Ethanol as an inducer of apoptotic process in cheek mucosae in rats

Katarzyna Borowska, Małgorzata Stępień

Department of Histology and Embryology with Experimental Cytology Unit, Medical University of Lublin, 11 Radziwiłłowska, 20–080 Lublin, Poland

 

Corresponding author: Prof. Katarzyna Borowska, E-mail: k_borowska@wp.pl

 

Submission: 19.11.2017; Acceptance: 23.11.2017

DOI: 10.7241/ourd.2017e.16

How to cite this article: Borowska K, Stępień M. Ethanol as an inducer of apoptotic process in cheek mucosae in rats. Our Dermatol Online. 2017;8(4e):e8.


ABSTRACT

Apoptosis is the process that plays a important role in development and tissue homeostasis. This  physiological process is regulated by caspases. The caspases are specific cysteine proteases. The aim of this study was to prove how ethanol induces apoptotic process in cheek mucosae cells in rats. Fifteen male Wistar rats were used in the research. They were divided into two treated groups (group A and group Abis) and control group. The biggest histological changes of cheek mucosae was observed in group with ethanol four weeks after last consumption. There is no indication of ability to regeneration in short time after treatment. The most marked was expression of caspase 8 in group A bis. In caspase 9 expression  group A was more visible. 

Key words: ethanol, apoptosis, caspases, rat, cheek mucosae

 


INTRODUCTION

The distribution of ethanol in the body is proportional to the water content in the particular tissue and rate of blood flow. The metabolism of alcohol can follow several pathways. The most important way is in the liver by acetic acid (90%) and sweat, urine, breath (10%). The primary enzymes involved are aldehyde dehydrogenase, alcohol dehydrogenase, cytochrome P450 and catalase.  Chronic ethanol consumption and ethanol metabolism are strongly linked to pathological consequences and tissue damage. We can observed short and long term effects of the body`s response to ethanol intake. The liver is the main organ responsible for metabolism of alcohol with the principal enzyme-alcohol dehydrogenase [1]. Ethanol can be also metabolized in extrahepatic tissue by the enzymes catalase and cytochrome P450. Metabolism of ethanol with alcohol dehydrogenase produces acetaldehyde which is a highly reactive and toxic product that may damage tissue. This is oxidative pathway which add oxygen or remove hydrogen and highly reduced cytosolic pH in liver cells. Acetaldehyde is changing mainly by aldehyde dehydrogenase in mitochondria to acetate. The nonoxidative metabolism of alcohol is minimal and it leads to molecules – fatty acid ethyl esters. These esters are detected long time after alcohol is eliminated. The other nonoxidative pathway requires the enzyme phospholipase. The different gens are associated with varying levels of enzymatic activity [2]. The hepatocytes are the first ones to show the evidences of damage from chronic alcohol consumption. And the body can recognize cells as foreign and generates immune molecules against them [3,4]. Acetaldehyde can induce neurotransmitters such as serotonin and dopamine. All physiological cell deaths proceed by process of apoptosis. The execution stage occurs through the activation of caspases, the family of cysteine proteases. Caspases are present in most cells in the cytosol as inactive enzymes. There are two independent apoptosis pathways. The first involves ligation of death receptors by their ligands, the active caspase 8 can activate downstream of caspases. The second pathway by stress signal in mitochondrion can release cytochrome c which binds to Apaf1 antibody and this complex turns procaspase 9 in active caspase 9, in apoptosome. There is no cross-talk between the pathways. These caspases 8 and 9 can activate cascade of caspases, the next step named execution and now the apoptotic process cannot be turn back. Apoptosis is characterized by nuclear condensation, cell shrinkage membrane blebbing and DNA fragmentation [5,6]. Cell survival requires the active of apoptosis which is accomplished by inhibiting the expression of proapoptic factors [7]. The aim of this study was to prove how ethanol induces apoptotic process in cheek mucosae cells in rats.  The aim of the study was to assess how alcohol induces apoptotic process in cheek mucosae cells.
The main objective of the experiment was how does the ethanol change or not cheek mucosae during short drug test? The next question was if there is a difference in the regeneration of mucosae after 4 weeks break of taking ethanol. And the last issue, if the alcohol treatment impacts on activity of expression caspase 8 and caspase 9. 

MATERIAL AND METHODS

Fifteen male Wistar rats, weighting 200 g each (at the beginning of the experiment) were used in this research. They were kept in the same environment and fed on standard diet. The study has been approved by Medical University in Lublin 5/2009. The experiment was divided in two groups. The first group was sacrificed 24 hours after the last feed containing ethanol (group A) and the second one four weeks after the last feed containing substances in question (A bis). The animals of groups A and A bis were fed for four weeks with addition of ethanol only. The control group consisted of five animals kept on typical diet without any additions. The specimens were obtained from the inside of the oral cavity – cheek, fixed, blocked and stained haematoxylin & eosin in standard procedure. The staining for immunohistochemically detection of caspase 8 and caspase 9 antigenicity was done on the same blocks. The slides were examined under the microscope at various magnifications (excluding oil immersion). Most of the digital photography documentation was taken at 200X or 400X magnfication. Histological evaluation concerns changes of number, quality and kind of cheek cells. The immunohistochemically examination of expression caspase 8 and caspase 9, was counting of apoptotic cells in the field of view. Histomorphological measurements were statistically evaluated and significance calculated by using one way ANOVA test followed by Kruskala –Wallis, Chi^2 Pearsona. All the results were expressed as mean p < 0,05, it was considered as statistically significant.

RESULTS

Histomorphological resultsThe process of apoptosis was noticed in all groups: treated and control (Fig. 1). Histologic examination with hemathoxylin and eosin stain (H&E), apoptosis involves signal cells or small cluster of cells. It is observed regular several layers in control group. The basal layer can be seen as 1-3 lines of basal cells which are mostly disposed longitudinal or cubic shape. Nucleus is round with marked chromatin and there is only one nucleus. The apoptotic cells appears as round, oval mass with eosinophilic cytoplasm and dens purple nuclear chromatin fragments. Apoptotic bodies consist of cytoplasm with tightly packed organelles and parts of nuclear material. Multiple layers of viable epithelial cells- basal layer, stratum spinosum, stratum granulosum were different than in control group. The first was noticed  the width of layers was wide and ridged. There were no regular sheets of cells. The basal cells were well-defined and it can be seen process of hyperplasia. Basal layer was disturbed most in group A bis but it can be noticed a lesser extent in the A group. The stratum spinosum was overgrown, wider like in the hypertrophy. We can see keratohialin in the cells of stratum granulosum. The stratum corneum was strongly marked, the weakest was in group A. The connective tissue contains more cellularity like macrophages, parenchymal cells and the capillary vessels were expanding.  Stromal lesions included aedema, presence of inflammatory cells were also observed.

Figure 1: Photographs showing H&E Treated groups and control.
 
Immunohistochemical examination
Positive reaction was observed in all groups but the expression for caspase 8 was not the same (Fig. 2). The week reaction was in the control group and it was cytoplasmatical type only. No significant discrepancies was noticed in statistical distribution of immunohistochemically reaction intensification between control group and group A and A bis (Table 1, Fig. 3, Table 2, Fig.4).
 
Figure 2: Photographs showing cheek mucosae treated caspase 8 antigenicity.
Table 1: Immunohistchemical reaction caspase 8 antigenicity groups A (A8) and A bis (A bis8).
Figure 3: Immunohistochemical reaction caspase 8 antigenicity groups A and A bis.
Table 2: Immunohistochemical reaction caspase 8 antigenicity groups A (A8) and control (K8).
Figure 4: Immunohistochemical reaction caspase 8 antigenicity groups A and control.

The expression for caspase 9 was also positive in all groups. More visible were the results in group A. No significant discrepancies was noticed in statistical distribution of immunohistochemically reaction intensification between groups A and A bis (Fig.5, Table 3, Fig.6).  
 
Figure 5: Photographs showing cheek mucosae treated caspase 9 antigenicity.
Table 3: Immunohistochemical reaction caspase 9 antigenicity groups A (A9) and A bis (A bis9).
Figure 6: Immunohistochemical reaction caspase 9 antigenicity groups A and A bis.
 
DISCUSSION
Metabolism of ethanol starts in the mouth immediately after consumption. Particles of ethanol diffuse to saliva and mucosae. Alcohol dehydrogenase is in mucosae cells and bacteria cells so the concentration of enzyme is higher [8]. Pathological changes can be cause contact with ethanol and mucosae. Toxic metabolites of alcohol such as acetaldehyde, active oxygen and esters change of permeability of mucosae cells [9]. Inflammatory is the quickest and generally complication poisoning of ethanol. In treated groups A and A bis there were infiltration of inflammatory cells. Mucosal atrophy is the result of the processes. It can be beginning of carcinoma (about 1% of patients) [10]. Toxic metabolites disturbs the process repairs of DNA so, apoptotic process intensifies [11]. There is important P53 protein in this moment which regulate apoptosis. Apoptosis is coordinated process and dependent of ATP or ADP. The activation of apoptotic process is depending on group of cysteine proteases called caspases. The enzymes can be observed inactive shape in each cell because apoptosis is homeostatic mechanism to maintain the population of cells in tissues. When cells are damaged by disease or noxious agents and in immune reactions, apoptosis is the basic process which regulate the number of healthy cells. The other process important in this moment is necrosis, uncontrolled and passive, affects large parts of cells. There are two main pathways of apoptosis extrinsic via the death receptor and intrinsic via mitochondrial pathway. There are  many stimulants which can activate procaspase and change into active caspase. There are several groups of caspase and they are divided into initiators – starters caspases 2,8,9; effectors- 3,6,7. One activation of caspase can activate the next and it starts cascade of caspases. In the beginning this process can be stop because some mediators can block reaction. The cells during apoptotic process are changing, cells shrink and they are smaller and organelles are tightly packed. Pykcnosis is the next process when is the condensation of chromatin (cytoplasm is dark and nuclear chromatin is dense purple). The separation of cells fragments into apoptotic bodies is called budding. These bodies are phagocytosed by macrophages and this is quickly act so there is no inflammatory. Results of examination show that the inflammatory process in connective tissue was observed. It was probably cause of toxic metabolites of alcohol. The changing of apoptotic reaction was better shown in group A bis. The reaction H&E in cheek mucosae were the same for A and A bis in nucleus and cytoplasm was more affinity to eosin cause there were many mitochondrias in cells which took part in metabolic process. In short time distribution of alcohol it was noticed influence in quality and quantity of mucosae cells. The biggest histological changes of cheek mucosae was observed in group with ethanol four weeks after last consumption. There is no indication of ability to regeneration in short time after treatment. The most marked was expression of caspase 8 in group A bis. In caspase 9 expression  group A was more visible. 
 

REFERENCES

1. Lee SL, Chau GY, Wu CW, Yin SJ. Functional assessment of human alcohol dehydrogenase family in ethanol metabolism: Significance of first-pass metabolism. Alcohol Clin Exp Res. 2006;30:1132-42.
2. Agarwal DP. Genetic poly morphisms of alcohol metabolizing enzymes. Pathol. Biol. 2001;49:703-9.
3. Barry RE, Williams AJ, McGovan JD. The detection of acetaldehyde/liver plasma membrane protein adduct formed in vivo by alcohol feeding. Liver. 1987;7:364-8.
4. Lin RC, Lumeng L, Shahidi S, Kelly T, Pound DC. Protein – acetaldehyde adducts in serum of alcoholic patients. Alcohol Clin Exp Res. 1990;14:438-43. 
5. Degterev A, Yuan J. Expansion and evolution of cell death programmes. Nat Rev Mol Cell Biol. 2008;9:378-90. 
6. Indran IR, Tufo G, Pervaiz S, Brenner C. Recent advances in apoptosis, mitochondria and drug resistance in cancer cells. Bioch Biop Acta. 2011;1807:735-45.
7. Pradelli LA, Bebeteau M, Ricci JE. Mitochondrial control of caspase-dependent and – independent cell death. Cell Mol Life Sci. 2010;67:1589-97. 
8. Bagan JV, Vera-Sempere F, Marzal C, Pellín-Carcelén A, Marti- Bonmati E, Bagan L. Cytological changes in the oral mucosae after use of a mouth rinse with alcohol. A prospective double blind control study. Med Oral Patol Oral Buccal. 2012;17:956-61.
9. Olney JW, Wozniak DF, Farber NB, Jevtovic-Todorovic V, Bittigau P, Ikonomidou C. The enigma of fetal alcohol neurotoxicity. Ann Med. 2002;34:109-19.
10. Topham CH, Taylor SS. Mitosis and apoptosis: who wish the balance set? Cur Opt Jn Cell Biol. 2013;25:780-5.
11. Jover R, Pansoda X, Gomez-Lechon MJ. Potentiation of heroin and methadone hepatotoxity by ethanol: an in vitro study using colored human hepatocytes. Xenobiotica. 1992;22:471-8.

Notes

Source of Support: Nil,

Conflict of Interest: None declared.


CONTENT

SERVICES

Other Resources

Our Dermatology Online

Home
Current Issue
All Issues
Instruction for authors
Submit Manuscripts
Ethics in Publishing
For Reviewers
Readers
About
Editors & Publishers 
Statistics
Copyright
Contact Us