Tramadol Biological Effects: I: Effective Therapeutic Efficacy of Lagenaria siceraria Preparation (Gamal & Aref1) and Melatonin on Cell Biological, Histochemical and histopathological Changes in Ileum of Tramadol-Induced Male Mice

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INTRODUCTION
The opioid is considered first-line treatment for hospitalized patients with moderate to severe pain (Holden et al., 2005). They are currently the most effective pain-relieving pharmaceuticals. Opioids are the most potent and effective analgesics available and became accepted as appropriate treatment for acute, cancer and noncancer chronic pain (Collet, 2001). However, they are also rewarding and their repeated use can lead to dependence and addiction.
Tramadol is used worldwide and is listed in many medical guidelines to treat both acute and chronic pain. There is growing evidence of abuse of tramadol in some African and West Asian countries. Tramadol has some side effects. The present study was designed a follow-up and treatment of the cell biological, histochemical and Pathological response which might be induced in the ileum of tramadol application. A total number of 70 adult males Swiss albino mice were obtained and survived during experiments from/in (Autoradiographic lab. of Cell Biology and Immunology studies, Faculty of Science, South Valley University), under supervision Dr. Abdel-baset Aref Mohamed. At the time of the experiments, the mice were aged 90  5 days and weighed 30  2 g. All male mice were kept under the same conditions of light-dark cycle (12h-12h), temperature (23  2 o c), and humidity (37-40%). They were supplied standard food and water ad libitum. The daily injections of tramadol for 20 reaches to 40 days highly changed the volume of nuclei and protein, RNA and DNA materials contained in the cells lining ileum, therefore, extensively and severely destructions showed in the normal histological architecture of ileum of male mice.
Finally, we could say, Although, tramadol has many cytological, histochemical and histopathological, side effects on ileum of male mice but the treatments with Lagenaria siceraria and melatonin have a more effective therapeutic effect on tramadol side effects.
In fact, addiction to opioid analgesics is a growing socioeconomic and health problem with potentially serious consequences documented by a rise in deaths due to overdose (Hall et al., 2008). In the United States, the medical use of opioids has increased dramatically in the past two decades (Okie, 2010).
A parallel rise in opioid-related adverse effects such as emergency room visits and overdose mortality has also occurred (Bohnert et al., 2011). In palliative care, the commonly prescribed opioids include tramadol, morphine, fentanyl, methadone, buprenorphine, diamorphine, oxycodone and codeine, and these drugs exhibit multi-system effects due to their interactions with receptors that are targeted by the endogenous opioid system. Tramadol is a synthetic, centrally acting analgesic, available in Europe since 1977 and in the United States since 1995 for treatment of pain syndromes previously amenable only to the opiate analogues (Moore et al., 1999).
In human and veterinary medicine, tramadol has been in clinical use for the relief of mild to moderate pain (Pypendop and Ilkiw, 2008). Also, tramadol is used perioperatively in veterinary anesthesia as it significantly reduces the requirements of volatile anesthetics and opioid agents (Seddighi et al., 2009).
Tramadol has a dual mode of action. Its analgesic efficacy is attributed to its partial affinity for the µ-opiate receptor and its inhibition of norepinephrine and serotonin reuptake (Shadnia et al., 2008). Tramadol is considered a safe drug devoid of many serious adverse effects of traditional opioids. However, recently, abuse and dependence of tramadol as well as toxicity and tramadol related deaths have been increasingly reported (Tjäderborn et al., 2007).
Melatonin, a hormone produced by the pineal gland, is a potent scavenger of reactive oxidative radicals . Thus, Melatonin can prevent free radicalinduced cellular oxidative damage and can contribute to the physiological functions of the antioxidant defensive system (Kumar et al., 1999). Also, it can stimulate several antioxidant systems, thereby facilitating more antioxidant activities and stabilizing cell membranes. In addition, it modulates the gene expression of several protective enzymes and reduces apoptosis and lipid peroxidation (Reiter, 1998).
Melatonin and its metabolites have an anti-inflammatory property and have been proven to be highly effective in a variety of disorders linked to inflammation and oxidative stress. (Carrillo et al., 2005) A melatonin supplement can decrease oxidative stress in renal tissues and attenuate kidney scarring. In a model of cyclosporine-induced nephropathy, pretreatment with melatonin decreased tubular and glomerular damage (Kumar et al., 1999). Treated animals were divided into 3 groups, including 10 males each. Group T1: animals were daily subcutaneously injected with tramadol Hcl (125ug / 100g b.w.) for 20 days. Group T2: animals were daily subcutaneously injected with tramadol Hcl (125ug / 100g b.w.) for 40 days. Group C: animals were daily subcutaneously injected with distilled water (0.25ml/ 30g b.w.) and served as control.

MATERIALS AND METHODS
All animals were sacrificed the day following the last injection. Experiment II:-All male mice were injected subcutaneously with a daily injection of tramadol (125ug / 100g b.w) for 40 days and subdivided into 4 groups, concluding 10 males each, designated as groups T, S, L and M as follows: Group T: Animals were treated with tramadol only. Group S: tramadol-treated animals were injected with a daily injection of distilled water (0.25ml / 30g b.w.) for 40 days. Group L: tramadol-treated animals were treated with a daily oral dose of a special preparation of Lagenaria siceraria (Gamal Yagteen1 & Abdelbaset Aref1) (0.5 mg/ b.w.) for 40 days. Group M: tramadol-treated animals were daily injected with a subcutaneous injection of Melatonin (100g / 100g b.w.) for 40days at 16h, 2h before the end of the light cycle for 40 days.
The mice of group T in experiment II were similar to mice of group T2 in experiment I, which were daily injected with tramadol for 40 days only before sacrificed.
All animals were sacrificed the day following the last injection or oral dose.
Experiments of this research were conducted in lab. achieve stability of environmental conditions, separation between treated animals and control ones and IACUC targets. IACUC Approval Project Number:

Tissues Preparation:
Tissues specimens of ileum excised, fixed in neutral formalin 10%, dehydrated in graded serious of ethanol, cleared, embedded in paraffin wax and sectioned at 5 microns thickness.
Karyometric studies were applied to H & E-stained ileum. The volume of cell nuclei was performed using camera program (LAS ZA). A total number of (200) nuclei were measured/animal. The measurements were carried out in two ways according to shape nucleus.
The first concerned rounded nuclei of cells where the formula of the sphere was applied: The second concerned oval nuclei of cells. The volume of the nucleus was obtained by means of revolution spheroid: Where: A -longer diameter & P-short diameter All methods were applied according to Pearse (1980), Carleton et al., 1980 andLewinski et al., 1984.

RESULTS Cell
Biological Changes (Karyometric Studies): -Experiment I: In the ileum of control mice, the value of mean volume nuclei of epithelial cells lining villi was 98 μ (Table 1). While in the ileum of the mice which daily received tramadol for 20 days (T1) or 40 days (T2), the value of mean volume nuclei of epithelial cells lining villi was 48 μ and 19 μ respectively.
From the quantitative point of view, the daily injection of tramadol for 20 or 40 days decreased the mean volume value of nuclei of epithelial cells lining villi in the ileum of mice by 51 % and 80% respectively versus those of control mice. While the daily injection of tramadol for 40 days (T2) decreased 60 % the mean volume value of nuclei of epithelial cells lining villi versus those of mice of (T1) ( Table 1).
From the cytological point of view, although the tramadol showed an inhibitory effect on the nuclei volume of epithelial cells lining villi in ileum of mice, but with time, this inhibitory effect decreased.

Experiment II:
The ileum of mice after 40 days from stop tramadol injection (TS) the nuclei of epithelial cells lining villi showed a mean volume value of 58 μ, while in ileum of the mice which received Lagenaria siceraria (TL) or melatonin (TM) for 40 days, the mean volume value of nuclei in both groups was 57µ and 63µ respectively, (Table  1).
From the quantitative point of view, the treatments with stopping tramadol or Lagenaria siceraria or melatonin increased by 202%, 200%, and 231 % respectively versus those of mice that receiving tramadol only for 40 days (Table 1).
From the cytological point of view, all previous treatments revealed an inhibitory effect on the changes induced by tramadol on volume nuclei of epithelial cells lining villi in ileum in mice but did not reach to control mice.    (Table  2).
Although the treatment with stop tramadol or Lagenaria siceraria increased slightly the DNA content in nuclei of epithelial cells lining ileum of mice compared with those of mice that received tramadol only for 40 days but melatonin treatment increased markedly the DNA content which reached to those of control mice.
From the histochemical point of view, the tramadol administration showed a remarkable inhibitory effect on the chemical material contents: protein, RNA and DNA content and stimulatory effect on collagen content epithelium cells lining ileum in mice, but the treatment with melatonin or Lagenaria siceraria reduced the inhibitory effect on tramadol side effects shown by histochemical methods. RNA Content (Toluidine Blue):-Experiment I: The ileum of the control mice (C) stained with RNA stain toluidine blue showed high RNA contents with deeply stained coloration in cytoplasm and nucleolus of epithelial cells lining ileum of mice (Figs. 13 & 14) and (  (Table  2).
Although the treatments with stopping tramadol or Lagenaria siceraria increased slightly the RNA materials contents in cytoplasm and nucleolus of epithelial cells lining ileum of mice compared with those of mice which received tramadol only for 40 days, but melatonin treatment increased markedly the RNA contents and reached to those of control mice.

Protein Content (Bromophenol Bleu Technique):-Experiment I:
The ileum of the control mice(C) stained with bromophenol blue showed high protein content inside epithelial cells lining ileum with deeply blue stainability (Figs.25 & 26) and (  (Table  2).
Except for treatment with stop tramadol injection, the treatments with both, Lagenaria siceraria and melatonin, decreased markedly collagen materials contents in ileum compare with those of mice which reserved melatonin for 40 days and also reached to normal level of collagen.
However, the ileum of the mice which received daily tramadol for 40 days (T2) recorded extensive necrosis and desquamation of the intestinal tips (Figs.55 & 56) and (Table 3). There was ileum vacuolation and degeneration were recorded (Fig.57).
Daily injection of tramadol for 20 and 40 days partially destroyed the normal histological architecture of ileum of mice Experiment II: Ileum of the mice after 40 days from stopping tramadol injection (TS) showed congestion of blood vessels (Fig.58) and (Table 3), besides the thickening of tunica muscularis with fibrous tissues (Fig.59). Also, desquamation and necrosis of the ileum villi were observed (Fig.60). The mice treated with Lagenaria siceraria (TL) exhibited normal arrangement of the ileum layers and villi (Figs.61) and (Table 3). Moreover, mice treated with melatonin (TM) showed normal ileum layers (Fig.62) with some inflammatory cell's infiltration (Fig.63&64) and (Table 3).
The daily treatments for 40 days with Lagenaria siceraria (TL) or melatonin (TM) repair the side effects of a daily injection of tramadol for 40 days on ileum of mice which nearly restored its normal histological architecture.
From the histopathological point of view, the tramadol administration induced histopathological side effects on the architecture of ileum in mice, but the Lagenaria siceraria or melatonin treatment showed an inhibitory effect on these tramadol side effects. The histochemical score of ileum of mice of groups control (C), injection of tramadol for 20 days (T1) and 40 days (T2), tramadol and stopping tramadol injection (TS), tramadol injection and treated with Lagenaria siceraria (TL) and tramadol injection and treated with melatonin (TM) stained with bromophenol blue for total protein, Mallory trichrome technique for collagen , toluidine blue for RNA, Feulgen reaction for DNA and PAS for polysaccharides were classified according to severity into severe (+++), moderate (++), mild (+) and absent (-).

DISCUSSION
Oxygen-free radicals produced from tramadol have shown to induce DNA breaks and DNA damage. The appearance or disappearance of protein fraction in the present study may be attributed to this damage or may be explained by the report of Bedwell et al. (1989), who claimed that the free radicals promote sulfhydral mediated cross-linking of the labile amino acids such as methionine, histidine, cysteine and lysine causing a fragmentation of polypeptide chains in the protein molecule. Tramadol increased lipid peroxidation, due to long term use (Atici et al., 2005). In living cells, when the formation of intracellular reactive oxygen species exceeds the cell's antioxidant capacity, oxidative stress can rise resulting in damage to cellular macromolecules such as proteins, lipids and DNA (Nordberg and Arner, 2001;Valko et al., 2007). Verma and Kaplowitz. (2009) stated that acetaminophen overdose is currently the most frequent cause of acute liver failure, evaluation of the mechanisms of drug-induced liver injury indicates that mitochondria are critical targets for drug toxicity, either directly or indirectly through the formation of reactive metabolites. The consequence of these modifications is generally a mitochondrial oxidant stress and peroxynitrite formation, which leads to structural alterations of proteins and mitochondrial DNA and, eventually, to the opening of mitochondrial membrane permeability transition (MPT) pores. MPT pore formation results in a collapse of mitochondrial membrane potential and cessation of adenosine triphosphate synthesis. In addition, the release of intermembrane proteins, such as apoptosis-inducing factor and endonuclease G, and their translocation to the nucleus, leads to nuclear DNA fragmentation.
From the cytological point of view, the present results indicated that the tramadol has continuously inhibitory effect on the nuclei volume of epithelial cells lining villi in ileum of male mice which resaved tramadol for 20 or 40 days. From the cytological point of view, all previous treatments, stopping tramadol or Lagenaria siceraria or melatonin, revealed an inhibitory effect on the changes induced by tramadol on volume nuclei of epithelial cells lining villi in ileum in mice but did not reach to control mice. Although, Tramadol has an inhibitory effect on cellular activity treatments with stopping tramadol or Lagenaria siceraria or melatonin decreased this tramadol effect.
The present work is a part from M. Sc. thesis (Mariam M. Jad et al.,In Press), the ileum of animals received tramadol for 40 days and left for a recovery period (40 days). Organs showed partial improvement in the total proteins and nucleic acids materials content versus mice which received tramadol for 40 days and then sacrificed or control mice.
The reduction of protein, nucleic acids and polysaccharides contents in cells of kidney (Mariam M. Jad et al.,In Press) and ileum recorded in this work were particularly restored approximately 40 days after the abstinence of tramadol, but such restorations were apparently relatively incomplete compared within mice previously treated for 40 days only. Regarding the possible recovery of investigated chemical materials content in animal kidney and ileum following tramadol drug withdrawal, no studies seem to have been carried out in this respect.
From the histochemical point of view, the results of the present work indicated that tramadol has similar side effects on chemical component materials (such as protein, nucleic acids) in the epithelial cells lining ileum of mail mice. In the present study, the daily administration of mice with the therapeutic dose of tramadol for 20 days or 40 days caused a remarkable reduction in the total proteins contents of investigated cells of the ileum to compare with those of the control mice. The reduction of protein content observed in this study may be attributed partially to the decrease of hepatic protein synthesis due to the hyperactivity of hydrolytic enzymes (Sivaprasada et al., 1983). Furthermore, this decrease in protein content can be explained by Chen et al. (1999), who stated that initiation of lipid peroxidation, necrosis and subsequent impairment in cellular metabolism collectively altered the major cellular components, including protein, and glycogen.
Experimental studies have also supported the toxic effects of chronic use of opioids on liver proteins. Hashiguchi et al. (1996) studied the central effects of morphine and morphine-6-glucuronide on tissue protein synthesis. They found that morphine and M6G suppress tissue protein synthesis through central mechanisms, mediated by opiateinduced respiratory In the present study, the daily administration of mice with the therapeutic dose of tramadol for 20 days or 40 days caused a remarkable increase of collagen content in investigated cells of kidney and ileum to compare with those of control mice. These results may be interpreted as a secondary event following tramadolinduced lipid peroxidation of renal tissues. Lipid peroxidation of cell membranes leads to loss of membrane fluidity, changes in membrane potential and an increase in membrane permeability, all of which lead to alteration of the chemical compound of the cells. The increased collagen fibers occur due to decreased collagen metabolism that may be related to oxidative stress (Altindag et al., 2008).
Despite, the beneficial reduction in Myeloperoxidase (MPO) activity caused by the opioid agents, in the current study, there was no corresponding decrease in histological verify score. However, tramadol as the most efficacious agent in reducing MPO, showed a trend toward restoration of villus height. It is noteworthy that this effect also occurred in healthy animals and is previously unreported. In order to fully understand the inflammatory processes occurring in chemotherapy-induced mucositis, their relation to histological repair and synergism between therapeutics, future studies should investigate inflammatory markers such as the pro-inflammatory cytokines and cyclooxygenase activity. Mechanisms of pain production by cytotoxic agents in gastrointestinal mucositis are poorly understood, (Cata et al., 2008). Yet pain remains one of the key doselimiting factors for patients (Gibson et al., 2015). However, research effort in this area is likely to increase since the discovery that: 1) chemotherapyrelated hyperalgesia can be reversed by immune-modulatory agents (Gibson et al., 2015) and there is a high level of μ-opioid receptor expression in the intestinal tract, particularly during inflammation (Philippe et al., 2003). Therefore, the ability to reliably measure affective pain as opposed to nociception is a much-needed animal model refinement. This study has indicated that behavioral pain assessment scoring may be one such tool but does require further validation. The present data do however raise some concerns with the ability of the model to replicate the human mucositis condition if human patients frequently require morphine omimetic agents to control pain Opioids are the most potent and effective analgesics available and have become accepted as an appropriate treatment for acute or cancer and noncancer chronic pain (Collet, 2001). However, they are also rewarding and their repeated use can lead to dependence and addiction. In fact, addiction to opioid analgesics is a growing socioeconomic and health problem with potentially serious consequences documented by a rise in deaths due to overdose (Hall et al., 2008).
In the present work, in ileum, tramadol showed necrosis and desquamation of the intestinal villi, intestinal vacuolation and degeneration, and congestion of blood vessels.
Tramadol administration induced histopathological side effects on the architecture of ileum in mice such as extensive necrosis, desquamation of the intestinal tips, ileum vacuolation, and degeneration. Similar results are reported by Whittaker et al. (2016).
Oxidative stress-induced by opiate exposure is a significant factor in the etiology of male infertility and can lead to increased DNA fragmentation (Lamont and Mathews, 2007). Also, Vuong et al. (2010) found that tramadol enhance testicular levels of nitric oxide and lipid peroxidation and decreased the activities of the antioxidant enzymes significantly compared with control.
Our results indicated that the daily injection of tramadol for 20 or 40 days decreased markedly DNA materials content in epithelial cells of renal tubules in kidney and lining epithelial cells of villi of ileum of mice versus control. These results may be attributed to the damage of DNA report Bedwell et al. (1989) and Vuong et al. (2010) described that tramadol decreases the activities of the antioxidant enzymes significantly. Also, oxidative stress induced by opiate exposure is a significant factor can lead to increased DNA fragmentation (Lamont and Mathews, 2007).
On the contrary, the administration of Lagenaria siceraria diminished and alleviated the pathological changes.
Since kidney showed slight degree of necrosis and degeneration with slight congestion in blood vessels, also, minimally mononuclear infiltration was detected. Ileum exhibited normal arrangement of the intestinal layers and villi. Lagenaria siceraria showed minimal collagen fibers distribution in kidney and ileum. Also by PAS and bromophenol techniques, kidneys and ileum showed high polysaccharides and protein contents with deeply stained red and blue color, respectively. Ghosh et al. (2009) displayed the antioxidant potential of Lagenaria siceraria which accounts for cytotoxic activity. Lagenaria siceraria fruits contain a water-soluble polysaccharide , which is composed of 3-O-acetyl methyl-á-d-galacturonate, methyl-á-dgalacturonate, and â-d-galactose in a ratio of 1:1:1. The polysaccharide possesses cytotoxic activity in vitro. Also, Lagenaria siceraria has radical scavenging activity (Deshpande et al., 2009). Lagenaria siceraria offered immunomodulatory effects of nbutanol soluble and ethyl acetate soluble fraction of the successive methanol extract of Lagenaria siceraria (Gangwal et al., 2008). So Lagenaria siceraria could reverse changes in the tissues.
Moreover, the administration of melatonin corrected and ameliorated the pathological changes induced by tramadol. The kidney showed slight necrosis and degenerative changes with sloughing in some renal epithelium, while other renal tubules detected regeneration and mild congestion of the blood vessels. Also, a mild degree of fibrous tissue infiltration was observed. Ileum demonstrated intestinal layers with some inflammatory cell infiltration.
Melatonin functions in scavenging free radicals might be classified in to four main categories: (1) as an antioxidant directly scavenges ROS (Reiter, 2000), (2) in stimulating the antioxidant enzymes production and activation (Rodriguez et al., 2004) and (3) in increasing the efficacy of mitochondrial functions by improving MPTP, inhibition of cytochrome c release and refining of oxidative phosphorylation in mitochondrial respiratory chain which further will decrease the peroxidation of membrane lipids (Acuna-Castroviejo et al., 2007).
Moreover , Permpoonputtana and Govitrapong (2013) revealed that the anti-neuroinflammatory effects of melatonin resulted from the inhibition of activated NF-κB in a cell signaling pathway. Melatonin as pre-treatment can reduce pro-inflammatory cytokines IL-1β and TNF-α and increases the serum levels of anti-inflammatory cytokine IL-4 (Carrasco et al., 2013). These results are consistent with the findings of acetic acid-Induced Colitis in rats. Melatonin completely improves the latter inflammation (Tahan et al., 2011). Melatonin inhibits peroxynitrite production and activation of poly ADP ribosesynthetase (nuclear enzyme) in macrophages of rats exposed to zymosan-induced shock (Maestroni, 1993). Inhibition of peroxynitrite production by melatonin due to antiinflammatory effects (Poeggeler et al., 1993).
Finally, we could say, from the cytological, histochemical and histopathological point of view, the present data showed tramadol administration for 20 or 40 days decreased the nuclei volume and cellular chemical contents; DNA, RNA and protein of epithelial cells lining villi in ileum of male mice. Thus, tramadol adversely affects cellular activities, including cell division and protein synthesis, which are finally reflected in histopathological changes. Although, Tramadol has inhibitory effect on cellular activity but treatments with stopping tramadol administration or Lagenaria siceraria or melatonin decreased this tramadol effect.