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International Journal of Radiation Biology
ISSN: 0955-3002 (Print) 1362-3095 (Online) Journal homepage: http://www.tandfonline.com/loi/irab20
Chronic Fatigue and Immune Deficiency Syndrome(CFIDS), cellular metabolism, and ionizingradiation: A review of contemporary scientificliterature and suggested directions for futureresearch
Andrej Rusin, Colin Seymour & Carmel Mothersill
To cite this article: Andrej Rusin, Colin Seymour & Carmel Mothersill (2018): Chronic Fatigue andImmune Deficiency Syndrome (CFIDS), cellular metabolism, and ionizing radiation: A review ofcontemporary scientific literature and suggested directions for future research, International Journalof Radiation Biology, DOI: 10.1080/09553002.2018.1422871
To link to this article: https://doi.org/10.1080/09553002.2018.1422871
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Short Title: Ionizing radiation effects on CFIDS, cell metabolism
REVIEW
Chronic Fatigue and Immune Deficiency Syndrome (CFIDS), cellular metabolism, and ionizing
radiation: A review of contemporary scientific literature and suggested directions for future
research
Andrej Rusin
McMaster University, Biology, Hamilton, Ontario, Canada
Colin Seymour
McMaster University, Medical Physics and Applied Radiation Sciences, Hamilton, Ontario,
Canada
Carmel Mothersill
McMaster University, Biology, 1280 Main street West, Hamilton, Ontario, Canada
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CONTACT Andrej Rusin Email: [email protected] McMaster University, Biology, 1280 Main
Street West, Hamilton, Ontario, L8S 4L8 Canada
Supplemental Material for this article can be accessed on the publisher’s website.
Abstract
Purpose: To investigate biochemical pathways known to be involved in radiation response and
in CFIDS to determine if there might be common underlying mechanisms leading to symptoms
experienced by those accidentally or deliberately exposed to radiation and those suffering from
CFIDS. If such a link were established, to suggest testable hypotheses to investigate the
mechanisms with the aim of identifying new therapeutic targets.
Conclusions: Evidence for involvement of the alpha-synuclein, cytochrome c oxidiase, αB-
crystallin, RNase L, and lactate dehydrogenase/STAT1 pathways is strong and suggests a
common underlying mechanism involving mitochondrial dysfunction mediated by ROS and
disruption of ATP production. The downstream effect of this is compromised energy
production. Testable hypotheses are suggested to investigate the involvement of these
pathways further.
Key Words: Chronic Fatigue Syndrome, Bystander effects of radiation, Reactive oxygen species
(ROS), Post-Radiation Syndrome, Atomic Veterans
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Introduction
Atomic Veterans and Chronic Fatigue
Victims of radiation accidents such as Chernobyl or Fukushima, and those exposed deliberately
in the Atomic Bombs, the Cold War nuclear detonations, and the more recent Gulf War
complain of diffuse symptoms many years later (Loganovsky 2000; Milano 2000; Durakoviæ
2003; Bertell 2006; Broinowski 2013). Generally, their contention that ionizing radiation
exposure led to their symptoms manifesting up to 60 years later are dismissed as non-causal
due to the allegedly low doses they received and the lack of sufficient numbers to establish
good epidemiology (McCauley et al. 2002; Coughlin et al. 2013; Mothersill and Seymour 2016).
Many of the symptoms and disease manifestations are similar to those seen in patients
diagnosed with Chronic Fatigue and Immune Deficiency Syndrome (CFIDS).
The onset of symptoms following acute radiation exposure has been termed Post-Radiation
Syndrome (PRS), an illness studied mainly in atomic bomb and Chernobyl survivors. A few
studies have noted that the pathology of PRS is very similar to a family of multisystem illnesses
including CFIDS, fibromyalgia, and multiple chemical sensitivity, according to Pall (2008). This
connection was mainly made due to a similar onset of the disease after a short-term stressor,
the same chemical markers appearing in both illnesses, and similar symptoms (Loganovsky
2000; Pall and Satterlee 2001; Pall 2007, 2008, 2009). According to the review published by Pall
(2008), while the pathways thought to be involved in PRS were mostly studied in patients
exposed to very high doses of radiation, other studies have demonstrated that much lower
doses can result in activation of the same pathways (Mohan and Meltz 1994; Prasad et al. 1994;
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Yin et al. 2003; Mitra et al. 2005; Rithidech et al. 2005). These include doses within the range
experienced by the Chernobyl Liquidators (Kumerova et al. 2000). The same study discussing
Chernobyl Liquidators indicated that the workers experienced symptoms consistent with CFIDS,
although it did not mention the disease by name. There have been many studies that have
linked low-dose radiation exposure to CFIDS directly, primarily due to neurophysiologic and
metabolic changes that occur in some patients; studies include those conducted on Chernobyl
survivors and Gulf War veterans. Among these are studies showing effects in patients receiving
total biological doses of 0-70 mSv (Pflugbeil et al. 2006; Pall 2008; Loganovsky 2009; Bazyka et
al. 2010; Loganovsky et al. 2016).
While no clinical study has directly linked CFIDS with radiation exposure, some symptoms
documented in victims of radiation accidents and in radiotherapy patients were shown to be
consistent with symptoms of CFIDS, including fatigue, immune system dysfunction, and
cognitive dysfunction (Pastel et al. 2002; Pflugbel et al. 2006; Pall 2008; Loganovsky 2016;
Mothersill and Seymour 2016). Such symptoms are often dismissed as inconsequent to
radiation exposure or as radiophobia mainly on the grounds that they do not appear to be dose
dependent and are reported by people who have been exposed to doses considered too low to
cause biological effects (Pastel 2002; Mothersill and Seymour 2016). What these authors are
ignoring is that non-targeted effects of radiation (NTE) have an exceedingly low threshold for
induction in the region of 2-5mGy (Liu Z et al. 2007; Schettino et al. 2005) and once turned on
they continue to be expressed over time, including over generations (Nagasawa and Little 1992;
Mothersill and Seymour 1997; Wright 1998; Limoli et al. 2000; Mothersill et al. 2000; Morgan
2003; Smith et al. 2007; Lorimore et al. 2008; reviewed in Nugent et al. 2010; Shi et al. 2016;
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Smith et al. 2016). The second point about NTE is that the effects saturate at relatively low
doses, being fully expressed after a 0.5Gy acute dose of gamma radiation (Seymour and
Mothersill 2000; Liu Z et al 2007). Therefore, it is not surprising that there is no correlation of
these symptoms with dose; this idea has been discussed and reviewed in Mothersill and
Seymour 2016 cited above.
Chronic Fatigue and Immune Deficiency Syndrome
CFIDS is a disease without a definitive cause or mechanism. CFIDS patients claim that low dose
exposure to ionizing radiation may be a common factor involved in their disease (Loganovsky
2000; Pastel 2002). While investigation into the disease has yielded many findings over the last
few decades, the full nature of the disease remains unresolved. The purpose of this review is to
examine the literature concerning a few core biochemical pathways, such as mitochondrial
dysfunction, the cellular oxidative stress response, and inflammatory processes, where
radiation exposure is known to cause perturbations and where there is evidence that the
pathway is aberrant in patients with CFIDS. By drawing the two largely independent fields of
CFIDS and radiation research together, it is hoped that common ground can be found which
could inform new testable hypotheses in the area.
CFIDS is a disease primarily characterized by persistent or recurring long-term fatigue and
susceptibility to infections (Holmes et al. 1988). Patients present a combination of other
symptoms in addition to the ubiquitous lassitude, including headache, muscle aches,
impairment of neurocognitive functions, and reduced quality of sleep (Evengård et al. 1999),
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symptoms usually encountered in patients following acute radiation exposure as well
(Kumerova et al. 2000; Pastel 2002). Considering that the disease has affected millions since it
was discovered, it is surprising that researchers are still uncertain about the exact cause of
CFIDS and the mechanisms behind its manifestation. Despite some stigma towards the pursuit
of research concerning CIFDS outside of a psychopathology (Anderson et al. 2013), recent
studies have shown that CFIDS almost certainly has a physiological basis. While it is important
to distinguish between the putative immunologic, physiologic, and neurologic models of CFIDS,
it is also important to understand, as eloquently argued by Whiteside & Friberg (1998), the
potential for interconnectedness among the three and work towards developing a
comprehensive model that explains all facets of the illness. Among the potential immunologic
and neurologic factors that may contribute to the onset and maintenance of CFIDS pathology,
one of the most exciting areas involves the investigation of the metabolic symptoms of the
disease.
CFIDS, Mitochondria, and Metabolism
A recent comprehensive study of the metabolomics of patients with CFIDS carried out by Dr.
Robert Naviaux et al. (2016) effectively showed that the disease can be characterized as a
“highly concerted hypometabolic response” to environmental stress, and one that can be easily
traced to mitochondria. Specifically, the study showed that the metabolic response was
homogeneous to heterogeneous stressors by examining a variety of biochemical pathways that
were consistently altered or abnormal in patients with CFIDS. The study also suggested that
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every metabolic abnormality observed in CFIDS patients was either regulated directly by redox
reactions or the availability of NADPH. In addition to this, they found that many of the NADPH-
dependent enzymes identified in the study had mitochondrial isoforms that were known to be
upregulated during periods of environmental stress. They describe NADPH as a “global
barometer of cellular fuel status” by interacting with both mitochondrial NADH consumption
and the availability of similar reducing agents in the cytosol. Considering the findings of this
paper, it is not only appropriate to propose that CFIDS may be intrinsically linked to metabolic
dysfunction, but also that it may be consequently inextricably linked to the function, or rather
the dysfunction, of mitochondria.
The idea that CFIDS is correlated to the improper functioning of mitochondria is not a radically
new one; in fact, it has been described by many authors in the past. When taking into account
the symptoms of CFIDS alone, they all appear to stand out as a hallmark of mitochondrial
dysfunction in terms of energy production and deficit in the cell (Filler et al. 2014). The same
review by Filler et al. (2014) proposed that significant downregulation of genes associated with
antioxidant mechanisms, aerobic energy production, and metabolism in peripheral blood
mononuclear cells of CFIDS patients when compared to control groups. The article also
described proper mitochondrial function as negatively correlated with fatigue symptoms and
another study determining the partial blockage of an ADP-ATP translocator in the mitochondria
in patients with CFIDS (Booth et al. 2012). Oxidative stress, which can be caused by the
presence of compounds such as radical oxygen species (ROS) and radical nitrogen species (RNS)
in a cell, is another feature indicative of mitochondrial dysfunction (Lin & Beal 2006) which is
also observed in patients with CFIDS, according to the literature. One study (R Kurup & P Kurup
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2003) showed that antioxidants, which include compounds such as superoxide dismutase that
are produced by cells to combat oxidative stress, were decreased in patients with CFIDS; in
addition to this, they suggested that, due to decreased free-radical scavenging, that ROS and
RNS were increased in plasma samples of CFIDS patients when compared to control groups
potentially leading to mitochondrial dysfunction. They proposed that free-radical generation
could be implicated in the pathogenesis of CFIDS, accounting for muscle pain and fatigability in
patients. ROS have been shown to be involved in a variety of diseases and therefore their
presence generally results in a greatly diminished quality of life if improperly regulated in
human cells; high concentrations of ROS in a cell can cause oxidative damage to integral
metabolic components (Panieri & Santoro 2016), DNA damage (Tada-Oikawa et al. 2000), and
can eventually induce apoptosis (Armeni et al. 2004). Interestingly, one clinical study (Fulle et
al. 2000) noted specific oxidative modifications and reduced oxidative metabolism in the
myocytes of patients with CFIDS. Another study (Wawrzyniak et al. 2016) showed impaired
biogenesis signalling and abnormal mitochondrial content in the myocytes of older individuals
with idiopathic, or rapid-onset, chronic fatigue. When taken as a whole, these findings suggest
that oxidative muscle damage and impaired mitochondrial function could be culprits in the
onset of CFIDS. Due to the presence of mitochondrial dysfunction in patients with CFIDS and
organelle’s tendency to produce damaging radical chemical species (RCS) if perturbed, the
mechanisms that contribute to oxidative stress and their relationship to the disease should be
of particular interest to researchers.
According to a study by Myhill et al. (2009), there is considerable evidence that mitochondrial
dysfunction is present in some sufferers of CFIDS. Biopsies of human muscles in CFIDS patients
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have shown abnormal mitochondrial degeneration. (Byrne et al. 1985; Behan et al. 1991;
Vecchiet et al. 1996). Further studies have shown that CFIDS patients contain deletions in
mtDNA genes associated with energy production, specifically in skeletal muscle (Zhang et al.
1995; Vecchiet et al. 1996). Increased activity of antioxidant enzymes and RCS-linked oxidative
damage has been observed in the muscles of CFIDS patients (Fulle et al. 2000). Decreased levels
of chemicals essential in metabolic reactions in mitochondria have also been shown to be
deceased in CFIDS patients. (Kuratsune et al. 1994; Plioplys and Plioplys 1995). Reduced
oxidative metabolism has also been observed in patients with CFIDS along with higher levels of
lactate and pyruvate (Arnold et al. 1984; Buist 1989; Wong et al. 1992; McCully et al. 1996; Lane
et al. 1998). Two studies showed that lipid replacement and antioxidant nutritional therapy
administered through a dietary regimen can help restore mitochondrial function and alleviate
fatigue symptoms (Nicolson 2003; Nicolson and Ellithorpe 2006).
The Evidence for Potential Involvement of Radiation
Many publications have linked ionizing radiation to the mitochondria-dependent generation of
ROS and RNS, primarily linking it to radiation-induced bystander effects and genomic instability
(reviewed in Szumiel 2015). According to Azzam et al. 2012, short and long-term radiation
induced ROS/RNS could result to damage to mitochondrial DNA in genes coding for electron-
transport subunits and their associated proteins. Cells without a functioning electron transport
chain do not experience ROS/RNS production (Leach et al. 2001). Cells with ROS/RNS induced
mtDNA damage could produce dysfunctional proteins associated with the electron transport
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chain, resulting in pro-oxidant chemical formation, which can eventually lead to de-novo
oxidative damage to biological structures long after radiation exposure with the mtDNA
potentially being passed on to daughter cells after division contributing to genomic instability
(Bogenhagen and Clayton 1977; Spitz et al. 2004; Gaziev and Shaĭkhaev 2007; Azzam et al.
2012). An in-vivo study using mice exposed to 0.2 Gy of x-rays found significant changes in
pyruvate metabolism and structural proteins persisting four weeks after exposure in heart cells.
Higher doses resulted in respiratory complex I and III partial deactivation as well (Barjaktarovic
et al. 2011). A few studies have shown that mitochondrial protein import could be used as a
marker for the long-term effects of ionizing radiation exposure, even at small doses, as this
could be the result of damage to components in translocational machinery or changes to
membrane potential (Pandey et al. 2006; Azzam et al. 2012).
After reviewing the literature, it is plausible to suggest that the production of ROS due an
endogenous or exogenous cause can contribute to the dysfunction of mitochondria in patients
suffering from CFIDS, among other potential causes. It is generally accepted that the primary
target for radiation effects in biological systems is DNA. However, in addition to the targeted
effects of ionizing radiation on DNA, the radiolysis of water and other compounds a human cell
result in the formation of ROS which can directly alter DNA through oxidative damage (Dandona
et al. 1996). Many RCS are known to damage many biological components in cells other than
DNA, including membranes that are essential to the homeostasis of the cell (Bonnekoh et al.
1990; Kryston et al. 2011; Martin et al. 2011). A high concentration of ROS in the cell can
critically compromise the mitochondrial membrane and affect the mitochondrial membrane
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potential, which is crucial for the production of the majority of aerobic energy in cells (Tada-
Oikawa et al. 2000). As reviewed earlier, many studies have linked the production of RCS to
mitochondrial dysfunction and to CFIDS, suggesting involvement of factors such as lipid
peroxidation, mtDNA damage, pyruvate metabolism, and protein import. Other studies outside
of the field have observed very similar events in cells exposed to ionizing radiation, both at high
and low doses. Considering the broader metabolic abnormalities in individuals suffering from
CFIDS and the link to ATP production (Myhill et al. 2009), there is some compelling evidence in
contemporary scientific literature to warrant the association of CFIDS and mitochondrial
dysfunction with ionizing radiation exposure and general RCS production. If these connections
are juxtaposed with the strong evidence for a biophoton bystander effect as described by Le et
al. in Radiation Research (2015) and Le et al. in Physics in Medicine and Biology (2015), it is
reasonable to suggest that CFIDS may be aggravated or even caused by low level radiation
exposure through a novel signalling cascade.
It is difficult to estimate how many different pathways are linked to mitochondria that might be
affected by ionizing radiation either directly or indirectly by bystander signaling. The approach
in this paper is to focus on pathways and proteins involved in low dose radiation response that
are also suggested as being involved in CFIDS. The particular focus is on involvement in the
phenomena of cellular oxidative stress and altered cellular energy metabolism and production. Accep
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Biochemical Pathways and Proteins of Interest
Alpha-synuclein
Alpha-synuclein (α-syn) is, by virtue of scientific debate, either an intrinsically unfolded protein
(Riedel et al. 2011) or a protein that might undergo folding with the help of heat shock proteins
(HSPs) to assume its native conformation in some circumstances (Bruinsma et al. 2011). In
humans, α-syn has no known function other than its deleterious role in disease (Riedel et al.
2011). α-syn is known to readily oligomerize, fibrillate, and aggregate, and is the major
component of inclusion bodies in cells of patients with Parkinson’s, Multiple System Atrophy,
and certain forms of Dementia (Bruinsma et al. 2011). Specifically, the same paper showed that
α-syn is known to interact with many HSPs, including αB-crystallin and sHspB8, with different
mutations altering the ability of HSPs to prevent fibrilization.
α-syn is thought to have two major forms: a membrane-bound and nonmembrane bound form.
This is believed due to the finding that α-syn has the capability to interact with poly-
unsaturated fats, which, in turn, may help promote the oligomerization process (Riedel et al.
2011). Following this finding, it was postulated that α-syn exists in its nucleating form when
bound to membranes and its monomeric form when in the cytosol (Riedel et al. 2011). Because
unsaturated fats are known to easily undergo lipid peroxidation after exposure to ionizing
radiation (Hammer & Wills 1979), it was also proposed that subsequent α-syn oxidative
modification by lipid peroxides and their radical intermediates generated from proximal
membrane material could promote its aggregation (Thomas et al. 2007; Riedel et al. 2011).
Studies concerning the tendency for α-syn to aggregate independent of cellular components
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after overexpression and ROS introduction respectively have also been conducted
(Andrekopoulos et al. 2004; Uversky et al. 2005). Aggregation of α-syn consequently promotes
further oxidative stress in the cell (Flower et al. 2005) and could lead to the compromising of
broader cellular metabolism. Indeed, increased α-syn levels have already been linked to
compromised mitochondrial function and altered cellular metabolism (Hu et al. 2009; Brown et
al. 2016a). A recent article (Brown et al. 2016a) demonstrated that α-syn binds to TOM20, a
mitochondrial outer membrane translocase receptor, causing deficient mitochondrial
respiration, increased ROS production, and loss of membrane potential. Other studies have
shown that mitochondrial dysfunction and ER stress can lead to α-syn-mediated cell death
(Smith et al. 2005). Another study (Booth et al. 2012) found that blockage of mitochondrial
membrane proteins is a potential biomarker of CFIDS. Therefore α-syn should be further
examined with regards to its potential role in CFIDS and other diseases.
One study (Saligan et al. 2013) also showed statistically significant correlations between α-syn
induction, ionizing radiation, and fatigue symptoms. The extensive study showed that cancer
patients exposed to gamma rays by external beam radiotherapy (EBRT) showed elevated α-syn
expression after the therapy. The paper also found that the fatigue experienced by patients
following the treatment was significantly correlated to α-syn plasma concentration, which
indicates that the EBRT produced the same effects on α-syn expression as it did on the fatigue
experienced by the patients. While this study alone does not conclusively link α-syn to fatigue
and radiation exposure, it raises some important questions concerning its role in cellular
metabolism and inflammation in relation to fatigue symptoms. The underlying mechanisms of
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this relationship could prove very insightful in the study of CFIDS and its association with
ionizing radiation.
In conclusion, α-syn could be a protein that is highly involved in the development or
aggravation of CFIDS in many ways. Its link to membranes and lipid peroxidation, mitochondria
and cellular metabolism, and radiation treatment-related fatigue symptoms warrant further
investigation into its role and relationship to ionizing radiation in the context of CFIDS (Figure
1).
Lactate Dehydrogenase and STAT1
Lactate Dehydrogenase (LDH) is an enzyme that is involved in the reaction that converts lactate
to pyruvic acid and back using NAD+ and NADH in the cell. Because of its abundance in the cell
and because it is mainly released following tissue damage, it is an important marker for
measuring alterations to the cell membrane (Bonnekoh et al. 1990; Armeni et al. 2004).
Presence of excess lactate has been linked to plasma acidosis and muscle fatigue (Lemire et al.
2008). Studies conducted in the past few years have shown that the importance of LDH may be
greater than previously thought, as lactate was identified as a major energy source for human
neurons (Hall et al. 2009; Boumezbeur et al. 2011; Wyss et al. 2011). Lactate concentrations are
positively correlated with radioresistance, which could indicate intrinsic antioxidant properties
(Hirschhaeuser et al. 2011).
LDH subtype A (LDH-A) is an isozyme that is highly expressed in various kinds of malignant
tumors and is probably heavily involved in the Warburg Effect (Fantin et al. 2006). The Warburg
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Effect is primarily used to describe the metabolism of tumor cells, wherein a switch is made in
generating the majority of energy between oxidative phosphorylation and aerobic glycolysis
(Vander Heiden et al. 2009). Reduction of oxidative phosphorylation efficiency has been
established in CFIDS patients in neutrophils, with a switch to glycolysis postulated after blood
samples were taken from CFIDS patients and assayed (Booth et al 2012). When taking into
consideration the fact that mitochondrial oxidative phosphorylation produces ROS, it is thought
that this effect limits the production of ROS in normal cells (Vander Heiden et al. 2009). LDH-A
is also considered a potential target of cancer suppression. A recent study (Kim et al. 2014)
showed that inhibition of LDH-A activity decreases the conversion rate from pyruvate to lactate,
and consequently mitochondrial membrane potentials and cellular ATP levels as well; this
ultimately leads to oxidative stress in the cell. Reduction of the production of ATP is one of the
many biomarkers that have recently been discovered in CFIDS patients’ blood and has been
tested extensively (Myhill et al. 2009). A few studies have further linked LDH to mitochondria by
indicating that LDH can exist inside mitochondria, contributing, much like cytosolic LDH, to
promoting a lactate balance in cells by dehydrogenating lactate produced in the cytosol (Brooks
et al. 1999). While there was some debate about the existence of LDH in mitochondria
(Rasmussen et al. 2002), a few recent studies have unequivocally confirmed its presence
(Lemire et al. 2008; Passarella et al. 2014).
Signal Transducer and Activator of Transcription 1 (STAT1) is a transcription factor in humans,
traditionally thought to be a protein involved in interferon signalling. Pitroda et al. (2009)
identified STAT1 as a transcription factor of LDH-A, among many other proteins involved in
metabolic energy pathways. They suggested that STAT1 works closely to regulate glycolysis and
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even demonstrated that STAT1 helps to rescue the expression of proteins essential to
metabolic energy pathways after x-ray induced suppression. Another paper studied the
exposure of STAT1 to UVA. After exposure to UVA, the modulation of STAT1 expression
assumed a biphasic characteristic, according to Mazière et al. (2000). They also found that this
effect was reduced with the introduction of antioxidants, indicating the presence of ROS and
oxidative stress probably play a role in the modulation of STAT1. Furthermore, the regulation
and activation of STAT1 is complicated by the need for tyrosine/serine phosphorylation and the
MAPK/ERK pathway, as inhibiting either phosphorylation or the MAPK/ERK pathway inhibits the
activation of STAT1. STAT1 is also particularly interesting because of its close involvement with
interferon signalling; pertinent to CFIDS, STAT1 is involved downstream of interferon gamma, a
signalling molecule modulated and typically decreased in lymphocytes of patients with CFIDS, in
one pathway (Klimas et al. 1990; Visser et al. 1998; Patarca 2001; Mulero et al. 2015; Baris et al.
2016). The potential role of STAT1 in regulating cellular metabolism and LDH-A could suggest its
involvement in a variety of diseases, in addition to its link to interferon signalling in the context
of CFIDS. Moreover, the ability of ionizing radiation to affect, let alone modulate, the
expression of STAT1 is cause enough for further research into the effects of ionizing radiation
on LDH and its regulators, the interplay of ROS, and ultimately the pathways’ possible
contribution to CFIDS. Further discussion of STAT1 in relation to the expression of RNase L is
discussed in the Ribonuclease L section.
Studies have also shown that radiation-induced senescence of human breast cancer cells also
caused the upregulation and activation of LDH (Liao et al. 2014). The study by Liao and his team
also showed that, by altering the monocarboxylate transporter 1 (MCT1), adenosine
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monophosphate-activated protein kinase (AMPK), and the nuclear factor kappa-light-chain-
enhancer of activated B cells (NF-κB) signalling pathways, radiation can effectively induce the
senescence of cells by metabolic alterations. The AMPK pathway is particularly interesting, as it
mediates the cellular response when the AMP/ATP ratio becomes too high as the cell
experiences energetic stress. The study also showed that invasion of surrounding cells and
radiation-induced bystander signalling was dependent on the MCT1, AMPK, and NF-κB
pathways. Recent studies have linked the NF-κB pathway and loss of p53 directly to CFIDS
(Morris & Maes 2012). A very interesting review proposed a common etiology between post-
radiation syndrome and CFIDS. Pall (2008) described post-radiation syndrome as a CFIDS-like
disease and linked the entire illness to the nitric-oxide/peroxynitrite cycle (NO/ONOO-). The it
was proposed that the cycle involves NF-κB and interferon gamma signalling and oxidative
stress, culminating in a “vicious cycle” that chronically elevates levels of nitric oxide. It was also
concluded that radiation can, even at relatively low doses such as those experienced by
Chernobyl recovery workers, can lead to the activation of this cycle, primarily through NF-κB.
Moreover, another study (Badalà et al. 2008) found elevated ventricular lactate in CFIDS
patients and implicated oxidative stress as a cause of the disease. These findings suggest that,
while CFIDS may be a complicated disorder with many different symptoms, more research must
be done in order to rule out the direct involvement of LDH and its associated proteins in the
illness.
LDH is an important protein with respect to cellular metabolism and energy production. The
involvement of LDH and its associated pathways in the Warburg Effect, oxidative stress,
mitochondrial structural and functional integrity, STAT1 regulation pathways, and radiation-
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induced activation and senescence all necessitate future endeavours to fully understand its
potential role in contributing to CFIDS symptoms (Figure 2).
Cytochrome c and Cytochrome c oxidase
Cytochrome c oxidase (CCO) is a large multi-protein complex located in the mitochondria of
eukaryotes. Also known as Complex IV, it is a main component of the electron transport chain,
where it donates four electrons to molecular oxygen and transfers four protons to it as well,
creating two molecules of water in the process. In addition to this, it pumps protons into the
intermembrane space of mitochondria to create the electrochemical gradient required to make
ATP (Denis 1986). Cytochrome c (CC) is a hemeprotein associated with the inner membrane of
mitochondria; essential for the electron transport chain as well, it has the capacity to carry a
single electron, to donate that electron during oxidative phosphorylation, and to shuttle
electrons between Complex III and Complex IV.
CC is crucial in many pathways in the cell. Its release from the mitochondria following
mitochondrial permeabilization is necessary for many pathways leading to apoptosis (Yang
1997). Its involvement in the electron transport chain makes its uncompromised role an
important factor in the viability of cells with respect to energy production. CC has the capability
to produce intramitochondrial ROS when interacting with several proteins, leading many to
believe that it is involved in signalling and mitochondrial damage through ROS-propagation
pathways (Giorgio et al. 2005; Marchi et al. 2012; Panieri & Santoro 2016).
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A study by Tirelli et al. (1994) indicated that there were no significant differences in CCO
reduction between control groups and CFIDS patients. Indeed, a few other older scientific
articles note no significant disparities between the amount of CCO reduction in healthy
individuals versus CFIDS patients, particularly in muscle tissue where the energy demand is high
for cells (Byrne & Trounce 1987). However, more recent publications have noticed some
important connections between CCO and CFIDS.
The aforementioned link that RCS have to mitochondrial dysfunction and CFIDS has been
studied extensively in the literature. A study (Brown 2001) found that CCO can be reversibly
impaired by a radical nitrogen species, NO in this case, as CCO competes with it for access to
oxygen. It noted that prolonged or high dosages of NO or any of its derivatives can cause
irreversible deleterious effects, such as CCO’s uncoupling, mitochondrial permeability, and even
cell death. Another article noted that CC can be modified by nitration of its tyrosine residues
after low doses of peroxynitrite are administered (Marchi et al. 2012). The authors found that
the nitration of CC also resulted in the upregulation of peroxidase activity for hydrogen
peroxide in the cell and the impairment of membrane potential formation, likely a result of
damaged subunits in the electron transport chain. Oxidative damage to mitochondrial proteins
and complexes crucial to oxidative phosphorylation not only results in impaired ATP
production, but also unregulated ROS production by the affected complexes, which leads to
amplification of ROS in mitochondria and increased release into the cell (Hosseini et al. 2014;
Zorov et al. 2014). It has been shown that the production of peroxynitrite and its derivatives,
NO included, can be induced by x-ray exposure in mice (Hanaue et al. 2007), which could
potentially lead to CC nitration, multi-complex corruption, RCS proliferation, mitochondrial
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membrane depolarization, and reduced ATP production leading to ROS contribution to fatigue
and even tissue insult as a result of mass cell death. The theme that the nitric oxide cycle and
elevation of NO has something to do with CFIDS in humans is pervasive in the literature; many
papers have been published on the topic, including a few linking the nitric oxide cycle to
radiation exposure and PRS (Pall and Satterlee 2001; Pall 2007, 2008, 2009).
The biggest problem with studying CC and CCO is their nature as incredibly complicated
macromolecules, particularly CCO. Be that as it may, the roles that they might have in CFIDS
should not be underestimated. Both molecules are already being studied heavily, and the fact
that have already been implicated in a number of diseases only further necessitates research.
The discovery of RCS signalling pathways and the involvement of CC in them, the potential for
exogenous RNS to affect the metabolism of mitochondria through direct covalent interactions
with CC and competition with CCO, and the demonstrated radiation-induced production of a
reactive species of nitrogen implicated in these cascades in vivo (Figure 3) beckons the need for
further research into CC and CCO.
αB-crystallin
αB-crystallin (αBC), encoded by the gene CRYAB, is a protein that is a member of the small heat
shock protein (sHsp) family and is involved in binding to unfolded proteins to inhibit their
aggregation and ultimately preventing apoptosis (Boelens 2014). αBC interacts with many
proteins, inhibiting the formation of mature α-syn fibrils (Hsu et al. 2000; Riedel et al. 2009),
reducing the ubiquitination and degradation of a pore-forming subunit of major cardiac voltage
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gated sodium channel (Huang et al. 2016), and binding to p53 in addition to several pro-
apoptotic proteins and preventing their translocation to mitochondria (Liu S et al. 2007). In fact,
the interplay between p53 and αBC shows tight regulation on a transcriptional and post-
translational level and affects oxidative stress and the Warburg Effect in human cells. It was
discovered that p53 binds directly to the promoter region of CRYAB to enhance its expression;
transfection of a p53 overexpression vector into MCF-7 cells increased expression of αBC,
reduced intracellular ROS, caused the Warburg Effect, and increased ROS after p53 was
depleted (Liu S et al. 2014). It was also shown, as stated previously in the LDH section, that
reduced levels of p53 is indicative of mitochondrial dysfunction and directly linked to CFIDS.
Many sHSPs are mobilized during oxidative stress (Dimauro et al. 2016), and in case of αBC, a
few crucial mitochondrial pathways involved in ROS production and cell damage might be one
of the major reasons for this.
αBC was shown to be modified heavily by the reactive nitrogen species peroxynitrite, where
peroxynitrite enhanced its chaperone and antioxidant abilities (Morris & Maes 2012). This is
very interesting when noting that peroxynitrite is heavily involved in compromising CC and CCO,
as discussed in the Cytochrome c section, and is thought to be central to the etiology of CFIDS
and PRS (Pall and Satterlee 2001; Pall 2007; Meeus et al. 2008; Pall 2008, 2009). Specifically,
there appears to be the potential for rescue in the Cytochrome c and Cytochrome c oxidase-
mediated ROS pathway after radiation exposure when considering the scientifically-
corroborated observation of radiation-induced formation of peroxynitrite (Zorov et al. 2014).
αBC was also shown to inhibit the release of Cytochrome c from mitochondria (Xu et al. 2013),
potentially by binding to mitochondrial transmembrane channels. As shown by Chis et al.
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(2012), Cytochrome c co-localizes with αBC during periods of oxidative stress, as are other pro-
apoptotic proteins. αBC was also shown to have antioxidant properties by binding directly to
Cu2+ ions and rendering them inactive (Ahmad et al. 2008). Crystallin genes have been shown
to be highly induced following radiation exposure in human cells and are closely linked to
mitochondria-stress pathways (Andley et al. 2000; Parcellier et al. 2005; Chaudhry 2006).
Could αBC be implicated in CFIDS? While the connections made to CFIDS and radiation may not
be conclusive, this chaperone is clearly involved with many aspects of proper cellular function.
The fact that αBC is involved in regulating every protein discussed in this paper, the fact that
increased αBC is correlated with increased lactate levels and the Warburg Effect, and the fact
that is relatively sensitive to ionizing radiation only speaks to the need to research αBC with
respect to radiation and CFIDS (Figure 4).
Ribonuclease L
Ribonuclease L (RNase L) is a ribonuclease that, upon activation, destroys RNA that resembles
viral RNA. RNase L is mobilized after either a type I interferon signalling cascade involving the
stimulation of JAK kinases and after activation of STAT1 and/or STAT2 results in transcription of
2,5A Synthase (2-5OAS); this synthase is activated by ss and dsRNA to create 2’-5’-
oligodendylates (2-5A), which bind to and activate RNase L (Stark et al. 1998; Silverman 2007a).
RNAse L is also believed to be involved in apoptosis, specifically during the cell’s efforts to enter
apoptosis following viral infection (Chakrabarti et al. 2011). Indeed, after a certain point of
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activation, RNAse L is capable of destroying essential RNA in the cell in an attempt to induce
apoptosis to prevent the infection from spreading (Silverman 2007b).
It has been believed for some time that RNase L cleavage is linked to CFIDS. The protein exists
in its functional 83 kDa form in both CFIDS and healthy groups. Various truncated forms of
RNase L also exist, albeit mostly in unremarkably similar concentrations in both groups.
Curiously, some studies suggest that the 37 kDa form of the protein exists preferentially in
CFIDS groups (Suhadolnik et al. 1997; De Meirleir et al. 2000). One study in particular
attempted to link the upregulated activity of elastase to the cleavage of RNase L (Meeus et al.
2008). The same study found significant amounts of truncated 37 kDa RNase L in patients with
CFIDS. Upregulation of 2-5OAS has been directly linked to CFIDS as well, and one study found a
significant correlation in the expression of 2-5OAS with fatigue and depression symptoms
stimulated by interferon alpha treatment in patients with Hepatitis C (Felger et al. 2012).
RNAse L has been shown to have capabilities to cleave 28s rRNA in mice as part of an attempt
to prevent viral proliferation (Iordanov et al. 2000); another study showed RNase L-
independent 28s cleavage can occur during viral infection (Banerjee et al. 2000). A study by
Iordanov et al. (2000) suggested that the degradation of 28s rRNA, which makes up the large
ribosomal subunit, could lead to overall reduced translation in the cell and induction of pro-
inflammatory cytokines to the cell as part of the innate immune response. Such an implication
would mean that the loss or dysregulation of RNase L in any way could lead to an inappropriate
response to viral infection and serious immune dysfunction, which could be a route to
developing the immune symptoms present in CFIDS.
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The possibilities for the involvement of radiation in this pathway are virtually limitless and
mostly unexplored in the literature. For instance, it has been shown that RNAse L plays an
important role in upregulating the cellular response to gamma radiation-induced DNA damage
by affecting the transcription of proteins such as p21 (Al-Haj et al. 2012). It has been shown
through several studies that various kinds of kinases, including MAP and ATM kinases, can be
activated by ionizing radiation to phosphorylate their targets (Chen et al. 1996; Canman et al.
1998; Iordanov et al. 2000). Results from the Maziere et al. paper (2000) imply that the biphasic
modulation of STAT1 following radiation exposure might very well have something to do with
MAPK, JAK, or other similar tyrosine kinases, as inhibition of tyrosine kinase activity inhibited
the UVA-effect on STAT1. STAT1 is required for the transcription of 2-5OAS, which is essential
for the activation of RNase L among other interferon response genes in the Type I pathway
expressed throughout the human body in a multitude of tissues during viral infection (Mullan et
al. 2005). STAT1 is also necessary and sufficient to induce transcription of interferon-stimulated
genes in the Type II pathway specific to Type II interferons released from activated T
lymphocytes (Durbin et al. 2000; Mullan et al. 2005).
As described in the STAT1 section, it is important to note that there are many potential
pathways that may link radiation to its pathway and eventually CFIDS. Dysregulation of the
RNase L pathway is an interesting possibility for the manifestation of CFIDS. Many questions
still remain unanswered in this pathway with regards to radiation and CFIDS, necessitating
further research.
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Potential New Areas for Research
The literature discussed above confirms that ionizing radiation has been shown to be capable of
perturbing various pathways thought to be involved in the manifestation of CFIDS. Some areas
for future research are discussed below in the hope of contributing to our understanding of
CFIDS and its potential connection to radiation exposure.
Involvement of the Bystander Effect
Because of the potential for signal propagation through cell populations (Nagasawa and Little
1992; Mothersill and Seymour 1998, reviewed in Mothersill et al. 2017a; 2017b) as a
mechanism which can magnify the size of the target for effects after low doses, the following
experiments might help to determine if the effects can be extrapolated to living multicellular
organisms following localized radiation exposure. Such studies could lead to a better
understanding of the potential for cellular symptoms of radiation to proliferate in a cell
population to affect the system as a whole. Comparison of results with and without the
compounds discussed above would enable us to determine if the radiation response pathway
were actually impacted by the interference with the biochemical pathway.
The Peroxynitrite Cycle
The effect of the presence of peroxynitrite on αBC, CC, and CCO (Brown 2001; Andrekopoulos
et al. 2004) could be a primary focus as a potential pathway in the proliferation of ROS,
mitochondrial and cell damage, energy deficiency, and apoptosis. As noted earlier, the
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formation of peroxynitrite can be induced by x-ray exposure, particularly in mice (Hanaue et al.
2007), and a few papers (Pall 2007, 2008, 2009) have noted that RNS pathways could be the
missing link between CFIDS and radiation exposure in humans.
The first step would be to identify a human cell line where the production of peroxynitrite
occurred. This would be of high interest with regard to CFIDS. A control with established
baseline concentrations of peroxynitrite and its derivatives would have to be determined,
before looking at the impact of ionizing radiation on other samples. The baseline production of
other ROS and RNS species could be assayed as well in this step. If initial results are promising
an experiment attempting to correlate the radiation dose, the peroxynitrite concentration, and
the nitration of Cytochrome c could be performed. Assaying the modification of Cytochrome c
could be done using sodium dodecyl sulfate polyacrylamide gel electrophoresis using a
molecular weight ladder and comparing the untreated lane to the varying radiation
dosages/concentrations of peroxynitrite. Using SDS-PAGE in this way could show a shift in mass
of cytochrome c between the untreated samples and the treated samples, answering the
question of whether increased radiation exposure leads to increased nitration of Cytochrome c.
If this does not work due to the change in mass being potentially too small to detect, other
methods could be discussed. Again, this experiment would only attempt to establish whether
increasing doses of gamma radiation exposure alter Cytochrome c through nitration
mechanisms.
Using immortalized human cell lines, preliminary clonogenic assays could potentially show the
reduction in viability of cells that undergo this kind of alteration to Cytochrome c, which could
potentially link the alteration to damaged electron transport chain (ETC) components.
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Bystander signals are already known to impact the ETC (Le et al. 2017) This step would
putatively link reduced viability caused by radiation exposure to cellular respiration based on
Cytochrome c’s intimate involvement in oxidative phosphorylation. A direct assay of the
alterations in each Complex by various methods might reveal some damage as well, which
would support the theory, although not directly. An assay of the functionality of Cytochrome c
in irradiated cells would have to be done to implicate it in reduced cell viability.
αB-crystallin could be involved in several ways in this process. It is suspected, based on
literature review, that alterations to αBC by peroxynitrite enhance its antioxidant and
chaperone properties. There is the potential to measure the rescue of Cytochrome c by
showing that αBC binds directly to Cytochrome c after irradiation and consequent nitration, as
it has been shown to co-localize with unaltered Cytochrome c (Chis et al. 2012). This could be
confirmed using a co-immunoprecipitation assay. This would involve isolating modified
Cytochrome c using an antibody, then identifying potential binding partners using a Western
Blot in the hopes of identifying αBC. It would also be interesting, assuming Cytochrome c is
bound by αBC after irradiation, if the affinity increases or decreases with more nitration and/or
exposure to radiation.
Because Cytochrome c oxidase is involved in competition with nitrogen species, this could be a
novel mechanism induced by ionizing radiation that could be linked to oxidative stress and
CFIDS in the future. Considering that ROS production, Cytochrome c, and αB-crystallin are
involved in cell metabolism, energy production, and have been linked to CFIDS in some way,
this pathway that involves the production of ROS, the loss of function of Cytochrome c, and αB-
crystallin is interesting when considering its ties to ionizing radiation. This experiment would
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clearly not determine if this pathway operates in vivo in those suffering from CFIDS, however
elucidation of these pathways and their associated proteins is most definitely a good start.
P53, αB-crystallin, and ROS
Some very interesting regulation can be found in the p53 and αBC pathway. It appears as
though αBC negatively regulates the apoptotic functionality of p53 (Liu S et al. 2007). P53
positively regulates the expression of αBC (Liu S et al. 2007; 2014) and a number of proteins
that probably interact with other proteins involved in cell metabolism in order to induce the
Warburg Effect (Morris & Maes 2012; Liu S et al. 2014; Panieri & Santoro 2016).
No published experiments have yet assayed αBC’s resilience to gamma radiation using small
doses. It would be interesting to irradiate a solution containing αBC in the soluble fraction at
low doses, run it on an SDS-PAGE gel, determine if there are any appreciable changes, and then
determine what percentage of αBC was lost due to the radiation compared to the control. The
same experiment could then be done, with cells. The cells could then be lysed, αBC isolated,
and then measured. This would be done alongside unirradiated controls to determine if the
fraction of functional αBC was appreciably altered. Clonogenic assays would be done at each
endpoint to determine if this effect can be correlated with survivability. If the results between
the experiments using the chemical alone or the chemical in cells are different, then we can
postulate that ROS had a role in the damage done to αBC in the cell. It could suggest that ROS
generated inside cells might have played a role in αBC’s reduction. This would suggest that
certain photosensitizers might help in the propagation of ROS and the damage that they do
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inside cells. If the converse was seen, then some compound or protein in living cells might be
protecting αBC from alterations caused by radiation that is not present in the chemical solution.
Further studies could be done to determine what radioprotector this might be, potentially by
reviewing the literature to identify putative binding partners and using coimmunoprecipitation
to validate them.
Considering that there are p53 +/+ and p53 -/- cell lines available, similar experiments can be
done on both cell lines. Firstly, the disparity between cells with no p53 and cells with p53
concerning expression of αBC would have to be established. Then, experiments could be done
in a similar fashion as above to try to link the reduction of αBC to pro-apoptotic pathways
involving p53, a shift to more glucose in the cell, and ROS production. Even though these
experiments would be performed on human cell lines and the results would not be directly
applicable to CFIDS patients, determining how these pathways behave in certain situations
could give researchers more potential biomarkers to observe in CFIDS patients after follow-up
studies testing for relevance in such patients.
Lactate Dehydrogenase and the Warburg Effect
In one reviewed study, it was shown that ventricular lactate levels were significantly higher in
people with CFIDS (Badalà et al. 2008). This raises the question of whether or not rising
intracellular and intercellular lactate levels have a role to play in CFIDS. Perhaps damaged
mitochondria due to ROS induce a cellular response to use more aerobic glycolysis in the
cytosol while some mitochondria shut down. The problem with LDH is that it can be involved in
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a number of ways in the onset or aggravation of CFIDS: LDH could protect the cell from
oxidative damage, facilitate the switch to aerobic glycolysis if the mitochondria are damaged, or
facilitate the switch indirectly through a number of different pathways.
The mechanisms of such a switch could be triggered by radiation exposure. It has been shown
that lactate could be a radioprotective molecule (Hirschhaeuser et al. 2011) and that LDH-A can
induce oxidative stress if it is downregulated in cancer cells (Panieri & Santoro 2016). If LDH is,
in fact, an indirect radioprotective element in normal cells, then its loss would render cells more
susceptible to radiation. To determine if ROS play a major role in the radioprotective nature of
LDH, an assay measuring the antioxidant properties of LDH might be appropriate where radical
chemical species’ levels are being measured. A simple experiment would be to perform an
assay in a solution where RCS can be generated by radiation. Different doses of radiation can be
applied to each flask. After irradiation and measuring of relative reactive species in the
solution, LDH can be added to each with varying concentrations. The assay would be performed
again to measure RCS. If the trials show reduced RCS, then it could suggested that LDH is
sufficient as an antioxidant and can protect against radiation-induced generation of RCS
irrespective of the presence of other proteins or chemicals. If no significant reduction is seen,
then LDH might not be an antioxidant at all; much more likely than this however, is that LDH
does not have intrinsic chemical antioxidant properties, but might reduce reactive species in
the cell in the proper chemical environment and when assisted with auxiliary proteins. The
same set of experiments can be done again, but this time with LDH present in the solution
before irradiation. If a significant difference in ROS is seen between the two groups, then LDH
might be adversely or positively affected by radiation directly, which contributes to its
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functionality in a cellular environment. If there is no significant difference between the two
groups, then it can be concluded that LDH is not directly affected by ionizing radiation at the
doses and wavelengths used.
Another set of experiments could follow testing the effects of ionizing radiation of LDH in living
cells. These sets of experiments would involve irradiating cells and will attempt to link ionizing
radiation and generation of RCS to radioprotective effects in vivo. This would involve irradiating
cells at the same dosages as in the first set of experiments mentioned above. Assays measuring
the loss of LDH and generation of RCS would then be used to attempt to correlate the two. If
the results are positive, then the loss of LDH should correspond to the increase of RCS in the
cell. This would imply that the loss of LDH as a radioprotector might lead to the increase of RCS
in cells. In order to confirm this, a knockdown of LDH by means independent of radiation would
have to be performed to directly implicate the loss of LDH to increased RCS caused by radiation
in vivo. There are a few methods that could accomplish this, but the most direct one would
involve the use of RNAi. If this method is chosen, then LDH would be knocked down in cells by
RNAi and then subjected to radiation. The levels of LDH and RCS in the dosage groups would
have to be measured. When this experiment is complete, one could compare the numbers to
the previous experiment where RNAi-induced knockdown of LDH was not performed. If
significant disparities are seen between the groups, then it can be better concluded that LDH
plays a direct role in reducing RCS in human cells, either intrinsically or through some pathway
involving other proteins or chemicals.
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To take these experiments further, clonogenic assays should also be done to correlate this
effect to survivability. Lactate levels can also be measured in cells which would be anticipated
to go up following knockdown of LDH. The effects of this on cellular metabolism can also be
measured; membrane potentials of mitochondria can be measured using the JC-1 dye or
another stain for membrane polarization, the functionality of different complexes can be
assayed before and after, and so on. Every subsequent experiment would attempt to link the
reduction of LDH and radiation to metabolic dysfunction and contribute to identifying the
potential causes of cellular markers of CFIDS.
Alpha-synuclein and ROS
Researchers believe that the aggregation of α-syn causes ROS formation (Hsu et al. 2000;
Uversky et al. 2005; Boelens 2014). In a similar fashion, ROS production causes α-syn
aggregation (Hsu et al. 2000; Uversky et al. 2005; Riedel et al. 2011). One article suggests that
the proximity of α-syn to lipid membranes contributes to its oxidative modification upon lipid
peroxidation after irradiation, causing it to aggregate (Riedel et al. 2011). A few tests could
correlate the two. The purpose of these experiments would be to determine if α-syn can
undergo aggregation after irradiation, what mechanisms underlie this aggregation process, and
what effects are produced on the cell.
The first step would be to measure lipid peroxidation by some kind of assay after irradiation in
cells. There are several assays available to measure lipid peroxidation, with one of them being
an LDH assay. This would be done in live cells. Next, one could extract α-syn and run it on a gel
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to determine how much was lost when compared to unirradiated controls. Next, one could
irradiate extracted α-syn in solution at different doses and run that on a PAGE gel to determine
if any significant alterations occurred. After this experiment is complete, an experiment should
be performed where membrane tissue is isolated from human cells. One could then incubate
extracted α-syn with membrane tissue, irradiate at different dosages, purify the α-syn solution,
run it on an SDS-PAGE gel, and determine if there are any differences between this gel and the
one with α-syn alone. This would tell researchers if presence of fatty tissue affects the
alteration of α-syn by radiation. This would directly link lipid peroxidation to α-syn alterations if
the results are positive. If the results are negative, the finding that radiation-induced lipid
peroxidation, radical propagation, and termination in α-syn is negligible, and alterations happen
either directly or through the presence of another protein or species that can oxidize α-syn.
After this experiment, another experiment would consider the radiation-induced alterations of
α-syn in vivo using similar methods as above. There are a few possibilities in the outcomes of
each experimental sequence. The following lists all of the possible results and conclusions
following the full sequence of experiments:
1. Incubating and irradiating α-syn together with lipids produces no significant change in
the alterations of α-syn
Conclusion: Lipid peroxidation mechanisms are negligible in radioactive alterations to
α-syn.
a. Irradiating live cells containing α-syn produced no significant change in the
alterations of α-syn compared to ex vivo trials
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Conclusion: If different than unirradiated controls, then α-syn probably
undergoes alterations caused by radiation directly. If the same as unirradiated
controls, then radiation probably has no significant effect on the alteration of
α-syn, both in vivo and ex vivo.
b. Irradiating live cells containing α-syn produced a significant change in the
alterations of α-syn compared to ex vivo trials
Conclusion: While lipid peroxidation mechanisms are negligible, some other
mechanism exists that affects the oxidation of α-syn. This probably occurs by
photosensitizer species and/or other proteins; further investigation is needed
to identify these.
2. Incubating and irradiating α-syn together with lipids produces a significant change in the
alterations of α-syn
Conclusion: Lipid peroxidation mechanisms are at play in radioactive alterations to α-
syn.
a. Irradiating live cells containing α-syn produced no significant change in the
alterations of α-syn compared to ex vivo trials
Conclusion: Lipid peroxidation is the primary pathway that is needed for
oxidative alterations of α-syn.
b. Irradiating live cells containing α-syn produced a significant change in the
alterations of α-syn compared to ex vivo trials
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Conclusion: Lipid peroxidation is one of the primary pathways that are needed
for oxidative alterations of α-syn. Other mechanisms affect the oxidation of α-
syn; further research is needed to identify these pathways.
It is important to note that the above results would not be mutually exclusive, as a dosage
threshold may have to be reached before any one of these deterministic effects are observed. It
is also important to note that, due to the findings by Saligan et al. (2013), radiation dosages can
cause elevated α-syn concentrations; this will need to be accounted for by using many controls
and measuring quantity of protein in the PAGE gels by comparing them to the concentration of
the molecular ladder. If this method is deemed too inaccurate, another assay measuring
protein expression must be done for the controls before the experiment and for the test groups
after the experiments.
Clonogenic assays would be done at each endpoint in the in vivo trials to determine if
survivability was affected. These assays would help correlate the survivability of cells with
reduced α-syn functionality in the trials. Further experimentation can be done to elucidate the
other mechanisms that might affect the radiation-induced dysfunction and aggregation of α-syn
where applicable, and implicate the effects of bystander signalling and radioprotectors in these
results in future studies.
Overall, the findings in these experiments would contribute to understanding one pathway in
which CFIDS could potentially manifest. The experiments described above would be relying on
the ideas described in Saligan et al. (2013), wherein the group described elevated fatigue
symptoms with radiation therapy and increased expression of α-syn. Considering that
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subsequent studies will be needed to determine if this effect is relevant to CFIDS patients and
even if α-syn radiation-induced transcriptional and post-translational alterations cannot be
linked to fatigue symptoms for quite some time, these experiments will be a crucial first step in
determining the effects of radiation on α-syn from a molecular perspective.
A multitude of other experiments must be pursued in order to further understand the
involvement of these pathways in CFIDS, as these experiments are merely suggested directions
for future research. No one study will conclusively validate or disprove the connections that
these proteins have to CFIDS. Again, the purpose of all of these proposed experiments would
not be to indisputably and unassailably link any of the mentioned proteins to CFIDS, but rather
to help elucidate these pathways further and potentially give other researchers background
studies to begin making connections that could not be made before, potentially to CFIDS and
the influence of radiation.
Conclusion
Alpha-synuclein, cytochrome c, cytochrome c oxidase, lactate dehydrogenase, STAT1, and αB-
crystallin are but a few proteins potentially involved in the development of Chronic Fatigue
Syndrome. When taking into consideration the very real and exciting possibility of signal
propagation in cell systems via bystander signalling, the effects of radiation on the disease
might involve a completely novel mechanism of radiation-induced pathogenesis that could have
drastic implications in not only Chronic Fatigue Syndrome, but a number of other diseases as
well. Numerous very interesting biochemical pathways have been identified through literature
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review that could shed further light on the mechanisms behind Chronic Fatigue Syndrome in
the context of ionizing radiation, radical chemical species formation, cellular damage, and
apoptosis.
The beauty of modern biology is that it allows scientists to investigate innumerable biochemical
pathways involved in diseases that will inevitably converge on a set of causes for the disorder.
While the proteins under investigation have a broad range of potential functions in the cell,
determining the connection between these proteins and the metabolic and cellular
characteristics of Chronic Fatigue Syndrome may provide invaluable insights into the disease.
Understanding the molecular biology and biochemistry behind a particular disorder paves the
road to patient studies, the development of targeted treatments specific to one or more
pathways, therapy options, clinical trials, and eventually a treatment. In this circumstance,
taking a broader approach by targeting processes and proteins shown to be involved in Chronic
Fatigue Syndrome as well as many cellular characteristics of disease will identify targets for
more research, and will, eventually, lead to a substantiated and comprehensive model for a
disease that has burdened too many for far too long.
Acknowledgments
The CFIDS Foundation Inc. who funded this review and in particular the director - Dr. Alan
Cocchetto who pointed the authors in the direction of many of the papers covered by the
review and provided encouragement and ideas.
Declaration of Interest
The authors declare no conflicts of interest.
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