Microbes, Metals and

Methylation
Dr. Amy Yasko
April 2005
 MICROBES

METALS METHYLATION
 At least 15% of the general population has a
genetic enzyme defect (C-to-T mutation of
nucleotide 677 of the MTHFr gene) that causes
elevated homocysteine. High levels of
homocysteine are correlated with heart disease, as
well as with Alzheimer’s disease. Moreover, higher
risks of neural tube malformations have been
attributed to this mutation. An additional genetic
change in the MTHFr gene, known as A1298C,
also results in a lowered enzymatic activity. It is
essential when screening, to use a test that looks
for BOTH of these mutations, the C677T and the
A1298C.
Saliva and blood tests are available to determine if
you have this mutation. According to Dr. Richard
Kunin up to 80%-90% of the patients he sees carry
one or the other of these MTHFr mutations. In Dr.
Yasko’s practice, she has seen an exceedingly
high correlation between children with this
mutation and autism and ADD, ADHD. The
pathways involved with this mutation are
understood, and nutritional supplements are
available that bypass the genetic defect.
(Presentation: Vitamin Related Mutations, Jan.,
2004).)
 Case study
• Severely autistic
• MTHFr
• Negative provoked urine
• RNA based protocol with EDTA
Provoked urine tests do not
always reflect the actual body
burden of heavy metals,
especially in the presence of
chronic infection.
DMSA Provoked Urine
June 4, 2001

NO MERCURY
DMSA Provoked Urine
July 16,2001

NO MERCURY
DMSA Provoked Urine
August 15, 2001

NO MERCURY
DMSA Provoked Urine
February 3, 2002

NO MERCURY
 “ D-Glucaric Acid marginally
low: A result lower than normal
range may be of no clinical
significance, or reflect (1) an
environment unusually free of
xenobiotics…

Mercapturic Acids Normal:

The level of mercapturic acids
in this patients urine sample is
within the normal range for
age and gender for individuals
that have not been exposed to
greater than average levels of
xenobiotics…”
Examples of the use
of RNA
T Aluminum (ug/g Creatinine)
h
e

E200 180

f
f150
e
c
120

t100

o 50
f 35

19 20

M 0 0 0 0

e
6/18/03 7/10/03 7/24/03 8/5/03
6/17/03 7/3/03 7/17/03 8/1/03

t
a Safe Range: 60
l

R
N
 Mercury (ug/g Creatinine)

24
25

20

15

15
12

10 9.8

10
7.1
6.4 6.5 6.6
5.4 5.3

5
3.9
2.8
2.3 2.3
1.8

0 0

0
7/10/03 8/15/03 10/1/03
6/17/03 8/1/03 9/3/03

Safe Range: 5
 Lead (ug/g Creatinine)

20
18

15
13

10
7.4
6.6
5.7

5
3.6
3.3
2.3
1.7
0.8 0.8 1 1 1 1.1
0.4 0.2 0.6 0.6 0.4
0.2 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

9/7/03 10/18/03 11/14/03 12/15/03 1/18/04 3/5/04 6/1/04

8/19/03 9/28/03 11/3/03 11/28/03 1/3/04 2/7/04 4/22/04 8/4/04

Safe Range: 5
 Tungsten (ug/g Creatinine)

12 12

10
8.8

6.1
6 5.7
5.4

2 1.6
1.4
1
0.8 0.8 0.8
0.5 0.6
0.4 0.4 0.3 0.3 0.3 0.3 0.3
0.2 0.2
0 0 0 0 0 0 0 0 0

9/7/03 10/18/03 11/14/03 12/15/03 1/18/04

8/19/03 9/28/03 11/3/03 11/28/03 1/3/04

Safe Range: 1.5

Prevalence of Myocarditis and Skeletal Muscle Injury
During Acute Viral Infection in Adults
JAMA April 2003

• Patients with serologically

confirmed acute influenza viral
infection
• Creatinine levels were elevated
 Creatinine in Urine

Creatinine

200 200

170 170

150

100 90 92
83
76
66
61
57
50 45
40
50 53

35
30
24 25 22 26
18

0
3/30/044/9/044/17/046/17/046/21/047/20/048/25/049/8/049/23/0410/7/04
3/23/044/5/044/12/046/1/046/20/047/12/048/4/048/30/049/13/049/29/0410/12/04

Mercury Excretion in Urine

Mercury (ug/g Creatinine)

6 5.9

3 2.7
2.5
2.2
2 1.9
1.5 1.5

1 0.70.7
0.3
0 0 0 0 0 0 0 0 0 0 0 0

3/30/044/9/044/17/046/17/046/21/047/20/048/25/049/8/049/23/0410/7/04
3/23/044/5/044/12/046/1/046/20/047/12/048/4/048/30/049/13/049/29/04
10/12/04
MICROBES: virus
“Viruses use a “Trojan horse” strategy in
which the victim assists the intruder. To
extract assistance from the host cell,
viruses use the detailed “inside
information” that they have acquired
during million of years of coevolution with
their hosts.”

Science Vol 304 April 9,2004

 Factors that may lead to Chronic Viral Infection

• Decreased T cell response due to MTHFr mutation that impairs DNA

synthesis needed for T cell clonal expansion
• Impaired B cell response due to lack of T helper cells, and T
regulatory cells.
• Further induction of IDO by interferon gamma
• Interferon gamma has been reported to increase intestinal
permeability and blood-brain permeability
• Increased IDO, impact on self versus non self
• Decreased vaccine efficiency, increased viral load
• Nature of retroviruses themselves
 Chronic Viral Infection
• Measles, Mumps, Rubella, HHV-6,
Herpes Zostar (Chicken Pox)
• Streptococcus, Herpes Virus, Measles Virus
use the same receptor to get into cells
• Increased susceptibility to all three
• Viruses glutamate
• Viruses host MT proteins bind metals
inside virally
infected cells
 Viruses as Parasites
• Induction of Metallothionein proteins by
viral infection
• Trapping of heavy metals by virus
 Viral infection leads to the
increased absorption or
retention of metals in the body.
 Relationship between Virus and Metals

J Toxicol Environ Health A. 2001 Aug 10;63(7):511-23.

Mortality in mice infected with an amyocarditic coxsackievirus and given a
subacute dose of mercuric chloride.

South PK, Morris VC, Levander OA, Smith AD.

Beltsville Human Nutrition Research Center, US Department of Agriculture, Agricultural Research Service, Maryland 20705-
2350, USA.

An amyocarditic strain of coxsackievirus B3 (CVB3/0) induces heart damage when inoculated into selenium
(Se)-deficient mice. Mercury (Hg), an Se antagonist, is known to aggravate viral infections. The experiments
reported here assessed the effect of prior Hg treatment in mice subsequently inoculated with an amyocarditic
strain of coxsackievirus. A pilot study showed that under our conditions the maximum tolerated dose of
HgCl2 in uninfected mice was 6 mg HgCl2/kg body weight. In the main study, doses of 0, 3 or 6 mg HgCl2/kg
body weight were administered intraperitoneally (ip) to 7-wk-old male mice fed a standard chow diet. Two
hours later, half the mice were inoculated ip with CVB3/0. Ten days postinoculation, no mortality was
observed in mice given only virus. In mice not given virus, 10% injected with 6 mg HgCl2/kg body weight
died. On the other hand, 64% of the mice given both virus and 6 mg HgCl2/kg body weight died. Fifteen
percent of the hearts from virus-infected mice given 3 mg HgCl2/kg body weight and 33% of the hearts from
virus-infected mice given 6 mg HgCl2/kg body weight exhibited a higher incidence of lesions than hearts from
mice-given virus alone. Moreover, viral heart titers were elevated in infected mice injected with 6 mg
HgCl2/kg body weight compared to infected mice receiving no Hg. Thus, an amyocarditic coxsackievirus
given to mice after a nonlethal subacute dose of Hg results in mortality, increased incidence of heart lesions,
and elevated viral heart titers. These results demonstrate the important role of toxic elements in determining
the severity of viral infections.
Relationship between Virus and Metals
Toxicol Lett. 1996 Dec;89(1):19-28

Effects of methyl mercury on cytokines, inflammation and virus clearance in a

common infection (coxsackie B3 myocarditis).

Ilback NG, Wesslen L, Fohlman J, Friman G.

Pharmacia and UpJohn, Helsingborg, Sweden.

A myocarditic coxsackievirus B3 (CB3) infection in Balb/c mice was used to investigate the effects of 12
weeks of methyl mercury (MeHg) exposure (3.69 mg/g diet) on inflammatory heart lesions, virus in the heart,
the cytokine response, i.e. cachectin/TNF-alpha and gamma-interferon (IFN-gamma) levels in plasma, and
on disease complications and mortality. This dose of MeHg did not influence mortality in this infection model.
The inflammatory and necrotic lesions in the ventricular myocardium 7 days after the inoculation covered
2.2% of the tissue section area in infected control mice. This damage was increased (n.s.) by 50% (to 3.3%
of the tissue section area) in MeHg-treated mice. The response pattern of lymphocyte subsets in situ in
myocardial inflammatory lesions was corroborated using an immune histological technique. MeHg treatment
tended to increase (2.2-fold, n.s.) the number of Mac 2+ cells (macrophages) in the heart muscle in this
infection. Plasma levels of both TNF-alpha and IFN-gamma increased on day 3 of the infection in MeHg-
treated as well as in non-MeHg-treated mice, but the mean IFN-gamma response was more pronounced in
the MeHg-treated mice. On day 7 of the infection, when most animals still showed clinical signs of disease,
cytokine levels were back to normal. MeHg-exposure in non-infected mice did not affect cytokine levels. In
situ hybridization of virus RNA in myocardial tissue showed remaining virus in those mice who had the lowest
plasma IFN-gamma levels. A 20% increased (P < 0.05) lymphoproliferative response to the T cell mitogen
Con A was observed as a result of the MeHg treatment. Even heart tissue lesions and virus persistence
tended to be influenced by MeHg in a direction compatible with the development of chronic disease.
 Relationship between Virus and Metals

Biol Trace Elem Res. 2003 Feb;91(2):111-24

Sequential changes in Fe, Cu, and Zn in target organs during early

Coxsackievirus B3 infection in mice.

Ilback NG, Benyamin G, Lindh U, Friman G.

Section of Infectious Diseases, Department of Medical Sciences, Uppsala University Hospital, Sweden.

In Coxsackievirus B3 (CB3) infection, the heart and pancreas are major target organs and, as a general host
response, an associated immune activation and acute phase reaction develops. Although iron (Fe), copper
(Cu), and zinc (Zn) are involved in these responses, sequential trace element changes in different target
organs of infection have not been studied to date. In the present study, Fe, Cu, and Zn were measured
through inductively coupled plasma mass spectrometry (ICP-MS) in the plasma, liver, spleen, heart, and
pancreas during the early phase (d 1 and 3) of CB3 infection in female Balb/c mice. The severity of the
infection was assessed through clinical signs of disease and histopathology of the heart and pancreas,
including staining of CD4 and CD8 cells in the pancreas. During infection, the concentrations of Fe, Cu, and
Zn changed in the plasma, liver, and pancreas, but not in the spleen and heart. The changes in plasma Cu,
Zn, and Fe seemed to be biphasic with a decrease at d 1 that turned into increased levels by d 3. Cu showed
similar biphasic changes in the liver, spleen, and pancreas, whereas, for Zn and Fe, this pattern was only
evident in the liver. In the pancreas, the reverse response occurred with pronounced decreases in Fe (23%, p
< 0.05) and Zn (64%, p < 0.01) at d 3. Although the pathophysiological interpretation of these findings requires
further research, the sequential determination of these elements may be of clinical value in enterovirus
infections in deciding the stage of disease development.
 Relationship between Virus and Metals

Biol Trace Elem Res. 2000 Aug;76(2):149-60

Trace element changes in the myocardium during coxsackievirus B3

myocarditis in the mouse.

Funseth E, Lindh U, Wesslen L, Friman G, Ilback NG.

Department of Medical Sciences, Uppsala University Hospital, Sweden.

During most infections plasma, concentrations of trace elements change, but it is unclear if this reflects
changes in infected target tissues. In coxsackievirus B3 (CB3) infection, the myocardium is a target in both
humans and mice. The concentrations of 12 trace elements were analyzed by inductively coupled plasma-
mass spectrometry (ICP-MS) in the myocardium of sham-inoculated controls and infected A/J mice 4 and 7 d
postinoculation. The size of the inflammatory lesion was positively correlated to the virus content of the heart,
as estimated by histopathology and in situ hybridization, respectively. Iron, cobalt, vanadium, and selenium
showed transient changes, whereas for the other elements, tendencies on d 4 were manifest on d 7. A three-
fold increase in calcium on d 7 suggests prestages of calcification, whereas increases in zinc, selenium, and
copper may be the result of the accumulation of immune cells. The magnesium decrease may contribute to the
increased sensitivity to cardiac arrhythmias in myocarditis.
 Relationship between Virus and Metals

Biol Trace Elem Res. 1998 Jul;63(1):51-66

Effects of selenium supplementation on virus-induced inflammatory heart

disease.

Ilback NG, Fohlman J, Friman G.

Toxicology Division, National Food Administration, Uppsala, Sweden.

The effects of 10 wk of selenium (Se) supplementation (5 ppm) in drinking water on immune responses and
resistance to a myocarditic Coxsackie virus B3 (CB3) infection were studied in female Balb/c mice. Se
supplementation reduced CB3-induced mortality: at day 14 postinoculation, survival was 58% in the Se-treated
group as compared to 25% in the untreated group. Whole-blood glutathione peroxidase (GSH-Px) activity was
elevated by 68% (p < 0.001) and Se content in the liver by 24% (p < 0.001). Red (RBC) and white blood cell
(WBC) counts, as well as the number of cells in the spleen and thymus, were unaffected. The cellular counts of
T-lymphocytes (CD4+, CD8+) and natural killer (NK+) cells in the blood were not affected. However, the
CD4+/CD8+ ratio (5.2) tended to increase after Se supplementation (5.9). The spleen lymphoproliferative
response to T- and B-cell mitogens were increased by 9 and 43%, respectively (ns), in the Se-supplemented
group. The total NK cell activity in blood and spleen showed minor increases, but when the activity in the blood
was expressed per cell, the increase amounted to 35% (ns) with Se supplementation. The inflammatory and
necrotic lesions in the ventricular myocardium at 7 and 14 d postinoculation were not significantly reduced by
Se treatment, probably owing to the increased survival with Se even of mice with the most pronounced heart
damage; comparable untreated mice were estimated to have died at day 14. Results indicate that modest
doses of Se can improve immune function, which may increase the general resistance to this viral infection.
 Relationship between Virus and Metals

Eur Heart J. 1995 Dec;16 Suppl O:20-4

New aspects of murine coxsackie B3 myocarditis--focus on heavy metals.

Ilback NG, Lindh U, Fohlman J, Friman G.

Kabi Pharmacia AB, Helsingborg, Sweden.

The magnitude of inflammatory lesions in the hearts of coxsackie B3 (CB3)-virus infected mice can be
affected by the potentially toxic heavy metals cadmium (Cd), nickel (Ni) and methyl mercury (MeHg). The
infection is associated with a changed distribution, such as Cd accumulation in the spleen and kidneys. New
target organs for Ni during the infection were the heart, pancreas and lungs in which inflammatory lesions
were present. This increased uptake was correlated with the disturbed function of immune cells and an
increased inflammatory reaction. Ni and MeHg appeared to have a direct effect on immune cells that resulted
in changed natural killer cell activity and decreased mobilization of macrophages, CD4+ and CD8+ cells into
the inflammatory lesions. Although MeHg increased spleen T cell activity and gamma-interferon (IFN-gamma)
levels, the inflammatory lesions in the heart increased. Another detrimental effect of MeHg treatment was
evident by an increased calcium and decreased zinc content in the inflamed heart, which may partly explain
the more severe inflammatory lesion. The host's response, CB3 infection, changed the distribution of each
metal in a specific way, a fact which may subsequently result in altered target organ toxicity and resistance to
the infection.
Relationship between Virus and Metals

Toxicol Appl Pharmacol. 1992 May;114(1):166-70

A common viral infection can change nickel target organ distribution.

Ilback NG, Fohlman J, Friman G.

Toxicology Laboratory, National Food Administration, Uppsala, Sweden.

The autoradiographic distribution of the toxic heavy metal nickel (Ni) was studied at 4 and 7 days post-
coxsackievirus B3 (CB3) infection in Balb/c mice. The distribution of the iv injected 63Ni was studied 10 min,
4 hr, and 24 hr after administration. Results clearly show that the site of 63Ni accumulation is greatly
changed during this viral infection. This newly discovered distribution was mainly visible as a greatly
increased accumulation in the pancreas and the wall of the ventricular myocardium. Healthy animals showed
almost no 63Ni accumulation in these tissues. These results for the first time show that an invading
microorganism can change the distribution of an environmental pollutant
 Relationship between Virus and Metals

Scand J Infect Dis Suppl. 1993;88:93-8

Altered distribution of heavy metals and lipids in coxsackievirus B3 infected

mice.

Ilback NG, Fohlman J, Friman G.

Kabi Pharmacia AB, Helsingborg, Sweden.

This report presents evidence that a micro-organism common in our environment, coxsackievirus B3 (CB3)
and the host responses it causes, can change the body distribution of heavy metals and lipids. The present
results show that the distributions of intravenously injected 109Cd, 63Ni and 14C-Cholesterol are changed
during infection, in a way that is specific for each of the studied compounds. Increased accumulation of 109Cd
in the spleen and kidneys, 63Ni in the pancreas and ventricular myocardium, and 14C-Cholesterol in the heart
and pancreas was observed during CB3 infection. This may affect the development of inflammatory lesions
and subsequently result in altered and/or increased target organ toxicity as well as lipid accumulation. Thus,
risk assessment in exposed populations may have to be evaluated depending on individual nutritional and
exposure status.
Relationship between Virus and Metals

Chemosphere. 1994 Sep;29(6):1145-54

Immune responses and resistance to viral-induced myocarditis in mice

exposed to cadmium.

Ilback NG, Fohlman J, Friman G, Ehrnst A.

Kabi Pharmacia AB, Helsingborg, Sweden.

The effects of 10 weeks of treatment with cadmium (Cd) on the immune function and resistance to
coxsackievirus B3 (CB3)-induced myocarditis in female Balb/c mice were investigated. A 2mM dose of Cd in
the drinking water did not influence mortality due to the CB3 infection. The inflammatory and necrotic lesions
in the ventricular myocardium seven days after inoculation (2.94% of tissue section area) were not increased
by Cd (2.82% of tissue section area). The response pattern of lymphocyte subsets in situ in myocardial
inflammatory lesions was elucidated by an immune histochemical staining technique. With Cd treatment the
number of cytotoxic T cells and B cells in these lesions decreased by 22% (n.s.) and 21% (p < 0.05),
respectively. Spleen weight and the lymphoproliferative response to the B-lymphocyte mitogen increased by
19% (p < 0.05) and 23% (n.s.), respectively. The titers of neutralizing antibodies increased by 22% (n.s.) with
Cd treatment. However, the activity of spleen T lymphocytes and spontaneous cell-mediated cytotoxicity
(NK-cell) was unchanged. Thymus weight and WBC count in peripheral blood tended to decrease. Thus, Cd
exposure seems to result in a decreased maturation and mobilization of T and B lymphocytes, but increased
humoral immune host responses.
 Relationship between Virus and Metals

Toxicology. 1992;71(3):193-202

Altered distribution of 109cadmium in mice during viral infection.

Ilback NG, Fohlman J, Friman G, Glynn AW.

Toxicology Laboratory, National Food Administration, Uppsala, Sweden.

The distribution of the toxic heavy metal cadmium (Cd) was studied in Coxsackie virus B3 (CB3)-infected
Balb/c mice by whole-body autoradiography and gamma-counting. The distribution of 109Cd was studied 4
days post CB3-inoculation and 10 min after intravenous injection of 0.21 microgram of Cd/kg body weight.
Whole-body autoradiography results showed that the distribution of 109Cd is greatly changed during this
viral infection. This newly discovered distribution was mainly visible as a greatly increased accumulation in
the renal and adrenal cortices. After impulse counting of selected organs it was found that the normal
accumulation of 109Cd in the kidneys (184,354 +/- 30,961 c.p.m.) was increased by 47% (P less than 0.05)
during CB3 infection (270,503 +/- 54,780 c.p.m.). In contrast to healthy animals, some infected mice
showed accumulation of 109Cd in the spleen. These results show for the first time that an invading micro-
organism can change the distribution of an environmental pollutant.
 Relationship between Virus and Metals
Biol Trace Elem Res. 2000 Winter;78(1-3):131-47

Trace element distribution in heart tissue sections studied by nuclear microscopy is

changed in Coxsackie virus B3 myocarditis in methyl mercury-exposed mice.
Ilback NG, Lindh U, Wesslen L, Fohlman J, Friman G.
Toxicology Division, National Food Administration, Uppsala, Sweden.

Methyl mercury (MeHg) has been shown to change Coxsackie virus type B3 (CB3) myocarditis in a direction
compatible with the development of chronic disease. Murine models of CB3 myocarditis closely mimic the
pathogenesis in humans. There are also indications that metals, such as mercury, and trace elements may
interact and adversely affect viral replication and development of inflammatory lesions. The effects of low-dose
MeHg exposure on myocardial trace element distribution, as determined by means of nuclear microscopy, was
studied in CB3 myocarditis. Balb/c mice were fed a MeHg-containing diet (3.9 mg/kg diet) for 12 wk prior to
infection. Areas of inflammatory lesions in the myocardium were identified by traditional histologic examination,
and serial tissue sections in these selected areas were used for immune histology (macrophages), in situ
hybridization of virus genomes, and nuclear microscopy of tissue trace element distribution. Areas with no
inflammation or virus were compared with areas of ongoing inflammation and viral replication. In the
inflammatory lesions of MeHg-exposed mice as compared to nonexposed mice, the myocardial contents of
calcium (Ca), manganese (Mn), and iron (Fe) were significantly increased, whereas the zinc (Zn) content was
decreased. The increased Ca and decreased Zn contents in the inflamed heart may partly explain a more
severe disease in MeHg-exposed individuals. Although not significant in the present study, with a limited
number of mice, the inflammatory and necrotic lesions in the ventricular myocardium on d 7 of the infection was
increased by 50% (from 2.2% to 3.3% of the tissue section area) in MeHg-exposed mice and, also, there was a
tendency of increased persistence of virus with MeHg exposure. No increased MeHg uptake, either in the
inflammatory lesions or in the areas of noninflamed heart tissue in infected mice, could be detected. The present
results indicate that a "competition" exists between potentially toxic heavy metals from the environment/diet and
important trace elements in the body and that a disturbed trace element balance adversely influences the
development of pathophysiologic changes in inflammatory heart disease.
 Relationship between Virus and Metals

Pancreas. 2003 Mar;26(2):190-6

Trace element changes in the pancreas during viral infection in mice.

Ilback NG, Benyamin G, Lindh U, Fohlman J, Friman G.

Section of Infectious Diseases, Department of Medical Sciences, Uppsala University, Sweden.

The trigger for some cases of juvenile diabetes has been suggested to be an interaction between a virus and
various trace elements. Infection with human coxsackievirus B3 (CB3) in the murine model results in viral
replication and inflammation in the pancreas. AIM: To determine how infection affects the trace element
balance in the pancreas. METHODOLOGY: Concentrations of the following trace elements were measured in
the serum and pancreas during the early phase (days 1 and 3) of CB3 infection in female Balb/c mice:
aluminium, arsenic, cadmium (Cd), calcium (Ca), cobalt (Co), copper (Cu), iron (Fe), lead (Pb), magnesium
(Mg), manganese (Mn), mercury (Hg), selenium, silver, vanadium (V), and zinc (Zn). The trace element
concentrations were measured through inductively coupled plasma mass spectrometry. The histopathology
was established by hematoxylin-eosin techniques and immunohistochemical staining of both CD4 and CD8
cells of the pancreas. RESULTS: Infected mice developed expected clinical signs of disease. The only
changes at day 1 occurred in the serum, with a pronounced decrease in the Zn concentration and a small
increase in the V concentration. At day 3, concentrations of several trace elements, including Cu, Zn, Fe, Ca,
V, and Mn, showed pronounced changes in both the serum and the pancreas. Ca, Cu, Mg, Mn, and V, but
none of the potentially toxic elements, accumulated in the pancreas. Cu and V concentrations increased in
the serum as well. CONCLUSION: Several trace element changes, preceding the development of
pancreatitis, occurred in the pancreas in this viral infection, the exact pathogenic interpretation of which
warrants further studies.
 Relationship between Virus and Metals

Biometals. 2000 Dec;13(4):361-7

Relation between trace element levels in plasma and myocardium during

coxsackievirus B3 myocarditis in the mouse.

Funseth E, Lindh U, Friman G, Ilback NG.

Department of Medical Sciences, Uppsala University Hospital, Sweden.

During most infections the plasma levels of trace elements change, but it is not clear if this reflects changes
in the infected tissues. Coxsackievirus B3 (CB3) infection may result in viral replication, subsequent
inflammation and changed trace element levels in the myocardium. In the present study, the trace element
levels in the plasma and heart of adult male A/J mice were determined during the pre-inflammatory stage
(day 4) of CB3 myocarditis for the following trace elements: aluminium (Al), arsenic (As), calcium (Ca),
cobalt (Co), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn), selenium (Se), silver (Ag), vanadium
(V) and zinc (Zn). The severity of the infection was assessed through clinical signs of disease and trace
element levels were measured through inductively-coupled plasma mass-spectrometry (ICP-MS). In the
heart, the levels decreased for V (59%; p < 0.01), Co (38%; p < 0.01), Al (81%; p < 0.01), As (66%; p < 0.01)
and Se (16%; p < 0.01). Increased levels were detected for Mn (13%; p < 0.05), Fe (48%; p < 0.01), Cu
(34%; p < 0.01) and Ag (46%; p < 0.01). In the plasma, decreases were detected in the level of Zn (32%; p <
0.05), whereas increases were seen in Mn (362%; p < 0.05), Fe (272%; p < 0.01), Co (71%; p < 0.05), Cu
(25%; n.s.) and Mg (43%; p < 0.01) levels. A correlation was found between the levels in plasma and
myocardium for Co (r(s) = -0.636; p < 0.05), Fe (r(s) = 0.764; p < 0.05), Mn (r(s) = 0.682; p < 0.05) and Mg
(r(s) = -0.791; p < 0.05). Thus, determination of some of these trace elements in the plasma may be useful
to indicate target tissue involvement in the early pre- inflammatory stage of an infectious disease. Some of
these elements are important nutrients for the immune system, while others may be associated with the
development of disease complications, such as cardiac arrhythmias.
 Relationship between Virus and Metals

Chem Biol Interact. 1998 May 1;113(1):79-89

The intestinal absorption of cadmium increases during a common viral

infection (coxsackie virus B3) in mice.

Glynn AW, Lind Y, Funseth E, Ilback NG.

Toxicology Division, National Food Administration, Uppsala, Sweden.

Murine intestinal absorption, tissue accumulation and redistribution of 109Cd during infection were studied
using the common human virus Coxsackie virus B3 (CB3) adapted to the mouse. Female Balb/c mice
were infected with CB3 and, on day 4 of the infection, dosed orally with 0.3 or 750 microgram Cd/kg body
weight, with 109Cd as a tracer, in order to study intestinal absorption and tissue distribution of Cd during
infection (Experiment 1). Other mice were dosed with 0.3 microgram Cd/kg body weight 3 days before
being infected and, on day 4 of the infection, Cd redistribution was studied (Experiment 2). In both
experiments non-infected control animals received the same treatment as infected animals. Results
showed that the infected animals had a higher gastrointestinal absorption of Cd than noninfected animals
when Cd was administered during infection. In the infected animals the absorption at the low Cd dosage
was increased by 70% and was tripled at the high dosage. The increased absorption enhanced the
accumulation of Cd in all organs studied. Moreover, the infection caused a Cd dose-dependent change in
the organ distribution of Cd, when Cd was administered during the infection. However, no redistribution of
previously accumulated Cd occurred during ongoing disease, indicating that Cd was not mobilised from
body stores by the infection. These results show, for the first time, that an invading micro-organism can
increase the intestinal absorption and concomitantly alter the tissue distribution of an environmental
pollutant (Cd) if exposure occurs during the course of viral infection.
Ability of viruses to trigger
host metallothionein
synthesis
 Virus Induced Metallothionein Synthesis

Toxicology. 2004 Jul 1;199(2-3):241-50

Metallothionein is induced and trace element balance changed in target
organs of a common viral infection.

Ilback NG, Glynn AW, Wikberg L, Netzel E, Lindh U.

Toxicology Division, Swedish National Food Administration, P.O. Box 622, Uppsala S-751 26, Sweden. nils-
gunnar.ilback@slv.se

In experimental studies on the common human coxsackievirus B type 3 (CB3) infection, administered
cadmium (Cd) is known to accumulate in the liver and kidneys. CB3 adapted to Balb/c mice was used to
study whether infection affects the Cd-binding protein, metallothionein (MT) and if this alters the normal
physiological trace element balance in the liver, kidney, spleen and brain. On day 3 of infection, degradation
of liver proteins (44%, P<0.01) occurred, whereas in the spleen, protein increased (63%, P<0.05). The
infection increased MT five-fold (P<0.01) in liver and kidneys, and in spleen by 34% (P<0.05). A redistribution
of Cd and copper (Cu) from the liver to the kidney was associated with this increase in MT, resulting in an
increased (P<0.01) kidney/liver ratio for both elements. The infection increased the zinc (Zn) concentration
more in the kidney than in the liver, but the kidney/liver ratio was not significantly affected. Results show that
MT is increased in several organs during the early phase of infection and is associated with redistribution of
both essential and non-essential trace elements. This may be a normal response in common infections that
could adversely influence the pathogenesis when the host is concomitantly exposed to potentially toxic trace
elements, even at levels in the physiological range.
 Virus Induced Metallothionein Synthesis
Sci Total Environ. 2002 Feb 4;284(1-3):37-47
Effects of coxsackievirus B3 infection on the acute-phase protein metallothionein
and on cytochrome P-4501A1 involved in the detoxification processes of TCDD in
the mouse.
Funseth E, Pahlman M, Eloranta ML, Friman G, Ilback NG.

During acute infections, the synthesis of acute-phase proteins and other proteins participating in the host
defence are stimulated in the liver and kidney. In previous studies of coxsackievirus B3 (CB3) infection in
mice, we found that cadmium (Cd) accumulates in the kidney, whereas 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD) accumulates in the liver. To study if CB3 infection affects the synthesis of the Cd-binding protein
metallothionein (MT) and the TCDD-binding/detoxifying cytochrome P-450 (CYP-450) isozyme CYP1A1, the
basal and TCDD-induced levels of serum MT and liver CYP1A1 isozyme were determined in healthy and
CB3-infected A/J mice. Furthermore, because interferons affect CYP450 activity, the serum levels of the
interferons alpha (IFN-alpha) and -beta (IFN-beta) were measured in CB3-infected mice and in mice treated
with the interferon-inducer polyinosinic/polycytidylic acid (poly I/C). Virus or poly I/C was administered
intraperitoneally (i.p.) on day 0 and 500 ng TCDD/kg bodyweight on day 1. On day 4, CB3 infection had
induced MT approximately 10-fold, regardless of TCDD treatment (P < 0.01 in infected mice and P < 0.001
in infected, TCDD-treated mice). TCDD alone induced a 10-fold increase in CYP1A1 activity (P < 0.001),
whereas infection alone suppressed the normal CYP1A1 activity by 75% (P < 0.001). Infection also
suppressed the TCDD-induced CYP1A1 activity by approximately 30% (n.s.). Poly I/C suppressed CYP1A1
by 20-25% (n.s.) at both basal and TCDD-induced levels. Serum IFN-alpha and IFN-beta levels were
undetectable in controls, in TCDD-treated and in the poly I/C-treated groups on day 4, probably because the
short IFN peak is detectable only hours after injection. Conversely, on day 4 of the infection, IFN-alpha and
IFN-beta were consistently raised in the TCDD-treated infected mice, whereas increased IFNs as a result of
infection alone could be detected in only one individual. These results suggest that the normal host
responses during acute infections down-regulate detoxifying processes in favour of acute-phase protein
synthesis. This may explain the observed changed pattern of accumulation, excretion and toxicity of the
environmental pollutants cadmium and TCDD during this common virus infection.
 Virus Induced Metallothionein Synthesis
Mol Cell Biol. 2001 Dec;21(24):8301-17
Influenza virus infection induces metallothionein gene expression in the
mouse liver and lung by overlapping but distinct molecular mechanisms.

Ghoshal K, Majumder S, Zhu Q, Hunzeker J, Datta J, Shah M, Sheridan JF, Jacob ST.
Department of Molecular and Cellular Biochemistry, College of Medicine, The Ohio State University, 333 Hamilton Hall, 1645
Neil Ave., Columbus, OH 43210, USA.

Metallothionein I (MT-I) and MT-II have been implicated in the protection of cells against reactive oxygen
species (ROS), heavy metals, and a variety of pathological and environmental stressors. Here, we show a
robust increase in MT-I/MT-II mRNA level and MT proteins in the livers and lungs of C57BL/6 mice exposed
to the influenza A/PR8 virus that infects the upper respiratory tract and lungs. Interleukin-6 (IL-6) had a
pronounced effect on the induction of these genes in the liver but not the lung. Treatment of the animals with
RU-486, a glucocorticoid receptor antagonist, inhibited induction of MT-I/MT-II in both liver and lung,
revealing a direct role of glucocorticoid that is increased upon infection in this induction process. In vivo
genomic footprinting (IVGF) analysis demonstrated involvement of almost all metal response elements, major
late transcription factor/antioxidant response element (MLTF/ARE), the STAT3 binding site on the MT-I
upstream promoter, and the glucocorticoid responsive element (GRE1), located upstream of the MT-II gene,
in the induction process in the liver and lung. In the lung, inducible footprinting was also identified at a unique
gamma interferon (IFN-gamma) response element (gamma-IRE) and at Sp1 sites. The mobility shift analysis
showed activation of STAT3 and the glucocorticoid receptor in the liver and lung nuclear extracts, which was
consistent with the IVGF data. Analysis of the newly synthesized mRNA for cytokines in the infected lung by
real-time PCR showed a robust increase in the levels of IL-10 and IFN-gamma mRNA that can activate
STAT3 and STAT1, respectively. A STAT1-containing complex that binds to the gamma-IRE in vitro was
activated in the infected lung. No major change in MLTF/ARE DNA binding activity in the liver and lung
occurred after infection. These results have demonstrated that MT-I and MT-II can be induced robustly in the
liver and lung following experimental influenza virus infection by overlapping but distinct molecular
mechanisms.
 Virus Induced Metallothionein Synthesis

J Virol. 2001 May;75(9):4321-31

Global impact of influenza virus on cellular pathways is mediated by both
replication-dependent and -independent events.

Geiss GK, An MC, Bumgarner RE, Hammersmark E, Cunningham D, Katze MG.

Department of Microbiology, School of Medicine, University of Washington, Seattle 98195, USA.

Influenza virus, the causative agent of the common flu, is a worldwide health problem with significant
economic consequences. Studies of influenza virus biology have revealed elaborate mechanisms by which
the virus interacts with its host cell as it inhibits the synthesis of cellular proteins, evades the innate antiviral
response, and facilitates production of viral RNAs and proteins. With the advent of DNA array technology it is
now possible to obtain a large-scale view of how viruses alter the environment within the host cell. In this
study, the cellular response to influenza virus infection was examined by monitoring the steady-state mRNA
levels for over 4,600 cellular genes. Infections with active and inactivated influenza viruses identified changes
in cellular gene expression that were dependent on or independent of viral replication, respectively. Viral
replication resulted in the downregulation of many cellular mRNAs, and the effect was enhanced with time
postinfection. Interestingly, several genes involved in protein synthesis, transcriptional regulation, and
cytokine signaling were induced by influenza virus replication, suggesting that some may play essential or
accessory roles in the viral life cycle or the host cell's stress response. The gene expression pattern induced
by inactivated viruses revealed induction of the cellular metallothionein genes that may represent a protective
response to virus-induced oxidative stress. Genome-scale analyses of virus infections will help us to
understand the complexities of virus-host interactions and may lead to the discovery of novel drug targets or
antiviral therapies.
 Virus Induced Metallothionein Synthesis

Hepatology. 2002 May;35(5):1237-46

Cellular response to conditional expression of hepatitis C virus core protein
in Huh7 cultured human hepatoma cells.

Li K, Prow T, Lemon SM, Beard MR.

Department of Microbiology and Immunology, The University of Texas Medical Branch at Galveston, Galveston, TX 77555-
0133, USA.

Data suggesting that the hepatitis C virus (HCV) core protein influences normal cellular processes remain
controversial. To determine the effects of core on cellular gene expression in hepatocytes, we developed a
human hepatoma (Huh7)-derived cell line with tightly regulated core expression under the control of a
tetracycline-regulated promoter. Cells expressing core did not have impaired proliferative abilities.
Changes in gene expression profiles in response to core expression were determined using commercial
oligonucleotide microarrays (Affymetrix GeneChip). Significant increases were observed in the abundance
of mRNA-encoding members of the metallothionein (MT) family, as well as nicotinamide N-
methyltransferase (NNMT) and glutathione peroxidase-like protein (GPLP). These changes did not result
from removal of tetracycline from growth media, and were confirmed in reverse-transcription polymerase
chain reaction (RT-PCR) assays. They suggest that core protein expression leads to intracellular oxidative
stress, and that vital cellular functions are, in turn, protected by up-regulation of cellular antioxidant
defense mechanisms. In conclusion, these findings can explain many potentially conflicting prior
observations concerning the effects of core on cellular physiology, and are of relevance to the role of core
protein in the pathogenesis of HCV-related fibrosis and hepatocellular carcinoma.
MICROBES:bacteria
Receptor:
• Strep
• Measles
• Herpes
Binding of aluminium ions by
Staphylococcus aureus 893.
Bradley TJ, Parker MS.
Experientia. 1968 Nov 15;24(11):1175-6.
Aluminum interferes
with glutamate
dehydrogenase
 Mercury inhibits
glutamine synthase
X
X
X
Glutamate pathway
“In the absence of glutamate, neurons are unaffected by
acute exposure to mercury, suggesting that neuronal
dysfunction is secondary to disturbances in astrocytes.”

“Coapplication of nontoxic concentrations of MeHG and

glutamate leads to the typical appearance of neuronal
lesions associated with excitotxin stimulation”

Brookes, 1992/Matyja and Albrecht, 1993

Aluminum inhibits the
activity of DHPR
resulting in decreased
BH4
Toxicol In Vitro. 2003 Oct-Dec;17(5-6):533-7.
Altindag ZZ, Baydar T, Engin AB, Sahin G.
Effects of the metals on dihydropteridine reductase activity.

Metals are the oldest toxins known to human. Particularly, occupational and
environmental exposure to aluminium, lead, mercury, cadmium, and manganese
cause serious health problems by interaction with biological systems. Cellular
targets of these metals are mostly specific biochemical processes (enzymes) and/or
membranes of cells and organelles. To prevent and/or reduce the untoward or
irreversible toxic effects of the metals by using biomarkers are as important as to
know and to understand of their toxicity mechanisms. Dihydropteridine reductase
(DHPR), which possessed essential thiol groups at the activity site, plays a crucial
role in the maintenance of tetrahydrobiopterin (BH4). BH4 is the cofactor in the
synthesis and regulation of neurotransmitters. A limited number of the evidences
have shown that DHPR may be a target for the metals. Therefore, the present study
was designed to assess possible in vitro effects of the commonly exposed metals
on the enzyme activity. It was found that aluminium, cadmium, mercury, di-phenyl
mercury, lead, diethyl lead, in chloride forms, and manganese, in sulphate form, led
to statistically significant decreases in DHPR activity, in a concentration-dependent
manner, in vitro.
N Engl J Med. 1987 Jul 9;317(2):80-4.
Altmann P, Al-Salihi F, Butter K, Cutler P, Blair J, Leeming R, Cunningham J, Marsh F
Serum aluminum levels and erythrocyte dihydropteridine reductase activity in
patients on hemodialysis.

Aluminum intoxication due to aluminum-containing antacids or dialysate can cause

encephalopathy in patients undergoing hemodialysis, but the biochemical mechanism
has not been defined. The enzyme dihydropteridine reductase (DHPR) is essential for
the maintenance of normal brain concentrations of tetrahydrobiopterin, which is itself
required for the synthesis of specific neurotransmitters. This enzyme is also present in
erythrocytes. We measured erythrocyte DHPR activity and concentrations of the
biopterin derivatives of its substrate and of aluminum in 38 patients on hemodialysis
who had no clinical evidence of encephalopathy. Serum aluminum levels ranged from
15 to 190 micrograms per liter (mean, 67.6 +/- 7.7) as compared with 4.9 +/- 0.99
micrograms per liter in normal subjects. DHPR activity was inversely related to the
serum aluminum concentration (r = -0.61, P less than 0.001) and was less than the
activity predicted from the hemoglobin concentration in these patients. Serum
concentrations of biopterin derivatives were markedly elevated. Eighteen patients were
given the aluminum-chelating agent deferoxamine in a single dose, after which DHPR
activity doubled. These studies suggest that aluminum inhibits DHPR activity in
erythrocytes and that aluminum chelation reverses this effect. Although we did not
directly measure DHPR activity in the brains of dialysis patients without
encephalopathy, we propose that the reduction in activity in erythrocytes may reflect a
similar reduction in the brain. Our findings could help to explain the encephalopathy
associated with aluminum intoxication.
Aluminum inhibits the
activity of
acetylcholinesterase
 Acetylcholinesterase Inhibition and
Neurological Inflammation

• Aluminum Alzheimers
• Organophosphates Parkinson’s
• Pyridostigmine ALS/Gulf War
Syndrome
• Aluminum Autism
Tetanus Toxin
 Acetylcholinesterase Inhibition

Muscarinic Stimulation Nicotinic Stimulation

• Pinpoint pupils
• Blurred vision • Muscle twitching
• Hypersecretion • Muscle weakness
• Bladder incontinence • Dilated pupils
 The Autonomic Nervous System
Structure Sympathetic Stimulation Parasympathetic Stimulation
Iris (eye muscle) Pupil dilation Pupil constriction
Salivary Glands Saliva production reduced Saliva production increased
Oral/Nasal Mucus production
Mucus production increased
Mucosa reduced
Heart rate and force
Heart Heart rate and force decreased
increased
Lung Bronchial muscle relaxed Bronchial muscle contracted
Gastric juice secreted; motility
Stomach Peristalsis reduced
increased
Small Intestine Motility reduced Digestion increased
Large Intestine Motility reduced Secretions and motility increased
Increased conversion of
Liver
glycogen to glucose
Kidney Decreased urine secretion Increased urine secretion
Norepinephrine and
Adrenal medulla
epinephrine secreted
Wall relaxed Wall contracted
Bladder
Sphincter closed Sphincter relaxed
• Magnesium will help protect against
aluminum effects on cellular
respiration

• Malic acid

• EDTA
METALS
 Consequences of
Increased Metal Burden

TNF alpha
Glutathione
Myelination
Memory
Cognition
Bridging of GSH :
GSH affect T cell activation
GSSH inhibits methionine synthase in E.coli (Hondrop, PLOS Bio. Nov.2004)
 Metals myelination

methylation is necessary for myelination

Metals myelination remyelination

lack of methylation
methylation and metals myelination

Without myelin, axons will continue

to grow and pruning will be
compromised.
 Metals Myelination

Myelination ensures the rapid conduction of

electrical impulses in the nervous system.
Neuregulins Myelin wrapping

Neuregulin is the signal for the regulation of

myelin sheath thickness

Neuregulin also induces GABA receptors

Constant activation of neuregulin

may cause demyelination
Proper myelination requires proper pruning
methylation and metals myelination

Without myelin, axons will continue

to grow and pruning will be
compromised.
 Glial Cells (affected by Ammonia) relationship to Pruning
Science News: April 22, 2004
Glia and neurons collaborate to degrade unwanted axons during brain development | By Helen Dell

Glial cells, whose function is poorly understood, seem to cooperate actively with neurons to sculpt neural circuits in the developing
brain, according to two studies published in the April 20 Current Biology.
During development, the controlled and programmed death of specific cells is as important as the survival of others; for example, the
webbing on embryonic hands degenerates to fashion fingers from a clump of tissue, according to V. Hugh Perry, professor of
experimental neuropathology at the University of Southampton, who was not involved in the studies. The brain is no exception. Brain
development involves the production of excess neurons and axonal branches, which then die back in a process dubbed “pruning.”
Both papers study the remodeling of neural networks that occurs during the metamorphosis of Drosophila, where unwanted larval
axons degrade to form adult neurons. The first paper, from Liqun Luo's lab at Stanford University, is “a particularly beautiful
ultrastructural study,” according to an accompanying commentary by Kendal Broadie, of the Department of Biological Science at
Vanderbilt University. The second paper, from Takeshi Awasaki and Kei Ito at the University of Tokyo, provides experimental
evidence of the importance of glia in the pruning process.
Luo and colleagues used a genetically encoded marker to highlight different cell types—neurons or glia—so they could watch the
pruning process using electron microscopy. They observed that during pruning, glia invade the surrounding area and absorb the
degenerating axons. They also found evidence that the glia might have an active role in the process, rather than just cleaning up
after axonal breakdown, as some features of the glial invasion happen independently of the axonal degeneration.
Awasaki and Ito adopted a different methodology, first examining the morphology of the axon branches during pruning. They
observed hole-like structures among the branches and suggest that each “hole” corresponds to a clump of glia infiltrating from
outside the bundle.
“This finding was very unexpected and was a breakthrough of this study,” said Awasaki. Inhibiting cellular functions in the glia also
inhibited glial infiltration into the axonal bundles and suppressed axonal pruning, suggesting that the glia actively break down axons
rather than just scavenge debris.
“Taken together, these papers suggest that glia definitely play an active role in engulfing the [axonal] fragments,” Luo told The
Scientist. However, whether the glia are actually regulating the process remains to be seen, he said.
“As yet, we just don't have a biology of synapse and axon degeneration,” said Perry. “So this is a very interesting subject.”
“These new papers are the first to report experimental evidence for the importance of neuron–glia interaction, and the indispensable
role of glia during developmentally regulated axon pruning,” wrote Broadie.
“The molecules required for the communication between degenerating axons and phagocytosing glia will be the next big prize,”
Broadie concluded
 5 amino
Glutamic acid heme
levulinic acid

Inhibited
by lead

red blood cells cytochromes

PBG Synthase (Porphobilinogen Synthase), also called ALA Dehydratase,
catalyzes condensation of two molecules of d-aminolevulinic acid (ALA) to
form porphobilinogen (PBG).
The binding sites for Zn++ in the homo-
octomeric mammalian Porphobilinogen
Synthase, which include cysteine S
ligands, can also bind Pb++ (lead).
Inhibition of Porphobilinogen Synthase
by Pb++ results in elevated blood
ALA, which may cause some of the
neurological effects of lead poisoning.
ALA (d-aminolevulinate) is toxic to the
brain. This may be due in part to the
fact that ALA is somewhat similar in
structure to the neurotransmitter GABA
(g-aminobutyric acid). In addition,
reactive oxygen species (oxygen
radicals) are generated during
autoxidation of ALA.
Uroporphyrinogen III is the precursor for synthesis of vitamin B12, chlorophyll,
and heme, in organisms that produce these compounds.
 CH3

CH3 HC S CH2 protein

N
H3C CH3
N Fe N
− protein
OOC CH2 CH2 CH S CH2
N
CH3

CH2 CH3

CH2

COO− Heme c

Heme is the prosthetic group of hemoglobin,

myoglobin, & cytochromes.
Figure 9
This is a schematic diagram illustrating the transfer of electrons from NADH, through the electron carriers in the electron transport
chain, to molecular oxygen. Please click on the pink button below to view a QuickTime animation of the functions of the proteins

embedded in the inner mitochondrial membrane that are necessary for oxidative phosphorylation.
Haem proteins by function

Function Protein class / family

Catalysis Oxidoreductases
•Haem, haem-thiolate, and sirohaem •Catalases
enzymes in ENZYME database •Haem-thiolate
•Haem enzymes in LIGAND database •Peroxidases
Electron transfer •Cytochromes
Oxygen transport and storage •Globins

Nitric oxide transport

•Nitrophorin
NOS heme groups
Animal haem peroxidases in enzyme databases

Alternative
ENZYME LIGAND BRENDA Official name
names
Eosinophil
peroxidase;
lactoperoxida
1.11.1.7 1.11.1.7 1.11.1.7 Peroxidase
se;
myeloperoxid
ase
Iodinase;
iodotyrosine
Iodide
1.11.1.8 1.11.1.8 1.11.1.8 deiodase;
peroxidase
thyroid
peroxidase
Cyclooxygena
se;
Prostaglandin
prostaglandin
1.14.99.1 1.14.99.1 1.14.99.1 -endoperoxide
G/H synthase;
synthase
prostaglandin
synthase
 IUBMB Enzyme Nomenclature
EC 1.11.1.8
Common name: iodide peroxidase

Reaction:
iodide + H2O2 = iodine + 2 H2O
Other name(s): iodotyrosine deiodase; iodinase; iodoperoxidase
(heme type); thyroid peroxidase; iodide peroxidase-tyrosine
iodinase; iodotyrosine deiodinase; monoiodotyrosine deiodinase;
thyroperoxidase; tyrosine iodinase
Systematic name: iodide:hydrogen-peroxide oxidoreductase
METHYLATION
 Methylation
• Importance of methylation
• Pathway
• What we can do about it
 Methylation
Methyl groups – CH3

H C

Bound to DNA, enzymes, vitamins

 In many cancer cells, inappropriate
methylation – the attachment of methyl (CH3)
groups to DNA’s cytosine bases – can silence
genes that supress tumor growth.
Effect of methylation on
Metallothionein genes
“Differences in methylation may
account for the differential
susceptibility of tissues to
cadmium toxicity”
 Methylation is related to neurotransmitter levels; methylation of intermediates in
tryptophan metabolism can affect the levels of serotonin. Intermediates of the
methylation pathway are also shared with the pathway involved in dopamine
synthesis. Consequently, imbalances in the methylation pathway will also affect the
neurotransmitter dopamine. In addition to its direct role as a neurotransmitter,
dopamine is involved in methylating phospholipids in the cell membranes.
Membrane fluidity is important for a variety of reasons including proper signaling of
the immune system as well as protecting nerves from damage. The building blocks
for DNA and RNA require the methylation pathway. Without adequate DNA and
RNA it is difficult for the body to synthesize new cells. This would result in a
decreased level of new T cell synthesis. De novo T cell synthesis is necessary to
respond to viral infection, as well as for other aspects of the proper functioning of
the immune system. T cells are necessary for antibody producing cells in the body
(B cells) as both T helpers and T suppressors to appropriately regulate the
antibody response. In addition, the decreased level of methylation can result in
improper DNA regulation. DNA methylation is necessary to prevent the expression
of viral genes that have been inserted into the bodys DNA. Loss of methylation can
lead to the expression of inserted viral genes. Proper levels of methylation are also
directly related to the body?s ability to both myelinate nerves and to prune nerves.
Myelin is a sheath that wraps around the neuronal wiring to insulate and facilitate
faster transmission of electrical potentials.
Without adequate methylation, the nerves cannot myelinate in the first place, or cannot
remyelinate after insults such as viral infection or heavy metal toxicity. A secondary
effect of a lack of methylation and hence decreased myelination is inadequate
?pruning? of nerves. Pruning helps to prevent excessive wiring, or unused neural
connections and reduces the synaptic density. Without adequate pruning the brain cell
connections are misdirected and proliferate into dense, bunched thickets.All of these
changes, when they occur in utero or in very young children, can alter brain
development, and can also set up metabolic changes that cause ongoing compromise
of brain function.
The metabolically caused changes in brain function can, however, be treated if
problems driving these metabolic changes are treated and corrected. This would
suggest that autism is a medical condition associated with a defect in the methylation
pathway, with brain and behavior changes that lead to observable autistic behaviors
occurring as a downstream consequence of these medical abnormalities. and that it
should be characterized as such. An analogy can be made to the medical condition of
cardiovascular disease. There too, a mutation in the MTHFr C677T allele establishes
sets up an individual to be a genetically susceptible individual. The combination of
infectious agents (IOM Report June 2004), the inflammatory cascade that is triggered
as a result of infection, along with this genetic mutation and other environmental
stressors leads to cardiovascular disease in individuals with the appropriate genetic
background. Continuing with this analogy, perhaps autism should be reclassified as a
defect in methylation that results in neuroinflammatory disease.
Dr. Amy Yasko Neurological Research Institute 2004
 Methylation
• DNA synthesis
• Megaloblastic anemia

• T cells

• Intestinal mucosa

• Involved in DNA regulation

• Host

• Viral

• Myelination and pruning

• Proper immune response to i.e. TB

• Membrane fluidity, phospholipid methylation

• Enzymatic reactions requiring methylation

• Melatonin

• Neurotransmitter levels : dopamine,norepinephrine

• Tryptophan methylation: serotonin,

 Methylation-dependent T cell immunity to Mycobacterium
tuberculosis heparin-binding hemagglutinin.
Temmerman S, Pethe K, Parra M, Alonso S, Rouanet C, Pickett T, Drowart A, Debrie AS, Delogu G, Menozzi
FD, Sergheraert C, Brennan MJ, Mascart F, Locht C.
Nat Med. 2004 Sep;10(9):935-41. Epub 2004 Aug 08.

• Methylation of the Mycobacterium tuberculosis

heparin-binding hemagglutinin (HBHA) by the
bacterium is essential for effective T cell
immunity to this antigen in infected healthy
humans and in mice.

• Post-translational modifications of proteins may

be crucial for their ability to induce protective T
cell-mediated immunity against infectious
diseases such as tuberculosis.
Methylation in Arsenic Detoxification
Arsenite methylation by methylvitamin B12 and glutathione
does not require an enzyme.
Toxicol Appl Pharmacol. 1999 Feb 1;154(3):287-91.
Zakharyan RA, Aposhian HV.
Department of Molecular and Cellular Biology, The University of Arizona, Tucson, Arizona, 85721-0106, USA.

Although inorganic arsenic is methylated enzymatically by arsenic methyltransferases, which

have been found in many mammalian livers, the detection of such enzymes has not been
successful in surgically removed human livers. Results of the present experiments
demonstrated that methylvitamin B12 (methylcobalamin, CH3B12) in the presence of thiols
and inorganic arsenite can produce, in vitro, substantial amounts of monomethylarsonic acid
(MMA) and small amounts of dimethylarsinic acid (DMA) in the absence of enzymes.
Furthermore, this nonenzymatic methylation of inorganic arsenite by CH3B12 was increased
substantially by the presence of dimercaptopropanesulfonate (DMPS) and/or sodium selenite.
The actions of DMPS and selenite together were additive. The methylation by CH3B12 was
neither inhibited nor stimulated by human liver cytosol. Since the amount of MMA produced
by the in vitro system described in this study was not small, these results emphasize the need
for a properly designed nutritional study in humans exposed to inorganic arsenic as to the
relationship between vitamin B12, selenium, and the metabolism of carcinogenic inorganic
arsenic. Copyright 1999 Academic Press.
 Methylation is related to neurotransmitter levels; methylation of
intermediates in tryptophan metabolism can affect the levels of
serotonin. Intermediates of the methylation pathway are also shared
with the pathway involved in dopamine synthesis. Consequently,
imbalances in the methylation pathway will also affect the
neurotransmitter dopamine. In addition to its direct role as a
neurotransmitter, dopamine is involved in methylating phospholipids
in the cell membranes. Membrane fluidity is important for a variety of
reasons including proper signaling of the immune system as well as
protecting nerves from damage. The building blocks for DNA and
RNA require the methylation pathway. Without adequate DNA and
RNA it is difficult for the body to synthesize new cells. This would
result in a decreased level of new T cell synthesis. De novo T cell
synthesis is necessary to respond to viral infection, as well as for
other aspects of the proper functioning of the immune system.
 DNA Methylation
“DNA methylation may maintain the large amount of
non-coding DNA in an inert state.”

“This process would help prevent the transcription of

large parts of the genome… INSERTED VIRAL
SEQUENCES.”

“…consequences of loss of methylation… could cause

the potentially harmful expression of inserted viral
genes.”

New England Journal of Medicine, November 20, 2003

 Serotonin
5-HT; 5-hydroxytryptamine

Melatonin
N-acetyl-5-methoxytryptamine
Abbreviations: CPT I, carnitine-palmitoyl transferase I; CT,
carnitine:acylcarnitine translocase; CPT II, carnitine-
palmitoyl transferase II; CAT, carnitine-acetyl transferase;
CoA, coenzyme A.
Figure 9
This is a schematic diagram illustrating the transfer of electrons from NADH, through the electron carriers in the electron transport chain, to
molecular oxygen. Please click on the pink button below to view a QuickTime animation of the functions of the proteins embedded in the
inner mitochondrial membrane that are necessary for oxidative phosphorylation.
 Biochem Biophys Res Commun. 2004 Dec 3;325(1):
RNase-L regulates the stability of mitochondrial DNA-encoded mRNAs in mouse embryo fibroblasts.

Chandrasekaran K, Mehrabian Z, Li XL, Hassel B.

Department of Anesthesiology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.

kchandra@anethlab.umm.edu

Accelerated decrease in the levels of

mitochondrial DNA-encoded mRNA (mt-
mRNA) occurs in neuronal cells exposed
either to the excitatory amino acid,
glutamate or to the sodium ionophore,
monensin, suggesting a role of mitochondrial
RNase(s) on the stability of mt-mRNAs.
Methylation in Arsenic Detoxification
Arsenite methylation by methylvitamin B12 and glutathione
does not require an enzyme.
Toxicol Appl Pharmacol. 1999 Feb 1;154(3):287-91.
Zakharyan RA, Aposhian HV.
Department of Molecular and Cellular Biology, The University of Arizona, Tucson, Arizona, 85721-0106, USA.

Although inorganic arsenic is methylated enzymatically by arsenic methyltransferases, which

have been found in many mammalian livers, the detection of such enzymes has not been
successful in surgically removed human livers. Results of the present experiments
demonstrated that methylvitamin B12 (methylcobalamin, CH3B12) in the presence of thiols
and inorganic arsenite can produce, in vitro, substantial amounts of monomethylarsonic acid
(MMA) and small amounts of dimethylarsinic acid (DMA) in the absence of enzymes.
Furthermore, this nonenzymatic methylation of inorganic arsenite by CH3B12 was increased
substantially by the presence of dimercaptopropanesulfonate (DMPS) and/or sodium selenite.
The actions of DMPS and selenite together were additive. The methylation by CH3B12 was
neither inhibited nor stimulated by human liver cytosol. Since the amount of MMA produced
by the in vitro system described in this study was not small, these results emphasize the need
for a properly designed nutritional study in humans exposed to inorganic arsenic as to the
relationship between vitamin B12, selenium, and the metabolism of carcinogenic inorganic
arsenic. Copyright 1999 Academic Press.
Methyl-donor deficiency due to chemically
induced glutathione depletion
Cancer Research, Vol 56, Issue 5 995-1005, Copyright © 1996 by American Association for Cancer Research
K Lertratanangkoon, RS Orkiszewski and JM Scimeca
Department of Pharmacology, University of Texas Medical Branch, Galveston 77555-1031, USA.

• Dietary deficiency of methionine (Met) is known to deplete cellular

Met and cause DNA hypomethylation, but depletion of Met and
impairment in methylation due to chemically induced glutathione
(GSH) depletion has escaped recognition.

• These results provide direct evidence that depletion of GSH leads to

Met depletion and also injures the methylation processes.

• In animal models, levels of glutathione remain constant in females

who have suffered a brain injury, but drop by as much as 80 percent
in males with the same injury. When glutathione levels drop, brain
cells die much more quickly. This suggests that boys with brain
injuries may require different life-saving treatments than girls.
(Researchers Find Brain Cells Die Differently in Males and Females; Pediatric Academic
Societies Press release; 21-Apr-2004 )
Methylation Reelin

Reelin GAD enzyme

 Detoxification
• Glucuronidation
• Sulfation
• Methylation
• Catechol O methyl transferase (flavinoids)
• COMT
• Phenol O methyltransferase(phenols)
• Thiol methyltransferase (IBD)
 Supplementation with
SAMe in the Methylation
Pathway
 S-adenosyl-methionine (SAM)

Some of the important reactions in which SAM is involved are:

Methylation of DNA and RNA. DNA- and RNA-methylases use SAM
as a source of methyl groups. A major target of methylases is the 5
position of cytosine of DNA. The degree of methylation correlates
with transcriptional activity. (Globin genes, for example, are highly
methylated in non-erythroid cells but not in erythroid cells.
The conversion of epinephrine to norepinephrine is also catalyzed
by an N-methyl transferase that uses SAM. Note that because
methionine is an essential amino acid, if it is limiting, choline could
also become a nutritional requirement
SAMe is involved in the synthesis of:

• Creatine
• Methylcobalimin
• Phosphatidylcholine
• Coenzyme Q10
• Carnitine

• Methylation by SAMe is a critical step in the

stabilization of many proteins including myelin.
 Decreased methylation creates a
compromised host for the virus.

“DNA methylation may maintain the large amount of

non-coding DNA in an inert state.”

“This process would help prevent the transcription of

large parts of the genome… INSERTED VIRAL
SEQUENCES.”

“…consequences of loss of methylation… could cause

the potentially harmful expression of inserted viral
genes

New England Journal of Medicine, November 20, 2003

Superfamily: S-adenosyl-L-methionine-dependent methyltransferases Lineage:

Catechol O-methyltransferase, COMT (1)

Plant O-methyltransferase, C-terminal domain (3)
RNA methyltransferase FtsJ (1)
Fibrillarin homologue (1)
Hypothetical protein MJ0882 (1)
Hypothetical protein HI0319 (YecO) (1)
Glycine N-methyltransferase (1)
Phenylethanolamine N-methyltransferase, PNMTase (1)
Histamine methyltransferase (1)
Guanidinoacetate methyltransferase (1)
Arginine methyltransferase, HMT1 (2)
lacks the last two strands of the common fold replaced with a beta-sandwich oligomerisation
subdomain
Protein-L-isoaspartyl O-methyltransferase (3)
Glucose-inhibited division protein B (GidB) (1)
Probable methyltransferase Rv2118c (1)
contains additional N-terminal beta-sandwich domain, res. 1-67
Chemotaxis receptor methyltransferase CheR, C-terminal domain (1)
contains additional N-terminal all-alpha domain, res. 11-91
RNA methylases (4)
DNA methylases (5)
Type II DNA methylase (3)
circularly permuted version of the common fold
Spermidine synthase (1)
contains additional N-terminal tetramerisation all-beta domain, res. 1-71
Mycolic acid cyclopropane synthase (2)
SAH as Antiviral Agents
 Inhibition of mRNA methylation: an approach to specific inhibition of viral
replication.
Sharma OK, Goswami BB, Borek E.
Eukaryotic mRNAs and most viral mRNAs contain very extensive modification on the 5'
end consisting of the "cap," part of which is a guanine which is methylated in the 7-position.
It is quite well established that the methylated cap is essential for the efficient translation of
the mRNA. In a search for an effective chemotherapeutic agent, Dr. Roland Robins
synthesized the compound ribavirin; this compound turned out to be an extraordinarily
effective virostatic agent against both RNA and DNA viruses. Given the capping of the
mRNAs produced by both types of virus, and, given the structure of ribavirin, it seemed to
us that it may be fruitful to explore whether this drug might act in both cases by blocking
the capping reaction. Such a mechanism indeed turned out to be a reality. We have shown
that ribavirin triphosphate acts as a competitive inhibitor for the capping of mRNAs. We
and others have shown that uncapped mRNAs are poorly translated. An interesting
corollary confirmation of these findings is that encephalomyocarditis virus (EMC) and polio
virus generate mRNAs which are not capped, and ribavirin is innocuous to these viruses.
Another agent which acts as an inhibitor of mRNA methylation emerged from subsequent
efforts. It is known from the work of Ian Kerr that extracts of interferon treated cells in the
presence of double stranded RNA synthesize a unique 2'-5'-linked oligo (adenylic
acid) 5'-triphosphate, a small trinucleotide of unusual structure, which inhibits protein
synthesis. We have explored its effect on the methylation of mRNA and found it to be a
potent inhibitor of methylation of the cap. Thus, two agents are known which inhibit
methylation of mRNA and they may serve as prototypes for designing other such inhibitors,
with a view to specific inhibition of mRNAs foreign to the host.
Physiol. Rev. 80: 1107-1213, 2000;
 Synthesis and biological activities of
chloroethylurea, methylurea, and nitrosourea
analogues of N-deacetylmethylthiocolchicine.
Lin TS, Shiau GT, Prusoff WH, Harmon RE.
J Med Chem. 1980 Dec;23(12):1440-2.

A series of urea and nitrosourea

analogues of N-
deacetylmethylthiocolchicine (1) has been
synthesized, and their antineoplastic and
antiviral activities were evaluated.
Physiol. Rev. 80: 1107-
1213, 2000;
How Does Methylation Control
Synthesis of Proteins?
One of the ways the cells control which genetic information
they will use is to chemically modify the DNA. The illustration
shows an enzyme (diagrammed in ribbons) adding methyl
groups to some of the DNA (balls in the form of a double
helix). This inactivates that part of the chromosome. It's as if
we were to put glue on the edges of some of the books in the
library; those pages would become unavailable to readers.
 CpG methylation, chromatin structure and gene
silencing-a three-way connection.
EMBO J. 1998 Sep 1;17(17):4905-8. Razin A.

• There is a three-way connection between

DNA methylation, gene activity and
chromatin structure .
• Methylation plays a pivotal role in
establishing and maintaining an inactive
state of a gene by rendering the chromatin
structure inaccessible to the transcription
machinery.
 Targeted Methylation
Introducing methylated DNA at specific genomic
loci affects local histone acetylation.
By Jonathan Weitzman
Methylation of DNA at CpG dinucleotides represses gene transcription.
Methylation plays an important role in development, imprinting, X-
chromosome inactivation and tissue-specific gene expression, but the
mechanisms of methylation-induced repression are still unclear. In the
December Molecular and Cellular Biology, Schubeler et al. show that
localized histone deacetylation can explain methylation-induced repression
(Mol Cell Biol 2000, 20:9103-9112). The authors used an elegant
technique called recombinase-mediated cassette exchange (RMCE) to
introduce in vitro-methylated DNA at defined chromosomal positions. They
used the Cre recombinase to insert methylated or unmethylated forms of
the human β-globin gene promoter driving a green fluorescent protein
(GFP) reporter gene. Methylation repressed GFP expression, and was
stable in cells over at least 12 weeks in culture. Methylation did not affect
DNA replication or global chromatin remodeling. However, methylation
caused a hypoacetylation of histones H3 and H4 within the transgene.
These observations support a model in which methylated DNA represses

local transcription by recruiting histone deacetylase activity .

 DNA methylation in genomic imprinting,
development, and disease
The Journal of Pathology, September 2001, vol. 195, no. 1, pp. 97-110(14)
Paulsen M.[1]; Ferguson-Smith A.C.[1]*, [1]University of Cambridge, Department of Anatomy, Cambridge CB2
3DY, UK [*]University of Cambridge, Department of Anatomy, Downing Street, Cambridge CB2 3DY, UK

Changes in DNA methylation profiles are common features

of development and in a number of human diseases, such
as cancer and imprinting disorders like Beckwith–
Wiedemann and Prader–Willi/Angelman syndromes. This
suggests that DNA methylation is required for proper gene
regulation during development and in differentiated tissues
and has clinical relevance. DNA methylation is also
involved in X-chromosome inactivation and the allele-
specific silencing of imprinted genes. This review describes
possible mechanisms by which DNA methylation can
regulate gene expression, using imprinted genes as
examples. The molecular basis of methylation-mediated
gene regulation is related to changes in chromatin structure
and appears to be similar for both imprinted and biallelically
expressed genes.
 The expression of many cellular
genes is modulated by DNA
methylation and histone
acetylation. These processes can
influence malignant cell
transformation and are also
responsible for the silencing of
DNA constructs introduced into
mammalian cells for therapeutic or
research purposes.
 Targeted Methylation
Introducing methylated DNA at specific genomic loci
affects local histone acetylation.
By Jonathan Weitzman

• Methylation of DNA at CpG dinucleotides

represses gene transcription.
• Localized histone deacetylation can explain
methylation-induced repression.
(Mol Cell Biol 2000, 20:9103-9112).

• Methylation caused a hypoacetylation of

histones.
• Methylated DNA represses local transcription by
recruiting histone deacetylase activity.
 Universal lack of methylation and
inability to produce nucleic acids
necessary for RNA synthesis result in a
situation where the body is lacking the
required regulatory elements for genetic
silencing. Silencing is a multistep
process that involves RNA as well as
methylation and deacetylation of
histones.
Histone deacylation

Turn off Genes

DNA methylation
 SIR2

Histone deacylation

Turn off Genes

DNA methylation

IGF
Model of how calorie restriction may extend life span in mammals. Effects occur at
two levels: (1) sensing CR to adjust hormonal levels and (2) executing a slowing of
aging on all organs. Roles for Sir2 genes are proposed at both levels.
Family of protein deacetylases (Sirtuins) are
nicotinamide adenine dinucleotide (NAD)-
dependent enzymes that hydrolyze one
molecule of NAD for every lysine residue
that is deacetylated. The Sirtuins are
phylogenetically conserved in eukaryotes,
prokaryotes, and Archeal species.
Prokaryotic and Archeal species usually
have one or two Sirtuin homologs, whereas
eukaryotes typically have multiple versions.
The founding member of this protein family
is the Sir2 histone deacetylase of
Saccharomyces Cerevisiae.
 Sir2-dependent activation of acetyl-CoA synthetase by
deacetylation of active lysine.
Starai VJ, Celic I, Cole RN, Boeke JD, Escalante-Semerena JC.
Department of Bacteriology, University of Wisconsin, Madison, WI 53706-1567, USA.
Science. 2002 Dec 20;298(5602):2390-2.

Acetyl-coenzyme A (CoA) synthetase (Acs) is an enzyme central to

metabolism in prokaryotes and eukaryotes. Acs synthesizes acetyl
CoA from acetate, adenosine triphosphate, and CoA through an
acetyl-adenosine monophosphate (AMP) intermediate.
Immunoblotting and mass spectrometry analysis showed that
Salmonella enterica Acs enzyme activity is posttranslationally
regulated by acetylation of lysine-609. Acetylation blocks synthesis of
the adenylate intermediate but does not affect the thioester-forming
activity of the enzyme. Activation of the acetylated enzyme requires
the nicotinamide adenine dinucleotide-dependent protein deacetylase
activity of the CobB Sir2 protein from S. enterica. We propose that
acetylation modulates the activity of all the AMP-forming family of
enzymes, including nonribosomal peptide synthetases, luciferase, and
aryl- and acyl-CoA synthetases. These findings extend our knowledge
of the roles of Sir2 proteins in gene silencing, chromosome stability,
and cell aging and imply that lysine acetylation is a common
regulatory mechanism in eukaryotes and prokaryotes.
Pycnogenol
Grape Seed Extract
Alpha lipoic acid
• “It is possible that the alteration of NAD levels by
manipulation of the NAD biosynthetic pathway, Sir2
protein activity, or other downstream effectors will
provide new therapeutic opportunities for the treatment
of diseases involving axonopathy and
neurodegeneration.”

• “These findings suggest that novel therapeutic strategies

directed at increasing the supply of NAD and/or Sir2
activation may be effective for treatment of diseases
characterized by axonopathy and neurodegeneration.”
• Valproic acid and similar fatty acids
can induce inhibition of HDAC or
inhibition of gene methylation and
thereby alter actions of transcription
factors.

• Valproic acid is a histone deacetylase

inhibitor.
Awareness of Roles Played by:

• Lysine
• Nadh
• Igf
• Telomerase
• Valproic Acid
ProLongevity NutriSwitch RNA™
Female, 72

Before Prolongevity After Prolongevity

Triglycerides 425 242
Total Cholesterol 218 185
Glucose 152 69
HDL 36 36
LDL 131 101
Modifications for Mutations
Regulation
• Ubiquitination • UBE3A
• Methylation • MTHFr, MeCP2…
• Acetylation • NAT
• (Phosphorylation) • (Casein Kinase 2, milk)