THE HISTOLOGICAL IMPACT OF SUB-CHRONIC ENERGY DRINK AND CAFFEINE CONSUMPTION ON LIVER FUNCTION IN SWISS ALBINO RATS.

246 Nigerian Journal of Pharmaceutical and Biomedical Research Vol. 8 Issue.3 December, 2024. p-ISSN: 2579-1419 e-ISSN: 2814-1423 THE HISTOLOGICAL IMPACT OF SUB-CHRONIC ENERGY DRINK AND CAFFEINE CONSUMPTION ON LIVER FUNCTION IN SWISS ALBINO RATS. Fatima S. Dibal, Yahaya Hassan A. Gandawa, Garba Uthman Sadiq and Shettima Yeli. Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Maiduguri, Borno State, Nigeria. http://doi.org/10.55639/607.phar.10601.006 Abstract Energy drinks (EDs) are special type of drinks that gives energy by the addition of many energy enhancing ingredients or boosters, most notably caffeine. The study’s primary focus is on evaluating histopathological changes on liver in Swiss Albino rats after administration of energy drinks and caffeine for 28 days. A sachet of caffeine was dissolve in each bottle of energy drink. Twenty rats weighing 90-200grams were grouped into five rats per group of four. Group one as the control given distilled water alone, group two as low dose which was given 5.7mL/kg of combined Coffee and Energy drink every day which is equivalent to one bottle per day for an average man weighing 70kg, group three as medium dose (7.5mL/kg) and group four as the high dose (10mL/kg) and the liver was excised for histological study. This study has demonstrated that oral administration of ED and caffeine to rats for 28 days resulted in varying degrees of liver damage. Results of the current study showed that ED and caffeine induced histopathological alterations in the hepatic cells. All hepatocytes were vacuolated with small darkly stained nuclei. Also, there were dilated and congested blood vessel and marked mononuclear cellular infiltration in portal tract area and the central vein appears congested in medium and high dose. It was concluded that energy drink and caffeine causes degenerative changes in the liver in the form of vacuolations in the cytoplasm of hepatocytes, apoptosis and congestion of the central vein. Keywords: energy drink, caffeine, liver, histology, albino rats. Introduction Energy drink (ED) is a special type of drink that gives energy by the addition of many energy enhancing ingredients or boosters, most notably caffeine and other ingredients including simple sugars, amino acids and artificial sweeteners in addition to herbal supplements and vitamin B complex (Akande and Banjouke., 2011). Nowadays, energy drinks are widely used among youth and athletes all over the world to increase their physical performance, alertness, attention and concentration. As the popularity of energy drinks continues to increase, their potential adverse effects become a major point of concern. Many studies revealed their side effects on liver and kidney in addition to their adverse effects on hematopoietic, cardiovascular and nervous systems (Olas and Bryśb., 2019). Reports of significant, adverse health problems due to ingestion of EDs have increased in recent years (AL Sunni., 2015). A major constraint in understanding the link between EDs and the adverse effect of their consumption is that very little is known about the toxicity of the various compounds present in them. However, based on reported cases of ED-associated health problems, and the well-established physiological effects of the active 247 ingredients of EDs, it is very likely that the observed adverse effects of EDs are linked to their compositions (Mansy et al., 2017). Growth in consumption is attributed to aggressive marketing strategies (Miller. 2008), their taste, the need for energy and combating sleepiness (ADEPIS., 2013). Increased consumption, ready availability, easy accessibility, and the limited regulation of EDs is increasingly a significant public health concern (Visram et al., 2016). Through positively affecting bodily functions, energy drinks are often marketed as ‘functional foods or dietary supplements (Finnegan, 2003), therefore bypassing legislation regarding caffeine content (Pomeranz, 2012). This could lead to problems as large doses of caffeine reportedly sometimes as high as 505mg per serving (Reissig et al., 2009) can put consumers at risk of intoxication (Hershorin and Lipschultz., 2011). In addition to this, EDs frequently contain a number of other often understudied and unregulated ingredients that may be of potential harm. The use of energy drinks has been associated with a number of shortterm benefits such as improvements in aerobic endurance, anaerobic performance, reaction time, concentration and memory (Seifert et al., 2011), as well as reductions in driver sleepiness (Mets et al.,2011). McLellan and Lieberman (2012), however, have concluded that there is little evidence to attribute such effects to any contents other than caffeine. Materials and method Materials Beakers, syringes (1ml, and 2ml), NGTube, weighing balance, hand Gloves, cotton wool, , Razor blade, sterile universal container, slide, cover slide, light microscope, permanent markers, methylated spirits, 10% formalin, Alcohol, hemoxylin, Eosin. Ethical approval Approval was sought and received from National Health Research Ethics Committee (NHREC) to conduct this research (NHREC/01/01/2007-22/01/2021) Animals and Adaptation Period Animals were obtained from Bayero University Kano, Faculty of Pharmaceutical Sciences weighing 90200grams. The rats were housed in conventional housing conditions in the faculty of Pharmacy Animal House, University of Maiduguri. They were fed on rat’s pellets (feeds) and water ad libidum. All the animals were maintained under standard laboratory conditions of temperature and optimum ventilation. Good hygiene was maintained by constant cleaning of feces and spilled feds. The Animals were exposed to the study environment for 10 days before the test period. Grouping of Experimental animals Twenty rats were grouped into five rats per group of four. Group one as the control, group two as 5.7ml/kg (low dose), group three as 7.5ml/kg (medium dose) and group four as 10ml/kg (high dose) for combined Coffee and Energy drink Energy drink and Coffee Energy drink and Coffee were purchased from the commercial area in the University of Maiduguri, Borno State. The Energy drink used for this experiment is a 400ml energy drink which contains: carbonated water, sucrose, acids (citric acid, tartaric acid), acidity regulator (sodium citrate), nature identical and artificial pineapple, and carbohydrate (56g), of which sugars were 54g, sodium (0.2g), niacin (12.8mg, 85%), 248 vitamin B6 (1.2mg, 92%), and caffeine (120mg), flavor, inositol (1mg/100ml), taurine (100mg/100ml), preservatives (potassium sorbate and sodium benzoate), negligible fat, saturates, and proteins. The Coffee with expiration date 0f 2025 is a product of an India 100% Instant coffee made from unique blend of Arabic and Robust. Calculation of dose and administration of ED and Coffee The volumes administered of the ED were equivalent to one bottle for a 70kg man (5.7mL/kg, Low Dose), two bottles (7.5mL/kg, Medium Dose) and three bottles (10mL/kg, High Dose) which adjusted (Ferreira et al., 2013). A single instant coffee/serving for an average man 3g was used which was dissolved in energy drink (350ml) to make mixture of 9mg/ml. Treatments were based on sub chronic toxicity test guidelines by the Organization for Economic Cooperation and Development (OECD 407). Treatment was administered orally for 28 days. Sample Collection After the specified duration of treatment was achieved, the rats were sacrificed by deception. Liver samples were immediately excised, weighted and transferred into sterile universal container containing 10% neutral formalin for fixation at 48 hours in room temperature. Tissue Processing Sample was transferred to the teaching hospital, university of Maiduguri, department of histology, tissue sample was dehydrated using series of alcohol solution, embedded in paraffin wax and thin section was cut from embedded tissue mounted on glass slide (Suvarna et al., 2018). Staining some sections with Hematoxylin and Eosin (H&E) mounted on glass slides. Sample was stained with Hematoxylin for 7 minutes, washed well in running tap water, transferred into eosin for 3 minutes and the excess stain was washed by water. The sections were dehydrated by alcohol, cleared by xylene and then mounted on glass slides and was viewed using light microscopy for histological analysis (Suvarna et al., 2018). Microscopy The stained sections were transferred to Faculty of Veterinary Medicine, University of Maiduguri and examined under a light microscope, photomicrograph was taken using digital camera and stream basic imaging software. Liver morphology, cellular changes, and any pathological findings were assessed. 249 Results (a) (b) (c) (d) (e) (f) Figure 1(a) Photomicrograph of control liver showing normal hepatocytes radiating away from the central vein (arrows) H&E x100 (b) normal hepatocytes (black arrows) radiating away from the central vein and clear sinusoids (green arrows) H&E x100 (c)Photomicrograph of liver treated with medium dose (7.5ml/kg) showing multifocal areas of coagulative necrosis of the hepatocytes (arrows) H&E x100 (d) Photomicrograph of liver treated with medium dose (7.5ml/kg) showing Focal coagulative necrosis of the hepatocytes H&E x100 (e)Photomicrograph of liver treated with high dose (10ml/kg) showing zone of mononuclear cell infiltration (yellow arrow) and mild congestion (black arrow) H&E x100 (f) 250 Photomicrograph of liver treated with high dose (10ml/kg) showing wide spread coagulation necrosis and massive aggregation of inflammatory cells(arrows) H&E x100 Figure 2. (a) Photomicrograph of low dose (5.7ml/kg) liver showing mononuclear cell infiltration around the portal trait (circle) H&E x200 (a) (b) (c) 251 (d) (e) Figure 3. (a) Photomicrograph of low dose (5.7ml/kg) liver showing area of coagulative necrosis of the hepatocytes showing hepatic degeneration (black arrows) H&E x400 (b) wide spread coagulative necrosis of the hepatocytes H&E x400 (c) mild congestion (CGX), mononuclear cell infiltration (arrows) H&E x400 . (d)Photomicrograph of liver treated with high dose (10ml/kg) showing zone of mononuclear cell infiltration (yellow arrow) and mild congestion (black arrow) H&E x100 (e) Severe wide spread coagulative necrosis of the hepatocytes H&E x400 Discussion This study has demonstrated that oral administration of ED and caffeine to rats for 28 days resulted in varying degrees of liver damage. Results of the current study showed that energy drink and caffeine induced histopathological alterations in the hepatic cells. All hepatocytes were vacuolated with small darkly stained nuclei. Also, there were dilated and congested blood vessel and marked mononuclear cellular infiltration in portal tract area and the central vein appears congested in medium and high dose. This result is confirmed by Khayat et al., (2014). Who explained hepatic cytoplasmic vacuolations due to presence of lipid droplets which were attributed to deteriorative changes within hepatocytes? Also, the current result agreed with Kasab and Tawfik, (2018), who stated that these changes were a manifestation of cell damage and were caused by lipid peroxidation (oxidative stress) and DNA damage as a sign of toxicity and were attributed to the preservatives or caffeine gradient that are present in the energy drinks. Also, there was vacuolated degeneration of hepatocytes. This was explained by Roodi et al., (2018) who considered the vacuolation of hepatocytes as ballooning degeneration and explained it as a type of cellular defense mechanism against toxins because these vacuoles collect the harmful elements and prevent them from affection of the biological activities of hepatocytes. As well, free radicals attack polyunsaturated fatty acids of plasma membranes leading to their degradation resulting in vacuolation. Another observed 252 finding was the vascular congestion which is previously mentioned by Raju et al., (2015). Previous studies demonstrated histopathological lesions in different organs following administration of energy drinks. These organs include liver (Khayyat et al.,2012). kidney, brain (Salih et al., 2018), testis (Dias et al., 2015; Ahmed, 2016), pancreas, fundus of stomach (Ayuob and ElBeshbeishy, 2016), gastric and duodenal mucosae (Mohamed et al., 2018). The findings from this study has revealed that energy drinks and caffeine may impart some debilitating effect on body organs if taken without caution. Which may include degenerative changes in the liver in the form of vacuolations in the cytoplasm of hepatocytes, apoptosis and congestion of the central vein. Acknowledgement The authors wish to acknowledge the contributions of those who supported and guided me and made this research possible without whom the work would have been a mere delusion, these include Prof. Timothy S. Yerima , Prof. Wazis H. Chama and Dr Hassan Braimah Yesufu who despite their tight schedules, were able to put me through and guided me effectively I appreciate the whole members of Borno State Substance Abuse Research Team (SART). I also wish to acknowledge the support of TETFund. References Abonar, M.M., AboRaya, A.A., El-Bakary, N.A., and Elwan, W.M. (2022). 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