Exposure of excess iodine for 30 days at relatively

Exposure of excess iodine for 30 days at relatively high doses 0.7mg/kg
bw(100EI 30D) and 35mg/kg
bw(500EI 30D) in rats, alters functional
status of PBL as evident with the changes in oxidative stress parameters, serum
cytokine levels, proliferation assay of lymphocytes and the urinary iodine
excretion pattern depending on the dose of iodine administered.

Lymphocytes are a
diverse group of white blood cells 60–80% of these comprise of cytotoxic
T-cells, killer of virally infected and cancerous self-cells, cytokine
secretory helper T-cells and remaining 20–40% are natural killer (NK) cells and
B-lymphocytes. However each
subpopulation has different functional characteristics (Moser et al., 2010). Mature B and T cells are continuously circulated from
one peripheral lymphoid tissue to another to recognize antigens and to respond
accordingly. This continuous migration process is known as lymphocyte
recirculation. When, these lymphocytes encounter a recognized antigen in one of
the peripheral lymphoid tissues, they would become activated and initiate an
adaptive response. (Abbas et al., 2009). Lymphocytes have the ability in intensifying a specific immune
response against any foreign antigen. Maturation of individual lymphocytes makes it possible,
bearing a distinctive variant of a prototype antigen receptor; so that the
populations of T and B lymphocytes collectively bear a huge range
of receptors that are highly diverse in their antigen-binding sites (Janeway
et al.,2001). Further, lymphocytes are the sensitive biomarkers of DNA damage
and oxidative stress in blood (Collins et al., 1998).

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In
the present study, excess iodine exposure markedly reduced the net body weight
gain of the excess iodine administered animals depending on the doses as
compared to the control group. Excess iodine exposure markedly reduced the net body
weight gain in the treated animals depending on the doses possibly due to
metabolic alteration as observed by Yang et al (2006). Moreover, excess
iodine caused a decrease in body weight gain of calves Paulíková et al., 2002,
young rats, and survival rate of pups Schone et al., 2006 as reported
earlier. It
has been reported by Lewis et al.,(2004) and Chakraborty et al., (2014) that
after suppression of thyroid hormone activity, there occurs a relevant
reduction in cellular oxidation that may lead to reduced body weight after
excess iodine supplementation.        

Iodine
nutritional status was measured determining the urinary iodine content, as 90%
of the body’s iodine is excreted through it (Dunn et
al., 1993).There are studies where in iodine
nutritional status has been evaluated by measuring the urinary iodine
concentration in experimental animals. Lupachik et al., 2007; Chandra et al.,
2015. In the earlier study of Palaniappan
et al., (2017) a relationship between excess iodine intake and excreted urinary
iodine concentration was established where the higher level of urinary iodine
was excreted than the control. In the present study, compared to
control, the excretion of iodine was found to elevated dose dependently in the groups,
administered with higher doses of iodide in comparison to control group.

         Lymphocytes, the
most numerous circulating WBC after neutrophils, are responsible for immune
responses to specific organisms. Increase in differential number of blood
lymphocytes indicates the first line defense system (Cesta et al., 2006; Swirski et al., 2009). Our study indicates
the occurrence of activation of the body’s first line defense system during
excess iodine treatment on dose dependent manner. Mannem et al (2014) reported that enhancement of WBC
production by immune system is due to toxicity in body. Accordingly significant
increase in WBC count in excess iodine treated experimental animals as observed
in the present study found to be due to toxicity after such treatment. Increase in number of blood
monocyte indicates the inflammatory effect on rat immune system. Evidence from
murine and human studies has suggested that monocytosis can be an indicator of
various inflammatory diseases such as tissue damage and infection. Furthermore,
monocytes can differentiate into inflammatory or anti-inflammatory
subsets-tissue macrophages and dendritic cells (Yang et al., 2014). Our results
showed significant elevation of blood eosinophil count in excess iodine treated
rats (100EI 30D and 500EI 30D both). Ekambaram et al., (2010) showed in their
study that eosinophilic infiltration of the thyroid gland has higher
association with Hashimoto thyroiditis. Thus excess iodine has the potentiality
to develop Hashimoto’s thyroiditis in the long run, though we have not studied
it.

        The oxidative stress may reflect in
particular immune cells by altering the potential mechanism of the
physiological responses (Preutt et al.,
2009). Environmental factors as selenium, zinc and
arsenic found to be implicated in the interference of immune function through
generation of oxidative stress leading to autoimmune disorders (MacGillivray et
al, 2014; Dangleben et al., 2013). A
pro-oxidant state occurs to oxidative stress when a disproportion between
oxidizing e.g. reactive oxygen species (ROS)
and reducing agents (e.g. antioxidants) ensues Sies, 1997. Macro-molecular impairment
in the form of protein carbonylation, lipid peroxidation and DNA oxidation can be developed by extensive and
obstinate oxidative stress. According to the immune status, generation of ROS by neutrophil and intracellular and
extracellular discharge can be beneficial as microbial defense against the
pathogens to destroy it Dahlgren et al., 1999. Excessive ROS generation in the presence of immune complexes
with auto-antigens induces macromolecular damage. T-cell proliferation can be
inhibited by extensive exposure to high ROS concentrations that lead to
apoptosis Thoren et al., 2007.

The immunomodulatory effects of
low-level, chronic polychlorinated biphenyl PCB exposure were investigated in
female rhesus (Macaca mulatta) monkeys; the results indicate that low-level,
chronic PCB exposure affect immune system components due to altered T-cell
and/or macrophage function (Tryphonas et
al.,1991). In
this study the cytotoxic effects of excess iodine may reflect on lymphocyte
immune function. Abnormal response to oxidative stress i.e. a decreased level
of SOD activity in excessively excess iodine treated rats than that of control
rats was observed in the present study. These findings are in general agreement
with abnormal responses to environmental stressors and the induction of immuno-toxicity
(Preutt et al., 2009).As, iodine
is a strong oxidant, so that the results of the present study are well in line
with the earlier report that excessive iodine intake for a long time could
decrease the activity of anti-oxidation enzyme-SOD and GPx of blood, brain, and
thyroid (Xiang et al., 1999).

Peroxides,
including hydrogen peroxide (H2O2), are one of the main
reactive oxygen species (ROS)
leading to oxidative stress (Halliwell et
al., 1999). H2O2 is continually generated by
several enzymes (including superoxide dismutase, glucose oxidase, and monoamine
oxidase) and must be degraded to prevent oxidative damage (Halliwell 1992).In
the present investigation, catalase activity was decreased in excessively
excess iodine treated rats. Both glutathione peroxidase and catalase, the two
main enzymes involved in H2O2 detoxification, have been
shown to be implicated in the disposal of exogenous H2O2
(Dringen et al., 1997).
Catalase is known to be of special importance when the passage of H2O2
in high concentrations is required. In physiological condition, thyroid
follicles continuously produce H2O2 for thyroid hormone
synthesis throughout lifetime, and excessive H2O2 is
degraded by GSH-Px, thus
protecting the thyroid from H2O2-induced damage (Xiang et al., 1999). When iodine intake is
excessive, the oxidative and anti-oxidative balance is upset by generation of enormous
H2O2 and other free radicals. If the imbalance persists
for a long time, a large amount of GSH-Px
is consumed. In our study after excessive iodine exposure according to dose, GSH-Px activity of peripheral blood lymphocytes was
reduced. The study of Corvilain et
al., (2000) reported that, H2O2 generation is
stimulated by iodine thereby on the level of the oxidative stress. Further,
these might lead to oxidative damage to some cells, which might result in
proliferation and differentiation of immune cells or alter the immune response
(Chen et al., 2007).It may thus
be speculated that excess iodine dose dependently produces oxidative stress in
peripheral blood lymphocytes which possibly are responsible for altered
lymphocyte proliferation and immune suppression as evident in the present
study.

Further, the result shows that excess iodine treatment decreased
GSH levels in peripheral blood lymphocytes. GSH is one of the body’s most
important antioxidants responsible for free radical scavenging in all cell types
(Kumaran et al., 2004). The
observed decrease in glutathione concentration may result from the decreased
availability of substrates needed for glutathione synthesis, particularly
cysteine, which is considered to be the most limiting amino acid (Grimble et al., 1992). The decrease in GSH
level in the lymphocytes of excess iodine treated rats establishes evidence of
strong oxidative stress and a breakdown of the redox balance in cells of the
experimental animals.

The
increase in lipid peroxidation as observed in this study possibly associated
with an increased H2O2 concentration in lymphocytes.
Being a major indicator of oxidative damage, lipid peroxidation is initiated by
ROS and causes impairment of membrane function (Selvakumar et al., 2006). The significant
increase in lipid peroxidation observed in this study may be endorsedas direct
effect of increased generation of ROS resulting from excess iodine treatment.
These results are well supported with the earlier finding that excessive iodide
leads to increase the level of MDA (Xiang et al., 1999).The present study shows that excess iodine induces
a remarkable oxidative response in the PBL as evidenced by an increase in the
concentrations of TBARS.

Significant
elevation of nitrite (NO) level in PBL was observed in excess iodine treated
rats than that of control. Zhang et
al. (2015) reported that excess iodine increased the activity of inhibitory
nitric oxide synthase (iNOS) of rat aorta endothelial cell. Also, NO is
emerging as a potential powerful mediator of T-cell responses. It can both
enhance and suppress T-cell functions and some subsets of T-cell clones can be
activated to produce NO (Liew et al.,
1995). The production of NO has been thoroughly reported in cattle immune cells
such as, lymphocytes (Kirk et al.,
1990) and blood leukocytes (Boulanger et
al., 2001). Excess production of NO that has been observed in several
inflammatory diseases is a potent inhibitor of lymphocyte proliferation which
is its one of the immune modulatory property.

Iodine-induced
thyroiditis has been observed in autoimmune-prone animals and is characterized
by lymphocytic infiltrations in the thyroid followed by increased cytokine
secretion. Excess iodine triggers the production of cytokines and chemokines
that can recruit immune-competent cells to the thyroid (Bonita et al., 2003).The TNF ? and IL-6 found in plasma are likely produced by various
tissues, including activated leukocytes, adipocytes, and endothelial cells.
Because the increased circulating cytokine levels found in excess iodine
treated rats  seem to originate from non-circulating
cells (Pickup et al., 2000) In our investigation pro-inflammatory
cytokines (IL6 and TNF ?) production in serum were emerged in excess iodine
treated rat. Oxidative stress affects the production of IL-6 and TNF-alpha
(Fern´andezet al., 1993). Pro-inflammatory stimuli like TNF-alpha enhance its
secretion without any influence of IL-6 (Berger et al., 2001). Free oxygen
radicals are general mediators of signal transduction pathways, which are able
to induce cytokine production from various cell types Haddad et al., 2001;
Kosmidou et al., 2002.Oxidative stress can increase cytokine production via
several different mechanisms by activating the transcription factors nuclear
factor kappa B (NF?B) and activator protein-1 (AP-1) leading to the
transcription of genes encoding cytokines growth factors, and ECM proteins
Nath et al.,1998 through oxygen derivatives, acting as second messengers. The
detrimental effects of NO in oxidative processes Davis et al., 2001 induced by
pro-inflammatory cytokines Nussler et al., 1992. Makay et al., (2009) hypothesized
that reactive oxygen intermediates, induced by excess iodine mediated cellular
toxicity, act as a signal for the release of pro-inflammatory cytokines like
IL-6 and TNF-alpha.

In
the present study, excess iodine supplementation through oral gavage to rats
has been resulted in a raised level of stimulation index as evidenced by the
results of lymphocyte proliferation assay in both treated groups (100EI 30D and
500EI 30D). Chen et al., (2007) reported
that auto-aggression T cells was activated followed by the pathogenesis of
autoimmune disease. From this study, it was found that the proliferation
response of lymphocytes was abnormal in excess iodine exposed rats, suggesting
that the lymphocyte immune function has been impaired.

Excess
iodine promotes DNA damage of lymphocytes through generation of free radicals (Halliwel
et al., 2007). Lymphocytes are the sensitive
biomarkers of DNA damage and oxidative stress in blood. Increased DNA damage of
lymphocytes in excess iodine treated rats in this study also favors the
findings of earlier studies (Collins et al., 1998; Majumdar et al., 2008). The
highly toxic superoxide anion (O2-) seriously disrupts normal
metabolism through oxidative damage to cellular components. Oxygen-free
radicals can also be transformed to reactive hydroxyl radicals that ultimately
can cause DNA damage. Both the bases and the sugar moieties are being attacked
by intracellular ROS by producing single and double-strand breaks in the
backbone, adducts of base, sugar groups and cross links to other molecules that
block replication(Sies,1993).Therefore,
elimination of superoxide anion is definitely necessary for the survival of
cellsYao et al., 2006.

Taken
together, we can summarize that excess iodine (both in moderate excess and
excessive excess dose) causes impairment in lymphocyte function as revealed by
altered generation of free radicals, diminished anti-oxidative enzymes and antioxidant,
enhanced production of pro-inflammatory cytokines and abnormal lymphocyte
proliferation in rats.

Conclusions

In conclusion, we speculate that excess iodine
altered the functional status of lymphocyte and can result in lymphocyte immune
dysfunction. Further results of this study presented the evidence of cytotoxic
effects of excess iodine on lymphocyte immune function which might lead to
generation and development of autoimmune diseases. 

Therefore, the iodine status in a
community should be carefully monitored to provide accurate data for
establishing a social strategy for nutrient ingestion. Iodine prophylaxis and
treatment programs remain important for iodine deficiency correction but
follow-up over time and continuous control of iodine intake must be sustained.

Acknowledgements

The entire work was financially supported by Rajiv Gandhi
National Fellowship to Miss Adipa Saha under University Grants Commission
(UGC), New Delhi, Govt. of India Sanction
No.F1-17.1/2013-14/RGNF-2013-14-SC-WES-41948 / (SAIII/Website) in the
Department of Physiology, University of Calcutta under the supervision of Prof
Amar K Chandra.

Declaration
of Interest statement

The
authors declared no potential conflicts of interest with respect to the
authorship, research, or publication of this article.