Ameliorative Effect of Turmeric and Cocoa Extract against Acute Second Hand Exposure of Tobacco Smoking on Hepatocytes and Enterocytes in Albino Rats: Ultrastructural Study

1Department of Medical Laboratory Sciences, Faculty of Pharmacy, Jadara University, Irbid, Jordan. 2Department of Biological Sciences, School of Science, The University of Jordan, Amman, Jordan. 3Department of Anatomy and Histology, School of Medicine, The University of Jordan, Amman, Jordan. 4Department of Medical Laboratory Science, College of Science, Komar University of Science and Technology, Sulaymaniyah, Kurdistan region, Iraq. 5Department of Pathology and Microbiology and Forensic Medicine, Faculty of Medicine, The University of Jordan, Amman, Jordan. 6Royal Medical Services, Zarqa., Jordan. 7Pharmacological and Diagnostic Research Center (PDRC), Faculty of Pharmacy, Al-Ahliyya Amman University, Amman 19328, Jordan. *Corresponding Author E-mail: m.alqaraleh@ammanu.edu.jo

Tobacco leaves are smoked as cigarettes, chewed or used in waterpipes (also known as narghile, or arghileh shisha, goza, hookah, and hubbly bubbly) 1 . It has been estimated that tobacco smoking was responsible for more than seven million deaths each year 2 , and it is expected that by 2030 the death rate will exceed millions per year in developing countries 3 .
Many toxicants found in waterpipe smoking such as tar, carbon monoxide, heavy metal 1,4 .The presence of these toxic substances in different concentrations may cause major lethal illnesses, including lung cancer, cardiovascular disease and chronic obstructive pulmonary disease 5 . Secondhand exposure to cigarette and waterpipe tobacco smoking can induce histopathological changes and inflammation on different organs in the experimental animals [6][7][8] . Nicotine, a constituent of tobacco, is reported to induce oxidative stress both in vitro and in vivo, as well as depleting antioxidant defense mechanisms 9 .
There are several important natural antioxidants which have a protective effect against free radicals 10,11 . Natural antioxidants with free radical-scavenging activity such as the cocoa bean have received much attention, due to their health-promoting properties. Cocoa is the mature fruit of the cacao tree (Theobroma cacao L.), which grows in tropical regions of Africa and South America 12 . The main components of cocoa powder that have an antioxidant capacity are polyphenol and methylxanthine compounds, where these could significantly contribute to their health-promoting activities 13 . Cocoa beans are one of the main sources of polyphenols, especially epicatechin. Epicatechin has been reported to have an antioxidant capacity 14 . High consumption of chocolate and cocoa was associated with a decrease in both blood pressure and the risk of cardiovascular disease 15 .
Turmeric (T) is a yellow powder, derived from the plant Curcuma longa. Pharmacological studies have demonstrated that it has antitumor, anti-inflammatory, anti-infection, and antioxidant activity, with very low toxicity 16 , Turmeric consists of a water-soluble component, turmeric (T; mol weight 24,000 daltons), and lipid-soluble component Curcumin (Cu; mol weight 369.89 daltons). Curcumin polyphenols (diferuloylmethane) inhibit toxin-mediated stress responses via their anti-inflammatory and antioxidant properties in addition to inducing the expression of cytoprotective proteins 17 . Curcumin neutralizes nitric oxide, provides continuity to antioxidant enzymes such as catalase and superoxide dismutase, and lowers lipid peroxidation, to protect tissues and organs from oxidative damage caused by nicotine 18 .
This study aims to assess the ultrastructural alterations of the liver and small intestine of albino rats induced by two type of tobacco smoking; and investigate the effect of using turmeric and cocoa extract to protect these two organs from damage induced by cigarette or waterpipe tobacco smoking.

Preparation of turmeric and cocoa extract
Cocoa blend and curcumin from Curcuma longa (turmeric) powder were purchased from (Sigma, Germany).Both antioxidants containing materials were macerated in 80% methanol for three days, according to 19 , then filtered and concentrated to a small volume to remove the entire methanol using a rotary evaporator. The low volume was later freeze-dried to obtain a crude extract and kept at 4°C until needed. The cocoa or curcumin crude extract were administered to rats via oral gavage with a dose of 200 mg/kg/day 19

. experimental design in vivo
Seventy-two adult male albino rats were purchased from animal's house / Jordan University of Science and Technology, Irbid, Jordan. Rats were randomly divided into nine groups; each contains eight rats, and left to acclimatized in room temperature, well-ventilated room with light/dark cycle of 12/12 hours, feed with standard animal chew and free-water access ad libitum.
This experiment was performed in Animals House / Department of Biological Sciences / The University of Jordan. All animal experiments were conducted according to the guidelines of European Commission Directive 86/609/EEC for laboratory animal care and use. The experimental groups were divided as following: Group 1 (Control): Rats were exposed to fresh air only. Group 2 (Curcumin): Rats were uptake curcumin via oral gavage with dose of 200 mg/kg/day. Group 3 (Cocoa): Rats were uptake cocoa crude via oral gavage with dose of 200 mg/kg/day. Group 4 (Cigarette smoking): Rats were exposed to cigarette smoking (1 cigarette/rat/day). Group 5 (Cigarette smoking + Curcumin): Rats were uptake curcumin with dose of 200 mg/kg/ day before one hour from exposure to cigarette smoking (1 cigarette/rat/day). Group 6 (Cigarette smoking + Cocoa): Rats were uptake cocoa crude extract with dose of 200 mg/ kg/day before one hour from exposure to cigarette smoking (1 cigarette/rat/day). Group 7 (Waterpipe smoking): Rats were exposed to waterpipe smoking (1 waterpipe tobacco head 5 g molasses/rat group/day). Group 8 (Waterpipe smoking + Curcumin): Rats were uptake curcumin with dose of 200 mg/kg/ day before one hour from exposure to waterpipe smoking (1 waterpipe tobacco head 5 g molasses/ rat group/day). Group 9 (Waterpipe smoking + Cocoa): Rats were uptake cocoa crude extract with dose of 200 mg/ kg/day before one hour from exposure to waterpipe smoking (1 waterpipe tobacco head 5 g molasses/ rat group/day).
The cigarettes (L&MRed, Philip Morris, Jordan) and tobacco molasses (two apples flavor, Mazaya, Jordan) were purchased from local markets.
Each group of rats exposed to tobacco smoking (cigarette or waterpipe) was placed inside transparent plexiglass box (0.6 x 0.5 x 0.1 m 3 ) acting as inhalation chamber to perform a smoking process using modified smoking machine described by [4]. The duration for experimental exposure lasts for 30 consecutive days.
All rats were euthanized by cervical dislocation and dissected to obtain the liver and parts of small intestine. Those organs were washed properly using phosphate buffered saline pH 7.2, 2.5 % glutaraldehyde for 72 hours prior to electron microscopy preparations.
Tissues of the liver, small intestine were cut into approximately (1 mm 3 ), then fixed with 2.5 % glutaraldehyde in PBS (0.1 M, pH 7.2) at 4 o C in glass vials for 72 hours. The tissue post-fixed with 1% osmium tetroxide in in PBS for 1 hr according  to [20]. Dehydration was done by immersing the samples for 5 min ascending grades of ethanol.

results
Ultrathin sections from the control, turmeric, and cocoa extract of experimental rats showed normal hepatocytes containing normal large, round nuclei with predominant euchromatin. Mitochondria, which filled the cytoplasm, were normal in shape and size containing visualized cristae. Abundant rough endoplasmic reticulum was normally distributed within the cytosol of hepatocytes ( Fig. 1A-C).
However, rats treated with cigarette smoking showed hepatocytes with polymorphic mitochondria with mild deterioration in cristae and matrix; nuclei contain some chromatin condensations adjacent to the inner margins of the nuclear envelope, the nuclear envelope appeared irregular in shape, rough endoplasmic reticulum appeared scattered and dilated cisternae, the cytosol continent was distorted with the presence of some vacuolization (Fig. 1D). Oral uptake of turmeric extract with cigarette exposure showed less vacuolization and better cellular architecture with regular nuclear envelope of hepatocytes in comparison with cigarette smoking only (Fig. 1E). Parallel treatment of Cocoa crude extract along with cigarette smoking, yet vacuolization presents with disrupt mitochondria (Fig. 1F).
The examined ultrathin section of hepatocytes in mice exposed to waterpipe smoking showed several abnormalities; increase in the number of mitochondria, and swollen in shape, thinning with disorganization of mitochondrial cristae, and the nucleus has heterochromatic DNA with intact nucleus envelope. Vacuolization and lipid droplets were evident, and some blebs were displayed from the plasma membrane of hepatocytes (Fig. 1G). Ultrathin sections of liver tissue from waterpipe smoke-exposed rats with turmeric showed better recovery results in hepatocytes in comparison to sections of hepatocytes of cocoa extract treatment with waterpipe tobacco smoking ( Fig. 1H & I). The treatment with these crude extracts showed some similarities like reduce or absence of blebbing, retain the normal shape and size of mitochondria, increase the proportion of euchromatic chromatin in nuclei. But turmeric extract showed better enhancement in term of reducing vacuolization.
Ultrathin sections from the control, turmeric, and cocoa extract of experimental rats (Fig. 2 A-C) showed normal morphology of enterocytes containing typical withstanding microvilli, rod-shaped mitochondria, and no abnormalities were noticed in their organelles.
The cigarette smoking exposed group showed atypical cellular morphology; mitochondria appear in polymorphic shapes containing partial disorientation in their cristae, and the matrix appeared to be diffused and electron-lucent ( Fig 2D). Also, it showed a slight shortening of microvilli, the organelles contained granular electron-dense inclusions, and vacuolization was observed in the cytoplasm of enterocytes. Parallel treatment with turmeric or cocoa crude extract (Fig.  2 E-F) preserve the typical length of microvilli with a uniform organization from the apical part of enterocytes and decreased vacuolization in the cytosol of enterocytes. However, mitochondria appeared less polymorphic in shapes with distinct cristae and matrix in enterocytes of the turmeric treated group than the cocoa once.
In waterpipe tobacco smoking, ultrathin sections of the enterocytes were seen to have edema and with slight deformity and dilation of cellular membranes resembling blebs. They contain excessive dense bodies with small vacuoles of lipidic nature distributed within the cytoplasm; rounded mitochondria with deteriorated cristae; microvilli baselines were irregular and shortening in their lengths. The enterocytes of this group slightly lost their sinuosity due to cellular swelling with the indication of moderate to severe structure injury (Fig. 2G).Turmeric or cocoa crude extract treatment show (Fig. 2 H-I) uniform of microvilli length organization and decreased in vacuolization in cytosol of enterocyte with less polymorphism of mitochondria, these appear in turmeric treatment more than cocoa.

discussion
Tobacco causes many of the world's major lethal illnesses, including chronic obstructive pulmonary disease, lung cancer, and cardiovascular disease 21 . The rising prevalence of smoking worldwide with its related risks made it mandatory to find remedies to ameliorate its serious consequences 22 . In this research, we study the effect of use natural products containing antioxidants(cocoa and turmeric) against the ultrastructural alterations in liver and small intestine in experimental rats induced by cigarette and waterpipe tobacco smoking.
This study showed that turmerichad chemopreventive ability to attenuate the oxidative damage induced by cigarette and waterpipe smoking through retaining the normal architecture of hepatocytes, decreasing the vacuolization within these cells, our results demonstrated pleomorphic mitochondria with electron dense condensations inside them and partially disrupted cristae. In addition, microvilli appeared disrupted. The changes in the mitochondria might explain the oxidative stress in the jejuna enterocytes.
Exposure to cigarette smoke, may affect both the function and structure of intestinal barrier, as it increases intestinal permeability, causes damage of microvilli, leads to bacterial translocation and abnormal tight junction proteins with destruction of tight junction 23 . Microvilli aid in transport molecule to blood stream and increase the surface area, contribute to absorption process, the destruction or any abnormality in microvilli structure or function may cause inflammation that effect absorption and breakdown of nutrient, which will result in malabsorption of nutrients.
Turmeric neutralizes nitric oxide, provides continuity to antioxidant enzymes such as catalase and superoxide dismutase, and lowers lipid peroxidation, to protect tissues and organs from oxidative damage caused by nicotine 18 . Previous studies stated that turmeric has gastroprotective effect the ability of turmeric to rises mucin secretion also minimize the gastric ulcer complications 24 . Our results showed that the impact of enterocyte with less vacuolization in cytosol, and the microvilli uniform in shape with more organization in apical part of enterocyte, the mitochondria less polymorphism with normal cristae.
Cocoa beans are one of the main sources of polyphenols, especially epicatechin. Epicatechin has been reported to have an antioxidant capacity 14 . These polyphenols content in cocoa have antimutagenic, anti-inflammatory and anti-cariogenic effects 25,26 .
Our result show that the treatment by cocoa extract of small intestine exposure to smoking the microvilli appear parallel, long on the apical part of epithelia cell, mitochondria with various shape cristae are prominent and decreased of vacuolization in cytosol of enterocyte.
Our results demonstrated using tobacco causes adverse impact in hepatocyte, mitochondria which appeared swollen and polymorphic shape with mild deterioration in cristae and matrix; and nucleusisheterochromatic with irregular shape of nucleus envelope.
Rough endoplasmic reticulum shows dispersed and dilated cisterns, the cytosol continent was blurred by the presence of some vacuolation. These results were consistent with earlier findings that presented exposure of rat for three months to cigarette smoking lead to decrease of matrix density with disrupted of mitochondrial cristae, increase size of endoplasmic reticulum, and rise and regular nuclear envelope in comparison with cigarette smoking only. while treatment with cocoa extract showed less recovery effect in hepatocyte than turmeric extract, still destruction and in number of mitochondria 20 . Treatment with turmeric extract with cigarette exposure showed less vacuolization and improved in hepatocyte structural with normal mitochondria pleomorphic shape of mitochondria.
Many study showed the protective effect of turmeric again toxic component such as copper, cadmium, mercury and lead which elevate heavy metal, the turmeric minimize the hepatotoxicity and maintain against mitochondrial dysfunction 27 . Also turmeric can have as antioxidantanti-inflammatory and antimicrobial effect 28 . Turmeric neutralizes nitric oxide, provides continuity to antioxidant enzymes such as catalase and superoxide dismutase, and lowers lipid peroxidation, to protect tissues and organs from oxidative damage caused by nicotine 18 .

conclusion
Our results showed that rats exposed to water pipe smoking, indicate extreme histological changes in mitochondria structure the appear swollen in shape and increase in number with disorganization of cristae, and the nucleus has heterochromatic DNA with intact nucleus envelope. This study demonstrated that the using of turmeric extract with waterpipe smoke to keep the hepatocyte architecture and normal structure of mitochondria also cell membrane in its correct state, While the use of cocoa extract reduced the cell-destructive effect of smoking toxicity. The main components of cocoa powder that have an antioxidant capacity are polyphenol and methylxanthine compounds, where these could significantly contribute to their health-promoting activities.
acknowledgMents This work was supported by the Deanship