Clinical and Experimental Pharmacology and Physiology (2014) 41, 265–269
doi: 10.1111/1440-1681.12218
RAPID COMMUNICATION
Cocaine induces DNA damage in distinct brain areas of female rats under different hormonal conditions Marilise F de Souza,* Tierre A Goncßales,* Aline Steinmetz,† Dinara J Moura,† Jenifer Saffi,† Rosane Gomez*‡ and Helena MT Barros* *Laboratory of Behavioral Neuroscience, †Laboratory of Toxicological Genetics, Federal University of Health Sciences of Porto Alegre (UFCSPA), and ‡Department of Pharmacology, Institute of Basic Health Sciences, Federal University of Rio Grande do Sul (UFRGS), Porto Alegre, Brazil
SUMMARY We evaluated levels of neuronal DNA damage after acute or repeated cocaine treatment in different brain areas of female rats after ovariectomy or sham surgery. Rats in the control and acute groups were given saline i.p., whereas in the repeated group were given 15 mg/kg, i.p., cocaine for 8 days. After a 10 day washout period, the control group was given saline i.p., whereas rats in the acute and repeated groups were given a challenge dose of 15 mg/kg, i.p., cocaine. After behavioural assessment, rats were killed and the cerebellum, hippocampus, hypothalamus, prefrontal cortex and striatum were dissected for the Comet assay. Acute cocaine exposure induced DNA damage in all brain areas. This effect persisted after repeated administration, except in the hypothalamus, where repeated treatment did not cause increased DNA damage. Sexual hormones exhibited a neuroprotective effect, decreasing cocaine-induced DNA damage in cycling rats in all brain areas. Key words: cocaine, DNA damage, oestrogen, ovariectomy, progesterone.
INTRODUCTION Hormonal differences between males and females influence cocaine use and abuse. Indeed, females experience more intense behavioural effects of cocaine than males, including cocaine sensitization.1 This phenomenon is defined by the augmented motor stimulant response that occurs with repeated intermittent exposure to a specific drug2 and is a marker of some facets of addiction.3 In female rats, behavioural sensitization to cocaine varies according to hormonal status.4 Oestrogen increases the risk of dependence, whereas progesterone protects females against cocaine dependence.5 Furthermore, oestrogen increases levels of reactive
Correspondence: Marilize F de Souza, Division of Pharmacology, St. Sarmento Leite, 245/317, UFCSPA, 90050-170, Porto Alegre, RS, Brazil. Email: [email protected] Received 31 October 2013; revision 6 February 2014; accepted 9 February 2014. © 2014 Wiley Publishing Asia Pty Ltd
metabolites and reactive oxygen species (ROS) associated with DNA damage,6 but progesterone behaves as an anti-oxidant.7 A single exposure to cocaine induces DNA damage in the brain of male mice.8,9 However, the effects of female hormones on DNA damage in different brain areas after acute or repeated cocaine treatment has not been studied.
RESULTS AND DISCUSSION Overall, we showed that cocaine increased DNA damage in each brain area examined in female rats (Fig. 1). Figure 2 shows representative increased nuclear DNA migration after treatment with cocaine in the hippocampus from ovariectomized (OVX) rats; acute cocaine administration (15 mg/kg, i.p.) significantly increased migration compared with control. The present study evaluated the neurotoxicity of cocaine in female rats, expanding on previous reports by Alvarenga et al.,8,9 who showed that cocaine increases DNA damage in the entire brain of male mice. Cocaine administration generates ROS in the brain10 and induces neuronal cell death.11 Interestingly, although acute cocaine administration produced DNA damage in the hypothalamus of female rats in the present study, repeated administration of 15 mg/kg, i.p., cocaine for 8 days, followed by a 10 day washout period and then challenge with 15 mg/kg, i.p., cocaine left no evidence of DNA damage (Fig. 1). Some have reported that repeated low-dose exposure to direct-acting genotoxins may be tolerated by cells through homeostatic mechanisms, such as DNA repair.12 However, we only found this adaptative mechanism in the hypothalamus of female rats, which may have the most efficient DNA repair compared with other areas of the brain. Coincidently, the hypothalamus is involved in reward and reinforcement processing by regulating dopamine responses to drugs of abuse.13 In this sense, we infer that cocaine sensitization can be correlated with lower DNA damage in female rats in some brain regions. Curiously, a recent study found that repeated cocaine administration induced a rapid increase in the formation and accumulation of new dendritic spines in the frontal cortex of male mice and this phenomenon was correlated with a preference for cocaine.14 Therefore, a greater behavioural response to repeated cocaine exposure, which is strongly related to addiction development, may be due to bigger and better neuronal activity or less cell
266
MF Souza et al.
Fig. 1 The damage index in different brain regions of different groups of ovariectomized (□) and sham-operated (■) rats. CTR, control (saline) treatment; ACT, acute cocaine administration; RPT, repeated cocaine administration. Data are the mean SEM. †P < 0.001 compared with CTR for the same brain region; ‡P < 0.001 compared with the RPT group for the hypothalamus; §P < 0.013 compared with all other brain structures in the RPT group. (Ftreat(2,07) = 81.715, P < 0.001; Fcond(1,07) = 7.945, P = 0.006; Ftreat 9 cond(2,107) = 15.701, P < 0.001; Ftreat 9 reg(8,107) = 2.580, P = 0.013). *P < 0.001, §P < 0.013. Cond, horomonal condition.
(a)
(b)
Fig. 2 Quantification of the DNA damage index by the Comet assay under alkaline conditions in the hippocampus of ovariectomized rats in (a) the saline group and (b) the group treated acutely with cocaine.
damage, at least in the initial phases of drug use. Longer cocaine treatment studies are needed to determine whether this effect is maintained. When we compared hormonal conditions in the control group, cycling females were found to have higher levels of DNA damage than OVX rats in all brain regions examined (Fig. 1), suggesting a crucial role for hormones in the induction of DNA damage in rats that did not receive cocaine. One may propose that although progesterone appears to activate the anti-oxidant defence system,7 oestrogen induces DNA damage by forming reactive metabolites and ROS.6 Therefore, although the levels of
oestrogen and progesterone in sham rats were not significantly higher than in OVX rats (Table 1), a slight increase in both hormones may produce DNA damage. Conversely, when we explored the effect of hormonal conditions on DNA damage in rats that received cocaine, cycling females were found to have a lower DNA damage index than OVX female rats in all brain areas (Fig. 3), suggesting that female gonadal hormones protect against DNA damage induced by cocaine. Evaluation of the phases of the oestrous cycle indicated that rats were cycling normally and were homogeneously distributed among the different cycle phases. In addition, analysis
© 2014 Wiley Publishing Asia Pty Ltd
Cocaine DNA damage in female rat brain Table 1 Oestrogen and progesterone levels in different treatment groups Oestrogen (pg/mL) SHAM CTR 91.2 0.8 Cocaine treatment Acute 113.6 0.5* Repeated 126 14*
Progesterone (ng/mL)
OVX
SHAM
OVX
83.8 2.3
58.2 10.9
39.6 8.9
88.6 1.9 66.0 5.6
45.2 0.8 35.7 0.3
44.1 7.1 38.3 8.2
CTR, saline-treated cocaine group. Data are the mean SEM. *P < 0.001 compared with the ovariectomized (OVX) group (Fint(2,14) = 22.016).
of hormonal levels indicated that blood serum oestrogen concentrations were higher in sham than OVX rats (105 5 vs 79.9 3.5 pg/mL, respectively; F1,18 = 15.61; P < 0.001) but there was no significant difference in progesterone levels between the two groups (46.4 6.0 vs 40.7 5.6 ng/mL, respectively; F1,11 = 0.477; P = 0.504). Analyses of hormonal levels according to treatments are given in Table 1. There was no difference in progesterone and oestrogen levels between sham and OVX rats treated with saline, probably because some of these female rats stayed in the cycle phases in which levels of oestrogen and progesterone are low. However,
Fig. 3 treated treated (1,67) =
267
cocaine treatment, both acute and repeated, increased oestrogen levels in sham-operated rats. A previous study reported that cocaine increases oestrogen levels in intact female rhesus monkeys.15 The effect of female hormones on the incidence of DNA damage is contradictory. Although some studies have shown that increased activity of oxidative enzymes results in an equilibrated redox state and decreased DNA damage,7 others have reported that hormones induce genomic instability.6 However, the results of the present study suggest that primarily oestrogen, but also progesterone or both hormones, may have neuroprotective effects and decrease DNA damage in the neurons of rats exposed to cocaine. The presence of hormones in normally cycling females appears to reduce the incidence of DNA damage induced by cocaine. This result contributes to the hypothesis that greater behavioural sensitization is associated with less DNA damage in female rats and, consequently, a better response in neural pathways involved in cocaine addiction.14 In conclusion, we have shown that cocaine administration increases DNA damage in several distinct brain areas in female rats and that female sexual hormones may act to protect neurons against cocaine damage. Additional studies need to be conducted to clarify whether exogenous administration of oestrogen, progesterone or both would decrease the neurotoxic effects of cocaine.
Damage index in different brain regions in rats treated acutely (□) or repeatedly (■) with cocaine, as a percentage change relative to the salinecontrol (CTR) group, following ovariectomy (OVX) or sham operation. Data are the mean SEM. *P < 0.001 compared with the repeatedly group for the same brain region; †P < 0.001 compared with the sham group for the same brain region (Ftreat(1,67) = 23.496, P < 0.001; Fcond 144.644, P < 0.001; Fcond(1,67) = 144.644).
© 2014 Wiley Publishing Asia Pty Ltd
MF Souza et al.
268 METHODS
The present study was approved by the Ethics Committee of the Federal University of Health Sciences of Porto Alegre (approval no. 1034/10) and was performed in adult female Wistar rats that were either OVX or sham operated (n = 4–5 per group). Rats were allowed to recover for at least 10 days after surgery and then divided into three experimental groups: (i) saline control; (ii) acute cocaine; and (iii) repeated cocaine. The sensitization protocol16 was performed with the control and acute administration groups receiving 1 mL/kg saline, i.p., whereas the repeated administration group received 15 mg/kg, i.p., cocaine hydrochloride for 8 consecutive days. After a 10 day washout period, the acute and repeated administration groups received a cocaine challenge dose (15 mg/ kg, i.p.), whereas rats in the control group received 1 mL/kg, i.p., saline. The oestrous cycle was monitored by examining the cellular characteristics of vaginal smears collected daily. Rats were killed by decapitation and trunk blood was collected and the serum used for analysis of hormone levels. Oestradiol (reference range 20– 3200 pg/mL) and progesterone (reference range 0.2–40 ng/mL) concentrations were determined by ELISA using commercial reagents (Diagnostics Biochem Canada ON, Canada) and Symbiosys (Sa~o Paulo, Brazil), respectively).
Immediately after rats had been killed, the cerebellum, hippocampus, hypothalamus, prefrontal cortex and striatum were dissected and frozen. These regions were selected because they are involved in drug addiction.1,17–19 The prefrontal cortex and striatum are a part of the limbic system,2 whereas the hippocampus is related to memory.17 Conversely, the hypothalamus commands the autonomic nervous system and endocrine responses during motivated behaviour,18 and the cerebellum is involved in the performance of cognitive tasks that require explicit and episodic memory.19 A single-cell suspension from different brains areas was obtained according to the method described by Hartmann et al.20 Briefly, 0.2 g each brain area was placed in a separate microtube with 1 mL chilled solution (phosphate-buffered saline (PBS) with 20 mmol/L EDTA and 10% dimethylsulphoxide (DMSO)) and chopped into pieces using scissors. The pieces were allowed to settle and the supernatant containing single cells was collected. The isolated cells were counted in a Neubauer chamber to determine cell concentration. The Comet assay was performed as reported by Singh et al.21 and Hartmann and Speit22 and shown in Fig. 4. Isolated cells (~ 104 cells/mL) were mixed with low melting point agarose solution and spread on agarose-precoated
Fig. 4 Representation of the Comet assay. PBS, phosphate-buffered saline; LMP, low melting point; DMSO, dimethylsulphoxide.
© 2014 Wiley Publishing Asia Pty Ltd
Cocaine DNA damage in female rat brain microscope slides for the Comet assay. For each treatment, three slides were made of the different brain areas. Slides were incubated in ice-cold lysis solution (2.5 mol/L NaCl, 10 mmol/L Tris, 100 mmol/L EDTA, 1% Triton X-100 and 10% DMSO, pH 10.0) at 4°C for at least 1 h to remove cell membranes. Then, slides were placed in a horizontal electrophoresis unit and incubated with fresh buffer solution (300 mmol/L NaOH, 1 mmol/L EDTA, pH 13.0) at 4°C for 15 min to allow DNA unwinding and the expression of alkali-labile sites. Electrophoresis was conducted for 15 min at 25 V (94 V/cm). All these steps were performed under yellow light or in the dark to prevent additional DNA damage. Slides were stained using silver nitrate. One hundred cells from each treatment were selected and analysed for DNA migration and the average of the three slides from each treatment group was used to determine the damage index. The damage index is an arbitrary score calculated for cells in different damage classes, which are scored visually according to the tail length of the “comet” into five classes: Class 0, undamaged, without a tail; Class 1, with a tail shorter than the diameter of the head nucleus; Class 2, with a tail length one- to twofold greater than the diameter of the head; Class 3, with a tail longer than twofold the diameter of the head; and Class 4, comets with no heads. The damage index ranges from 0 (no tail; 100 cells 9 0) to 400 (maximum migration: 100 cells 9 4). Two- or three-way ANOVA followed by a Tukey post hoc test was used to determine the significance of differences (P < 0.05) in the DNA damage index; cocaine treatment, hormonal condition and different brain areas were considered as independent variables. One- or two-way ANOVA followed by a Tukey post hoc test was used to determine the significance of differences (P < 0.05) in progesterone and oestrogen levels in female rats according to cocaine treatment and hormonal condition.
ACKNOWLEDGEMENTS
4.
5.
6. 7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
This project was financed by Coordination of Improvement of Higher Education Personnel (CAPES), National Council for Scientific and Technological Development (CNPq) and Federal University of Health Sciences of Porto Alegre (UFCSPA).
17. 18.
DISCLOSURE 19.
The authors declare no conflicts of interest.
20.
REFERENCES 1. Becker JB, Hu M. Sex differences in drug abuse. Front. Neuroendocrinol. 2008; 29: 36–47. 2. Steketee JD, Kalivas PW. Drug wanting. Behavioral sensitization and relapse to drug-seeking behavior. Pharmacol. Rev. 2011; 63: 348–65. 3. Vanderschuren LJ, Kalivas PW. Alterations in dopaminergic and glutamatergic transmission in the induction and expression of
21.
22.
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behavioral sensitization: a critical review of preclinical studies. Psychopharmacology 2000; 151: 99–120. Sell SL, Thomas ML, Cunningham KA. Influence of estrous cycle and estradiol on behavioral sensitization to cocaine in female rats. Drug Alcohol Depen. 2002; 67: 281–90. Fox HC, Sofuoglu M, Morgan PT, Tuit KL, Sinha R. The effects of exogenous progesterone on drug craving and stress arousal in cocaine dependence: Impact of gender and cue type. Psychoneuroendocrinology 2013; 29: 456–8. Roy D, Liehr JG. Estrogen, DNA damage and mutations. Mutat. Res. 1999; 424: 107–15. Verma Y, Rana SVS. Modulation of phase-II enzyme activities in benzene treated ovariectomized rats. Environ. Toxicol. Pharmacol. 2011; 31: 371–7. Alvarenga TA, Andersen ML, Ribeiro DA et al. Single exposure to cocaine or ecstasy induces DNA damage in brain and other organs of mice. Addict. Biol. 2009; 15: 96–9. Alvarenga TA, Ribeiro DA, Araujo P et al. Sleep loss and acute drug abuse can induce DNA damage in multiple organs of mice. Hum. Exp. Toxicol. 2011; 30: 1275–81. Dietrich JB, Mangeol A, Revel MO, Burgun C, Aunis D, Zwiller J. Acute or repeated cocaine administration generates reactive oxygen species and induces antioxidant enzyme activity in dopaminergic rat brain structures. Neuropharmacology 2005; 48: 965–74. Lepsch LB, Munhoz CD, Kawamoto EM et al. Cocaine induces cell death and activates the transcription nuclear factor kappa-b in pc12 cells. Mol. Brain 2009; 2: 1–15. Jenkins GJ, Za€ır Z, Johnson GE, Doak SH. Genotoxic thresholds, DNA repair, and susceptibility in human populations. Toxicology 2010; 278: 305–10. Calipari ES, Espa~na RA. Hypocretin/orexin regulation of dopamine signaling: Implications for reward and reinforcement mechanisms. Front. Behav. Neurosci. 2012; 6: 1–13. Mu~noz-Cuevas FJ, Athilingam J, Piscopo D, Wilbrecht L. Cocaineinduced structural plasticity in frontal cortex correlates with conditioned place preference. Nat. Neurosci. 2013; 16: 1367–9. Mello NK, Mendelson JH, Negus SS, Kelly M, Knudson I, Roth ME. The effects of cocaine on gonadal steroid hormones and LH in male and female rhesus monkeys. Neuropsychopharmacology 2004; 29: 2024–34. Scheggi S, Raone A, De Montis MG, Tagliamonte A, Gambarana C. Behavioral expression of cocaine sensitization in rats is accompanied by a distinct pattern of modifications in the PKA/DARPP-32 signaling pathway. J. Neurochem. 2007; 103: 1168–83. Koob G, Volkow ND. Neurocircuitry of addiction. Neuropsychopharmacol. Rev. 2010; 35: 217–38. Vasconcelos SMM, Feitosa LB, Felix PA, Aguiar LMV, Sousa FCF, Viana GSB. Motivacß~ao, vias neuronais e drogas de abuso. Rev. Psiq. Clın. 2002; 29: 130–4. Grant S, London ED, Newlin DB et al. Activation of memory circuits during cue-elicited cocaine craving. Proc. Natl Acad. Sci. USA 1996; 93: 12040–5. Hartmann A, Agurell E, Beevers C et al. Recommendations for conducting the in vivo alkaline Comet assay. Mutagenesis 2003; 18: 45–51. Singh NP, McCoy MT, Tice RR, Scheider EL. A simple technique for quantification of low levels of DNA damage in individual cells. Exp. Cell Res. 1988; 175: 184–91. Hartmann A, Speit G. The contribution of cytotoxicity to DNAeffects in the single cell gel test (Comet assay). Toxicol. Lett. 1997; 90: 183–8.
© 2014 Wiley Publishing Asia Pty Ltd
doi: 10.1111/1440-1681.12218
RAPID COMMUNICATION
Cocaine induces DNA damage in distinct brain areas of female rats under different hormonal conditions Marilise F de Souza,* Tierre A Goncßales,* Aline Steinmetz,† Dinara J Moura,† Jenifer Saffi,† Rosane Gomez*‡ and Helena MT Barros* *Laboratory of Behavioral Neuroscience, †Laboratory of Toxicological Genetics, Federal University of Health Sciences of Porto Alegre (UFCSPA), and ‡Department of Pharmacology, Institute of Basic Health Sciences, Federal University of Rio Grande do Sul (UFRGS), Porto Alegre, Brazil
SUMMARY We evaluated levels of neuronal DNA damage after acute or repeated cocaine treatment in different brain areas of female rats after ovariectomy or sham surgery. Rats in the control and acute groups were given saline i.p., whereas in the repeated group were given 15 mg/kg, i.p., cocaine for 8 days. After a 10 day washout period, the control group was given saline i.p., whereas rats in the acute and repeated groups were given a challenge dose of 15 mg/kg, i.p., cocaine. After behavioural assessment, rats were killed and the cerebellum, hippocampus, hypothalamus, prefrontal cortex and striatum were dissected for the Comet assay. Acute cocaine exposure induced DNA damage in all brain areas. This effect persisted after repeated administration, except in the hypothalamus, where repeated treatment did not cause increased DNA damage. Sexual hormones exhibited a neuroprotective effect, decreasing cocaine-induced DNA damage in cycling rats in all brain areas. Key words: cocaine, DNA damage, oestrogen, ovariectomy, progesterone.
INTRODUCTION Hormonal differences between males and females influence cocaine use and abuse. Indeed, females experience more intense behavioural effects of cocaine than males, including cocaine sensitization.1 This phenomenon is defined by the augmented motor stimulant response that occurs with repeated intermittent exposure to a specific drug2 and is a marker of some facets of addiction.3 In female rats, behavioural sensitization to cocaine varies according to hormonal status.4 Oestrogen increases the risk of dependence, whereas progesterone protects females against cocaine dependence.5 Furthermore, oestrogen increases levels of reactive
Correspondence: Marilize F de Souza, Division of Pharmacology, St. Sarmento Leite, 245/317, UFCSPA, 90050-170, Porto Alegre, RS, Brazil. Email: [email protected] Received 31 October 2013; revision 6 February 2014; accepted 9 February 2014. © 2014 Wiley Publishing Asia Pty Ltd
metabolites and reactive oxygen species (ROS) associated with DNA damage,6 but progesterone behaves as an anti-oxidant.7 A single exposure to cocaine induces DNA damage in the brain of male mice.8,9 However, the effects of female hormones on DNA damage in different brain areas after acute or repeated cocaine treatment has not been studied.
RESULTS AND DISCUSSION Overall, we showed that cocaine increased DNA damage in each brain area examined in female rats (Fig. 1). Figure 2 shows representative increased nuclear DNA migration after treatment with cocaine in the hippocampus from ovariectomized (OVX) rats; acute cocaine administration (15 mg/kg, i.p.) significantly increased migration compared with control. The present study evaluated the neurotoxicity of cocaine in female rats, expanding on previous reports by Alvarenga et al.,8,9 who showed that cocaine increases DNA damage in the entire brain of male mice. Cocaine administration generates ROS in the brain10 and induces neuronal cell death.11 Interestingly, although acute cocaine administration produced DNA damage in the hypothalamus of female rats in the present study, repeated administration of 15 mg/kg, i.p., cocaine for 8 days, followed by a 10 day washout period and then challenge with 15 mg/kg, i.p., cocaine left no evidence of DNA damage (Fig. 1). Some have reported that repeated low-dose exposure to direct-acting genotoxins may be tolerated by cells through homeostatic mechanisms, such as DNA repair.12 However, we only found this adaptative mechanism in the hypothalamus of female rats, which may have the most efficient DNA repair compared with other areas of the brain. Coincidently, the hypothalamus is involved in reward and reinforcement processing by regulating dopamine responses to drugs of abuse.13 In this sense, we infer that cocaine sensitization can be correlated with lower DNA damage in female rats in some brain regions. Curiously, a recent study found that repeated cocaine administration induced a rapid increase in the formation and accumulation of new dendritic spines in the frontal cortex of male mice and this phenomenon was correlated with a preference for cocaine.14 Therefore, a greater behavioural response to repeated cocaine exposure, which is strongly related to addiction development, may be due to bigger and better neuronal activity or less cell
266
MF Souza et al.
Fig. 1 The damage index in different brain regions of different groups of ovariectomized (□) and sham-operated (■) rats. CTR, control (saline) treatment; ACT, acute cocaine administration; RPT, repeated cocaine administration. Data are the mean SEM. †P < 0.001 compared with CTR for the same brain region; ‡P < 0.001 compared with the RPT group for the hypothalamus; §P < 0.013 compared with all other brain structures in the RPT group. (Ftreat(2,07) = 81.715, P < 0.001; Fcond(1,07) = 7.945, P = 0.006; Ftreat 9 cond(2,107) = 15.701, P < 0.001; Ftreat 9 reg(8,107) = 2.580, P = 0.013). *P < 0.001, §P < 0.013. Cond, horomonal condition.
(a)
(b)
Fig. 2 Quantification of the DNA damage index by the Comet assay under alkaline conditions in the hippocampus of ovariectomized rats in (a) the saline group and (b) the group treated acutely with cocaine.
damage, at least in the initial phases of drug use. Longer cocaine treatment studies are needed to determine whether this effect is maintained. When we compared hormonal conditions in the control group, cycling females were found to have higher levels of DNA damage than OVX rats in all brain regions examined (Fig. 1), suggesting a crucial role for hormones in the induction of DNA damage in rats that did not receive cocaine. One may propose that although progesterone appears to activate the anti-oxidant defence system,7 oestrogen induces DNA damage by forming reactive metabolites and ROS.6 Therefore, although the levels of
oestrogen and progesterone in sham rats were not significantly higher than in OVX rats (Table 1), a slight increase in both hormones may produce DNA damage. Conversely, when we explored the effect of hormonal conditions on DNA damage in rats that received cocaine, cycling females were found to have a lower DNA damage index than OVX female rats in all brain areas (Fig. 3), suggesting that female gonadal hormones protect against DNA damage induced by cocaine. Evaluation of the phases of the oestrous cycle indicated that rats were cycling normally and were homogeneously distributed among the different cycle phases. In addition, analysis
© 2014 Wiley Publishing Asia Pty Ltd
Cocaine DNA damage in female rat brain Table 1 Oestrogen and progesterone levels in different treatment groups Oestrogen (pg/mL) SHAM CTR 91.2 0.8 Cocaine treatment Acute 113.6 0.5* Repeated 126 14*
Progesterone (ng/mL)
OVX
SHAM
OVX
83.8 2.3
58.2 10.9
39.6 8.9
88.6 1.9 66.0 5.6
45.2 0.8 35.7 0.3
44.1 7.1 38.3 8.2
CTR, saline-treated cocaine group. Data are the mean SEM. *P < 0.001 compared with the ovariectomized (OVX) group (Fint(2,14) = 22.016).
of hormonal levels indicated that blood serum oestrogen concentrations were higher in sham than OVX rats (105 5 vs 79.9 3.5 pg/mL, respectively; F1,18 = 15.61; P < 0.001) but there was no significant difference in progesterone levels between the two groups (46.4 6.0 vs 40.7 5.6 ng/mL, respectively; F1,11 = 0.477; P = 0.504). Analyses of hormonal levels according to treatments are given in Table 1. There was no difference in progesterone and oestrogen levels between sham and OVX rats treated with saline, probably because some of these female rats stayed in the cycle phases in which levels of oestrogen and progesterone are low. However,
Fig. 3 treated treated (1,67) =
267
cocaine treatment, both acute and repeated, increased oestrogen levels in sham-operated rats. A previous study reported that cocaine increases oestrogen levels in intact female rhesus monkeys.15 The effect of female hormones on the incidence of DNA damage is contradictory. Although some studies have shown that increased activity of oxidative enzymes results in an equilibrated redox state and decreased DNA damage,7 others have reported that hormones induce genomic instability.6 However, the results of the present study suggest that primarily oestrogen, but also progesterone or both hormones, may have neuroprotective effects and decrease DNA damage in the neurons of rats exposed to cocaine. The presence of hormones in normally cycling females appears to reduce the incidence of DNA damage induced by cocaine. This result contributes to the hypothesis that greater behavioural sensitization is associated with less DNA damage in female rats and, consequently, a better response in neural pathways involved in cocaine addiction.14 In conclusion, we have shown that cocaine administration increases DNA damage in several distinct brain areas in female rats and that female sexual hormones may act to protect neurons against cocaine damage. Additional studies need to be conducted to clarify whether exogenous administration of oestrogen, progesterone or both would decrease the neurotoxic effects of cocaine.
Damage index in different brain regions in rats treated acutely (□) or repeatedly (■) with cocaine, as a percentage change relative to the salinecontrol (CTR) group, following ovariectomy (OVX) or sham operation. Data are the mean SEM. *P < 0.001 compared with the repeatedly group for the same brain region; †P < 0.001 compared with the sham group for the same brain region (Ftreat(1,67) = 23.496, P < 0.001; Fcond 144.644, P < 0.001; Fcond(1,67) = 144.644).
© 2014 Wiley Publishing Asia Pty Ltd
MF Souza et al.
268 METHODS
The present study was approved by the Ethics Committee of the Federal University of Health Sciences of Porto Alegre (approval no. 1034/10) and was performed in adult female Wistar rats that were either OVX or sham operated (n = 4–5 per group). Rats were allowed to recover for at least 10 days after surgery and then divided into three experimental groups: (i) saline control; (ii) acute cocaine; and (iii) repeated cocaine. The sensitization protocol16 was performed with the control and acute administration groups receiving 1 mL/kg saline, i.p., whereas the repeated administration group received 15 mg/kg, i.p., cocaine hydrochloride for 8 consecutive days. After a 10 day washout period, the acute and repeated administration groups received a cocaine challenge dose (15 mg/ kg, i.p.), whereas rats in the control group received 1 mL/kg, i.p., saline. The oestrous cycle was monitored by examining the cellular characteristics of vaginal smears collected daily. Rats were killed by decapitation and trunk blood was collected and the serum used for analysis of hormone levels. Oestradiol (reference range 20– 3200 pg/mL) and progesterone (reference range 0.2–40 ng/mL) concentrations were determined by ELISA using commercial reagents (Diagnostics Biochem Canada ON, Canada) and Symbiosys (Sa~o Paulo, Brazil), respectively).
Immediately after rats had been killed, the cerebellum, hippocampus, hypothalamus, prefrontal cortex and striatum were dissected and frozen. These regions were selected because they are involved in drug addiction.1,17–19 The prefrontal cortex and striatum are a part of the limbic system,2 whereas the hippocampus is related to memory.17 Conversely, the hypothalamus commands the autonomic nervous system and endocrine responses during motivated behaviour,18 and the cerebellum is involved in the performance of cognitive tasks that require explicit and episodic memory.19 A single-cell suspension from different brains areas was obtained according to the method described by Hartmann et al.20 Briefly, 0.2 g each brain area was placed in a separate microtube with 1 mL chilled solution (phosphate-buffered saline (PBS) with 20 mmol/L EDTA and 10% dimethylsulphoxide (DMSO)) and chopped into pieces using scissors. The pieces were allowed to settle and the supernatant containing single cells was collected. The isolated cells were counted in a Neubauer chamber to determine cell concentration. The Comet assay was performed as reported by Singh et al.21 and Hartmann and Speit22 and shown in Fig. 4. Isolated cells (~ 104 cells/mL) were mixed with low melting point agarose solution and spread on agarose-precoated
Fig. 4 Representation of the Comet assay. PBS, phosphate-buffered saline; LMP, low melting point; DMSO, dimethylsulphoxide.
© 2014 Wiley Publishing Asia Pty Ltd
Cocaine DNA damage in female rat brain microscope slides for the Comet assay. For each treatment, three slides were made of the different brain areas. Slides were incubated in ice-cold lysis solution (2.5 mol/L NaCl, 10 mmol/L Tris, 100 mmol/L EDTA, 1% Triton X-100 and 10% DMSO, pH 10.0) at 4°C for at least 1 h to remove cell membranes. Then, slides were placed in a horizontal electrophoresis unit and incubated with fresh buffer solution (300 mmol/L NaOH, 1 mmol/L EDTA, pH 13.0) at 4°C for 15 min to allow DNA unwinding and the expression of alkali-labile sites. Electrophoresis was conducted for 15 min at 25 V (94 V/cm). All these steps were performed under yellow light or in the dark to prevent additional DNA damage. Slides were stained using silver nitrate. One hundred cells from each treatment were selected and analysed for DNA migration and the average of the three slides from each treatment group was used to determine the damage index. The damage index is an arbitrary score calculated for cells in different damage classes, which are scored visually according to the tail length of the “comet” into five classes: Class 0, undamaged, without a tail; Class 1, with a tail shorter than the diameter of the head nucleus; Class 2, with a tail length one- to twofold greater than the diameter of the head; Class 3, with a tail longer than twofold the diameter of the head; and Class 4, comets with no heads. The damage index ranges from 0 (no tail; 100 cells 9 0) to 400 (maximum migration: 100 cells 9 4). Two- or three-way ANOVA followed by a Tukey post hoc test was used to determine the significance of differences (P < 0.05) in the DNA damage index; cocaine treatment, hormonal condition and different brain areas were considered as independent variables. One- or two-way ANOVA followed by a Tukey post hoc test was used to determine the significance of differences (P < 0.05) in progesterone and oestrogen levels in female rats according to cocaine treatment and hormonal condition.
ACKNOWLEDGEMENTS
4.
5.
6. 7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
This project was financed by Coordination of Improvement of Higher Education Personnel (CAPES), National Council for Scientific and Technological Development (CNPq) and Federal University of Health Sciences of Porto Alegre (UFCSPA).
17. 18.
DISCLOSURE 19.
The authors declare no conflicts of interest.
20.
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