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Probing the In Vitro Cytotoxicity of the Veterinary Drug Oxytetracycline


The study investigated the effect of oxytetracycline (OTC) on the anti-oxidative defense system, the structure (hemolysis rate and morphology) and function (ATP enzyme activity) of human red blood cells (hRBCs) to investigate the possible toxic mechanism of OTC to hRBCs. The experimental results indicate that OTC can cause a decline in the function of the antioxidant defense system of hRBCs, resulting in oxidative stress. OTC can bring about morphological changes to hRBCs, and further leads to hemolysis, when the concentration of OTC is over 861025 M (about 164 mg/ml). At a low OTC concentration, below 461025 M (82 mg/ml), OTC can enhance the activity of ATP enzyme of hRBCs, known as hormesis. However, at a high concentration, above 461025 M (about 82 mg/ml), the ATP enzymatic activity was inhibited, affecting the function of hRBCs. The estalished mechanism of toxicity of OTC to hRBCs can facilitate a deeper understanding of the toxicity of OTC in vivo.


Oxytetracycline (OTC, structure shown in Fig. 1), a type of tetracycline antibiotics with broad-spectrum antibiotic activity [1], [2], is extensively applied for therapeutic purposes in humans as well as an antibiotic and growth promoter in intensive farming systems and aquaculture [3]. Effective therapy should be obtained when plasma OTC concentrations are above 4 mg/ml declared by the National Committee for Clinical Laboratory Standards (NCCLS) to achieve that the target bacteria are not resistant to OTC [4]. At the therapy dose of 20 mg/kg, the maximum serum concentration of OTC can reach 4.10 mg/ml for calves [5]. For different animals, the peak concentration and terminal half-life of OTC in blood can be achieved 4,16 hours and 21,63 hours after administration, respectively [6–10]. OTC concentrations in plasma can be kept at or above 0.5 mg/ml (minimum inhibitory concentration) for approximately 6 days [11]. The bioavailability of OTC is low [3], so the ingested OTC in animal body is metabolized partially and the residual OTC is excreted and released into soils, surface water and groundwater [3], [12]. OTC has been detected at nanogram to low-microgram per liter levels in wastewater effluents and natural waters [13]. OTC can bioaccumulate in various organisms including invertebrates and fish in food chain and enter human bodies by water drinking and food intake such as milk, meat and eggs (at nanogram to microgram per liter levels) [14–16], posing a threat to human health [13]. OTC can also be taken up by blood cells [17]. The Joint FAO/WHO Expert Committee of Food Additives andContaminants (JECFA), at its 50th Meeting in 1998, established a group acceptable daily intake (ADI) of 0,0.03 mg kg21 body weight for the tetracyclines (oxytetracycline (OTC), tetracycline (TC) and chlortetracycline (CTC)), alone or in combination. The committee also recommended maximum residue limits (MRLs) of 100 mg L21 in milk and muscle of all food-producing species [18], [19]. The toxicity of OTC residues in the environment including animal food, soils, surface and ground water, has attracted widespread attention [1], [2], [20], [21]. OTC can inhibit the antibody levels in fish (dose rate equivalent to 75 mg/kg body weight/day) [22], induces DNA damage in carp kidney cells (exposure dose 250 mg/l) [23], has teratogenic effects on carp embryos (exposure dose 127.5,364.1 mg/l) [24], interacts with cytoplasmic protein synthesis (incubation with 100,500 mg/ml of OTC) [25], and induce blood disorder in Juvenile Nile Tilapia Oreochromis niloticus (fish fed with 1.25% OTC and above) [26]. OTC also has effect on the secretion kinetics of the rat exocrine pancreas and can lead to the decrease in trypsin level in male wistar rats (OTC dose 30 mg/kg/d) [27], [28]. Red blood cells (RBCs), also referred to as erythrocytes, are the most abundant cells in the bloodstream [29]. The primary function of RBCs is to transport oxygen from the lungs to various parts of the body [30]. In addition, RBCs are also a key player in getting waste carbon dioxide from the tissues to the lungs of the body where it is expelled [30], and regulate blood pH [31]. However, intake of hazardous substances may result in injury of RBCs, affecting its functions.

OTC can cause significant reductions in several blood parameters including erythrocyte, hematocrit, and hemoglobin values, for Juvenile Nile Tilapia Oreochromis niloticus [26]. However, the effect of OTC on the anti-oxidative defense system of RBCs is still unknown. The changes in the activities of anti-oxidative defense system have been regarded as one of the toxic mechanisms of many hazardous substances. We also have little knowledge about the influence of OTC on the structure and function of RBCs. In this work, we investigated the toxicity indexes of OTC to human red blood cells (hRBCs) including the antioxidant capacity, the hemolysis rate and morphology (structure), and the ATP enzyme activity (function). We consider the work as a worst case scenario in view of the common concentration of OTC residues in the environment. The study is helpful for understanding the effect of OTC on hRBCs during the blood transportation process and its toxicity in vivo.

Materials and Methods 

2.1. Reagents and apparatus

EDTA dipotassium salt dihydrate (Tianjin Kermel Chemical Reagent Co., Ltd.) stabilized human blood samples were freshly obtained from Hospital of Shandong University (Jinan). A stock solution of OTC (1.061023 M) was prepared by dissolving 0.0497 g oxytetracycline hydrochloride (Sigma) in 100 ml of saline solution. 2,3-naphthalenedicarboxaldehyde (NDA) was obtained from Nippon Kasei Chemical Co., Ltd. Glutaraldehyde (Tianjin Kermel Chemical Reagent Co., Ltd.) was used as fixative. Isoamyl acetate was obtained from Tianjin Chemagent Research Co., Ltd. PBS buffer (10x, pH 7.4) was purchased from Shanghai Biocolor BioScience & Technology Company. All other reagents were of analytical grade. All UV-visible absorption spectra and absorption value were measured on a UV-2450 spectrophotometer (SHIMADZU, Kyoto, Japan). Automatic balance centrifuge (LDZ4-2, Jiangsu Jintan Medical Instrument Factory) was used for centrifugation. Vortex mixer (vortex-6) was purchased from Kylin-Bell Lab Instruments Co., Ltd. Digital dry bath incubator (HB-100, Hangzhou Bioer Technology Co., Ltd) was used to control temperature of the samples.

2.2. Ethics statement

The study was approved by the Ethics Committee of Hospital of Shandong University (Jinan). Written informed consent was obtained from all study participants.

2.3. Determination of the activities of SOD, CAT and GSHPx

SOD is a potent protective enzyme that can selectively scavenge O2.2 by catalyzing its dismutation to H2O2 and molecular oxygen (O2) to protect the cells from being injured [32]. CAT catalyzes the degradation of H2O2 to H2O and O2: 2H2O2R2H2O+O2 [33]. The GSH-Px is an important enzyme extensively existing in vivo, which can specifically catalyze the reduction of H2O2 by GSH to protect the integrity of the structure and function of the membrane. With the activities of SOD, CAT and GSH-Px as the indicators, we studied the effect of OTC on the antioxidant defense of hRBCs.

2.3.1. SOD activity determination. 



Test principle: superoxide anion radical (O2.2), produced in xanthine and xanthine oxidase reaction systems, can oxidize hydroxylamine to nitrite, which shows violet under the effect of color reagent. The absorbance can be measured with visible or UV-vis spectrophotometer. When the measured sample contains superoxide


dismutase (SOD), its specific inhibition of O2.2 can reduce the formation of nitrite, the absorbance value is lower than that of the control with colorimetry. The SOD activity was determined by utilizing the SOD detection kit (Nanjing Jiancheng Bioengineering Institute). The freshly obtained blood sample (1 ml) was added to 2 ml of PBS, mixed by vortexing, and then the hRBCs were isolated from serum by centrifugation at 2000 rpm for 5 min. After being washed two times with 2 ml of PBS solution, the purified hRBCs were diluted to 2 ml with PBS. 0.2 ml of the diluted cell suspension was added to 0.8 ml OTC solutions of different concentrations and mixed by vortexing, then incubated for 3 hours under gentle shaking. Following incubation, the samples were centrifuged (2000 rpm65 min), and the supernatant was discarded and the pellet was resuspended in 1 ml PBS. The SOD activity of the sample was measured according to the procedure of the detection kit. The relative SOD activity was calculated using the following formula [34]:  


Relative SOD activity~(Acontrol{A1)=(Acontrol{A0)|100%  


Where Acontrol is the absorbance of the control tube, A1 and A0 are the absorbances of the testing tube of RBCs with and without OTC, respectively.



2.3.2. CAT activity determination.

Test principle: the decomposition of hydrogen peroxide (H2O2) by catalase (CAT) can be rapidly stopped by ammonium molybdate. The residual H2O2 interacts with ammonium molybdate to produce a yellowish complex. The amount formed can be determined at 405 nm to calculate the CAT activity. The freshly obtained blood sample (1 ml) was washed three times with 2 ml PBS by centrifugation for 5 min at 2000 rpm. The purified hRBCs were diluted to 10 ml with PBS. 0.2 ml of the diluted cell suspension was mixed with 0.8 ml OTC solutions of different concentrations by vortexing, and then incubated for 3 hours under gentle shaking. Following incubation, the samples were centrifuged (2000 rpm65 min), and the supernatant was discarded and the pellet was resuspended in 0.3 ml ultrapure water to achieve hemolysis. The hemolytic blood was used to measure the CAT activity according to the procedure of the detection kit (Nanjing Jiancheng Bioengineering Institute)

.2.3.3. GSH-Px activity determination.

Test principle: glutathione peroxidase (GSH-Px) can promote the reaction of H2O2 with reduced glutathione (GSH) to produce H2O and GSSG. The GSH-Px activity can be expressed by the speed of the enzymatic reaction, detected by the consumption of GSH. In the experiment, the GSH-Px activity was determined by utilizing the GSH-Px detection kit (Nanjing Jiancheng Bioengineering Institute) with spectrophotometry method. The freshly obtained blood sample (1 ml) was washed, diluted, incubated with OTC and centrifuged according to the procedure in Section 2.3.2. The supernatant was discarded and the pellet was resuspended in 1 ml ultrapure water to achieve hemolysis. 0.2 ml of the hemolytic blood was used to measure the GSH-Px activity according to the procedure of the detection kit (Nanjing Jiancheng Bioengineering Institute).

2.4. Determination of GSH

Test principle: GSH is an important nonenzymatic antioxidant. It participates in the maintaining of redox equilibrium which may alleviate cellular oxidative injury. In this work, NDA was used to label the intracellular GSH of hRBCs. Nonfluorescent NDA can readily penetrate the cell membrane, interacting with the nonfluorescent GSH to produce a green fluorescent GSH-NDA derivative [35]. The fluorescence intensity of GSH-NDA derivative was directly proportional to the intracellular content of GSH by fluorescence image analysis. NDA can react concomitantly with the amino and thiol groups of GSH, and no additional nucleophile reagent is required [36]. So the bifunctional reaction is rapid, nonenzymatic, and highly selective [36]. The freshly obtained blood sample (1 ml) was washed, diluted, incubated with OTC and centrifuged according to the procedure in Section 2.3.2. The supernatant was discarded and the pellet was resuspended in 1 ml PBS. For the determination of GSH, 0.3 ml cell suspension was incubated with 100 ml 0.02 mol/L NDA solution and 0.6 ml PBS for 0.5 hour in the dark at room temperature. 0.05 ml of the suspension was placed on the no-clean cover glass for investigation (24650 mm, Citotest Labware Manufacturing Co., Ltd). An inverted microscope (Model IX81, Olympus, Tokyo, Japan) equipped with a 106objective (PlanApo, Olympus, Tokyo, Japan), a mercury lamp, a mirror unit consisting of 470–490 nm excitation filter (BP470–490), a 505 nm dichromatic mirror (DM 505), a 510–550 nm emission filter (BA510–550) and a 16-bit thermoelectrically cooled EMCCD (Cascade 512B, Tucson, AZ, USA) were used for epifluorescence measurements. The derivatized GSH was excited with a 470–490 nm light ray through the objective. The fluorescence emitted by these molecules was collected by the same objective and the fluorescence images were acquired by the EMCCD. Image acquisition was controlled by the MetaMorph software (Universal Imaging, Downingtown, PA, USA). The micrographs of hRBCs without derivatization were observed by the inverted microscope under bright field and epifluorescence illumination conditions.



2.5. Determination of Malondialdehide (MDA) 

Test principle: MDA is the degradation product of lipid peroxidation which can indicate the level of oxidative stress. It can condense with TBA to form a coloured MDA-TBA complex, with the maximum absorption peak at 532 nm that can be measured by visible absorption spectrophotometry. The freshly obtained blood sample (1 ml) was washed, diluted, incubated with OTC and centrifuged according to the procedure in Section 2.3.1. The supernatant was discarded and the pellet was resuspended in 1 ml ultrapure water to achieve hemolysis. 0.1 ml of the hemolytic blood was used to measure the MDA concentration according to the procedure of the detection kit (Nanjing Jiancheng Bioengineering Institute).

2.6. Hemolysis assay 


The freshly obtained blood sample (1 ml) was washed, diluted and mixed with OTC according to the procedure in Section 2.3.2. RBC incubation with ultrapure water and PBS were used as the positive and negative controls, respectively. All the sample tubes were kept in static condition at room temperature for 3 h. Finally, the mixtures were centrifuged at 2000 rpm for 5 min, and 500 ml of supernatant of all samples was diluted with 2.5 ml ultrapure water. The absorbance values of the diluted supernatants (3 ml) at 540 nm were determined by the UV-2450 spectrophotometer [37]. The hemolysis rate of hRBCs was calculated using the following formula [38]:

Hemolysis rate~(Asample{Anegative)=(Apositive{Anegative)|100%

Where Asample, Apositive and Anegative are the absorbances of sample, the positive and negative controls, respectively.

2.7. Scanning electron microscopy (SEM) studies of hRBCs

The freshly obtained blood sample (1 ml) was washed according to the procedure in Section 2.3.1. The purified hRBCs were diluted by 25 times with PBS. 0.2 ml of the diluted cell suspension was incubated with 0.8 ml OTC solutions of different concentrations for 3 hours under gentle shaking. Following incubation, the samples were centrifuged (2000 rpm65 min) and the supernatant was discarded. Fixation was performed by addition of 2.5% glutaraldehyde and 12 h incubation. The fixed samples were washed with 0.1 M phosphate buffer for more than 3 h. Then, the sample was fixed in osmium tetroxide (1%) for 1,1.5 h and washed in double-distilled water for 2 h. Dehydration was done with increasing concentrations of ethanol (50%, 70%, 80%, 90%and 100%) twice. The ethanol was displaced by isoamyl acetate (100%) for 15 min (twice). Finally, the sample was dried with the conventional critical point drying method, platinum-coated with ion sputtering coater (Eiko, IB-5) and then observed with a scanning electron microscope (Hitachi, S-570) to investigate the effect of OTC on the morphology of hRBCs.

2.8. ATPase activity assay determination
ATPase is an important enzyme existing in the membrane of cells in vivo [39]. It functions in maintaining ionic and osmotic balance inside and outside the cell, maintaining transmembrane electrochemical gradients, and in cellular energy metabolism [39],  [40]. The reduction in ATPase activity can result in the disorder of the functions, which affects the normal function of cells [39], [40]. For example, a rise in internal Na+ , K+ or Ca2+ , Mg2+ ions of RBCs can cause changes in cell shape and volume, increase in cellular rigidity and hemolysis. ATPase can cause the decomposition of ATP into ADP and inorganic phosphorus. The activity of ATPase can be determined by measuring the amount of inorganic phosphorus. The freshly obtained blood sample (1 ml) was washed with PBS, incubated with OTC, and then centrifuged according to the procedure in Section 2.3.1. The supernatant was discarded and the pellet was resuspended in 0.5 ml ultrapure water to achievehemolysis. 0.2 ml of the hemolytic blood was used to measure the ATPase activity according to the procedure of the detection kit (Nanjing Jiancheng Bioengineering Institute).
2.9. Statistical analysis
Data are from three independent experiments and presented as mean 6 SD in the study of the effect of OTC on the activities of SOD, CAT, GSH-Px and ATPase, content of GSH and MDA, and hemolysis rate of hRBCs.
Results and Discussion
3.1. Effects of OTC on antioxidant capacity of hRBCs

Radicals are present in tissues in vivo as free and bound forms. The bound radical is necessary for normal physiological activity. However, the free radical is highly reactive, easily combining with electrons from tissue macromolecules to achieve more stability through electron pairing. In toxicology, there is interest in free radicals and reactive oxygen species (ROS), which can be produced by the normal metabolism of cells or by the exogenous factors (e.g. smoking, radiation, dust). ROS are free radicals and non-free radical oxygen-containing molecules that have higher chemical reactivity than ground state molecular oxygen [41], including species such as O2.2, hydroxyl radicals (?OH), nitric oxide (NO?), singlet oxygen (1O2) and H2O2 [32]. ROS, within physiological concentrations, play an important role in regulating the body’s normal physiological functions such as apoptosis, gene expression and signal transduction [42]. However, when free radicals and antioxidative processes are not balanced, that is, when the production of ROS exceeds the scavenge ability of the body’sdefense system, or the damaged body’s defense system can not function properly, free radicals can produce oxidative stress, inducing the oxidation of biological macromolecules (nucleic acids, proteins, lipids et al), damage to the structure and function of cells, and diseases such as pulmonary fibrosis, epilepsy, hypertension and atherosclerosis [43], [44]. Antioxidants, being mainly natural molecules, can prevent the uncontrolled formation of ROS or inhibit reactions of ROS with biological structures. Enzymes such as SOD, CAT and GSH-Px provide the main antioxidant defense [45]. There must be a balance between the three enzymes to remove ROS from the body properly [46]. As non-enzymatic defense, the role of dietary supplements such as GSH, vitamins (C, E) and carotenoids, is also very important to control oxidative injury [33], [47]. In this section, we studied the effect of OTC on the antioxidant defense of hRBCs including SOD, CAT, GSH-Px and GSH.