Effects of acute and chronic administration of ... - Springer Link

Mol Cell Biochem (2013) 380:171–176 DOI 10.1007/s11010-013-1670-2

Effects of acute and chronic administration of fenproporex on DNA damage parameters in young and adult rats Cinara L. Gonçalves • Gislaine T. Rezin • Gabriela K. Ferreira • Isabela C. Jeremias Mariane R. Cardoso • Samira S. Valvassori • Bruna J. P. Munhoz • Gabriela D. Borges • Bruno N. Bristot • Daniela D. Leffa • Vanessa M. Andrade • Joaõ Quevedo • Emilio L. Streck

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Received: 30 January 2013 / Accepted: 17 April 2013 / Published online: 1 May 2013 Ó Springer Science+Business Media New York 2013

Abstract Obesity is a chronic and multifactorial disease, whose prevalence is increasing in many countries. Pharmaceutical strategies for the treatment of obesity include drugs that regulate food intake, thermogenesis, fat absorption, and fat metabolism. Fenproporex is the second most commonly consumed amphetamine-based anorectic worldwide; this drug is rapidly converted in vivo into amphetamine, which is associated with neurotoxicity. In this context, the present study evaluated DNA damage parameters in the peripheral blood of young and adult rats submitted to an acute administration and chronic administration of fenproporex. In the acute administration, both young and adult rats received a single injection of fenproporex (6.25, 12.5 or 25 mg/kg i.p.) or vehicle. In the chronic administration, both young and adult rats received

one daily injection of fenproporex (6.25, 12.5, or 25 mg/kg i.p.) or Tween for 14 days. 2 h after the last injection, the rats were killed by decapitation and their peripheral blood removed for evaluation of DNA damage parameters by alkaline comet assay. Our study showed that acute administration of fenproporex in young and adult rats presented higher levels of damage index and frequency in the DNA. However, chronic administration of fenproporex in young and adult rats did not alter the levels of DNA damage in both parameters of comet assay. The present findings showed that acute administration of fenproporex promoted damage in DNA, in both young and adult rats. Our results are consistent with other reports which showed that other amphetamine-derived drugs also caused DNA damage. We suggest that the activation of an efficient DNA

C. L. Gonçalves G. K. Ferreira I. C. Jeremias M. R. Cardoso E. L. Streck (&) Laborato´rio de Bioenerge´tica, Programa de Po´s-graduaçaõ em Cieˆncias da Sau´de, Universidade do Extremo Sul Catarinense, Avenida Universita´ria, 1105, Criciu´ma, SC 88806-000, Brazil e-mail: [email protected]

S. S. Valvassori J. Quevedo Laborato´rio de Neurocieˆncias, Programa de Po´s-graduaçaõ em Cieˆncias da Sau´de, Universidade do Extremo Sul Catarinense, Avenida Universita´ria, 1105, Criciu´ma, SC 88806-000, Brazil

C. L. Gonçalves G. K. Ferreira I. C. Jeremias M. R. Cardoso S. S. Valvassori J. Quevedo E. L. Streck National Institute for Translational Medicine (INCT-TM), Criciu´ma, Brazil

B. J. P. Munhoz G. D. Borges B. N. Bristot D. D. Leffa V. M. Andrade Laborato´rio de Biologia Celular e Molecular, Programa de Po´sgraduaçaõ em Cieˆncias da Sau´de, Universidade do Extremo Sul Catarinense, Avenida Universita´ria, 1105, Criciu´ma, SC 88806000, Brazil

C. L. Gonçalves G. K. Ferreira I. C. Jeremias M. R. Cardoso S. S. Valvassori J. Quevedo E. L. Streck Center of Excellence in Applied Neurosciences of Santa Catarina (NENASC), Criciu´ma, Brazil G. T. Rezin Laborato´rio de Fisiopatologia Clıńica e Experimental, Programa de Po´s-graduaçaõ em Cieˆncias da Sau´de, Universidade do Sul de Santa Catarina, Avenida Jose´ Acaćio Moreira, 787, Tubaraõ, SC 88704-900, Brazil

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repair mechanism may occur after chronic exposition to fenproporex. Our results are consistent with other reports that showed some amphetamine-derived drugs also caused DNA damage. Keywords Fenproporex DNA damage Peripheral blood Rats

Mol Cell Biochem (2013) 380:171–176

Several studies suggest that amphetamine and its derivatives are neurotoxic and associated with increased risk of diseases. In this context, in the present study we investigated the effects of acute and chronic administration of fenproporex on the parameters of DNA damage in young and adult rats, evaluated by alkaline comet assay.

Materials and methods Introduction Animals The growing number of overweight and obese people worldwide represents one of the largest public health concerns at the beginning of the 21st century. According to the projections for the year 2015, approximately 2.3 billion adults will be overweight, and more than 700 million will be obese [1, 2]. Obesity is a chronic and multifactorial disease, whose prevalence is increasing in many countries [3]. For a long time the pharmacological treatment of obesity was seen as a controversial therapeutic option, and subject to considerable criticism. This type of treatment is now being reassessed, especially concerning the emerging concept of long-term anti-obesity medications in conjunction with other therapies for weight loss, or even more importantly, aiming at helping to maintain body weight over an extended period of time [4]. Pharmaceutical strategies in the treatment of obesity include drugs that regulate food intake, thermogenesis, fat absorption, or fat metabolism [5]. Very few drugs of this group are approved for clinical use due to their potential for abuse and dependency [6]. The fenproporex is the second most commonly consumed amphetamine-based anorectic worldwide [7]. It acts by releasing or blocking neuronal reuptake of noradrenaline and dopamine [8, 9]. Monoamine neurotransmitters, such as noradrenaline, dopamine, and serotonin, have been implicated as effectors in the CNS control of energy homeostasis [10]. Initially developed to provide appetite suppression without stimulant effects, fenproporex has since been found to have addictive potential [11]. Although tests are recommended by regulatory agencies worldwide, many of these commercialized drugs have not been investigated regarding their possible genotoxic effect [12]. The studies of these drugs have evaluated the efficacy of reducing body mass, without much interest in their adverse effects. Indiscriminate use associated with increased periods of therapeutic use, suggests that the pattern of use may increase the rates of genetic damage. As increase in genetic damage is positively correlated with carcinogenesis [13], anti-obesity drugs can increase the cancer index in the exposed population, but in turn, overweight or obesity shows an incidence of cancer greater than in persons with a normal body mass index [14, 15].

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Adult (60 days) and young (30 days) male Wistar rats were obtained from Central Animal House of Universidade do Extremo Sul Catarinense. For adult and young rats, we used four groups (three groups were received fenproporex; the control group received vehicle) (n = 5). The animals were caged in groups of five with free access to food and water, maintained on a 12 h light–dark cycle (lights on 7:00 a.m.), at a temperature of 23 ± 1 °C. All experimental procedures were carried out in accordance with the National Institutes of Health Guide for the care and use of laboratory animals and the Brazilian Society for Neuroscience and Behavior (SBNeC) recommendations for animal care, with the approval of UNESC Ethics Committee (protocol number 43/2009). Moreover, all efforts were made to minimize animal suffering, as well as to reduce the number of animals. Acute administration of drugs Animals received a single injection of fenproporex (6.25, 12.5 or 25 mg/kg, intraperitoneal) or Tween 2 % intraperitoneal. The range of doses used was based on previous studies of Rezin et al. [16, 17]. 2 h after the injection, the rats were killed by decapitation and their peripheral blood was removed. Chronic administration of drugs Animals received a single injection daily of fenproporex (6.25, 12.5 or 25 mg/kg, intraperitoneal) or Tween 2 % intraperitoneal for 14 days. The range of doses used was based on previous studies of Rezin et al. [16, 17]. 2 h after the last injection, the rats were killed by decapitation and their peripheral blood was removed. Comet assay The alkaline comet assay in peripheral blood was performed as described by Singh et al. [18], with modifications suggested by Tice et al. [19]. Samples of 5 lL of blood were embedded in 95 lL of 0.75 % low-melting


point agarose, and added to a microscope slide precoated with normal agarose (1.5 % buffer solution). When the agarose solidified, the slides were placed in a lysis buffer (2.5 M NaCl, 100 mM EDTA, and 10 mM Tris; pH 10.0–10.5) containing freshly added 1 % (v/v) Triton X-100 and 10 % (v/v) dimethylsulfoxide (DMSO) for a minimum of 1 h. After treatment with a lysis buffer to allow DNA unwinding, slides were incubated in a freshly made alkaline electrophoresis buffer (0.3 M NaOH and 1 mM EDTA; pH [13) for 20 min in a horizontal electrophoresis tank; DNA was then submitted to electrophoresis for 20 min at 25 V (0.90 V/cm) and 300 mA. Every step was carried out under indirect yellow light. After electrophoresis, slides were washed three times in a neutralization buffer (0.4 M Tris; pH 7.5) for 5 min, rinsed three times in distilled water, and left to dry overnight at room temperature. Slides were stained with silver nitrate, as described previously by Villela et al. [20]; the slides were fixed for 10 min in trichloroacetic acid 15 % w/v, zinc sulfate 5 % w/v, and glycerol 5 % v/v, rinsed three times in distilled water, and dried for 2 h at 37 °C. The dry slides were rehydrated for 5 min in distilled water, stained (sodium carbonate 5 % w/v, ammonium nitrate 0.1 % w/v, silver nitrate 0.1 % w/v, tungstosilicic acid 0.25 %, and formaldehyde 0.15 % w/v, freshly prepared in the dark), and then constantly shaken for 35 min. The stained slides were rinsed twice with distilled water, submerged in the stop solution (acetic acid 1 %), rinsed again, and then immediately coded for analysis with an optic microscope. Images of 100 randomly selected cells were analyzed for each individual. The cells were scored visually into five classes, according to tail size and shape (from undamaged 0 to maximally damaged 4), and a value (damage index) was assigned to each comet according to its class (see Fig. 1). Damage index ranged from 0 (completely undamaged: 100 cells 9 0) to 400 (with maximum damage: 100 cells 9 4. Damage frequency (%) was calculated based on the percentage of damaged cells (0–100 %). International guidelines and recommendations for the comet assay consider visual scoring of comets a well-validated evaluation method. It has a high correlation with computer-based image analysis [21].

Fig. 1 Comet assay for evaluation of DNA damage (9400). The cells are assessed visually and received scores from 0 (undamaged) to 4 (maximally damaged), according to the size and shape of the tails

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Statistical analysis For the analyses of DNA damage parameters, all data were expressed as mean ± SD. Statistical analyses for damage index and damage frequency, measured by comet assay, were carried out using one-way analysis of variance (ANOVA), with non-normal distribution observed; comparisons were made using the Kruskal–Wallis test, with Dunn’s test as post hoc.

Results and discussion Acute administration of 25 mg/kg of fenproporex in young rats presented higher levels of DNA damage in the peripheral blood in both parameters of the comet assay, damage index (Fig. 2a) and damage frequency (Fig. 2b) (P \ 0.05). Acute administrations of 12.5 and 25 mg/kg of fenproporex in adult rats also presented higher levels of DNA damage in the peripheral blood in both parameters, damage index (Fig. 2c) and damage frequency (Fig. 2d) (P \ 0.05). However, chronic administration of fenproporex in young (Fig. 3a, b) and adult (Fig. 3c, d) rats did not alter the levels of DNA damage in the peripheral blood in both parameters of comet assay: damage index and damage frequency (P \ 0.05). Moreira et al. [22] performed a study in mice applying a daily rate dose of 15 mg/kg, and concluded that the drug, at this concentration, exhibits low teratogenic potential. As DNA damage or failure in repair produces mutations which are important for the development of carcinogenesis, this information is very useful in programs of risk evaluation, even though there is no evidence that a high level of damage to white blood cells increases cancer risk [23–26]. Fenproporex is an amphetamine-derived anorectic used in the treatment of obesity [8, 9], and is rapidly converted in vivo into amphetamine [27], which can be detected in urine for up to 60 h after ingestion [28]. Amphetamine screening tests are commonly considered positive if the concentration of this drug in urine exceeds 1.0 ng/mL [7]. Consumption of a low daily dose of 10 mg of fenproporex leads to the detection of amphetamine within 3 h in urine, with peak urinary levels above 4.0 ng/mL [27]. Bengel et al. [29] showed that fenfluramine, an amphetamine analog used in the treatment of obesity is neurotoxic. D-Fenfluramine was observed to have a cytotoxic effect on serotonin transporter, because it caused DNA fragmentation and apoptosis. Another study demonstrated that acute administrations of sibutramine and fenproporex increased the frequency of genotoxic damage in Swiss mice, but did not present cytotoxic effects toward the polychromatic erythrocytes in the bone marrow of these animals [30]. These data corroborate with our findings; acute exposition to an

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Fig. 2 Mean (±SD) values of damage index and damage frequency observed in peripheral blood of young (a, b) and adult (c, d) rats after acute administration of fenproporex. *Different from control group, P \ 0.05 (Kruskal–Wallis, Dunn)

Fig. 3 Mean (±SD) values of damage index and damage frequency observed in peripheral blood of young (a, b) and adult (c, d) rats after chronic administration of fenproporex. *Different from control group, P \ 0.05 (Kruskal–Wallis, Dunn)

amphetamine derivative is able to affect DNA structure. Besides, Andreazza et al. [31] showed that amphetamine increased peripheral DNA damage. DNA damage can include chemical and structural modifications to purine and pyrimidine bases, 20 -deoxyribose and the formation of single- and double-strand breaks. Strand

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breaks within DNA can occur directly, due to damage from reactive oxygen species exposure, or indirectly, due to cleavage of the DNA backbone during DNA base excision repair [32, 33]. A study developed with other psychostimulants, such as cocaine and 3,4-methylenedioxymethamphetamine (MDMA), evaluated genetic damage in several


organs. All doses of these drugs were able to induce DNA damage in blood cells, as well as extensive genotoxic damage in the liver and brain cells [34]. On the other hand, duloxetine administration, a potent inhibitor of serotonin and noradrenaline reuptake with effects on dopamine reuptake (similar to sibutramine mechanism), did not induce DNA damages in the brain and blood [35]. The DNA damage found in these studies and in the present study may be due to oxidative stress. There are several studies demonstrating that amphetamine-derived drugs induce oxidative stress. Oxidative stress may occur by excessive formation of free radicals, which may cause oxidative damages to proteins, lipids, and DNA [30, 36– 39]. Moreover, it is established that the impairment of respiratory chain in mitochondria leads to an increase of free radicals generation, especially superoxide, and that these reactive oxygen species inhibit the mitochondrial respiratory chain, resulting in the generation of more reactive species, forming a cyclic phenomenon [40, 41]. Another study performed by Valvassori et al. [42] showed that D-amphetamine inhibited mitochondrial respiratory chain activity in the brains of adult rats. Moreover, a previous study of our laboratory showed an increase in the brain energy metabolism of young rats by acute and chronic administration of fenproporex [16]. Frenzilli et al. [43] showed that MDMA administration-induced oxidative stress and DNA single- and double-strand breaks. Acute administration of fenproporex caused increase in the damage frequency and damage index of the peripheral blood, and chronic administration of fenproporex did not alter DNA damage parameters. It remains unclear why chronic administration of fenproporex did not cause damage on DNA. We suggest that the activation of an efficient DNA repair mechanism may occur after chronic exposition to fenproporex.

Conclusions Our results showed that an acute administration of fenproporex promoted damage in DNA, in both young and adult rats. We suggest the activation of an efficient DNA repair mechanism that acts after chronic exposition to fenproporex. Our results are consistent with other reports that showed some amphetamine-derived drugs also caused DNA damage. Acknowledgments This research was supported by grants from Programa de Po´s-graduaçaõ em Cieˆncias da Sau´de—Universidade do Extremo Sul Catarinense (UNESC), Nućleo de Exceleˆncia em Neurocieˆncias Aplicadas de Santa Catarina NENASC project, Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (PRONEX— FAPESC/CNPq), and Instituto Nacional de Cieˆncia e Tecnologia Translacional em Medicina (INCT).

175 Conflict of interest

The authors declare no conflict of interest.

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