211
Microbes, Metals and Methylation Dr. Amy Yasko April 2005
Microbes, Metals and Methylation
Dr. Amy YaskoApril 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 notalways reflect the actual body
burden of heavy metals, especially in the presence of
chronic infection.
DMSA Provoked UrineJune 4, 2001
NO MERCURY
DMSA Provoked UrineJuly 16,2001
NO MERCURY
DMSA Provoked UrineAugust 15, 2001
NO MERCURY
DMSA Provoked UrineFebruary 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 acidsin 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
19 20
35
0 0
180
0
120
6/17/03
6/18/03
7/3/03
7/10/03
7/17/03
7/24/03
8/1/03
8/5/03
0
50
100
150
200
Aluminum (ug/g Creatinine)
Safe Range: 60
The
Effect
of
Metal
RN
2.3 2.3
0
6.4 6.5
10
15
12
5.4 5.3
24
6.67.1
2.8
9.8
3.9
1.8
0
6/17/03
7/10/03
8/1/03
8/15/03
9/3/03
10/1/03
0
5
10
15
20
25
Mercury (ug/g Creatinine)
Safe Range: 5
0.2 0 00.8
0.40.8
0.2 0 0
6.6
0 0 0
5.7
18
0.6
3.6
0.60 0
3.3
0 0 0 0 0
13
0 0 0
1
0 0 0 0 00.4
7.4
1.71
0
2.3
0
1 1.1
8/19/039/7/03
9/28/0310/18/03
11/3/0311/14/03
11/28/0312/15/03
1/3/041/18/04
2/7/043/5/04
4/22/046/1/04
8/4/04
0
5
10
15
20
Lead (ug/g Creatinine)
Safe Range: 5
0.50
12
6.1
0.4 0.4
1 0.8
5.7
0.2 0.2 0
8.8
5.4
0.8
1.6
0.80.3
0
0.6
00.3
0
1.4
0.3 0.30
0.30 0
8/19/03
9/7/03
9/28/03
10/18/03
11/3/03
11/14/03
11/28/03
12/15/03
1/3/04
1/18/040
2
4
6
8
10
12
Tungsten (ug/g Creatinine)
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
200
45 40
1824 25 22
50
83
6653
170
26
90
170
76
61
30
57
35
92
3/23/043/30/04
4/5/044/9/04
4/12/044/17/04
6/1/046/17/04
6/20/046/21/04
7/12/047/20/04
8/4/048/25/04
8/30/049/8/04
9/13/049/23/04
9/29/0410/7/04
10/12/04
0
50
100
150
200
Creatinine
Creatinine in Urine
0
1.5
0
5.9
1.5
0
2.22.5
0.7
2.7
0 0 0 00.3
0.7
0 0
1.9
0 0
3/23/043/30/04
4/5/044/9/04
4/12/044/17/04
6/1/046/17/04
6/20/046/21/04
7/12/047/20/04
8/4/048/25/04
8/30/049/8/04
9/13/049/23/04
9/29/0410/7/04
10/12/04
0
1
2
3
4
5
6
Mercury (ug/g Creatinine)
Mercury Excretion in Urine
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
• 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 virallyinfected cells
Chronic Viral Infection
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.
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 amyocarditicstrain 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 coxsackievirusgiven 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 enterovirusinfections 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 lymphoproliferativeresponse 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, Cdexposure 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.
Relationship between Virus and Metals
Ability of viruses to trigger host 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. [email protected]
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.
Virus Induced Metallothionein Synthesis
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 andglutamate 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 FSerum 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 hemodialysiswho 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
• Pinpoint pupils• Blurred vision• Hypersecretion• Bladder incontinence
• Muscle twitching• Muscle weakness• Dilated pupils
Muscarinic Stimulation Nicotinic Stimulation
The Autonomic Nervous System
Structure Sympathetic Stimulation Parasympathetic StimulationIris (eye muscle) Pupil dilation Pupil constrictionSalivary Glands Saliva production reduced Saliva production increasedOral/Nasal Mucosa
Mucus production reduced Mucus production increased
Heart Heart rate and force increased Heart rate and force decreased
Lung Bronchial muscle relaxed Bronchial muscle contracted
Stomach Peristalsis reduced Gastric juice secreted; motility increased
Small Intestine Motility reduced Digestion increasedLarge Intestine Motility reduced Secretions and motility increased
Liver Increased conversion ofglycogen to glucose
Kidney Decreased urine secretion Increased urine secretion
Adrenal medulla Norepinephrine andepinephrine secreted
Bladder Wall relaxedSphincter closed
Wall contractedSphincter relaxed
• Magnesium will help protect against aluminum effects on cellular respiration
• Malic acid
• EDTA
METALS
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)
Consequences of Increased Metal Burden
Metals myelination
methylation is necessary for myelination
Metals myelination remyelination
lack of methylation
methylation metals myelinationand
Without myelin, axons will continue to grow and pruning will be
compromised.
Metals
Myelination ensures the rapid conduction of electrical impulses in the nervous system.
Myelination
Neuregulins
Neuregulin is the signal for the regulation of myelin sheath thickness
Myelin wrapping
Neuregulin also induces GABA receptors
Constant activation of neuregulinmay cause demyelination
Proper myelination requires proper pruning
methylation metals myelinationand
Without myelin, axons will continue to grow and pruning will be
compromised.
Glial Cells (affected by Ammonia) relationship to PruningScience News: April 22, 2004Glia 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
Glutamic acid5 amino
levulinic acid heme
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 PorphobilinogenSynthase, 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.
Heme is the prosthetic group of hemoglobin, myoglobin, & cytochromes.
N
N
N
N
CH3 HC
CH3
S CH2
CH3
CH S CH2
CH3
CH2
CH2
COO−
CH3
H3C
CH2CH2−OOC
protein
protein
Fe
Heme c
Figure 9This 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 / familyCatalysis •Haem, haem-thiolate, and sirohaemenzymes in ENZYME database •Haem enzymes in LIGAND database
Oxidoreductases•Catalases•Haem-thiolate•Peroxidases
Electron transfer •CytochromesOxygen transport and storage •Globins
Nitric oxide transport •Nitrophorin
NOS heme groups
Animal haem peroxidases in enzyme databases
ENZYME LIGAND BRENDA Official name Alternative names
1.11.1.7 1.11.1.7 1.11.1.7 Peroxidase
Eosinophilperoxidase; lactoperoxidase; myeloperoxidase
1.11.1.8 1.11.1.8 1.11.1.8 Iodide peroxidase
Iodinase; iodotyrosinedeiodase; thyroid peroxidase
1.14.99.1 1.14.99.1 1.14.99.1Prostaglandin-endoperoxidesynthase
Cyclooxygenase; prostaglandin G/H synthase; prostaglandin synthase
IUBMB Enzyme NomenclatureEC 1.11.1.8
Common name: iodide peroxidase
Reaction:iodide + H2O2 = iodine + 2 H2OOther name(s): iodotyrosine deiodase; iodinase; iodoperoxidase
(heme type); thyroid peroxidase; iodide peroxidase-tyrosine iodinase; iodotyrosine deiodinase; monoiodotyrosine deiodinase;
thyroperoxidase; tyrosine iodinaseSystematic name: iodide:hydrogen-peroxide oxidoreductase
METHYLATION
Methylation
• Importance of methylation• Pathway• What we can do about it
Methyl groups – CH3Methylation
Bound to DNA, enzymes, vitamins
CH
H
H
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, MenozziFD, 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.
Arsenite methylation by methylvitamin B12 and glutathionedoes 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 in Arsenic Detoxification
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 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
DNA Methylation
MelatoninN-acetyl-5-methoxytryptamine
Serotonin5-HT; 5-hydroxytryptamine
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 9This 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. [email protected]
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.
Arsenite methylation by methylvitamin B12 and glutathionedoes 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 in Arsenic Detoxification
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.
Methyl-donor deficiency due to chemically induced glutathione depletion
• 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 )
Reelin GAD enzyme
Methylation Reelin
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.
“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
Decreased methylation creates a compromised host for the virus.
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 oligomerisationsubdomain
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. 1-67Chemotaxis receptor methyltransferase CheR, C-terminal domain (1)
contains additional N-terminal all-alpha domain, res. 11-91RNA methylases (4) DNA methylases (5) Type II DNA methylase (3)
circularly permuted version of the common foldSpermidine synthase (1)
contains additional N-terminal tetramerisation all-beta domain, res. 1-71Mycolic 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 nitrosoureaanalogues 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 nitrosoureaanalogues 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 MethylationIntroducing 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 histoneacetylation. 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 MethylationIntroducing methylated DNA at specific genomic loci
affects local histone acetylation.By Jonathan Weitzman
• Methylation of DNA at CpG dinucleotidesrepresses 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.
Turn off Genes
Histone deacylation
DNA methylation
SIR2
IGF
Turn off Genes
Histone deacylation
DNA methylation
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 deacetylaseactivity 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 deacetylaseinhibitor.
Awareness of Roles Played by:
• Lysine• Nadh
• Igf• Telomerase
• Valproic Acid
Before Prolongevity After ProlongevityTriglycerides 425 242
Total Cholesterol 218 185Glucose 152 69
HDL 36 36LDL 131 101
ProLongevity NutriSwitch RNA™Female, 72
Modifications forRegulation
• Ubiquitination• Methylation• Acetylation• (Phosphorylation)
Mutations
• UBE3A• MTHFr, MeCP2…• NAT• (Casein Kinase 2, milk)
