The Effect of Cigarette Smoke Exposure on Histology Liver in Male Mice

When smoking Reactive Oxygen Species (ROS), which is a reactive oxidizing agent, can cause damage to the DNA structure of liver cells and increase the occurrence of liver necrosis. The research aims to determine the histopathological picture of the liver in male mice (Mus musculus) exposed to cigarette smoke. This research is an experimental laboratory with the sample used is male mice (Mus musculus). The sample with the most damage in treatment I obtained 5 (55.6%) samples with a score of 2 who had damage to hepatocytes. , 7 (77.8%) samples with a score of 3 had central venous dilation, 3 (33.3%) samples with a score of 1 had fatty liver, and 3 (33.3%) samples with a score of 1 had liver necrosis. While in treatment II, 5 (55.6%) samples with a score of 2 had hepatocyte cell damage, 8 (88.9%) samples with a score of 3 had central vein dilation, 5 (55.6%) samples with a score of 3 had fatty liver, and 5 (55.6%) samples with a score of 2 had liver necrosis. Based on these results, the histopathological changes in the liver of male mice (Mus musculus) were exposed to cigarette smoke.


Introduction:
Cigarettes have become an endless phenomenon even though the World Health Organization (WHO) has warned about the dangers of cigarettes and is the most dangerous killer, but with warnings in fact there are still many consumers who continue to grow and cigarette production continues to spread throughout the world. (1) Cigarettes contain many substances that are harmful to the body, tobacco contains 2.500 known chemicals and 4.800 chemical substances in cigarette smoke that can threaten and endanger human health, including NO, tar, nicotine, and CO gas. present in tobacco and other materials such as pesticides, fertilizer residue, NTRM (nontobacco related material), TSNA (tobacco-specific nitrosamine), and Bap (benzo-a-pyrene) which are present during the processing, planting, and presentation in trade. The content that can cause disruption of oxygen exchange in the body so that smokers become easily tired is carbon monoxide, this substance will be and settle in the lungs, causing lung work to decrease then nicotine content can cause dependence and addiction to cigarettes. ( The liver is the largest organ after skin and the heaviest gland in humans with a weight of 1500 grams, equivalent to 2.5% of an adult's body weight. The liver fills almost all of the right hypochondrium and epigastrium, the location of the liver extends into the left hypochondrium next to the inferior diaphragm to separate the lungs, pleura, perichondrium, and heart.(5) This organ is important for the body as a detoxifier of toxins that are no longer needed by the body, this process works with the help of one of the enzymes, namely the glucuronyltransferase enzyme, besides that the liver also produces catalase enzymes which are useful for the body to ward off free radicals. If there are fewer antioxidants in the liver cells than the free radicals in the body, over time the liver cells will degenerate, resulting in liver necrosis. Smoking is a risk factor that can cause damage to the shape of liver cells. When smoking Reactive Oxygen Species (ROS), which is a reactive oxidizing agent produced by cigarettes, will increase and can cause damage to the DNA structure of liver cells and can increase the occurrence of liver necrosis and oxidative stress.(6) Oxidative stress describes an imbalance between antioxidant and oxidant produced in the body, so it can cause damage, especially damage to cells in the body. Under conditions of oxidative stress, there is nitric oxide (NO) which forms oxidized molecules with one of the ROS, namely Superoxide anion, to Peroxynitrite which can cause changes in gene transcription, protein formation, and trigger DNA damage. (7) The most dangerous substance in cigarettes is nicotine, the nicotine content that comes from cigarettes that have been inhaled will pass through the lungs and later by the blood will be carried to the liver because the liver is an organ that can metabolize nicotine. The result of nicotine metabolism is the metabolite compound Nnitrosamine in the form of 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), this metabolite is a strong carcinogenic compound found in the liver that can affect liver damage. and interfere with the metabolic function of the liver.(8) Continuous exposure to cigarette smoke will cause a toxic effect that is getting bigger and slowly causes liver damage such as hepatocyte necrosis with a microscopic appearance, namely cells that have dead, dense, and smaller nuclei with chromatin and reticular fibers that are folded. The cells can become eosinophilic or cariolytic, the toxic effects of smoking can also cause disruption of liver function. (9) Methods: This research was carried out at the Pharmacology Laboratory of Andalas Padang University, the Anatomical Pathology Laboratory of the Siti Rahmah Islamic Hospital, and the Laboratory of the Faculty of Medicine, Baiturrahmah University, Padang from August to December 2021. The sample of this study was male mice (Mus musculus) aged 25-30 weeks, with a bodyweight of 25-30 grams. The number of mice (Mus musculus) was 27 grouped into 3 groups, namely the negative control group (KN), treatment group I (KP I), and treatment group II (KP II) where each group consisted of 9 male mice.
This type of research is descriptive categorical with laboratory experimental methods. The treatment group I was the group that was given exposure to cigarette smoke for 14 days, treatment group II was for 28 days, and the negative noncontrol group was the group that was not exposed to cigarette smoke. Liver organs were taken and made preparations with hematoxylin-eosin staining to check for damage to the liver. Cigarette smoke exposure was carried out in a wire cage covered with plastic with a size of 40 x 40 x 35 and with holes at the top and bottom exposed as many as 3 cigarettes per day for 30 minutes.

Results:
This study used 9 mice in each treatment group based on the inclusion criteria, from the results of data collection and analysis obtained:  Based on table 3, it was found that in the treatment group I out of 9 samples there were 3 samples (33.3%) most of which were included in the score 1, namely there was fatty liver < 1/3 of the entire field of view. Meanwhile, in the second treatment group, there were 5 samples (55.6%) with a score of 3, which was fatty liver >2/3 of the entire field of view.  Based on table 4, it was found that in the treatment group I out of 9 samples there were 3 samples (33.3%) most of which were included in the score 1, namely there was liver necrosis <1/3 of the entire field of view. Meanwhile, in the second treatment group, there were 5 samples (55.6%) with a score of 2, which was experiencing hepatic necrosis 1/3-2/3 of the entire field of view.

Histopathological description of the liver in mice (Mus musculus) by observing the presence of hepatocyte cell damage
Caryorexic cells have irreversible properties because there is damage to the plasma membrane, lysosomes, cell membrane dysfunction, mitochondrial dysfunction, and loss of DNA. The picture seen in cariorexic cells is that there is a fragmented cell nucleus. (10) The occurrence of hepatocyte cell damage due to cigarette smoke can affect the metabolic work of macrophages which release leukotrienes TNF-α, B4, and IL-8 and in turn, can increase the production of superoxide and H2O2 and form free radicals that cause oxidative stress in the liver and trigger lipid peroxidation in the liver. Cell membranes can eventually cause damage to hepatocytes. Liver cell damage can trigger excessive cell proliferation so that in the long term it will cause liver cancer or tumor formation.(11)

Histopathological description of the liver in mice (Mus musculus) by observing the presence of dilation of the central vein
Free radicals contained in cigarettes can increase fat peroxidation and cause sinusoid damage which results in pressure on the sinusoid walls due to dams in the veins in the form of blood accumulation caused by toxic substances contained in cigarettes. The blood flow to the liver will flow through the sinusoids and into the central vein.(12) Blood will carry metabolic products from hepatocytes including metabolic products that are toxic, the central vein will accumulate metabolic products and the number of toxic substances contained in the blood can cause lysis of central venous endothelial cells. This lysis process causes an increase in the diameter of the central vein.(13)

Histopathological description of the liver in mice (Mus musculus) by observing the presence of fatty liver
The process of the fatty liver occurs due to the presence of ROS, one of which is found in cigarettes, causing an imbalance between the breakdown and formation of triglycerides eventually causing an increase in triglyceride transport and an increase in lipolysis, especially in adipose in the middle of the body, which then results from lipolysis will be carried through the portal vein. to the liver for processing and causes increased levels of fat in the liver. (14) High levels of fat in the liver will cause adipose accumulation in hepatocyte cells and can potentially lead to liver cirrhosis and non-alcoholic fatty liver disease. (15) Fat levels in the blood will increase within 2 hours after smoking in the amount of 2-5 sticks. (16) Fatty liver in humans is reversible (recoverable) supported by a healthy lifestyle. Factors that can cause NAFLD are obesity, insulin resistance usually in type 2 DM patients, and high cholesterol levels. (