Table 1: Effects of pefloxacin and amoxicillin on plasma, HDL and LDL-VLDL lipid profile of the animals

Plasma Cholesterol (mg/dl) Triacylglycerols (mg/dl) Phospholipid (mg/dl) Free Fatty acid (mg/dl) HDL Cholesterol (mg/dl) Triacylglycerols (mg/dl) Phospholipid (mg/dl) LDL-VLDL Cholesterol (mg/dl) Triacylglycerols (mg/dl) Phospholipid (mg/dl) Control 56.602.43a 63.185.19a 75.3315.41a 14.102.25a 39.062.77cd 26.833.65a 68.531.42ab 25.942.31ab 22.431.72a 86.992.81bc Pefloxacin day 5 83.434.82b 70.606.80ab 131.2210.89c 18.850.84bc 27.514.35b 46.162.59c 56.8610.04a 29.285.06ab 29.142.22b 76.795.13b Pefloxacin day 10 80.009.90ab 93.007.41b 173.0218.01d 20.400.44c 29.073.52bc 49.934.32cd 86.994.44b 35.5710.50bc 45.435.75c 94.2812.30c Pefloxacin day 15 56.448.84a 93.269.88b 80.6811.25 ab 16.000.36ab 21.062.87ab 42.604.55bc 88.946.90b 39.433.43bc 39.947.34bc 63.181.54a Amoxicillin day 5 97.0212.01b 93.0810.60b 144.8319.36cd 18.050.94bc 15.022.68a 34.261.81ab 58.8111.07 a 38.456.55bc 27.651.60ab 72.416.02ab Amoxicillin day 10 96.996.06b 71.304.98ab 108.3817.90b 16.001.47ab 47.514.63d 59.324.17d 56.3812.80a 45.123.88c 26.474.89ab 83.113.55 bc Amoxicillin day 15 71.3314.24ab 58.789.40a 100.1220.29b 15.600.81ab 20.413.21ab 26.044.29a 86.026.18b

AMOXICILLIN- AND PEFLOXACIN-INDUCED CHOLESTEROGENESIS AND PHOSPHOLIPIDOSIS IN RAT TISSUES

1

Abstract ID
2907 Poster Board - 153

Ademuyiwa O.1, Rotimi S. O.3, Ojo D. A.2, Balogun E. A.1 and Talabi O. A.4
Department of Biochemistry, 2Department of Microbiology, University of Agriculture, Abeokuta, Nigeria, 3Department of Biological Sciences, Covenant University, Ota, Nigeria and 4State Hospital, Abeokuta, Nigeria.
ABSTRACT

14.402.71a 34.108.27b 58.811.79a

Dyslipidemia is currently becoming a confounding factor in assessing the safety of new and existing drugs. In order to investigate whether amoxillin (an extended-spectrum penicillin) and pefloxacin (a third generation fluoroquinolone) perturb lipid metabolism, rats were treated with therapeutic doses of each antibiotic for 5 and 10 days respectively. Twenty four hours and 5 days after antibiotic withdrawal, blood and other tissues (liver, kidney, brain, heart and spleen) were removed from the animals after an overnight fast and analysed for their lipid contents. Both antibiotics produced various degrees of compartment-specific dyslipidemia in the animals. While plasma and erythrocyte dyslipidemia was characterised by up-regulation of the concentrations of the major lipids (cholesterol, triglycerides, phospholipids and free fatty acids), hepatic and renal dyslipidemia was characterised by cholesterogenesis and phospholipidosis. Splenic dyslipidemia was characterised by cholesterogenesis and decreased phospholipid levels. Cardiac and brain cholesterol was not affected by the antibiotics. A transient phospholipidosis was observed in the brain whereas cardiac phospholipids decreased significantly (p < 0.05). At the membrane level, phospholipidosis alone was the hallmark of dyslipidemia in the erythrocyte membrane while lipoprotein abnormalities were reflected as downregulation of HDL cholesterol. Furthermore, the two antibiotics increased triglyceride levels in all compartments in addition to increasing the activity of hepatic HMG-CoA reductase. Although erythrocyte phospholipidosis was resolved 5 days after withdrawing the antibiotics, dyslipidemia observed in other compartments was still not reversible after withdrawing the antibiotics. Our findings suggest that induction of cholesterogenesis and phospholipidosis might represent additional adverse effects of amoxillin and pefloxacin and these might be contributing factors in the toxicity of these antimicrobials. Introduction Amoxillin [(2S, 5R, 6R)-6-{[(2R)-2-amino-2-(4-hydroxyphenyl)-acetyl] amino}-3, 3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0] heptane2-carboxylic acid] and pefloxacin [1-ethyl-6-fluoro-7-(4-methylpiperazin-1-yl)-4-oxoquinoline-3-carboxylic acid] are broad spectrum antimicrobial agents used in the treatment of typhoid infection (Chambers, 2007) . While amoxillin is a -lactam aminopenicillin (Fig. 1a), pefloxacin is a fluoroquinolone (Fig. 1b). Both are active against gram-negative bacteria. Amoxillin acts by inhibiting the synthesis of bacterial cell walls. It inhibits cross-linkage between the linear peptidoglycan polymer chains that make up a major component of the cell walls of both gram-positive and gram-negative bacteria. Pefloxacin on the other hand owes it antibiotic activity to its blocking of bacterial DNA synthesis (Chambers, 2007).

Each value represents the meanS.E.M of 5 rats. Values within the same row with different superscripts are significantly different at p<0.05.

Table 2: Effects of pefloxacin and amoxicillin on erythrocyte and erythrocyte ghost lipid profile of the animals
Erythrocyte Cholesterol (mg/dl) Triacylglycerols (mg/dl) Phospholipid (mg/dl) Free Fatty acid (mg/dl) Erythrocyte Ghost Cholesterol (mg/g) Triacylglycerols (mg/g) Phospholipid (mg/g) Control 123.4319.54a 38.1610.76a 156.494.71a 26.562.31a 7.030.27a 3.940.27a 7.910.26a Pefloxacin day 5 78.2212.90g 52.582.41c 179.827.37a 31.460.85d 7.350.54a 4.860.29b 14.851.42c Pefloxacin day 10 127.337.47c 55.875.57c 187.606.98a 33.000.42c 5.560.10c 3.820.15a 10.930.56b Pefloxacin day 15 183.8516.98d 39.762.28a 156.493.89a 28.600.36b 6.580.81a 4.270.64a 11.091.21 b Amoxicillin day 5 45.413.95c 50.594.59c 173.9920.64a 30.660.95bc 8.170.90b 4.940.54b 13.361.09c Amoxicillin day 10 145.6917.55e 64.481.35b 166.2125.55a 29.161.51e 6.010.66c 3.590.36c 10.381.11 b Amoxicillin day 15 104.6815.57f 48.866.73c 161.359.40a 28.160.88b 7.440.64a 4.850.22b 12.791.01c

Each value represents the meanS.E.M of 5 rats. Values within the same row with different superscripts are significantly different at p<0.05.

Table 3: Effects of pefloxacin and amoxicillin on the lipid profiles of liver, kidney, brain, spleen and heart of the animals
Liver Control
a

Cholesterol (mg/g) 2.340.07 Triacylglycerols (mg/g) 1.560.19a Phospholipid (mg/g) 14.290.95a Kidney Cholesterol (mg/g) 2.580.35a 3.420.09b 3.140.26c 2.870.22c 2.840.21c 2.930.15c 3.440.09b Triacylglycerols (mg/g) 4.250.29a 4.410.40b 4.200.45a 6.670.78c 3.480.11 a 5.770.46c 6.210.56c a d b c d c Phospholipid (mg/g) 10.010.79 6.900.28 12.152.05 14.480.68 7.290.80 13.901.84 12.251.10b Brain Cholesterol (mg/g) 12.240.59a 12.721.09a 12.870.45a 11.651.60 a 13.351.81a 15.821.26a 14.092.06a Triacylglycerols (mg/g) 0.600.06a 0.590.13a 2.160.24c 0.940.06d 0.730.18d 2.110.04 c 1.050.04b Phospholipid (mg/g) 39.462.00a 44.325.64a 38.302.84a 39.663.61a 37.623.83a 51.903.67b 40.821.63a Spleen Cholesterol (mg/g) 1.090.11 a 0.820.07a 1.150.17d 0.950.07a 1.310.15d 1.880.36b 1.670.10c Triacylglycerols (mg/g) 0.560.16a 1.220.21b 1.580.30b 1.080.24b 1.270.10b 1.360.14b 1.060.20b Phospholipid (mg/g) 12.491.35a 9.530.38d 8.800.52c 10.500.48b 11.370.16 b 9.040.63c 9.770.36d Heart a a a a a a Cholesterol (mg/g) 0.580.05 0.660.07 0.680.09 0.520.10 0.770.12 0.620.16 0.610.07a Triacylglycerols (mg/g) 2.810.48a 6.730.79b 6.790.60b 5.360.54b 6.600.60b 4.860.73b 5.320.78b Phospholipid (mg/g) 10.350.46a 5.640.49c 7.050.86b 9.530.33d 8.021.36c 9.870.32d 9.910.34d Each value represents the meanS.E.M of 5 rats. Values within the same row with different superscripts are significantly different at p<0.05.

Pefloxacin day 5 2.350.04a 1.760.07a 14.090.74a

Pefloxacin day 10 2.390.03a 2.650.11 b 25.172.49b

Pefloxacin day 15 2.930.21b 2.510.13b 24.691.53b

Amoxicillin day 5 2.160.04a 1.640.06a 10.790.56a

Amoxicillin day 10 2.990.15b 2.500.08b 20.803.51b

Amoxicillin day 15 3.230.04b 2.720.05b 20.020.50b

Fig. 1a: Chemical structure of amoxillin

Fig. 1b: Chemical structure of pefloxacin

Although amoxillin and pefloxacin are generally considered safe and well tolerated, they have been associated with a wide range of adverse effects. Adverse effects for both antibiotics range from fever, nausea, diarrhea and vomiting to major allergic reactions including photosensitivity and skin rash. Mental changes, light-headedness, confusion and anxiety, have also been reported for both antibiotics. Tendinopathy, sometimes resulting in spontaneous rupture of tendons, has also been reported as a side effect of treatment with pefloxacin (Brodgen et al., 1975; Simonin et al., 2000; Leone et al., 2003).
Figure 2: Effects of pefloxacin and amoxicillin on hepatic HMG CoA/Mevanolate ratios as an index of HMG-CoA reductase activity of the animals Each bar represents the meanS.E.M of 5 rats. Bars with different alphabets are significantly different at p<0.05.

Although these data about amoxillin and pefloxacin have been known for sometime, nothing has been published on the interaction of these antibiotics with lipids. Interest in the interaction of drugs with lipids derives from the observation that lipids play a major role in the pathogenesis and progression of various disease conditions. Furthermore, dyslipidemia is currently becoming a confounding factor not only in new drug development, but also in assessing safety of earlier approved drugs so as to uncouple dyslipidemia from toxic manifestations of the drug. Whether amoxillin (an extended-spectrum penicillin) or pefloxacin (a third generation fluoroquinolone) induce any dyslipidemia, was the basis of the present study. .

mevalonate. The ratio of HMG-CoA to mevalonate is taken as an index of the activity of HMG-CoA reductase. An increase in this ratio indicates inhibition of cholesterogenesis while a decrease indicates enhanced cholesterogenesis. Materials and methods Chemicals Pefloxacin was a product of Lek Pharmaceutical and Chemical Company, Ljubljana, Slovenia, while amoxillin was obtained from Beecham Pharmaceuticals, Brentford, England. Animals and treatment Thirty five male albino Sprague-Dawley rats (bred in the animal holding of Nigerian Institute of Medical Research (NIMR), Lagos, Nigeria) with a mean body weight of 230g were used for the experiment. They were housed in an animal room with normal controlled temperature (222C) and a regular 12h light-dark cycle (06:00-18:00h). They were allowed 14 days to acclimatise before the commencement of antibiotic exposure. The animals were maintained on a standard pellet diet. Animals were divided into 7 groups of 5 animals each. While 1 group served as control group, three groups were treated with amoxillin (7.14mg/kg body weight, 8 hourly) and the remaining three groups with pefloxacin (5.71mg/kg body weight, 12 hourly) for 5 and 10 days respectively. The antibiotics were constituted in 5% dextrose and were prepared fresh before each administration. They were administered in a total volume of 0.1ml. Control animals received equivalent volume of 5% dextrose. All drug administration was by the intraperitoneal route. During the experiment, the animals were allowed free access to food and distilled water. At the end of the antibiotic treatment and 5 days after the discontinuation of the antibiotics, blood was collected from the animals into heparinised tubes by cardiac puncture under light ether anaesthesia after an overnight fast. Liver, kidney, brain, heart and spleen were removed from the animals for biochemical analyses. Blood samples were centrifuged to separate plasma and red blood cells. All samples were stored at -20C until analysed. Biochemical analyses Plasma and lipoprotein lipid profiles Plasma concentrations of total cholesterol and triglycerides were determined with commercial kits (Spin React S.A., Santa Colona, Sant Esteve De Bas, Spain). HDL cholesterol and triglycerides were determined in plasma with same commercial kits for total cholesterol and triglycerides after very low density lipoproteins (VLDL) and LDL were precipitated with heparin-MnCl2 solution as described by Gidez et al. (1982). Total phospholipids in plasma were extracted with chloroform-methanol mixture (2:1, v/v) as described by Folch et al. (1957). Phospholipid content was then determined as described by Stewart (1980). Free fatty acids (FFA) in plasma were determined according to the method of Soloni and Sardina (1973). Erythrocyte lipid profile Lipids were extracted from erythrocytes using chloroform-isopropanol (7:11, v/v) as described by Rose and Oklander (1965). For the determination of cholesterol, an aliquot of the chloroform-isopropanol extract was evaporated to dryness at 60C. Triton X-100/chloroform mixture (1:1, v/v, 20l) was added to resolve the lipids and again the solvent was evaporated. Then 1ml of commercially available cholesterol kit reagent (Spin React S.A., Santa Colona, Sant Esteve De Bas, Spain) was added and vortexed. After incubation in the dark at room temperature for 30 minutes, cholesterol content was determined by colorimetry. Determination of total phospholipids and free fatty acids in the chloroformisopropanol extract of the erythrocyte followed the same procedure as described for plasma. Organ lipid profiles Lipids were extracted from the organs (liver, kidney, brain, heart and spleen) as described by Folch et al. (1957). After washing with 0.05M KCl solution, aliquots of the chloroform-methanol extract were then used for the determination of cholesterol, triglycerides and phospholipids concentrations. Cholesterol was determined in an aliquot of the chloroform-methanol extract of each organ as described for erythrocytes while determination of phospholipids followed the same procedure as described for plasma. Triglyceride concentrations in aliquots of the chloroformmethanol extracts of each organ were determined following the procedure described by Kriketos et al. (2003). Isolation of erythrocyte ghost and determination of its lipid profile Erythrocyte membranes were prepared according to the method described by Hanahan and Ekholm (1974). Lipids were extracted from the membrane suspensions and determined as described for erythrocytes. Determination of hepatic HMG-CoA reductase activity This was determined according to the method of Rao and Ramakrishnan (1975) by measuring the hepatic concentrations of HMG-CoA and Soloni FG, Sardina LC. Colorimetric micro-determination of free fatty acids. Clin Chem 1973; 19 (4): 419 24. Rose HG, Oklander M. Improved procedure for the extraction of lipids from human erythrocytes. J Lipid Res 1965; 6: 428 31. Simonin M-A, Gegout-Pottie P, Minn A, Gillet P, Netter P, Terlain B. Pefloxacin-induced Achilles tendon toxicity in rodents: biochemical changes in proteoglycan synthesis and oxidative damage to collagen. Antimicrob Agents Chemother 2000; 44(4): 867 72. Rao AV, Ramakrishnan S. Indirect assessment of hydroxymethylglutaryl- CoA reductase (NADPH) activity in liver tissue. Clin Chem 1975; 21 (10). 1523 25. Kriketos AD, Furler SM, Gan SK, Poyten AM, Chisholm DJ, Campbell lV. Multiple indexes of lipid availability are independently related to whole body insulin action in healthy humans. J Clin Endocr Met 2003; 88 (2): 993-8. Hanahan DJ, Ekholm JE. The preparation of red cell ghosts (membranes). In: Fleischer S, Packer L, editors. Methods in Enzymology vol. 31. Biomembranes, Part A. New York: Academic Press 1974, p. 168 72. Folch J, Lees M, Sloane SGH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 1957; 226: 497 509. Gidez LT, Miller GH, Burnstein M, Slagle S, Eder HA. Separation and quantitation of subclasses of human plasma high density lipoproteins by a simple precipitation procedure. J Lipid Res 1982; 23: 1206 23. Chambers HF. Beta-Lactam and other cell wall and membrane active antibiotics. In:Katzung BG, editor. Basic and Clinical Pharmacology. 10th ed. New York: McGraw-Hill Companies Inc.; 2007, p.726 44. Brodgen, RN, Speight TN, Avery CS. Amoxillin: a review of its antibacterial and pharmacokinetic properties and therapeutic use. Drug 1975; 9: 88 140. Statistical analysis Data are expressed as meanS.E.M. One way analysis of variance (ANOVA) followed by Duncan Multiple Range Test was used to analyse the results with p < 0.05 considered significant. Results Administration of the two antibiotics resulted in up-regulation of the major lipids (cholesterol, triglycerides, phospholipids, FFA) in plasma and erythrocytes of the animals (Tables 1 & 2). Lipoprotein dyslipidemia was characterised by decreased HDL cholesterol and increased concentrations of both triglycerides and phospholipids. Results also showed that amoxillin and pefloxacin induced a phospholipidosis in erythrocyte ghost (Table 2). In all the organs, administration of the antibiotics resulted in a significant increase (p < 0.05) in triglyceride concentrations (Table 3). In the brain and heart, antibiotic treatment did not affect cholesterol concentrations, whereas in the spleen, 10 days of antibiotic treatment resulted in the induction of cholesterogenesis. While a transient phospholipidosis was observed in the brain, both cholesterogenesis and phospholipidosis were induced in the liver and kidney (Table 3). In addition to these, the two antibiotics decreased the hepatic HMGCoA/mevalonate ratio, thus suggesting an increase in the activity of HMG-CoA reductase Conclusions Administration of amoxillin and pefloxacin even at therapeutic doses, results in the induction of cholesterogenesis and phospholipidosis in tissues. While cholesterogenesis may be as a result of increased HMG-CoA reductase activity and reduced cholesterol-7-hydroxylase activity, phospholipidosis might be mediated by increased availability of FFA and inhibition of phospholipase. References Baselt R. Disposition of toxic drugs and chemicals in man. 8th ed. Foster City: Biomedical Publications; 2008, p. 81-83. (Fig. 2).