|Year : 2019 | Volume
| Issue : 1 | Page : 7-13
Histopathological and histochemical effects of nicotine on the liver and kidney of adult male rats
Medhat M Menshawy PhD 1, Walid M Sharaf2, Reham S.E. Esmail3, Abdel R.H. Farrag2
1 Department of Biology, Center of Basic Sciences and College of Pharmaceutical Sciences and Drug Manufacturing, Misr University for Science and Technology, Almotamyez District, 6th October City, Egypt
2 Department of Pathology, National Research Centre, Dokki, Cairo, Egypt
3 Department of Pathology, Faculty of Medicine, Fayoum University, Fayoum, Egypt
|Date of Submission||19-Nov-2018|
|Date of Acceptance||04-Feb-2019|
|Date of Web Publication||27-Jun-2019|
Medhat M Menshawy
Center of Basic Sciences (CBS), Department of Biology, Misr University for Science and Technology, Almotamyez District, 6th October City
Source of Support: None, Conflict of Interest: None
Background/aim Nicotine is the more abundant alkaloid component contained in tobacco. Smoking causes numerous diseases that are associated with anemia. Nicotine is metabolized primarily by the liver, and to a lesser extent, by the kidney. The aim of this study was to investigate the iron distribution and histopathological changes in liver and kidney tissues of rats administered with nicotine.
Materials and methods This study was performed on thirty adult male albino rats. Animals were divided into three groups (10 each). Group I served as control rats, which were injected subcutaneously with 1 ml of normal saline; group II rats were injected subcutaneously with nicotine at a dose of 0.25 mg/kg/day; and group III rats were injected subcutaneously with nicotine at a dose of 0.5 mg/kg/day for 8 weeks. At the end of the experiment, the animals were killed under ether anesthesia. Liver and kidney tissues specimens were prepared by routine histological procedures and examined under a light microscope.
Results Injection with nicotine showed iron deposition inside the liver sinusoids, seen as green bluish stain. In addition, scattered iron intracellular deposition appeared as scattered bluish dots in kidney. Histopathological examination of liver specimens from rats injected subcutaneously with nicotine showed mononuclear cell infiltration and that some of the hepatocytes had a hyperchromatic nucleus and enlarged sinusoids as compared with the control ones. However, kidney specimens showed some vacuolated tubular cells, few dilated tubules, and the presence of intraluminal casts in some tubules.
Conclusion This study indicated that subcutaneous injection of nicotine causes iron deposition in the liver and kidney and is associated with structural damage.
Keywords: iron, kidney, liver, nicotine, rats, tissues
|How to cite this article:|
Menshawy MM, Sharaf WM, Esmail RS, Farrag AR. Histopathological and histochemical effects of nicotine on the liver and kidney of adult male rats. J Arab Soc Med Res 2019;14:7-13
|How to cite this URL:|
Menshawy MM, Sharaf WM, Esmail RS, Farrag AR. Histopathological and histochemical effects of nicotine on the liver and kidney of adult male rats. J Arab Soc Med Res [serial online] 2019 [cited 2020 Jan 24];14:7-13. Available from: http://www.new.asmr.eg.net/text.asp?2019/14/1/7/261615
| Introduction|| |
Nicotine is the more abundant alkaloid component, and it is believed to be the reason for cigarette smoking in many people, particularly as they derive satisfaction and pleasant impression from inhaling nicotine .
Available data showed that nicotine affects several biological activities. Occasionally, it causes bronchitis and interferes with alveoli structure, causing progression of emphysema-like injury and lung cancer . Moreover, it decreases exercise tolerance and causes coronary thrombosis ,.
Smoking also has negative effects on renal structure and function. Tobacco compounds are discharged through the kidneys after metabolizing in the liver, which plays an important role in the development of renal diseases, and their incidence and progression . As smokers are constantly exposed to chemical substances such as cadmium, lead, and mercury, the effects of these chemicals may aggravate signs of nephrotoxicity in the presence of renal diseases .
Nicotine is a major tobacco alkaloid, which causes liver inflammation owing to the adverse actions of its secondary metabolites . Additionally, it has been found that there is a relationship between smoking and liver’s ability to detoxify dangerous substances  and kidney dysfunction . Nicotine is metabolized primarily by the liver, and to a lesser extent, the lung and the kidney, with the primary metabolite being cotinine .
Anemia represents a decrease in one or more of the chief red blood cell measurements. Cigarette smoking has been related with several chronic diseases that can be complicated by anemia. A number of different malignancies ,, as well as gastric or duodenal ulcers , are accountable for a significant amount of anemia between smokers; anemia may be the end result of blood loss or owing to inhibition of erythropoiesis by a malignant tumor or chemotherapeutic cure.
Iron is an essential micronutrient and has important roles in oxygen transport, oxidative phosphorylation, and other enzymatic functions ,. There is tight control of systemic iron levels by means of iron absorption, storage, and recycling. Iron deficiency causes a decrease in hemoglobin production and can consequently lead to anemia, whereas iron overload burdens the body and results in excess tissue iron, which can cause cell damage ,.
Iron overload also known as hemochromatosis is a phenomena that indicates iron accumulation in the body from any cause. The most important causes are hereditary hemochromatosis, a genetic disorder, and transfusional iron overload, which can result from repeated blood transfusions ,. Hemochromatosis may be associated with chronic liver disease and cirrhosis of the liver, heart failure, irregular heart rhythm, diabetes, hypogonadism, and arthritis ,. Liver, heart, and endocrine glands are the most commonly affected organs by hemochromatosis . Therefore, the aim of this study was to investigate the iron distribution and histopathological changes in liver and kidney tissues of rats administered with nicotine.
| Materials and methods|| |
Nicotine hydrogen tartrate with product number 36733-1 G (99% nicotine) was purchased from Sigma Chemical Corporation (Sigma Aldrich, St. Louis, Missouri, USA). All experimental procedures were performed according to the institutional committee of the animal’s care and use guidelines, National Research Centre (Egypt). The dose of nicotine was delivered to each rat via the subcutaneous injection daily throughout the period of experimentation (8 weeks). The nicotine dosage freshly prepared in normal saline for each group of animals was injected via a subcutaneous route with 0.25 mg/kg (low dose) and 0.50 mg/kg (high dose) body weight/day. The working solutions were stored in foil wrapped glass bottle at 4°C for no longer than 10 days.
Thirty healthy male albino rats weighing 150–200 g obtained from the animal house of National Research Centre, Dokki, Cairo, Egypt, were used in the study. The animals were housed under standard laboratory conditions of light, temperature, and humidity with a 12 h light/dark cycle and had access to food and water ad libitum throughout the study. Animals were acclimatized to the conditions for 7 days before the experiment initiation. The studies were performed in accordance with the guidelines for the humane treatment of animals as set forth by the Association of Laboratory Animal Sciences and the Center for Laboratory Animal Sciences at National Research Center. The animal were randomly divided according to their weight into three groups, with 10 rats each, as follows:
- Group Ι (control): rats injected subcutaneously with 1 ml of normal saline for 8 weeks.
- Group II: rats injected subcutaneously with nicotine (0.25 mg/kg/day) for 8 weeks.
- Group III: rats injected subcutaneously with nicotine (0.5 mg/kg/day) for 8 weeks.
Histochemical and histological studies
At the end of the experimentation, the animals were killed after light ether anesthesia. Liver and kidney tissues specimens were fixed in 10% formaldehyde solution for 24 h, dehydrated through a series of graded alcohol, and cleared in xylene. Tissue specimens were embedded in paraffin wax and sectioned (thickness, 5 μm). For light microscopic evaluation, paraffin sections were stained with Prussian blue for iron detection  and hematoxylin and eosin for histopathological study , and then were examined using Olympus CX 51 microscope and photomicrographed using C100 camera (Olympus, Tokyo, Japan).
| Results|| |
Histochemical study of iron
Examination of liver sections, stained with Prussian blue stain, of control rats shows negative staining of iron as indicated by the absence of the bluish stain in the hepatocytes and blood sinusoids ([Figure 1]a).
|Figure 1 Liver section of (a) a control rat showing negative staining of iron as indicated by the absence of the bluish stain, (b) a rat given low dose of nicotine showing iron deposition inside the liver sinusoids, seen as green bluish stain (arrows), (c) a rat given high dose of nicotine showing iron deposition inside the sinusoids, seen as green bluish stain (arrows), and (d) a rat given high dose of nicotine showing iron deposition intrasinusoidal and in between the inflammatory cells in the portal tracts, seen as green bluish stain (arrows) (Prussian blue stain, ×400).|
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Histochemical evaluation of liver sections, stained with Prussian blue stain, of rats after subcutaneous injection with 0.25 mg/kg/rat/day of nicotine for 8 weeks showed iron deposition inside the liver sinusoids that appeared as green bluish stain ([Figure 1]b).
Investigation of liver sections of rats after subcutaneous injection with (0.50 mg/kg/rat/day) of nicotine for 8 weeks showed iron deposition inside the sinusoids, seen as green bluish stain ([Figure 1]c). On the contrary, some sections indicated iron deposition intrasinusoidal and in between the inflammatory cells in the portal tracts, which appear as green bluish stain ([Figure 1]d).
Microscopic examination of kidney sections of control rats, stained with Prussian blue stain, showed regular glomeruli and tubules, and no iron deposition was seen ([Figure 2]a).
|Figure 2 A kidney section of (a) a control rat showing regular glomeruli and tubules, no iron deposition seen, (b) a rat given low dose of nicotine showing minimal iron deposition inside the cells, seen as light bluish (arrows), and (c) a rat given high dose of nicotine showing scattered iron intracellular deposition, seen as scattered bluish dots (arrows) (Prussian blue stain, ×400).|
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On the contrary, histochemical investigation of kidney sections of rats after subcutaneous injection with 0.25 mg/kg/rat/day of nicotine for 8 weeks and stained with Prussian blue showed minimal iron deposition inside the cells of the tubules, appearing as light bluish color ([Figure 2]b), whereas investigation of kidney of rats after subcutaneous injection with 0.50 mg/kg/rat/day of nicotine for 8 weeks showed scattered iron intracellular deposition, which appears as scattered bluish dots ([Figure 2]c).
Histopathological investigation of sections of control liver shows normal the architecture of hepatic lobules, the central veins lie at the center of the lobules, surround by the hepatocytes. The hepatocytes appear with strongly eosinophilic granulated cytoplasm and distinct nuclei. Between the strands of hepatocytes, the hepatic sinusoids are shown, as seen in [Figure 3]a.
|Figure 3 A photomicrograph of sections of liver of (a) normal architecture of a hepatic lobule, (b) rat injected with low dose of nicotine showing portal tract free of inflammation, (c) liver section of a rat injected with low dose of nicotine showing degenerative feature of the hepatocytes associated with mild inflammatory cell aggregated and in the portal tract (arrow), and (d) liver section of a rat injected with a high dose of nicotine shows marked degenerative feature of the hepatocytes that is associated with inflammation in the portal areas and bile duct proliferation (arrows) (hematoxylin and eosin, ×400).|
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Histopathological investigation of liver sections of rats injected subcutaneously with a dose equivalent to 0.25 mg/kg of nicotine for 8 weeks showed portal tract free of inflammation ([Figure 3]b). In some rats, examination of liver section of rats injected subcutaneously with a dose equivalent to 0.25 mg/kg of nicotine for 8 weeks showed mild inflammatory cell aggregated in the portal tract ([Figure 3]c).
Microscopic examination of liver section of rats injected subcutaneously with a dose equivalent to 0.50 mg/kg of nicotine for 8 weeks showed degeneration of hepatocytes, marked portal tract inflammation, and bile duct proliferation ([Figure 3]d).
Examination of kidney sections from control rats stained with hematoxylin and eosin show normal architecture of the renal corpuscles and tubules ([Figure 4]a).
|Figure 4 Sections of kidney from (a) a control rat showing normal architecture of the renal corpuscles and tubules (hematoxylin and eosin, ×200), (b) a rat injected with a low dose of nicotine showing tubular cells vacuolization (arrows) and tubular dilatation, (d and c) a rat injected with a high dose of nicotine showing vacuolated tubular cells, few dilated tubules, and showing intraluminal casts in some tubules (hematoxylin and eosin, ×400).|
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Histopathological investigation of kidney sections of rats after subcutaneous injection with 0.25 mg/kg of nicotine showed renal tubular cell vacuolization and tubular dilatation ([Figure 4]b), whereas examination of kidney of rats after subcutaneous injection with 0.50 mg/kg of nicotine showed some vacuolated tubular cells, few dilated tubules, and some tubules showed intraluminal casts ([Figure 4]c).
| Discussion|| |
Nicotine, a pharmacologically active ingredient in tobacco, is generally regarded as a primary risk factor in the development of cardiovascular disorders, myocardial infarction, stroke, kidney cancer, pulmonary diseases, and certain immunological dysfunction . This highly addictive alkaloid has been reported to induce oxidative stress in both in vivo and in vitro . The mechanisms of free radical generation by nicotine are not clear. However, it has been reported that nicotine disrupts the mitochondrial respiratory chain, leading to an increased generation of superoxide anions and hydrogen peroxide . Previous studies have suggested that superoxide anion and hydrogen peroxide are the main source of causing nicotine-induced free radicals, depleting the cellular antioxidants .
In the present study, histochemical examination of iron deposition in liver and kidney showed highly significant amount in rats administered with nicotine as compared with the healthy ones. The body lacks mechanisms for increasing excretion of the accumulated amount of iron , leading to iron overload, most of which is stored in the liver. However, some author investigated that iron may also accumulate in other organs such as the spleen, the kidneys, or the bone marrow 
The liver seemed to be the appropriate organ to give a true reflection to the extent of the toxicity caused by nicotine injection. As shown in the present study, nicotine administration resulted in degeneration of the hepatocytes and expansion of portal tracts associated with chronic infiltrating inflammatory cells. The previous alterations were dose dependent. This obvious hepatic toxicity following nicotine injection was previously reported in the work of Siegelman et al. . The progressive dilatation of the blood vessels in the liver could be considered as a reaction change that might be related to increased levels of prostaglandin synthesis, which led to induced smooth relaxation and consequent vasodilatation either directly or through releasing other vasodilator substances in blood . Backhle et al.  explained the dilatation and congestion of the portal veins by the portal hypertension caused by obstruction at the sinusoidal level because of degenerating ballooning hepatocyts. The work of Walter and Israel  stated that the presence of infiltrating lymphocytes, mainly in the portal areas in addition to the peripheral parts of the lobules, might be explained as a defense reaction of the lobule in response to the toxicity of injected nicotine, being metabolized in the liver.
These histopathological results were confirmed exactly with the histochemical examination. Nicotine intake in rats disturbed the antioxidant defense in rats, increased the lipid peroxidation, and reduced the antioxidants in tissues . The liver is affected by the oxidative stress caused by iron-overload toxicity. When transferrin-bound iron saturation is increased by more than 75%, no transferrin-bound iron (NTBI) begins to accumulate . NTBI is potentially toxic owing to its high propensity to induce reactive oxygen species (ROS), and it can cause cellular damage both at the plasma level and in intracellular organelles. NTBI and iron deposits may cause liver injury during iron overload ,. ROS are produced by metabolic functions in the cell. Iron, in conjunction with ROS, leads to an increase in hydroxyl radicals and, consequently, cellular damage. Through Haber Weiss and Fenton reactions, iron generates ROS ,.Histopathological examination of kidney sections of rats after subcutaneous injection with low dose (0.25) of nicotine showed renal tubular cells vacuolization and tubular dilatation, but examination of kidney of rats after subcutaneous injection with high dose (0.50) of nicotine showed some tubules dilatation, vacuolated tubular cells, and intraluminal casts in some tubules.
Many studies report that cigarette smoking leads to lung, larynx, oropharynx, kidney, urinary bladder, and breast cancers. Smoking has also been reported to produce alterations in histological structure and functional changes in the liver. Previous studies showed that smoking causes necrosis, cellular proliferation, fibrosis, and focal nodular hyperplasia in the liver. Smoking has also been found to affect the glomeruli structure and function in patients with type 2 diabetes, which has been reported to play an important role in nephropathy development and progression .
In conclusion, the present study indicated that subcutaneous injection of nicotine causes iron overload and structural damage of the liver and kidney, which seemed to be the appropriate organs to give a proper sign to the extent of the toxicity caused by nicotine injection.
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Conflicts of interest
There are no conflicts of interest.
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