Major metabolic homocysteine-derivative, homocysteine thiolactone, exerts changes in pancreatic β-cell glucose-sensing, cellular signal transduction and integrity

Archives of Biochemistry and Biophysics 461 (2007) 287–293 www.elsevier.com/locate/yabbi

Major metabolic homocysteine-derivative, homocysteine thiolactone, exerts changes in pancreatic -cell glucose-sensing, cellular signal transduction and integrity Steven Patterson ¤, Peter R. Flatt, Neville H. McClenaghan School of Biomedical Sciences, University of Ulster, Coleraine BT52 1SA, UK Received 9 January 2007, and in revised form 2 February 2007 Available online 2 March 2007

Abstract Homocysteine can be converted to its reactive thioester, homocysteine thiolactone. Cytotoxic properties of these amino thiols have been attributed to protein homocysteinylation, increased oxidative stress, DNA damage and apoptosis. This study used pancreatic BRINBD11 -cells to examine functional defects caused by acute and long-term exposure to homocysteine thiolactone in comparison with homocysteine. Acute and long-term exposure to both agents caused concentration-dependent inhibitions of glucose-induced insulin secretion while impairing the insulin-secretory responses to alanine, KCl, elevated Ca2+, forskolin and PMA. Acute exposures also caused signiWcant reduction in the amplitude of KCl-induced membrane depolarisation but no eVects on changes of intracellular Ca2+ induced by alanine or KCl. Cellular insulin content and DNA damage were not altered following culture, however, there were early signs of apoptosis consistent with impaired cellular integrity. In conclusion, exposure to homocysteine thiolactone, like homocysteine, induced -cell dysfunction and demise by mechanisms independent of changes in membrane potential and [Ca2+]i. © 2007 Elsevier Inc. All rights reserved. Keywords: Apoptosis; Insulin secretion; Homocysteine thiolactone; Pancreatic -cells

Homocysteine is an independent risk factor for premature coronary heart disease and atherosclerosis, which are the most common causes of mortality in type 2 diabetes [1–3]. Indeed, homocysteine may be directly linked to the pathogenesis of coronary disease in individuals with type 2 diabetes [4,5], with a higher level of mortality compared to nondiabetic subjects [6]. The possible link between plasma homocysteine levels and diabetes is complex and as yet unclear, with impaired kidney function and glomerular Wltration rate being perhaps the most important contributor to hyperhomocysteinaemia in type 2 diabetes [7]. Furthermore, hyperinsulinaemia, insulin resistance and obesity have been suggested to be inextricably linked to elevations of plasma homocysteine [8–10].

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0003-9861/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2007.02.011

Homocysteine has been shown to damage endothelial cells via intracellular generation of reactive oxygen species [11]. In addition, homocysteine has been demonstrated to increase DNA synthesis and proliferation of neural crestderived vascular smooth muscle cells [1], which may play a key role in the development atherosclerotic plaques. Furthermore, homocysteine has been reported to cause neurotoxicity [12], principally through over-stimulation of NMDA receptors and generation of free radicals [13,14], and induce neuronal apoptosis, via DNA damage, PARP activation, and p53 induction [15,16]. Hyperhomocysteinaemia promotes the conversion of homocysteine to its cyclic thioester, homocysteine thiolactone, due to an error editing function of some aminoacyl-tRNA synthetases [17]. Homocysteine thiolactone is able to homocysteinylate proteins at the lysine residue and alter protein structure and function [17–19] and this may play a key role in atherosclerosis. Exposure to homocysteine thiolactone has been reported to impair

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insulin signalling, which could be reversed by glutathione, perhaps indicating a role of increased oxidative stress [20]. In this regard, homocysteine thiolactone has been demonstrated to induce apoptotic DNA damage in a human leukaemia HL-60 cell line, associated with intracellular production of hydrogen peroxide and caspase 3 activation [21]. In recent studies, we have extended the reported detrimental actions of homocysteine to include pancreatic -cells [22,23]. These studies demonstrated that acute or prolonged homocysteine-exposure caused demise of -cell function, which could not simply be attributed to a decrease in cell viability [22,23]. This prompted the present study examining the impact of acute or long-term exposure to metabolic derivative, homocysteine thiolactone on key regulatory pathways modulating insulin secretion, cellular ionic Xux and -cell function and integrity. Materials and methods Chemicals Reagents of analytical grade and deionised water (Purite) were used. RPMI-1640 tissue culture medium, foetal bovine serum, and antibiotics were from Gibco, 125I-bovine insulin was purchased from Amersham Biosciences (Bucks, UK). In situ cell death detection kit (TUNEL assay) was from Roche Diagnostic Ltd. (West Sussex, UK). Membrane potential kit and calcium assay kit were purchased from Molecular Devices (Sunnyvale, CA, USA). All other chemicals were from Sigma–Aldrich (Poole, UK) or BDH chemicals (Poole, UK).

Cell culture Pancreatic BRIN-BD11 cells established by electrofusion of New England Deaconess Hospital (NEDH) rat pancreatic islet cells and RINm5F cells [24], were grown in RPMI-1640 tissue culture medium containing 11.1 mM glucose and 0.3 g/L of L-glutamine, supplemented with 10% (v/v) foetal calf serum, 100 IU/ml penicillin and 0.1 mg/ml streptomycin at 37 °C with 5% CO2 and 95% air. The cells were gently washed with Hanks’ balanced saline solution (HBSS) before detachment from the culture Xask with the aid of 0.025% (w/v) trypsin containing 1 mM EDTA and utilised immediately. The properties and responsiveness of BRINBD11 cells to a wide range of physiological and pharmacological agents have been described elsewhere [24–26]. The BRIN-BD11 cells used in this study were from passages 19–32.

Insulin release and cellular content Insulin release from BRIN-BD11 cells was determined using cell monolayers. BRIN-BD11 cells were harvested, resuspended in culture medium and seeded in each well of 24-well multiplates at a density of 1.5 £ 105 cells per well. After overnight culture at 37 °C to allow attachment of cells, the culture medium was removed and 1 ml Krebs–Ringer bicarbonate (KRB)1 buVer consisting of NaCl, KCl, MgSO 4, CaCl2, KH2 PO4, Hepes (115, 4.7, 1.2, 1.28, 1.2 and 25 mM, respectively) and 8.4% (w/v) NaHCO3 (pH 7.4 with NaOH) supplemented with 0.1% (w/v) bovine serum albumin and 1.1 mM D-glucose, was carefully added to each well. The cells were preincubated at 37 °C for 40 min, after which the buVer was removed and replaced with 1 ml of the test buVer supple1 Abbreviations used: KRB, Krebs–Ringer bicarbonate; PFA, paraformaldehyde; RT, room temperature.

mented with glucose, amino acids and other known modulators of pancreatic -cell function as indicated in the Wgures. Following acute 20-min incubation at 37 °C, aliquots of test buVer were removed from each well and stored at ¡20 °C for subsequent determination of insulin by radioimmunoassay. Cells were extracted for insulin content measurement by addition of 1 ml of ice-cold acid-ethanol solution (75% v/v ethanol, 1.5% v/v concentrated HCl). After overnight incubation and cellular disruption at 4 °C, acid-ethanol aliquots were removed for subsequent determination of cellular insulin content. Insulin was measured by dextran-charcoal radioimmunoassay [27], using guinea-pig antiporcine insulin serum, and rat insulin standard.

Measurement of membrane potential and intracellular Ca2+ Membrane potential and intracellular calcium ([Ca2+]i) were determined using monolayers of BRIN-BD11 cells [28]. Cells were seeded into 96-well black-walled, clear bottom microplates (Greiner Bio-One, Gloucestershire, UK) at a density of 1.0 £ 105 cells per well and allowed to attach overnight, 100 l of KRBB was added to each well and incubated for 10 min after which 100 l of either FLEX membrane potential assay kit [29] or FLEX calcium assay kit (Molecular Devices, CA, USA), was added to wells at 37 °C. Fluorometric data were acquired using the FLEXstation™, a scanning Xuorometer and integrated Xuid transfer workstation (Molecular Devices, CA, USA). The cells were exposed to excitation light from a xenon-arc Xashlamp at a wavelength of 530 nm (membrane) or 485 nm (calcium) and subsequent Xuorescence emission measured at 565 nm (membrane) or 525 nm (calcium) using a bottom read mode. Emission cut-oV Wlters were set at 550 nm for membrane potential or 515 nm for intracellular calcium.

Measurement of apoptosis by TUNEL assay BRIN-BD11 cells were harvested, and resuspended in tissue culture medium to a density of 1.0 £ 105 cells/ml, and added to polylysine-coated slides in tissue culture Petri dishes (Iwaki). Following overnight attachment, cells were cultured for a further 18 h in the absence or presence of homocysteine thiolactone, homocysteine or streptozotocin. Culture media was removed and the cells Wxed with 4% paraformaldehyde (PFA) for 1 h at room temperature (RT). Cells were then blocked with 0.3% (v/v) H2O2 in methanol (1 h), prior to permeabilization using 0.1% (v/v) Triton X-100 in 0.1% (w/v) sodium citrate buVer (2 min, 4 °C) and addition of TUNEL reaction mix for 1 h at 37 °C. Finally cells were washed in PBS, mounted in 50:50 glycerol/PBS and stored in the dark until analysis under a Xuorescence microscope with a FITC Wlter.

Assessment of DNA damage using Comet assay Measurement of DNA strand breakage in BRIN-BD11 cells exposed to various reagents was carried out using the single cell gel electrophoresis assay [30]. Cells were suspended in pre-warmed (37 °C) 1% low melting point agarose at a concentration of 1 £ 104–5 £ 104 cells/100 l. This was added promptly to a slide coated with an initial agarose layer, covered with a coverslip and allowed to cool (5 min, 4 °C). Cells were lysed in pre-chilled lysis buVer (1% Triton X-100, 2.5 M NaCl, 100 mM EDTA, 10 mM Tris, pH 10.0) for 1–2 h at 4 °C. Slides were then placed in a gel electrophoresis tank (Bio-Rad, Richmond, CA, USA), submerged in pre-chilled electrophoresis buVer (NaOH 300 mM and EDTA 1 mM) for 20 min to allow unwinding of the DNA, and subjected to electrophoresis at 25 V, and a current of 300 mA (20 min, 4 °C). Slides were washed in pre-chilled neutralizing buVer (Tris 0.4 M, pH 7.5) at 4 °C for three 5 min intervals. DNA was then stained by the addition of 10 l ethidium bromide (20 g/ml diluted in PBS) and slides were stored with cover-slip in a moisture chamber at 4 °C. Measurements of DNA damage were carried out using the epiXuorescence microscope (Nikon, Kingston, Surrey, UK) and analysed using the Komet 5.0 software package (Kinetic imaging, Liverpool, UK). DNA damage was expressed as the % of Comet tail DNA.

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Statistical analysis Results are presented as means § SEM. Groups of data were compared by one-way ANOVA in conjunction with Bonferroni’s post hoc test. DiVerences were considered signiWcant if P < 0.05.

Results Acute eVects of homocysteine thiolactone and homocysteine on glucose-induced insulin release The eVects homocysteine thiolactone or homocysteine on insulin secretion from BRIN-BD11 cells at 16.7 mM glucose are shown in Table 1. Both agents exerted concentrationdependent inhibitory eVects at 50–1000 M. As shown in Table 2, acute exposure to 250 M homocysteine thiolactone or homocysteine similarly inhibited the insulin response to a variety of established secretagogues when tested at 16.7 mM glucose. This included stimulatory concentrations of alanine, KCl, Ca2+, forskolin and PMA. Acute eVects of homocysteine thiolactone and homocysteine on membrane potential and intracellular Ca2+ The eVects of homocysteine thiolactone or homocysteine on [Ca2+]i and membrane potential in the presence of Table 1 Acute eVects of homocysteine thiolactone and homocysteine on insulin release at stimulatory (16.7 mM) glucose Concentration of HCTL or HC (M)

Insulin release (% of control) HCTL

HC

0 (control) 50 250 1000

100.0 § 0.6 70.8 § 5.2¤¤¤ 59.4 § 3.6¤¤¤ 18.9 § 1.4¤¤¤

100.0 § 0.6 68.4 § 4.8¤¤¤ 56.7 § 2.8¤¤¤ 8.2 § 0.9¤¤¤

Values are means § SEM (n D 6). ¤¤¤ P < 0.001 compared with control insulin release at 16.7 mM glucose (100% D 2.75 ng/106 cells/20 min). Abbreviations: HC, homocysteine; HCTL, homocysteine thiolactone. Table 2 Acute eVects of 250 M homocysteine thiolactone or homocysteine on insulin release at 16.7 mM glucose in the presence of a range of established secretagogues Addition

Insulin release (% of control none) Control

None Ala (20 mM) KCl (30 mM) Ca2+ (7.68 mM) Forsk (25 M) PMA (10 nM)

100.0 § 1.4 448.0 § 15.8¤¤¤ 566.1 § 19.2¤¤¤ 193.6 § 8.7¤¤¤ 428.3 § 8.6¤¤¤ 507.0 § 15.8¤¤¤

HCTL 59.4 § 2.8 348.0 § 15.6 494.5 § 4.5 148.5 § 7.4 310.5 § 5.8 379.1 § 22.6

HC 64.6 § 4.5 290.6 § 7.1 438.9 § 11.2 95.4 § 1.8 303.2 § 6.2 354.3 § 11.9

Values are means § SEM (n D 6). ¤¤¤ P < 0.001 compared with eVects of control in the absence of the secretagogue (None). P < 0.01, P < 0.001 compared with control insulin release for the same secretagogue. None refers to 16.7 mM glucose. Normal KRB buVer contained 1.28 mM Ca2+. Abbreviations: Ala, alanine; KCl, potassium chloride; Forsk, forskolin; PMA, phorbol 12-myristate 13-acetate.

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alanine (20 mM) and a depolarizing concentration of KCl (30 mM) are shown in Fig. 1. Both agents had little eVect on alanine-induced changes of [Ca2+]i and membrane potential (Fig. 1a and b). There was a modest but insigniWcant increase in the initial elevation of Ca2+ in the presence of alanine and homocysteine thiolactone. Both homocysteine thiolactone and homocysteine signiWcantly (P < 0.01– P < 0.001) lowered the amplitude of KCl-induced membrane depolarization compared to depolarization in the presence of KCl alone (Fig. 1c). Interestingly, these eVects appeared to be independent of any noticeable changes to KCl-induced Ca2+ inXux (Fig. 1d). EVects of long-term exposure to homocysteine thiolactone or homocysteine on insulin secretory responses and cellular insulin content Insulin secretion induced by acute exposure to alanine, KCl, Ca2+, forskolin or PMA at 16.7 mM glucose following 18 h culture in the absence or presence of homocysteine thiolactone or homocysteine are shown in Fig. 2. Prior exposure to homocysteine thiolactone caused signiWcant reductions in the secretory responsiveness of the cells to all agents tested by 12–39% (P < 0.01–P < 0.001). Homocysteine exposure also lowered subsequent secretory responses to all test agents by 8–48%, however, only glucose, alanine, KCl and Ca2+ were signiWcantly reduced (P < 0.001) (Fig. 2). Culture with 250 M homocysteine thiolactone or homocysteine did not signiWcantly aVect insulin content (74.7 § 7.3 ng/106 cells or 90.3 § 4.3 ng/106, n D 4, respectively) compared with control cells (89.7 § 3.8 ng/106 cells, n D 4). EVects of long-term exposure to homocysteine thiolactone and homocysteine on DNA damage and apoptosis As shown in Fig. 3, 18 h culture with 250 M homocysteine thiolactone or homocysteine did not cause any signiWcant DNA damage. Although DNA damage was not evident, there was evidence of BRIN-BD11 cell apoptosis following 18 h exposure to homocysteine thiolactone (5.0fold increase, P < 0.01) or homocysteine (3.0-fold increase, P < 0.05) consistent with a decline in cellular integrity (Fig. 4). Streptozotocin (10 mM), used as a positive control, caused signiWcant DNA damage (58.5 § 1.4% tail DNA, P < 0.001) and there was a 9.0-fold increase (P < 0.001) in the number of apoptotic BRIN-BD11 cells following streptozotocin culture (Figs. 3 and 4). Discussion The present study investigated the impact of acute or prolonged exposure to homocysteine thiolactone on various aspects of insulin secretion and pancreatic -cell function. As reported previously [31], homocysteine and metabolically-derived homocysteine thiolactone caused concentration-dependent inhibition of insulin secretion at

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Fig. 1. EVects of acute (3 min) exposure to 250 M homocysteine thiolactone or homocysteine on alanine (a, b) and KCl (c, d) induced changes in membrane potential and intracellular Ca2+ in BRIN-BD11 cells at 5.6 mM glucose. Each data point represents means § SEM (n D 6). ¤¤P < 0.01, ¤¤¤P < 0.001 compared with eVects on membrane potential with KCl alone. Abbreviations: HC, homocysteine; HCTL, homocysteine thiolactone; RFU, relative Xuorescent units.

stimulatory (16.7 mM) glucose. Furthermore, at 16.7 mM glucose the acute insulinotropic eVects of alanine, a depolarizing concentration of KCl, elevated Ca2+, and modulators of adenylate cyclase and protein kinase C pathways,

namely forskolin and PMA, were impaired by homocysteine thiolactone or homocysteine. Since homocysteine thiolactone impaired the acute insulinotropic responses to the various agents in a similar fashion to homocysteine,

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Fig. 2. EVects of 18 h exposure to 250 M homocysteine thiolactone or homocysteine on insulinotropic responses to a range of established secretagogues. BRIN-BD11 cells were cultured (18 h) in either standard culture medium alone or containing 250 M of HCTL or HC. Following culture, cells were preincubated (40 min) in buVer containing 1.1 mM glucose and the insulinotropic actions of alanine, KCl, Ca2+, forskolin and PMA at 16.7 mM glucose were tested during a 20-min incubation period. Values are means § SEM (n D 6). ¤¤¤P < 0.001 compared with eVects in the absence of secretagogues. P < 0.01, P < 0.001 compared with the same secretagogue following culture in the absence of HCTL or HC. None refers to 16.7 mM glucose alone. Normal KRB buVer contained 1.28 mM Ca2+. Abbreviations: Ala, alanine; KCl, potassium chloride; Forsk, forskolin; PMA, phorbol 12-myristate 13-acetate.

perhaps homocysteine may exert its detrimental actions through conversion to the reactive thiolactone. Interestingly, homocysteine thiolactone has previously been

Fig. 3. EVects of long-term (18 h) exposure to 250 M homocysteine thiolactone or homocysteine on BRIN-BD11 cell DNA damage. Cells cultured for 18 h in standard culture medium alone or containing STZ (10 mM), HCTL (250 M) or HC (250 M), were analysed for DNA damage by Comet assay. Values are means § SEM (n D 50). ¤¤¤P < 0.001 compared to DNA damage in control cells cultured (18 h) in the absence of HC, HCTL, or STZ. Abbreviations: HC, homocysteine; HCTL, homocysteine thiolactone; STZ, streptozotocin.

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Fig. 4. EVects of long-term (18 h) exposure to 250 M homocysteine thiolactone or homocysteine on BRIN-BD11 cell death by apoptosis. Cells cultured (18 h) in either standard culture medium alone or containing STZ (10 mM), HCTL (250 M) or HC (250 M), were assessed for cellular apoptosis using the TUNEL assay. Values are means § SEM for eight separate Welds of observations. ¤P < 0.05, ¤¤P < 0.01, ¤¤¤P < 0.001 compared to number of apoptotic cells from control slides. Control refers to apoptosis in cells cultured (18 h) in the absence of homocysteine, homocysteine thiolactone, or streptozotocin. Abbreviations: HC, homocysteine; HCTL, homocysteine thiolactone; STZ, streptozotocin.

reported to cause homocysteinylation of proteins at lysine residues, causing a concentration-dependent denaturation of the protein, thereby altering protein function [17,18]. As such, it is possible that homocysteinylation of key proteins involved in pancreatic -cell signalling pathways underlie the detrimental eVects on insulin secretion, but this remains to be clariWed. In order to understand the mechanisms through which homocysteine thiolactone and homocysteine exert their inhibitory eVects on insulin secretion, changes in membrane potential and [Ca2+]i were examined. When the acute eVects of KCl-induced changes in membrane potential and [Ca2+]i were studied, the amplitude of KCl-induced depolarisation was reduced, without any noticeable eVects on [Ca2+]i. This is reminiscent of the Wndings of Pacher et al. [32], who reported that homocysteine caused a slight decrease in action potential amplitude in ventricular papillary muscles and atria. Furthermore, homocysteine was found to acutely inhibit transient outward currents in ventricular myocytes [33]. In the present study, alanine-induced changes in membrane potential and [Ca2+]i were not signiWcantly altered by homocysteine though homocysteine thiolactone caused a slight increase in initial alanine-induced Ca2+ inXux rate and reduced amplitude of membrane repolarization. Since KCl-, alanine-, and Ca2+-induced insulin secretion was inhibited by both homocysteine thiolactone and homocysteine, the present data suggest that they exert their eVects largely independent of alterations of membrane potential or [Ca2+]i. As the insulinotropic eVects of forskolin, a potent activator of adenylate cyclase [34], and PMA, which activates protein kinase C [35], were also impaired by homocysteine thiolactone and homocysteine, an inhibitory eVect at a later KATP channel-independent stage of the insulin secretion coupling pathway may be involved.

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Prolonged exposure of pancreatic -cells to hyperglycaemic and hyperlipidaemic conditions can induce initial desensitization of responses to key secretagogues, with eventual glucotoxic and lipotoxic eVects. Such actions appear to be mediated by chronic oxidative stress, decreased insulin gene expression, increased levels of cellular triglycerides which promotes steatosis and can lead to eventual lipoapoptosis [36–40]. Previous reports on the cytotoxic eVects of homocysteine thiolactone or homocysteine on a number of diVerent cell types, suggest that increased oxidative stress may be a major factor [13,21,41]. Insulin secretion studies following 18 h exposure of BRINBD11 cells to 250 M of homocysteine or metabolically related homocysteine thiolactone, once again showed impaired eVects of insulin secretagogues, despite no signiWcant diVerences in cellular insulin content. Previous studies demonstrated that acute exposure to homocysteine thiolactone or homocysteine did not signiWcantly alter BRIN-BD11 cell viability [22,31] and the present data further revealed that 18 h exposure did not signiWcantly aVect DNA damage. However, interestingly the present data demonstrated cells showing early signs of apoptosis, suggesting that an even longer duration of exposure may exert additional adverse eVects culminating in cell death, prompting further research. In this regard, it is notable that there was increased evidence of apoptosis following 18 h culture with homocysteine thiolactone compared with homocysteine, consistent with observations on human umbilical vein endothelial cells [42]. Apoptosis of HL-60 cells and other cell lines [15,16,43] has also been reported as a result of exposure to homocysteine thiolactone, which may relate to promotion of apoptotic cell death by caspase 3 activation [21]. Notably, a 3- to 10-fold increase in the rate of pancreatic -cell apoptosis has been reported in type 2 diabetes [44] giving rise to decreased -cell mass and increased insulin demand. Since these primary data suggest that homocysteine thiolactone and homocysteine may increase the rate of -cell demise, further studies are warranted to test whether these thiols contribute to decreased -cell mass in vivo. In conclusion, the thioester homocysteine thiolactone, like its metabolic precursor, homocysteine, exerts detrimental acute and long-term eVects on pancreatic -cells. Thus, it appears from the present data that the homocysteineinduced demise of the -cell may be mediated by intracellular conversion to this major thiol-derivative, with adverse eVects on insulin-secretory function and cellular integrity. In a recent report, “The Hordaland Homocysteine Study”, plasma homocysteine levels of up to 70 M were measured in patients with hyperhomocysteinaemia [45]. While the concentrations of homocysteine thiolactone and homocysteine used in this ‘short-term’ in vitro study are rarely observed clinically, chronic exposure of pancreatic -cells to pathophysiological levels of homocysteine in vivo merit investigation. In this regard, the present data prompt additional studies in islets and animal models of hyperhomocy-

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