Abstract
Our group previously reported that arsenic (As) exposure induced apoptosis in hippocampus neurons. The aim of the present study was to clarify the protective capacity of taurine (Tau) on As-induced neuronal apoptosis and the related mechanism in mouse hippocampus. Mice were divided into: control group, Tau control group, As exposure group and Tau protective group, randomly. The apoptotic rate of mouse hippocampus was determined by TUNEL staining. The levels of Bcl-2 and Bax gene and protein were analyzed by real time RT-PCR and WB, respectively. Furthermore, cytochrome c (Cyt C) release, and the activity of caspase-8 and caspase-3 were also determined. The results showed that Tau treatment induced the decrease of TUNEL-positive cells, prohibited the disturbance of Bcl-2 and Bax expression, and inhibited Cyt C release and caspase-8 and caspase-3 activation significantly. The results indicated that Tau supplement markedly ameliorates As-induced apoptosis by mitochondria-related pathway in mouse hippocampus.
$Shuangyue Li and Lijun Yang are contributed equally to this work.
Access provided by CONRICYT-eBooks. Download conference paper PDF
Similar content being viewed by others
Keywords
1 Introduction
Arsenic (As), a naturally toxicant existing in both organic and inorganic forms, is universal found in food, groundwater, ambient air and dust. As toxin, being inorganic and/or organic compound, is widely spread in water and environment which was resulted by the increasing pollution. Several researches indicated that inhalated- or ingested-exposure of As involved in the more and more chronic diseases which include diabetes mellitus, cardiovascular disease, peripheral vascular disease and various cancers, and one of the major targets is nerve system (Ferreccio et al. 2000, Chen and Ahsan 2004, Meliker et al. 2007, Huang et al. 2008, Das et al. 2012a, b). It was illustrated that As exposure induces dysfunctions in nervous system or severe signs of neuropathy in animal and human models (Meliker et al. 2007; Sinczuk-Walczak 2009; Zhang et al. 2013). Impaired learning ability and neural behaviors were observed in many As-intoxicated rodents at environmental relevant levels (Sinczuk-Walczak 2009; Zhang et al. 2013).
The hippocampus, one of the most important components of brain, is responsible for several vital functions, including behaviours, mental and intellectual activities, in both rodents and human. Moreover, the hippocampus is very susceptible to the toxicities of some neurotoxins such as ethanol, lead, MnCl2 and CuO (Sharifi et al. 2002; Oliveira-da-Silva et al. 2009; Sinczuk-Walczak 2009; Wang et al. 2012; Zhang et al. 2013). It was reported that the gene levels of caspase-3 and 9, the effectors of apoptosis, were markedly elevated in hippocampus of As-intoxicated rats (Zhang et al. 2013). Our group recently found that As intoxication caused apoptosis in hippocampus neurons by showing the increasing number of Tunel-positivity cells (Wang et al. 2015). These researches indicated abnormal apoptosis induced by As exposure in hippocampus may involve in the neurotoxicity induced by As exposure.
Apoptosis, the basic process under both physiological and pathological condition, regulates cell death according to surrounding environmental (Namgung and Xia 2000). Disruption or over-activation of this process, the programmed cell death, may lead to the pathogenesis of nervous system. Thus, minimizing neuronal apoptosis and promoting injured neuron recovery may be the most effective and direct therapeutic approach to reduce the neurotoxic effects of As.
Taurine, a natural amino acid, exists in mammalian brain and acts as a functionally neurotransmitter or neuromodulator. It modifies protein phosphorylation, regulates calcium transport and maintains the structural integrity of membrane in nerve tissue (Zhou et al. 2011). It has been shown that Tau protected nervous system against injury, acting as an antioxidant and apoptosis inhibitor. However, the protective capacity of Tau on As-induced apoptosis in hippocampus and its related mechanism is unknown.
In the present study, Tunel assay was used to examine the capacity of Tau on As-induced apoptosis in hippocampus. To clarify the mechanism of anti-apoptotic potential of Tau, Bax and Bcl-2 expression in hippocampus was examined by real-time PCR and Western blotting (WB). The release of cytochrome c (Cyt C) was examined by WB, and the activity of caspase-8 and caspase-3 were analyzed by commercial kits. The study aimed at illustrating the neuroprotection of Tau on As-induced apoptosis in mouse hippocampus and exploring its related mechanism.
2 Materials and Methods
2.1 Animal and Treatment
19.2 ~ 24.7 g male mice were provided by Animal Center, Dalian Medical University. During experiments, all mice were raised under 20 ~ 24 °C temperature, 55% humidity, 12 h dark-light cycle environment with an ad libitum diet and water. The 30 mice were divided into control group, As exposure group and Tau protection group randomly. As exposure group exposed to 4 mg/L As2O3 in double-distilled water orally; tau protection group received 4 mg/L As2O3 in double-distilled water orally and 150 mg/kg Tau once daily by gavage; control group only received double-distilled water. After 60-day treatment, all model mice were sacrificed and samples were collected carefully. The animal experiments were carried out according to the guidelines of the committee of Dalian Medical University.
2.2 Tunel Assay
In Situ Cell Death Detection Kit (Roche, Germany) was used to perform Tunel assay according to instructions. DAPI staining was performed to counter the number of cells. To quantify the apoptotic cells, 6 fields were randomly selected per slide under fluorescence microscope and calculated the apoptosis index (AI). AI score equals the percentage of number of Tunel-positive cells on total number of cells.
2.3 Real Time RT-PCR
Trizol® reagent (Takara, China) was used to extract RNA sample according to the instructions. Transcriptor First Strand cDNA Synthesis Kit (Roche,\USA) was used to perform RT reactions. TP800 System and SYBR Green PCR kit (Takara, Japan) were used to carry out real time RT-PCR. 95″C 5 min, followed by 95″C for 30 s, 40 cycles, then 55″C 30 s, 72″C 30 s were used as reaction conditions. The primers for genes of interest and β-actin are as followed: Bcl-2, GACTGAGTACCTGAACCGG CATC, CTGAGCAGCGTCTTCAGAGACA; Bax, CGAATTGGCGAT GAACTGGA, CAAACATGTCAGCTGCCACAC; β-actin, GGAGAT TACTGCCCTGGCTCCTA, GACTCATCGTACTCCTGCTTGCTG.
2.4 Western Blot
Total proteins were extracted from liver tissue with lysis buffer. BCA method was used to qualify protein concentration. SDS-polyacrylamide gel electrophoresis was carried out with same gram of loading sample protein, and the protein samples were transferred to a PVDF membrane. After blocking, the membrane was incubated with Bax, Bcl-2 (1:800), Cyt C (1:1000) and β-actin (1:500) primary antibodies, respectively. The blots were treated with HRP-conjugated secondary antibodies, and then detected by Bio-Rad imaging system (Bio-Rad, USA), and then qualified with the Gel-Pro software.
2.5 Caspase-8 and Caspase-3 Activity Detection
Caspase-8 and Caspase-3 Colorimetric Assay Kit (Beyotime, China) was used to detect caspase activities of liver according to the manufacturer’s manipulations. The liver lyses were incubated in ice-cold lysis buffer for 20 min, then centrifuged at 10,000 × g 2 min. The related results were showed as a ratio to control.
2.6 Statistical Analysis
Statistical analysis was performed with SPSS 11.0 statistical software. Data were analyzed using one-way ANOVA and expressed as means ± SD in triplicate.
3 Results
3.1 Protective Capacity of Tau on As-Induced Apoptosis in Mouse Hippocampus
Tunel staining was performed to detect the apoptotic cells in hippocampus. As shown in Fig. 1, an elevated quantity of TUNEL-positive cells were observed in As-intoxicated mouse hippocampus, whereas almost no Tunel-positive cells was observed in both saline and Tau alone-treated mouse hippocampus, indicating As intoxication induced apoptosis in hippocampus. Interestingly, compared with As alone group, Tau treatment markedly reduced the quantity of Tunel-positive cells. The increased apoptosis induced by As was significantly reversed by Tau treatment, suggesting Tau attenuates As-induced apoptosis in mouse hippocampus.
3.2 Protective Capacity of Tau on Bax and Bcl-2 Expression in As-Exposed Hippocampus
Level of Bax and Bcl-2 were examined by both real time RT-PCR (Fig. 2a, b) and WB (Fig. 2c, d). As Fig. 2a showed that Bax gene expression was markedly increased in As group than that in control group. However, after treating with Tau, the gene expression of Bax was markedly decreased. On the other hand, compared with control group, As intoxication markedly decreased the gene expression of Bcl-2 in hippocampus, which was significantly reversed in protective groups (Fig. 2b). The similar effects also observed in protein level of Bax and Bcl-2 in Tau protect group (Fig. 2c, d).
3.3 Protective Capacity of Tau on Cyt C Expression in Hippocampus of As-Exposed Mice
As Fig. 3a showed the protein level of Cyt C in mitochondria significantly decreased in As-exposed hippocampus compared to control group. On the other hand, the protein level of Cyt C in cytosol significantly increased in As-exposed hippocampus (Fig. 3b). These results indicate As exposure caused mitochondrial Cyt C release. Interestingly, the release was abolished when As-intoxicated mice were treated with Tau (Fig. 3a, b).
3.4 Protective Capacity of Tau on Activities of Caspase-8 and Caspase-3 in As-Intoxicated Hippocampus
Activities of caspase-8 and caspase-3 in As-intoxicated hippocampus were shown in Fig. 4. Compared with control group, caspase-8 activity in As-exposed hippocampus was markedly higher. Interestingly, Tau reversed the activation of caspase-8 in As-treated mice compared with As alone group. Consistent with that of caspase-8, As intoxication also induced the activation of caspase-3, which was blocked by Tau treatment (Fig. 4b).
4 Discussion
Increasing evidences showed that taking an apoptosis inhibitor may be a practical approach for the protection against nervous system disorders. Currently, the anti-apoptotic potential of Tau has been highlighted in many disorders (Hsu et al. 2008; Leon et al. 2009; Das et al. 2010; Taranukhin et al. 2010; Gao et al. 2011; Das et al. 2012a, b). Moreover, it was reported that Tau treatment could also prevent from apoptosis induced by various toxicants in vitro (Leon et al. 2009; Zhou et al. 2011; Chang et al. 2014). In the present study, the anti-apoptotic capacity of Tau was assayed in hippocampus of mice received As alone or with Tau by Tunel staining. As expected, As-induced elevation of Tunel-positivity cells in hippocampus was markedly blocked by Tau treatment, indicating that Tau has the efficacy to protect against As-induced apoptosis in hippocampus of mice.
Intrinsic pathway is the major intracellular signaling leading to apoptosis, which is accompanied by the dysregulation of Bcl-2 family protein and Cyt C release, where Cyt C eventually activates caspase-3 (Mikhailov et al. 2003; Wang et al. 2013). Bax and Bcl-2 are the representative regulators of Bcl-2 family, which play a vital role in mediating apoptosis process (Braun 2012). It was reported that As intoxication increased Bax level and decreased Bcl-2 level in cultured mesenchymal cells (Zhou et al. 2007). However, there are no reports that the MSCs or Tau treatment mediates the disturbed expression of Bax and Bcl-2 in As-intoxicated nerve cells. In the present study, the results suggested that Tau significantly inhibited the disrupted expression of Bax and Bcl-2 in hippocampus. Bax/Bcl-2 ratio was also assessed in this study, which is markedly decreased in hippocampus once As-intoxicated mice were treated with Tau. These results indicate a potential link between mediating the disturbed expression of Bax and Bcl-2 and the anti-apoptotic capacity of Tau in hippocampus of As-exposed mice.
Several evidences illustrated that the disturbance of Bax and Bcl-2 expression led to the efflux of mitochondrial Cyt C, which activates the downstream caspase cascade (Wang et al. 2013). Among the identified caspases, caspase-8 and caspase-3 are the important enzymes that induce the activation of apoptosis process (Braun 2012; Kadeyala et al. 2013). It was reported that Tau treatment significantly inhibited endosulfan-induced the activation of caspase-3 in rat testis (Aly and Khafagy 2014). In the present stud, we found that Tau significantly suppressed Cyt C release and the activation of caspases in As-intoxicated mouse hippocampus, indicating the trigger of apoptosis in As-intoxicated hippocampus was blocked by Tau. These studies and our results indicate that Tau represses intrinsic apoptosis pathway and the inhibited intrinsic pathway may take part in the prevention of Tau against As-induced apoptosis in mouse hippocampus.
5 Conclusion
In conclusion, the results indicated that taruine inhibits As-induced apoptosis in hippocampus of mice. Moreover, treatment of Tau significantly inhibited the disturbed expression of Bax and Bcl-2, the release of Cyt C and the activated caspases in As-exposed hippocampus. These results indicate that Tau may protect against apoptosis in hippocampus via mediating As-disturbed intrinsic pathway. As for the more precise mechanism, further studies are required.
Abbreviations
- As:
-
Arsenic
- Cyt C:
-
Cytochrome c
- Tau:
-
Taurine
References
Aly HA, Khafagy RM (2014) Tau reverses endosulfan-induced oxidative stress and apoptosis in adult rat testis. Food Chem Toxicol 64:1–9
Braun RJ (2012) Mitochondrion-mediated cell death: dissecting yeast apoptosis for a better understanding of neurodegeneration. Front Oncol 2:182
Chang CY, Shen CY, Kang CK, Sher YP, Sheu WH, Chang CC, Lee TH (2014) Tau protects HK-2 cells from oxidized LDL-induced cytotoxicity via the ROS-mediated mitochondrial and p53-related apoptotic pathways. Toxicol Appl Pharmacol 279:351–363
Chen Y, Ahsan H (2004) Cancer burden from arsenic in drinking water in Bangladesh. Am J Public Health 94:741–744
Das J, Ghosh J, Manna P, Sil PC (2010) Protective role of Tau against arsenic-induced mitochondria-dependent hepatic apoptosis via the inhibition of PKCdelta-JNK pathway. PLoS One 5:e12602
Das J, Ghosh J, Manna P, Sil PC (2012a) Tau protects rat testes against doxorubicin-induced oxidative stress as well as p53, Fas and caspase 12-mediated apoptosis. Amino Acids 42:1839–1855
Das N, Paul S, Chatterjee D, Banerjee N, Majumder NS, Sarma N, Sau TJ, Basu S, Banerjee S, Majumder P, Bandyopadhyay AK, States JC, Giri AK (2012b) Arsenic exposure through drinking water increases the risk of liver and cardiovascular diseases in the population of West Bengal, India. BMC Public Health 12:639
Ferreccio C, Gonzalez C, Milosavjlevic V, Marshall G, Sancha AM, Smith AH (2000) Lung cancer and arsenic concentrations in drinking water in Chile. Epidemiology 11:673–679
Gao X, Yang X, Zhang B (2011) Neuroprotection of Tau against bilirubin-induced elevation of apoptosis and intracellular free calcium ion in vivo. Toxicol Mech Methods 21:383–387
Hsu TC, Chiang SY, Wu JH, Tsai CC, Huang CY, Chen YC, Tzang BS (2008) Treatment with Tau attenuates hepatic apoptosis in NZB/W F1 mice fed with a high-cholesterol diet. J Agric Food Chem 56:9685–9691
Huang YK, Huang YL, Hsueh YM, Yang MH, Wu MM, Chen SY, Hsu LI, Chen CJ (2008) Arsenic exposure, urinary arsenic speciation, and the incidence of urothelial carcinoma: a twelve-year follow-up study. Cancer Causes Control 19:829–839
Kadeyala PK, Sannadi S, Gottipolu RR (2013) Alterations in apoptotic caspases and antioxidant enzymes in arsenic exposed rat brain regions: reversal effect of essential metals and a chelating agent. Environ Toxicol Pharmacol 36:1150–1166
Leon R, Wu H, Jin Y, Wei J, Buddhala C, Hu WJ (2009) Protective function of Tau in glutamate-induced apoptosis in cultured neurons. J Neurosci Res 87:1185–1194
Meliker JR, Wahl RL, Cameron LL, Nriagu JO (2007) Arsenic in drinking water and cerebrovascular disease, diabetes mellitus, and kidney disease in Michigan: a standardized mortality ratio analysis. Environ Health 6:4
Mikhailov V, Mikhailova M, Degenhardt K, Venkatachalam MA, White E, Saikumar P (2003) Association of Bax and Bak homo-oligomers in mitochondria. Bax requirement for Bak reorganization and cytochrome c release. J Biol Chem 278:5367–5376
Namgung U, Xia Z (2000) Arsenite-induced apoptosis in cortical neurons is mediated by c-Jun N-terminal protein kinase 3 and p38 mitogen-activated protein kinase. J Neurosci 20:6442–6451
Oliveira-da-Silva A, Vieira FB, Cristina-Rodrigues F, Filgueiras CC, Manhaes AC, Abreu-Villaca Y (2009) Increased apoptosis and reduced neuronal and glial densities in the hippocampus due to nicotine and ethanol exposure in adolescent mice. Int J Dev Neurosci 27:539–548
Sharifi AM, Baniasadi S, Jorjani M, Rahimi F, Bakhshayesh M (2002) Investigation of acute lead poisoning on apoptosis in rat hippocampus in vivo. Neurosci Lett 329:45–48
Sinczuk-Walczak H (2009) Nervous system disorders induced by occupational exposure to arsenic and its inorganic compounds: a literature review. Med Pr 60:519–522
Taranukhin AG, Taranukhina EY, Saransaari P, Podkletnova IM, Pelto-Huikko M, Oja SS (2010) Neuroprotection by Tau in ethanol-induced apoptosis in the developing cerebellum. J Biomed Sci 17(Suppl 1):S12
Wang L, Ohishi T, Shiraki A, Morita R, Akane H et al (2012) Developmental exposure to manganese chloride induces sustained aberration of neurogenesis in the hippocampal dentate gyrus of mice. Toxicol Sci 127:508–521
Wang Y, Wu Y, Luo K, Liu Y, Zhou M, Yan S, Shi H, Cai Y (2013) The protective effects of selenium on cadmium-induced oxidative stress and apoptosis via mitochondria pathway in mice kidney. Food Chem Toxicol 58:61–67
Wang Y, Bai C, Guan H, Chen R, Wang X, Wang B, Jin H, Piao F (2015) Subchronic exposure to arsenic induces apoptosis in the hippocampus of the mouse brains through the Bcl-2/Bax pathway. J Occup Health 57:212–221
Zhang J, Liu X, Zhao L, Hu S, Li S, Piao F (2013) Subchronic exposure to arsenic disturbed the biogenic amine neurotransmitter level and the mRNA expression of synthetase in mice brains. Neuroscience 241:52–58
Zhou J, Wang W, Wei QF, Feng TM, Tan LJ, Yang BF (2007) Effects of arsenic trioxide on voltage-dependent potassium channels and on cell proliferation of human multiple myeloma cells. Chin Med J 120:1266–1269
Zhou J, Li Y, Yan G, Bu Q, Lv L, Yang Y, Zhao J, Shao X, Deng Y, Zhu R, Zhao Y, Cen X (2011) Protective role of Tau against morphine-induced neurotoxicity in C6 cells via inhibition of oxidative stress. Neurotox Res 20:334–342
Acknowledgements
This work was supported by National Natural Science Foundation of China (grant numbers 81273038 and 81102160) and China Postdoctoral Science Foundation Funded Project (No. 2015 M581338).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media B.V.
About this paper
Cite this paper
Li, S. et al. (2017). Taurine Ameliorates Arsenic-Induced Apoptosis in the Hippocampus of Mice Through Intrinsic Pathway. In: Lee, DH., Schaffer, S.W., Park, E., Kim, H.W. (eds) Taurine 10. Advances in Experimental Medicine and Biology, vol 975. Springer, Dordrecht. https://doi.org/10.1007/978-94-024-1079-2_16
Download citation
DOI: https://doi.org/10.1007/978-94-024-1079-2_16
Publisher Name: Springer, Dordrecht
Print ISBN: 978-94-024-1077-8
Online ISBN: 978-94-024-1079-2
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)