Histopathological of Mice ( Mus musculus ) Liver Induced by Lead (Pb) Orally

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INTRODUCTION
The number of motorized vehicles in Indonesia is increasing rapidly, which harms the concentration of air emissions produced (1,2).Motor vehicles are the main cause of all toxic air emissions.Around 70% of air pollution in Indonesia is currently caused by vehicle emissions that emit smoke containing 173 hazardous substances which harm body health (3) Other pollution factors caused by lead, namely mining, coal burning, and environmental pollution from the air such as smoke, dust, industrial installations, and even poor food hygiene, are factors that support lead poisoning in living things (4).
Lead (Pb) exposure has been reported to be associated with liver-related diseases.
Lead is toxic to living things, even in small amounts (1).Lead can enter the body through various intermediaries, like the respiratory system and be absorbed directly through the skin.In the body, it acts on enzymes related to heme synthesis, DNA transcription and neurotransmitter release, which regulate cellular growth and memory (5).The more lead enters the body over a long period, it will accumulate to harm human health and cause progressive poisoning (6).The liver metabolizes heavy metals and excretes them into the intestines through bile.About 5% of the bile substance is removed through the faeces and the rest can be reabsorbed through the enterohepatic circulation.As a result,

MATERIALS AND METHODS
The study was conducted at the

Dosage Calculation
The determination of the lead dose in this study was derived from the conversion of the maximum dose relevant to human subject to that suitable for mice.This conversion was accomplished using an animal dose calculation table based on Laurence and

Experimental Animal Treatment
In this study, 25 male mice were prepared for in vivo experiment.The mice were divided into five treatment groups, consisting of 5 Swiss Webster strain mice for each group.The distribution of treatment groups is explained in Table 1.

Adaptation
The experimental procedure involved the daily provision of 0.5 mL of lead acetate solution for a duration of four weeks.Following this extended exposure, the livers of mice were collected to the histological images.

Histopathological Examination
The collected samples were fixed in 10% NBF solution for a period 24 hours.Subsequently, they underwent a dehydration process using a series of graded alcohol solutions, starting from 70% and gradually progressing to 80% and 96%.Post dehydration, the sample were cleared using

Histology of Lead-Induced Mice Liver
Following the treatment procedure, liver tissue samples were collected and subsequently preserved.
The tissue processing consists of 8 stages, fixation, dehydration, clearing, embedding, blocking, cutting, colouring and mounting.Following these preparatory steps, the tissue samples underwant staining using the HE method.
Figure 1 shows the histology of lead-induced mice liver, with HE staining conducted at 40x magnification.The histological images represent the following groups: KN (negative control), K2 (dose group II), K3 (dose group III), K4 (dose group IV), K5 (dose group V).

The Measure of Degree of Damage
The degree of damage according to the Mitchel method ( 15) is the degree of 0 is no cell damage; 1 is liver cell damage has reached 0.1-5%; 2 is liver cell damage has reached 6-25%; 3 is liver cell damage has reached 26-50%; and 4 is liver cell damage reaches 50%.The score of each liver histology preparation in the treatment group, namely the negative control, no damage occurred, the mean was 0; groups K2 and K3 experienced damage with an average score of 1; group K4 experienced damage with an average score of 2; then finally group K5 experienced damage with an average score of 3 (Table 2).
The results of histopathology observations of the mice's liver under a microscope showed damage to cells that experience hydropic degeneration, however, the rest are normal.The average percentage of liver cell damage is calculated and presented in Figure 2.
liver cells are exposed to chemicals, which leads to liver dysfunction, cell damage, and organ failure.Considering that aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gammaglutamyl transferase (GGT) from damaged hepatocytes are released into the blood, these are useful markers for liver injury.Some effects of exposure to lead can be permanent.If caught early, healthcare providers, and communities can act to prevent further exposure and reduce damage.The most important thing that parents and caregivers, healthcare providers, and public health professionals should be aware of is the higher the level of lead poisoning, the more severe the liver damage will be.The liver is the largest gland in the body and is located at the top of the abdominal cavity under the diaphragm (7).The liver acts as a metabolic and detoxification process to neutralize toxic compounds that enter the body (8).Most of the toxic substances that enter the body after being absorbed by the epithelial cells of the small intestine will be transported to the liver through the portal vein (9).Chronic exposure to subtoxic concentrations of lead produced changes in the hepatocytes, portal triads and the sinusoids.The hepatocyte alterations were mainly anisokaryosis, nuclear vesiculation, binucleation, cytoplasmic inclusions, cytoplasmic swelling, hydropic degeneration, necrosis and reduction in glycogen content.In addition, portal triads mild chronic inflammation, Kupffer cells hyperplasia and occasional fatty change were seen together with hemosiderosis (10).The purpose of this study was to determine the effect of lead on the appearance of the liver of mice so that the mechanism of damage caused by Lead in the liver could be clearly described.
of the Bandung Health Polytechnic in July 2022.This study uses experimental research methods.The study focused on Swiss Webster strain male mice (Mus musculus) aged between 8-12 weeks, with an average weighed o approximately 30 grams.Mice were selected as the experimental subjects due to their physiological similarities to humans, compact size and body weight, and easy to handle (11).There were 25 mice used in this study, which were divided into five groups, namely the negative control (KN), dose 2 (K2), dose 3 (K3), dose 4 (K4) and dose 5 group (K5).The mice were adapted for one week to their new environment and did not experience stress.Sample calculation obtained by Federer formula.The sampling technique used was non-probability sampling with consecutive sampling technique (12).The mice used were healthy males with normal activities, aged 8-12 weeks and weighing 20-35 grams (13).The primary data analysed in this study was the score of the degree of liver damage from all treatment groups.In all groups, adaptation was carried out for 7 days by being given mice food ratio.The following week, the KN group only received normal feed without any treatment, while the K2, K3, K4 and K5 groups received treatment with induction of lead via oral with a dose of 0.5 ml, once a day for 35 days.Each group will undergo surgery to harvest liver organs weekly.The surgery for the control group was carried out together with the K5 group in the last week (on the 28 th day).The liver that has been taken was put into a 10% neutral buffered formalin (NBF) solution and will be made for histological preparations.The preparation and colouring of these preparations were carried out at the Bandung Health Polytechnic Integrated Laboratory.The preparation is done by using fixation, dehydration, clearing, embedding, and impregnation, blocking, trimming, cutting and mounting.Meanwhile, the preparation was stained by Hematoxylin Eosin (HE) staining (14).The obtained results are presented in the form of an overview.The data was statistically analysed using descriptive, in the form of information from researchers and several literature sources (15).Data was obtained from the results of liver histology scores.The score was determined by observing the damage to the histological appearance of the mice's liver.The anatomical pathologist would give a score of 0 for no liver cell damage occurs; 1: Liver cell damage reaches 0-1.5%; 2: Liver cell damage reaches 6-25%; 3: Liver cell damage reaches 26-50%; and 4: Liver cell damage reaches 50% (13).175Furthermore, the data from the statistical percentage test results were analyzed using the Kruskal-Wallis test and then continued with the homogeneity test.If the results of the Kruskal-Wallis test show a significant difference, then proceed with the Mann-Whitney test (α = 0.05) at a 95% confidence level.All experiments were performed with ethical approval from the Health Research Ethics Committee Politeknik Kesehatan Kementrian Kesehatan Bandung (No.78/KEPKEC/ III//2023).
Bacharach's method(1964).The World Health Organization (WHO) states that the tolerance limit for lead (Pb) in the human body is 50 mg/adult body weight in a week.The conversion factor of 70 kg human to 20 g mice = 0.0026.The dosage calculation for the lead dose = 50 mg/BB with route of administration = oralmaximum volume of 1 mL is as below: Conversion factor = 0.0026 (mice mice) xylol and then subjected to the impregnation stage, being soaked in liquid paraffin for a duration of 12 hours.Tissue were cleaned with xylene and embedded in paraffin.Using a rotary microtome, sections were cut at a nominal thickness of 5-6 m and stained with hematoxylin and eosin to observe microscopic histopathological alterations and their severity (16).Histopathological observations of the liver were performed after staining using Hematoxylin and Eosin staining (HE staining) by taking 400x and 200x magnification photos under a trinocular microscope Olympus U TV0.5XC-3, T7 Tokyo, Japan.The analysis of 400x magnification photos was followed by observations from pathological anatomical specialist, dr.Komala, Sp.PA., an anatomical specialist from Cimacan Hospital, Indonesia.Data Analysis The obtained results were organized and presented in the form of an overview.The data was statistically analysed using descriptive, in the form of information from researchers and several literature sources.The data was statistically analyzed using a descriptive approach, primarily focusing on the liver histology scores.The statistical analysis encompassed the utilization of the Kruskal-Wallis test, a nonparametric method for comparing multiple independent samples.In instances where Kruskal-Wallis indicated a notable difference, the analysis further involved the application of the Mann-Whitney test (α = 0.05) at a 95% degree of confidence (17).

Figure 2 .Figure 1 .
Figure 2. Average score of liver cell damage degrees

Table 1 .
Distribution of Treatment Groups

Table 2 .
Processing of Liver Histology Preparatory Scores