Original Article Alterations in Lipid Profile, Oxidative Stress and Hepatic Function in Rat Fed with Saccharin and Methyl-Salicylates

Int J Clin Exp Med 2015;8(4):6133-6144 www.ijcem.com /ISSN:1940-5901/IJCEM0006657

Original Article Alterations in lipid profile, oxidative stress and hepatic function in rat fed with saccharin and methyl-salicylates

Kamal Adel Amin1,2, Hessah Mohammed AlMuzafar1

1Department of Chemistry Biochemistry, Science College, University of Dammam, KSA; 2Department of Biochem- istry, Faculty of Veterinary Medicine, Beni Suef University, Egypt Received February 3, 2015; Accepted April 3, 2015; Epub April 15, 2015; Published April 30, 2015

Abstract: Background: Food additives attract consumers, improve foods quality, control weight, and replace sugar in foods, while it may affect seriously children and adults health. Aim: To investigate the adverse effects of saccharin and methylsalicyltaes on lipid profile, blood glucose, renal, hepatic function, and oxidative stress/antioxidant (lipid peroxidation, Catalase and reduced glutathione (GSH) in liver tissues). Methods: 46 young male albino rats were used. Saccharin and methylsalicylate were giving orally as low and high dose for 30 days. Rats were divided into 5 groups, 1st control group, 2nd and 3rd low and high saccharin-treated groups and 4th and 5th low and high methylsalicylate-treated group. Results: Serum total cholesterol, triglyceride, glucose levels and body weight gain were decreased in saccharin high dose compared to control. Rats ingested high dose of saccharin presented a significant reduction in serum triglycerides, cholesterol and LDL levels. Low and high doses of saccharin exhibited a significant increase in liver function marker of ALT, AST, ALP activity, total proteins and albumin levels and renal function test (urea and creatinine levels) in comparison with control group. Saccharin high dose induce a significant decline in hepatic GSH levels, catalase and SOD activities while increased in hepatic MDA level. Conclusion: It could be concluded that, saccharin affects harmfully and alters biochemical markers in hepatic and renal tissues not only at greater doses but also at low doses. Whereas uses of metylsalicylates would not pose a risk for renal function and hepatic oxidative markers.

Keywords: Food additives, liver, oxidative biomarkers, saccharin, methyl-salicylates

Introduction is a very stable compound with respect to heat and time so that it can be used in hot beverag- In the last decades, the rising concern about es, canned vegetables, bakery products and health and life quality have encouraged societ- reduced sugar jams [5]. Saccharin is an impor- ies to exercise, eat healthy food and reduce the tant sweetener, especially for diabetics, as it consumption of food rich in sugar, salt and fat. goes directly through the human digestive sys- With increased consumer interest in reducing tem without being digested. Although saccharin sugar intake, food products made with sweet- thus has no food energy, it can trigger the eners rather than sugar have become more release of insulin in rats, apparently as a result common [1]. The low-calorie artificial sweeten- of its taste [6]. There are different forms of sac- ers, such as aspartame, saccharin, acesul- charin including sodium saccharin, calcium sac- fame-K and cyclamate, have become sugar charin, potassium and acid saccharin. Sodium alternatives to replace sucrose [2], and have saccharin is more often used as it is more pal- been widely used in dairy products, energy con- atable. Saccharin has the chemical formula trol diet and diabetes in Africa, Asia, Europe C H NO S, can be produced in various ways and and in the USA. 7 5 3 can be used to prepare exclusively disubstitut- Saccharin is the non-nutritive, non-caloric ed amines from alkyl halides via a Gabriel syn- intense artificial sweeteners, as it has 300-500 thesis. In the European Union, saccharin is also times the sweetness of sucrose, but it has a known under the E number (additive code) slightly bitter aftertaste [3]. It still of the most E954. The accepted daily intake of saccharin is important and widely used sweeteners [4] as it 2.5 mg/kg body weight [7]. Saccharin and methyl-salicylates on oxidative and hepatic function

The degree of saccharin absorption occurs rap- excreted via urine as salicyluric acid and acyl idly and dependent on food intake, after its and phenolic glucuronides. The probable meta- removal from the diet, almost complete tissue bolism of the salicylates does not provide toxi- clearance resulted in 3 days. Saccharine of cological concerns [15]. Moderate oral toxicity renal, bladder, liver and muscle tissues and of the salicylates found in acute case, with tox- plasma levels increased in diets containing icity commonly reducing with increasing size of 7.5% or 10% saccharin for 22 days. This was the ester R-group. The aromatic salicylates are attributed to the animals’ reduced ability to of low to moderate acute oral toxicity [1300 to eliminate saccharin [8]. So cause its accumula- > 5000 mg/kg body weight). Differences in tion in the tissue when it consumed 2-3 times a acute oral toxicity are correlated to the virtual week. amount of the molecular weight produced as salicylic acid after hydrolysis [16]. From the The renal and hepatic effects of in vivo treat- genetic toxicity data (2 years investigation of ment with saccharin have been determined as oral methyl salicylate) and the metabolism of increased DNA elutability in the range 130- the salicylates (simple alcohols and acids 210% compared with controls [9]. Consumption metabolites), it seems that the salicylates are of large amounts of saccharin might produce improbably to be oncogenic [17]. hypoglycemia [10], reduce hyperinsulinemia, decrease the insulin resistance and improve Uses of salicylates and salicylic acid in cosmet- glycemic control during saccharin intake in ic products would not produce a risk for repro- hyperglycemic obese mice [11]. In contrast ductive or developmental effects in human intake of foods or fluids containing non-nutri- being according to the Cosmetic Ingredient tive sweeteners was accompanied by increased Review Board. Synthetic sweetener and flavor- food intake, body weight gain, accumulation of ing materials are routinely widely used due to body fat, and weaker caloric compensation their taste and sensory pleasure fragrance [12]. In the US, food containing saccharin must properties to food and beverage [18], consis- be labeled with a warning that use of this prod- tency, stability and low price, however many of uct may be hazardous to health and has caused them become toxic after extended use, initiat- cancer in laboratory animals. Some studies ing health complications such as indigestion, showing a correlation between saccharin con- anemia and allergic reactions as urticarial, sumption and increased frequency of cancer asthma and, pathological injuries in the brain, [especially bladder cancer] and others finding kidney, spleen and liver, tumors, paralysis, no such correlation, some studies have shown mental retardation, nervous manifestations, a correlation between consumption and cancer abnormalities in offsprings, growth retardation incidence [13]. There are shortages in data and eye faults [19, 20]. From the previous liter- about saccharine and its oxidative stress action ature there are contradictory suggestion on the that may correlate to carcinogenic effects. role of saccharin and methylsalicyltaes on health. Salicylate esters, a chemicals extensively used as flavor and fragrance additives in foods, bev- Natural food free from pesticide residue and erages and a wide variety of consumer prod- pollution or additives is the best choice for ucts, are suspected to have estrogenic activity human. Our goal is to maintain the people [14]. Dermal exposures to methyl salicylate, healthy during their life. Drinking juice, meat oral exposures to salicylic acid and sodium food products, some milk products etc, all con- salicylate, are all associated with reproductive tain synthetic additives, to let us eat much. We and developmental toxicity. An exposure to cos- choose and ingest food to get its benefit, what metic product would be only 20 % of the level about taking food with additives that may seen with ingestion of a “baby” aspirin [81 mg] increase its consumption. We face environmen- on a daily basis. The oral bioavailability of salic- tal pollution, many diseases, daily stresses, ylates is supposed to be 100% and well metabolic changes during life and aging, so we absorbed by mouth. The salicylates are under- need to have natural, functional food to keep going extensive hydrolysis, primarily in the us withstand, these stressors not another hepatic tissues, to salicylic acid that conjugat- stress from food additives specially when the ed with either glucuronide or glycine and is consumer pay for that.

6134 Int J Clin Exp Med 2015;8(4):6133-6144 Saccharin and methyl-salicylates on oxidative and hepatic function

Therefore, the aim of the present work was to ous, heavy using of these additive threatening evaluate the toxic harmful effect of some syn- the life of many peoples, some countries has thetic food additives of sweeteners and odor- regulation to avoid using children and patient ant or flavors on liver, renal and metabolic bio- from using those additive and have law for the markers in rats. company, other have nothing to do with the market and companies. Material and methods The dose that we used is high compared with Animals and diet ADI to show the safety margin of these compound, the substance with higher safety border A total of 46 young male Rattus Norvegicus consider safer than those with low. I mean if albino rats weighting about 70-80 g, of about 5 the chosen saccharine is safe, have non-signif- weeks old, were used in the current study. They icant effect on the body markers (liver function, were obtained from National Research Center, kidney function and oxidative stress) in double Cairo, Egypt. Animals were retained under or triple or 4 times than ADI, it consider more observation for 7 days before the start of the safe. investigation to eliminate any intercurrent infec- tion. They were preserved in stainless steel We choose higher dose to determine the safety cages at ordinary atmospheric temperature of border, you will say the children will not con- 27 ± 5°C and in good ventilation. This study sume that much amount. The kids or consumer was carried out in agreement with the rules of use too much of synthetic sweetener because Beni Suef University for animal uses and these many additives have been provided here. If you animals were used for the studied food take in the morning biscuit which contain additives. sweeteners, with tea having saccharin and in the work you got cookie having sweetener and Rats were nourished on the regular basal diet flavor, in your car taste some fried. and supplied with tap water and the contents of experimental diets were according to Kim et al., We like to say most food in the supermarket [21], it composed of: lipid 5%, carbohydrates contain flavor, sweetener, and coloring to 6%, protein 20.3%, fiber 5%, salts 3.7%, and attract the consumer. Even Fanta have tartra- vitamins 1%. zine that have serious effect on the body organs. Compounds and chemicals We choose saccharine because many people Chemicals utilized in the experiment included use it especially diabetic who having metabolic saccharin (food sweeteners) that is found in its disturbances include changes in oxidative sodium salt, white crystalline powder, and sodi- stress so saccharine exaggerates the condi- um saccharin content is 99.62%, arsenic less tion. We like to aware the consumer about the than 2 ppm, foreign substance less than 10 effectiveness of saccharine on the oxidative ppm, from El-Goumhoria Company (Egypt) pur- stress and its toxicity limit. chased from Taianjin North Food Co. LTD (China). Manufacturer aimed to attract the consumer for each product and this would accumulate Methylsalicylates (food odorant) is a clear col- through days, so we need to aware the consum- orless liquid, its concentration is 99.8% pur- er. Food additive is not a medication to give ADI chased from El-Goumhoria Company (Egypt). or dose, it take with food and when we eat food Saccharine and methylsalicylate were in a solid we did not look at its dose ADI or small, we condition and both prepared as solutions of 2 (people) just eat until satisfy, the condition is doses low and high through dissolving in dis- serious. tilled water. The low and high doses of Saccharin were 10 and 500 mg/kg bw, while doses Experimental design and animal grouping Methylsalicylates were 80 and 250 mg/kg bw respectively. Rats were classified into the following: Group 1), Control (10 rats) didn’t administrate any Normal ingestion of these compound may be chemicals. Group 2), Saccharin low dose (8 safe at ADI, however abnormal much continu- rats) were administered 10 mg/kg bw per day

6135 Int J Clin Exp Med 2015;8(4):6133-6144 Saccharin and methyl-salicylates on oxidative and hepatic function

Table 1. Effect of saccharin (10 and 500 mg/kg bw) and methyl-salicylate (80 and 250 mg/kg bw) on body weight, serum lipid profile and glucose High Saccha- Low Methyl- High methyl- Control Low Saccharin rin salicylate salicylate Initial Body weight (g) 78.0 ± 6.04a 76.6 ± 4.28a 74.4 ± 2.4a 74.1 ± 2.74a 75.25 ± 1.97a Final Body weight (g) 145.4 ± 7.3a 130.0 ± 5.9b 98.0 ± 2.98c 125.6 ± 3.94b,e 111 ± 3.17c,e Body weight gain (g) 65.4 ± 3.17a -54.6 ± 2.66a,b -22.8 ± 2.27c -50.8 ± 1.98b -35.2 ± 3.5c Growth rate (%) 83.85 71.28 30.64 68.56 46.78 Cholesterol (mg/dl.) 143.41 ± 4.32a 125.13 ± 2.84b 121.6 ± 1.71b 149.2 ± 3.59a 139.65 ± 2.78a,b Triglycerides (mg/dl.) 130.6 ± 3.98a 125.16 ± 4.15a 101 ± 3.19b 136.16 ± 2.72a 137 ± 1.23a HDL (mg/dl.) 87.11 ± 1.47a 75.51 ± 1.42a 66.11 ± 2.24c 78.6 ± 2.83a 95.68 ± 3.33a LDL (mg/dl.) 34.18 ± 1.52a 31.0 ± 1.83a 26.23 ± 2.55b 33.63 ± 1.19a 33.80 ± 1.44a Glucose (mg/dl.) 87.37 ± 6.18a 52.62 ± 3.57b 40.75 ± 2.4b 96.87 ± 3.25a 84.0 ± 5.12a Data expressed as MEAN ± SE. Means with the same superscript letters (s) a, b, c, e are not significantly different. Means having different letters are significantly different (P < 0.05). for 30 days per os using stomach tube. Group cholesterol, triglycerides and HDL-cholesterol 3), Saccharin high doses (10 rats) were admin- were estimated by enzymatic colorimetric istered 500 mg/kg bw per day for 30 days methods. Serum level of glucose was analysed orally using stomach tube. Group 4), Methyl- using enzymatic colorimetric method consis- salicylates low doses (8 rats) were adminis- tent with Trinder [25]. Catalase activity was tered 80 mg/kg bw/day for thirty days per os assessed in hepatic homogenate according to using stomach tube. Group 5), Methylsalicylates Cohen et al. [26]. Hepatic GSH and lipid peroxi- high dose (10 rats) were administered 250 mg/ dation levels was evaluated by using the proce- kg bw per day for 30 days per os using stomach dure of Beutler et al. [27] and Presuss [28] tube. respectively.

Sampling and tissue preparation Statistical analysis

At the completion of the experimental time, The statistical analysis performed by GraphPad venous blood samples were harvested from the Instat software [version 3, ISS-Rome, Italy]. orbital sinus of control and tested rats by glass Data were analysed using one-way analysis of capillaries after fasting overnight. The blood variance (ANOVA) followed by Tukey-Kramer samples were collected in clean dry centrifuge (TK) multiple comparisons post-test. Values of tubes and permitted to coagulate at room tem- P < 0.05 were considered as significant. The perature then centrifuged at 3500 rpm for 15 data recorded in tables as mean ± standard min. for getting the serum. Hepatic tissues error (SE). homogenates were set by dissolving 0.25 g of hepatic tissue in 5 ml saline (0.9% NaCl) then Results minced by homogenizer for 15 min followed by centrifugation using centrifuge for 10 min at Effect of saccharin and methyl-salicylate on 3000 rpm, the formed supernatants were body weight gain and growth rat collected for measuring the oxidative stress biomarkers. Data showing the effect of low and high dosag- es of food sweeteners and odorant on final Serum and tissue biochemical analysis body weight and body weight gain were expressed at Table 1. Serum ALT and AST activities were determined according to Reitman and Frankel [22] using Rats consumed saccharin high dose, exhibited kits obtained from Randox Company UK. Total a significant reduction, while low dose indicat- protein was measured in agreement with the ed a non-significant changes in final body method of Henry [23], serum urea according to weight in comparison with normal control method of Patton and Crouch [24], serum total group.

6136 Int J Clin Exp Med 2015;8(4):6133-6144 Saccharin and methyl-salicylates on oxidative and hepatic function

Table 2. Effect of saccharin (10 and 500 mg/kg bw) and methyl-salicylate (80 and 250 mg/kg bw) on serum liver function and renal function markers Low Methyl- High methyl- Control Low Saccharin High Saccharin salicylate salicylate ALT (U/L) 8.91 ± 0.27a 13.5 ± 1.25b 17.58 ± 0.47c 12.16 ± 0.61b 12.4 ± 0.48b AST (U/L) 47.85 ± 0.79a 63.70 ± 2.41b 86.36 ± 2.05c 47.23 ± 0.70a 49.51 ± 0.99a ALP (U/L) 87.56 ± 2.79a 117.01 ± 1.14b 117.55 ± 0.98b 108.95 ± 2.68c 112.38 ± 1.59c,b T.Protein (g/dl) 5.59 ± 0.02a 6.5 ± 0.16 b 6.70 ± 0.24b 4.41 ± 0.34b 4.14 ± 0.25b Albumin (g/dl) 3.81 ± 0.04a 4.42 ± 0.27b 4.59 ± 0.33b 2.73 ± 0.27d 2.82 ± 0.33d Urea (mg/dl) 17.54 ± 0.30a 43.95 ± 0.69b 44.53 ± 1.07b 15.96 ± 0.17a 16.9 ± 0.54a Creatinine (mg/dl) 1.32 ± 0.03a 1.96 ± 0.04b 2.82 ± 0.11c 1.34 ± 0.06a 1.39 ± 0.02a Data expressed as MEAN ± SE. Means with the same superscript letters (s) a, b, c, d are not significantly different. Means having different letters are significantly different P( < 0.05).

Rats consumed low dose of methylsalicylates Effect of saccharin and methyl-salicylate on and that consumed high dose of the same food hepatic function markers flavor, produced a significant decline in final body weight comparing with normal rats, and Low and High dose of both Saccharin and extraordinary methylsalicylates exhibited a sig- Methyl-salicylate exhibited a significant in- nificant reduction than low dose of methylsalic- crease in serum ALT, ALP activity when com- ylates on final body weight. Little and high pared to control rats and high dose of Saccharin doses of methylsalicylates induced a signifi- showed the highest level among the groups cant decrease in body weight gain in compari- (Table 2). son with control group. Rats ingested low and high doses of saccharin Effect of saccharin and methyl-salicylate on exhibited a significant increase in liver function lipid profile and serum glucose marker of serum ALT, AST, ALP activity, total proteins and albumin concentration in compari- Data showing the effect of food sweeteners son with control group (Table 2). (saccharin) both low and high doses on lipid profile (serum total cholesterol, triglycerides, Rats consumed low dose or high dose of meth- HDL and LDL-Cholesterol) and fasting serum ylsalicylate presented a significant decrease in glucose were expressed in Table 1. total protein level of serum comparing to normal control group, while serum albumin was Rats consumed saccharin in low and high not significantly changed (Table 2). doses revealed a significant decrease in serum total cholesterol and glucose level when com- Effect of saccharin and methyl-salicylate on pared to control rats (Table 1). renal function tests

Rats consumed high dose of saccharin showed Saccharin low and high dose produced a signifi- a significant decrease in serum triglycerides, cant elevation in blood urea and creatinine lev- cholesterol and LDL levels when compared to els and the higher concentration recorded with control rats. high dose, whereas low and high methylsali- cyltes not induce any significant changes with High dose saccharin showed the maximum urea and creatinine when compared with con- reduction in serum fasting glucose in all groups. trol rats (Table 2).

Methylsalicyaltes food odors both low and high Effect of saccharin and methyl-salicylate on doses showed a non significant changes in lipid hepatic oxidative stress markers profile (serum total cholesterol, triglycerides, HDL and LDL-Cholesterol) and fasting serum Saccharin high dose induced a significant glucose when consumed orally by young rats reduction in hepatic GSH levels, catalase and daily for 30 days (Table 1). SOD activities while increased in hepatic MDA

6137 Int J Clin Exp Med 2015;8(4):6133-6144 Saccharin and methyl-salicylates on oxidative and hepatic function

Table 3. Effect of Saccharin (10 and 500 mg/kg bw) and Methyl-salicylate (80 and 250 mg/kg bw) on oxidative stress biomarkers (GSH, MDA, SOD and catalase) in liver homogenate Low Methyl- High Methyl- Control Low Saccharin High Saccharin salicylate salicylate GSH (nmol/100 mg) 72.9 ± 1.41a 68.13 ± 0.87a 58.12 ± 0.48b 73.10 ± 1.44a 72.24 ± 1.24a MDA (nmol/g/h) 4.82 ± 0.41a 5.03 ± 0.19a 7.64 ± 0.62b 5.08 ± 0.23a 4.91 ± 0.25a SOD (u/g) 70.68 ± 1.50a 73.58 ± 0.98a 42.33 ± 0.86b 70.27 ± 0.82a 73.22 ± 1.02a Catalase (kx102) 47.03 ± 0.41a 48.85 ± 0.84a 29.65 ± 2.62b 44.56 ± 0.43a 46.2 ± 0.65a Data expressed as MEAN ± SE. Means with the same superscript letters (s) a, b, are not significantly different. Means having different letters a, b, are significantly different P( < 0.05). level. However biomarkers of oxidative stress in cholesterolemic effect as revealed by a liver homogenate were not significantly changed decrease in total serum cholesterol especially with low saccharin dose and both low and high with the high dose treated groups [29, 30]. methylsalicylates (Table 3). These results are in agreement with the results Discussion obtained by Dib et al., [31] who reported a significant reduction in body weight of rats The synthetic and some of the naturally present [50%] after administration of a 14-day sodium food additives, have been reviewed for toxicity. saccharin. Limit values have been evaluated for dietary intake by humans on the basis of conclusion of Though, the present results are in contrary with data attained in experimental animals. that obtained by Polyák et al., [32] who reported that body weight increase in saccharin con- In our work we tried to examin the side toxic sumed rats. However, some works suggests effects and biochemical variations in blood and that frequent consumers of Saccharin as sugar tissues of experimental rats treated with low substitutes may also be at increased risk of and high dose of two compounds saccharine excessive weight gain, metabolic syndrome, and methylsalicylates that are commonly used type 2 diabetes, and cardiovascular disease in Egyptian and saudian field in different prod- [33], while this study did not provide sufficient uct (grocery, companies and many hyper and evidence for increased body weight. supermarket) of food additives. The poor body weight gain in case of animals We considered low dose (double of ADI) consumed methylsalicylates may attribute to because young children in Egypt can consume uncoupling oxidative phosphorylation, inhibit- a double of ADI (or more) daily in several prod- ing Krebs cycle enzymes (succinate and ucts without control, in addition we used the α-ketoglutarate dehydrogenase) by salicylate, high dose (a much higher than ADI) to evaluate and inhibiting amino acid synthesis; this can the toxicity and health hazards of these addi- lead to decreasing in tissue build and reduce tives on biochemical assay and oxidative the growth rate of these animals [34]. stress. Body weight loss is considered by some authors Food additives is not antibiotic have ADI dose, to be a good reliable sensitive toxicity indicator not exceed that dose but people eat what they [35]. Thus the body weight loss in the present taste, smell and look good, this is the issue so study may represent the primary marker of higher dose should be examined as it related to food additive bad effect. These data indicated uncontrolled food consumption, not drugs. that artificial sweeteners saccharin assist in Once again it is food not tablet take it with care weight management, control of blood glucose and our role is to explore the reality. of diabetics, and also can be used to replace sugar in foods. The significant body weight losses with high saccharine may be attributed to reduce food The hypoglycemia observed in saccharin dosed consumption per day and may be a conse- rats are in accordance with Abdallah [30] and quence to the hypotriglyceredemia and hypo- Horwitz et al., [10] who reported that feeding of

6138 Int J Clin Exp Med 2015;8(4):6133-6144 Saccharin and methyl-salicylates on oxidative and hepatic function high quantities of saccharin might reduce blood Cholesterol is a soft waxy substance in the glucose level. Moreover, Osfor & Elias [36] blood stream and in the body’s cells. It is used reported that blood glucose levels of rats given to form cell membranes and to produce certain saccharin significantly dropped after 12 weeks hormones. Its total body content depends on of oral administration. the balance between body synthesis plus that absorbed from diet. The intestinal cholesterol Although saccharin has no food energy, it can pool comes from dietary cholesterol and the trigger the release of insulin in rats, apparently majority from biliary excretion. Approximately as a result of its taste; this may lead to reduc- 50% of the intestinal cholesterol pool is reab- tion in blood glucose level so it was believed sorbed by the intestines [40] and recirculated that saccharin is beneficial to diabetic people. via the enterohepatic circulation, with the Saccharin causes a reflex release of insulin remainder excreted in feces. The deviation mediated by the gustatory nerves and hypogly- from normal serum cholesterol levels is consid- cemia is produced as readily after washing the ered as symptoms of liver diseases [41]. In the mouth with saccharin as by drinking the sac- present study the decreased cholesterol level charin solution. It is the “sweet taste” of sac- implies liver damage which is in accordance charin which is responsible for its hypoglycemic with increased alkaline phosphatase level. effect, a property not common to the sulfonamides as a group [6]. The mechanism of hypocholesterolemic and hypolipidemic effect may be attributed to In addition, Bailey et al. [11] reported a reduc- reduced total cholesterol synthesis by the sac- tion of hyperinsulinemia, decrease the insulin charin suppressed in vivo liver enzymatic activ- resistance and improve glycemic control during ity of acetyl-CoA synthetase, citrate lyase, and saccharin consumption in hyperglycemic obese mitochondrial citrate exchange leading to a mice. reduction of available cytoplasmic acetyl-CoA, Saccharin causes a reflex release of insulin which is required for the synthesis of choles- mediated by the gustatory nerves. This theory terol and fatty acids [42]. Moreover liver acetyl- has been proposed by Kun and Hormath [37] CoA carboxylase, phosphatidate phosphohy- and is supported by their observation that the dralase, and glycerol-3-phosphate acyl trans- hypoglycemia is produced as readily after ferase activities were markedly reduced by the washing the mouth with saccharin as by drink- saccharin analogues. Suppression of these ing the saccharin solution. It is the “sweet enzymes would lead to a reduction of triglycer- taste” of saccharin which is responsible for its ide synthesis. hypoglycemic effect, a property not common to Cyclic AMP (cAMP) is formed from ATP by ade- the sulfonamides as a group. nylyl cyclase at the inner surface of cell mem- In contrast Swithers et al., [12] suggested that branes and acts as an intracellular second one mechanism by which exposure to high- messenger. The mechanism of action of the intensity sweeteners that interfere with a pre- non-nutritive sweetener, saccharin, was to dictive relation between sweet tastes and calo- stimulate significantly the activity of adenylate ries may impair energy balance which could cyclase in membranes derived from skeletal alter glucose homeostasis and reduce satiety. muscle of rat; in addition Sodium saccharin We concluded that if the satiety reduced how enhanced adenylate cyclase activity in a dose the body weight gain increased. So our study related manner [43]. cAMP activates phosphor- provides evidence that reduced blood glucose ylase that triggers glycogenolysis, gluconeo- level and hypotriglyceridemia induced low body genesis so induce hyperglycemia. Also adenylat weight gain. cyclase, activates hormone-sensitive lipase that produces lipolysis and converting triglycer- High and low saccharin induced hypocholester- ide into free fatty acid and glycerol. olemia and high saccharin induce hypotriglyceridemia when compared to control groups in The saccharin analogues accelerated bile agreement with results recorded by Sharma et excretion of cholesterol metabolites and in- al., [38] and Ashour & Abdelaziz [39]. Moreover, creased the fecal excretion of the cholesterol, Osfor & Elias [36] reported the same effect for triglycerides, neutral lipids, and phospholipids 12 weeks. thus, the liver and plasma lipoprotein lipid con-

6139 Int J Clin Exp Med 2015;8(4):6133-6144 Saccharin and methyl-salicylates on oxidative and hepatic function tents including, cholesterol, triglycerides, and increase in ALT activity after both 6 and 12 neutral lipids were markedly reduced by the weeks of administration. saccharin. Thus the studied food additives saccharin can significantly reduce serum total cho- AST levels were significantly higher in saccha- lesterol, triglycerides and LDL-cholesterol and rine treated group and that chronic saccharin act as antihyperlipidemic, so it consider of intake reflects various metabolic, hormonal health benefit. and neural responses in males and females [49]. Also, Shakoori et al. [50] reported that Both oral low and high methylsalicylates (win- both low and high concentrations of saccharin tergreen odor) reduce serum albumin signifi- caused hypertrophy of hepatic cell, its nucleus cantly and as a result the total serum protein and nucleoli in addition to excessive vacuola- was also fall. It has been reported that aspirin tion, in male albino rats at a dose of 65 mg/kg and sodium salicylates act as potent inhibitors body weight/day (weak dose) for a total period of protein synthesis in guinea pig gastric muco- of 39 weeks. The elevation in serum amino- sal tissue and rat islets [44]. transferase activities could be due to drastic effects caused by free radicals interaction with Inhibition of protein synthesis by salicylates cellular membranes or may be related to break- through inducing phosphorylation of the sub- down of liver parenchyma [51]. unit of eukaryotic translation initiation factor 2, resulting in the inhibition of mRNA translation The changes in liver function attributed to in the cells [45]. Thus, the food odor methyl- hepatocellular impairment which subsequently salicylates induced a significant hypoprotein- caused leakage and the release of greater than emia when given orally for 30 days. This decre- normal levels of intracellular enzymes into the ment may be due to loss of protein formation blood. Elevation in the activities of aminotrans- from the alimentary tract, or to decrease forma- ferases indicated an early diagnosis of hepato- tion of protein in the liver (impaired ability of the toxicity and considers as tissue damage liver to form albumin) this depression may be biomarkers. due to an alternation in the intracellular protein synthesis mechanism and the oxidative enzyme Our results revealed that both low and high change were probably secondary in altering doses of methylsalicylates showed a significant proteins [46]. elevation in serum ALT and ALP activities when compared to control group. The elevation of Artificial sweeteners are widely used nowadays, ALT, and ALP activities in groups dosed with when the sensitivity of the people to the gener- methylsalicylates are in agreement with al health increased, the subject of sweeteners Humphreys [52] who reported that methylsalic- as food additives will take dangerous and effec- ylates, showed some enlargement of the liver tive dimension. It is almost difficult to follow a of dogs when given orally. The elevation in diet which is completely free from sweeteners serum alkaline phosphatase (ALP) in low and and other food additives. The toxicity of sweet- high saccharin and methylsalicylate may be an eners has become increasing and has attract- evidence of obstructive damage in the liver tis- ed the concern of many scientists to study their sue due to saccharin and methylsalicylate toxic effect. Several previous studies have administration. revealed that the use of artificial sweeteners may entail some hazards to the users [47]. The liver cells play an important role in both synthesis and secretion of ALP into the bile. Low and high dose of Saccharin exhibited a sig- Therefore, the alterations in ALP activity caused nificant increase in serum ALT, AST and ALP by saccharin may be attributed to early choles- activities when compared with control rats. Our tatic liver damage which primarily affects the results correlate well with Abdallah [30]. In liver parenchyma, thus making ALP a sensitive addition Negro et al. [48] reported elevated index in the diagnosis of infiltrative diseases serum activity of liver enzymes after the oral [53]. administration of three different drugs, of which saccharin was the only common constituent. It appears that in lower animals the toxicity of Furthermore Osfor & Elias [36] reported that methyl salicylate is essentially identical with saccharin treated rats showed a significant that of salicylic acid, although it is conceivable

6140 Int J Clin Exp Med 2015;8(4):6133-6144 Saccharin and methyl-salicylates on oxidative and hepatic function that in man the small proportion of unhydro- Oxidative stress is an imbalance between oxi- lyzed ester might have a more toxic action [54]. dants and antioxidants in favor of the oxidants, After ingestion of salicylates, salicylic acid is potentially causing damage to cells or cellular formed, and readily absorbed in the stomach components [59]. and small bowel. Salicylate poisoning is mani- fested clinically by disturbances of several A particularly destructive aspect of oxidative organ systems, including the CNS and the car- stress is the production of reactive oxygen spe- diovascular, pulmonary, hepatic, renal, and cies (ROS), which include free radicals, H2O2 metabolic systems. and peroxides that can cause extensive cellular damage [60]. Most of these oxygen-derived When the acetates administered orally, they species are produced at a low level by normal hydrolyzed rapidly in liver into acetic acid and aerobic metabolism and the damage they their corresponding alcohol. The corresponding cause to cells is constantly repaired. However, alcohol in case of isoamylacetates is isopenta- under the severe levels of oxidative stress that nol; liberation of that alcohol can affect the liver cause necrosis, the damage causes ATP deple- cells by increasing the cytotoxicity lead to tion, preventing controlled apoptotic death and increase release of hepatic enzymes like ALT, damage on DNA [61, 62]. and ALP into circulation [55]. Our study revealed that high dose of saccharin Thus, saccharin and methylsalicylates, have a induced a significant reduction in liver Catalase, risky effect on liver and alter hepatic function SOD, and GSH, while showed a significant by elevating serum ALT, AST and ALP in both low increase in liver MDA level when compared to and high doses and this effect is more appre- control rats, while methylsalicylate dosed rats ciable at high doses. didn’t show any significant changes when compared to control rats (Table 3). Significant elevated serum creatinine and urea with high and low dose of saccharin, may be We have not been able to find studies targeting attributed to the toxic effects of saccharin on the effect of saccharin on hepatic biomarkers the kidney especially with high dose that can of oxidative stress. We attributed the oxidative lead to disorders in the renal function and stress induced by high dose of saccharin to the hence reduced glomerular filtration rate fol- inflammation initiated to the liver cells; this lowed by retention of urea and creatinine in the opinion can be supported by observation of blood [36, 56]. Abdallah [30] who stated that livers of rats Saccharin administartion caused a reduction in administrated saccharin showed a portal infil- the renal slice steady-stable accumulation of teration with mononuclear inflammatory cells p-aminohippurate and tetraethylammonium mainly lymphocytes and macrophages, the and at 60 days of age, increased urine volume, same observation was reported by Hassanin a decrease in urine osmolality, and increased [56] who noted that the portal tract in liver sec- potassium excretion were also found [57]. tions of rats received saccharin for six weeks showed moderate infiltration by mononuclear A combination of saccharin and aspirin had a inflammatory cells, mainly lymphocytes. high incidence of renal papillary necrosis and calcification. These toxic effects of aspirin and Consequently inflammatory processes induce saccharin are independent responses, and oxidative or nitrosative stress and lipid peroxi- administration of both greatly accentuates dation, thereby generating excess ROS, reac- these responses [58]. tive nitrogen species, and DNA-reactive alde- hydes [63]. Stimulated inflammatory cells The present data revealed that there is no sig- undergo a respiratory burst and release ROS nificant change in serum creatinine or urea lev- such as superoxide anion, hydrogen peroxide els between the groups of rats dosed with and numerous secondary oxidants as well as methylsalicylates and control groups. From the the arachidonic acid cascade [64]. previous data we postulated that saccharin affect the kidney leading to disturbed renal So liberation of ROS by inflammatory cells function and increasing serum creatinine and induced lipid peroxidation by depletion of GSH urea levels. content and decrease catalase, and SOD activi-

6141 Int J Clin Exp Med 2015;8(4):6133-6144 Saccharin and methyl-salicylates on oxidative and hepatic function ties and increased the formation of malondial- Saccharin induces changes in hepatic and dehyde as a product of lipid peroxidation by renal function at dose dependent manner and high dose of saccharin, these free radicals become more risky at higher doses because of can also lead to damage of the hepatic cells its ability to induce oxidative stress by forma- and increase the liberation of AST and ALT tion of free radicals may be due to liver function enzymes into serum as we observed in saccha- disturbances. Food flavoring agent methylsalic- rin treated rats specially those consumed high ylates have a bad effect on liver function and dose (500 mg/kg.bw.). Increased oxidative induce an elevation to serum transaminases. markers in saccharine group may not due to Therefore, it is necessary to create consumer lipid profile elevation but due to liver function awareness regarding the ill effects of these disturbances. food additives, and mention the type and concentration of each material added to food spe- Regarding hepatic antioxidant system there cially that consumed frequently by children. was a significant inhibition of the antioxidant Finally, it is advisable to limit the uses and con- defense system during saccharin administra- sumption of these food odorant or food flavor tion, specifically a decrease of catalase, and additives especially by young age. SOD activities and equivalent fall in GSH content which prevents the cell death by the toxic Disclosure of conflict of interest radicals so their levels in the tissue homogenate were decreased. on the other hand MDA None. level was increased as a product of lipid peroxidation arisen by the ROS action on lipids of cel- Address correspondence to: Dr. Kamal Adel Amin, lular membrane. Department of Chemistry Biochemistry, University of Dammam Faculty of Science, P.O. Box 1982 Our study provide novel data and conclusion Dammam 31441, KSA. E-mail: [email protected]. that saccharin can produce ROS and lipid per- sa oxidation to the lipid of hepatic cell membranes and these ROS lead to depletion of the compo- References nents of cellular antioxidant system GSH, SOD [1] Rolls BJ. Effects of intense sweeteners on hun- and catalase because these components are ger, food intake, and body weight: a review. Am used by the cells to reduce the toxic action of J Clin Nutr 1991; 53: 872-8. liberated oxygen radicals. [2] Bandyopadhyay A, Ghoshal S, Mukherjee A. Genotoxicity testing of low-calorie sweeteners: The oxidative stress produced by saccharine aspartame, acesulfame-K, and saccharin. provide an explanation of its role in initiation of Drug Chem Toxicol 2008; 31: 447-457. carcinoma specially when accumulated with [3] Cook-Fuller CC. Low-calorie Sweeteners. In: large dose in the bladder and other tumors in Ann, editor. Nutrition. 11th edition. USA; 2000. different tissues as well as reports of induction pp. 15-16. of hyperplasia. [4] Spillance WJ. Molecular Structure and Sweet Taste. In: Grenby TH, editor. Advances in When we choose higher dose, it means we Sweeteners. London, Glasgow, New York; examine its safety margin and if it have even 1996. pp. 1-25. wide safety limit it should be taken with care as [5] Mitchell ML, Pearson RL. Saccharin. In: Lyn it is food not tablet. Saccharine and methyl- O’Brien Nabors, editor. Alternative Sweeten- salicylates to certain extent are not safe on ers. 3rd edition. New York: Basel Hong Kong; lipid profile, glucose and hepatic and renal 1991. pp. 127. [6] Ionescu E, Rohner F, Proietto J, Rivest RW, function with our examined dose. So break tak- Jeanrenaud B. Taste-induced changes in plas- ing food additives, and back to natural food. ma insulin and glucose turnover in lean and genetically obese rats. Diabetes 1988; 37: In conclusion, frequent consumption of saccha- 773-779. rine have the effect of inducing metabolic [7] Fowlkes KD and Carter G. Atlernative sweeten- derangements that affect body weight, glucose ers. Drug Consults 1994; 86: 162-16. and lipids levels. These results question the [8] Sweatman TW, Renwick AG. The tissue distri- effect of saccharine on weight-maintenance or bution and pharmacokinetics of saccharin in growth rate.

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