8679 http://annalsofrscb.ro
Histopathological and Immunohistochemical Study in Liver of Mice Muse Musculus Treated with Β-Carotene
Khalid A. S. Alsalami*, Ali A.A. Al-Ali, Ahmed Muhssin Athbi
Department of Biology, college of Education for pure science, University of Basrah, Basrah, Iraq
*Email: [email protected]
Abstract
β-Carotene is one of the natural bioactive products that has received attention as additives to some cancer-suppressing drugs, so the current study aims to detect the relationship of this substance in stimulating the pathways of apoptosis. The present study evaluated the role of β- Carotene in vivo mice, it included detection of histopathological changes in the liver of mice Mus musculus treated with β-Carotene, and immunological detection of apoptosis proteins (caspase 8, caspase 9 and p53) in mentioned organ. There are variety of reversible and irreversible histopathological changes were observed in liver of mice treated with 300 µg/ml of β-Carotene while the samples that treated with 150 µg/ml did not suffered irreversible changes. The immunohistochemistry assay of apoptotic proteins (caspase 8, caspase 9 and P53) showed staining caspase8 protein in Large areas of liver tissue, of mice treated with high concentrations of β-Carotene, with brown of DAB stain, while the caspase9 and p53 proteins were stained in small separate area of tissue. In conclusion, the β-Carotene has a toxic effect with high concentrations on liver tissue and have no toxic effect in low concentration. The injury of tissue as a result the potential of the β-Carotene to induce apoptosis.
Key word: Histopathology, immunohistochemistry, liver, mice, β-Carotene Introduction
The β-Carotene is one type of the carotenoids, It has an orange color, it is available in fruits and vegetables, and in human body at low concentrations. Many studies have indicated that β- Carotene is an antioxidant factor (Teodoro et al., 2012). Gumpricht et al.(2004) observe that a β- carotene have potent to decreased generation of reactive oxygen species(ROS) by >50% in rat hepatocyte that were exposed to the toxic hydrophobic bile acidglycochenodeoxycholic acid (100 or 500 µM) and β-Carotene (100 µM). Lyama et al.(1996) showed that oral administration of β- Carotene for 10 days led to its accumulation in mouse various tissues and excrete a protective role
against oxidative stress. Park et al. ( 2020) observe that Low-β-carotene concentration have an active role in reducing the level of ROS. Also, hepato protective effect of β-carotene against cadmium toxicity in rats has been reported, It reduced the concentration of H2O2 in rat blood (Bashandy and Alhazza, 2008). Other researchers showed to the pro-oxidant properties of β- carotene and trigger ROS-mediated apoptosis (Shin et al. 2020), Which gives it a clear role to inhibiting growth of some cancers by inducing p27kip1 expression and decreasing SKp2 expression (Freedman et al., 2008). β-carotene-induced increases in ROS may lead to caspase-3 activation causing apoptosis (Park et al., 2015). Treatment with β-carotene lead to increased intracellular ROS level, which then triggere caspase activation (Prasad et al., 2006). The β-carotene can be picked up from the bloodstream by body tissues, to be stored or metabolized directly to vitamin A. In mammalian , liver is the main organ for storage a large quantities of β-carotene (Sy et al., 2012). An accumulation of β-carotene in liver may cause injury in liver tissue.
The current study aimed to detection the β-carotene histopathological effects in mice liver, and its relationship in inducing apoptosis proteins (caspase8,9 and p53).
Material and methods
Male mice were divided into three groups A, B and C. Group A was injected (inter peritoneal) with 150μg/ml of β-Carotene, B was injected with 300μg/ml of β-Carotene and C injected with normal saline 0.9 .
The mice were dissected at two periods (15, 30 D), then extracted their livers and fixed with formalin 10% for 48h to study histopathological changes and immunhistochemistry of apoptosis proteins (caspase8, caspase9 and P53).
Histopathological study
Humason (1972) assay was adopted to preparation of tissue sections for study histopathological changes, which is briefly:
After fixation, the tissues are washed with tap water for 24h. Then dehydrated by passing through series of diluted ethanol (70% for 4h, 85% for 2h, 100% for 2h). Then the tissues were cleared by xylene, and infiltrated with paraffin wax at 50 C° for 2h, then embedded with paraffin wax. The tissues were sectioned at 5 µm , then the section were floated and mounted on the slides. After the sections dried up, it was stained with Haematoxylin and Eosin after it passed with a series of solutions(xylene for 20 minute, ethanol (100%, 90% and 70% respectively) for 5minute for each one, D.W. for 2 minute, Haematoxylin stain for 5 minute, T.W. for 2 minute, Eosin stain for 5
8681 http://annalsofrscb.ro
minute, D.W. for 2 minute, ethanol (70%, 90% and 100% respectively) for 5minute for each one and xylene for 5 minute). Then the sections were coverd with cover slid by Canada balsam and examined by light microscope and visualization by digital camera.
Emmunohistochemistry study
For immunohistochemical detection of apoptosis proteins (caspase8, caspase9 and P53) in mice liver tissue we have used Ultra Cruz® kit (Eckle et al., 2004), according to the following steps:-
1- Deparaffinization tissue sections by placing it in xylene for 30 minutes.
2- Rehydration the tissue sections by passing it with a series of ethanol dilutions (100%, 90%, 80% and 70%) for 15 minutes each one and then washed with distilled water (W.D.) for 1 minute.
3- Soaking the sections with H2O2 1% W.D. for 5 minutes, then wash with PBS for 5 minutes.
4- Soaking the sections with Ultra Cruz® Blocking Reagent for 1h.
5- Incubation the sections in 4µg/ml of diluted primary antibody with blocking reagent at 4°C for 24h, then wash with phosphate buffer saline (PBS) for 5 minutes.
6- Expose sections to secondary antibody diluted by blocking reagent (25-1) for 1 hour, then wash with PBS for 5 minutes.
7- Expose sections to DAB-H2O2 stain (2% DAB with 0.015 H2O2) for 10 minutes, then wash with W.D.
8- Counterstaining for 5second.
9- Dehydrating by passing with sires ethanol (70% 90% and 100%,) for 5minute each one.
10-Clearing with xylene for 10 minutes.
11- Stabilizing with mounting medium.
12- Examining by light microscope, then visualization by digital camera.
Results
The microscopic examination of mice livers tissue treated with 150μg/ml of β-Carotene for 15 days (Figure1(B)) showed no histopathological changes compared to the control group (Figure1(A)), while minor histopathological changes were observed in mice treated at the same concentration for 30 days, it represented by a slight expansion of sinusoids and a vacuolar degeneration of a few hepatic cells (Figure 1 (C)).
8682 http://annalsofrscb.ro
Histopathological changes have become more clear in mice's liver treated with 300μg/ml of β- Carotene, the vacuolar degeneration become surrounding the hepatic cell nuclei (Figure 1 (D)), many hepatic cells were suffered necrosis(Figure 1 (D, E)), and lyses their nucleus , and inflammation adjacent the central vein were observed (Figure 1(E).
Figure 1: H&E staining sections of mice's liver treated with β-Carotene. (400x magnification) A- Mice treated with normal saline. B-Mice treated with 150 µg/ml for 15 days , showing hepatic cells are not affected. C- Mice treated with 150µg/ml for 30 days, showing degeneration (arrows), sinusoids expansion (arrow heads). D- Mice treated with 300µg/ml for 15 days, showing degeneration (arrows), necrosis (arrow heads). E- Mice treated with 300µg/ml for 30 days, showing degeneration (arrows), necrosis (arrow heads), lyses nucleus(ring) and inflammation(stares).
8683 http://annalsofrscb.ro
Immunohistochemical detection showed that caspase8 proteins antibodis were stained with a brown DAB stain in large areas of mice liver tissue treated with 300μg/ml of β-Carotene for 30 days (Figure 2 (B)) compared to the control group (Figure 2 (A)).
While the immunohistochemical detection of caspase9 (Figure 2 (C)) and P53 protein antibodies (Figure D) 2) showed that its antibody was stained in small areas of liver's tissue.
Discussion
Microscopic examination of liver sections of mice treated with β-Carotene showed minor changes that increased in a concentration-dependent manner. Mice liver treated with low concentration (150µg/ml) suffered very minor histopathological changes, while severe histopathological changes were observed in the liver of mice treated with high concentration, This may indicate the role of low concentration of β-Carotene, in reducing the level of oxidative potential(Das et al., 2014(, while the high concentration of β-Carotene has transformed its role Figure 2: mice liver sections stained with Immunohistochemical stain (DAB-H2O2). (400x magnification). A- Control group. B- Mice treated with β-Carotene at 300 µg/ml for 30 days showing staining of caspase8 protein (arrows). C - Mice treated with β-Carotene at 300 µg/ml for 30 days showing staining of caspase9 protein (arrows). D - Mice treated with β-Carotene at 300 µg/ml for 30 days showing staining of caspase9 protein (arrows).
from an antioxidant to an oxidant agent, due to the toxicity of the high concentration of β- Carotene on tissues (Bjelakovic et al., 2013). Inflammation is one of the histopathological changes seen in mice liver treated with high concentration of β-Carotene, which may be due to the role of this material in cell damage, which caused attracting inflammatory cells to the damage area (Premanathan et al., 2011). Other histopathological changes such as degeneration and necrosis were observed, which may have occurred due to the effect of reactive oxygen species(ROS) which activated by materials(Monteiro et al., 2005). Excessive production of ROS will lead to disturbances in various bio reactions in the cell (Mekkawy et al.,2013) , May causing lipid peroxidation in plasma membranes, which led to permeability disruption, and causing degeneration(Stevens et al., 2009) . The continued effect of the material will lead to greater damage, such as cell death and necrosis (Levison et al., 2008), this explain the degeneration and necrosis which observed in large area of liver tissue (Thophon et al.,2003). The damage caused by the high concentration of β-Carotene may be due to the disorder of enzymatic activity of liver, and then the hepatic enzyme system of prevent toxicity will loss, causing histopathological changes in liver (Athikesavan et al.,2006).
Immuonohistochemistry assay was used to detect apoptosis proteins (caspase8, caspase9, p53) in mice liver tissue treated with β-Carotene. The results showed a variation in β-Carotene's ability to induce this proteins production , It has been observed that liver tissue is stained brown for DAB dye in varying areas. The caspase8 protein has been stained in large areas of tissue, while caspase9 and p53 proteins were stained in small areas of tissue, this suggests that β-Carotene has a role in stimulating the expression of caspase8 protein larger than its role in caspase9 and p53 proteins. This demonstrates the role of β-Carotene in inducing caspase-dependent apoptosis in tissue by extrinsic pathway (Palozza et al., 2009; Yu et al., 2012).
References
1. Athikesavan, ,S.; Vincent, S.; Ambrose, T. and Velmurugan, B (2006). Nickel induced histopathological changes in the different tissues of freshwater fish, Hypophthalmich thysmolitrix , J. Environ. Bio. Lucknow (India), 27(2): 391-395.
2. Bashandy,S. A.; and Alhazza, I. M.(2008).The Hepatoprotective Effect of β-carotene against cadmium toxicity in Rats. Journal of pharmacology and toxicology .3(6):457-463.
3. Bjelakovic, G. Nikolova, and D. Gluud, C. (2013).Meta-Regression Analyses, Meta- Analyses, and Trial Sequential Analyses of the Effects of Supplementation with Beta- Carotene, Vitamin A, and Vitamin E Singly or in Different Combinations on All-Cause Mortality: Do We Have Evidence for Lack of Harm?. https://doi.org/10.1371/journal.
pone. 0074558
4. Das, R . Das, A. Roy, A. Kumari, U. Bhattacharya, S. and Haldar, P.(2014). β- Carotene Ameliorative arsenic-induced toxicity in albino mice.Biological Trace Elemnt Research, 164(2):226-233.
8685 http://annalsofrscb.ro
5. Eckle, V. ; Buchmann, A.; Bursch, W.; Schulte-Hermann, R. ;and Schwarz, M.(2004). Immunohistochemical Detection of Activated Caspases in Apoptotic Hepatocytes in Rat Liver. Toxicologic Pathology, 32:9–15.
6. Freedman, N.D.; Park, Y.; Subar, A.F.; Hollenbeck, A.R.; Leitzmann, M.F.;
Schatzkin, A. and Abnet, C.C. (2008). Fruit and vegetable intake and head and neck cancer risk in a large United States prospective cohort study. Int. J. Cancer, 122, 2330–
2336.
7. Gumpricht, E.; Dahl, R.; Devereaux, W. M. and Sokol, J. R.(2004). β-Carotene Prevents Bile Acid-Induced Cytotoxicity in the Rat Hepatocyte: Evidence for an Antioxidant and Anti-Apoptotic Role of β-Carotene In Vitro. International Pediatric Research Foundation , Inc. Vol. 55, No. 5.
8. Humason, G. L. (1972). Animal tissue techniques. 3rd ed. W. H. Freeman and company, Son Fran. 614 p.
9. Levison, D. A.; Reid, R.; Harrison, D.J. and Fleming, S. (2008). Muirs text book of pathology . 4th ed., Hodder Arnold, P 570.
10. Lyama T, Takasuga A, Azuma M. Beta-carotene accumulation in mouse tissues and a protective role against lipid peroxidation. Int J Vitam Nutr Res. 1996;66(4):301–5.
11. Mekkawy, I.A.A.; Mahmoud, U.M.; Wassif .E.K. and Nageib .M. (2013). Effect of Cadmium on some histopathological and histochemical characteristics of the kidney and gills tissues of Oreochromis niloticus dietary supplement with tomato paste and vitamin E, Egypt, J. of Fishies and Aqua. Sci., 8(5): 553-580.
12. Monteiro, S.M.; Mancera, J.M.; Fontaínhas-Fernandes, A. and Sousa M. (2005).
Copper induced alterations of biochemical parameters in the gill and plasma of
Oreochromis niloticus. Comp. Biochem. Physiol. C. 141:375-383.
13. Palozza, P.; Bellovino, D.; Simone, R.; Boninsegna, A. and Cellini, F. (2009). Effect of beta-carotene-rich tomato lycopene beta-cyclase (tlcy-b) on cell growth inhibition in HT- 29 colon adenocarcinoma cells. Br J Nutr102: 207–214.
14. Park, Y. Choi, J. Lim, J. W. Kim, H. β-Carotene-induced apoptosis is mediated with loss of Ku proteins in gastric cancer AGS cells. Genes Nutr (2015) 10:17
15. Park, H.A.; Hayden, M. M.; Bannerman, S. ; Jansen, J. and Kristi M. (2020). Anti- Apoptotic Effects of Carotenoids in Neurodegeneration. Molecules, 25, 3453.
16. Prasad, R.Y. k.; Wallace, H.; Tennant, T. k.; Kitchin, D. and Kligerman, C.F. (2010).
The Genotoxicity of Titanium Dioxide and Nanoceria in Vitro, Presented at Genetics &
Environmental Mutagenesis Society, Chapel Hill, NC, 03/15/2010.
17. Prasad V, Chandele A, Jagtap JC, Sudheer Kumar P, Shastry P (2006) ROS-triggered caspase 2 activation and feedback amplification loop in beta-carotene-induced apoptosis.
Free Radic Biol Med 41:431–442
18. Premanathan, M.; Karthikeyan, K.; Jeyasubramanian, K. and Manivannan, G.
(2011). Selective Toxicity of Zno Nanoparticles toward Gram-Positive Bacteria and Cancer Cells by Apoptosis through Lipid Peroxidation. Nanomedicine: Nanotechnology, Biology and Medicine 7, no. 2: 184-92.
19. Pebriani, R. ., Jafar, N. ., Wahiduddin, Hidayanti, H. ., Burhanuddin, & Ummu Salamah. (2021). The Effect of Extract of Canarian Nuts on Reduction of Total Cholesterol Levels of Hyperglicemic Rat. Journal of Scientific Research in Medical and Biological Sciences, 2(1), 19-29. https://doi.org/10.47631/jsrmbs.v2i1.128
20. Shin, J. Song, M. Oh , J. Keum, Y. and Saini, R. K. Pro-oxidant Actions of Carotenoids in Triggering Apoptosis of Cancer Cells: A Review of
21. Emerging Evidence. Antioxidants. 2020, 9, 532.
22. Stevens, A.; Lowe, J.; Scott, I. and Damjanov, I. (2009). Core pathology.3thed. Mosby ,Elsvier, P 632.
23. Sy, C.; Gleize, B.; Dangles, O.; Landrier, J.F.; Veyrat, C.C. and Borel, P. (2012).
Effects of physicochemical properties of carotenoids on their bioaccessibility, intestinal cell uptake, and blood and tissue concentrations. Mol. Nutr. Food Res., 56, 1385–1397.
24. Teodoro, A.J.; Oliveira, F.L.; Martins, N.B.; Maia, G.A. and Martucci, R.B. (2012).
Effect of lycopene on cell viability and cell cycle progression in human cancer cell lines.
Cancer Cell Int12: 36–44.
25. Thophon, S.; Kruatrachue, M.; Upathan, E. S. and Pokethitiyook, P.(2003).
Histopathological alterations of white seabass, Lates calcarifer in acute and subchronic cadmium exposure. Environ. Poll., 121: 307-320.
26. Yu, C.C.; Ko, F.Y.; Yu, C.S.; Lin, C.C.; Huang, Y.P. and Yang, J.S. (2012).
Norcantharidin triggers cell death and DNA damage through S-phase arrest and ROS- modulated apoptotic pathways in TSGH 8301 human urinary bladder carcinoma cells. Int J Oncol 41:1050-60.
