Biol Trace Elem Res DOI 10.1007/s12011-015-0399-z
Zinc Chloride and Lead Acetate-Induced Passive Avoidance Memory Retention Deficits Reversed by Nicotine and Bucladesine in Mice Kaveh Tabrizian 1,2 & Abdolmajid Yazdani 1 & Behnam Baheri 1 & Borna Payandemehr 3 & Mehdi Sanati 3 & Mahmoud Hashemzaei 1 & Abdolhossein Miri 2,4 & Majid Zandkarimi 5 & Maryam Belaran 6 & Sahar Fanoudi 7 & Mohammad Sharifzadeh 3
Received: 22 March 2015 / Accepted: 3 June 2015 # Springer Science+Business Media New York 2015
Abstract It is very important to investigate the neurotoxic effects of metals on learning and memory processes. In this study, we tried to investigate the effects and time course properties of oral administration of zinc chloride (25, 50, and 75 mg/kg, for 2 weeks), lead acetate (250, 750, 1,500, and 2, 500 ppm for 4, 6 and 8 weeks), and their possible mechanisms on a model of memory function. For this matter, we examined the intra-peritoneal injections of nicotine (0.25, 0.5, 1, and 1.5 mg/kg) and bucladesine (50, 100, 300, and 600 nM/ mouse) for 4 days alone and in combination with mentioned metals in the step-through passive avoidance task. Control animals received saline, drinking water, saline, and DMSO (dimethyl sulfoxide)/deionized water (1:9), respectively. At the end of each part of studies, animals were trained for 1 day in step-through task. The avoidance memory retention
alterations were evaluated 24 and 48 h later in singular and combinational studies. Zinc chloride (75 mg/kg) oral gavage for 2 weeks decreased latency times compared to control animals. Also, lead acetate (750 ppm oral administrations for 8 weeks) caused significant lead blood levels and induced avoidance memory retention impairments. Four-days intraperitoneal injection of nicotine (1 mg/kg) increased latency time compared to control animals. Finally, findings of this research showed that treatment with intra-peritoneal injections of nicotine (1 mg/kg) and/or bucladesine (600 nM/mouse) reversed zinc chloride- and lead acetate-induced avoidance memory retention impairments. Taken together, these results showed the probable role of cholinergic system and protein kinase A pathways in zinc chloride- and lead acetate-induced avoidance memory alterations.
* Mohammad Sharifzadeh [email protected]
Keywords Zinc chloride . Lead acetate . Nicotine . Bucladesine . Avoidance memory
1
2
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Zabol University of Medical Sciences, Zabol, Iran Medicinal Plants Research Center, Zabol University of Medical Sciences, Zabol, Iran
3
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran, Iran
4
Department of Pharmacognosy, Faculty of Pharmacy, Zabol University of Medical Sciences, Zabol, Iran
5
Department of Pharmaceutics, Faculty of Pharmacy, Zabol University of Medical Sciences, Zabol, Iran
6
Department of Physiology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
7
Department of Pharmacology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
Introduction According to considerable body of evidence, exposure to high concentrations of heavy metals induces neurotoxic effects in mammals [1]. Understanding of the associated mechanistic effects of lead and zinc on learning and memory alterations in central nervous system (CNS) is very important to be investigated [2–5]. From such experimental studies, exposure to lead (Pb) can cause serious neurotoxic effects at the molecular [6], cellular [7], and behavioral levels [8, 9]. On the other hand, there are reports about the memory impairments, apoptosis, and cytotoxicity induced by excess levels of zinc consumption [10]. Therefore, its balanced concentration is required for normal behavior, CNS development, and
Tabrizian et al.
preventing of neurological disorders such as Alzheimer’s disease [10, 11]. The critical role of acetylcholine in learning, memory, and attention processes has been shown by several investigators [12–14]. Various behavioral and molecular approaches have focused on the involvement of neuronal nicotinic acetylcholine receptor in memory and cognition processes [15, 16]. Protective role of nicotine on memory impairments induced by different brain lesions has been shown in numerous experimental studies [15, 17]. cAMP-dependent protein kinase A (PKA) is activated by cyclic adenosine 3′,5′-monophosphate (cAMP) as an intracellular second messenger and has an important role in the induction of different phases of memory formation [15, 18–20]. PKA has an important role in phosphorylation of downstream targets such as regulatory and transcription factors. Regulations of activity-dependent changes in neural morphology and transcription are modulated by ERK (extracellular signal-regulated kinase) and phosphorylation of transcription factors such as CREB (cAMP response-element binding protein), respectively [15, 21, 22]. Taking all these together, there is no study on the effect of zinc and lead on memory changes in avoidance memory retention and their possible mechanisms. This experimental study was designed for the first time to evaluate the effects of intra-peritoneal injections of bucladesine, a membranepermeable selective activator of PKA, and nicotine on avoidance memory retention alterations induced by zinc chloride and lead acetate in male mice. Step-through passive avoidance learning task was used for training and evaluation of the avoidance memory retention.
Materials and Methods Animals In this experimental study, all animal manipulations were carried out according to the guidelines for the care and use of laboratory animals of Zabol University of Medical Sciences. Mice (20–25 g) from the Faculty of Pharmacy, Zabol University of Medical Sciences were used in this study. All animals were maintained under controlled conditions (12-h light/dark cycle at room temperature of 20–22 °C). They were housed in groups of five per each cage, having free access to food and water. All animal experiments (n=7) were done during the light cycle. Drugs Zinc chloride (ZnCl2) (Merck, Germany) and lead acetate (Merck, Germany) were dissolved in saline (0.9 %) and animal’s drinking water, respectively, to obtain desirable
concentrations. Nicotine and bucladesine (Sigma, St. Louis, MO, USA) were dissolved in saline (0.9 %) and dimethyl sulfoxide (DMSO)/deionized water (1:9), respectively. Step-Through Avoidance Learning Task The details of the passive avoidance apparatus was described in previous studies [23]. In this study, each animal was gently placed in the light chamber, the door was opened after 10 s and the time that animal waited before crossing to the dark chamber was recorded as the latency (300 s was determined as a cut-off point of study). Electric shocks (0.2-mA intensity for 2 s) were delivered to the grid floor of the dark compartment. All training and testing trials were carried out at a similar time in each day. During the retention test sessions, no electric shock was applied. Experiments Experiment 1 Part A: Fresh solutions of zinc chloride in normal saline were prepared daily. Different concentrations of zinc chloride (25, 50, and 75 mg/kg) were administered via oral gavage needles once a day for two weeks. On day 14th, one training trial was done in step-through avoidance inhibitory learning task. Avoidance memory retentions were evaluated 24 and 48 h later to training trials. Control animals received saline via oral gavage needles for the same period of time. Part B: In another part of this experiment, the time-course (4, 6, and 8 weeks) oral administration of different doses of lead acetate (250, 750, 1,500, and 2,500 ppm concentrations of lead in animal’s drinking water) on avoidance memory retention were conducted in step-through avoidance inhibitory learning task. On the final day of each study, training of animals was done. Avoidance memory retention was tested similar to zinc-treated animals. Control animals received drinking water for the same periods of time.
Experiment 2 The aim of this experiment was to assess the effects of 4 days intra-peritoneal injections of nicotine (0.25, 0.5, 1, and 1.5 mg/kg, Part A) and bucladesine (50, 100, 300, and 600 nM/mouse, Part B) on avoidance memory retention. On the final day of each study (after the last injection), one training trial was done. Avoidance memory retentions were tested 24 and 48 h later to training trials. Control animals received saline and DMSO/deionized water (1:9), respectively.
Zinc Chloride and Lead Acetate-Induced Passive Avoidance Memory
a
Experiment 3 The aim of this experiment was to assess the effects of nicotine (1 mg/kg, i.p. for 4 days) on ZnCl2- (75 mg/kg, oral gavage for 2 weeks; part A) and lead acetate- (750 ppm, oral administration for 8 weeks; part B) induced avoidance memory retention alterations in step-through passive avoidance task. The 4-days intra-peritoneal injections of nicotine were started on days 11th of ZnCl2 and 53th of lead acetate oral administration. On the final day of each study (after the last injection), one training trial was done. Avoidance memory retentions were tested 24 and 48 h later to training trials.
Latency (sec)
300
200
100
0 control
250
750
1500
2500
Lead Acetate concentration (ppm)
b Experiment 4 The aim of this experiment was to assess the effect of bucladesine (600 nM/mouse, i.p. for 4 days) on ZnCl2(75 mg/kg, oral gavage for 2 weeks; part A) and lead acetate- (750 ppm, oral administration for 8 weeks; part B) induced avoidance memory retention alterations in stepthrough avoidance learning task. The 4-days intra-peritoneal injections of bucladesine were started on days 11th of ZnCl2 and 53th of lead acetate treatments. On the final day of each study (after the last injection), one training trial of animals was done. Avoidance memory retentions were tested 24 and 48 h later to training trials.
Latency (sec)
200
*
*
*
100
* 0 control
250
750
1500
2500
Lead Acetate concentration (ppm)
c
Zinc and Lead Assessments in Blood After completion of avoidance memory retention evaluations in step-through passive avoidance task, heart blood was collected in an Eppendorf tube and centrifuged at 3,000 rpm for 10 min. After dilution in distilled deionized water on ratio 1:10, zinc concentration were measured by atomic absorption spectrophotometer (WFX-210, Beijing, China ) at wavelength of 213.9 nm, bounpass of 1 nm, and lamp current of 3 mA [4]. Also, determination of lead in heart blood samples was done by atomic absorption spectrophotometer (WFX-210, Beijing, Table 1 Average of latency time during the retention tests in stepthrough passive avoidance task at different oral doses of ZnCl2 (oral gavages for 2 weeks) Treatment
Latency (sec)
200
*
*
*
1500
2500
100
** 0 control
250
750
Lead Acetate concentration (ppm)
Fig. 1 Time-course effects of pre-training oral administration of different concentrations of lead acetate on latency time in step-through passive avoidance task. Each value represents the mean±S.E.M. of seven mice. Significantly, *p
Zinc Chloride and Lead Acetate-Induced Passive Avoidance Memory Retention Deficits Reversed by Nicotine and Bucladesine in Mice Kaveh Tabrizian 1,2 & Abdolmajid Yazdani 1 & Behnam Baheri 1 & Borna Payandemehr 3 & Mehdi Sanati 3 & Mahmoud Hashemzaei 1 & Abdolhossein Miri 2,4 & Majid Zandkarimi 5 & Maryam Belaran 6 & Sahar Fanoudi 7 & Mohammad Sharifzadeh 3
Received: 22 March 2015 / Accepted: 3 June 2015 # Springer Science+Business Media New York 2015
Abstract It is very important to investigate the neurotoxic effects of metals on learning and memory processes. In this study, we tried to investigate the effects and time course properties of oral administration of zinc chloride (25, 50, and 75 mg/kg, for 2 weeks), lead acetate (250, 750, 1,500, and 2, 500 ppm for 4, 6 and 8 weeks), and their possible mechanisms on a model of memory function. For this matter, we examined the intra-peritoneal injections of nicotine (0.25, 0.5, 1, and 1.5 mg/kg) and bucladesine (50, 100, 300, and 600 nM/ mouse) for 4 days alone and in combination with mentioned metals in the step-through passive avoidance task. Control animals received saline, drinking water, saline, and DMSO (dimethyl sulfoxide)/deionized water (1:9), respectively. At the end of each part of studies, animals were trained for 1 day in step-through task. The avoidance memory retention
alterations were evaluated 24 and 48 h later in singular and combinational studies. Zinc chloride (75 mg/kg) oral gavage for 2 weeks decreased latency times compared to control animals. Also, lead acetate (750 ppm oral administrations for 8 weeks) caused significant lead blood levels and induced avoidance memory retention impairments. Four-days intraperitoneal injection of nicotine (1 mg/kg) increased latency time compared to control animals. Finally, findings of this research showed that treatment with intra-peritoneal injections of nicotine (1 mg/kg) and/or bucladesine (600 nM/mouse) reversed zinc chloride- and lead acetate-induced avoidance memory retention impairments. Taken together, these results showed the probable role of cholinergic system and protein kinase A pathways in zinc chloride- and lead acetate-induced avoidance memory alterations.
* Mohammad Sharifzadeh [email protected]
Keywords Zinc chloride . Lead acetate . Nicotine . Bucladesine . Avoidance memory
1
2
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Zabol University of Medical Sciences, Zabol, Iran Medicinal Plants Research Center, Zabol University of Medical Sciences, Zabol, Iran
3
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran, Iran
4
Department of Pharmacognosy, Faculty of Pharmacy, Zabol University of Medical Sciences, Zabol, Iran
5
Department of Pharmaceutics, Faculty of Pharmacy, Zabol University of Medical Sciences, Zabol, Iran
6
Department of Physiology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
7
Department of Pharmacology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
Introduction According to considerable body of evidence, exposure to high concentrations of heavy metals induces neurotoxic effects in mammals [1]. Understanding of the associated mechanistic effects of lead and zinc on learning and memory alterations in central nervous system (CNS) is very important to be investigated [2–5]. From such experimental studies, exposure to lead (Pb) can cause serious neurotoxic effects at the molecular [6], cellular [7], and behavioral levels [8, 9]. On the other hand, there are reports about the memory impairments, apoptosis, and cytotoxicity induced by excess levels of zinc consumption [10]. Therefore, its balanced concentration is required for normal behavior, CNS development, and
Tabrizian et al.
preventing of neurological disorders such as Alzheimer’s disease [10, 11]. The critical role of acetylcholine in learning, memory, and attention processes has been shown by several investigators [12–14]. Various behavioral and molecular approaches have focused on the involvement of neuronal nicotinic acetylcholine receptor in memory and cognition processes [15, 16]. Protective role of nicotine on memory impairments induced by different brain lesions has been shown in numerous experimental studies [15, 17]. cAMP-dependent protein kinase A (PKA) is activated by cyclic adenosine 3′,5′-monophosphate (cAMP) as an intracellular second messenger and has an important role in the induction of different phases of memory formation [15, 18–20]. PKA has an important role in phosphorylation of downstream targets such as regulatory and transcription factors. Regulations of activity-dependent changes in neural morphology and transcription are modulated by ERK (extracellular signal-regulated kinase) and phosphorylation of transcription factors such as CREB (cAMP response-element binding protein), respectively [15, 21, 22]. Taking all these together, there is no study on the effect of zinc and lead on memory changes in avoidance memory retention and their possible mechanisms. This experimental study was designed for the first time to evaluate the effects of intra-peritoneal injections of bucladesine, a membranepermeable selective activator of PKA, and nicotine on avoidance memory retention alterations induced by zinc chloride and lead acetate in male mice. Step-through passive avoidance learning task was used for training and evaluation of the avoidance memory retention.
Materials and Methods Animals In this experimental study, all animal manipulations were carried out according to the guidelines for the care and use of laboratory animals of Zabol University of Medical Sciences. Mice (20–25 g) from the Faculty of Pharmacy, Zabol University of Medical Sciences were used in this study. All animals were maintained under controlled conditions (12-h light/dark cycle at room temperature of 20–22 °C). They were housed in groups of five per each cage, having free access to food and water. All animal experiments (n=7) were done during the light cycle. Drugs Zinc chloride (ZnCl2) (Merck, Germany) and lead acetate (Merck, Germany) were dissolved in saline (0.9 %) and animal’s drinking water, respectively, to obtain desirable
concentrations. Nicotine and bucladesine (Sigma, St. Louis, MO, USA) were dissolved in saline (0.9 %) and dimethyl sulfoxide (DMSO)/deionized water (1:9), respectively. Step-Through Avoidance Learning Task The details of the passive avoidance apparatus was described in previous studies [23]. In this study, each animal was gently placed in the light chamber, the door was opened after 10 s and the time that animal waited before crossing to the dark chamber was recorded as the latency (300 s was determined as a cut-off point of study). Electric shocks (0.2-mA intensity for 2 s) were delivered to the grid floor of the dark compartment. All training and testing trials were carried out at a similar time in each day. During the retention test sessions, no electric shock was applied. Experiments Experiment 1 Part A: Fresh solutions of zinc chloride in normal saline were prepared daily. Different concentrations of zinc chloride (25, 50, and 75 mg/kg) were administered via oral gavage needles once a day for two weeks. On day 14th, one training trial was done in step-through avoidance inhibitory learning task. Avoidance memory retentions were evaluated 24 and 48 h later to training trials. Control animals received saline via oral gavage needles for the same period of time. Part B: In another part of this experiment, the time-course (4, 6, and 8 weeks) oral administration of different doses of lead acetate (250, 750, 1,500, and 2,500 ppm concentrations of lead in animal’s drinking water) on avoidance memory retention were conducted in step-through avoidance inhibitory learning task. On the final day of each study, training of animals was done. Avoidance memory retention was tested similar to zinc-treated animals. Control animals received drinking water for the same periods of time.
Experiment 2 The aim of this experiment was to assess the effects of 4 days intra-peritoneal injections of nicotine (0.25, 0.5, 1, and 1.5 mg/kg, Part A) and bucladesine (50, 100, 300, and 600 nM/mouse, Part B) on avoidance memory retention. On the final day of each study (after the last injection), one training trial was done. Avoidance memory retentions were tested 24 and 48 h later to training trials. Control animals received saline and DMSO/deionized water (1:9), respectively.
Zinc Chloride and Lead Acetate-Induced Passive Avoidance Memory
a
Experiment 3 The aim of this experiment was to assess the effects of nicotine (1 mg/kg, i.p. for 4 days) on ZnCl2- (75 mg/kg, oral gavage for 2 weeks; part A) and lead acetate- (750 ppm, oral administration for 8 weeks; part B) induced avoidance memory retention alterations in step-through passive avoidance task. The 4-days intra-peritoneal injections of nicotine were started on days 11th of ZnCl2 and 53th of lead acetate oral administration. On the final day of each study (after the last injection), one training trial was done. Avoidance memory retentions were tested 24 and 48 h later to training trials.
Latency (sec)
300
200
100
0 control
250
750
1500
2500
Lead Acetate concentration (ppm)
b Experiment 4 The aim of this experiment was to assess the effect of bucladesine (600 nM/mouse, i.p. for 4 days) on ZnCl2(75 mg/kg, oral gavage for 2 weeks; part A) and lead acetate- (750 ppm, oral administration for 8 weeks; part B) induced avoidance memory retention alterations in stepthrough avoidance learning task. The 4-days intra-peritoneal injections of bucladesine were started on days 11th of ZnCl2 and 53th of lead acetate treatments. On the final day of each study (after the last injection), one training trial of animals was done. Avoidance memory retentions were tested 24 and 48 h later to training trials.
Latency (sec)
200
*
*
*
100
* 0 control
250
750
1500
2500
Lead Acetate concentration (ppm)
c
Zinc and Lead Assessments in Blood After completion of avoidance memory retention evaluations in step-through passive avoidance task, heart blood was collected in an Eppendorf tube and centrifuged at 3,000 rpm for 10 min. After dilution in distilled deionized water on ratio 1:10, zinc concentration were measured by atomic absorption spectrophotometer (WFX-210, Beijing, China ) at wavelength of 213.9 nm, bounpass of 1 nm, and lamp current of 3 mA [4]. Also, determination of lead in heart blood samples was done by atomic absorption spectrophotometer (WFX-210, Beijing, Table 1 Average of latency time during the retention tests in stepthrough passive avoidance task at different oral doses of ZnCl2 (oral gavages for 2 weeks) Treatment
Latency (sec)
200
*
*
*
1500
2500
100
** 0 control
250
750
Lead Acetate concentration (ppm)
Fig. 1 Time-course effects of pre-training oral administration of different concentrations of lead acetate on latency time in step-through passive avoidance task. Each value represents the mean±S.E.M. of seven mice. Significantly, *p
