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Effects of post mortem interval and gender in DNA base excision repair activities in rat brains
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Effects of post mortem interval and gender in DNA base excision repair activities in rat brains Daniela Tathiana Soltys, Carolina Parga Martins Pereira, Gabriela Naomi Ishibe, Nadja Cristhina de Souza-Pinto ∗ Departmento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP 05508-900, Brazil
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Article history: Received 20 August 2014 Accepted 9 January 2015 Available online xxx Keywords: Base excision repair In vitro incision assay Post mortem interval Gender
a b s t r a c t Most human tissues used in research are of post mortem origin. This is the case for all brain samples, and due to the difficulty in obtaining a good number of samples, especially in the case of neurodegenerative diseases, male and female samples are often included in the same experimental group. However, the effects of post mortem interval (PMI) and gender differences in the endpoints being analyzed are not always fully understood, as is the case for DNA repair activities. To investigate these effects, in a controlled genetic background, base excision repair (BER) activities were measured in protein extracts obtained from Wistar rat brains from different genders and defined PMI up to 24 hours, using a novel fluorescent-based in vitro incision assay. Uracil and AP-site incision activity in nuclear and mitochondrial extracts were similar in all groups included in this study. Our results show that gender and PMI up to 24 hours have no influence in the activities of the BER proteins UDG and APE1 in rat brains. These findings demonstrate that these variables do not interfere on the BER activities included in these study, and provide a security window to work with UDG and APE1 proteins in samples of post mortem origin. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The field of DNA repair has advanced greatly in the past decade. Several studies have established an essential role for DNA repair in genomic stability and in processes such as aging and several diseases. Associations of lower DNA repair capacity with disease state have been found in several cancers [1] and in neurodegeneration [2]. Changes in DNA repair activity have also been observed during normal aging in several model organisms, including humans [3–6]. Most of these studies rely on comparing DNA repair, using various in vitro and cellular assays, in biological samples from different individuals with or without a specific pathology or other condition (life style/nutrition). When using model organisms, such as rodents, fly, worms or yeast, obtaining samples is relatively easy and interindividual variations are minimized by an adequate experimental group design. When using humans, however, the biological samples become an important issue when designing and interpreting the experiments. Most human studies use samples that can be obtained
∗ Corresponding author: Dept. of Biochemistry, IQ, USP, Av. Prof. Lineu Prestes, 748, Bloco 10 Superior, sala 1065, Cidade Universitária, São Paulo, SP 05508-900, Brazil. Tel.: +55 11 3091 1387; Fax: +55 11 3091 3811. E-mail address: [email protected] (N.C. de Souza-Pinto).
through non-invasive, such as saliva and urine, or minimally invasive, such as blood, methods. For functional studies, such as DNA repair activity measurements, however, the biological relevance of the findings has always to be interpreted in light of tissue specificity, as DNA repair protein expression levels and activity vary significantly from tissue to tissue [7,8]. In some cases, small tissue biopsies can be obtained, but the amount of tissue can limit the assays that can be carried out. This is relevant when studying mitochondrial DNA repair activities, as the amount of tissue required for isolation of mitochondria is relatively large. In the case of brain, this issue becomes particularly relevant, as brain biopsies are difficult to obtain, and limited to certain conditions. An alternative that has been used in a wide range of areas in biomedical sciences is the use of post mortem tissue, obtained from cadavers after authorization by family members. This approach has the advantage of allowing looking at tissue-specific variations, and may provide more biologically relevant results. Nonetheless, it is well recognized that degradative process initiate shortly after death [9–11], which could hamper sample integrity. For instance, if the tissues are not properly preserved RNA is quickly degraded and this degradation is correlated with PMI [12,13], and the samples are no longer suitable for gene expression studies. Human tissue banks store and provide samples obtained after varying PMI. In cases where sample availability is limited, like
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Please cite this article in press as: D.T. Soltys, et al., Effects of post mortem interval and gender in DNA base excision repair activities in rat brains, Mutat. Res.: Fundam. Mol. Mech. Mutagen. (2015), http://dx.doi.org/10.1016/j.mrfmmm.2015.01.003
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the cases for rare or age-associated diseases with low prevalence, limiting sample selection to those of low PMI could hinder the study. Thus, knowing whether PMI specifically affects the activity of interest may increase the safety window for sample selection and sample availability. Another issue is that in such cases, differentiating between male and female subjects may not be an option, again due to the small number of available samples. Base excision repair (BER) is the main repair pathway for small base modifications, abasic sites and single strand breaks, which are the quantitatively most relevant types of DNA lesions [14]. BER is accomplished in four sequential steps as follows: damage recognition and removal (performed by DNA glycosylases), abasic site processing with cleavage of the phosphodiester backbone (activity of APE1, or the -lyase activity of bifunctional DNA glycosylases), end-trimming and DNA resynthesis (pol  or pol ␦ in nucleus, pol ␥ in mitochondria), and ligation (DNA ligase I or III) [15]. The DNA glycosylases provide the substrate specificity of the repair pathway, as each of the 13 known DNA glycosylases have a defined set of modified bases as substrates. Among those, uracil-DNA glycosylase (UDG) is responsible for removing uracils from DNA [16], which are products of spontaneous deamination of cytosine. A lower activity of the DNA glycosylase OGG1 was found in peripheral blood mononuclear cells of lung cancer patients when compared with normal subjects [17], as lower UDG, OGG1 and pol  activities were found in post mortem brains of Alzheimer’s disease patients when compared with age-matched normal individuals [18]. While these results and others suggest that changes in BER activity are associated with the pathophysiology of the diseases, the sample type and source limit the interpretation as lung cells may have different levels/modulation of OGG1 activity in the first case and PMI could differentially affect controls and AD brains in the latter. Thus, the goal of this work is to investigate the impact of gender and PMI in BER activities using a controlled genetic background to minimize inter-individual variations. A novel fluorescent-based incision assay was developed to measure UDG and APE1 activities, and employed to measure incision levels in nuclear and mitochondrial extracts from male and female rat brains and from male rat brains collected after increasing PMI. Our results indicate that UDG and APE1 activities are preserved in both cellular compartments up to 24 h after death, and are similar between the two genders. These results provide experimental evidence to justify the use of human samples with up to 24 h of PMI and to group together male and female subjects in comparative BER capacity studies. 2. Materials and methods
another centrifugation. The pellets were pulled and stored at −80 ◦ C for nuclear extract preparation. The supernatant was centrifuged at 8000 × g for 15 min. The pellets were suspended in 5 mL of 3% Ficoll 400 in 0.5× MSHE buffer (0.21 M mannitol, 70 mM sucrose, 10 mM HEPES pH 7.4, 1 mM EGTA, 2 mM EDTA, 0.15 mM spermine, and 0.75 mM spermidine), and layered onto 5 mL of 6% Ficoll/0.5× MSHE. The gradients were centrifuged at 10,500 × g for 30 min. The pellet (crude mitochondria) was suspended in MSHE and incubated with 0.1% digitonin for 30 min, in ice. The final mitochondrial pellet was obtained after centrifugation at 9,000 × g and two washes with MSHE. Pellets were stored at −80 ◦ C for mitochondrial extract preparation. Protein extracts were obtained by suspending the fractions in buffer containing 20 mM HEPES (pH 7.0), 150 mM KCl, 2 mM EDTA, 1% Triton X-100 and protease inhibitors (Roche). The samples were incubated for 1 h at 4 ◦ C with shaking and centrifuged at 50,000 × g for 1 h. Supernatants were collected and glycerol added to a final concentration of 10%. All centrifugation steps were at 4 ◦ C. Protein concentrations were measured by Bradford method with bovine gamma globulin (Bio-Rad Laboratories Inc., Hercules, CA, USA) as standard. 2.3. Western blot Nuclear and mitochondrial protein levels were measured by standard western blot techniques with 50 g per lane of subcellular fractions and antibodies against Lamin B2 (X223), TFAM (E-16) or COX4 (20E8), at a dilution of 1:500 (antibodies were purchased from Santa Cruz Biotechnology, Santa Cruz, USA). 2.4. Preparation of fluorescent-labeled substrates Oligonucleotides containing uracil (U), tetrahydrofuran abasic site analog (AP) or the control without lesion (Ctrl) were obtained commercially (U and Ctrl from Invitrogen, Life Technologies, NY, USA; AP from Midland Certified Reagent Company, Midland, USA). Sequences of oligonucleotides were the same as used in [18]. The lesion-containing and control oligonucleotides were labeled at the 3 -end using terminal transferase – TdT (New England Biolabs, Ipswich, MA, USA) and Alexa Fluor 647-aha-dCTP (Molecular Probes, Life Technologies), following manufacturer’s instructions. Labeled oligonucleotides were gel-purified to remove products of incomplete synthesis and unincorporated AlexaFluor-dCTP, and annealed to the complementary strand, in a 1:5 ratio, by heating at 95 ◦ C and allowing to cool down slowly. The resulting duplex were further purified by gel-filtration with illustra MicroSpinTM G-25 Columns (GE Healthcare Life Sciences, Pittsburgh, PA, USA).
2.1. Animals 2.5. In vitro measurement of base excision repair activities Wistar rats were obtained from the Chemistry Institute Animal Facility and were kept under standard conditions. Three-monthold animals were euthanized by CO2 inhalation. For the gender experiments, brains were extracted from males and females immediately after the sacrifice and flash-frozen in liquid N2 . For the PMI experiments, males were sacrificed and the brains removed and flash-frozen at 0 h, 6 h, 12 h or 24 h after death. Brains were maintained at −80 ◦ C until use. All experiments were performed in accordance with the Guidelines for the Use and Care of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee (process #05/2011). 2.2. Isolation of nuclear and mitochondrial proteins Nuclear and mitochondrial fractions were obtained from approximately 500 mg of frozen brain tissue as described in [7], with the following modifications. The brain homogenates were centrifuged at 1200 × g for 12 min, and the supernatant subjected to
Recombinant proteins UDG and APE1 were purchased from New England Biolabs. Uracil and AP-site incision activities were measured as described in [19], replacing the radioactive labeled substrates for 50 fmoles of fluorescent-labeled substrate per reaction. Determination of protein concentration to use in the assays was performed with increasing amounts of nuclear extracts, incubated for 15 min with the AP duplex or 1 h with the U duplex. Measurement of APE1 activity was performed by incubating the AP duplex with 10 ng of extract for 5 min (nuclear) or 15 min (mitochondrial) at 37 ◦ C. Reactions were stopped by adding formamide and heating for 10 min at 90 ◦ C. For UDG activity, the U duplex was incubated with 5 g of extract for 1 h at 37 ◦ C. Reactions were stopped by adding NaOH to a final concentration of 50 mM and heated for 15 min at 75 ◦ C, followed by formamide addition. The products were resolved under denaturing conditions (23% acrilamida/bis-acrilamida 19:1, Urea 7 M, TBE 1×) and visualized in the laser scanner Typhoon Trio (GE Healthcare Life Sciences). The
Please cite this article in press as: D.T. Soltys, et al., Effects of post mortem interval and gender in DNA base excision repair activities in rat brains, Mutat. Res.: Fundam. Mol. Mech. Mutagen. (2015), http://dx.doi.org/10.1016/j.mrfmmm.2015.01.003
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software ImageQuant (GE Healthcare Life Sciences) was used for quantification. 2.6. Statistical analysis The assays were performed twice, in duplicates, and the results were plotted as mean ± standard deviation. Differences between experimental groups were analyzed by the Student’s t-test and a P < 0.05 was considered significantly different. 3. Results 3.1. Optimizing assay conditions with fluorescent-labeled substrates In vitro BER assays using radioactive-labeled substrates are extensively used in the literature [19]. These, however, generate radioactive waste and pose a risk to the experimenter when handling the source vials. To avoid these risks we replaced radioactiveby fluorescent-labeled substrates using Alexa Fluor-dCTP to introduce the fluorophore at the 3 -end with TdT. The integrity of the substrates after labeling was tested using recombinant proteins, APE1 and UDG. Incision products, resolved under denaturing PAGE, were within the limit of detection and were visualized in a laser scanner, even when low amounts of substrate (50 fmoles) were used (Fig. S1). In order to test these substrates with complex biological samples, and to investigate the influence of PMI and gender in the BER activities, nuclear and mitochondrial extracts were obtained from rat brains. Nuclear contamination of mitochondrial extracts could difficult the interpretation of the results, as nuclear BER activities are higher than mitochondrial in rodent brains [6,7]. Thus, the purity of the mitochondrial fractions was checked by western blot detecting the exclusively nuclear protein Lamin B2 and a mitochondrial protein – COX4 or TFAM (Fig. S2). All mitochondrial extracts used in this study were either free or showed less than 5% contamination with nuclear proteins. In order to compare the incision activities in the experimental groups, we determined the linear detection range of the fluorescent-based incision assays using increasing amounts of nuclear extracts from male rat brains with the abasic sitecontaining duplex (Fig. 1A and B) or the uracil-containing substrate (Fig. 1C and D). For the AP-substrate we observed a linear increase in incision between 0 and 25 ng of extract, while for the U substrate, a linear increase was observed between 0 and 7.5 g. Thus, we chose 10 ng of extract for the AP assays and 5 g for the U assays. 3.2. UDG and APE1 activities are preserved in rat brains up to 24 h after death To get a better understanding on the effects of PMI in BER activities, male rats were sacrificed and their brains were removed and processed within different periods after death. Nuclear and mitochondrial extracts were obtained from these tissues and employed in incision assays, using the conditions described above. As the main DNA glycosylase for U incision in duplex DNA in both nuclear and mitochondrial extracts is UDG [20], we are, therefore, assigning the U incision activity to UDG. Likewise, as APE1 is the major abasic site endonuclease in mammalian cells [21], we are assigning the AP incision activity to APE1. No changes in UDG activities up to 24 h after sacrifice were detected in incision assays with duplex U as substrate (Fig. 2A and C), in both nuclear and mitochondrial compartments. Similar results were obtained with the AP substrate, indicating that APE1 activity is also maintained for this time frame (Fig. 2B and D). These results indicate that both UDG and APE1 activities are preserved up to 24 h of PMI in rat brains, and establish
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this period as a safe window for use of post mortem brains in BER studies. 3.3. UDG and APE1 activity are similar in male and female brains To investigate the effect of gender in BER activities, males and female rats were sacrificed and the brains immediately collected. These samples were used to obtain nuclear and mitochondrial proteins. We detected no difference in uracil incision levels between males and females (Fig. 3A and C) for both nuclear (P = 0.3429) and mitochondrial (P = 0.6571) extracts. Similar results were obtained when we measured AP-site incision levels (Fig. 3B and D), for both nuclear (P = 0.2000) and mitochondrial (P = 0.3429) proteins. Thus, gender does not influence UDG and APE1 activities, at least in rat brains. 4. Discussion Studies comparing DNA repair capacity between healthy individuals and those suffering from different pathologies have yield a significant body of knowledge on the role of DNA repair in human diseases, such as cancer [1,22,23] and neurodegeneration [2,24]. In the case of neurodegenerative diseases, however, this approach is limited to the use of cellular models, such as primary fibroblasts from skin biopsies, or post mortem samples. While the fibroblasts and other cells types easily obtained, such as lymphocytes and muscle cells, have been used to identify genetic associations [17,25–27], it is clear that DNA repair activities vary among different cell types [7,8], and as such, their use for comparing DNA repair capacity in disease state is limited. Thus, post mortem samples are commonly used for these studies. However, the PMI of individual samples may vary significantly, and how this impacts the activities been studied has not been addressed yet in a controlled experiment, with homogeneous samples. Here we show, using fluorescent-based in vitro assays, that both nuclear and mitochondrial activities of the BER proteins UDG and APE1 obtained from Wistar rat brains are preserved up to 24 h after sacrifice, establishing a safety window to work with BER activities in post mortem brain samples. A few studies have sporadically addressed the issue of the role of post mortem interval in specific DNA repair activities. Comparing BER activity and PMI a posteriori in human brain samples, Canugovi et al. [28], found no correlation between PMI and BER activities in the inferior parietal regions from control and Alzheimer’s disease individuals. In another study, the DNA binding activity of Ku, a heterodimer required for non-homologous end joining DNA repair, was not significantly correlated with PMI in human brain extracts, also in a posteriori analysis [29]. DNA fragmentation patterns (assessed by in situ end-labeling technique) were also found to be independent of PMI in human cortex and up to 24 h after death in rat brains [30]. However, it is well established that other cellular and whole-organism physiological parameters, e.g. transcriptional profile [31] and metabolites concentrations in the blood [32], change extensively with the extension of the time after death. Thus, when working with post mortem samples, it is paramount to determine if and how PMI can affect the endpoint being investigated, and, in that respect, the results presented here will contribute by establishing a security window to study BER activities in post mortem brain samples. Another issue that is often faced when using post mortem samples from tissue banks is that samples from both genders have to be grouped together due to the limited number of available samples. Again, the relationship between gender and DNA repair activities has not been comprehensively addressed. Analyses of the background levels of DNA damage in lymphocytes of young and healthy
Please cite this article in press as: D.T. Soltys, et al., Effects of post mortem interval and gender in DNA base excision repair activities in rat brains, Mutat. Res.: Fundam. Mol. Mech. Mutagen. (2015), http://dx.doi.org/10.1016/j.mrfmmm.2015.01.003
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Fig. 1. Determination of protein concentration to use in the in vitro assays. Increasing concentrations of rat brain nuclear extracts were incubated with the AP duplex for 15 min (A) or with the U duplex for 1 h (C), and the products resolved using denaturing PAGE. A control duplex (without lesion) was used as a negative control. Representative blots showing the resolution of the products are shown in B and D. The values represent the mean ± SD of two independent experiments in duplicate.
Fig. 2. Effect of PMI in U and AP incision activities in rat brain. Uracil (A and C) incision in nuclear and mitochondrial extracts was measured using 5 g extract from brains removed immediately or 6, 12 and 24 after sacrifice. AP site (B) incision in nuclear and mitochondrial extracts was measured using 10 ng of extract from the same samples. Representative blots are shown in (C) and (D). As incision control, the substrates were incubated with recombinant UDG and APE1 (+ lanes). The values represent the mean ± SD of two independent experiments in duplicate. n = 3 animals per time-point.
individuals indicate higher levels of alkali labile sites and single strand breaks (SSBs) in males when compared to females, and similar levels of oxidized DNA lesions (FPG-sensitive sites and 8-oxodG) [25]. A population study with twins samples (40–77 yo) observed no differences between genders in any of the parameters analyzed in peripheral blood mononuclear cells: endogenous SSBs, SSB repair capacity, ␥-H2AX response and double strand break repair capacity
[26]. Similarly, no differences were detected in OGG1 activity in protein extracts prepared from human blood cells obtained from both genders of several ages, with the exception of a slightly lower activity in the group of males older than 55 yo compared to younger males [27]. However, no study had yet compared BER activities in brains from males and females, as specific gender differences are clearly observed in brain morphology [33,34]. Using Wistar rat
Please cite this article in press as: D.T. Soltys, et al., Effects of post mortem interval and gender in DNA base excision repair activities in rat brains, Mutat. Res.: Fundam. Mol. Mech. Mutagen. (2015), http://dx.doi.org/10.1016/j.mrfmmm.2015.01.003
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Fig. 3. Effect of gender in U and AP incision activities in rat brain. Uracil (A) incision in nuclear and mitochondrial extracts was measured using 5 g extract from brains from female or male rats. AP site (B) incision in nuclear and mitochondrial extracts was measured using 10 ng of extract from the same samples. Representative blots are shown in (C) and (D). As incision control, the substrates were incubated with recombinant UDG and APE1 (+ lanes). Averages of two independent measurements for each animal were plotted and SD for each group is shown. n = 4 animals per gender.
brains, we found no differences in the activities measured here, in both nuclear and mitochondrial extracts, indicating that, at least for BER activities, gender does not influence the DNA repair capacity. In conclusion, our results demonstrate that, when evaluating BER activities, it is safe to work with brain post mortem samples processed up to 24 h after death, and that grouping males and females together does not introduce gender-specific variability in the sample. Conflict of interest statement The authors declare no conflict of interest. Acknowledgments This work was supported by FAPESP grants INCT/Redoxoma (08/57721-3) and CEPID/Redoxoma (2013/07937-8) and grant 10/51906-1 to NCS-P. DTS has a FAPESP postdoctoral fellowship (2012/11889-9) and was previously supported by a CNPq postdoctoral fellowship (150974/2011-6). CPMP had a CNPq MSc. fellowship and GNI was supported by a CNPq-PIBIC fellowship (RUSP). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.mrfmmm.2015.01.003. References [1] F. Dietlein, L. Thelen, H.C. Reinhardt, Cancer-specific defects in DNA repair pathways as targets for personalized therapeutic approaches, Trends Genet. 30 (2014) 326–339.
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Effects of post mortem interval and gender in DNA base excision repair activities in rat brains Daniela Tathiana Soltys, Carolina Parga Martins Pereira, Gabriela Naomi Ishibe, Nadja Cristhina de Souza-Pinto ∗ Departmento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, SP 05508-900, Brazil
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Article history: Received 20 August 2014 Accepted 9 January 2015 Available online xxx Keywords: Base excision repair In vitro incision assay Post mortem interval Gender
a b s t r a c t Most human tissues used in research are of post mortem origin. This is the case for all brain samples, and due to the difficulty in obtaining a good number of samples, especially in the case of neurodegenerative diseases, male and female samples are often included in the same experimental group. However, the effects of post mortem interval (PMI) and gender differences in the endpoints being analyzed are not always fully understood, as is the case for DNA repair activities. To investigate these effects, in a controlled genetic background, base excision repair (BER) activities were measured in protein extracts obtained from Wistar rat brains from different genders and defined PMI up to 24 hours, using a novel fluorescent-based in vitro incision assay. Uracil and AP-site incision activity in nuclear and mitochondrial extracts were similar in all groups included in this study. Our results show that gender and PMI up to 24 hours have no influence in the activities of the BER proteins UDG and APE1 in rat brains. These findings demonstrate that these variables do not interfere on the BER activities included in these study, and provide a security window to work with UDG and APE1 proteins in samples of post mortem origin. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The field of DNA repair has advanced greatly in the past decade. Several studies have established an essential role for DNA repair in genomic stability and in processes such as aging and several diseases. Associations of lower DNA repair capacity with disease state have been found in several cancers [1] and in neurodegeneration [2]. Changes in DNA repair activity have also been observed during normal aging in several model organisms, including humans [3–6]. Most of these studies rely on comparing DNA repair, using various in vitro and cellular assays, in biological samples from different individuals with or without a specific pathology or other condition (life style/nutrition). When using model organisms, such as rodents, fly, worms or yeast, obtaining samples is relatively easy and interindividual variations are minimized by an adequate experimental group design. When using humans, however, the biological samples become an important issue when designing and interpreting the experiments. Most human studies use samples that can be obtained
∗ Corresponding author: Dept. of Biochemistry, IQ, USP, Av. Prof. Lineu Prestes, 748, Bloco 10 Superior, sala 1065, Cidade Universitária, São Paulo, SP 05508-900, Brazil. Tel.: +55 11 3091 1387; Fax: +55 11 3091 3811. E-mail address: [email protected] (N.C. de Souza-Pinto).
through non-invasive, such as saliva and urine, or minimally invasive, such as blood, methods. For functional studies, such as DNA repair activity measurements, however, the biological relevance of the findings has always to be interpreted in light of tissue specificity, as DNA repair protein expression levels and activity vary significantly from tissue to tissue [7,8]. In some cases, small tissue biopsies can be obtained, but the amount of tissue can limit the assays that can be carried out. This is relevant when studying mitochondrial DNA repair activities, as the amount of tissue required for isolation of mitochondria is relatively large. In the case of brain, this issue becomes particularly relevant, as brain biopsies are difficult to obtain, and limited to certain conditions. An alternative that has been used in a wide range of areas in biomedical sciences is the use of post mortem tissue, obtained from cadavers after authorization by family members. This approach has the advantage of allowing looking at tissue-specific variations, and may provide more biologically relevant results. Nonetheless, it is well recognized that degradative process initiate shortly after death [9–11], which could hamper sample integrity. For instance, if the tissues are not properly preserved RNA is quickly degraded and this degradation is correlated with PMI [12,13], and the samples are no longer suitable for gene expression studies. Human tissue banks store and provide samples obtained after varying PMI. In cases where sample availability is limited, like
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Please cite this article in press as: D.T. Soltys, et al., Effects of post mortem interval and gender in DNA base excision repair activities in rat brains, Mutat. Res.: Fundam. Mol. Mech. Mutagen. (2015), http://dx.doi.org/10.1016/j.mrfmmm.2015.01.003
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the cases for rare or age-associated diseases with low prevalence, limiting sample selection to those of low PMI could hinder the study. Thus, knowing whether PMI specifically affects the activity of interest may increase the safety window for sample selection and sample availability. Another issue is that in such cases, differentiating between male and female subjects may not be an option, again due to the small number of available samples. Base excision repair (BER) is the main repair pathway for small base modifications, abasic sites and single strand breaks, which are the quantitatively most relevant types of DNA lesions [14]. BER is accomplished in four sequential steps as follows: damage recognition and removal (performed by DNA glycosylases), abasic site processing with cleavage of the phosphodiester backbone (activity of APE1, or the -lyase activity of bifunctional DNA glycosylases), end-trimming and DNA resynthesis (pol  or pol ␦ in nucleus, pol ␥ in mitochondria), and ligation (DNA ligase I or III) [15]. The DNA glycosylases provide the substrate specificity of the repair pathway, as each of the 13 known DNA glycosylases have a defined set of modified bases as substrates. Among those, uracil-DNA glycosylase (UDG) is responsible for removing uracils from DNA [16], which are products of spontaneous deamination of cytosine. A lower activity of the DNA glycosylase OGG1 was found in peripheral blood mononuclear cells of lung cancer patients when compared with normal subjects [17], as lower UDG, OGG1 and pol  activities were found in post mortem brains of Alzheimer’s disease patients when compared with age-matched normal individuals [18]. While these results and others suggest that changes in BER activity are associated with the pathophysiology of the diseases, the sample type and source limit the interpretation as lung cells may have different levels/modulation of OGG1 activity in the first case and PMI could differentially affect controls and AD brains in the latter. Thus, the goal of this work is to investigate the impact of gender and PMI in BER activities using a controlled genetic background to minimize inter-individual variations. A novel fluorescent-based incision assay was developed to measure UDG and APE1 activities, and employed to measure incision levels in nuclear and mitochondrial extracts from male and female rat brains and from male rat brains collected after increasing PMI. Our results indicate that UDG and APE1 activities are preserved in both cellular compartments up to 24 h after death, and are similar between the two genders. These results provide experimental evidence to justify the use of human samples with up to 24 h of PMI and to group together male and female subjects in comparative BER capacity studies. 2. Materials and methods
another centrifugation. The pellets were pulled and stored at −80 ◦ C for nuclear extract preparation. The supernatant was centrifuged at 8000 × g for 15 min. The pellets were suspended in 5 mL of 3% Ficoll 400 in 0.5× MSHE buffer (0.21 M mannitol, 70 mM sucrose, 10 mM HEPES pH 7.4, 1 mM EGTA, 2 mM EDTA, 0.15 mM spermine, and 0.75 mM spermidine), and layered onto 5 mL of 6% Ficoll/0.5× MSHE. The gradients were centrifuged at 10,500 × g for 30 min. The pellet (crude mitochondria) was suspended in MSHE and incubated with 0.1% digitonin for 30 min, in ice. The final mitochondrial pellet was obtained after centrifugation at 9,000 × g and two washes with MSHE. Pellets were stored at −80 ◦ C for mitochondrial extract preparation. Protein extracts were obtained by suspending the fractions in buffer containing 20 mM HEPES (pH 7.0), 150 mM KCl, 2 mM EDTA, 1% Triton X-100 and protease inhibitors (Roche). The samples were incubated for 1 h at 4 ◦ C with shaking and centrifuged at 50,000 × g for 1 h. Supernatants were collected and glycerol added to a final concentration of 10%. All centrifugation steps were at 4 ◦ C. Protein concentrations were measured by Bradford method with bovine gamma globulin (Bio-Rad Laboratories Inc., Hercules, CA, USA) as standard. 2.3. Western blot Nuclear and mitochondrial protein levels were measured by standard western blot techniques with 50 g per lane of subcellular fractions and antibodies against Lamin B2 (X223), TFAM (E-16) or COX4 (20E8), at a dilution of 1:500 (antibodies were purchased from Santa Cruz Biotechnology, Santa Cruz, USA). 2.4. Preparation of fluorescent-labeled substrates Oligonucleotides containing uracil (U), tetrahydrofuran abasic site analog (AP) or the control without lesion (Ctrl) were obtained commercially (U and Ctrl from Invitrogen, Life Technologies, NY, USA; AP from Midland Certified Reagent Company, Midland, USA). Sequences of oligonucleotides were the same as used in [18]. The lesion-containing and control oligonucleotides were labeled at the 3 -end using terminal transferase – TdT (New England Biolabs, Ipswich, MA, USA) and Alexa Fluor 647-aha-dCTP (Molecular Probes, Life Technologies), following manufacturer’s instructions. Labeled oligonucleotides were gel-purified to remove products of incomplete synthesis and unincorporated AlexaFluor-dCTP, and annealed to the complementary strand, in a 1:5 ratio, by heating at 95 ◦ C and allowing to cool down slowly. The resulting duplex were further purified by gel-filtration with illustra MicroSpinTM G-25 Columns (GE Healthcare Life Sciences, Pittsburgh, PA, USA).
2.1. Animals 2.5. In vitro measurement of base excision repair activities Wistar rats were obtained from the Chemistry Institute Animal Facility and were kept under standard conditions. Three-monthold animals were euthanized by CO2 inhalation. For the gender experiments, brains were extracted from males and females immediately after the sacrifice and flash-frozen in liquid N2 . For the PMI experiments, males were sacrificed and the brains removed and flash-frozen at 0 h, 6 h, 12 h or 24 h after death. Brains were maintained at −80 ◦ C until use. All experiments were performed in accordance with the Guidelines for the Use and Care of Laboratory Animals, and were approved by the Institutional Animal Care and Use Committee (process #05/2011). 2.2. Isolation of nuclear and mitochondrial proteins Nuclear and mitochondrial fractions were obtained from approximately 500 mg of frozen brain tissue as described in [7], with the following modifications. The brain homogenates were centrifuged at 1200 × g for 12 min, and the supernatant subjected to
Recombinant proteins UDG and APE1 were purchased from New England Biolabs. Uracil and AP-site incision activities were measured as described in [19], replacing the radioactive labeled substrates for 50 fmoles of fluorescent-labeled substrate per reaction. Determination of protein concentration to use in the assays was performed with increasing amounts of nuclear extracts, incubated for 15 min with the AP duplex or 1 h with the U duplex. Measurement of APE1 activity was performed by incubating the AP duplex with 10 ng of extract for 5 min (nuclear) or 15 min (mitochondrial) at 37 ◦ C. Reactions were stopped by adding formamide and heating for 10 min at 90 ◦ C. For UDG activity, the U duplex was incubated with 5 g of extract for 1 h at 37 ◦ C. Reactions were stopped by adding NaOH to a final concentration of 50 mM and heated for 15 min at 75 ◦ C, followed by formamide addition. The products were resolved under denaturing conditions (23% acrilamida/bis-acrilamida 19:1, Urea 7 M, TBE 1×) and visualized in the laser scanner Typhoon Trio (GE Healthcare Life Sciences). The
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software ImageQuant (GE Healthcare Life Sciences) was used for quantification. 2.6. Statistical analysis The assays were performed twice, in duplicates, and the results were plotted as mean ± standard deviation. Differences between experimental groups were analyzed by the Student’s t-test and a P < 0.05 was considered significantly different. 3. Results 3.1. Optimizing assay conditions with fluorescent-labeled substrates In vitro BER assays using radioactive-labeled substrates are extensively used in the literature [19]. These, however, generate radioactive waste and pose a risk to the experimenter when handling the source vials. To avoid these risks we replaced radioactiveby fluorescent-labeled substrates using Alexa Fluor-dCTP to introduce the fluorophore at the 3 -end with TdT. The integrity of the substrates after labeling was tested using recombinant proteins, APE1 and UDG. Incision products, resolved under denaturing PAGE, were within the limit of detection and were visualized in a laser scanner, even when low amounts of substrate (50 fmoles) were used (Fig. S1). In order to test these substrates with complex biological samples, and to investigate the influence of PMI and gender in the BER activities, nuclear and mitochondrial extracts were obtained from rat brains. Nuclear contamination of mitochondrial extracts could difficult the interpretation of the results, as nuclear BER activities are higher than mitochondrial in rodent brains [6,7]. Thus, the purity of the mitochondrial fractions was checked by western blot detecting the exclusively nuclear protein Lamin B2 and a mitochondrial protein – COX4 or TFAM (Fig. S2). All mitochondrial extracts used in this study were either free or showed less than 5% contamination with nuclear proteins. In order to compare the incision activities in the experimental groups, we determined the linear detection range of the fluorescent-based incision assays using increasing amounts of nuclear extracts from male rat brains with the abasic sitecontaining duplex (Fig. 1A and B) or the uracil-containing substrate (Fig. 1C and D). For the AP-substrate we observed a linear increase in incision between 0 and 25 ng of extract, while for the U substrate, a linear increase was observed between 0 and 7.5 g. Thus, we chose 10 ng of extract for the AP assays and 5 g for the U assays. 3.2. UDG and APE1 activities are preserved in rat brains up to 24 h after death To get a better understanding on the effects of PMI in BER activities, male rats were sacrificed and their brains were removed and processed within different periods after death. Nuclear and mitochondrial extracts were obtained from these tissues and employed in incision assays, using the conditions described above. As the main DNA glycosylase for U incision in duplex DNA in both nuclear and mitochondrial extracts is UDG [20], we are, therefore, assigning the U incision activity to UDG. Likewise, as APE1 is the major abasic site endonuclease in mammalian cells [21], we are assigning the AP incision activity to APE1. No changes in UDG activities up to 24 h after sacrifice were detected in incision assays with duplex U as substrate (Fig. 2A and C), in both nuclear and mitochondrial compartments. Similar results were obtained with the AP substrate, indicating that APE1 activity is also maintained for this time frame (Fig. 2B and D). These results indicate that both UDG and APE1 activities are preserved up to 24 h of PMI in rat brains, and establish
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this period as a safe window for use of post mortem brains in BER studies. 3.3. UDG and APE1 activity are similar in male and female brains To investigate the effect of gender in BER activities, males and female rats were sacrificed and the brains immediately collected. These samples were used to obtain nuclear and mitochondrial proteins. We detected no difference in uracil incision levels between males and females (Fig. 3A and C) for both nuclear (P = 0.3429) and mitochondrial (P = 0.6571) extracts. Similar results were obtained when we measured AP-site incision levels (Fig. 3B and D), for both nuclear (P = 0.2000) and mitochondrial (P = 0.3429) proteins. Thus, gender does not influence UDG and APE1 activities, at least in rat brains. 4. Discussion Studies comparing DNA repair capacity between healthy individuals and those suffering from different pathologies have yield a significant body of knowledge on the role of DNA repair in human diseases, such as cancer [1,22,23] and neurodegeneration [2,24]. In the case of neurodegenerative diseases, however, this approach is limited to the use of cellular models, such as primary fibroblasts from skin biopsies, or post mortem samples. While the fibroblasts and other cells types easily obtained, such as lymphocytes and muscle cells, have been used to identify genetic associations [17,25–27], it is clear that DNA repair activities vary among different cell types [7,8], and as such, their use for comparing DNA repair capacity in disease state is limited. Thus, post mortem samples are commonly used for these studies. However, the PMI of individual samples may vary significantly, and how this impacts the activities been studied has not been addressed yet in a controlled experiment, with homogeneous samples. Here we show, using fluorescent-based in vitro assays, that both nuclear and mitochondrial activities of the BER proteins UDG and APE1 obtained from Wistar rat brains are preserved up to 24 h after sacrifice, establishing a safety window to work with BER activities in post mortem brain samples. A few studies have sporadically addressed the issue of the role of post mortem interval in specific DNA repair activities. Comparing BER activity and PMI a posteriori in human brain samples, Canugovi et al. [28], found no correlation between PMI and BER activities in the inferior parietal regions from control and Alzheimer’s disease individuals. In another study, the DNA binding activity of Ku, a heterodimer required for non-homologous end joining DNA repair, was not significantly correlated with PMI in human brain extracts, also in a posteriori analysis [29]. DNA fragmentation patterns (assessed by in situ end-labeling technique) were also found to be independent of PMI in human cortex and up to 24 h after death in rat brains [30]. However, it is well established that other cellular and whole-organism physiological parameters, e.g. transcriptional profile [31] and metabolites concentrations in the blood [32], change extensively with the extension of the time after death. Thus, when working with post mortem samples, it is paramount to determine if and how PMI can affect the endpoint being investigated, and, in that respect, the results presented here will contribute by establishing a security window to study BER activities in post mortem brain samples. Another issue that is often faced when using post mortem samples from tissue banks is that samples from both genders have to be grouped together due to the limited number of available samples. Again, the relationship between gender and DNA repair activities has not been comprehensively addressed. Analyses of the background levels of DNA damage in lymphocytes of young and healthy
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Fig. 1. Determination of protein concentration to use in the in vitro assays. Increasing concentrations of rat brain nuclear extracts were incubated with the AP duplex for 15 min (A) or with the U duplex for 1 h (C), and the products resolved using denaturing PAGE. A control duplex (without lesion) was used as a negative control. Representative blots showing the resolution of the products are shown in B and D. The values represent the mean ± SD of two independent experiments in duplicate.
Fig. 2. Effect of PMI in U and AP incision activities in rat brain. Uracil (A and C) incision in nuclear and mitochondrial extracts was measured using 5 g extract from brains removed immediately or 6, 12 and 24 after sacrifice. AP site (B) incision in nuclear and mitochondrial extracts was measured using 10 ng of extract from the same samples. Representative blots are shown in (C) and (D). As incision control, the substrates were incubated with recombinant UDG and APE1 (+ lanes). The values represent the mean ± SD of two independent experiments in duplicate. n = 3 animals per time-point.
individuals indicate higher levels of alkali labile sites and single strand breaks (SSBs) in males when compared to females, and similar levels of oxidized DNA lesions (FPG-sensitive sites and 8-oxodG) [25]. A population study with twins samples (40–77 yo) observed no differences between genders in any of the parameters analyzed in peripheral blood mononuclear cells: endogenous SSBs, SSB repair capacity, ␥-H2AX response and double strand break repair capacity
[26]. Similarly, no differences were detected in OGG1 activity in protein extracts prepared from human blood cells obtained from both genders of several ages, with the exception of a slightly lower activity in the group of males older than 55 yo compared to younger males [27]. However, no study had yet compared BER activities in brains from males and females, as specific gender differences are clearly observed in brain morphology [33,34]. Using Wistar rat
Please cite this article in press as: D.T. Soltys, et al., Effects of post mortem interval and gender in DNA base excision repair activities in rat brains, Mutat. Res.: Fundam. Mol. Mech. Mutagen. (2015), http://dx.doi.org/10.1016/j.mrfmmm.2015.01.003
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Fig. 3. Effect of gender in U and AP incision activities in rat brain. Uracil (A) incision in nuclear and mitochondrial extracts was measured using 5 g extract from brains from female or male rats. AP site (B) incision in nuclear and mitochondrial extracts was measured using 10 ng of extract from the same samples. Representative blots are shown in (C) and (D). As incision control, the substrates were incubated with recombinant UDG and APE1 (+ lanes). Averages of two independent measurements for each animal were plotted and SD for each group is shown. n = 4 animals per gender.
brains, we found no differences in the activities measured here, in both nuclear and mitochondrial extracts, indicating that, at least for BER activities, gender does not influence the DNA repair capacity. In conclusion, our results demonstrate that, when evaluating BER activities, it is safe to work with brain post mortem samples processed up to 24 h after death, and that grouping males and females together does not introduce gender-specific variability in the sample. Conflict of interest statement The authors declare no conflict of interest. Acknowledgments This work was supported by FAPESP grants INCT/Redoxoma (08/57721-3) and CEPID/Redoxoma (2013/07937-8) and grant 10/51906-1 to NCS-P. DTS has a FAPESP postdoctoral fellowship (2012/11889-9) and was previously supported by a CNPq postdoctoral fellowship (150974/2011-6). CPMP had a CNPq MSc. fellowship and GNI was supported by a CNPq-PIBIC fellowship (RUSP). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.mrfmmm.2015.01.003. References [1] F. Dietlein, L. Thelen, H.C. Reinhardt, Cancer-specific defects in DNA repair pathways as targets for personalized therapeutic approaches, Trends Genet. 30 (2014) 326–339.
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Please cite this article in press as: D.T. Soltys, et al., Effects of post mortem interval and gender in DNA base excision repair activities in rat brains, Mutat. Res.: Fundam. Mol. Mech. Mutagen. (2015), http://dx.doi.org/10.1016/j.mrfmmm.2015.01.003