This document outlines a proposed thesis on analyzing metal exposure through hair samples. It discusses the roles of various metals like iron, zinc, arsenic, and nickel in the body. Zinc is important for immune function and deficiency can increase apoptosis of immune cells. Arsenic and nickel are carcinogenic and can negatively impact the immune system. Nickel specifically can activate genes like HIF-1 that promote tumor growth. The document proposes using hair samples to measure long-term metal exposure in people, as hair levels reflect chronic intake over months to years. Factors like hair color and sex can influence metal content measured in hair.

1. 1
Hairy Research
Firhan A. Malik
(BSc. Biochemistry)
January 24, 2005
Laurentian University

2. 2
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

3. 3
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

4. 4
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

5. 5
RBC Production
Martini FH. 2004. Fundamentals of Anatomy and Physiology. Sixth Edition. Pearson/Benjamin Cummings.

6. 6
Hemoglobin
Martini FH. 2004. Fundamentals of Anatomy and Physiology. Sixth Edition. Pearson/Benjamin Cummings.

7. 7
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
http://www.acu-cell.com

8. 8
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
http://www.acu-cell.com

9. 9
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
http://www.acu-cell.com

10. 10
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.

11. 11
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

12. 12
Zn and the Immune System
Zn
Pro to Pre-Lymphocyte
Maturation of
Lymphocytes
Inhibit apoptosisErythropoiesis
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

13. 13
Low Zn = High Death
Fraker PJ and King LE. 2004. Reprogamming of the immune system during zinc deficiency. Annu. Rev.
Nutr. 24:277-298

14. 14
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
Fraker PJ and King LE. 2004. Reprogamming of the
immune system during zinc deficiency. Annu. Rev. Nutr.
24:277-298
ZA = zinc adequate diet
ZD = zinc deficient diet

15. 15
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

16. 16
As and the Immune System

17. 17
As and Cell Death
Ø Increased levels of As
leads to lower numbers of
viable cells
Ø As does cause apoptosis
of lymphoma cells
Beyersmann D. 2002. Effects of carcinogenic metals on
gene expression. Toxicol. Letters. 127:63-68

18. 18
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.

19. 19
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.

20. 20
Ni: The Bad News
Beyersmann D. 2002. Effects of carcinogenic metals on gene expression. Toxicol.
Letters. 127:63-68

21. 21
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

22. 22
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.

23. 23
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.

24. 24
Ni activates 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.

25. 25
Go on…
With Oxygen:
α Pro564
Fe
Pro564
OH
Deactivates
Hypoxia
Pathway
Ni2+ Ni2+
Activates
Hypoxia
Pathway
HIF-1
Prolyl
hydroxylase

26. 26
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.
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.

27. 27
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.

28. 28
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.

29. 29
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.

30. 30
Ni Uptake
Cangul H et al. 2002. Molecular mechanisms of nickel carcinogenesis. Toxicol. Letters. 127:69-75.

31. 31
Ni Uptake
Oller AR. 2002. Respiratory Carcinogenicity Assessment of Soluble Nickel Compounds. Environ. Health Pers. 110(5):
841-44.

32. 32
Ni causes fragmentation of
heterochromatin
Cangul H et al. 2002. Molecular mechanisms of nickel carcinogenesis. Toxicol. Letters. 127:69-75.

33. 33
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)

34. 34
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)

35. 35
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)

36. 36
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

37. 37
Structure of Hair
Porter RM. 2003. Review: Molecular mouse models of hair loss. J. Anat. 202:123-131

38. 38
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

39. 39
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.

40. 40
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.

41. 41
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
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.

42. 42
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
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.

43. 43
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 > 1Non-
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

44. 44
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.

45. 45
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

46. 46
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
Shamberger RJ. 2002. Validity of hair mineral testing. Biol. Trace. Elem. Res. 87:1-28.
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.

47. 47
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
Shamberger RJ. 2002. Validity of hair mineral testing. Biol. Trace. Elem. Res. 87:1-28.
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.

48. 48
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.

49. 49
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.

50. 50
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.

51. 51
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.

52. 52
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

53. 53
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)
Billoni N. et al. 2000. Thyroid hormone receptor beta1 is expressed in the human hair follicle. British J. Derm. 142:645-52.

54. 54
Zn made me bald?!?
Billoni N. et al. 2000. Thyroid hormone receptor beta1 is expressed
in the human hair follicle. British J. Derm. 142:645-52.

55. 55
ICP-MS Crash Course
PHILIPS. 2002. Inductively Coupled Plasma Mass Spectrometry: ICP-MS

56. 56
ICP-MS Crash Course
PHILIPS. 2002. Inductively Coupled Plasma Mass Spectrometry: ICP-MS

57. 57
ICP-MS Crash Course
PHILIPS. 2002. Inductively Coupled Plasma Mass Spectrometry: ICP-MS

58. 58
Let’s bring it all together
Trace Metals in Hair
Nutrition and Diet
Health StatusArea of
Residence
Age
Sex
Type of Shampoo Hair Dye
Employment

59. 59
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.

60. 60
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.

61. 61
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

62. 62
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)

63. 63
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

64. 64
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