Toxicology and Applied Pharmacology 232 (2008) 119–124

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Toxicology and Applied Pharmacology

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Methadone ameliorates multiple-low-dose streptozotocin-induced type 1 diabetesin mice

K. Amirshahrokhi a, A.R. Dehpour a, J. Hadjati b, M. Sotoudeh c, M. Ghazi-Khansari a,⁎a Department of Pharmacology School of Medicine, Tehran University of Medical Sciences, Tehran, Iranb Department of Immunology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iranc Department of Pathology, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

⁎ Corresponding author. Fax: +9821 6640 2569.E-mail addresses: ghazikha@sina.tums.ac.ir, khansag

(M. Ghazi-Khansari).

0041-008X/$ – see front matter © 2008 Elsevier Inc. Aldoi:10.1016/j.taap.2008.06.020

a b s t r a c t

a r t i c l e i n f o

Article history:

Type 1 diabetes is an autoim Received 30 January 2008Revised 23 June 2008Accepted 30 June 2008Available online 11 July 2008

Keywords:MethadoneMultiple-low-dose-streptozotocinType 1 diabetesCytokines

mune disease characterized by inflammation of pancreatic islets and destructionof β cells by the immune system. Opioids have been shown to modulate a number of immune functions,including T helper 1 (Th1) and T helper 2 (Th2) cytokines. The immunosuppressive effect of long-termadministration of opioids has been demonstrated both in animal models and humans. The aim of this studywas to determine the effect of methadone, a μ-opioid receptor agonist, on type 1 diabetes. Administration ofmultiple low doses of streptozotocin (STZ) (MLDS) (40mg/kg intraperitoneally for 5 consecutive days) to miceresulted in autoimmune diabetes. Mice were treated with methadone (10mg/kg/day subcutaneously) for24days. Blood glucose, insulin and pancreatic cytokine levels were measured. Chronic methadone treatmentsignificantly reduced hyperglycemia and incidence of diabetes, and restored pancreatic insulin secretion inthe MLDS model. The protective effect of methadone can be overcome by pretreatment with naltrexone, anopioid receptor antagonist. Also, methadone treatment decreased the proinflammatory Th1 cytokines[interleukin (IL)-1β, tumor necrosis factor-α and interferon-γ] and increased anti-inflammatory Th2cytokines (IL-4 and IL-10). Histopathological observations indicated that STZ-mediated destruction of β cellswas attenuated by methadone treatment. It seems that methadone as an opioid agonist may have aprotective effect against destruction of β cells and insulitis in the MLDS model of type 1 diabetes.

© 2008 Elsevier Inc. All rights reserved.

Introduction

Insulin-dependent diabetes mellitus (IDDM) is an autoimmunedisease. Type 1 diabetes results from specific destruction of theinsulin-producing β-cells in the pancreatic islets of Langerhans by theimmune system (Bach, 1994). It is believed that destruction ofpancreatic islet β cells and consequent IDDM is the result of perturbedimmune regulation. Multiple environmental and genetic factors makethe immune cells, particularly T lymphocytes to invade islet β cellsand cause pancreatic inflammation (Rabinovitch and Suarez-Pinzon,1998). This inflammatory response is known as insulitis (Pukel et al.,1988). Cytotoxicity of T cells towards islet β cells is generated bycytokines and free radicals. Autoreactive T helper 1 (Th1) cells andproinflammatory cytokines [interferon (IFN)-γ, interleukin (IL)-1 andtumor necrosis factor (TNF)-α] are pathogenic to islet β cells, whereasT helper 2 (Th2) cells and their cytokines (IL-4 and IL-10) areprotective against destruction of β cells (Rabinovitch, 1998). Blockingthe function of type 1 cytokines and increasing type 2 cytokines can

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reduce the development of IDDM in rodent models (Rabinovitch andSuarez-Pinzon, 1998).

Multiple lowdoses of streptozotocin (STZ) (MLDS) canbe used as ananimal model for type 1 diabetes. STZ is a potent alkylating agent anddamages islet β cells selectively by two different mechanisms. First,when given in a single high dose, it rapidly destroys islet β cells bydirect cytotoxic action, most probably due to DNA alkylation. Second,when STZ is given inmultiple lowdoses, it induces inflammation of theislets by immune cells, with subsequent destruction of β cells andprogressive hyperglycemia within a few days. This model of diabetesshares many histological and clinical features with human type 1diabetes and requires the participation of macrophages and T cells(Like and Rossini, 1976; Rossini et al., 1978; Papaccio et al., 2000).Therefore, the MLDS model has been applied extensively to study theimmune pathways (e.g. cytokine signaling, Fas ligand transduction andcytokine-induced β cell apoptosis) involved in destruction of islet βcells (Kawasaki et al., 2004; Rees and Alcolado, 2005).

It is believed that the endogenous opioid system regulates immunefunctions. Previous studies have established the presence of opioidreceptors (μ, κ and δ) on cells of the immune system (Mehrishi andMills, 1983). Many animal and human studies have shown that opioidsinduce immunosuppressive effects, and chronic opioid use has beenassociated with an increased incidence of infection (Budd, 2006).

Fig. 1. Effect of methadone treatment on MLDS-induced hyperglycemia (A) andincidence of diabetes (B) in mice. STZ (40 mg/kg/day ip) was injected during fiveconsecutive days. Methadone (10 mg/kg/day sc) was administered from 3 days beforethe first STZ injection for 24 days. Day 1 was defined as the day of the first injection ofSTZ. Blood glucose was measured on days 0, 7, 14 and 21. The incidence of diabetes wasexpressed as an accumulative percentage of mouse blood glucose levels greater than200 mg/dl. Values are means±SEM for eight mice. ⁎Pb0.05; ⁎⁎Pb0.01 vs. vehicle-treated mice; †Pb0.05 vs. MLDS-treated mice.

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These drugs can suppress NK-cell, macrophage and T-lymphocytefunctions (Bryant and Roudebush, 1990; McCarthy et al., 2001). It hasbeen shown that chronic morphine treatment inhibits Th1 cytokinesand upregulates Th2 cytokines. Moreover chronic morphine treatmentin vivo or in vitro decreases IFN-γ, IL-1 and TNF-α production andincreases IL-4 and IL-10protein levels (Royet al.,1998, 2001, 2004, 2005).

Previous pharmacological studies have suggested that the inhibi-tory action of opioids on immune responses is mediated by opioidreceptors, mostly μ-opioid receptors. Immunosuppressive actions ofopioids are absent in μ-opioid receptor-deficient mice and are alsoblocked by opioid antagonists (Budd 2006; Gaveriaux-Ruff et al.,1998). It has been demonstrated that, like other opioids, methadonehas immunosuppressive effects (Li et al., 2002). Methadone is a potentμ-opioid receptor agonist and widely used for the treatment of drugusers with opioid dependence. We hypothesized that methadone, dueto its immunosuppressive activity, may affect autoimmune diseasessuch as type 1 diabetes. Therefore, in this study, we decided toinvestigate whether methadone treatment could prevent the devel-opment of type 1 diabetes in mice.

Methods

Animal and chemicals. Male BALB/c mice 6–7weeks old, weighing20–25g, were purchased from the Pasteur Institute of Iran. The micehad free access to tap water and ad libitum food, and were housed in aroomwith a 12-h light/dark cycle. All animal procedures were carriedout in accordance with the National Institute of Health's Guide for theCare and Use of Laboratory Animals (NIH publication 85-23, revised1985). All the chemicals were purchased from Sigma.

Animal treatments. Male BALB/c mice were randomly divided intofour groups. The mice in the first and third groups receivedsubcutaneous (sc) injections of methadone (10mg/kg/day). Three dayslater, the mice in the first and second groups were treated with STZ at40mg/kg/day intraperitoneally for 5 consecutive days. STZwas adminis-teredwithin 10min of its dissolution (Like andRossini 1976;Karabatas etal., 2005). The mice in the fourth group received citrate buffer (vehicle)and were used as untreated controls. Blood glucose levels weremeasured on days 0, 7, 14 and 21 (in relation to the first STZ dose). Theblood samples were obtained from the tail vein of non-fasted mice andglucose wasmeasured by a blood glucose meter (Accu-Chech Active). Inour study, multiple low doses of STZ did not lead to animal death.

Mice were considered diabetic when non-fasting blood glucoselevels were N200 mg/dl for two consecutive days. Mice wereanesthetized and pancreata were removed on day 21 for cytokineand histological analysis. Blood was also collected and centrifuged.The plasma was separated and stored at − 70 °C until insulin assay.

Cytokine assay. Pancreas tissue samples were removed from miceand snap frozen in liquid nitrogen. The samples (100mg) werehomogenized in 800μl Tris–HCl buffer containing protease inhibitors(Mabley et al., 2002, 2003). All homogenized samples were centri-fuged (16,000g, 4 °C) in a refrigerated centrifuge for 30min, and thesupernatant was taken and frozen at −70 °C. Supernatant sampleswere analyzed for murine cytokine concentrations using specificELISA kits (Bender MedSystems GmbH). Cytokine levels in thepancreas were expressed as pg cytokine/mg protein, which weredetermined using the Bradford method (Bradford, 1976).

Plasma insulin determination. Non-fasting blood samples werecollected on day 21 in heparinized tubes. Plasma was separated andassayed for insulin concentration. The insulin level of plasma wasdetermined using a commercial Mouse Insulin ELISA kit (Mercodia).

Histological examination. Histological examination of mouse pan-creas from each study groupwas carried out to evaluate the severity of

insulitis at day 21. Pancreatawere removed and a half of each pancreaswas fixed with 10% formalin, embedded in paraffin and stained withhematoxylin and eosin (H&E).

Statistical analysis. Data are expressed as means±SEM. Statisticalanalysis was performed using one-way ANOVA followed by Student's ttest. P b 0.05 was considered significant.

Results

Effects of methadone on MLDS-induced hyperglycemia and diabetes

In order to determine whether methadone prevents STZ-induceddiabetes, blood glucose level was measured in all four groups of mice.Whenmicewere injectedwithmultiple sub-diabetogenic doses of STZ,they presented a progressive rise in blood glucose concentration (Fig.1A) and the increased incidence of diabetes (Fig.1B). Administration ofmethadone (10mg/kg) from 3days before the first STZ injection for24days reduced incidence of hyperglycemia and diabetes. Control andmethadone-treated mice remained normoglycemic throughout thestudy. Experiments with various doses of methadone showed that thethreshold dose for a significant inhibitory effect on blood glucose level

Fig. 2. Naltrexone antagonized the suppressive effect of methadone on MLDS-inducedhyperglycemia. Mice were administered naltrexone (10 mg/kg/day sc) 60 min prior tomethadone. Day 1 was defined as the day of the first injection of STZ. Blood glucose wasmeasured on days 0, 7, 14 and 21. Values are means±SEM for six mice. ⁎Pb0.05compared with MLDS+methadone group.

Fig. 3. Effect of methadone treatment on serum insulin levels in four study groups.Methadone (10 mg/kg/day sc) significantly decreased the effect of MLDS on serum insulinlevels. The insulin level of plasma was determined on day 21. Values are means±SEM.(n=4–6). ⁎Pb0.01 vs. vehicle-treated mice; †Pb0.01 vs. MLDS-treated mice.

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was 10mg/kg. Lower doses of methadone (3 and 6mg/kg/day) did notsignificantly decreaseMLDS-induced hyperglycemia (data not shown).We used naltrexone as a non-selective opioid receptor antagonist todetermine thatmethadone exerts its effect through an opioid receptor.The half-life of methadone is ∼ 2h in mice (Levier et al., 1995).Naltrexone is a long-acting opioid antagonist with a half-life of 4h.Naltrexone is metabolized to its activemetabolite 6-naltrexol, which isa weaker antagonist but has a longer half-life. It has been shown thatnaltrexone at a dosage of 10mg/kg/day blocks opioid receptorsblockade that lasts 24h (Zagon et al., 1983). Treatment of mice withopioid receptor antagonist naltrexone (10mg/kg/day sc, 1h prior tomethadone) prevented the effect of methadone on MLDS-inducedhyperglycemia (Fig. 2).

Determination of serum insulin levels on day 21 showed that inMLDS-treated mice, serum insulin level was dramatically decreased(Fig. 3). Methadone prevented the MLDS-induced decrease in seruminsulin. Administration of vehicle and methadone alone had no effecton serum insulin levels.

Effects of methadone and MLDS on pancreatic cytokines

To study the mechanism of methadone-induced protection againstMLDS-induced diabetes, we measured the levels of Th1 and Th2cytokines in supernatants of pancreatic tissues from each study groupon day 21. As shown in Fig. 4, MLDS injections caused significantelevation in the pancreatic levels of TNF-α, IFN-γ and IL-1β (Th1cytokines) as compared with those in the control group. Treatment ofmicewithmethadonemarkedly reducedMLDS-induced production ofTNF-α, IFN-γ and IL-1β, as compared with that in MLDS-treated mice.Administration of methadone alone did not influence Th1 cytokinelevels when compared to mice receiving vehicle. The decreasing effectof MLDS on pancreatic levels of IL-4 and IL-10 (Th2 cytokines) was notstatistically significant. There was remarkable enhancement of IL-4cytokine level in mice treated with methadone plus MLDS. There wasalso a trend toward an increase in IL-10 cytokine level in mice treatedwith methadone plus MLDS. However, this effect was not statisticallysignificant. These results suggest that methadone protects islets fromautoimmune attack.

Histological studies

Histological analysis of pancreatic tissue indicated that, in MLDSmice, STZ caused β-cell destruction and severe insulitis (islet

leukocyte infiltration). In control mice, islets had intact β cells andinsulitis was absent (Fig. 5). In mice treated with methadone plusMLDS, moderate insulitis was obvious, in contrast to control andMLDSalone mice. Prevention of insulitis in methadone-treated mice wassignificant but not complete. These observations demonstrate thatMLDS-induced destruction of β cells and the degree of inflammationwere ameliorated by methadone in pancreatic tissue.

Discussion

Our study demonstrated that methadone, as an opioid agonist, hasprotective effect against MLDS-induced type 1 diabetes in BALB/cmice. The ability of methadone to attenuate MLDS-induced diabeteswas demonstrated by modulating the activity of the immune system.

Diabetes induced by MLDS in mice is considered to be anexperimental animal model of autoimmune diabetes. MLDS-induceddiabetes exhibits clinical and immunological similarities to type 1diabetes in humans, which are characterized by infiltration of T cellsand macrophages in Langerhans islets, with selective destruction of βcells. It is generally thought that immune cells, cytokines, free radicalsand production of nitric oxide have an important role in thepathogenesis of the disease (Kolb, 1994; O'Brien et al., 1996). TheTh1 subset of CD4T cells and proinflammatory cytokines, such as TNF-α, IFN-γ, IL-1β, IL-2, IL-12 and IL-18, have a direct β-cell cytotoxiceffect in mice. In contrast, there is inhibition of β-cell damage by Th2anti-inflammatory cytokines, such as IL-4, IL-5 and IL-10 (Kawasaki etal., 2004; Suarez-Pinzon and Rabinovitch, 2001). The balance betweenTh1 and Th2 cytokines has been suggested to be the determiningfactor in diabetes progression.

The cytokines TNF-α, IFN-γ and IL-1β have a central and importantregulatory role in β-cell destruction in the pancreas (Lamhamedi-Cherradi et al., 2003). During insulitis, TNF-α is secreted frommacrophages and induces production of several other inflammatorycytokines, such as IL-1β and IL-6. It has been reported that inhibitorsof TNF-α prevent diabetes in mouse diabetes models (Beales, 1998;Holstad and Sandler, 2001). IFN-γ also mediates β-cell death and anti-IFN-γ mAb prevents insulitis and hyperglycemia in STZ-induceddiabetes (Gysemans et al., 2005; Herold et al., 1996). Treatment withneutralizing antibodies against IL-1β has been shown to preventdiabetes in NOD mice (Cailleau et al., 1997).

The role of Th2 cytokines (IL-4 and IL-10) has been extensivelyinvestigated in prevention of insulitis and β-cell destruction. Both IL-4and IL-10 have been shown to suppress insulitis and IDDM in NOD

Fig. 4. Pancreatic levels of Th1 and Th2 cytokines in four study groups on day 21. Methadone (10 mg/kg/day sc) prevented the MLDS-induced increase in TNF-α (A), IL-1β (B) and IFN-γ (C) in pancreata of BALB/c mice, while methadone treatment increased the level of IL-4 (D). Methadone treatment did not significantly increase pancreatic IL-10 levels (E). Resultsare means±SEM (n=4–6). ⁎Pb0.05 compared with vehicle-treated mice; †Pb0.05 compared with MLDS-treated mice.

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mice (Cameron et al., 1997; Hancock et al., 1995; Pennline et al., 1994).As mentioned above, it is postulated that any interruption of Th1cytokines production and enhancement of Th2 cytokines may haveprotective effects against type 1 diabetes.

There have been several studies on prevention of β-cell damage ortype 1 diabetes by anti-inflammatory and immunosuppressive agents(Rydgren et al., 2007; Yang et al., 2003; Maksimovic-Ivanic et al.,2002; Tabatabaie et al., 2000). Several other studies have indicatedthat opioids interact with the immune system and modulate a varietyof immunological parameters (Vallejo et al., 2004; Budd, 2006;McCarthy et al., 2001). In vivo administration of exogenous opioids atpharmacological concentrations has suppressive effects on antibodyand cellular immune responses, cytokine expression and graft-

versus-host responses (Bryant and Roudebush, 1990). These effectsare mediated by opioid receptors, because they are blocked by opioidantagonists (Sacerdote, 2000). Another study has shown thatsystemic administration of morphine inhibits LPS-induced TNF-αproduction (Bencsics et al., 1997). Morphine inhibition of phagocy-tosis and secretion of TNF-α is mediated through μ-opioid receptors(Roy et al., 1998). It is speculated that chronic stimulation of the μ-opioid receptor results in an increase in intracellular cAMP. Opioid-induced increase in intracellular cAMP leads to a decrease in NF-κBactivation and suppression of IFN-γ gene expression (Wang et al.,2003). Investigators have reported that morphine treatment in vivoand in vitro decreases both Th1 cytokines (IL-2 and IFN-γ) andincreases Th2 cytokines (IL-4, IL-5 and IL-10). They have concluded

Fig. 5. Histological evaluation demonstrated the ability of methadone to ameliorate β-cell destruction and the degree of inflammation in pancreatic islets. (A) vehicle-treated;(B) MLDS; (C) MLDS+methadone (Original magnification, 400×).

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that μ-opioid agonists can direct T cells toward Th2 differentiation (ashift from Th1 to Th2 differentiation) (Roy et al., 2004). Another studyhas also shown that continuous injection of β-endorphin, as a μ-receptor agonist, induces a shift from Th1 to Th2 cytokine productionin mice. While the opioid receptor antagonist naloxone increases Th1responses (Sacerdote et al., 1998, 2000). Also in vivo and in vitrostudies have demonstrated that methadone, as another opioidagonist, has inhibitory effects on function of T lymphocytes andmononuclear phagocytes and natural killer cell cytotoxicity (Li et al.,2002).

According to the mechanisms of β-cell destruction leading todiabetes and immunosuppressive activities of methadone, we haveinvestigated the immunological effects of chronic methadone treat-ment in a murine model of type 1 diabetes. Our study showed thatmethadone treatment ameliorated MLDS-induced hyperglycemia andincidence of diabetes, and increased pancreas insulin secretion.Methadone has high affinity for μ receptors and lower affinity for δand κ receptors (Kristensen et al., 1995). The protective effect ofmethadone against hyperglycemia was reversed by naltrexone, whichindicates a contribution from opioid receptors, mostly of the μ type, inthe protective effect of methadone against diabetes. Therefore, theprotective role of methadone is likely to be mediated by opioidreceptor signaling.We showed that chronicmethadone treatment hadthe ability to reduceMLDS-induced Th1 or proinflammatory cytokines,including TNF-α, IFN-γ and IL-1β in the pancreas of mice. Thisobservation is very important, because these mediators play a centralrole in β-cell damage. In addition, we demonstrated that chronicmethadone treatment induced at least one of the Th2 cytokines (IL-4)in the pancreas of MLDS-treated mice. All these actions of methadonemay explain how it ameliorates the development of type 1 diabetes.Also, our histological analysis demonstrated the ability of methadoneto attenuate β-cell destruction and insulitis development by inflam-matory cells.

To the best of our knowledge, the current study is the first tosuggest that chronic methadone treatment possesses protectiveactivity against immune-mediated type 1 diabetes in an MLDSmouse model.

Acknowledgments

This study was supported by a grant from Vice Chancellor ofResearch of Tehran University of Medical Sciences (132/11662, 18March 2005). We also thank Ali Mohammadi-Karakani, for skillfultechnical assistance in preparing pathological samples.

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