- Email: [email protected]
Effect of melatonin administration on DNA damage and repair responses in lymphocytes of rats subchronically exposed to lead
Mutation Research 742 (2012) 37–42
Contents lists available at SciVerse ScienceDirect
Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Effect of melatonin administration on DNA damage and repair responses in lymphocytes of rats subchronically exposed to lead Minerva Martínez-Alfaro a,∗ , Daniel Hernández-Cortés a , Katarzyna Wrobel b , Gustavo Cruz-Jiménez a , a ˜ Julio Cesar Rivera-Leyva a , Rosa María Pina-Zentella , Alfonso Cárabez Trejo c a
Department of Pharmacy, University of Guanajuato, Guanajuato, México 36050, Mexico Department of Chemistry, University of Guanajuato, Guanajuato, México 36050, Mexico c INB, Mexico b
a r t i c l e
i n f o
Article history: Received 17 June 2011 Received in revised form 17 November 2011 Accepted 21 November 2011 Available online 28 November 2011 Keywords: DNA repair DNA damage Apoptosis Lead acetate Melatonin
a b s t r a c t Lead exposure induces DNA damage, oxidative stress, and apoptosis, and alters DNA repair. We investigated the effects of melatonin co-administered to rats during exposure to lead. Three doses of lead acetate (10, 50 and 100 mg/kg/day) were administered to rats during a 6-week period. Lymphocytes were analyzed. Lead exposure decreased glutathione (GSH) levels in blood, and at doses of 100 mg/kg/day and 50 mg/kg/day without melatonin, caused high levels of DNA damage, induced apoptosis, and altered DNA repair. Melatonin co-treatment did not attenuate the effects of lead at 100 mg/kg/day, indicating that the effect of melatonin on GSH reduction is not sufficient to reduce the genotoxic effects of lead at this high dose. After 6 weeks of treatment, decreased weight gain was observed in high lead-dose groups (100 mg/kg/day), with or without melatonin, and in medium-dose groups (50 mg/kg/day) with melatonin, compared with the control group. The protective action of melatonin against lead toxicity is dependent on the dose of lead. Further pharmacological studies are needed to determine whether melatonin acts via melatonin membrane receptors on lymphocytes. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Lead, used in pipes, paints, lead acid batteries, solders, refining, smelting, and as an additive in gasoline, is found at high concentrations in the environment. Lead induces many adverse health effects, including nephrotoxicity [1], procoagulant activation of erythrocytes [2], behavioral alterations [3], and neurotoxicological effects [4]. One of the suggested molecular mechanisms of lead toxicity is binding to sites in a variety of proteins, thereby competing with endogenous cations, principally calcium and zinc. Biophysical studies have revealed that lead tightly binds zinc and calcium sites and alters the structural conformation and biological activity of the corresponding proteins. The ability of lead to substitute for divalent ions affects many cellular functions [5]. The International Agency for Research on Cancer (IARC) has classified inorganic lead compounds as probable human carcinogens (group 2A) and organic lead compounds as possible human carcinogens (group 2B). Epidemiological studies of lead-exposed workers have shown an excess of renal, lung, stomach, and brain
∗ Corresponding author at: Department of Pharmacy, University of Guanajuato, Guanajuato, México 36050, Mexico. Tel.: +52 4737320006x8031. E-mail address: [email protected] (M. Martínez-Alfaro). 1383-5718/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2011.11.011
cancers [6]. The mean lead concentration in the blood of battery workers (248.3 g/L) was significantly higher than that in control individuals (27.49 g/L) [7]. The genotoxic effects of this exposure include positive results in the micronucleus and comet assays. Consistent with these observations, lead acetate induces DNA damage (both single- and double-strand breaks) as well as DNA–protein crosslinks in human lymphocytes in vitro at 1–10 M concentrations [8]. In another in vitro study on human lymphocytes, lead nitrate induced DNA damage at doses of 2.7 and 3 mM [9]. In mice, lead acetate induces oxidative stress, DNA damage, and apoptosis in vivo [10]. There is little evidence that lead interacts directly with DNA at normal blood lead concentrations [11]. The best documented mechanisms of lead genotoxicity are indirect, and include inhibition of DNA repair and production of free radicals [12]. A recent in vitro study in human cells [13] reported that lead exposure interferes with non-homologous end-joining repair processes by inhibiting DNA-PK kinase activity. Moreover, in AA8 cell extracts, lead induces a dose-dependent inhibition of the activity of AP endonuclease, which is the major endonuclease involved in repairing mutagenic and cytotoxic abasic sites in DNA [14]. The level of DNA damage determines the cellular response to the genotoxic agent [15]. Production of free radicals by lead exposure has been documented both in vivo and in vitro [16,17]. In one such study, lead acetate induced DNA strand breaks in a time- and
38
M. Martínez-Alfaro et al. / Mutation Research 742 (2012) 37–42
dose-dependent manner. Singlet oxygen, generated by Fenton-like reactions, was suggested to be the principal species involved in the DNA breakage. This lead-induced DNA breakage was inhibited by oxygen scavengers [18]. Alteration of antioxidant enzymes by lead exposure is another mechanism suggested for oxidative stress [19,20]. Melatonin, a hormone produced by the pineal gland, has many physiological functions, including chronomodulation of biological systems and regulation of immune functions, acting as either an inhibitor or activator of immune responses [21]. Moreover, melatonin has free radical scavenger and antioxidant chemical properties [22]. Experimental evidence suggests that melatonin exerts a protective action against lead effects. Administration of melatonin in conjunction with lead treatment reduced the hepatic and renal toxicity in rats treated with 100 mg/kg lead for 30 days in vivo [23]. Melatonin protects neuroblastoma cells against lead-induced apoptosis [24], and prevents oxidative DNA damage in blood lymphocytes in vitro by scavenging reactive oxygen species [25]. Despite numerous reports of the beneficial effects of melatonin against lead toxicity, little is known about the influence of melatonin on subchronic lead treatment in vivo. The aim of this study was to investigate the effects of melatonin on DNA damage induced by lead acetate. DNA damage, DNA repair, and apoptosis were assessed in lymphocytes from rats treated for 6 week with three different doses of lead acetate (10, 50 and 100 mg/kg/day) in the presence or absence of melatonin. DNA damage and repair were assayed by single-cell gel electrophoresis (comet assay); apoptosis was assessed by detecting nucleosomal fragments (180–200 bp) using gel electrophoresis. In addition, body weight and blood GSH and lead levels were evaluated. Melatonin co-administration reduced oxidative stress, decreased the level of DNA damage, and had an antiapoptotic effect in lymphocytes from rats treated with 10 or 50 mg/kg/day lead acetate. These effects of melatonin were correlated with the DNA damage-repair capacity of these lymphocytes. However, melatonin co-administration was not able to reduce DNA damage in lymphocytes from rats treated with the 100-mg/kg/day dose of lead, despite similar levels of GSH at 50 and 100 mg/kg/day doses. Moreover, lymphocytes from rats treated with high doses of lead acetate showed a high level of DNA damage and low DNA damage-repair capacity, effects that were likely linked to the presence of apoptosis. Thus, at high lead doses, mechanisms other than oxidative stress probably play a more important role in genotoxicity. The possible involvement of melatonin receptor effects will require further exploration. 2. Materials and methods 2.1. Chemicals The repair endonuclease Fpg was obtained from New England Bio-Labs, DAPI from Vectashield, and Histopaque 1077 and all other compounds from the Sigma Chemical Company (Toluca, México).
2.2. Animals and experimental design Male 5-week-old Sprague-Dawley rats were used in these experiments. Rats were housed in steel cages with an illumination period of 12 h/day, and provided a commercial diet and purified water ad libitum. After a 1-week acclimation period, animals were divided into eight groups (0, 10, 50 or 100 mg Pb/kg body weight ± melatonin) of five rats each; all groups were very similar in age and body weight. The number of animals used was the minimum necessary to obtain relevant results. All animals were handled in accordance with the guidelines of our institute and followed international norms of animal care and maintenance.
2.3. Body weight The body weights of all animals were measured weekly.
2.4. Melatonin administration Melatonin (10 mg/kg body weight) was administered daily to animals by intraperitoneal injection during the designated night period. 2.5. Lead administration Rats were treated orally (gavage) with lead acetate at doses of 0, 10, 50 or 100 mg/kg body weight 5 days a week for 6 weeks. 2.6. Lead quantification For lead determination, internal standard (115 In and 209 Bi), 100 L, was added to blood samples (400 L), and a wet digestion was carried out with concentrated nitric acid (500 L) for 2 h at 120 ◦ C. In the second step, an aliquot of H2 O2 (30%, 200 L) was added and the mixture was again incubated at 120 ◦ C for 1 h. Finally, the sample was cooled to room temperature, the volume was brought to 5 mL with deionized water, and the sample was centrifuged and introduced into an ICP-MS system. Each analysis was carried out in triplicate, and blanks were run in parallel. An inductively coupled plasma mass spectrometer (Model 7500ce; Agilent Technologies, Tokyo, Japan) with a Meinhard nebulizer and Peltier-cooled spray chamber (2 ◦ C) was used. The instrumental operating conditions were as follows: forward power, 1500 W; plasma gas flow rate, 15 L/min; carrier gas flow rate, 0.89 L/min; make-up gas flow rate, 0.15 L/min; sampling depth, 10 mm; dwell time, 300 ms per isotope; platinum sampling and skimmer cones were used. The isotopes 206 Pb, 207 Pb, 208 Pb were monitored and standardized to 115 In and 209 Bi. Calibration was performed with Agilent commercial standards at lead concentrations of 0, 0.2, 0.4, 1.0, 2.0, 5.0 and 10 g/L, with the internal standards, Bi and In (5.0 g/L each). The lead detection limit was 0.02 g/L. 2.7. GSH measurement A GSH assay kit that utilizes an enzymatic recycling method was used to measure GSH levels in pooled lymphocytes (106 cells); GSH reductase was used to quantify GSH. In this method, the sulfhydryl group of GSH reacts with DTNB (5,5 -dithio-bis2-nitrobenzoic acid) and produces a yellow-colored product, 5-thio-2-nitrobenzoic acid (TNB), which is detected by measuring absorbance at 405 nm. The rate of TNB production is directly proportional to the concentration of GSH in the sample. This assay was carried out in accordance with manufacturer’s protocol; samples were first concentrated and deproteinated before assaying. 2.8. Comet assay The comet assay used was based primarily on a previously described protocol [26], with modifications to detect oxidized bases. In brief, four folded comet slides were made for each treatment: one for basal and three for repair treatment; all slides were treated with Fpg formamidopyrimidine (fapy)-DNA glycosylase. The same numbers of slides were made without enzyme. Lymphocytes (20,000) in 10 L PBS with 90% viability (estimated by exclusion of trypan blue) were mixed with 75% agarose, 100 L, and the mixture was transferred onto a frosted slide (ES 370; Erie Scientific) pre-coated with normal-melting agarose (1%). A coverslip was added and the slide was cooled on ice to allow the agarose to harden. After the embedding procedure, three slides were exposed to experimental (DNA-damaging) treatments and one control (no DNA damage) slide. DNA damage was induced by exposing rat lymphocytes to 10 M H2 O2 for 5 min on ice. The initial level of induced DNA damage was estimated immediately after H2 O2 treatment. Following H2 O2 exposure, cells were either placed in lysis buffer (10 mM Tris pH 10, 2.5 M NaCl, 100 mM EDTA, 200 mM NaOH, 1% [v/v] Triton X-100, 10% [v/v] DMSO) for 1 h on ice, or were incubated in RPMI 1640 at 37 ◦ C for 15 or 45 min to allow repair of DNA damage and then transferred to lysis buffer for 1 h at 4 ◦ C. For enzyme treatment, the slides were removed from lysis buffer and incubated with enzyme reaction buffer (40 mM HEPES, 0.1 M KCl, 0.5 mM EDTA, 0.2 mg/mL BSA, adjusted to pH 8 with KOH) for 10 min. Fpg (1:105 dilution, 50 L) was added to the slides and incubated for 35 min at 37 ◦ C. Slides were immersed in cold alkaline unwinding electrophoresis solution (0.3 M NaOH and 1 mM Na2 EDTA in deionized water, pH 13.5) for 30 min and subjected to electrophoresis for 30 min at a constant voltage of 0.74 V/cm. All procedures were carried out in the dark. The slides were stained with DAPI (4 ,6-diamidino-2phenylindole) and analyzed with image analysis software (AutoComet; Tritek Corp.). Triplicate samples, each containing 50 cells, were quantified for each condition. The tail moment was used as a DNA damage parameter. 2.9. DNA repair kinetics DNA repair kinetics were calculated by determining the net number of Fpgsensitive sites induced by H2 O2 treatment over 45 min. The mean Olive tail moment obtained without enzyme treatment was subtracted from values obtained with enzyme incubation under the same experimental conditions (Table 3). OTM is
M. Martínez-Alfaro et al. / Mutation Research 742 (2012) 37–42
Table 1 Mean body weight ± SE in rats at the beginning and end of experimental period (6 weeks). Statistical analysis by Tukey HSD showed a significant difference between control and treatment groups.
2.4 F D
Ln (Tail Moment)
G
1.6 E
B
0.8
39
Condition
Initial weight
Control 10 Pb 10 Pb Me 50 Pb 50 Pb Me 100 Pb 100Pb Me
203 192 204 199 196 194 199
*
C
± ± ± ± ± ± ±
4 4 5 4 5 4 5
Final weight 321 272 303 302 265 242 269
± ± ± ± ± ± ±
Weight gain (g)
15 15 17 13 17* 15* 17*
118 80 99 103 69* 48* 70*
p < 0.05.
A
0.0
0
20
40
Time (min) Fig. 1. Kinetics of DNA repair in lymphocytes exposed in vitro to H2 O2 . Natural logarithm of values from Table 3 was used as Olive tail moment values. Significance of differences in slopes compared to the control group were determined by the Kruskal–Wallis test (p < 0.05).
calculated for the distance in microns between the center of gravity in the head and the center of gravity in the tail, multiplied by the % Tail. M TOlive = (CG − CG)
DNA 100
In order to evaluate kinetic repair log natural of each value from Table 3 was used in Fig. 1. Statistical analyses of DNA repair kinetics were based on statistical differences in slopes, between different experimental conditions by ANOVA and Kruskal–Wallis Tests (Fig. 1). 2.10. DNA laddering DNA fragmentation, in which DNA is cleaved into large fragments (50–200 kb) followed by cleavage into smaller fragments called nucleosomal units (180–200 bp), is a hallmark of apoptosis. The DNase responsible for this DNA cleavage into smaller fragments is a caspase-3-activated DNase. The result of endonuclease digestion manifests as a DNA ladder (multimers of approximately 180–200 bp) in agarose gels. DNA laddering on gels was detected using a DNA ladder isolation kit that separates apoptotic DNA from intact, high molecular weight genomic DNA. The method involves lysis of cells and inactivation of nucleases. 2.11. Statistical analysis Body weight data were analyzed using a matched-pairs test. Dunnet’s Method was used to analyze difference between GSH values. For comet assays, data were expressed as means ± standard errors. Differences with a p-value < 0.05 were considered statistically significant. Calculations were carried out using JMP V.7 Software (SAS Inc., 2008).
3. Results A Tukey HSD analysis showed no statistically significant differences in mean body weight among initial groups. In contrast, final mean body weights and weight gain were significantly lower (p < 0.05) in the groups receiving a high lead acetate dose, with or without melatonin (100 mg/kg/day), or a medium lead dose (50 mg/kg/day) with melatonin (Table 1). The greatest difference after 6 weeks of treatment was between rats in the control group, which reached 158.1% of their initial body weight during treatment (203 g initial mean body weight; 321 g final mean body weight), and rats in the 100 mg/kg/day lead acetate group, which reached 124.7% of their initial body weight (194 g initial mean body weight; 242 g final mean body weight) (Table 1). Blood lead levels in control rats were in the range of 18–22 g/L, whereas in rats treated with 10 mg Pb/kg/day, the lead concentration ranged from 48 to 53 g/L, a level that was not toxic. In contrast, the levels of lead in the blood of animals treated
with higher doses of lead acetate were toxic. In these animals, blood lead level ranges were 125–151 and 192–221 g/L for treatment with 50 and 100 mg Pb/kg/day, respectively. Differences in blood levels between animals treated with or without melatonin (10 mg/kg/day) were not significant. GSH levels in lymphocytes from rats treated (100 mg/kg/day), with or without melatonin, medium dose 50 mg/kg/day without melatonin were significantly different from those in controls. Lymphocytes from control animals and rats treated with low lead dose with or without melatonin and medium dose with melatonin were not significantly different (Table 2). Basal DNA damage in lymphocytes from controls and rats treated with a lead dose of 10 mg/kg/day (i.e., non-toxic levels), with or without melatonin, were similar. In contrast, basal DNA damage in lymphocytes from rats with toxic blood levels (50 and 100 mg/kg/day groups) was significantly higher compared to controls (Table 3). Unexpectedly, the lymphocytes from rats treated with 100 mg/kg/day plus melatonin showed lower initial levels of DNA damage than lymphocytes from rats treated with 50 mg/kg/day plus melatonin. An analysis of tail moments over the 45-min repair period (experimental points 0, 15 and 45 min) showed that DNA damage in lymphocytes decreased with increasing repair time for all experimental conditions (Table 3). A statistical analysis of repair kinetics by linear regression analysis of slopes showed a significant difference between two groups. Specifically, the second group, with subgroups exposed to lead doses of 100 mg/kg/day, with or without melatonin, or 50 mg/kg/day without melatonin, exhibited a significant delay in DNA repair (p < 0.05) compared to the control group, consisting of subgroups exposed to lead doses of 10 mg/kg/day (with and without melatonin) and 50 mg/kg/day with melatonin (Fig. 1). Effect of GSH level on DNA basal damage. Basal DNA damage was correlated with the GSH levels. Effect of GSH level on DNA repair. The rate of DNA damage repair in lymphocytes was correlated on GSH levels for all lead acetate treatments (Fig. 2). Apoptosis.
Table 2 Comparisons of mean GSH levels with those in controls Dunnett’s method. Exposure and co-treatment conditions Control Pb 10 mg/kg/day Pb 10 mg/kg/day and melatonin 10 mg/kg/day Pb 50 mg/kg/day Pb 50 mg/kg/day and melatonin 10 mg/kg/day Pb 100 mg/kg/day Pb 100 mg/kg/day and melatonin 10 mg/kg/day *
p < 0.05.
Mean GSH/106 lymphocytes (mole) 7.56 7.30 7.03 4.25* 6.48 3.60* 4.10*
± ± ± ± ± ± ±
0.6 0.75 0.69 0.46 0.7 0.59 0.56
40
M. Martínez-Alfaro et al. / Mutation Research 742 (2012) 37–42
Table 3 Net Olive tail moments were obtained by subtraction of Olive tail moment values without enzyme treatment from those with enzyme treatment. Net Fpg DNA glycosylasesensitive sites were measured in lymphocytes at a specific time during repair after exposure to 10 M H2 O2 . Values represent the mean and standard deviation (shown in parenthesis; n = 15). Repairing time (min)
Treatments Control
Basal t0 t15 t45 *
1.28(0.17) 2.4(0.43) 1.6(0.31) 1.1(0.21)
Pb10 1.5(0.16) 3.54(0.32) 1.9(0.21) 1.7(0.22)
Pb10Me 1.41(0.18) 3.06(0.28) 1.72(0.23) 1.44(0.23)
Pb50
Pb50Me *
4.5(0.51) 5.28(0.63)* 6.22(0.58)* 6.42(0.66)*
Pb100 *
2.6(0.33) 6.2(0.74)* 4.3(0.25)* 2.84(0.37)*
Pb100Me *
3.06(1.38) 11.5(0.96)* 8.44(0.82)* 8.27(0.80)*
2.46(0.32)* 7.52(0.72)* 6.46(0.65)* 6.16(0.65)*
p < 0.05 for comparisons between treatment and control at specific repair times (Kruskal–Wallis test).
DNA fragmentation was used as an indicator of apoptosis. Lymphocytes from rats receiving a 100-mg/kg/day lead dose, with or without melatonin, and lymphocytes from rats treated with 50 mg Pb/kg/day showed apoptosis (Fig. 3). 4. Discussion The mean body weight gains of rats in groups receiving a high lead dose, with or without melatonin, or a medium lead dose with melatonin during treatment were significantly lower than those in the control group. This effect of lead on body weight is well documented [19]. Under our experimental conditions, only rats administered lead acetate at doses of 50 and 100 mg/kg/day had toxic blood lead levels. The present results show a significant reduction in blood GSH levels in rats treated for 6 weeks with lead acetate at doses of 100 mg/kg/day and 50 mg/kg/day without melatonin. This reduction in GSH levels is an indicator of oxidative stress and is a well-established action of lead [16,17]. Analysis of the high lead acetate dose (100 mg/kg/day) showed a significant reduction in blood GSH levels independent of melatonin co-treatment (Table 2). Considering the mechanisms involved in the antioxidant effects of melatonin, there are two possible explanations for this behavior. First, the scavenger power of melatonin at the dose used (10 mg/kg/day) may be overwhelmed by the oxidative stress induced at the high lead dose. Alternatively, this dose of melatonin may induce insufficient activation of antioxidant enzymes to counteract the oxidative stress induced by the high lead dose. Even though beyond the scope of this paper, elucidating the specific mechanism involved in this response remains an interesting research issue.
Fig. 2. Correlation between GSH level and DNA repair rate. Inverse correlation between GSH plasma level and basal DNA damage (inset).
The data from this study demonstrated DNA damage in the lymphocytes of rats exposed to toxic blood levels of lead; these results are in agreement with other studies [7–9,13]. The initial level of DNA damage in lymphocytes depended on the lead acetate dose and the level of GSH in the blood (inset, Fig. 2). The slope of repair kinetics in lymphocytes (the value used to assess DNA repair) depended on the degree of oxidative stress (GSH level) (Fig. 1). In addition, our results established that melatonin co-treatment for 6 weeks in vivo significantly attenuated the effects of lead treatment on DNA repair in lymphocytes at 50 mg/kg/day dose (Fig. 2). Unexpectedly, we found a significantly lower level of initial DNA damage in lymphocytes from rats treated with a lead dose of 100 mg/kg/day than in those treated with 50 mg/kg/day, although it is not clear whether this difference has biological implications (Table 3). This observation was made in lymphocytes from rats treated with melatonin and lead. We suggest that the ability of lead acetate to induce DNA–DNA or DNA–protein crosslinks at high lead concentration could be responsible for this behavior [8]; however, it is not clear why this would be observed only in the presence of melatonin. Another possible explanation of the DNA migration decrease is cytotoxicity. One study showed that melatonin stimulated the repair of oxidative DNA damage, inactivating H2 O2 in lymphocytes in vitro [25]. However, in our conditions, in which no additional melatonin
Fig. 3. DNA fragmentation in lymphocytes from rats treated with lead acetate for 6 weeks. Lane 1, molecular markers; lane 2, positive apoptosis controls from kit; lane 3, negative apoptosis control lymphocytes (untreated); lane 4, treated with 100 mg/kg/day for 6 weeks; lane 5, treated with 100 mg/kg/day plus melatonin (10 mg/kg/day); lane 6, treated with 50 mg/kg/day for 6 weeks; lane 7, treated with 50 mg/kg/day plus melatonin (10 mg/kg/day) for 6 weeks.
M. Martínez-Alfaro et al. / Mutation Research 742 (2012) 37–42
was used for in vitro repair assays, the lower level of basal DNA damage is one factor that contributes to more rapid DNA repair. Another factor to consider is that the 6-week exposure to this lead dose could maximally stimulate the repair enzymes in lymphocytes without exerting toxic effects on enzyme activity. However, additional studies will be necessary to explain this result. This more efficient DNA repair in lead-treated lymphocytes at a dose of 50 mg/kg/day plus melatonin is probably also a factor in inhibiting apoptosis in these lymphocytes. The effects of melatonin on apoptosis imply a proapoptotic activity in tumor cells and an antiapoptotic activity in normal cells. Two probable mechanisms can be invoked to account for the apoptosis effect of melatonin: a receptor-mediated mechanism and direct scavenging of free radicals. Melatonin exerts many functions by binding to specific receptors (MT1 and MT2) expressed on the plasma membrane of target cells. These receptors have recently been shown to mediate the anti-apoptotic effects of melatonin [27], a mechanism that was not explored in this work. The other mechanism proposed for the inhibitory effect of melatonin on lead toxicity-induced apoptosis is a reduction in oxidative stress. Oxidative stress has been linked to apoptosis in primary cultures of rat proximal tubular cells exposed to lead [17]. Activation of caspase-3 has been documented in human cells exposed to lead in vitro [28,29]. Moreover, another study has shown a close relation between oxidative stress, DNA damage, and apoptosis induced by lead in mice [10]. Cellular responses to DNA damage include alterations in cell-cycle checkpoint control, DNA repair, and apoptosis. One widely accepted mechanism of lead genotoxicity is oxidative stress, and melatonin may reduce the level of oxidative stress. We addressed this issue and demonstrated in vivo that melatonin reduces the genotoxic effects of lead in a lead-dose-dependent manner. The protective action of antioxidant compounds against lead-induced toxicity in rats provides evidence that free radical damage is one mechanism involved in lead toxicity. Our studies showed that, at high doses of lead, melatonin does not exert beneficial effects, despite GSH reduction by melatonin. More studies using other indicators of oxidative stress and higher melatonin doses are needed to clarify these results. In conclusion, we showed that lead acetate induces oxidative stress in an experimental in vivo model of subchronic lead exposition (6 weeks). Melatonin administration is capable of reducing toxic lead effects, but its efficacy depends on the lead dose administered. The lead acetate dose determined the level of lead in the blood and the extent of basal DNA damage in lymphocytes. Moreover, oxidative stress is one factor in the DNA repair rate, and both DNA repair rate and the level of DNA damage are important factors in the cellular “decision” to repair DNA damage and survive or undergo programmed cell death (apoptosis). We assume that apoptosis is initiated whenever the damage is too severe to be repaired or the repair capacity is insufficient. Thus, the level of DNA damage induced by a genotoxic substance depends on the dose of the damaging agent and the presence of other agents. Conflict of interest None. Acknowledgements ˜ We would like to recognize the assistance of Dr. René Antano, Martín García, Evelyn Flores and Omar González. This research was supported by PROMEP-SEP project 103.5/10/7310.
41
References [1] V.M. Weaver, B.G. Jaar, B.S. Schwartz, A.C. Todd, K.D. Ahn, S.S. Lee, J. Wen, P.J. Parsons, B.K. Lee, Associations among lead dose biomarkers, uric acid, and renal function in Korean lead workers, Environ. Health Perspect. 113 (2005) 36–42. [2] J.H Shin, K.M. Lim, J.Y. Noh, O.N. Bae, S.M. Chung, M.Y. Lee, J.H. Chung, Leadinduced procoagulant activation of erythrocytes through phosphatidylserine exposure may lead to thrombotic diseases, Chem. Res. Toxicol. 20 (2007) 38–43. [3] E.G. Moreira, I. Vassilieff, V.S. Vassilieff, Developmental lead exposure: behavioral alterations in the short and long term, Neurotoxicol. Teratol. 23 (2001) 489–495. [4] L.D. White, D.A. Cory-Slechta, M.E. Gilbert, E. Tiffany-Castiglioni, N.H. Zawia, M. Virgolini, A. Rossi-George, S.M. Lasley, Y.C. Qian, M.R. Basha, New and evolving concepts in the neurotoxicology of lead, Toxicol. Appl. Pharmacol. 225 (2007) 1–27. [5] H.A. Godwin, The biological chemistry of lead, Curr. Opin. Chem. Biol. 5 (2001) 223–227. [6] H. Fu, P. Boffetta, Cancer and occupational exposure to inorganic lead compounds: a meta-analysis of published data, Occup. Environ. Med. 52 (1995) 73–81. [7] Z. Chen, J. Lou, S. Chen, W. Zheng, W. Wu, L. Jin, H. Deng, J. He, Evaluating the genotoxic effects of workers exposed to lead using micronucleus assay, comet assay and TCR gene mutation test, Toxicology 223 (2006) 219–226. [8] K. Wozniak, J. Blasiak, In vitro genotoxicity of lead acetate: induction of single and double DNA strand breaks and DNA–protein cross-links, Mutat. Res. 535 (2003) 127–139. [9] A. Pasha Shaik, S. Sankar, S.C. Reddy, P.G. Das, K. Jamil, Lead-induced genotoxicity in lymphocytes from peripheral blood samples of humans: in vitro studies, Drug Chem. Toxicol. 29 (2006) 111–124. [10] J. Xu, L.J. Lian, C. Wu, X.F. Wang, W.Y. Fu, L.H. Xu, Lead induces oxidative stress, DNA damage and alteration of p53, Bax and Bcl-2 expressions in mice, Food Chem. Toxicol. 46 (2008) 1488–1494. [11] M. Valverde, C. Trejo, E. Rojas, Is the capacity of lead acetate and cadmium chloride to induce genotoxic damage due to direct DNA–metal interaction? Mutagenesis 16 (2001) 265–270. [12] D. Beyersmann, A. Hartwig, Carcinogenic metal compounds: recent insight into molecular and cellular mechanisms, Arch. Toxicol. 82 (2008) 493–512. [13] J. Gastaldo, M. Viau, Z. Bencokova, A. Joubert, A.M. Charvet, J. Balosso, N. Foray, Lead contamination results in late and slowly repairable DNA double-strand breaks and impacts upon the ATM-dependent signaling pathways, Toxicol. Lett. 173 (2007) 201–214. [14] D.R. McNeill, H.K. Wong, A. Narayana, D.M. Wilson 3rd, Lead promotes abasic site accumulation and co-mutagenesis in mammalian cells by inhibiting the major abasic endonuclease Ape1, Mol. Carcinog. 46 (2007) 91–99. [15] T.T. Su, Cellular responses to DNA damage: one signal, multiple choices, Annu. Rev. Genet. 40 (2006) 187–208. [16] N. Aykin-Burns, A. Laegeler, G. Kellogg, N. Ercal, Oxidative effects of lead in young and adult Fisher 344 rats, Arch. Environ. Contam. Toxicol. 44 (2003) 417–420. [17] L. Wang, H. Wang, M. Hu, J. Cao, D. Chen, Z. Liu, Oxidative stress and apoptotic changes in primary cultures of rat proximal tubular cells exposed to lead, Arch. Toxicol. 83 (2009) 417–427. [18] J.L. Yang, L.C. Wang, C.Y. Chang, T.Y. Liu, Singlet oxygen is the major species participating in the induction of DNA strand breakage and 8hydroxydeoxyguanosine adduct by lead acetate, Environ. Mol. Mutagen. 33 (1999) 194–201. [19] J.K. Kang, D. Sul, J.K. Kang, S.Y. Nam, H.J. Kim, E. Lee, Effects of lead exposure on the expression of phospholipid hydroperoxidase glutathione peroxidase mRNA in the rat brain, Toxicol. Sci. 82 (2004) 228–236. [20] R.P. Prasanthi, C.B. Devi, D.C. Basha, N.S. Reddy, G.R. Reddy, Calcium and zinc supplementation protects lead (Pb)-induced perturbations in antioxidant enzymes and lipid peroxidation in developing mouse brain, Int. J. Dev. Neurosci. 28 (2010) 161–167. [21] D. Bonnefont-Rousselot, F. Collin, Melatonin: action as antioxidant and potential applications in human disease and aging, Toxicology 278 (2010) 55–67. [22] D.X. Tan, R.J. Reiter, L.C. Manchester, M.T. Yan, M. El-Sawi, R.M. Sainz, J.C. Mayo, R. Kohen, M. Allegra, R. Hardeland, Chemical and physical properties and potential mechanisms: melatonin as a broad spectrum antioxidant and free radical scavenger, Curr. Top. Med. Chem. 2 (2002) 181–197. [23] G.H. El-Sokkary, G.H. Abdel-Rahman, E.S. Kamel, Melatonin protects against lead-induced hepatic and renal toxicity in male rats, Toxicology 213 (2005) 25–33. [24] C. Suresh, A.O. Dennis, J. Heinz, M.C. Vemuri, C.S. Chetty, Melatonin protection against lead-induced changes in human neuroblastoma cell cultures, Int. J. Toxicol. 25 (2006) 459–464. [25] T. Sliwinski, W. Rozej, A. Morawiec-Bajda, Z. Morawiec, R. Reiter, J. Blasiak, Protective action of melatonin against oxidative DNA damage: chemical inactivation versus base-excision repair, Mutat. Res. 634 (2007) 220–227. [26] A.R. Collins, S.J. Duthie, V.L. Dobson, Direct enzymic detection of endogenous oxidative base damage in human lymphocyte DNA, Carcinogenesis 14 (1993) 1733–1735.
42
M. Martínez-Alfaro et al. / Mutation Research 742 (2012) 37–42
[27] A. Das, M. McDowell, M.J. Pava, J.A. Smith, R.J. Reiter, J.J. Woodward, A.K. Varma, S.K. Ray, N.L. Banik, The inhibition of apoptosis by melatonin in VSC4.1 motoneurons exposed to oxidative stress, glutamate excitotoxicity, or TNFalpha toxicity involves membrane melatonin receptors, J, Pineal Res. 48 (2010) 157–169.
[28] J. Xu, L.D. Ji, L.H. Xu, Lead-induced apoptosis in PC 12 cells: involvement of p53, Bcl-2 family and caspase-3, Toxicol. Lett. 166 (2006) 160–167. [29] C.G. Yedjou, J.N. Milner, C.B. Howard, P.B. Tchounwou, Basic apoptotic mechanisms of lead toxicity in human leukemia (HL-60) cells, Int. J. Environ. Res. Public Health 7 (2010) 2008–2017.
Contents lists available at SciVerse ScienceDirect
Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Effect of melatonin administration on DNA damage and repair responses in lymphocytes of rats subchronically exposed to lead Minerva Martínez-Alfaro a,∗ , Daniel Hernández-Cortés a , Katarzyna Wrobel b , Gustavo Cruz-Jiménez a , a ˜ Julio Cesar Rivera-Leyva a , Rosa María Pina-Zentella , Alfonso Cárabez Trejo c a
Department of Pharmacy, University of Guanajuato, Guanajuato, México 36050, Mexico Department of Chemistry, University of Guanajuato, Guanajuato, México 36050, Mexico c INB, Mexico b
a r t i c l e
i n f o
Article history: Received 17 June 2011 Received in revised form 17 November 2011 Accepted 21 November 2011 Available online 28 November 2011 Keywords: DNA repair DNA damage Apoptosis Lead acetate Melatonin
a b s t r a c t Lead exposure induces DNA damage, oxidative stress, and apoptosis, and alters DNA repair. We investigated the effects of melatonin co-administered to rats during exposure to lead. Three doses of lead acetate (10, 50 and 100 mg/kg/day) were administered to rats during a 6-week period. Lymphocytes were analyzed. Lead exposure decreased glutathione (GSH) levels in blood, and at doses of 100 mg/kg/day and 50 mg/kg/day without melatonin, caused high levels of DNA damage, induced apoptosis, and altered DNA repair. Melatonin co-treatment did not attenuate the effects of lead at 100 mg/kg/day, indicating that the effect of melatonin on GSH reduction is not sufficient to reduce the genotoxic effects of lead at this high dose. After 6 weeks of treatment, decreased weight gain was observed in high lead-dose groups (100 mg/kg/day), with or without melatonin, and in medium-dose groups (50 mg/kg/day) with melatonin, compared with the control group. The protective action of melatonin against lead toxicity is dependent on the dose of lead. Further pharmacological studies are needed to determine whether melatonin acts via melatonin membrane receptors on lymphocytes. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Lead, used in pipes, paints, lead acid batteries, solders, refining, smelting, and as an additive in gasoline, is found at high concentrations in the environment. Lead induces many adverse health effects, including nephrotoxicity [1], procoagulant activation of erythrocytes [2], behavioral alterations [3], and neurotoxicological effects [4]. One of the suggested molecular mechanisms of lead toxicity is binding to sites in a variety of proteins, thereby competing with endogenous cations, principally calcium and zinc. Biophysical studies have revealed that lead tightly binds zinc and calcium sites and alters the structural conformation and biological activity of the corresponding proteins. The ability of lead to substitute for divalent ions affects many cellular functions [5]. The International Agency for Research on Cancer (IARC) has classified inorganic lead compounds as probable human carcinogens (group 2A) and organic lead compounds as possible human carcinogens (group 2B). Epidemiological studies of lead-exposed workers have shown an excess of renal, lung, stomach, and brain
∗ Corresponding author at: Department of Pharmacy, University of Guanajuato, Guanajuato, México 36050, Mexico. Tel.: +52 4737320006x8031. E-mail address: [email protected] (M. Martínez-Alfaro). 1383-5718/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.mrgentox.2011.11.011
cancers [6]. The mean lead concentration in the blood of battery workers (248.3 g/L) was significantly higher than that in control individuals (27.49 g/L) [7]. The genotoxic effects of this exposure include positive results in the micronucleus and comet assays. Consistent with these observations, lead acetate induces DNA damage (both single- and double-strand breaks) as well as DNA–protein crosslinks in human lymphocytes in vitro at 1–10 M concentrations [8]. In another in vitro study on human lymphocytes, lead nitrate induced DNA damage at doses of 2.7 and 3 mM [9]. In mice, lead acetate induces oxidative stress, DNA damage, and apoptosis in vivo [10]. There is little evidence that lead interacts directly with DNA at normal blood lead concentrations [11]. The best documented mechanisms of lead genotoxicity are indirect, and include inhibition of DNA repair and production of free radicals [12]. A recent in vitro study in human cells [13] reported that lead exposure interferes with non-homologous end-joining repair processes by inhibiting DNA-PK kinase activity. Moreover, in AA8 cell extracts, lead induces a dose-dependent inhibition of the activity of AP endonuclease, which is the major endonuclease involved in repairing mutagenic and cytotoxic abasic sites in DNA [14]. The level of DNA damage determines the cellular response to the genotoxic agent [15]. Production of free radicals by lead exposure has been documented both in vivo and in vitro [16,17]. In one such study, lead acetate induced DNA strand breaks in a time- and
38
M. Martínez-Alfaro et al. / Mutation Research 742 (2012) 37–42
dose-dependent manner. Singlet oxygen, generated by Fenton-like reactions, was suggested to be the principal species involved in the DNA breakage. This lead-induced DNA breakage was inhibited by oxygen scavengers [18]. Alteration of antioxidant enzymes by lead exposure is another mechanism suggested for oxidative stress [19,20]. Melatonin, a hormone produced by the pineal gland, has many physiological functions, including chronomodulation of biological systems and regulation of immune functions, acting as either an inhibitor or activator of immune responses [21]. Moreover, melatonin has free radical scavenger and antioxidant chemical properties [22]. Experimental evidence suggests that melatonin exerts a protective action against lead effects. Administration of melatonin in conjunction with lead treatment reduced the hepatic and renal toxicity in rats treated with 100 mg/kg lead for 30 days in vivo [23]. Melatonin protects neuroblastoma cells against lead-induced apoptosis [24], and prevents oxidative DNA damage in blood lymphocytes in vitro by scavenging reactive oxygen species [25]. Despite numerous reports of the beneficial effects of melatonin against lead toxicity, little is known about the influence of melatonin on subchronic lead treatment in vivo. The aim of this study was to investigate the effects of melatonin on DNA damage induced by lead acetate. DNA damage, DNA repair, and apoptosis were assessed in lymphocytes from rats treated for 6 week with three different doses of lead acetate (10, 50 and 100 mg/kg/day) in the presence or absence of melatonin. DNA damage and repair were assayed by single-cell gel electrophoresis (comet assay); apoptosis was assessed by detecting nucleosomal fragments (180–200 bp) using gel electrophoresis. In addition, body weight and blood GSH and lead levels were evaluated. Melatonin co-administration reduced oxidative stress, decreased the level of DNA damage, and had an antiapoptotic effect in lymphocytes from rats treated with 10 or 50 mg/kg/day lead acetate. These effects of melatonin were correlated with the DNA damage-repair capacity of these lymphocytes. However, melatonin co-administration was not able to reduce DNA damage in lymphocytes from rats treated with the 100-mg/kg/day dose of lead, despite similar levels of GSH at 50 and 100 mg/kg/day doses. Moreover, lymphocytes from rats treated with high doses of lead acetate showed a high level of DNA damage and low DNA damage-repair capacity, effects that were likely linked to the presence of apoptosis. Thus, at high lead doses, mechanisms other than oxidative stress probably play a more important role in genotoxicity. The possible involvement of melatonin receptor effects will require further exploration. 2. Materials and methods 2.1. Chemicals The repair endonuclease Fpg was obtained from New England Bio-Labs, DAPI from Vectashield, and Histopaque 1077 and all other compounds from the Sigma Chemical Company (Toluca, México).
2.2. Animals and experimental design Male 5-week-old Sprague-Dawley rats were used in these experiments. Rats were housed in steel cages with an illumination period of 12 h/day, and provided a commercial diet and purified water ad libitum. After a 1-week acclimation period, animals were divided into eight groups (0, 10, 50 or 100 mg Pb/kg body weight ± melatonin) of five rats each; all groups were very similar in age and body weight. The number of animals used was the minimum necessary to obtain relevant results. All animals were handled in accordance with the guidelines of our institute and followed international norms of animal care and maintenance.
2.3. Body weight The body weights of all animals were measured weekly.
2.4. Melatonin administration Melatonin (10 mg/kg body weight) was administered daily to animals by intraperitoneal injection during the designated night period. 2.5. Lead administration Rats were treated orally (gavage) with lead acetate at doses of 0, 10, 50 or 100 mg/kg body weight 5 days a week for 6 weeks. 2.6. Lead quantification For lead determination, internal standard (115 In and 209 Bi), 100 L, was added to blood samples (400 L), and a wet digestion was carried out with concentrated nitric acid (500 L) for 2 h at 120 ◦ C. In the second step, an aliquot of H2 O2 (30%, 200 L) was added and the mixture was again incubated at 120 ◦ C for 1 h. Finally, the sample was cooled to room temperature, the volume was brought to 5 mL with deionized water, and the sample was centrifuged and introduced into an ICP-MS system. Each analysis was carried out in triplicate, and blanks were run in parallel. An inductively coupled plasma mass spectrometer (Model 7500ce; Agilent Technologies, Tokyo, Japan) with a Meinhard nebulizer and Peltier-cooled spray chamber (2 ◦ C) was used. The instrumental operating conditions were as follows: forward power, 1500 W; plasma gas flow rate, 15 L/min; carrier gas flow rate, 0.89 L/min; make-up gas flow rate, 0.15 L/min; sampling depth, 10 mm; dwell time, 300 ms per isotope; platinum sampling and skimmer cones were used. The isotopes 206 Pb, 207 Pb, 208 Pb were monitored and standardized to 115 In and 209 Bi. Calibration was performed with Agilent commercial standards at lead concentrations of 0, 0.2, 0.4, 1.0, 2.0, 5.0 and 10 g/L, with the internal standards, Bi and In (5.0 g/L each). The lead detection limit was 0.02 g/L. 2.7. GSH measurement A GSH assay kit that utilizes an enzymatic recycling method was used to measure GSH levels in pooled lymphocytes (106 cells); GSH reductase was used to quantify GSH. In this method, the sulfhydryl group of GSH reacts with DTNB (5,5 -dithio-bis2-nitrobenzoic acid) and produces a yellow-colored product, 5-thio-2-nitrobenzoic acid (TNB), which is detected by measuring absorbance at 405 nm. The rate of TNB production is directly proportional to the concentration of GSH in the sample. This assay was carried out in accordance with manufacturer’s protocol; samples were first concentrated and deproteinated before assaying. 2.8. Comet assay The comet assay used was based primarily on a previously described protocol [26], with modifications to detect oxidized bases. In brief, four folded comet slides were made for each treatment: one for basal and three for repair treatment; all slides were treated with Fpg formamidopyrimidine (fapy)-DNA glycosylase. The same numbers of slides were made without enzyme. Lymphocytes (20,000) in 10 L PBS with 90% viability (estimated by exclusion of trypan blue) were mixed with 75% agarose, 100 L, and the mixture was transferred onto a frosted slide (ES 370; Erie Scientific) pre-coated with normal-melting agarose (1%). A coverslip was added and the slide was cooled on ice to allow the agarose to harden. After the embedding procedure, three slides were exposed to experimental (DNA-damaging) treatments and one control (no DNA damage) slide. DNA damage was induced by exposing rat lymphocytes to 10 M H2 O2 for 5 min on ice. The initial level of induced DNA damage was estimated immediately after H2 O2 treatment. Following H2 O2 exposure, cells were either placed in lysis buffer (10 mM Tris pH 10, 2.5 M NaCl, 100 mM EDTA, 200 mM NaOH, 1% [v/v] Triton X-100, 10% [v/v] DMSO) for 1 h on ice, or were incubated in RPMI 1640 at 37 ◦ C for 15 or 45 min to allow repair of DNA damage and then transferred to lysis buffer for 1 h at 4 ◦ C. For enzyme treatment, the slides were removed from lysis buffer and incubated with enzyme reaction buffer (40 mM HEPES, 0.1 M KCl, 0.5 mM EDTA, 0.2 mg/mL BSA, adjusted to pH 8 with KOH) for 10 min. Fpg (1:105 dilution, 50 L) was added to the slides and incubated for 35 min at 37 ◦ C. Slides were immersed in cold alkaline unwinding electrophoresis solution (0.3 M NaOH and 1 mM Na2 EDTA in deionized water, pH 13.5) for 30 min and subjected to electrophoresis for 30 min at a constant voltage of 0.74 V/cm. All procedures were carried out in the dark. The slides were stained with DAPI (4 ,6-diamidino-2phenylindole) and analyzed with image analysis software (AutoComet; Tritek Corp.). Triplicate samples, each containing 50 cells, were quantified for each condition. The tail moment was used as a DNA damage parameter. 2.9. DNA repair kinetics DNA repair kinetics were calculated by determining the net number of Fpgsensitive sites induced by H2 O2 treatment over 45 min. The mean Olive tail moment obtained without enzyme treatment was subtracted from values obtained with enzyme incubation under the same experimental conditions (Table 3). OTM is
M. Martínez-Alfaro et al. / Mutation Research 742 (2012) 37–42
Table 1 Mean body weight ± SE in rats at the beginning and end of experimental period (6 weeks). Statistical analysis by Tukey HSD showed a significant difference between control and treatment groups.
2.4 F D
Ln (Tail Moment)
G
1.6 E
B
0.8
39
Condition
Initial weight
Control 10 Pb 10 Pb Me 50 Pb 50 Pb Me 100 Pb 100Pb Me
203 192 204 199 196 194 199
*
C
± ± ± ± ± ± ±
4 4 5 4 5 4 5
Final weight 321 272 303 302 265 242 269
± ± ± ± ± ± ±
Weight gain (g)
15 15 17 13 17* 15* 17*
118 80 99 103 69* 48* 70*
p < 0.05.
A
0.0
0
20
40
Time (min) Fig. 1. Kinetics of DNA repair in lymphocytes exposed in vitro to H2 O2 . Natural logarithm of values from Table 3 was used as Olive tail moment values. Significance of differences in slopes compared to the control group were determined by the Kruskal–Wallis test (p < 0.05).
calculated for the distance in microns between the center of gravity in the head and the center of gravity in the tail, multiplied by the % Tail. M TOlive = (CG − CG)
DNA 100
In order to evaluate kinetic repair log natural of each value from Table 3 was used in Fig. 1. Statistical analyses of DNA repair kinetics were based on statistical differences in slopes, between different experimental conditions by ANOVA and Kruskal–Wallis Tests (Fig. 1). 2.10. DNA laddering DNA fragmentation, in which DNA is cleaved into large fragments (50–200 kb) followed by cleavage into smaller fragments called nucleosomal units (180–200 bp), is a hallmark of apoptosis. The DNase responsible for this DNA cleavage into smaller fragments is a caspase-3-activated DNase. The result of endonuclease digestion manifests as a DNA ladder (multimers of approximately 180–200 bp) in agarose gels. DNA laddering on gels was detected using a DNA ladder isolation kit that separates apoptotic DNA from intact, high molecular weight genomic DNA. The method involves lysis of cells and inactivation of nucleases. 2.11. Statistical analysis Body weight data were analyzed using a matched-pairs test. Dunnet’s Method was used to analyze difference between GSH values. For comet assays, data were expressed as means ± standard errors. Differences with a p-value < 0.05 were considered statistically significant. Calculations were carried out using JMP V.7 Software (SAS Inc., 2008).
3. Results A Tukey HSD analysis showed no statistically significant differences in mean body weight among initial groups. In contrast, final mean body weights and weight gain were significantly lower (p < 0.05) in the groups receiving a high lead acetate dose, with or without melatonin (100 mg/kg/day), or a medium lead dose (50 mg/kg/day) with melatonin (Table 1). The greatest difference after 6 weeks of treatment was between rats in the control group, which reached 158.1% of their initial body weight during treatment (203 g initial mean body weight; 321 g final mean body weight), and rats in the 100 mg/kg/day lead acetate group, which reached 124.7% of their initial body weight (194 g initial mean body weight; 242 g final mean body weight) (Table 1). Blood lead levels in control rats were in the range of 18–22 g/L, whereas in rats treated with 10 mg Pb/kg/day, the lead concentration ranged from 48 to 53 g/L, a level that was not toxic. In contrast, the levels of lead in the blood of animals treated
with higher doses of lead acetate were toxic. In these animals, blood lead level ranges were 125–151 and 192–221 g/L for treatment with 50 and 100 mg Pb/kg/day, respectively. Differences in blood levels between animals treated with or without melatonin (10 mg/kg/day) were not significant. GSH levels in lymphocytes from rats treated (100 mg/kg/day), with or without melatonin, medium dose 50 mg/kg/day without melatonin were significantly different from those in controls. Lymphocytes from control animals and rats treated with low lead dose with or without melatonin and medium dose with melatonin were not significantly different (Table 2). Basal DNA damage in lymphocytes from controls and rats treated with a lead dose of 10 mg/kg/day (i.e., non-toxic levels), with or without melatonin, were similar. In contrast, basal DNA damage in lymphocytes from rats with toxic blood levels (50 and 100 mg/kg/day groups) was significantly higher compared to controls (Table 3). Unexpectedly, the lymphocytes from rats treated with 100 mg/kg/day plus melatonin showed lower initial levels of DNA damage than lymphocytes from rats treated with 50 mg/kg/day plus melatonin. An analysis of tail moments over the 45-min repair period (experimental points 0, 15 and 45 min) showed that DNA damage in lymphocytes decreased with increasing repair time for all experimental conditions (Table 3). A statistical analysis of repair kinetics by linear regression analysis of slopes showed a significant difference between two groups. Specifically, the second group, with subgroups exposed to lead doses of 100 mg/kg/day, with or without melatonin, or 50 mg/kg/day without melatonin, exhibited a significant delay in DNA repair (p < 0.05) compared to the control group, consisting of subgroups exposed to lead doses of 10 mg/kg/day (with and without melatonin) and 50 mg/kg/day with melatonin (Fig. 1). Effect of GSH level on DNA basal damage. Basal DNA damage was correlated with the GSH levels. Effect of GSH level on DNA repair. The rate of DNA damage repair in lymphocytes was correlated on GSH levels for all lead acetate treatments (Fig. 2). Apoptosis.
Table 2 Comparisons of mean GSH levels with those in controls Dunnett’s method. Exposure and co-treatment conditions Control Pb 10 mg/kg/day Pb 10 mg/kg/day and melatonin 10 mg/kg/day Pb 50 mg/kg/day Pb 50 mg/kg/day and melatonin 10 mg/kg/day Pb 100 mg/kg/day Pb 100 mg/kg/day and melatonin 10 mg/kg/day *
p < 0.05.
Mean GSH/106 lymphocytes (mole) 7.56 7.30 7.03 4.25* 6.48 3.60* 4.10*
± ± ± ± ± ± ±
0.6 0.75 0.69 0.46 0.7 0.59 0.56
40
M. Martínez-Alfaro et al. / Mutation Research 742 (2012) 37–42
Table 3 Net Olive tail moments were obtained by subtraction of Olive tail moment values without enzyme treatment from those with enzyme treatment. Net Fpg DNA glycosylasesensitive sites were measured in lymphocytes at a specific time during repair after exposure to 10 M H2 O2 . Values represent the mean and standard deviation (shown in parenthesis; n = 15). Repairing time (min)
Treatments Control
Basal t0 t15 t45 *
1.28(0.17) 2.4(0.43) 1.6(0.31) 1.1(0.21)
Pb10 1.5(0.16) 3.54(0.32) 1.9(0.21) 1.7(0.22)
Pb10Me 1.41(0.18) 3.06(0.28) 1.72(0.23) 1.44(0.23)
Pb50
Pb50Me *
4.5(0.51) 5.28(0.63)* 6.22(0.58)* 6.42(0.66)*
Pb100 *
2.6(0.33) 6.2(0.74)* 4.3(0.25)* 2.84(0.37)*
Pb100Me *
3.06(1.38) 11.5(0.96)* 8.44(0.82)* 8.27(0.80)*
2.46(0.32)* 7.52(0.72)* 6.46(0.65)* 6.16(0.65)*
p < 0.05 for comparisons between treatment and control at specific repair times (Kruskal–Wallis test).
DNA fragmentation was used as an indicator of apoptosis. Lymphocytes from rats receiving a 100-mg/kg/day lead dose, with or without melatonin, and lymphocytes from rats treated with 50 mg Pb/kg/day showed apoptosis (Fig. 3). 4. Discussion The mean body weight gains of rats in groups receiving a high lead dose, with or without melatonin, or a medium lead dose with melatonin during treatment were significantly lower than those in the control group. This effect of lead on body weight is well documented [19]. Under our experimental conditions, only rats administered lead acetate at doses of 50 and 100 mg/kg/day had toxic blood lead levels. The present results show a significant reduction in blood GSH levels in rats treated for 6 weeks with lead acetate at doses of 100 mg/kg/day and 50 mg/kg/day without melatonin. This reduction in GSH levels is an indicator of oxidative stress and is a well-established action of lead [16,17]. Analysis of the high lead acetate dose (100 mg/kg/day) showed a significant reduction in blood GSH levels independent of melatonin co-treatment (Table 2). Considering the mechanisms involved in the antioxidant effects of melatonin, there are two possible explanations for this behavior. First, the scavenger power of melatonin at the dose used (10 mg/kg/day) may be overwhelmed by the oxidative stress induced at the high lead dose. Alternatively, this dose of melatonin may induce insufficient activation of antioxidant enzymes to counteract the oxidative stress induced by the high lead dose. Even though beyond the scope of this paper, elucidating the specific mechanism involved in this response remains an interesting research issue.
Fig. 2. Correlation between GSH level and DNA repair rate. Inverse correlation between GSH plasma level and basal DNA damage (inset).
The data from this study demonstrated DNA damage in the lymphocytes of rats exposed to toxic blood levels of lead; these results are in agreement with other studies [7–9,13]. The initial level of DNA damage in lymphocytes depended on the lead acetate dose and the level of GSH in the blood (inset, Fig. 2). The slope of repair kinetics in lymphocytes (the value used to assess DNA repair) depended on the degree of oxidative stress (GSH level) (Fig. 1). In addition, our results established that melatonin co-treatment for 6 weeks in vivo significantly attenuated the effects of lead treatment on DNA repair in lymphocytes at 50 mg/kg/day dose (Fig. 2). Unexpectedly, we found a significantly lower level of initial DNA damage in lymphocytes from rats treated with a lead dose of 100 mg/kg/day than in those treated with 50 mg/kg/day, although it is not clear whether this difference has biological implications (Table 3). This observation was made in lymphocytes from rats treated with melatonin and lead. We suggest that the ability of lead acetate to induce DNA–DNA or DNA–protein crosslinks at high lead concentration could be responsible for this behavior [8]; however, it is not clear why this would be observed only in the presence of melatonin. Another possible explanation of the DNA migration decrease is cytotoxicity. One study showed that melatonin stimulated the repair of oxidative DNA damage, inactivating H2 O2 in lymphocytes in vitro [25]. However, in our conditions, in which no additional melatonin
Fig. 3. DNA fragmentation in lymphocytes from rats treated with lead acetate for 6 weeks. Lane 1, molecular markers; lane 2, positive apoptosis controls from kit; lane 3, negative apoptosis control lymphocytes (untreated); lane 4, treated with 100 mg/kg/day for 6 weeks; lane 5, treated with 100 mg/kg/day plus melatonin (10 mg/kg/day); lane 6, treated with 50 mg/kg/day for 6 weeks; lane 7, treated with 50 mg/kg/day plus melatonin (10 mg/kg/day) for 6 weeks.
M. Martínez-Alfaro et al. / Mutation Research 742 (2012) 37–42
was used for in vitro repair assays, the lower level of basal DNA damage is one factor that contributes to more rapid DNA repair. Another factor to consider is that the 6-week exposure to this lead dose could maximally stimulate the repair enzymes in lymphocytes without exerting toxic effects on enzyme activity. However, additional studies will be necessary to explain this result. This more efficient DNA repair in lead-treated lymphocytes at a dose of 50 mg/kg/day plus melatonin is probably also a factor in inhibiting apoptosis in these lymphocytes. The effects of melatonin on apoptosis imply a proapoptotic activity in tumor cells and an antiapoptotic activity in normal cells. Two probable mechanisms can be invoked to account for the apoptosis effect of melatonin: a receptor-mediated mechanism and direct scavenging of free radicals. Melatonin exerts many functions by binding to specific receptors (MT1 and MT2) expressed on the plasma membrane of target cells. These receptors have recently been shown to mediate the anti-apoptotic effects of melatonin [27], a mechanism that was not explored in this work. The other mechanism proposed for the inhibitory effect of melatonin on lead toxicity-induced apoptosis is a reduction in oxidative stress. Oxidative stress has been linked to apoptosis in primary cultures of rat proximal tubular cells exposed to lead [17]. Activation of caspase-3 has been documented in human cells exposed to lead in vitro [28,29]. Moreover, another study has shown a close relation between oxidative stress, DNA damage, and apoptosis induced by lead in mice [10]. Cellular responses to DNA damage include alterations in cell-cycle checkpoint control, DNA repair, and apoptosis. One widely accepted mechanism of lead genotoxicity is oxidative stress, and melatonin may reduce the level of oxidative stress. We addressed this issue and demonstrated in vivo that melatonin reduces the genotoxic effects of lead in a lead-dose-dependent manner. The protective action of antioxidant compounds against lead-induced toxicity in rats provides evidence that free radical damage is one mechanism involved in lead toxicity. Our studies showed that, at high doses of lead, melatonin does not exert beneficial effects, despite GSH reduction by melatonin. More studies using other indicators of oxidative stress and higher melatonin doses are needed to clarify these results. In conclusion, we showed that lead acetate induces oxidative stress in an experimental in vivo model of subchronic lead exposition (6 weeks). Melatonin administration is capable of reducing toxic lead effects, but its efficacy depends on the lead dose administered. The lead acetate dose determined the level of lead in the blood and the extent of basal DNA damage in lymphocytes. Moreover, oxidative stress is one factor in the DNA repair rate, and both DNA repair rate and the level of DNA damage are important factors in the cellular “decision” to repair DNA damage and survive or undergo programmed cell death (apoptosis). We assume that apoptosis is initiated whenever the damage is too severe to be repaired or the repair capacity is insufficient. Thus, the level of DNA damage induced by a genotoxic substance depends on the dose of the damaging agent and the presence of other agents. Conflict of interest None. Acknowledgements ˜ We would like to recognize the assistance of Dr. René Antano, Martín García, Evelyn Flores and Omar González. This research was supported by PROMEP-SEP project 103.5/10/7310.
41
References [1] V.M. Weaver, B.G. Jaar, B.S. Schwartz, A.C. Todd, K.D. Ahn, S.S. Lee, J. Wen, P.J. Parsons, B.K. Lee, Associations among lead dose biomarkers, uric acid, and renal function in Korean lead workers, Environ. Health Perspect. 113 (2005) 36–42. [2] J.H Shin, K.M. Lim, J.Y. Noh, O.N. Bae, S.M. Chung, M.Y. Lee, J.H. Chung, Leadinduced procoagulant activation of erythrocytes through phosphatidylserine exposure may lead to thrombotic diseases, Chem. Res. Toxicol. 20 (2007) 38–43. [3] E.G. Moreira, I. Vassilieff, V.S. Vassilieff, Developmental lead exposure: behavioral alterations in the short and long term, Neurotoxicol. Teratol. 23 (2001) 489–495. [4] L.D. White, D.A. Cory-Slechta, M.E. Gilbert, E. Tiffany-Castiglioni, N.H. Zawia, M. Virgolini, A. Rossi-George, S.M. Lasley, Y.C. Qian, M.R. Basha, New and evolving concepts in the neurotoxicology of lead, Toxicol. Appl. Pharmacol. 225 (2007) 1–27. [5] H.A. Godwin, The biological chemistry of lead, Curr. Opin. Chem. Biol. 5 (2001) 223–227. [6] H. Fu, P. Boffetta, Cancer and occupational exposure to inorganic lead compounds: a meta-analysis of published data, Occup. Environ. Med. 52 (1995) 73–81. [7] Z. Chen, J. Lou, S. Chen, W. Zheng, W. Wu, L. Jin, H. Deng, J. He, Evaluating the genotoxic effects of workers exposed to lead using micronucleus assay, comet assay and TCR gene mutation test, Toxicology 223 (2006) 219–226. [8] K. Wozniak, J. Blasiak, In vitro genotoxicity of lead acetate: induction of single and double DNA strand breaks and DNA–protein cross-links, Mutat. Res. 535 (2003) 127–139. [9] A. Pasha Shaik, S. Sankar, S.C. Reddy, P.G. Das, K. Jamil, Lead-induced genotoxicity in lymphocytes from peripheral blood samples of humans: in vitro studies, Drug Chem. Toxicol. 29 (2006) 111–124. [10] J. Xu, L.J. Lian, C. Wu, X.F. Wang, W.Y. Fu, L.H. Xu, Lead induces oxidative stress, DNA damage and alteration of p53, Bax and Bcl-2 expressions in mice, Food Chem. Toxicol. 46 (2008) 1488–1494. [11] M. Valverde, C. Trejo, E. Rojas, Is the capacity of lead acetate and cadmium chloride to induce genotoxic damage due to direct DNA–metal interaction? Mutagenesis 16 (2001) 265–270. [12] D. Beyersmann, A. Hartwig, Carcinogenic metal compounds: recent insight into molecular and cellular mechanisms, Arch. Toxicol. 82 (2008) 493–512. [13] J. Gastaldo, M. Viau, Z. Bencokova, A. Joubert, A.M. Charvet, J. Balosso, N. Foray, Lead contamination results in late and slowly repairable DNA double-strand breaks and impacts upon the ATM-dependent signaling pathways, Toxicol. Lett. 173 (2007) 201–214. [14] D.R. McNeill, H.K. Wong, A. Narayana, D.M. Wilson 3rd, Lead promotes abasic site accumulation and co-mutagenesis in mammalian cells by inhibiting the major abasic endonuclease Ape1, Mol. Carcinog. 46 (2007) 91–99. [15] T.T. Su, Cellular responses to DNA damage: one signal, multiple choices, Annu. Rev. Genet. 40 (2006) 187–208. [16] N. Aykin-Burns, A. Laegeler, G. Kellogg, N. Ercal, Oxidative effects of lead in young and adult Fisher 344 rats, Arch. Environ. Contam. Toxicol. 44 (2003) 417–420. [17] L. Wang, H. Wang, M. Hu, J. Cao, D. Chen, Z. Liu, Oxidative stress and apoptotic changes in primary cultures of rat proximal tubular cells exposed to lead, Arch. Toxicol. 83 (2009) 417–427. [18] J.L. Yang, L.C. Wang, C.Y. Chang, T.Y. Liu, Singlet oxygen is the major species participating in the induction of DNA strand breakage and 8hydroxydeoxyguanosine adduct by lead acetate, Environ. Mol. Mutagen. 33 (1999) 194–201. [19] J.K. Kang, D. Sul, J.K. Kang, S.Y. Nam, H.J. Kim, E. Lee, Effects of lead exposure on the expression of phospholipid hydroperoxidase glutathione peroxidase mRNA in the rat brain, Toxicol. Sci. 82 (2004) 228–236. [20] R.P. Prasanthi, C.B. Devi, D.C. Basha, N.S. Reddy, G.R. Reddy, Calcium and zinc supplementation protects lead (Pb)-induced perturbations in antioxidant enzymes and lipid peroxidation in developing mouse brain, Int. J. Dev. Neurosci. 28 (2010) 161–167. [21] D. Bonnefont-Rousselot, F. Collin, Melatonin: action as antioxidant and potential applications in human disease and aging, Toxicology 278 (2010) 55–67. [22] D.X. Tan, R.J. Reiter, L.C. Manchester, M.T. Yan, M. El-Sawi, R.M. Sainz, J.C. Mayo, R. Kohen, M. Allegra, R. Hardeland, Chemical and physical properties and potential mechanisms: melatonin as a broad spectrum antioxidant and free radical scavenger, Curr. Top. Med. Chem. 2 (2002) 181–197. [23] G.H. El-Sokkary, G.H. Abdel-Rahman, E.S. Kamel, Melatonin protects against lead-induced hepatic and renal toxicity in male rats, Toxicology 213 (2005) 25–33. [24] C. Suresh, A.O. Dennis, J. Heinz, M.C. Vemuri, C.S. Chetty, Melatonin protection against lead-induced changes in human neuroblastoma cell cultures, Int. J. Toxicol. 25 (2006) 459–464. [25] T. Sliwinski, W. Rozej, A. Morawiec-Bajda, Z. Morawiec, R. Reiter, J. Blasiak, Protective action of melatonin against oxidative DNA damage: chemical inactivation versus base-excision repair, Mutat. Res. 634 (2007) 220–227. [26] A.R. Collins, S.J. Duthie, V.L. Dobson, Direct enzymic detection of endogenous oxidative base damage in human lymphocyte DNA, Carcinogenesis 14 (1993) 1733–1735.
42
M. Martínez-Alfaro et al. / Mutation Research 742 (2012) 37–42
[27] A. Das, M. McDowell, M.J. Pava, J.A. Smith, R.J. Reiter, J.J. Woodward, A.K. Varma, S.K. Ray, N.L. Banik, The inhibition of apoptosis by melatonin in VSC4.1 motoneurons exposed to oxidative stress, glutamate excitotoxicity, or TNFalpha toxicity involves membrane melatonin receptors, J, Pineal Res. 48 (2010) 157–169.
[28] J. Xu, L.D. Ji, L.H. Xu, Lead-induced apoptosis in PC 12 cells: involvement of p53, Bcl-2 family and caspase-3, Toxicol. Lett. 166 (2006) 160–167. [29] C.G. Yedjou, J.N. Milner, C.B. Howard, P.B. Tchounwou, Basic apoptotic mechanisms of lead toxicity in human leukemia (HL-60) cells, Int. J. Environ. Res. Public Health 7 (2010) 2008–2017.