Related protein in cancer cells. Environ. Health. Pers. 110(5):783-88.

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Ni activates Cap43


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Go on… With Oxygen:

α Pro564

Fe

Pro564

OH

Deactivates Hypoxia Pathway

Ni2+ Ni2+

Activates Hypoxia Pathway

HIF-1

Prolyl hydroxylase

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Go on…

Salnikow K. et al. 2003. The involvement of hypoxia-inducible transcription factor-1-dependent pathway in nickel carcinogenesis. Canc. Res. 63:3524-30.


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Get to the point! Ø  This is a suggested mechanism for nickel’s carcinogenic behaviour Ø  Hypoxic stress leads to the induction of p53 (apoptosis) and Nip3 (a

proapoptotic gene) Ø  Hypoxia selects for cells with enhanced glycolytic activity, causing

production of large amounts of lactic acid (known as the Warburg effect), commonly seen in tumor cells.

Ø  Moreover, enzymes of glycolysis (hexokinase I and glucose-6-phosphate) were also activated by nickel. This makes sense because it would increase the amount of lactic acid.

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Ni activates glycolytic enzymes by HIF-1 pathway

Salnikow et al. 2003. The involvement of hypoxia-inducible transcription factor-1-dependent pathway in nickel carcinogenesis. Canc. Res. 63:3524-30.

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HIF-dependant and independent genes activated by Ni

Salnikow K et al. 2003. The involvement of hypoxia-inducible transcription factor-1-dependent pathway in nickel carcinogenesis. Canc. Res. 63:3524-30.

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Ni Uptake

Cangul H et al. 2002. Molecular mechanisms of nickel carcinogenesis. Toxicol. Letters. 127:69-75.

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Ni Uptake

Oller AR. 2002. Respiratory Carcinogenicity Assessment of Soluble Nickel Compounds. Environ. Health Pers. 110(5):841-44.

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Ni causes fragmentation of heterochromatin

Cangul H et al. 2002. Molecular mechanisms of nickel carcinogenesis. Toxicol. Letters. 127:69-75.

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Ni, Hair and Sudbury Ø  Study conducted in 1975 by Dr. D.E. Goldsack and team

at Laurentian University Ø  Found that the distance from stack directly proportional

[Ni] in hair Ø  Those who lived less than 5 km had over 30 ppm of Ni in

hair

Goldsack DE and Niebor E. 1975. Nickel content of human hair as an index of environmental exposure to nickel in the Sudbury basin. (unpublished)

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Ni, Hair and Sudbury

Goldsack DE and Niebor E. 1975. Nickel content of human hair as an index of environmental exposure to nickel in the Sudbury basin. (unpublished)

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Why use hair? Ø  Less subjective to internal biochemical changes

(stability) Ø  Can track long-term and chronic exposure (months to

years) Ø  More economical for larger scale epidemiological studies Ø  Used extensively in forensics and drug analysis Ø  No pain (well depends on sobriety of researcher)

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Hair facts Ø  Over 140,000 individual hairs on an average human

head Ø  Each strand is 60 µm in diameter Ø  Contains keratin (fibrous protein) Ø  Melanocytes produces an indole pigment (melanin)

formed by the oxidation of tyrosine Ø  Hair colour is directly proportional to the number of

melanin granules and amount of melanin in granules

http://www-biol.paisley.ac.uk/courses/stfunmac/reading/keratin.htm

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Structure of Hair

Porter RM. 2003. Review: Molecular mouse models of hair loss. J. Anat. 202:123-131

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Hair Chemistry and Growth Ø  3 phases of hair growth:

l  Anagen – lasts between 2 to 8 years - growth cells in the papilla (hair root) rapidly divide

- hair shaft is keratinized and pushed out through pore - the follicle grows down into the deeper levels of the dermis

(now receives blood) Catagen - transitional phase (2 to 4 weeks)

- renewal process (hair stops growing) - follicle shrinks and breaks from the papilla

- the bulb detaches from blood supply and hair shaft is pushed up as the follicle disintegrates

Telogen - resting phase (2 to 4 months) - hair does not grow but still attached to the follicle

- dermal papilla is in a resting phase - at given time, 10-15% of all hairs are in this phase

http:/www.inhairit.com/hair-growth-cycle.html

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Hair growth stages and signals

Costarils G and Miller SE. 2001. Towards a molecular understanding of hair loss and its treatment. TRENDS in molecular medicine. 7(7):293-301.

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Are women superior? Ø  Ah… No Ø  Females have higher amounts

of certain trace metals in their hair

Ø  Possibly related to hormonal differences

Sturaro A et al. 1994. The influence of color, age, and sex on the content of zinc, copper, nickel, manganese and lead in human hair. Biol. Trace. Elem. Res. 40:1-8.

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The darker the better? Ø  There is a definite relationship

between the colour of hair and levels of trace metals

Ø  Typically darker colours have more trace metal content

Ø  An overall comparison: black > red > brown > blond


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The older the better? Ø  Zn content tends to increase

with age Ø  Cu content tends to decrease

with age Ø  Mn increases with age in blond

hair, but decreases in brown hair


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Hair and Epilepsy Ø  Epileptics have lower

concentration of Zn and Mg, but higher concentration of Cu

Ø  This suggests abnormal utilization and uptake of these trace metals

Ø  Interestingly:

Epileptics

Mg : Zn > 1 Non-Epileptics

Mg : Zn < 1

Ilhan et al. 1999. Serum and hair trace metal levels in patients with epilepsy and healthy subjects: does the antiepileptic treatment affect the element concentrations in hair? Eur. J Neu. 6:705-709

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Hair and Epilepsy Ø  After long-term anti-convulsant therapy:

•  Male and female epileptics had reduced Cu in their hair strands

•  Male and female epileptics had increased Mg in their hair strands

•  Zn increases with use of antiepileptic drugs

So, anti-convulsant therapy alters metabolism of trace elements and minerals, by altering brain enzymes.

Shamberger RJ. 2002. Validity of hair mineral testing. Biol. Trace. Elem. Res. 87:1-28.

Doretto MC et al. 2002. Zinc, magnesium and copper profiles in three experimental models of epilepsy. Brain Res. 956:166-172.

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Hair and Epilepsy

Ilhan et al. 1999. Serum and hair trace metal levels in patients with epilepsy and healthy subjects: does the antiepileptic treatment affect the element concentrations in hair? Eur. J Neu. 6:705-709

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Behavior Ø  Mentally handicapped children found to have elevated Cd and Pb in

hair Ø  Increased Mg and P related to emotional disturbances Ø  High Pb and Cd in hair of violent incarcerated male criminals Ø  High Pb and Cd in hair of children with ADHD and behavioral

problems Ø  High Al, Cu, Cd, and Mg in hair of dyslexic children

Ø  Exposure to Pb has deleterious effects on intelligence

High Pb in hair correlates with low IQ


http://www.cpp.org.pk/articles/metal_toxicology.html

Contiero E and Folin M. 1994. Trace elements nutritional status: use of hair as a diagnostic tool. 40:151-160.

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Hair and Breast Cancer Ø  Cr levels are higher in breast cancer patients’ hair Ø  Mn levels are lower in breast cancer patients’ hair relative to control

groups Ø  Malignant breast cancer tissue had high levels of Zn, Cu, Se and Fe Ø  Cu/Zn ratio higher in breast cancer tissue relative to healthy tissue


Kuo HW et al. 2002. Serum and tissue trace elements in patients with breast cancer in Taiwan. Biol. Trace Elem. Res. 89(1):1-11.

Killic E et al. 2004. Chromium and manganese levels in scalp hair of normals and patients with breast cancer. Biol. Trace Elem. Res. 102(1-3):19-26.

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Hair and the Heart Ø  Zn and Cr levels are lower in

hypertension and coronary disease patients’ hair than in healthy patients

Ø  Zn/Cu ratio lower in hypertension and coronary disease patients

AD = aged hypertension and coronary disease

AC = aged healthy controls

Tang Y-R. et al. 2003. Studies of five microelement contents in human serum, hair and fingernails correlated with aged hypertension and coronary artery disease. 92:97-103.

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Hair Loss Ø  Human hair found to have

receptor for thyroid hormone (β1)

Ø  In TRβ1 knockout mice, there is hearing loss and elevated levels of TH and TSH

Billoni N. et al. 2000. Thyroid hormone receptor beta1 is expressed in the human hair follicle. British J. Derm. 142:645-52.

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Zn, Melatonin and Thyroid Hormones

Ø  Zn supplementation increases the level of free 3,3’,5-Triido-L-thyronine (T3) in serum

Ø  Melatonin decreases the level of T3 in serum by increasing the absorption of Zn from the digestive system

Baltaci AK et al. 2004. Opposite effects of zinc and melatonin on thyroid hormones in rats. Toxicol. 195:69-75.

Bediz CS et al. 2003. Both zinc deficeincy and supplementation affect plasma melatonin levels in rats. Acta. Physiol. Hung. 90(4):335-9.

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Zn, Melatonin and Thyroid Hormones

Baltaci AK et al. 2004. Opposite effects of zinc and melatonin on thyroid hormones in rats. Toxicol. 195:69-75.

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Zn made me bald?!? Ø  Zn activates thyroid hormones (TH) Ø  TH will then bind to thyroid hormone receptors (TRs),

such as β1 in the scalp Ø  3,3’,5-Triido-L-thyronine (T3) interacts with TRs and

modulates gene transcription, cell growth, differentiation, development, metabolism, and thermogenesis

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Zn made me bald?!? Ø  T3 promotes growth and survival of hair follicles in-vitro Ø  Patients with hyperthyroidism were found to have greater

number of follicles in S-phase than patients with hypothyroidism

Ø  Whoa fellas, don’t break out those zinc bars just yet. There is an upper limit.

Ø  Too much zinc can also cause hair-loss! (suggests feedback mechanism)


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Zn made me bald?!?


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ICP-MS Crash Course

PHILIPS. 2002. Inductively Coupled Plasma Mass Spectrometry: ICP-MS

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ICP-MS Crash Course


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ICP-MS Crash Course


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Let’s bring it all together

Trace Metals in Hair

Nutrition and Diet

Health Status Area of Residence

Age Sex

Type of Shampoo Hair Dye

Employment

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Proposed Thesis Objectives: 1.  To perform an updated trace metal in hair analysis

using the 1975 study as a basis. Expand to include 30 elemental trace metals.

2.  Examine how health status, biological identity (age, sex) and nutrition correlate with trace metal concentration.

3.  Examine how area of residence correlates with trace metal concentration.

4.  Form a standardized procedure for biological sample analysis using ICP-MS.

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Proposed Thesis Procedure: 1.  Skin at nape of neck is sterilized with alcohol swabs. Equipment is

sterilized as well. 2.  Small strands of hair (1-2 g, 0.2-0.5 cm) are clipped off. 3.  Hair is stored in sterile, airtight containers. 4.  Participants fill out a questionnaire and signed consent is taken. 5.  Hair is digested in nitric acid, to remove all organics and proteins (only

trace metals remain behind). 6.  Dilutions are made. 7.  Samples are run in ICP-MS. This is done in triplicates. 8.  Concentration of 30 trace metals is found. 9.  The levels of trace metals are correlated with questionnaire responses

using multivariate statistics.

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Proposed Thesis Control Group: Ø  Used to determine length of time required for trace

metals to accumulate in hair Ø  25 participants chosen at random Ø  Have hair snipped every 20 days, for a total of three

times Ø  Each session they are asked to record any differences in

diet, residence, health, and hair status

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How far have we come? Ø  Over 500 participants from a variety of backgrounds Ø  Involvement of local hair salons Ø  Input from area health professionals Ø  Press attention Ø  Hair samples from areas outside of Sudbury (e.g., Elliot

Lake)

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What lies ahead? Ø  Performing the complete statistical analysis to determine

correlations Ø  Possibly link the results with trace metal in urine study

(N. Graham) Ø  Create distribution plots Ø  Take a long, long vacation

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Acknowledgements Ø  Dr. Graeme Spiers Ø  Dr. Douglas Goldsack Ø  Mr. Al Douglas Ø  Mr. Francois Prévost Ø  Mr. Troy Maki Ø  Mr. Neil Graham Ø  All the graduate students at CEM Ø  All the staff and students at MIRARCO Ø  Ms. Jane Djivré Ø  Ms. Andrea Poryckji (especially for business cards) Ø  All the hair salons Ø  Elliot Lake Research Field Station and their families and friends Ø  Laurentian University Public Affairs Office Ø  All my family and friends for preventing the rapid expansion of my ego Ø  Every single person who took part in the study