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VACCINE INGREDIENT TOXICITY – Unraveling the initiation of
oxidative damage to myelin and role of mast cells, microglia and
Schwan cells in neurogenic inflammation and inhibition of
remyelination
Beldeu Singh
Keywords: adjuvants, heavy metals, mercury, aluminum, solubility of mercury,
neurotoxic, adverse reactions, inflammation, mast cells, degranulation,
microglia, Schwan cells, oxidative damage, lipid peroxidation, Ca2+ signaling,
mitochondrial damage, demyelination, remyelination, histamine, chemokines,
autism
ABSTRACT
Since their first description in 1878 by Paul Ehrlich in 1878, mast cells have
been mostly viewed as effectors of allergy. Research over the past two
decades show that mast cells are involved in other physiological and
pathological processes. A mastocyte or a labrocyte is a type of white
blood cell, commonly known as mast cell that is a part of the immune and
neuroimmune systems. Mast cells appear to have a complex role and are also
involved in wound healing and may stimulate growth of blood vessels at sites
of injury. The mast cell is derived from the myeloid stem cell. They originate in
the bone marrow and migrate into all tissues of the body. The mast cell
contains many different granules containing powerful biologically active
molecules called mediators and histamine and heparin. Mast cells (MCs) are
an integral part of a highly regulated immune system that plays a protective
role and defense against pathogens. Through a process of cellular
communication, the presence of pathogens triggers the release of
antimicrobial cytotoxic or other molecules such as chemokines from secretory
vesicles called granules found inside mast cells. This process is used by
several different granulocytes of the immune system, including neutrophils,
basophils, and eosinophils. Such release leads to degranulation of these
cells. Mature mast cells can recognize and respond to various stimuli through
the release of an array of biologically active mediators. They can be activated
by foreign bodies to release the proinflammatory mediators. Oxidative stress
induced by heavy metals can cause demyelination. When triggers are present
in large amounts, they activate a large number of mast cells to degranulate,
thereby secreting excessive amounts of proinflammatory molecules that can
mediate allergic reactions and promote inflammation. Excessive activation
can precipitate dysregulation leading to disorder through inflammatory (IL-4
and IL-6) suppression of microglia near nerves that inhibit remyelination. The
mechanisms of heavy metal neurotoxicity are discussed for a better
1
understanding of oxidative damage, inflammation, demyelination and
inhibition of remyelination in connection with mast cell inflammation that
suppresses microglia or alter Schwan cells.
INTRODUCTION
Thimerosal is added to a vaccine as preservative or used in the
manufacturing process to suppress bacterial growth. Thimerosal is sodium
ethylmercury thiosalicylate, an organic compound of ethyl mercury, included in
certain vaccines to protect multiple dose ampules from bacterial and fungal
contamination. Adjuvants such as aluminum salts are added to enhance the
immune system’s response. Adjuvants used today make it possible to reduce
the amount of antigens (weak or dead viruses) in a vaccine. Formaldehyde is
used to inactivate bacterial products and perhaps pesticides are now added
too. Mercury and aluminum trigger cells of the immune system and being
inorganic, cause abnormal activation of cells of the immune system and
trigger inflammation. Aluminum (Al), the most commonly used vaccine
adjuvant, is a demonstrated neurotoxin and a strong immune stimulator.
Hence, adjuvant Al has the potential to induce neuroimmune
disorders.1 Adverse reactions in the neuroimmune system are expected.
Heavy metals have potential to produce the highly reactive chemical entities
such as free radicals having the ability to cause lipid peroxidation, DNA
damage, oxidation of sulfhydryl groups of proteins, depletion of protein and
several other effects.2 Heavy metals have been found to generate reactive
oxygen species (ROS), which in turn results in their toxic effects in the form of
hepatotoxicity and neurotoxicity as well as nephrotoxicity in both animals and
humans.3
Heavy metals such as mercury result in neurological injury that may lead to
developmental defects, peripheral neuropathies, and enhanced
neurodegenerative changes. Inorganic mercury in concentrations as low as
0.1 μM can induce VEGF and IL-6 release from human cultured mast cells.
Activated brain mast cells can disrupt the blood-brain barrier (BBB) and
further increase brain mercury levels. Mercury increases cytosolic calcium
levels in PC12 cells, and thimerosal does so in thymus lymphocytes. Mercury
may also increase cellular oxidative stress since neurons are highly
susceptible to reactive oxygen species (ROS) and neuronal mitochondria are
especially vulnerable to oxidative damage. Mercury also induces mast cells to
release inflammatory mediators.4 The disruption by thimerosal or aluminum of
Ca2+ in thymus in certain individuals can lead to various extents of immune
suppression.
2
Mercury and aluminum are considered to be neurotoxic metals, and they are
often linked to the onset of neurodegenerative diseases. The most toxic is
methylmercury and the most sensitive cell line was the neuroblastoma cell
line SH-SY5Y. Furthermore, there was marked mitochondrial activation,
especially in connection with aluminum and methyl mercury at low
concentrations. All the metals tested induced apoptosis, but with a different
time-course and cell-line specificity. The study emphasized the toxicity of
methylmercury (MeHg) to neural cells and showed that aluminum alters
various cellular activities.5 Within the brain, MeHg produces injury that is
pathologically characterized by atrophy of cerebral cortex and white matter as
well as cerebellum.6 Methylmercury is a neurotoxic compound which is
responsible for microtubule destruction, mitochondrial damage, lipid
peroxidation and accumulation of neurotoxic molecules such as serotonin,
aspartate, and glutamate.7
Aluminium interferes with most physical and cellular processes. Some of
these are mediated through free radicals since some symptoms of aluminium
toxicity can be detected in seconds and others in minutes after exposure to
aluminium.8 Aluminium toxicity probably results from the interaction between
aluminium and plasma membrane, apoplastic and symplastic targets.9 In
humans Mg2+ and Fe3+ are replaced by Al3+, which causes many disturbances
associated with intercellular communication, cellular growth and secretory
functions and degenerative lesions in nerves. Aluminium showed adverse
effects on the nervous system and resulted in loss of memory, problems with
balance and loss of co-ordination.10 Mercury and aluminum can therefore
establish varied toxic mechanisms that generate oxidative stress and trigger
inflammation and dysregulate the inflammatory cascade to precipitate
different problems in various tissues and organs leading to a spectrum of
disorders and serious multisystem developmental disorders in different people
that is also dependent on the their blood antioxidant levels and nutrition.
Mast cells can be found throughout normal connective tissue, often next to
blood vessels or nerves or beneath epithelial surfaces, where these cells are
exposed to the environment via the respiratory and gastrointestinal
tracts. Mast cells are found resident in tissues throughout the body,
particularly in association with structures such as blood vessels and nerves,
and in proximity to surfaces that interface the external environment.11 Mast
cells are distributed predominantly at the interface between the host and the
external environment and are preferentially located at nerve endings. This
feature in conjunction with its ability to recognize pathogens and to become
activated by foreign particles establishes mast cells to act as both first
responders in harmful situations as well as to respond to changes in their
environment by communicating with a variety of other cells implicated in
3
physiological and immunological responses. In a well-coordinated
inflammatory cycle, mast cells secrete pro-inflammatory cytokines during the
initial phase and promote the recruitment of macrophages; later anti-
inflammatory cytokines are expressed after this recruitment and downregulate
the production of all cytokines, thus determining the end of the process.
However, mast cell dysfunction is also the main offenders in several chronic
allergic/inflammatory disorders, cancer and autoimmune diseases. The
accumulating knowledge shows that mast cell function in the normal mode for
a protective role but under sustained and extensive activation, mast cells are
triggered to release excessive amounts of the proinflammatory molecules
stored in the granular sacs and such extensive activation trips mast cells into
dysfunction and dysregulate them into a pathological role.
“On one hand, MCs can also be beneficial for its host, for example, by
contributing to the defense against insults such as bacteria, microparasites
and snake venom toxins. When the MC is challenged by an external stimulus,
it may respond by degranulation. In this process, a number of powerful
preformed inflammatory "mediators" are released, including cytokines,
histamine, serglycin proteoglycans, and several MC-specific proteases:
chymases, tryptases, and carboxypeptidase A.”12 This critical role of mast
cells in both innate and adaptive immunity is clearly also modulated by certain
phytochemicals in food that can downregulate its secretory molecules and
reduce inflammation13 which will come to be recognized in therapeutic science
as a process that promotes the beneficial role of mast cells in the host.
It is now known that MCs have very broad and varied roles in both physiology
and disease. Interestingly, although MCs are part of the immune system, the
most quickly developing area of understanding is the involvement and
contribution MCs make in the progression of a variety of diseases from some
of the most common diseases to the more obscure.14 Two potential outcomes
of dysregulated immunity are allergy and autoimmunity. Both are
characterized by localized inflammation that leads to the injury and/or
destruction of target tissues. Until recently, it was generally accepted that the
mechanisms that govern these disease processes are quite disparate;
however, new discoveries suggest that the mast cell may underlie much of the
pathology in both these disease syndromes. Although well established as
effector cells in allergic inflammation, the location of mast cells and the wealth
of inflammatory mediators they express make it likely that they have profound
effects on many other autoimmune processes.15 For instance, serine
proteases in mast cell granules, such as chymase, atypical chymase, and
tryptase, which are major proteins in the granules, may play important roles in
the process of immunoglobulin E (IgE)-mediated degranulation and in
pathobiological alterations in tissues.16
4
Mast cells have an important immunoregulatory function, particularly at the
mucosal border between the body and the environment. Several studies have
noted an increased number of mast cells in the mucosa of patients with
gastrointestinal diseases such as irritable bowel syndrome, mastocytic
enterocolitis, and systemic mastocytosis.17 “Mast cells originate from a distinct
precursor in the bone marrow and migrate into most tissues, where they
acquire distinct characteristics in response to different micro-environmental
influences, such as stem cell factor, nerve growth factor, or the cytokines,
interleukin (IL)-3, LI-4 and IL-6. Mast cells are responsible for allergic
reactions but mounting evidence indicates that they also participate in
inflammation and homeostasis. They are present in the meninges, especially
the dura mater, which contains a large proportion of the total intracranial
histamine.”18 Mast cells play an increasingly important role in the
pathophysiology of gastrointestinal diseases, airways and inflammatory
problems in the CNS and brain.
NEUROGENIC INFLAMMATION – histamine from mast cells
The role of mast cells in allergic inflammatory reactions is well documented in
literature. A better understanding of this mechanism is vital in therapy and
healthcare. The term ‘neurogenic inflammation’ has been adopted to describe
the local release of inflammatory mediators that lead to inflammatory damage
to cells in the immediate vicinity. Neurogenic inflammation also occurs in brain
and visceral organs. The inflammatory response is evoked by the activation of
mast cells that trigger mast cell degranulation and release of chemokines and
histamine. It is brought about by several factors including allergens and
foreign bodies that can activate mast cells. Other cells of the immune system
can also contribute to neurogenic inflammation.
Neutrophils, whose responsiveness to SP has been repeatedly demonstrated,
contribute to the inflammatory soup following neurogenic inflammation. In fact,
neutrophils express the SP receptors NK1, NK2 and NK3, and when stimulated
with substance P (SP) induce the expression of COX-2 and PGE2 release in
the nanomolar range.19 The role of histamine in neurogenic inflammation and
in particular with regard to nociceptive pain is now recognized. Neurogenic
inflammation may be accompanied by pain. The biochemistry of such pain
must be understood to treat and manage pain.
Experimental data generally show that histamine plays a key role in the
complex physiopathological mechanism known as neurogenic inflammation.
Initial observations on the role of histamine in the neurogenic inflammation in
the dura matter and in the gut have been further elucidated to note the
involvement of mast cells.20 Persistent cough may be related to a cough reflex
hypersensitivity caused by neurogenic airway inflammation21 and this view is
5
in keeping with research that showed an increased histamine content in the
sputum of patients with idiopathic chronic cough and cough variant
asthma/eosinophilic bronchitis in comparison with normal subjects.22
Interstitial cystitis (IC) and painful bladder syndrome (PBS) are both causes of
neurogenic inflammation; in fact, an increased density of nerve fibers has
been reported in these conditions.23 It has also been demonstrated that PRV-
induced pelvic pain is not dependent on TNF-mediated effects but is mediated
by mast cell histamine.24 Due to increased histamine production in systemic
mastocytosis, symptoms can also include esophagitis, gastric ulcer disease
and intestinal malabsorption.25
Antihistamine therapy could be effective in pain relief when the pain is
associated with histamine release from mast cells but a better therapeutic
model can be developed based on edible substances that downregulate
histamine and other proinflammatory mediators, including IgE.
ACTIVATION OF MAST CELLS - gastroinflammation and
neuroinflammation
Mast cells appear to be highly engineered cells with multiple critical biological
functions. They may be activated by a number of stimuli that are both Fc
epsilon RI dependent and Fc epsilon RI independent. Activation through
various receptors leads to distinct signaling pathways. After activation, mast
cells may immediately secrete granule-associated mediators and generate
lipid-derived substances that induce immediate allergic inflammation. Mast
cell activation may also be followed by the synthesis of chemokines and
cytokines. Cytokine and chemokine secretion, which occurs hours later, may
contribute to chronic inflammation. Biological functions of mast cells appear to
include a role in innate immunity, involvement in host defense mechanisms
against parasitic infestations, immunomodulation of the immune system and
tissue repair and angiogenesis.26
Mast cells often are found in a perivascular location but especially in
mucosae, where they may response to various stimuli. They typically
associate with immediate hypersensitive responses and are likely to play a
critical role in host defense. Mast cells are sensitivity to certain glycoproteins
which are found in food leading to food allergies and glycoproteins of
pathogens that trigger a series of interactions among T cells, B cells and
antigen-presenting cells to mount an immune response. Mastocytosis is the
accumulation and proliferation of mast cells at various sites or tissues,
including skin, airways, brain and gut. Symptoms result from the infiltrated
organ and mast cell mediator release upon activation and can affect the
respiratory, digestive, neuropsychiatric and hematologic systems.
Management of systemic mastocytosis includes avoidance of triggers (eg,
6
alcohol, nonsteroidal anti-inflammatory drugs) and control of symptoms
related to mast-cell mediator release.27 Naturally, therapeutic science aims at
removal of allergens and mast cell activating agents to prevent mastocytosis
while simultaneously providing other non-toxic substances that serve to
stabilize mast cells and downregulate their mediator release to prevent
symptoms of mediator-inflammatory damage to cells.
Mast cells are also affected by both acute and chronic stress. When
stimulated by substance P, these mast cells release inflammatory mediators,
such as serotonin and proteases, as well as proinflammatory cytokines. Other
mediators are important for the function and regulation of the gastrointestinal
tract. For example, the release of histamine and prostaglandin D2 is important
for chloride and water secretion as well as control of intestinal motility. 28 Due
to the location of mast cells in the gut, in between nerve cells, they may play a
role in visceral sensitivity. Reaction from motor neurons secondary to
degranulation can result in gastro-inflammation for hypersecretion and power
propulsion, causing diarrhea and abdominal pain.29 Activation of mast cells
has been implicated in many types of neuro-inflammatory responses, and
related disturbances of gut motility, via direct or indirect mechanisms that
involve several mechanisms relevant to disease pathogenesis such as
changes in epithelial barrier function or activation of adaptive or innate
immune responses.30
Mast cells are both sensors and effectors in communication among nervous,
vascular, and immune systems. In the brain, they reside on the brain side of
the BBB, and interact with astrocytes, microglia, and blood vessels via their
neuroactive stored and newly synthesized chemicals. Upon action, mast cells
are the first responders, to release stored molecules that are also
inflammatory in nature and act to recruit more cells of the immune system that
initiate, amplify and prolong other immune and nervous responses until proper
downregulation in a controlled fashion, failing which the uncontrolled
inflammation through excessive secretion of inflammatory molecules
damages cells or cellular components in the vicinity of these molecules. Mast
cells both contribute to normative behavioral functioning and promote
deleterious outcomes in brain function, particularly cognition and emotion.
Prolonged and sustained activation of mast cells can lead cellular damage
and pathologic consequences. Sustained inflammation resulting in tissue
pathology implies persistence of an inflammatory stimulus or a failure in
normal resolution mechanisms. Mast cells may play a key role in treating
systemic inflammation or blockade of signaling pathways from the periphery
to the brain.31
There is extensive communication between the immune system and the
central nervous system (CNS) that occurs in a well regulated and controlled
7
mode involving proinflammatory cytokines to organize and orchestrate proper
immune responses to protect the host. Well regulated immune system
responses involving chemokines is directed at foreign bodies and pathogens
and is appropriately donwregulated in due course. Fundamentally, such
controlled inflammation is a physiologic response to injury and infection and is
necessary for removing detrimental stimuli and initiating tissue healing. A
similar process that occurs in the CNS in response to injury or disease is
termed neuroinflammation. In acute neuroinflammation, microglia become
activated and limit the area of injury through phagocytosing dying cells and
releasing pro-inflammatory cytokines.32 However, under abnormal or
excessive activation, it triggers neuroinflammation that is prolonged and it
overwhelms and dysregulates the biochemical limits of physiological control
and produces deleterious effects. At this stage, symptoms of neurogenic
inflammation appear and the natural biochemical must be augmented by non-
toxic phytomolecules from edible substances that help to remove the
activation factors and bind to the excess proinflammatory factors released by
mast cells (eg IgE and histamines) and downregulate mast cells.
Microglia cells are the major resident immune cells in the brain and play a
pivotal role in the immune surveillance of the central nervous system
(CNS). The initiation and propagation of neuroinflammation appear to rely
very much on the interaction between glia, immune cells, and neurons.
Emerging evidence now points to a more active role of neuroinflammation in
pathophysiology onset and progression, with glia having key roles in
neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s
disease, and Huntington’s disease, and may even contribute to multiple
sclerosis, amyotrophic lateral sclerosis, stroke and neuropsychiatric disorders.
This glia-immune cell connection now attracts special interest in
neuroinflammation and healthy brain function.33
Mast cell activation syndrome (MCAS) is a condition with signs and symptoms
involving the skin, gastrointestinal, cardiovascular, respiratory and neurologic
systems. It can be classified into primary, secondary and idiopathic.34 Patients
may have episodes of mast cell mediator release. The term mast cell
activation disease (MCAD) denotes a collection of disorders characterized by
(A) accumulation of pathological mast cells in potentially any or all organs and
tissues and/or (B) aberrant release of variable subsets of mast cell
mediators.35
ACTIVATION OF MAST CELLS BY MERCURY
Heavy metals such as mercury result in neurological injury that may lead to
developmental defects, peripheral neuropathies, and enhanced
neurodegenerative changes. Mercury can activate mast cells. Mercury's
8
activation of mast cell inflammatory mediator release may enhance allergic
reactions in atopic individuals and exacerbate IgE-dependent diseases36
Allergic symptomatology is often present in Autistic Spectrum Disorder (ASD)
patients37 and a survey of children with ASD in Italy reported that the strongest
association was with a history of allergies.38
Mercury (II) salts are usually more toxic than their mercury (I) counterparts
because of their greater solubility in water and rapid absorption by the
gastrointestinal tract. Mercury (II) chloride, primarily affects gastrointestinal
tract and kidneys. Since mercury salts cannot cross the BBB easily, mercury
salts inflict little neurological damage without continuous or heavy exposure.39
Again, it is obvious that the varied toxic mechanisms precipitate different
problems in various tissues and organs leading to a spectrum of disorders
and serious multisystem developmental disorders, depending on the
formation of salts and ions and blood antioxidant levels.
Many patients with Autism Spectrum Disorders (ASD) have "allergic"
symptoms; moreover, the prevalence of ASD in patients with mastocytosis,
characterized by numerous hyperactive mast cells in most tissues, is 10-fold
higher than the general population suggesting mast cell involvement. It was
found that mercuric chloride (HgCl2) can activate human mast cell, stimulates
VEGF and trigger release of IL-6. This phenomenon could disrupt the blood-
brain-barrier and lead to brain inflammation. Such activation provides a
biological mechanism for how low levels of mercury may contribute to ASD
pathogenesis.40 “Autism is a complex disorder. A plethora of research has also
discovered those with ASD have many abnormalities in the brain including …
loss of cell function, signaling dysfunction, loss of purkinje cells and
astrocytes, metabolism disorders, mitochondrial disorders, oxidative damage,
inflammation of the brain, mast cell activation, reduction of protein synthesis,
overstimulation of some areas of the brain involved in memory, alteration in
the excitatory/inhibitory imbalance of glutamatergic/GABAeric system and
systemic immune dysfunction all connected to the functional behavioral
problems seen in autism.” Neurobiological research in ASD has highlighted
pathways involved in neural development, synapse plasticity, structural brain
abnormalities, cognition and behavior.41
The neurochemical abnormalities of the brain in autistic children is a common
developmental disorder associated with structural abnormalities with cells and
reduced connectivity that may also be associated or due to Purkinje cell loss
and cerebellar white matter thinning and demyelination.42 Demyelination is a
critical issue in autism. In ethyl mercury toxicity in children, nerve cell loss was
widely present but most marked in calcarine cortex, and there was diffuse
proliferation of glia, demyelination of ninth and tenth cranial nerve roots and
9
atrophy of the cerebellar granule cell layer with relative sparing of Purkinje
cells.43
Autism spectrum disorders (ASDs) also known as pervasive developmental
disorders (PDD) are a behaviorally defined group of neurodevelopmental
disorders that are usually diagnosed in early childhood. ASDs
disproportionately affect male children. Autism spectrum disorders (ASDs)
also known as pervasive developmental disorders (PDD) are a behaviorally
defined group of neurodevelopmental disorders that are usually diagnosed in
early childhood. ASDs disproportionately affect male children.44 In some
experiments, the addition of aluminumhydroxide (often in vaccines),
antibiotics, thimerosal (sometimes in vaccines) and testosterone increased
the toxicity of mercury.45 The synergistic toxicity of oxidized testosterone
explain the observation, that much more males than females suffers from
autism or amyotrophic lateral sclerosis. Also females may have relatively
more glutathione in their brains.
Cytokines are proteins involved in cell signaling and are produced by a variety
of cells including macrophages, B and T lymphocytes, mast cells and
fibroblasts. They are important in modulating the immune system,
inflammation, infection, cancer and reproduction.46 “Groundbreaking work by
Pardo has shown an inflammatory-like state in postmortem autism brains of
all ages as indicated by elevated cytokines and activated microglia and
astrocytes which indicate that innate neuroimmune reactions play a
pathogenic role in an undefined proportion of autistic patients.”47 Smith looked
at maternal immune stimulation in mice and found a specific cytokine IL-6 that
accurately reproduces the abnormal autistic/schizophrenic like behavior in
offspring by changing gene expression.48 There are significant increases of a
variety of plasma cytokines in ASD patients which indicate that elevated
inflammatory molecular secretions by mast cells are linked to immune
dysfunction and disturbances in behavior and require confirmation in larger
replication studies.”49
Mercury can cause immune, sensory, neurological, motor and behavioral
dysfunction similar to those associated with Autism Spectrum Disorders
(ASD).50 Heavy metals such as mercury result in neurological injury that may
lead to developmental defects, peripheral neuropathies, and enhanced
neurodegenerative changes.51 87% of children included in the US Vaccine
Adverse Event Reporting System (VAERS) had ASD.52 Mercury has been
shown to induce proliferation and cytokine production from T lymphocytes.53
Mercuric chloride (HgCl2) in very low (nontoxic) doses induces the release of
histamine and cytokines, such as IL-4 and tumor necrosis factor-alpha (TNF-
α), from a murine mast cell line and from mouse bone marrow-derived
cultured mast cells.54 This information from research supports the conclusion
10
of a paper based on computerized medical records in the Vaccine Safety
Data-link that there was "significantly increased rate ratios for ASD with
mercury exposure from thimerosal-containing vaccines."55
SOLUBILITY OF MERCURY COMPOUNDS – cause demyelination
Compounds of mercury have low solubility. Dimethylmercury, a very toxic by-
product of the chemical synthesis of MeHg56 also has relatively low water
solubility (1.0 g/L at 21° C) that increases with temperature. At 25° C,
elemental Hg has a water solubility of 5.6×10-5 g/L. Mercuric chloride is
considerably more soluble, having a solubility of 69 g/L at 20° C. Due to its
low water solubility, MeHg chloride is considered to be relatively lipid soluble.57
At body temperature, which is about 37 degrees Celsius, it will have higher
solubility and will tend to bioaccumulate in nerve sheaths and fatty tissue.
Despite mercury exposure below "safety limits", significant adverse health
effects were found in most studies in workers exposed occupationally to
mercury, even several years after the exposure had ceased.58
Inorganic mercury levels of 0.02 μg Hg/g (2 μl of 0.1 μMolar Hg in 2 ml
substrate) led to the total destruction of intracellular mircrotubuli and to the
degeneration of axons.59 In other experiments inorganic mercury levels of 36
ng Hg/g (0.18 μMol Hg) led to increased oxidative stress as a prerequisite for
further cell damage.60 When 0.25-25 micrograms of an aqueous solution of
either mercuric chloride or methyl mercuric acetate was injected directly into
the sciatic nerve of 28 adult Wistar rats, the predominant effect of mercuric
chloride was on Schwann cells which showed cytoplasmic swelling and
necrosis, associated with extensive segmental demyelination. In contrast
methyl mercuric acetate caused axonal degeneration in many of the large
myelinated fibers but only minor alterations were observed in Schwann cells. 61
When demyelinating lesions appear, Schwann cells migrate to participate in
the important mechanism of myelin repair62 but altered Schwan cells may
suffer inhibition in myelin repair.
The second source of mercury in health care is multidose activated vaccines
containing ethyl mercury as a preservative. Ethyl mercury is one of the
metabolites of thimerosal. Acute or chronic mercury exposure can cause
adverse effects during any period of development. Mercury is a highly toxic
element; there is no known safe level of exposure. Ideally, neither children nor
adults should have any mercury in their bodies because it provides no
physiological benefit. Metallic mercury is lipophilic and is stored in fatty
tissues. Inorganic ions of mercury vary in water solubility. In general, divalent
mercuric salts are soluble in water. The high toxicity of mercuric ions can be
explained by the high affinity to sulfhydryl groups of amino acids, which are
building blocks for enzymes.63 Methyl and ethyl mercury cross cell
11
membranes as complexes with small molecular weight thiol compounds,
entering the cell in part as a cysteine complex on the large neutral amino acid
carriers and exiting the cell in part as a complex with reduced glutathione on
endogenous carriers and can cross the blood-brain barrier. Although methyl
mercury (MeHg) is considered a hazardous substance that is to be avoided
even at small levels when consumed in foods such as seafood and rice (in
Asia), the World Health Organization considers small doses of thimerosal safe
regardless of multiple/repetitive exposures to vaccines that are predominantly
taken during pregnancy or infancy.
In its liquid form, the elemental mercury (Hg0) is poorly absorbed and presents
little health risk. However, because of its soluble characteristics, elemental
mercury is highly diffusible and is able to pass through cell membranes as
well as the blood-brain and placental barriers to reach target organs. Once in
the bloodstream, mercury undergoes catalase and peroxidase-mediated
oxidation in red blood cells and tissues and is transformed into inorganic
mercuric mercury (Hg++) and mercurous mercury (Hg+), a process that limits
its absorption64 but this ion can initiate free radical chain reactions.
Methylmercury and its metabolized salts are primarily responsible for the
neurological alterations present in humans and experimental animals. The
toxic properties and target organs of Hg are dependent upon its chemical
speciation. Many of its toxic effects are related to the toxic increase in reactive
oxygen species (ROS). Oxidative stress is associated with the etiology of
neurodegenerative diseases such as amyotrophic lateral sclerosis,
Parkinson's disease, and Alzheimer's disease.65
The elemental or metabolic mercury is oxidized to divalent mercury after
absorption into the body tissues but a portion is transported as metallic
mercury to more distant tissues, particularly the brain. Neuropathologic
observations have shown that cortex of the cerebrum and cerebellum is
selectively involved in focal necrosis of neurons, lysis, and phagocytosis and
replacement by supporting glial cells which may be due to the promotion of
lipid peroxidation by mercury. In some individuals, the delayed observed
toxicity upon exposure to elementary mercury (Hg0) where effects may be
noted several months after exposure. In others, mercury compounds may be
rapidly oxidized in the blood, and Hg ions are transported via blood and the
symptoms appear in hours or days. Delayed effects may also depend on
glutathione status and selenium stores in the liver. Because of its lipid
solubility, mercury vapor penetrates cell membranes and is easily absorbed
and rapidly oxidized to Hg2+. This ion is likely to bind to sulfhydryl or
selenohydryl groups on proteins and has limited mobility. Mercury binding to
sulfhydryl groups can change the tertiary structure of proteins and block
receptor binding.66
12
Once inside the cells, mercury vapor is oxidized to Hg2+, the very toxic form of
mercury which binds covalently to thiol groups of proteins inhibiting their
biological activity67 depleting glutathione. Depletion of glutathione in nerves
leads to oxidative damage of nerve endings and myelin. Understanding the
molecular and cellular mechanisms of selective neuronal vulnerability (SNV)
to oxidative stress (OS) is important in the development of future intervention
approaches to protect such vulnerable neurons from the stresses of the aging
process and the pathological states that lead to neurodegeneration. Oxidative
stress (OS) contributes to the selective vulnerability of neurons (SVN) and can
lead deficiency in DNA damage repair and low calcium-buffering capacity.
Oxidative damage to lipids also modifies lipid molecules. Many of the
modifications of lipids, nucleic acids and proteins result in structural changes
in the respective macromolecules and lead to either dysfunction or loss of
activity of these molecules.68 OS may damage mitochondria that may produce
more free radicals. Dysfunctional mitochondria in vulnerable neurons can
release more ROS and thus maintain a vicious cycle of oxidative modification
of mitochondrial proteins leading to further OS. Currently available evidence
shows that mitochondria isolated from CA1 neurons release more ROS than
those from CA3 neurons.69
Glutathione is important in nerve growth and regeneration and development of
the Central Nervous System (CNS). The high affinity of heavy metal ions,
including mercury ions for sulfhydryl (SH) groups in tubulin, methylmercury
inhibits the organization of microtubules that are important in CNS
development.70 The binding to SH groups also interferes with the intracellular
signaling of multiple receptors (e.g., muscarinic, nicotinic, and dopaminergic)
and promotes the blockade of Ca2+ channels in neurons71 which may inhibit
myelin repair after oxidative damage.
Based on the direct chemical binding between sulfhydryl groups of glutathione
(GSH) and MeHg leads to glutathione depletion72 which can exacerbate
oxidative damage to myelin and non-toxic chelating therapy can significantly
increase Hg excretion to reduce symptoms of heavy metal-induced
inflammation and disease states. The depletion of glutathione associated with
disruption in Ca2+ signaling can lead to increased inflammation-induced
problems in the brain and in the gut.
HEAVY METAL INDUCED OXIDATIVE STRESS – disrupts Ca2+ signaling
in nerve cells
Calcium (Ca2+) signaling plays an important role in regulating and maintaining
normal neuronal function including neurotransmitter release, excitability,
neurite outgrowth, synaptic plasticity, gene transcription, and cell survival.
During OS, (by ROS/RNS) free radicals usually activate Ca2+ channels and
13
repress Ca2+ pumps,73 resulting in elevation of [Ca2+]i and initiation of
downstream events mentioned above. Because of the intricate relationship
between OS and Ca2+ dysfunction, Ca2+ dysfunction is another mechanistic
factor that may underlie the selective vulnerability of CNS neurons to OS. 74
The damaging effects of rapidly rising [Ca2+]i in cells, provide a direct link
between Ca2+dysregulation and the phenomenon of selective nerve
vulnerability (SNV). In human PD brain, neurons containing calbindin D-28K
are less vulnerable than those without this protein.75 Ca2+ signaling disruption
is detrimental to normal cell function and can lead to deleterious effects in the
mitochondria and energy production by disruption in the ADP->ATP cycle
which, in turn may damage the cell and excessive disruption can lead to nerve
cell death. The toxicology of metallic mercury on motor neurons and their
processes axonal atrophy, degenerative loss, and hypertrophy as distinct
pathological processes in the large caliber axon subgroup that are selectively
vulnerable to metallic toxins such as mercury.76
OXIDATIVE DAMAGE TO NERVES – myelin abnormalities
Damage to developing and mature brains, represented by myelin
abnormalities are often found in neurological diseases with evidence of
inflammatory infiltration and microglial activation. Cytokine and chemokine
activity in the CNS that causes demyelinating disease has relevance to
clinical conditions of neonatal and adult, brain trauma, and mental disorders
with observed white matter defects.77 Chemokines are small-molecular-weight
chemotactic cytokines that attract leukocytes to sites of infection and
inflammation.78 Cytokines are small proteins initially thought to be components
of the immune system, but have since been found to play a much broader role
in physiology. IL-6 is not a factor only involved in the protective immune
response. Animal models strongly suggest that IL-6 could have a role in the
observed neuropathology79 but the exact mechanism is not clearly
established. Both glial and neuronal cells expressed IL-6 upon activation.80
IL4 delivery in the central nervous system (CNS) is associated with clinical
and pathological protection from disease. IL-6 is also a multifunctional
cytokine, capable of affecting a wide range of cells outside and inside of the
CNS. IL-6 cytokine under certain conditions after nerve injury is involved in a
characteristic series of molecular and cellular events that facilitate axon
regeneration and re-innervation in target tissues.81 It does not directly cause
demyelination of nerves but excess IL-4 or IL-6 in chronic inflammation such
as in abnormal activation of mast cells suppresses glial cells and astrocytes
for myelin repair and nerve growth. Microglial cells serve as the resident
mononuclear phagocytes of the brain and are highly heterogeneous within the
healthy CNS, comprising 10% of the total cell population of the brain. Injury
can activate microglia to remove cellular debris may serve to remove aberrant
14
proteins, oxidized proteins, lipids and apoptotic cells to prevent subsequent
tissue inflammation.82 Upon normal activation, microglia can also release
cytokines can then act on astrocytes to induce a secondary inflammatory or
growth factor repair response.83 Glial cells remove oxidized molecules cellular
and debris in a systematic and controlled fashion. The accumulation of glial
cells at sites of damaged myelin caused by oxidative stress is important for its
regeneration. Studies show reduced microglial cell accumulation was
associated with inefficient removal of degraded myelin. Suppressed or
senescent glial cells or glial cells that have been abnormally activated by
heavy metals fail to remove damaged or degraded myelin which blocks
regeneration. Chronic neuroinflammation and aging associated with CNS
diseases has been proposed as a factor that could lead to dysfunctional or
senescent microglia.84 Abnormal activation of microglial cells may inhibit their
role in removing cellular debris and oxidized molecules or aberrant proteins.
Dysfunctional microglial cells and astrocytes and altered Schwan cells are the
key factors that precipitate neurodegeneration as a result of inhibition in
remyelination after injury via oxidative damage by heavy metals. Pro-
inflammatory molecules are mediators. They do not cause the damage during
chronic inflammation directly but they contribute to the degenerative problems
and development of pathological states by suppressing other cells that may
be involved in immune function or regeneration, repair and growth.
Overproduction of proinflammatory cytokines as a contributor to
pathophysiology of chronic neurodegenerative disorders can be seen as
suppressants of microglial cells even leading to their senescence that disrupt
remyelination but abnormal activation by heavy metal ions or dysfunctional
microglial cells may trigger microglial phagocytosis of neurons85 which
explains demyelination and cell loss in autism.
Accordingly, in ASD, some of the common characteristics associated with
chronic or excessive inflammation or brain inflammation are:-
1. All of the children examined showed a proinflammatory profile of
cytokines,
2. Which is related to oxidative stress, with
3. Significant increase in NF-κB DNA binding activity in the peripheral
blood samples, and
4. Demyelination and various extents of neurodegeneration with neuronal
cell loss,
5. Reduced connectivity found in the brains, followed by
6. Microglial suppression by sustained neuroinflammatory process, and
7. Abnormal activation of microglial cells that phagocytize neuronal cells,
and
8. Altered Schwan cells that fail in myelin repair.
15
The metabolism and excretion of these heavy metals depend on the presence
of antioxidants and thiols that aid arsenic methylation and both arsenic and
cadmium metallothionein-binding. S-adenosylmethionine, lipoic acid,
glutathione, selenium, zinc, N-acetylcysteine (NAC), methionine, cysteine,
alpha-tocopherol, and ascorbic acid have specific roles in the mitigation of
heavy metal toxicity86 and also depend on folate status.
CONCLUSION
Mercury and aluminum are global environmental pollutant with significant
adverse effects on human health when ingested or when introduced into the
body. Vaccine toxicity induced by the heavy metals that constitute the
ingredients is mediated through their solubility in the body, diffusion and
translocation in the body and bioaccumulation in fatty tissue that include the
nerves and its myelin sheath. The varied reactions in individuals depend on
the formation of salts of mercury and aluminum and extent of oxidative stress
and trigger of proinflammatory mediators by various cells, including mast cells
residing in the vicinity of nerve. Oxidative stress can cause lipid peroxidation
and damage to the myelin which attracts microglial for removing oxidatively
damaged lipid molecules and Schwann cells for myelin repair but this process
is disrupted by excessive and chronic inflammation that suppress the
microglial cells or alter the Schwan cells via abnormal activation by heavy
metal ions. A better understanding with accumulating knowledge can help to
develop better vaccines to meet challenges in managing viral epidemics.
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