Ethidium bromide for agarose preparation and agarose were purchased from Sigma-Aldrich (St. Louis, MO, USA). A total of 40 male Wistar rats were purchased from King Abdel-Aziz University, King Fahd Center for Scientific Research (Jeddah, Saudi Arabia). Catalase, malondialdehyde (MDA), glutathione peroxidase (GSH-Px), glutathione reductase (GR), creatinine and urea kits were purchased from Bio Diagnostic Co. (Dokki, Egypt). Coca-Cola, Pepsicola and 7-Up were purchased from Taif markets in Saudi Arabia. The 100 bp DNA ladder marker was purchased from Fermentas (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Oligo dT primer and Qiazol reagent were purchased from Qiagne (Valencia, CA, USA).

The present study was approved by the Ethics Committee for the College of Applied Medical Sciences (Taif University; no. 3792/34/1). A total of 40 male Wistar rats (12-weeks-old; weight, 200–280 g). The rats were monitored daily and were maintained under observation for 1 week for complete acclimatization. The rats were maintained at 12 h light/dark cycles and received free access to food and water for the first week. The rats were then divided into four groups: i) Control without any treatment; ii) Coca-cola group; iii) Pepsi group; iv) 7-Up group. The rats in groups 2–4 received free access to the respective soft drinks for three consecutive months. At the end of experiment, all rats were anesthetized by diethyl ether inhalation. Blood and tissues were collected from anesthetized rats in sterilized vacutainer tubes. The serum was extracted following centrifugation of clotted blood for 15 min at 3,000 × g and was maintained at −20°C until use. For gene expression analysis, hepatic and renal tissues were maintained at −80°C in Qiazol reagent for RNA extraction. For histopathological examination, sections from the bone, liver and kidney were immersed overnight in neutral buffered formalin (NBF; 10%) at room temperature.

MDA, GSH-Px, GSH-reductase (GSH-R), catalase, glucose, alkaline phosphatase (ALP), urea, bilirubin, creatinine, alanine transaminase (ALT), uric acid, aspartate transaminase (AST), creatine kinase (CK) and CKM were assayed spectrophotometrically using commercial kits purchased from Bio Diagnostic Co., according to the manufacturer's protocol. The levels of osteocalcin levels were assayed using enzyme-linked immunosorbent assay kits purchased from MyBioSource, LLC, San Diego, CA, USA). Various minerals, triglycerides (TG), cholesterol and high-dentisty lipoproteins (HDL) were assayed spectrophotometrically using kits imported from HUMAN Gesellschaft für Biochemica und Diagnostica mbH (Wiesbaden, Germany), according to the manufacturer's protocol. Serum lipase was measured using a lipase activity assay kit imported from Abcam (Cambridge, MA, USA).

The total RNA from tissue samples was extracted, as previously described (19). The integrity of RNA was visualized and confirmed following running on a denatured agarose gel (1.5%) stained with ethidium bromide. Oligo dT primer (0.5 ng) was added to 2 µg total RNA to induce denaturation and was subsequently used for cDNA synthesis (19). For semi-quantitative gene expression analysis, specific primers were designed for certain genes, as illustrated in Table I, using Oligo 4 computer program (version 7; Molecular Biology Insights, Inc., Colorado Springs, CO, USA) and synthesized by (Macrogen Company, Geumcheongu. Korea). PCR was performed in a total volume of 25 µl (1 µl cDNA, 1 µl 10 pM each primer, 12.5 µl PCR master mix and 9.5 µl sterilized, deionized water). A Bio-Rad T100™ Thermal Cycle machine (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used for PCR, which was performed at 95°C for 4 min, followed by 33 cycles of denaturation at 95°C for 50 sec, annealing as shown in Table I and extension at 72°C for 1 minute, with an additional final extension at 72°C for 5 min. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as internal standard and as a reference gene. The PCR products were electrophoresed in a 1.5% agarose gel, followed by staining with ethidium bromide in Tris-Borate-EDTA buffer. The bands were visualized using an InGenius 3.0 gel documentation system (Syngene, Frederick, MD, USA) under UV light.

Tissues from the bone, liver and kidney were dissected following diethyl ether inhalation and the sacrificing of the rats. The samples were immersed and fixed overnight in a 10% NBF solution. Bone, liver and kidney tissues were processed, washed and preserved in ethanol (70%). The samples were subsequently dehydrated in ethanol solution in ascending grades, cleared with xylene, paraffin wax embedded, casted and cut into 5 µm sections using microtome (Leica Microsystems, Inc., Buffalo Grove, IL, USA). Mayer's hematoxylin and eosin, and Masson's trichrome staining were used to stain the slides with the fixed tissue samples (20). The slides with tissue were examined using a Wolfe S9-0982 microscope (Carolina Biological Supply Company, Burlington, NC, USA) and images were captured using a Canon Power-Shot SX500 IS digital camera.

The data are presented as the mean ± standard error of the mean. Analysis of variance was used to analyze data together with post hoc descriptive tests using SPSS 11.5 for Windows (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference. The intensities of the bands were quantified densitometrically using Image J software (version 1.47) and were matched compared to the control group and expressed as a percent (http://imagej.en.softonic.com/).

SDC for 3 months revealed an increase in MDA levels (Table II) with a highly significant effect on Pepsi-administered rats compared with the control rats. By contrast, the antioxidant activity of GSH-Px, catalase and GSH-R in the rats were decreased significantly in all SDC groups (Table II).

SDC for 3 months, significantly increased the serum levels of glucose and moderately increased uric acid and bilirubin in the Coca-Cola, Pepsi and 7-up groups compared with the control group (Table III).

SDC for 3 months significantly increased the levels of liver biomarkers, with a more marked effect on AST and a moderate effect on ALT. Additionally, ALP increased in all groups compared with the control rats (Table IV).

SDC decreased both calcium and iron levels in the rats. By contrast soft drinks increased serum phosphorous levels. In addition, soft drinks revealed no effect sodium and chloride levels. Notably, soft drinks increased the levels of osteocalcin (Table V).

SDC increased serum lipase levels, which resulted in a decrease in cholesterol and TG levels in the SDC groups compared with the control (Table VI). By contrast, HDL was decreased in the Coca-Cola and Pepsi groups compared with the control group; however, 7-Up caused no effect.

SDC for 3 months downregulated the gene expression levels of GST and SOD (Fig. 1). The expression percentage of GST was ~80, 60 and 40% decreased in Coca-Cola, Pepsi and 7-Up groups, respectively. All groups exhibited a slight downregulation in the mRNA expression of SOD in hepatic tissue at a percentage of 30, 18 and 15%, respectively, compared with the control rats.

SDC significantly upregulated the mRNA expression of α1-AGP in the liver tissues (Fig. 2). The percentage of upregulation in the expression of α1-AGP was 66, 70 and 75% for Coca-Cola, Pepsi and 7-Up, respectively. By contrast, the expression of α2-MG was downregulated by Coca-Cola (45%) and moderately upregulated in the Pepsi and 7-Up groups by 15 and 20%, respectively, compared with the control rat (Fig. 2).

Next, the present study tested the effect of SDC on the mRNA expression levels of certain genes associated with normal kidney function and stability. The expression of desmin was upregulated in the Coca-Cola-, Pepsi- and 7-Up-administered rats at a percentage of 75, 50 and 60%, respectively (Fig. 3). Interleukin (IL)-1β was only increased in the Pepsi and 7-Up groups by 30 and 50%, respectively. The expression of homooxygenase (HO)-1 was only increased in the Coca-Cola and 7-Up groups by 90 and 25%, respectively. The present study also examined the genes associated with blood pressure, including angiotensinogen (AT) and its receptor (AT-R). The expression of AT was upregulated in the Coca-Cola group (55% increase compared with the control). The expression of AT-R was also upregulated by 64 and 10% for Cola and Pepsi, respectively, compared with the control and 7-Up groups (Fig. 3).

The bone consisted of numerous bony lamellae, and bone marrow extended between these lamella. The blood progenitor cells occupied the architecture of the bone marrow. The structure unit of the bone consisted of osteocytes arranged around the haversian canal (Fig. 4A). In the Coca-Cola and Pepsi groups, the bone lamella exhibited dark acidophilic areas in the bone architecture due to calcium withdrawal, while the other parts exhibited faint basophilic patches (Fig. 4B and C). In the 7-Up group, the bone lamella exhibited little histological changes in the form of small patches of faint basophilic areas (Fig. 4D).

The liver consisted of a central vein, which was surrounded by numerous hepatic cords. The hepatic cords were synthesized from polygonal hepatic cells with acidophilic cytoplasm and basophilic nuclei, which were centrally located. Von Kupffer cells were located between the hepatic cords (Fig. 5A). In the Coca-Cola group, the liver exhibited activation of Von Kupffer cells and congestion in the blood vessels (Fig. 5B). The liver of the Pepsi group exhibited congestion in blood vessels (Fig. 5C). In the 7-up group, the liver exhibited hydropic degeneration, in addition to congestion of the blood vessels (Fig. 5D).

The kidney of the control group consisted of renal corpuscle, surrounded by proximal and distal convoluted tubules (Fig. 6A). In all soft drink groups, the renal tissues exhibited mild congestion in the renal blood vessels (Fig. 6B–D).