EFFECTS OF LEAD EXPOSURE ON HEPATOSIT CELLS IN MICE Mus Muscullus
Novie Elvinawaty Mauliku, Arry Gustian noviemauliku@gmail.com
Department of Public Health, School of Health Sciences Jenderal Achmad Yani Cimahi, Indonesia ABSTRACT
Background: Lead (Pb) is a soft metal that found in nature which in certain level dangerous for the health.
Lead exposure was accumulated in the body can cause acute or chronic poisoning. Toxic effects of lead may cause cell damage, damage of nervous system, bones, kidneys, brain, decreased IQ, and liver. Objectives: The purpose of this research is to investigate the influence of chitosan and lead acetate to liver histopathology mice Mus muscullus.
Methods: The research is laboratory experimental with post-test control group design was conducted for 31 days. In this study, 10 mice Mus muscullus were randomly divided into two groups, control and treatment.
Resuluts: Based on analyzed data with T- test showed there is a significant influence of lead exposure to an increase in the number of cells parenchymal and hydrophic degeneration as well as the appearance of necrotic cells in mice induced with an average degree of damage of 1.7..
Keywords: Lead, degrees of damage, cell adaptation, Mus muscullus, necrosis.
INTRODUCTION
Various kinds of metals was found in the environment are very toxic substances in humans and animals. These metals such lead, mercury, and arsenic which have toxic properties and have been known to humans for years. Accumulate of the metal in the environment enter the food chain and makes its more dangerous to health. The spread of heavy metals in ecosystems comes from the modern industries so that we can be found in water, air, and even on the toys and office equipment (Flora, Gupta, & Tiwari, 2012). Lead is a xenobiotic substance for the body, which comes from the environment, both in natural and artificial forms.
Lead is a heavy metal that is naturally present in the earth’s crust, and spreads due to human activities that causes environmental pollution and have impact on surrounding living things (Sembel, 2015;
Soemirat & Ariesyadi, 2015).
Plumbum (Pb) or namely as lead was metals group IV-A in the periodic table of chemical elements. Lead is one of the elements that cannot be broken down into the other substances by chemical reactions. Lead has four kinds’ isotopes of bluish or silvery grey with a meal ting point on 327,50C boiling point at 17400C in the atmosphere.
Chemically, it’s is a heavy metal that has a low vapor point and can stabilize other compounds and becomes eenvironmental contamination and health problems in the world (Canfield & Jusko, 2008;
Naqi, 2015; Sembel, 2015; Soemirat & Ariesyadi, 2015).
The effect of lead for humans is to affect cognitive function, learning ability, shorten height, decrease hearing functions, affect behavior and intelligence, intelligence, damage bodily functions, such as the kidneys, nervous system, and reproduction, increase blood pressure and affect brain development. Can also cause anemia and for pregnant women who are exposed to lead will affect their breastfeeding children and accumulate in milk (Wakefield, 2002).
In organs, lead will accumulate in the bones.
Because in the Pb2+ ions, lead will replace the existence of Ca2+ ions in bone tissue. Lead in a pregnant woman can pass through the placenta and then it will go in the circulatory system of the fetus and then after the baby is born. Lead will be issued together with the milk. Although the amount of lead absorbed by the body only a few turns it’s very dangerous. That is because lead compounds can have toxic effects on various functions of the body's organs (Gidlow, 2015; J et al., 2008; World Health Organization, 2010).
Results of research conducted in Jakarta is known that a quarter of school children in Jakarta has a lead content in blood ranges from 10-14.9 ug/dL, which go beyond the limits set by the Centers for Disease Control and Prevention of the United States at less than 10 ug/dL which are classified as
non-toxic (Naria, 2005). Other research states that, the highest blood content of more than 10 ug/dL has been found in children who live in areas that are congested with traffic. Meanwhile, children who live near roads with low traffic density have been shown to have lower blood lead levels. Test results on the blood content among children in Jakarta quite a bit higher and consistent in comparison with other countries that have leaded gasoline. This is thought to occur because the blood lead levels in children in Jakarta declined since use of lead-free gasoline in Indonesia (Gusnita, 2012; Wagiu & Wulur, 2016).
Bogor, West Java, where the traffic is very high compared to other areas, was found the vegetables and the tea plant has been contaminated with lead from gasoline. The high concentration of lead found in the plant in this area is likely due to the use of chemical fertilizers. It was noted that the lead concentration in vegetables and plants in Bogor was greater than the WHO standard limit for vegetables (WHO set threshold: 2 ppm for wet weight and 2.82 ppm for dry weight) (Raharjo, Raharjo, & Setiani, 2018)
Lead enter the human body through various methods such mouth (oral), i.e. through food or drink, breathing, and skin (Ardillah, 2016; Aziz RA
& Marianti, 2014). Lead (Pb) that enters the body can cause acute and chronic poisoning. Acute toxicity of Pb is characterized by a burning sensation of the mouth, the stimulation of the gastrointestinal accompanied by diarrhea and chronic poisoning symptoms such as nausea, anemia, and pain around the stomach and cause paralysis. While the chronic effects are damage to the nervous system, bones, kidneys, brain and liver (Zidi I, 2014). Lead in the body will undergo metabolism (chemical changes), in the human body and heart into an organ which is particularly noticeable in these events. Lead is metabolized in 2 phases, cytochrome-catalyzed hydroxylation p450, then the hydroxylase compound is converted by the glutathione enzyme into various polar metabolites through conjugation with glutathione. High lead levels that would interfere with the metabolism of enzymes that can increase free radicals and cause damage to the cell membrane (Gidlow, 2015).
The liver is an important organ which secretes material for digestion, and is very susceptible to the influence of chemicals so that it becomes a major target organ of the toxic effects of chemical substances (toxicant). Most of the toxins that enter the body after being absorbed by small intestinal epithelial cells will be carried to the liver by the portal vein of the liver (“Lead poisoning and
health,” 2013). Lead entering the liver causes physiological disorders, so the liver tries to remove it as part of detoxification. By changing substances into a form of compounds that are easily removed from the body. Liver which accumulate metallic lead (Pb) will damage liver tissue, cause the fat degeneration and necrosis. The higher concentration of lead will make the higher of damage. Toxins that enter too large are toxic to the liver, and will cause degeneration of liver tissue, then necrosis can damage the liver tissue (Wakefield, 2002).
This is in accordance with Naqi's (2015) study of cerebral histological comparisons of changes in cortex, hippocampus, cerebellum, punch and amp; medulla of albino rats given the treatment that 4% lead acetate solution in water within 17 days with a dose of 1000 ppm in adlibitum mice showed damage to the liver, stomach, kidney and brain (Naqi, 2015). Another study conducted by Jannah, et al (2017) showed the changes in hepatocyte cell microscopic picture that given length of lead exposure make parenchyma degeneration, hydropic degeneration and necrosis (Jannah et al., 2017). The purpose of this study was to determine the effect of lead exposure (Pb) on hepatocyte cell damage index in Mus musculus mice due to lead exposure with 1000 ppm concentration in adlibitum.
METHODS
This research is experimental with posttest only group control design. This study consisted of 2 groups in which group 1 was the control group that was only given aquadest, and group 2 was the treatment group by administering Pb acetate solution by adlibitum. The observations were made for 30 days. The number of samples of 5 animals followed the minimum rules of animal testing which followed animal reduce. The animals used in this study were 10 Mus musculus mice aged 6-8 weeks male sex with body weight 30-40 grams, healthy (active, agile, good appetite and no visible anatomical abnormalities).
Preparat of liver using paraffin then haematoxylin eosin staining was done microscopically and then examined microscopically. The degree of liver cell damage was seen from the liver preparations of mice that were given hematoxilin-eosin (HE) staining, then observed changes in the structure of liver cell histopathology according to Manja Reonigk scoring by calculating the amount of liver mice preparations that were given Hematoxilin-Eosin (HE) staining.
Normal hepatocyte cells and hepatocyte cells
undergoing parenchymal degeneration, hydroponic degeneration and necrosis as much as 5 visual fields in each sample group treated using a light microscope at 400x magnification. Scores Degree of damage = 1 (normal cell count) + 2 (number of Parenchymal Degreneration cells) + 3 (number of hydropolic degeneration cells) +4 (number of Necrosis cells). Data was analysis using T independent test
RESULTS
The results of microscopic observations of the prevents of mice liver preparations are listed in table 1. Tabel 1 shows the average of the group with the administration of lead acetate is relatively higher than the negative control group
Table 1. Average of field view of mice liver
Mice Field 1 Field 2 Field 3 Field 4 Field 5 Mean
Negative Control 1 1.10 1.15 1.20 1.10 1.30 1.17
2 1.10 1.10 1.20 1.10 1.25 1.15
3 1.05 1.15 1.11 1.10 1.20 1.12
4 1.10 1.15 1.05 1.20 1.15 1.13
5 1.15 1.00 1.00 1.20 1.25 1.12
Exposure of lead 1 1.55 2.20 1.45 1.65 2.15 1.80
2 1.75 1.70 1.40 1.60 1.75 1.64
3 1.65 1.80 1.50 1.75 1.55 1.65
4 1.75 1.90 1.60 2.00 1.65 1.78
5 1.85 1.70 1.30 1.70 1.85 1.68
In the negative control group the minimum value was found to have a value of 1 which means that all cells were observed under normal conditions. The maximum value in the control group shows a value of 1.3 which means that of the 20 cells observed can be found a maximum of 2 cells that are necrosis. In the group whom exposed with lead, the average value of each visual field was at a minimum of 1.4 and a maximum of 2.2. This indicates that the lead acetate group can alter the cell morphology of hepatocytes towards a higher level of damage than the control group. Figures 1.4 indicate that of the 20 observed hepatocyte cells;
necrosis cells were found as many as 3 cells or 5
cells that experienced hydrographic degeneration or 7 cells that experienced parenchymal degeneration or a combination of all three with a decreased number. The maximum value of the lead acetate group of 2.2 indicates that of the 20 hepatocyte cells observed all experienced morphological changes in paremkin degeneration or from 20 hepatocyte cells, 8 necrotic cells or 12 cells undergoing hydorphic degeneration or a combination of all three with a decreased number. The results of the average degree of damage are then reprocessed to determine the effect of lead exposure on hepatocyte cell damage to the control group can be seen in Table 2. below.
Tabel 2 T Independent test Levene's Test for
Equality of Variances t-test for Equality of Means Mean Diff
F Sig T df Sig.
Degree of hepatocyte
Equal variances assumed
10.578 0.002 -12.75 48 <0,001 -0.57
Equal variances not assumed
-12.75 29.99 <0,001 -0.57
From table 2 above shows the significance value in the levene's test indicates that the research
group is not homogeneous in its group variation.
This shows that the selection of significance values
in the t test group is in the line of Equal variances not assumed that is sig <0.001 (sig <0.05) which means there is a significant difference between the lead acetate group and the negative control group for the degree of hepatocyte cell damage by the difference 0.57.
DISCUSSION
Lead that enters the body through oral, respiratory, or contact, will be absorbed by the blood and bind the active group of the ALAD enzyme (Amino Levulinic Acid Dehydrates), so that the process of synthesis of blood cells is disrupted (Palar, 2008). It’s distributed to extracellular fluid and soft tissue such liver, kidney, and nerves; and hard tissue (bones and teeth)(Wagiu
& Wulur, 2016). The concentration of lead residues in animal tissue depends on the pathway of entry and the time period of exposure to pollutants in the environment (air, water and plants) (Aziz RA &
Marianti, 2014).
Based on the results of this study, showed that mice exposed to lead had an average change in liver function compared to controls. in the group exposed to lead, sinusoids appear dilated and an increase in the number of degenerated cells and necrosis due to accumulation of fat in liver cells. Vakoula in the cytoplasm enlarges and forces the cell nucleus and undergoes changes in liver morphology. Lead toxicity that accumulates in tissues causes direct and indirect disruption of molecular physiological processes. Lead has the ability to cause oxidative damage to tissues and increase fat peroxidation, DNA damage and increase the production of ROS (Reactive Oxygen Species). Cellular, it’s known to cause excessive production of Reactive Oxygen Species (ROS) and consequently increases lipid peroxidation, decreases saturated fatty acids, and increases the content of unsaturated fatty acids in cell membranes. The increase in ROS production is a result of cell oxidative stress (Jannah et al., 2017).
ROS generated through the degenerative process of tissue will cause damage to cell components and affect the membrane li [id, protein, and DNA.
Increased ROS also has implications for increased hydroxyl radicals and causes structural damage to protein cells, nucleic acids, cell membranes and lipids (Ibrahim, Eweis, El-Beltagi, & Abdel- Mobdy, 2012; Jaishankar, Tseten, Anbalagan, Mathew, & Beeregowda, 2014).
This is what supports in research that the group which exposure lead causes damage to cells ranging from perenkin degeneration, hydrophic degeneration to necrosis. The results of the study
mention the common changes that occur in the liver are degenerative fat, hepatic lobular parenchymal necrosis and loss of normal architecture of hepatocytes. Cell damage in the exposed group occurs through various mechanisms, one of which is the ionic mechanism. This mechanism causes significant changes in biological processes, such as cell adhesion, intracellular and extracellular signals, interfering with protein mutation activities, apoptosis, ionic transport, enzyme regulation, and neurotransmitter release resulting in increased ROS levels and decreased antioxidant levels (Jaishankar et al., 2014).
This is consistent with the results of research that states that heavy metal exposure can cause cell changes in the form of cell degeneration (vacuola).
This study is in accordance with Suprijono's research (2017)showed that giving oral lead as much as 10 mg / day influences the liver histopathology picture of male rats and causes an increase in liver cell degeneration and necrosis (Suprijono & Banun, 2017). This happens due to cellular defense mechanisms against substances that enter the cell. Vacuolation in the cytoplasm is a consequence of the call to lipid inclusion and fat metabolism, or interference with oxidative phosphorylation in mitochondia by suppressing ATP production and failure of sodium ATP pumps that depend on cell membranes, resulting in intracellular sodium accumulation, causing water to enter into various cellular compartments and cell swelling occurs (Aziz RA & Marianti, 2014; Zidi I, 2014).
The process of liver damage caused by lead inhibits the action of the enzyme and increases the production of free radicals and maintains antioxidants in tissues and cells that cause organ damage (Dewanjee, Sahu, Karmakar, &
Gangopadhyay, 2013). Lead entering the liver will accumulate in tissues and damage the metabolic system in hepatotic cells. The administration of lead doses to animals results in the highest accumulation of lead in the kidneys, liver, bone marrow, brain and heart muscle. Chronic oral administration of lead in low doses causes accumulation of bone marrow, kidney bibs, and bones (Lin, Zengyong, & Jianzhu, 2010).
CONCLUSION
This study showed that the given of 1000 ppm lead acetate for 30 days by adibitum caused parenchymal degeneration, hydrophic degeneration and necrosis in the form of karyolysis and picnotics with significantly different results when compared
with negative controls with damage index was 1.7 compared to controls.
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