Abstract
BACKGROUND:
Tributyltin chloride (TBTC) is an organotin (OT) compound, a known neurotoxin and an endocrine disruptor, but it is inadequately characterized for its short-term exposure. This study aimed to explore the role of N-acetylcysteine (NAC) and Eplerenone in TBTC induced neurotoxicity in rats.
MATERIALS AND METHODS:
Rats were exposed to a single dose of TBTC and were behaviorally assessed on open field test, light and dark box, Morris water maze, and rota rod test. Thiobarbituric acid reactive substances (TBARS) and nitrite were estimated as a measure of oxidative and nitrosative stress. Evans blue extravasation was used to assess the blood–brain barrier (BBB) integrity and morphological changes were assessed by histopathology. NAC (50 mg/kg) was used as a standard treatment, and eplerenone (50 and 100 mg/kg) was used as test interventions.
RESULTS:
Short-term TBTC exposure produced significant anxiety-like behavioral alterations, accompanied by elevated brain TBARS and serum nitrite levels, indicating enhanced oxidative and nitrosative stress. BBB integrity was disrupted, and histopathological examination revealed early structural changes. Treatment with NAC and Eplerenone significantly attenuated behavioral disturbances, oxidative stress markers, BBB disruption, and histopathological damage. In contrast, no significant deficits were observed in spatial memory or motor coordination, likely due to the limited exposure duration.
CONCLUSION:
TBTC induced early neurotoxic changes in the brain, which were attenuated by NAC and eplerenone administration. The beneficial effects support for further mechanistic studies on mineralocorticoid receptor modulation as a potential target for OT-induced neurotoxicity.
Keywords: Behavior, biochemistry, mineralocorticoid, neurotoxicity, organotin
Introduction
Any adverse effect that can impact the anatomical and physiological aspects of the central and peripheral nervous systems is referred to as neurotoxicity, and it can result from exposure to a wide variety of biological, chemical, or physical agents,[1] the neurotoxic damages are characterized by neurocognitive impairments. A wide range of natural as well as man-made chemicals have been proven to cause a wide range of organ toxicities, neurotoxicity in particular. Hippocampus, basal ganglia,[2] limbic system, and prefrontal cortex are deeply involved in learning and cognition but show high vulnerability toward chemical insults.[3] Damage to blood–brain barrier (BBB) through numerous chemicals exposes the hippocampus to a host of noxious substances, as the hippocampal BBB is more fragile than other brain areas.[4] The occurrence of these events translates to a variety of neurological disorders such as brain damage, behavioral anomalies, and neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, and Schizophrenia.[5]
Organotins (OTs) are considered to be some of the most toxic chemicals introduced into the aquatic environments by human activities. OTs belong to the organometallic compounds family; structurally different organic chains bind covalently with an atom of Tin (Sn).[6] OTs have made their way into the food chain through their extensive use in industry and agriculture, and humans are exposed to OTs via contaminated fish and seafood due to the accumulation of these compounds in marine organisms.[7] OTs are primarily used in anti-fouling paints, biocides, and pesticides in ship paints and marine structures. OTs, besides being carcinogenic and neurotoxic, are well-known reproductive and endocrine disruptors.[8]
Among the OTs, trimethyltin and tributyltin chloride (TBTC) are strongly elliptogenic and potent to cause morphological changes characterized by neurodegeneration in the hippocampus and other brain areas.[9] Individuals exposed to OT compounds demonstrate impaired memory, psychomotor slowing, loss of balance[10] along with biochemical changes including reactive oxygen species (ROS) production, lipid peroxidation, overburdened ROS scavenging mechanisms,[11] and excitotoxicity credited to high Ca2+ levels in different brain areas.[12] TBTC is highly lipophilic and has a long half-life; due to this, it gets extensively accumulated in marine sediments and organisms.[13] Human exposure to TBT comes from contaminated food from marine sources and water, besides TBT is also used as a stabilizer in PVC, which find their way in lining the water supply pipes.[14] TBTC exposure leads to BBB and endocrine disruptions and elevated oxidative stress in animals, depletion of ROS scavenging enzymes, microglial activation, and upregulation of neuroinflammatory markers (interleukin-6 [IL-6], nuclear factor-kappa [NF-κB]), which translate into activation of pro-apoptotic proteins.[3,15] TBT also produces its toxicity through mitochondrial dysfunction and excitotoxicity.[16,17]
Mineralocorticoid receptor (MR) is a ligand-activated transcription factor and interacts with several pro-inflammatory (NF-κB and IL-6) and stress-associated genes;[18,19] MR also fuels the pro-oxidative pathways and mitochondrial dysfunction.[20] Animal models showing high levels of aldosterone show increased hippocampal oxidative stress, microglial activation, and pro-inflammatory genes’ upregulation.[21]
MR is highly expressed in hippocampal pyramidal and DG neurons and controls basal excitability, appraisal of stress, neurogenesis, and memory encoding.[22] MR shows high affinity toward both mineralocorticoids and glucocorticoids, and its activation has been associated with several pathologies of cardiovascular, renal, and immune system.[23] MR activation triggers a downstream pro-inflammatory pathway that induces damage in hippocampus.[24] Endothelial MR in brain vessels participates in local oxidative stress and inflammation, linking systemic aldosterone-MR signaling to neurovascular dysfunction.[21] MR antagonism has shown to be beneficial in cerebral ischemia-induced oxidative stress.[25] Eplerenone administration improved cognition related to neuroinflammation in a rodent model of PCOD[26] and in hypertensive rats.[27] MR modulators have been studies in a wide spectrum of neurological conditions, but their possible benefits in OT toxicity remain unexplored. Based on the literature in hand, we hypothesize that blockade of MR modulation can offer neuroprotective outcomes in TBTC-induced neurotoxicity.
Materials and Methods
Animals
In this study, male adult Wistar albino rats weighing 140–160 g were used. The animals were maintained and cared for at the Institutional Animal House Facility. All animals were exposed to a natural light cycle, and temperatures were maintained between 25 + 2°C at 30%–60% relative humidity. Animals were fed a standard pellet diet and filtered water ad libitum; animal care was performed as per the guidelines of CCSEA, and the study’s experimental protocol was approved by the Institutional Animal Ethics Committee.
Drugs and chemicals
Eplerenone I.P. (EPL) was received as a kind gift sample from Akums Drugs & Pharmaceuticals Ltd, Haridwar, India. 1,1,3,3 Tetramethoxypropane was purchased from Tokyo Chemical Industry (India) Pvt. Ltd., TBTC, carboxymethyl cellulose (CMC), corn oil, Evans blue (EB), Folin-ciocalteau reagent, bovine serum albumin, N-acetylcysteine (NAC), Copper sulphate, zinc sulphate, sodium potassium tartrate, sodium phosphate monobasic, sodium phosphate dibasic, and formaldehyde were procured from CDH Fine Chemicals, India. All other chemicals used were of analytical grade, and all the solutions and suspensions were prepared freshly before use.
Drug administration
Animals were given TBTC (20 mg/kg in corn oil), EPL (50 and 100 mg/kg suspended in 1% CMC) via oral gavage, and NAC (50 mg/kg in saline) intraperitoneally. All the doses were selected on the basis of previously published reports.[14,27,28]
Tributyltin chloride induced neurotoxicity
Animals were administered TBTC (20 mg/kg) orally in corn oil (1 ml/kg) once, and the drug treatments started 24 h before TBTC administration.
Experimental protocol
The study employed five experimental groups; each group consisted of 6 male animals.
Group I – Control: Animals received 1 ml/kg corn oil orally via oral gavage once
Group II – Induction TBTC only: Animals received TBTC (20 mg/kg) in corn oil via oral gavage (1 ml/kg) once
Group III – Standard treatment NAC + TBTC: Animals received NAC 50 mg/kg in 0.1M PBS for 4 days, and TBTC (20 mg/kg) in corn oil (1 ml/kg) was administered once on day 2, 2 h after NAC administration
Group IV and V – Treatment groups EPL (50/100) + TBTC: Animals received EPL (50 and 100 mg/kg), respectively, suspended in 1% w/v CMC prepared in distilled water for 4 days and TBTC (20 mg/kg po) in corn oil (1 ml/kg) was administered once on day 2, 2 h after EPL administration.
Behavioral assessment
All the behavior parameters were assessed on day 5 between 8:30 am and 1:00 pm in a semi-soundproof laboratory under dimly lit conditions. All animals were acclimatized for 3 days in laboratory conditions and human handling before initiation of experiments.
Open field test
An open field test (OFT) was conducted to assess the exploratory behavior in rats. Test animal was placed individually in the center of the apparatus, and its activities were observed for 10 min namely time spent in the center, crossings made in the center, and rearings. After observation, the animal was gently removed and kept in its home cage, and the apparatus was sprayed and cleaned with 70% ethanol to address any olfactory cues from the previous animal.[29]
Light and dark box test
Rats were tested in a light and dark box for assessing their anxiety-like behaviors. Briefly, rats were placed individually in the dark half of the box and were observed for 10 min to assess time spent in light compartment, time taken for the first head poke (risk assessment), and light and dark transitions.[30] After assessment, the animal was placed back in its home cage, and the apparatus was cleaned with 70% ethanol.
Rota rod test
The Rota-rod test is employed widely for assessing motor coordination in animals. Animals were briefly trained on the apparatus by putting them on the rotating rod (25 rpm) for a duration of 5 min; if the animal fell off, they were again placed on the rod. For assessing motor coordination, animals were individually placed on the rod rotating at 25 rpm, and the time for falling off the rod was noted. A total of 3 trials were performed with a gap of 5 min for fatigue recuperation.[31]
Morris water maze
MWM test was performed as per the previously described method by Aggarwal et al.[32] Parameters assessed were time spent in target quadrant and Elapsed Latency.
Biochemical estimations
Blood and tissue collection
Under anesthesia, through cardiac puncture, blood was drawn in sterile polypropylene tubes and was allowed to clot for 1 h; serum was collected by centrifugation at 4000 g for 15 min. The separated serum was aspirated into fresh tubes and was stored at − 20°C till analysis. After collecting the blood, the brains were quickly extracted on ice. Brains were washed with pre-chilled PBS, weighed, and were homogenized in 0.1M PBS (10%w/v) in a glass mortar and pestle previously kept in a refrigerator. Surprenant was obtained by centrifugation of the homogenate at 3000 g for 15 min and used for biochemical estimations.
Assessment of brain total protein
Brain total protein was estimated by the previously described methods of Lowry et al.[33] Brains’ total protein content was measured at 750 nm spectrophotometrically (Shimadzu UV-1800). Protein content was expressed as mg/mL of supernatant.
Assessment of brain thiobarbituric acid reactive substances
The thiobarbituric acid reactive substances (TBARS) level was assessed as a measure of damage to the membrane lipids through peroxidation due to ROS production. TBARS assessment was done by the previously described method of Ohkawa et al.[34]
Assessment of serum nitrite
Serum nitrite was assessed for the nitrosative stress estimation by the previously described method of Sastry et al.[35]
Assessment of blood–brain barrier integrity by Evans blue extravasation
EB is a hydrophilic compound that does not cross the BBB under intact BBB. We assessed BBB integrity as per the method of Lee et al.,[36] with modifications. Briefly, 2% EB solution prepared in normal saline was administered through the tail vein (3 ml/kg) and was allowed to circulate for 4 h. Animals under deep anesthesia were perfused transcardially with 0.1M PBS to clear free dye. The brains were removed, weighed, and homogenized in 1 mL formamide; the homogenate was kept overnight at 37°C for dye extraction. The next day, the homogenate was centrifuged at 10,000 g for 30 min. The absorption of the supernatant was measured at 610 nm with a spectrophotometer (Shimadzu UV-1800).
Histopathological analysis
Animals under deep anesthesia were perfused transcardially with ice-cold saline, followed by with 10% v/v formaldehyde in normal saline for tissue fixation. The brains were carefully removed and were kept in 10% formaldehyde for H and E staining and histopathological analysis.
Statistical analysis
All the data were presented as mean ± standard deviation and analyzed by one-way analysis of variance followed by Tukey’s multiple comparison test. P <0.05 was considered statistically significant.
Results
Effect of eplerenone and N-acetylcysteine administration on exploratory behavior assessed by open field test
The time spent in center, center crossings, and rearings reflect the exploratory behavior in novel environments. TBTC significantly reduced the exploratory behavior when compared to the non-exposed animals, and these behaviors were significantly restored by EPL (50 and 100 mg/kg) and NAC (50 mg/kg) as compared to the TBTC-treated rats Figure 1a-c.
Figure 1.
Effect of eplerenone and N-acetylcysteine on exploratory behaviour in tributyltin chloride-induced neurotoxicity in rats in the open-field test during a 10-min trial (a) time spent by the animals in the center of the open field test apparatus; (b) number of center crossings; (c) number of rearings. Data were statistically analyzed by using one-way ANOVA followed by Tukey’s multiple range test, and values are expressed as mean ± standard deviation with n = 6 in each group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus compared group
Effect of eplerenone and N-acetylcysteine administration on anxiety-like behavior assessed by light and dark box test
Treatment with EPL (50 and 100 mg/kg) and NAC significantly reduced the anxiety-like behavior in the TBTC animals. Treated animals showed lesser time for the first head poke into the light chamber (risk assessment), spent more time in the light compartment with a greater number of transitions between the dark and light compartments Figure 2a-c.
Figure 2.
Effect of eplerenone and N-acetylcysteine on anxiety-like behavior in tributyltin chloride-induced neurotoxicity in rats in light and dark box test during a 10-min trial. (a) Risk assessment time (first head poke); (b) time spent in the light compartment; (c) light-dark transitions. Data were statistically analyzed by using one-way ANOVA followed by Tukey’s multiple range test, and values are expressed as mean ± standard deviation with n = 6 in each group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus the compared group
Effect on the muscle coordination and memory by eplerenone and N-acetylcysteine administration
No significant differences were observed between the muscle coordination and memory as assessed by the rota-rod test apparatus and Morris water maze, respectively (Results not shown).
Blood–brain barrier integrity by Evans blue extravasation
TBTC administration compromised the BBB integrity, reflected by a significantly high amount of EB in the brain. Treatment with EPL (50 and 100 mg/kg) and NAC (50 mg/kg) prevented the BBB disruption by TBTC [Figure 3].
Figure 3.
Effect of eplerenone and N-acetylcysteine on blood–brain barrier integrity
Effect of eplerenone on the oxidative stress
TBTC administration produced a significant rise in brain TBARS levels and serum nitrosative stress. EPL (50 and 100 mg/kg) reduced the levels of TBARS and nitrite in the brain and serum, respectively [Figure 4].
Figure 4.
Effect of eplerenone and N-acetylcysteine on oxidative stress (a) brain thiobarbituric acid reactive substances expressed as nanomoles/mg of protein; (b) serum nitrosative stress expressed as µm/L. Data were statistically analyzed by using one-way ANOVA followed by Tukey’s multiple range test, and values are expressed as mean ± standard deviation with n = 4 in each group, ****P < 0.0001 versus compared group
Effect of eplerenone and N-acetylcysteine on morphological changes in the hippocampal region
As seen with H and E staining, TBTC induced damage in the hippocampal region, and a greater number of cells showed chromatin condensation. EPL and NAC prevented the morphological changes in the cells and offered neuroprotection [Figure 5a-e].
Figure 5.
Effect of eplerenone (EPL) and N-acetylcysteine (NAC) on early neurotoxic changes in the brain (hippocampal areas). Exposure to tributyltin chloride induced early neurotoxic changes in the neurons, which were ameliorated by NAC and EPL treatment (50 and 100 mg/kg). (a) Control; (b) TBTC only (induction); (c) TBTC+NAC (standard); (d) TBTC+EPL 50mg/kg; (e) TBTC+EPL 100mg/kg
Discussion
The present study shows that short-term exposure to TBTC can cause measurable neurobehavioral deficits and biochemical changes reflecting early neurotoxicity in rats. TBTC administration was able to induce anxiety-like phenotype and marked reduction in exploratory behavior of the animals, along with elevation in oxidative stress markers in the brain and serum. TBTC disrupted the BBB and also precipitated morphological changes in the brain tissue, evident from histopathology. Treatment with EPL – a MR antagonist and NAC prevented the early neurotoxicity symptoms, behavioral and morphological anomalies. However, there was no significant impact on the spatial memory and muscle coordination, which can be possibly due to short duration of exposure.
On the OFT and light and dark box test, animals displayed reduced exploration and a higher degree of anxiety, which falls in line with previously studies.[37,38] Anxiety-like behavior is considered to be an early behavioral marker of neurotoxicity before structural changes appear.[39] Elevated oxidative stress enhances anxiety-like behavior and fuels the neuroinflammatory pathways by increasing the levels of IL-1β and IL-6,[40,41,42] besides oxidative stress, the reduction in motor and exploratory activity can also be attributed to TBTC’s ability to alter monoaminergic neurotransmitters and norepinephrine in the brain.[43] NAC is a potent antioxidant and restores GSH levels[14] and could have restored the exploratory behaviors through an antioxidant mechanism. MR interacts with neuroinflammatory pathways and activates stress-related mechanisms;[18] MR activation also triggers oxidative stress and mitochondrial dysfunction.[20] EPL’s blockade of upregulated MR could be causally associated with the restoration of exploration and reduction in anxiety-like behavior in animals.
OTs being highly lipophilic easily penetrate and accumulate in the lipid-rich tissue like the brain and elevate the exposure of the cellular membranes to them, alongside disrupting and modifying biological barriers (BBB).[44] The brain has a very high metabolic rate and oxygen consumption; neuronal membranes containing high amounts of polyunsaturated fatty acids are highly susceptible to ROS-mediated lipid peroxidation.[45] Increased nitrosative stress causes disruption of the BBB and exposes the brain to a wider range of toxins; higher nitrosative stress also activates pro-inflammatory pathways (IL-1, IL-6, and TNF-α).[46,47] Modulation of MR signaling mitigates stress-induced oxidative burden by suppressing ROS generation. MR overactivation promotes expression of pro-oxidant pathways (e.g. NADPH oxidases) and inflammatory genes, which also elevate ROS production. EPL administration mitigated ROS burden and BBB disruption, which can be attributed to EPL-suppressed MR-mediated pro-inflammatory pathways.[48,49]
BBB disruption is an early event of TBTC neurotoxicity; this event can be attributed to oxidative and nitrosative damage caused to tight junction proteins forming the BBB and endothelial barriers. Damage to tight junctions increases the permeability and causes intracellular leakages, and EPL and NAC mediated protection from oxidative insults and modulation of MR signaling might have a stabilizing impact on the BBB.[15,50,51,52]
TBTC induces histopathological changes evident by cytoskeletal disturbance and early neurodegeneration, it also increases the markers of neuronal stress and reduces the expression of neurofilaments.[11,15] Damage to the hippocampus and cortex has been associated with anxiety and a reduction in exploration.[3] In the present study, chromatin condensation was seen in the hippocampal region of TBTC-treated animals, which was reduced by NAC and EPL treatment owing to their different mechanisms of action, offering neuroprotection.
The absence of cognitive and motor deficits in the Morris Water Maze and rotarod tests is possibly due to the short duration of TBTC exposure, and a higher intensity of functional impairment is warranted on these tests.[53] Literature shows that spatial learning deficits emerge after prolonged damage that impair long term potentiation and dendritic density.[54] The preservation of spatial learning and motor coordination despite evident ROS burden, BBB disruption, and behavioral anomalies points toward an early stage of neurotoxic insult rather neurodegeneration.
MRs are highly expressed in cortex, hippocampus, and other brain regions, where they tend to regulate neuronal excitability and stress responses. MR upregulation during stress leads to ROS burden, neuroinflammation and adverse morphological changes which can be accounted for the behavioral alterations in the animals. Present study explored the role of EPL in short-term exposure of TBTC induced neurotoxicity. EPL showed promising results by restoring the behavioral, biochemical and histopathological changes in TBTC-treated rats. Hence, EPL can be seen as a novel therapeutic target for neurotoxicity beyond an antioxidant agent.
This study has its limitations, such as restricted exploration to male rats, no molecular studies or pathway exploration, which should be explored in future.
Conclusion
The study investigated the role of mineralocorticoid recpetor modulation in TBTC induced early neurotoxicity in rats. Administartion of TBTC induce early behavioural changes in the animals refected through elevated anxiety and reduced exploration. TBTC also increased the oxidative and nitrosative burden in the animals. TBTC also disrupted the blood brain barrier and induced early morphological changes in the hippocampal region. The behavioural, biochemical and morphological changes were addressed by adminstration of NAC and EPL in a dose dependent manner. The finding are suggestive of mineralocorticoid receptors of being a contributor in TBTC induced neurotoxicity and more studies are advocated for exploring the mechanisms engaging in the pathological pathway.
Conflicts of interest
There are no conflicts of interest.
Acknowledgments
The authors would like to acknowledge Bharat Institute of Technology, Meerut, India, and Space Age Research and Technical Foundation Charitable Trust, Bharat Institute of Technology, Meerut, India, for providing all the necessary facilities to conduct this research work. Authors also acknowledge Akums Drugs & Pharmaceuticals Ltd., Haridwar, India for providing Eplerenone API as a a gift.
Funding Statement
Nil.
References
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