ToxSci Advance Access originally published online on November 10, 2006
Toxicological Sciences 2007 95(2):391-400; doi:10.1093/toxsci/kfl164
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
The Effect of 3-Methyladenine DNA Glycosylase–Mediated DNA Repair on the Induction of Toxicity and Diabetes by the ß-Cell Toxicant Streptozotocin
,1
* Eppley Institute for Research in Cancer and Allied Diseases
Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198–6805
1 To whom correspondence should be addressed at Department of Pharmaceutical Sciences, 512 Salk Hall, University of Pittsburgh, Pittsburgh, PA 15261. Fax: (412) 383-7436. E-mail: goldbi{at}pitt.edu.
Received July 26, 2006; accepted October 25, 2006
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Type 1 diabetes in humans arises from the autoimmune destruction of pancreatic ß-cells and typically presents in childhood. Genetic susceptibility is an underlying cause, but environmental agents, that is, toxins and viruses, are postulated to be initiating factors. The underlying role of ß-cell death in response to environmental or physiologic events has been investigated as a critical event in diabetes onset. A well-studied rodent model for type 1 diabetes utilizes streptozotocin (STZ) to induce ß-cell death. STZ is a selective ß-cell genotoxicant, and when administered in a single high dose it induces rapid onset of diabetes by generating DNA adducts, including N3-methyladenine and O6-methylguanine adducts, and subsequently ß-cell death by necrosis. In the present work, we have extended previous studies in which mice deficient in the repair of N3-methyladenine adducts, 3-methyladenine DNA glycosylase (alkyladenine DNA glycosylase [Aag]) null mice, were reported to be resistant to the direct cytotoxic effect of STZ, but later developed autoimmune diabetes (J. W. Cardinal et al., 2001, Mol. Cell. Biol. 231, 5605–5613). We found that Aag–/– mice treated with a single high dose of STZ were protected from widespread ß-cell necrosis and diabetes. However, moderate levels of ß-cell apoptosis were observed in the Aag–/– STZ-treated mice. While mice became glucose impaired for the duration of study (14 months after STZ injection), overt diabetes did not develop. We conclude that an autoimmune response is not initiated in Aag–/– mice in response to ß-cell apoptosis. Furthermore, tumor development is not observed in Aag–/– treated mice, suggesting that N3-methyladenine adducts that accumulate in the genome may not be promutagenic in ß-cells.
Key Words: type 1 diabetes; streptozotocin; apoptosis; 3-methyladenine; base excision repair.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Type 1 diabetes is an organ-specific autoimmune disease characterized by destruction of pancreatic ß-cells. Susceptibility is influenced by both genetic and environmental factors; however, the critical event that eventually leads to loss of tolerance remains unknown. The potential role of ß-cell death has been investigated as an early event leading to T-cell activation and loss of tolerance (Mathis et al., 2001
). In particular, toxins, such as N-nitroso compounds, and viral infections have been investigated for their potential role in triggering ß-cell death and inducing autoimmunity. Herein, we have employed a 3-methyladenine DNA glycosylase null mouse to explore the role base excision repair (BER) plays in ß-cell death induced by streptozotocin (2-deoxy-2-[3-methyl-3-nitrosourea]-1-D-glucopyranose; STZ), a ß-cell genotoxicant, and the resulting immune response after ß-cell death.
Two experimental models are commonly used to induce diabetes with STZ, and both rely on its DNA-damaging properties. The first approach consists of a single high-dose STZ (SHDS), which causes massive ß-cell necrosis and rapid induction of diabetes by 48 h. SHDS generates sufficient levels of DNA adducts to cause overactivation of poly(adenosine 5' diphosphate-ribose) polymerase (PARP) in the BER pathway. The extensive PARP activation results in the rapid depletion of cellular NAD+ and cell death through necrosis. The second method for inducing diabetes in mice uses multiple low-dose STZ (MLDS) for five consecutive days. In this model, apoptosis is the predominant form of ß-cell death (O'Brien et al., 1996), and onset of hyperglycemia is delayed and marked by immune cell infiltration into islets followed by their autoimmune destruction. In both models, STZ exerts its effects through induction of DNA damage, including N7-methylguanine (7-MeG), N3-methyladenine (3-MeA), and O6-methylguanine (6-MeG) (reviewed in Bolzan and Bianchi, 2002). However, ß-cell cytotoxicity via necrosis after a SHDS is dependent on activation of the BER pathway in response to 3-MeA (Murata et al., 1999). In the MLDS model, cell death is by apoptosis due to 3-MeA and/or 6-MeG (Hickman and Samson, 1999).
In a previous study it was reported that mice deficient in repair of 3-MeA adducts, 3-methyladenine DNA glycosylase (alkyladenine DNA glycosylase [Aag]) null mice, are initially protected from ß-cell damage induced by a SHDS (140 mg/kg); however, these mice developed delayed onset of diabetes with complete ß-cell loss by 8 months (Cardinal et al., 2001). In Aag–/– mice, the mechanism of STZ-induced ß-cell death switched from predominantly necrosis to apoptosis, which peaked at 48 h. After STZ treatment, Aag–/– mice retained 60% of their normal insulin level; however, by 8 months mice were considered diabetic based on hyperglycemia and insulin deficiency. Infiltration of CD4+ T cells was seen in islets, suggesting an autoimmune response as the cause for delayed onset of diabetes. This, as well as other studies, provides support for the role of ß-cell apoptosis in initiating loss of ß-cell tolerance and autoimmunity (Liadis et al., 2005; Mensah-Brown et al., 2002). It is also been shown that ß-cell apoptosis initiates priming of diabetogenic T cells (Turley et al., 2003).
To understand the mechanism for loss of ß-cell tolerance as a result of not repairing 3-MeA, we initiated studies to correlate DNA damage, cell death, and induction of an autoimmune response in STZ-treated Aag–/– mice. We confirmed that Aag–/– mice are partially protected from SHDS-induced toxicity and that ß-cell death occurs through apoptosis rather than necrosis. During the course of our studies, we were not able to reproduce the original results in which Aag–/– mice developed diabetes (Cardinal et al., 2001). However, our STZ-treated Aag–/– mice were glucose impaired throughout the study. T-cell depletion did not affect the response of Aag–/– mice to STZ implying that autoimmunity is not the underlying cause of insulin deficiency. We also showed that mice do not develop ß-cell tumors over the course of 14 months, further demonstrating that the 3-MeA adducts that accumulate in the islets of Aag null mice are cytotoxic but not promutagenic.
|
MATERIAL AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Hazardous material.
STZ is highly toxic and should be handled as a human carcinogen.
Mice.
Aag and methylguanine DNA methyltransferase (MGMT) null C57Bl/6 mice were obtained from Leona Samson (Engelward et al., 1997). Wild-type (WT) mice were obtained from the NCI laboratories (Bethesda, MD). Male mice (6–8 weeks) were used in all experiments. Mice were maintained in microisolator cages in a temperature- and humidity-controlled barrier facility and provided with autoclaved water and food ab libitum (Harland Teklad, 8656). All experiments were approved by the Institutional Animal Care and Use Committee at the University of Nebraska Medical Center.
STZ treatment.
Mice were injected ip with STZ (Fisher Scientific, Waltham, MA) solutions, prepared immediately before use, in citrate buffer (pH 4.5). The purity of the STZ was confirmed by 1H-nuclear magnetic resonance (NMR) spectrometry. Mice were injected with 140 mg/kg STZ in all experiments, while control mice received citrate buffer alone.
Blood glucose determination.
Blood glucose levels were determined by taking a drop of blood from an incision in the tip of the tail and testing with a hand-held glucometer (Lifescan Surestep Basic, J & J Lifescan, Inc. [Milpitas, CA]). When indicated, mice were fasted overnight (16–18 h) prior to taking blood glucose readings. Mice with fasting blood glucose levels above 150 mg/dl for two consecutive readings were considered diabetic. With nonfasting blood glucose readings, levels greater than 250 mg/dl were used to determine diabetes.
T-cell depletion.
CD4- and CD8-specific T cells were depleted in vivo using ip injections of anti-CD4 (GK1.5) and anti-CD8 (53-6.72) antibodies provided by Michael Hollingsworth (University of Nebraska Medical Center). Mice were injected with 0.5 mg of purified anti-CD4 and anti-CD8 antibody for three consecutive days. On the sixth day, 200 µl of blood was collected from the tail vein, and peripheral blood mononuclear cells were used to confirm depletion of CD4+ and CD8+ T cells. Cells were prepared by a standard FACs protocol (Current Protocols in Immunology, unit 5.3) and stained with antimouse CD3
(145-2C11), CD4 (H129.19), and CD8a (53-6.7) (BD Pharmingen, San Jose, CA). Samples were analyzed on a Becton Dickinson FACStarPlus flow cytometer and analyzed to confirm depletion. All control animals received rat IgG antibody (SFR3-DR5) on the same schedule as T-cell–depleted mice. One week after the T-cell depletion protocol started, mice were injected with a SHDS. The following day, mice received a final dose of anti-CD4 and anti-CD8 antibody or control antibody. Depletion was confirmed five days after STZ injection. After this time, T cells were allowed to repopulate.
Glucose tolerance test.
Prior to the glucose tolerance test, mice were fasted overnight. D-Glucose was dissolved in sterile water and filtered through a 2-µm filter before injection. Baseline blood glucose levels were measured as described above, and mice were then injected ip with 1.5 mg D-glucose/g body weight. Blood glucose levels were subsequently measured by tail vein sampling at 20 or 30, 60, 90, and 120 min after glucose loading.
Immunohistochemistry.
Mice were sacrificed by CO2 affixation, and the whole pancreas was removed and fixed overnight in 10% buffered formalin. Tissues were embedded in paraffin and sectioned (5 µm–thick sections) for immunohistochemistry. Prior to staining, sections were boiled for 10 min in 10mM citrate buffer. Staining was carried out using a VECTASTAIN ABC-AP kit (Vector Labs, Burlingame, CA) according to the manufacturer's instructions. For anti-insulin immunohistochemistry, sections were incubated in blocking serum for 20 min and immediately afterward incubated with guinea pig anti-insulin at 1:200 (Dako, Carpinteria, CA) for 1 h at room temperature. Sections were washed and incubated with secondary antibody for 1 h followed by ABC-reagent incubation for 30 min (Vector Labs). Sections were visualized with Vector Red alkaline phosphate substrate (Vector Labs) and counterstained with hematoxylin. Anti-CD3 immunohistochemistry sections were blocked with protein-free serum (Dako) for 5 min and incubated with rabbit anti-CD3 (Dako). Slides were visualized using the Dako EnVison+ System following manufacturer's instructions.
Morphometric analysis.
Insulin-immunostained pancreatic sections (5 µm thick) were examined and cross-sectional islet area analyzed. Sections were imaged using a Nikon Eclipse 90i microscope and camera, and islet area was determined using ACT-2U software. Up to 10 islets per section were measured, and three nonconsecutive sections randomly selected were examined for each mouse. Small islets consisting of less than 10 ß-cells were not included in the analysis. Overall, 261 islets were measured in the T-cell–depleted group, 234 in the isotype group, and 181 in the untreated group. On average 26 islets per mouse in each group were examined.
Apoptosis.
The location of ß-cell apoptosis in STZ-treated Aag–/– mice was identified using dual immunofluorescence for Terminal Deoxynucleotidyl Transferase–Mediated dUTP Nick End Labeling (TUNEL) and insulin staining. Paraffin-embedded tissue sections were dewaxed and rehydrated using xylene and ethanol baths. Sections were labeled for TUNEL-positive cells and visualized using Texas Red dye according to manufacturer's instructions (Roche Applied Science, Indianapolis, IN). Slides were subsequently labeled with guinea pig anti-insulin at 1:200 (Dako) for 1 h at room temperature. Insulin staining was visualized using Alexa Fluor 488 dye at a 1:200 dilution (Molecular Probes, Carlsbad, CA). Sections were mounted and examined using confocal microscopy. TUNEL-positive ß-cells were identified by the presence of red nuclei and green cytoplasm. At the 24-h time point, four STZ-treated and four untreated control mice were examined. At the 48- and 96-h time point, eight STZ-treated and seven control mice were examined: four to five islets per section were examined per mouse.
Statistics.
Blood glucose levels are reported as mean ± SEM. Values were compared using the Student's t-test or with one-way ANOVA when three groups were present. Values obtained from the morphometric analysis were analyzed using a Mann–Whitney U-test. Statistical calculations were carried out using Prism 4.0 (GraphPad, Inc.) software. Statistical significance was considered at p < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Aag–/– Mice Are Protected from the Cytotoxic Effect Induced by an SHDS
The positive control group, WT C57BL/6 mice, developed rapid onset of diabetes after a SHDS, with blood glucose levels averaging 409 mg/dl at 48 h (Fig. 1A). STZ-treated Aag–/– mice had significantly lower blood glucose levels at the same time averaging 249 mg/dl, although treated Aag–/– mice were still hyperglycemic in comparison to untreated Aag–/– mice, averaging 150 mg/dl (Fig. 1A). We observed that nonfasting blood glucose levels in Aag–/– mice were elevated shortly after STZ treatment, recovered after a few days, and at 6–10 weeks rose to levels exceeding 250 mg/dl (Fig. 1B). Upon development of hyperglycemia at 6–10 weeks, mice were sacrificed and the presence of ß-cells was determined by immunohistochemistry. Insulin staining in the pancreas of hyperglycemic mice was positive, indicating mice retained some level of functioning ß-cells (Fig. 2). No evidence of T-cell infiltration, a hallmark of the early stages of autoimmune-related diabetes, was observed between 6 and 10 weeks.
|
|
To determine the immediate effects of STZ on cell death and insulin content, Aag–/– mice were treated with STZ, and blood glucose levels and pancreas histology were examined at 24, 48, and 96 h. Apoptotic ß-cells were identified in mice as both TUNEL- and insulin-positive cells. At 24 h, apoptotic ß-cells were infrequently observed. By 48 h, a higher frequency of ß-cell apoptosis was seen in STZ-treated mice. From an average islet consisting of 200–500 cells, two to three ß-cells were positive for apoptosis (Fig. 3A). Not all TUNEL+ cells were identified as ß-cells in treated mice, and this indicates cells of other lineages also undergo increased apoptosis due to STZ exposure. Very few apoptotic ß-cells were observed in treated mice at 96 h. At all time points, no TUNEL+ ß-cells were seen in untreated control mice. In treated mice, the low frequency of TUNEL+ ß-cells indicates ß-cell apoptosis is rapid and transient. This is consistent with previous observations in which the clearance rate of apoptotic ß-cells was estimated to be 1.7 min (Kurrer et al., 1997
). While ß-cell apoptosis was observed at a relatively low frequency, the intensity of insulin staining was greatly decreased from 48 to 96 h (Fig. 3B), implying ß-cell apoptosis occurs around 48 h. These results show that Aag–/– mice are partially protected from the direct cytotoxic effect of STZ on pancreatic ß-cells and that ß-cell death occurs by apoptosis.
|
SHDS in Aag–/– Mice Results in the Development of Impaired Glucose Tolerance but Not Overt Diabetes
Aag–/– mice are protected from the early and direct necrotic effect of a SHDS; however, it was previously reported that Aag–/– mice presented with diabetes from an autoimmune response against pancreatic ß-cells 8 months after the initial treatment (Cardinal et al., 2001
). This suggested that Aag–/– mice are susceptible to developing autoimmunity during ß-cell death when self-antigens are available for cross-presentation and particularly during ß-cell apoptosis, which is most apparent 48 h after STZ injection. In order to examine if an autoimmune response was generated by STZ-induced cell death in Aag–/– mice, T-cell depletion was combined with a SHDS. Flow cytometry analysis showed mice were depleted of T cells the day before and at 1 week after STZ injection (data not shown). Shortly after an SHDS, T-cell–depleted and isotype control mice exhibited similar response patterns. Blood glucose levels were elevated starting at 48 h after STZ injection and returned to near normal levels after 1 week. Mice had intermittent hyperglycemia throughout the first 6 weeks, but no difference was observed between the T-cell–depleted and isotype control groups. To minimize variation between mice, fasting blood glucose levels were checked once a month after 6 weeks to diagnosis overt diabetes. Mice with fasting blood glucose levels greater than 150 mg/dl were considered overtly diabetic. In the STZ-treated mice, both T-cell–depleted and isotype control groups, overt diabetes was not observed over a period of 14 months after a SHDS. Blood glucose levels were slightly, but significantly, elevated compared with untreated controls; however, none of the mice showed blood glucose levels exceeding 150 mg/dl (Fig. 4A). A glucose tolerance test was done at 6 and 12 months, and it was determined that the STZ-treated Aag–/– mice had impaired glucose tolerance since both the T-cell–depleted and isotype control groups show an increased hyperglycemic response after the glucose bolus. Additionally, blood glucose levels in both the STZ-treated Aag–/– groups did not recover as quickly as control mice and remained above baseline levels after 120 min (Figs. 4B and C). For the duration of the study, which lasted 14 months, STZ-treated Aag–/– mice exhibited mild fasting hyperglycemia and impaired glucose tolerance. However, no difference was observed between the T-cell–depleted and isotype control groups, indicating an autoimmune response was not the underlying cause of insulin deficiency.
|
Islet Size Is Reduced in STZ-Treated Aag–/– Mice
Upon termination of the experiment at 14 months, histology from Aag–/– STZ-treated mice demonstrated the presence of well-defined islets in the pancreas. Anti-insulin immunohistochemistry showed the overall percentage of ß-cells within islets was similar in STZ-treated and untreated control mice (Fig. 5). However, from morphometric analysis it was determined the average cross-sectional area of islets in STZ-treated mice was significantly reduced in comparison to untreated controls (Fig. 6). The median islet area in STZ-treated mice was 44,488 and 44,131 µm2 in the T-cell–depleted and the isotype groups, respectively, compared with 77,152 µm2 in untreated mice. These results imply the glucose-impaired state observed in STZ-treated mice is due to smaller islets, which correlates to fewer ß-cells. No evidence of insulinomas was observed at 14 months, based upon blood glucose readings and histological analysis.
|
|
Pancreas-Specific Autoimmune Lesions Are Observed in Aged Aag–/– Mice
While we did not confirm that Aag–/– mice develop diabetes in response to SHDS, we did observe that as Aag–/– mice aged, an autoimmune destruction of the pancreas occurred that was independent of STZ treatment. In 16-month-old STZ-treated and untreated Aag–/– mice, pockets of activated lymphocytes were found in association with islet and acinar tissue. Pancreatic sections were stained with anti-CD3, and infiltrating lymphocytes were shown to consist primarily of T cells (Fig. 7). We also observed pockets of infiltrating immune cells in 10-month-old untreated Aag–/– mice. The severity of immune infiltrate increased with age, indicating further immune dysregulation occurs with aging in C57BL/6 mice. Our findings are in agreement with a previous report demonstrating that organ-specific autoimmune lesions, including pancreatic, increase with age in C57BL/6 mice and are observed in conjunction with islet cell autoantibodies (Hayashi et al., 1989
). Although pockets of infiltrating T cells are observed in STZ-treated Aag–/– mice at a late time point, this phenomenon does not appear to contribute to the observed hyperglycemia as untreated mice also have autoimmune lesions but remain euglycemic.
|
MGMT–/– Mice Are Sensitive to the Cytotoxic Effects of an SHDS
The role of 6-MeG adducts in ß-cell death was investigated because environmental toxins such as N-nitroso compounds (e.g., STZ) that generate 6-MeA have been postulated to be involved in diabetes development (reviewed in Akerblom et al., 2002
). It was found that levels of MGMT repair protein are low in pancreatic ß-cells; however, mRNA levels for MGMT were significantly elevated in response to DNA damage and lesions were effectively repaired, indicating ß-cells respond to 6-MeG adduct formation (Nelson et al., 1993). Since 6-MeG adducts induce apoptosis via a MGMT/mismatch repair–dependent pathway, we hypothesized unrepaired 6-MeG adducts may induce an enhanced apoptotic response in ß-cells. At 24 h after SHDS injection, blood glucose levels in MGMT–/– mice were normal; however, at 48 h blood glucose levels in both MGMT–/– and WT C57BL/6 mice exceeded 300 mg/dl (Fig. 8). Histology showed MGMT–/– mice responded similarly to WT mice, where islet size was decreased and insulin staining was nearly absent (data not shown). The lack of increased sensitivity in STZ-treated MGMT–/– mice indicates necrotic ß-cell death induced by repair of 3-MeA masks any effect of MGMT deficiency. Further complicating this study is the acute bone marrow toxicity that SHDS induced in MGMT–/– mice. In comparison to WT mice, MGMT–/– mice have increased sensitivity to various alkylating agents, resulting in bone marrow toxicity (Glassner et al., 1999). This complication eliminates the possibility of examining the effects of SHDS treatment in double Aag–/–/MGMT–/– mice. Further studies in this model would require bone marrow transplantation into STZ-treated mice, which has been shown to be effective in protecting MGMT–/– mice against N-methyl-N-nitrosourea (MNU)–induced toxicity (Reese et al., 2001).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Apoptosis of ß-cells has been suggested to be a critical event in the pathogenesis of type 1 diabetes (O'Brien et al., 1996
, 1997). To further probe the relationship between DNA damage, ß-cell apoptosis, and onset of autoimmunity, we used Aag–/– mice that are defective in the repair of 3-MeA, a cytotoxic DNA adduct (Varadarajan et al., 2003) formed as a result of STZ exposure. Our goal was to understand how induction of apoptosis in response to a ß-cell toxicant leads to the loss of tolerance and diabetes as reported by Cardinal et al., since this situation may be analogous to induction of diabetes by environmental cytotoxic agents, in particular, viruses and toxins. We also hoped to gain insight into how cytotoxic DNA alkylating agents, many of which are used in cancer chemotherapy, could be employed against tumor cells to generate antitumor-specific immune responses.
In the studies reported herein, we confirmed a previous report that ß-cells in Aag–/– mice are partially protected from the cytotoxic effect of a SHDS and that the mechanism of toxicity switches from necrosis in WT mice to apoptosis in the repair mutant. However, in our studies the switch to ß-cell apoptosis in the Aag–/– mice resulted in impaired glucose tolerance, not overt diabetes. The fact that Aag–/– mice are partially protected from the cytotoxic effects of STZ is counterintuitive since Aag–/– embryonic stem cells (ES) cells are more sensitive to killing by methyl methanesulfonate and MeOSO2(CH2)2-lexitropsin, the latter a methylating agent that selectively generates 3-MeA adducts (Engelward et al., 1996; Kelly et al., 1998). In addition, unrepaired 3-MeA adducts induced multiple genotoxic events, encompassing sister chromatid exchange and chromatid and chromosome gaps. These events lead to S phase arrest, p53 induction, and cell apoptosis (Engelward et al., 1998). However, while Aag–/– ES cells and mouse embryo fibroblasts are more sensitive to the cytotoxic effect of alkylating agents, it is known that bone marrow cells from Aag–/– mice become more resistant. Furthermore, the effect was lineage specific in Aag–/– mice: myeloid cells were protected while lymphoid cells were sensitive (Roth and Samson 2002). These results, and others showing that ß-cells are protected from alkylation-induced damage, demonstrate that the knockout of Aag glycosylase confers a survival advantage in these cells, in comparison to BER competent cells. Since ß-cells are protected from STZ-induced cytotoxicity in Aag–/– mice, we reconfirmed that 3-MeA adducts are, at least, partially tolerated by pancreatic ß-cells and that cell death induced by a SHDS in islets of WT mice requires repair intermediates generated downstream of 3-MeA.
The effects of STZ treatment in Aag–/– mice contrasts with results observed in PARP-inhibited rats, although both models behave similarly early on. PARP, which is activated in response to DNA-strand breaks formed during BER, catalyzes poly(adenosine 5' diphosphate–ribosyl)ation of various proteins associated with DNA metabolism (D'Amours et al., 1999). Inhibition of PARP initially results in a similar phenotype to STZ-treated Aag–/– mice. Both rats and mice treated with a SHDS and nicotinamide, a PARP inhibitor, are protected from the rapid onset of diabetes. Also, both models develop a type 2 diabetes phenotype characterized by mild hyperglycemia, decreased pancreatic insulin content, and abnormal glucose tolerance (Masiello et al., 1998; Nakamura et al., 2006). The detrimental effects of PARP inhibition are only seen starting 12 months after STZ injection. In rat models treated with STZ and different PARP inhibitors, rats are mildly hyperglycemic until 12 months when hypoglycemia was observed (Rakieten et al., 1971; Yamagami et al., 1985). In both studies, a high incidence of insulinomas was observed (64% and 80%), and these studies are cautionary to the use of nicotinamide to prevent diabetes in clinical trials. STZ-treated PARP–/– mice are also protected from the onset of diabetes (Burkart et al., 1999; Masutani et al., 1999; Pieper et al., 1999); however, no information is available regarding tumor incidence in PARP–/– mice since animals were followed for only 60 days in the longest study. When STZ-treated Aag–/– mice in our study did not develop autoimmunity after 8 months, we followed the mice for an additional 6 months to monitor for the development of ß-cell tumors. Tumors were not found upon autopsy and histopathological analysis. Considering that the time frame for tumor development in rats is between 10 and 13 months (Rakieten et al., 1971; Yamagami et al., 1985), our study period of 14 months should be sufficient to rule out the possible development of insulinomas, and we conclude that 3-MeA is not a promutagenic lesion in vivo.
This raises the question concerning the DNA adduct(s) that contributes to cancer in STZ-treated rats. STZ induces renal and liver tumors at (50 mg/kg by iv) doses (Arison and Feudale, 1967; Iwase et al., 1989; Rakieten et al., 1968), and insulinomas at 37.5–150 mg/kg ip, depending on rat strain, in combination with PARP inhibitors (Rakieten et al., 1971; Yamagami et al., 1985). The quantitatively predominant DNA lesion produced by STZ, 7-MeG, is only slowly repaired by Aag and appears to be excised by other glycosylases (Smith and Engelward, 2000). Another adduct generated by STZ, 6-MeG, is recognized specifically by MGMT, and is postulated to be the major promutagenic lesion generated by STZ. While MGMT levels are very low in ß-cells, there is limited ex vivo evidence indicating 6-MeG adducts are effectively repaired in MNU-treated ß-cells (Nelson et al., 1993). Since the formation and repair of 6-MeG would be unaffected in the STZ-treated Aag–/– mice, it seems unlikely that 6-MeG is responsible for insulinomas. Alternatively, BER intermediates, that is, abasic sites, 3'-hydroxyl and 5'-deoxyribose-5-phosphate residues, generated from repair of 3-MeA, which are cytotoxic (Sobol et al., 2003), have also been shown to be promutagenic in mammalian cells (Simonelli et al., 2005). Promutagenic BER intermediates that are downstream of 3-MeA are not generated in the Aag–/– mouse model, while they would be in PARP-inhibited animals. If this is so, BER intermediates may contribute to tumor development in PARP-inhibited rats, in the presence of functional 6-MeG repair.
The other issue that our work raises is why STZ-treated Aag–/– mice do not develop autoimmunity in response to apoptotic ß-cell death? We do not believe the impaired glucose intolerance observed in Aag–/– mice was due to a partial autoimmune response because the hallmark of autoimmune diabetes is complete destruction of ß-cells. Furthermore, recent evidence suggests ß-cells have limited replication capacity and new ß-cells arise from existing cells (Dor et al., 2004). Our work implies that Aag–/– mice were never able to fully compensate for the STZ-induced loss of ß-cells, which led to glucose intolerance.
Our failure to observe an autoimmune response in SHDS-treated Aag–/– mice could be due to several factors. First, C57BL/6 mice do not harbor highly penetrant diabetes susceptibility genes as indicated by the lack of spontaneous disease. This suggests that to break tolerance in C57BL/6 mice, a greater stimulus is required than in more susceptible strains. Second, multiple insults may be required to induce diabetes when using cytotoxic agents. Notably, Aag–/– mice, similar to WT mice, do develop autoimmune diabetes in response to MLDS treatment (Cardinal et al., 2001). Additionally, in the C57BL/6 mouse, combined single low-dose STZ treatment and Coxsackie virus infection induces diabetes, whereas neither treatment on its own is sufficient (Toniolo et al., 1980). Lastly, an SHDS has been shown to have immunosuppressive properties (Nichols et al., 1979). Supporting this finding, we have preliminary evidence that T cells are slightly depleted after a SHDS in Aag–/– mice. Considering STZ elicits immunosuppressive effects, it would be unlikely that an autoimmune response could be generated under these conditions.
An alternative explanation for the lack of autoimmunity in our studies focuses not on the initial damage, but on the cumulative damage sustained. Prolonged periods of hyperglycemia can result in glucose toxicity to ß-cells, where hyperglycemia leads to downregulation of insulin gene expression and eventually to the loss of ß-cells (Robertson, 2004). Glucose toxicity is a viable explanation for the development of autoimmunity observed by Cardinal et al., although, they reported no blood glucose levels between the initial STZ injection and development of diabetes at 8 months. If the mechanism of ß-cell loss over time was due to glucose toxicity, retention of ß-cells in our model could be explained in two ways. First, it is possible that the degree of initial toxicity induced in our Aag–/– mice, which were created independently of those used by Cardinal et al., could have been somewhat reduced, leading to more stable blood glucose levels throughout the study and lower glucose toxicity. Second, dietary factors can stress ß-cells, impacting glucose tolerance and insulin secretion. Accordingly diets higher in fat or caloric content can have detrimental consequences on ß-cells while lower fat or lower calorie diets are protective. Mice in our study were maintained on a standard 4% fat diet. Unfortunately no information was available regarding diet in the Cardinal et al., study, so we were unable to compare possible consequences due to diet.
Partial protection against STZ-induced ß-cell toxicity in the Aag null mice confirms that unrepaired 3-MeA adducts in ß-cells confer a survival advantage by preventing necrosis while causing only moderate levels of apoptosis. Clearly, our data show that the one-time induction of even significant levels of apoptosis is not sufficient to trigger loss of ß-cell tolerance. Additionally, it was shown that persistent 3-MeA lesions do not sensitize the mice to the development of islet cell tumors. This would suggest that cytotoxic agents that selectively generate 3-MeA may have a clinical advantage to current agents that produce multiple lesions, including 6-MeG. These results, therefore, have implications in both the search for environmental agents that contribute to diabetes and in the development of tumor immunotherapies.
|
ACKNOWLEDGMENTS |
|---|
We are grateful to Leona Samson for the Aag null mice and Michael Hollingsworth for hybridoma cell lines. This work was supported by the National Institutes of Health (RO1 CA 29088, P30 CA36727, and T32 CA09476).
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Akerblom HK, Vaarala O, Hyoty H, Ilonen J, Knip M. (2002) Environmental factors in the etiology of type 1 diabetes. Am. J. Med. Genet. 115:18–29.[CrossRef][ISI][Medline]
Arison RN and Feudale EL. (1967) Induction of renal tumour by streptozotocin in rats. Nature 214:1254–1255.[CrossRef][Medline]
Bolzan AD and Bianchi MS. (2002) Genotoxicity of streptozotocin. Mutat. Res. 512:121–134.[CrossRef][ISI][Medline]
Burkart V, Wang ZQ, Radons J, Heller B, Herceg Z, Stingl L, Wagner EF, Kolb H. (1999) Mice lacking the poly(ADP-ribose) polymerase gene are resistant to pancreatic beta-cell destruction and diabetes development induced by streptozocin. Nat. Med. 5:314–319.[CrossRef][ISI][Medline]
Cardinal JW, Margison GP, Mynett KJ, Yates AP, Cameron DP, Elder RH. (2001) Increased susceptibility to streptozotocin-induced beta-cell apoptosis and delayed autoimmune diabetes in alkylpurine-DNA-N-glycosylase-deficient mice. Mol. Cell. Biol. 21:5605–5613.
D'Amours D, Desnoyers S, D'Silva I, Poirier GG. (1999) Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions. Biochem. J. 342:Pt 2249–268.
Dor Y, Brown J, Martinez OI, Melton DA. (2004) Adult pancreatic beta-cells are formed by self-duplication rather than stem-cell differentiation. Nature 429:41–46.[CrossRef][Medline]
Engelward BP, Allan JM, Dreslin AJ, Kelly JD, Wu MM, Gold B, Samson LD. (1998) A chemical and genetic approach together define the biological consequences of 3-methyladenine lesions in the mammalian genome. J. Biol. Chem. 273:5412–5418.
Engelward BP, Dreslin A, Christensen J, Huszar D, Kurahara C, Samson L. (1996) Repair-deficient 3-methyladenine DNA glycosylase homozygous mutant mouse cells have increased sensitivity to alkylation-induced chromosome damage and cell killing. EMBO J. 15:945–952.[ISI][Medline]
Engelward BP, Weeda G, Wyatt MD, Broekhof JL, de Wit J, Donker I, Allan JM, Gold B, Hoeijmakers JH, Samson LD. (1997) Base excision repair deficient mice lacking the Aag alkyladenine DNA glycosylase. Proc. Natl. Acad. Sci. U.S.A. 94:13087–13092.
Glassner BJ, Weeda G, Allan JM, Broekhof JL, Carls NH, Donker I, Engelward BP, Hampson RJ, Hersmus R, Hickman R, et al. (1999) DNA repair methyltransferase (Mgmt) knockout mice are sensitive to the lethal effects of chemotherapeutic alkylating agents. Mutagenesis 14:339–347.
Hayashi Y, Utsuyama M, Kurashima C, Hirokawa K. (1989) Spontaneous development of organ-specific autoimmune lesions in aged C57BL/6 mice. Clin. Exp. Immunol. 78:120–126.[ISI][Medline]
Hickman MJ and Samson LD. (1999) Role of DNA mismatch repair and p53 in signaling induction of apoptosis by alkylating agents. Proc. Natl. Acad. Sci. U.S.A. 96:10764–10769.
Iwase M, Nunoi K, Sadoshima S, Kikuchi M, Fujishima M. (1989) Liver, kidney and islet cell tumors in spontaneously hypertensive and normotensive rats treated neonatally with streptozotocin. Tohoku J. Exp. Med. 159:83–90.[ISI][Medline]
Kelly JD, Shah D, Chen FX, Wurdeman R, Gold B. (1998) Quantitative and qualitative analysis of DNA methylation at N3-adenine by N-methyl-N-nitrosourea. Chem. Res. Toxicol. 11:1481–1486.[CrossRef][ISI][Medline]
Kurrer MO, Pakala SV, Hanson HL, Katz JD. (1997) Beta cell apoptosis in T cell-mediated autoimmune diabetes. Proc. Natl. Acad. Sci. U.S.A. 94:213–218.
Liadis N, Murakami K, Eweida M, Elford AR, Sheu L, Gaisano HY, Hakem R, Ohashi PS, Woo M. (2005) Caspase-3-dependent beta-cell apoptosis in the initiation of autoimmune diabetes mellitus. Mol. Cell. Biol. 25:3620–3629.
Masiello P, Broca C, Gross R, Roye M, Manteghetti M, Hillaire-Buys D, Novelli M, Ribes G. (1998) Experimental NIDDM: development of a new model in adult rats administered streptozotocin and nicotinamide. Diabetes 47:224–229.[Abstract]
Masutani M, Suzuki H, Kamada N, Watanabe M, Ueda O, Nozaki T, Jishage K, Watanabe T, Sugimoto T, Nakagama H, et al. (1999) Poly(ADP-ribose) polymerase gene disruption conferred mice resistant to streptozotocin-induced diabetes. Proc. Natl. Acad. Sci. U.S.A. 96:2301–2304.
Mathis D, Vence L, Benoist C. (2001) Beta-cell death during progression to diabetes. Nature 414:792–798.[CrossRef][Medline]
Mensah-Brown EP, Stosic Grujicic S, Maksimovic D, Jasima A, Shahin A, Lukic ML. (2002) Downregulation of apoptosis in the target tissue prevents low-dose streptozotocin-induced autoimmune diabetes. Mol. Immunol. 38:941–946.[CrossRef][ISI][Medline]
Murata M, Takahashi A, Saito I, Kawanishi S. (1999) Site-specific DNA methylation and apoptosis: Induction by diabetogenic streptozotocin. Biochem. Pharmacol. 57:881–887.[CrossRef][ISI][Medline]
Nakamura T, Terajima T, Ogata T, Ueno K, Hashimoto N, Ono K, Yano S. (2006) Establishment and pathophysiological characterization of type 2 diabetic mouse model produced by streptozotocin and nicotinamide. Biol. Pharm. Bull. 29:1167–1174.[CrossRef][ISI][Medline]
Nelson JW, Ledoux SP, Wilson GL. (1993) Repair of O6-methylguanine in rat pancreatic beta-cells after exposure to N-methyl-N-nitrosourea. Diabetes 42:1187–1194.[Abstract]
Nichols WK, Spellman JB, Vann LL, Daynes RA. (1979) Immune responses of diabetic animals. Direct immunosuppressant effects of streptozotocin in mice. Diabetologia 16:51–57.[CrossRef][ISI][Medline]
O'Brien BA, Harmon BV, Cameron DP, Allan DJ. (1996) Beta-cell apoptosis is responsible for the development of IDDM in the multiple low-dose streptozotocin model. J. Pathol. 178:176–181.[CrossRef][ISI][Medline]
O'Brien BA, Harmon BV, Cameron DP, Allan DJ. (1997) Apoptosis is the mode of beta-cell death responsible for the development of IDDM in the nonobese diabetic (NOD) mouse. Diabetes 46:750–757.[Abstract]
Pieper AA, Brat DJ, Krug DK, Watkins CC, Gupta A, Blackshaw S, Verma A, Wang ZQ, Snyder SH. (1999) Poly(ADP-ribose) polymerase-deficient mice are protected from streptozotocin-induced diabetes. Proc. Natl. Acad. Sci. U.S.A. 96:3059–3064.
Rakieten N, Gordon BS, Beaty A, Cooney DA, Davis RD, Schein PS. (1971) Pancreatic islet cell tumors produced by the combined action of streptozotocin and nicotinamide. Proc. Soc. Exp. Biol. Med. 137:280–283.[Medline]
Rakieten N, Gordon BS, Cooney DA, Davis RD, Schein PS. (1968) Renal tumorigenic action of streptozotocin (NSC-85998) in rats. Cancer Chemother. Rep. 52:563–567.[ISI][Medline]
Reese JS, Qin X, Ballas CB, Sekiguchi M, Gerson SL. (2001) MGMT expression in murine bone marrow is a major determinant of animal survival after alkylating agent exposure. J. Hematother. Stem Cell Res. 10:115–123.[CrossRef][ISI][Medline]
Robertson RP. (2004) Chronic oxidative stress as a central mechanism for glucose toxicity in pancreatic islet beta cells in diabetes. J. Biol. Chem. 279:42351–42354.
Roth RB and Samson LD. (2002) 3-Methyladenine DNA glycosylase-deficient Aag null mice display unexpected bone marrow alkylation resistance. Cancer Res. 62:656–660.
Simonelli V, Narciso L, Dogliotti E, Fortini P. (2005) Base excision repair intermediates are mutagenic in mammalian cells. Nucleic Acids Res. 33:4404–4411.
Smith SA and Engelward BP. (2000) In vivo repair of methylation damage in Aag 3-methyladenine DNA glycosylase null mouse cells. Nucleic Acids Res. 28:3294–3300.
Sobol RW, Kartalou M, Almeida KH, Joyce DF, Engelward BP, Horton JK, Prasad R, Samson LD, Wilson SH. (2003) Base excision repair intermediates induce p53-independent cytotoxic and genotoxic responses. J. Biol. Chem. 278:39951–39959.
Toniolo A, Onodera T, Yoon JW, Notkins AL. (1980) Induction of diabetes by cumulative environmental insults from viruses and chemicals. Nature 288:383–385.[CrossRef][Medline]
Turley S, Poirot L, Hattori M, Benoist C, Mathis D. (2003) Physiological beta cell death triggers priming of self-reactive T cells by dendritic cells in a type-1 diabetes model. J. Exp. Med. 198:1527–1537.
Varadarajan S, Shah D, Dande P, Settles S, Chen FX, Fronza G, Gold B. (2003) DNA damage and cytotoxicity induced by minor groove binding methyl sulfonate esters. Biochemistry 42:14318–14327.[CrossRef][Medline]
Yamagami T, Miwa A, Takasawa S, Yamamoto H, Okamoto H. (1985) Induction of rat pancreatic B-cell tumors by the combined administration of streptozotocin or alloxan and poly(adenosine diphosphate ribose) synthetase inhibitors. Cancer Res. 45:1845–1849.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||