|Year : 2016 | Volume
| Issue : 3 | Page : 176-182
Influence of ethanol on aluminum-induced alterations in oxidative stress of rat thalamic area
Prasunpriya Nayak1, Shiv Bhushan Sharma2, Nadella Vijaya Subbaraya Chowdary3
1 Department of Physiology, NRI Medical College and General Hospital, Chinakakani, Mangalagiri Mandal, Guntur District, Andhra Pradesh, India
2 Department of Physiology, Chettinad Hospital and Research Institute, Kelambakkam, Tamil Nadu, India
3 Department of Biochemistry, NRI Medical College and General Hospital, Chinakakani, Mangalagiri Mandal, Guntur District, Andhra Pradesh, India
|Date of Web Publication||10-Oct-2016|
Department of Physiology, NRI Medical College and General Hospital, Chinakakani, Mangalagiri Mandal, Guntur District, Andhra Pradesh - 522 503
Source of Support: ICMR Ad-hoc Research Grant to P Nayak,
Institutional Support., Conflict of Interest: None
Background: Neurotoxic impacts of aluminum are associated with oxidant imbalance and implicated in many senile neurodegenerative disorders. Thalamus is relatively protected from aging-related issues, however, seldom studied.
Aims: The study is aimed to find out the aluminum-induced oxidative stress in thalamic area and the influence of ethanol on that.
Settings and Design: Influence of aluminum on oxidative stress parameters in the thalamic area has been studied in the presence of varied levels of ethanol exposures.
Materials and Methods: Male Wistar rats were exposed to aluminum (10 mg/kg bw) and ethanol (0.8-1.6 g/kg bw). Thalamic levels of reduced glutathione (GSH) and lipid peroxidation thiobarbituric acid reactive substances (TBARS) were studied, along with the activities of superoxide dismutase (SOD), catalase, glutathione peroxidise (GPx), and glutathione reductase (GR).
Statistical Analysis Used: The data were statistically analyzed using Kruskal-Wallis test for variance and the significance of the difference between groups was studied using Mann-Whitney U test.
Results: Lone aluminum exposure failed to produce any alterations in all the tested parameters, except the GPx activity of thalamic area. Nevertheless, concomitant ethanol exposure caused significant alterations in those thalamic parameters barring GSH level and SOD activity. Maximum response was observed with the highest dose of ethanol exposure.
Conclusions: Though thalamic area is reported to be selectively susceptible to aluminum-induced oxidative stress, concomitant presence of pro-oxidant dominance might have augmented the aluminum-induced oxidative stress there. The observation may help to understand the mechanistic riddle of oxidative stress created by aluminum, a redox-inactive metal.
Keywords: Aluminum, catalase, glutathione peroxidase, glutathione reductase, lipid peroxidation, reduced glutathione, superoxide dismutase, thalamic area
|How to cite this article:|
Nayak P, Sharma SB, Chowdary NV. Influence of ethanol on aluminum-induced alterations in oxidative stress of rat thalamic area. J NTR Univ Health Sci 2016;5:176-82
|How to cite this URL:|
Nayak P, Sharma SB, Chowdary NV. Influence of ethanol on aluminum-induced alterations in oxidative stress of rat thalamic area. J NTR Univ Health Sci [serial online] 2016 [cited 2020 Apr 6];5:176-82. Available from: http://www.jdrntruhs.org/text.asp?2016/5/3/176/191837
| Introduction|| |
Presence of aluminum in the body is only undesirable as it has no useful biological function. Several neuropathological conditions have been reported with presence of aluminum in brain and, accordingly, the causative roles of aluminum for neurodegenerative changes have been ascribed. Because of its exceptional physico-chemical properties and abundance in ecology, exposure to aluminum is inevitable. There are many routes and sources of aluminum's entry into the body, but its cellular access is only via opportunistic entry through the means not intended for aluminum. However, presence of aluminum has been reported in several organs, enabling it to be involved in various systemic toxicities, with neurotoxicity being the mightiest. Aluminum gets accumulated in different brain regions. Both during development  and in adults, the thalamic area of rats (experimental) remains an easy target of aluminum accumulation. This is further evidenced in humans on the basis of a typical case of dialytic dementia in a patient treated with aluminum gels. In addition, a human autopsy case of aluminum encephalopathy demonstrated nerve cell atrophy and mild loss with stromal spongiosis, proliferation of astrocytes, and microglia in thalamus. On the other hand, oxidant balance in thalamic area could be easily targeted by aluminum exposure, as the most prominent increase in superoxide dismutase (SOD) activity was with thalamus and hippocampus upon acute administration of aluminum.
Aluminum is a redox-inactive neurotoxic metal; nevertheless, aluminum-induced neurotoxicity is always associated with some degree of oxidative stress. Deterioration of redox homeostasis has been reported in chronic and acute aluminum exposures,, though the mechanism(s) of aluminum-induced oxidative stress is still unclear. While aging could markedly enhance the aluminum-induced oxidative stress, aluminum has been found to enhance the lipid peroxidation and inhibit some key enzymes in the thalamus where normal aging does not have any effect. Using ethanol as pro-oxidant, regional specificity and pro-oxidant dependency of aluminum-induced oxidative stress was already observed., In addition, ethanol has been suggested as a positive inducer of age-related deterioration of neuronal cells. Therefore, the current study was planned to find out the oxidant dependency of aluminum-induced oxidative stress in thalamic area of rats.
| Materials and Methods|| |
All chemicals were of analytical grade and purchased from Sigma India, Bengaluru, SRL India, Mumbai, Merck India, Bengaluru, and Qualigen India, Mumbai.
Animal maintenance and treatments
The experimental protocol was approved by the institutional animal ethics committee. The animals were obtained, maintained, and treated in the registered animal house of the institute and the procedures were performed according to the guidelines of Committee for the Purpose of Control and Supervision on Experiments on Animals (CPCSEA, India).
Male albino Wistar rats weighing 100-120 g were used in the study. The animals were maintained under standard conditions. After 1 week of acclimatization, the rats were randomly divided (with the help of Random Allocation Software Version 1.0, May 2004) into eight groups [Table 1]. Both ethanol and aluminum treatments (daily for 4 weeks) were given as gavage. Ethanol or distilled water was given in the morning session, while aluminum or vehicle was given in the evening session daily. Because of inconclusive toxicokinetic interactions of ethanol and aluminum, different treatment sessions were maintained. Morning sessions were preferred for ethanol exposures to avoid impact of ethanol on food intake.
Tissue collection and biochemical assays
After the period of treatment, overnight fasted rats were sacrificed by cervical dislocation. The whole brain was removed and washed with ice-cold saline. Under dissection microscope, the thalamic area was separated immediately and preserved in the ice chamber for biochemical processing. The homogenized brain tissues were used for the determination of reduced glutathione (GSH) content, lipid peroxidation [thiobarbituric acid reactive substances (TBARS)], and activities of catalase, SOD, glutathione reductase (GR) and glutathione peroxidase (GPx), as described elsewhere.
The data were statistically analyzed using Kruskal-Wallis test for variance and the significance of the difference between groups was studied using Mann-Whitney U test keeping probability (P) value <0.05 as the cut-off value.
| Results|| |
Significant chi-square values of Kruskal-Wallis tests for all the parameters confirm that the observed alterations were because of the treatment protocol used. Though the differentiation of influencing element between aluminum exposure and ethanol exposure is not possible, lone aluminum exposure (comparison between C and CA groups) failed to produce any significant effect in almost all the tested parameters of thalamic oxidative stress, except the GPx activity of thalamus. On the other hand, the influence of ethanol (comparison of C vs E1/E2/E3) was also found to be dose specific and was found only in case of certain parameters.
[Figure 1] (Panel A) depicts decreases in median values of thalamic GSH content of all the treatment groups in comparison to control group (group C). Aluminum-treated groups demonstrated higher level of decrements in all the ethanol-exposed groups (31%, 25%, and 45% in E1A, E2A, and E3A groups, respectively) in comparison to aluminum-treated group without ethanol exposure (11% in group CA). When compared with their respective ethanol-exposed group having no aluminum treatment, the decrements were relatively lesser (26%, 21%, and 21% in E1A, E2A, and E3A groups, respectively); however, they were greater than the groups with no ethanol exposure. On the other hand, the median values of thalamic TBARS were found to be increased greatly in all the groups exposed to ethanol (with gradual increase along with rise in exposure dose). The percentage increment of median TBARS value was (Panel B, [Figure 1]) found to be raised in aluminum-exposed groups in each set of ethanol-exposed groups (19% vs 27%, 40% vs 45%, and 72% vs 91% in E1, E1A, E2, E2A, E3, and E3A, respectively).
|Figure 1: Box and whisker plot of thalamic levels of reduced glutathione (GSH µmoles of GSH/100 mg protein; Panel A) and lipid peroxidation (TBARS, µmoles of TBARS/100 mg protein; Panel B) of groups of animals (as detailed in text). Each data point represents six observations. KW indicates chi-squared value of Kruskal-Wallis test. “S” indicates significant difference, while “NS” indicates insignificant difference between the column and row in tables for Mann-Whitney U tests|
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The median values of thalamic SOD activities (Panel A, [Figure 2]) were found to be altered more in only ethanol-exposed groups (−8%, −24%, and −41% in E1, E2, and E3 groups, respectively) compared to ethanol-exposed groups concomitantly exposed to aluminum also (5.72%, −7%, and −17% in E1A, E2A, and E3A groups, respectively). Except for the group that received only aluminum treatment, all other groups demonstrated rise in thalamic catalase activities (Panel B, [Figure 2]). However, on comparing the groups with aluminum treatment and without aluminum treatment, aluminum-exposed groups showed decrement in catalase activities in all the groups except with lowest ethanol dose (E1 and E1A).
|Figure 2: Box and whisker plot of thalamic activities of superoxide dismutase (SOD, units/mg protein; Panel A) and catalase (µmoles of H2O2 decomposed/min/mg protein; Panel B) of groups of animals (as detailed in text). Each data point represents six observations. KW indicates chi-squared value of Kruskal-Wallis test. “S” indicates significant difference, while “NS” indicates insignificant difference between the column and row in tables for Mann-Whitney U tests|
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The median values of thalamic GPx activities were found to be lowered in all the aluminum-exposed groups in comparison to both aluminum-unexposed group of same ethanol dosing and the control group (Panel A, [Figure 3]). However, the groups receiving concomitant ethanol exposure had higher difference (>40%) than the groups receiving only aluminum exposure (13%). Similarly, lowering median level of thalamic GR activities was also demonstrated in all the treatment groups in comparison to that of group C.
|Figure 3: Box and whisker plot of thalamic activities of glutathione peroxidase (GPx, nmoles NADPH oxidized/min/mg protein; Panel A) and glutathione reductase (GR, nmoles NADPH oxidized/min/mg protein; Panel B) of groups of animals (as detailed in text). Each data point represents six observations. KW indicates chi-squared value of Kruskal-Wallis test. “S” indicates significant difference, while “NS” indicates insignificant difference between the column and row in tables for Mann-Whitney U tests|
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| Discussion|| |
Atrophy of thalamus has been reported in neurodegenerative disorders, and oxidative stress and glutamate-mediated excitotoxicity were suggested as possible mechanisms for the observation. Even though reports about aluminum accumulation in the thalamic area are obscure,, the region is reported to be vulnerable to aluminum-induced oxidative stress. Suggested mechanisms of thalamic atrophy in many neurodegenerative diseases  where aluminum toxicity has been implicated  bespoken possible involvement of thalamic area in aluminum-induced neurodegeneration. Then again, pro-oxidant-based escalation of aluminum-induced oxidative stress has been observed in cerebrum  and cerebellum. With this background, the present study was undertaken to identify the role of varied doses of prooxdiants on aluminum-induced oxidative stress in thalamic area.
Aluminum exposure at a dose used in the present study has been demonstrated to oxidative stress in different regions of brain only when they are concomitantly exposed to ethanol., In the same line, current investigation also did not demonstrate decrease in thalamic GSH level or increase in thalamic TBARS level in response to lone aluminum exposure [Figure 1]. Similarly, only ethanol exposure at any of the used doses was not able to cause significant reduction in thalamic GSH level. However, concomitant exposure to the highest dose of ethanol and aluminum caused significant reduction in thalamic GSH levels. Therefore, in the presence of exposure to higher doses of ethanol, aluminum could compromise one of the important antioxidant defense mechanisms of thalamus microenvironment, as abundance and capability to reduce peroxides through non-enzymatic process have made GSH the first choice of protection against oxidative events. On the other hand, except exposure to the lowest dose of ethanol (group E1), lone ethanol exposures (groups E2 and E3) also were able to enhance the lipid peroxidation significantly (compared to group C). Interestingly, group E1A demonstrated significant increase in thalamic TBARS level, compared to group CA [Figure 1]. Therefore, aluminum exposure could enhance the impact of ethanol exposure in terms of thalamic TBARS level.
Decreased brain SOD activity was reported by Aydin et al. in ethanol-exposed rats, while Jyoti and Sharma  showed that aluminum could reduce the brain SOD activity. Thus, both these treatments were expected to reduce the brain SOD activity and make the neurons more vulnerable to superoxide attack. From the observations of the current study, it appears that thalamic SOD activity is significantly elevated by aluminum exposure only when there is little pro-oxidant dominance or fall in thalamic SOD activity (group E1A), but either absence of pro-oxidant dominance or excess of pro-oxidant dominance has different responses. Though statistically insignificant, the group without ethanol exposure demonstrated aluminum-induced reduction while groups with higher doses of ethanol exposure showed aluminum-induced elevation of the thalamic SOD activity (Panel A, [Figure 2]). Therefore, the presence of pro-oxidant dominance might be very crucial to decide the impact of aluminum on the thalamic SOD activity. In this context, it is noteworthy that recently, superoxide speciation of aluminum has been proposed  and could be linked with the observed responses of thalamic SOD activity.
Enhanced thalamic catalase activity in the presence of ethanol exposure was prominently observed in the current investigation and aluminum was not able to produce any significant alteration even in a single occasion (Panel B, [Figure 2]). Similar enhancement in catalase activities in brain due to ethanol had been demonstrated in animal studies , as well as in cell cultures studies. On the other hand, aluminum-induced reduction in catalase activity was reported in cerebrum , and cerebellum. In the present investigation also, aluminum was found to trim back the ethanol-induced acclivity of thalamic catalase activity.
Ethanol-exposed groups that also received concomitant aluminum exposure in the present investigation demonstrated significant reduction in thalamic GPx activity, while majority of them that did not receive aluminum exposure failed to show such alteration (Panel A, [Figure 3]). Aluminum exposure caused reduction in GPx activity of hippocampus, liver, testis, and epididymis, while enhancement of cerebellar GPx activity was observed in response to aluminum exposure. Therefore, the response of GPx activity to aluminum insult is region-specific and the current investigation indicates that it could also depend on the oxidant status of the region. Specificities in aluminum-induced responses of GPx activity could be based on the level of available aluminum. Kohila et al. did not find significant alteration in aluminum absorption, even though they reviewed that ethanol could increase the neural membrane permeability for aluminum, possibly through increasing the membrane fluidity. Therefore, it is most likely that ethanol would have not changed the available aluminum in the thalamic area; however, because of alteration in the oxidant status brought about by ethanol exposure, the role of different chemical species of aluminum on the altered thalamic GPx activity cannot be ruled out.
The data obtained in the present investigation suggest that the thalamic GR activity was reduced by both aluminum and ethanol exposures (Panel B, [Figure 3]). Though aluminum exposure alone (group CA) and low dose of ethanol exposure (group E1) were separately not sufficient to produce significant reduction in thalamic GR activity, animals concomitantly exposed to aluminum along with ethanol (groups E1A, E2A, and E3A) showed obvious diminution in thalamic GR activity in the same line as higher doses of only ethanol exposure (groups E2 and E3). These observations indicate that aluminum exposure augments the ethanol-induced inhibition of thalamic GR activity and, thereby, makes thalamus more susceptible to oxidant-mediated degenerative changes by restricting the supply of GSH.
| Acknowledgments|| |
The work was partially supported by the grant from Indian Council of Medical Research, New Delhi (IRIS ID No. 2010-20650). Authors wish to thankfully acknowledge the support received from Department of Biochemistry, Department of Pharmacology, NRI MC & GH and the Management of NRI Academy of Sciences to carry out the work.
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[Figure 1], [Figure 2], [Figure 3]