Original Contribution

Alpha-PHENYL-TERT-BUTYLNITRONE (PBN) INHIBITS NFkB ACTIVATIONOFFERING PROTECTION AGAINST CHEMICALLY INDUCED DIABETES

EMILY HO,* GOUMAN CHEN,† and TAMMY M. BRAY**The Ohio State University, Columbus, OH, USA, and†University of Guelph, Guelph, ON, Canada

(Received26 August1999;Revised19 October1999;Accepted7 December1999)

Abstract—Alpha-phenyl-tert-butylnitrone (PBN) is an effective spin trapping agent by reacting with and stabilizingfree radical species. Reactive oxygen species (ROS) have been implicated in pancreaticb cell death and thedevelopment of insulin-dependent diabetes mellitus (IDDM). We speculate that treatment with the PBN, will protectagainst diabetes development in two distinct chemically induced models for IDDM. Pretreatment with PBN (150 mg/kgip) significantly reduced the severity of hyperglycemia in both alloxan- and streptozotocin (STZ) induced diabetes. Todetermine the mechanism by which PBN prevents hyperglycemia, we examined the ability of PBN to inhibit NFkBactivation and to stabilize alloxan- and STZ-induced radicals. Both alloxan and STZ induced NFkB activation in thepancreas 30 min after their injection (50 mg/kg iv). PBN pretreatment inhibited both alloxan- and STZ-inducedactivation of NFkB and nitric oxide production. EPR studies showed that PBN could effectively trap alloxan-inducedfree radicals. It is clear that PBN can inhibit NFkB activation in the pancreas and reduce hyperglycemia in two distinctdiabetogenic compounds. This research indicates that NFkB activation may be a key signal leading tob cell death andIDDM. Understanding the cellular pathways leading tob cell death may help in developing effective preventive ortherapeutic targets for IDDM. © 2000 Elsevier Science Inc.

Keywords—IDDM, NF-kB, Antioxidants, Spin trap, Free radicals

INTRODUCTION

Type 1 diabetes or insulin dependent diabetes mellitus(IDDM) is known to be a multifactorial disease [1–3].Despite different triggering factors, the final outcome ischaracterized by profound destruction of the insulin-producingb cells. There is growing evidence implicatingfree radical species in the destruction ofb-cells [4–7];however, the precise cellular mechanisms leading topancreaticb cell death have not yet been fully eluci-dated.

Although IDDM is generally considered to be im-mune disease of the pancreas, there are several knownchemicals used to induce IDDM. Two commonly useddiabetogenic agents are alloxan and streptozotocin(STZ). These structurally diverse compounds have a longhistory of use in diabetes research and are known to bespecifically toxic to the pancreaticb cells. Alloxan isthought to produce reaction oxygen species (ROS) dur-

ing its metabolism [8]. STZ is thought to release nitricoxide (NO) in its metabolism [9–11]. The inherent lackof antioxidant protection and the selectivity of uptake inthe pancreatic islets [12,13], may increase their sensitiv-ity to diabetogenic agents. However, there is no directevidence to demonstrate that the initial concentrations ofROS produced by alloxan or STZ are high enough toinduce massive oxidative damage to islet cells. It ispossible that other factors may be involved in amplifyingtheir cytotoxicity.

We propose that a critical determinant inb cell deathleading to IDDM may be the activation of nuclear tran-scription factorkB (NFkB). NFkB is a transcriptionfactor that can be activated by a variety of stresses suchas oxidants, xenobiotics, viruses and pro-inflammatorycytokines [14,15]. Once activated, NFkB translocates tothe nucleus and binds to DNA and upregulates the ex-pression of several genes involved in inflammatory andimmune responses. Target proteins include adhesionmolecules such as selectins, ICAM-1 and VCAM-1 [16]and various proinflammatory cytokines such as IL-2[17], IL-6 [18], TNF-a [19] and inducible NO synthase

Address correspondence to: Tammy M. Bray, 1787 Neil Ave.,Campbell Hall 347, Columbus, OH 43210, USA; Tel: (614) 292-4885;Fax: (614) 292-8880; E-Mail: bray.21@osu.edu

Free Radical Biology & Medicine, Vol. 28, No. 4, pp. 604–614, 2000Copyright © 2000 Elsevier Science Inc.Printed in the USA. All rights reserved

0891-5849/00/$–see front matter

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(iNOS) [20]. It is speculated that NFkB activation ini-tiates and propagates the immune/inflammatory re-sponse, resulting in the invasion immune cells that pro-duce and amplify free radical production, whichultimately destroys theb cells. NFkB activation mayserve as a common mechanism for the selective cytotox-city of structurally diverse diabetogenic compounds.Thus, compounds that can inhibit NFkB activation in-duced by oxidative stress may modulate the severity ofIDDM.

Alpha-phenyl-tert-butylnitrone (PBN) is a commonlyused spin trapping agent used in electron paramagneticresonance (EPR) studies. PBN acts as an effective trap-ping agent by reacting with and stabilizing free radicalspecies so they can be visualized by an EPR spectrom-eter. However, this stabilization property may also re-duce the reactivity of the free radical species, conse-quently making PBN an effective antioxidant. Thepotential therapeutic value of PBN has been examined inother free-radical mediated diseases such as ischemia-reperfusion damage, septic shock and aging [21–23].

The objectives of this study were (i) to determine theefficacy of PBN supplementation in modulating the de-velopment of chemically induced diabetes in CD-1 mice;and (ii) to investigate the ability of PBN in inhibitingNFkB activation and stabilizing free radicals producedby diabetogenic chemicals. Alloxan and STZs specificityto the pancreatic islets, combined with their ability toproduce free radicals provides us excellent models fordelineating the role of oxidative stresses onb cell dam-age and the development of IDDM.

MATERIALS AND METHODS

Animals

Weanling male CD-1 mice (Harlan, Indianapolis, IN,USA), weighing 20–25 g were used in all experiments.All animals were housed in individual cages in a tem-perature controlled environment (226 2°C) with a lightperiod between 0600 to 1800. Animals were allowed freeaccess to the diet with the exception of fasting periods forblood glucose determination. Animal protocol was ap-proved by the OSU Institutional Laboratory Animal Careand Use Committee.

Effect of PBN on alloxan-induced diabetes

To determine the dose-response of alloxan-induceddiabetes, weanling CD-1 mice were injected with 150,200, or 300 mg/kg alloxan (ip) following an overnightfast and the fasting blood glucose levels were monitoreddaily for 9 d. To examine the effect of PBN on thedevelopment of alloxan-induced diabetes, PBN (150

mg/kg body weight) dissolved in saline was administered30 min before alloxan injection. Control animals re-ceived sham saline injection. Blood was obtained fromthe intra-orbital sinus following an 8 h fast using a 10mLcapillary tube. Glucose concentrations were measuredusing the ONE-TOUCH II (LifeScan, Canada) completeblood glucose monitoring kit. To minimize the effects ofdiurnal fluctuations, blood samples were collected at thesame time every day.

Effect of PBN on STZ-induced diabetes

Multiple low doses of STZ, a commonly used methodto induce diabetes were used [24]. STZ (40 mg/kg)dissolved in citrate buffer (pH 4.0), was immediatelyinjected (ip) into mice (n 5 5) for 5 consecutive days.Control animals received a sham saline injection. Fastingblood glucose levels were monitored over 25 d. Glucoseconcentrations were measured using the ONE-TOUCHII complete blood glucose monitoring kit.

To examine the effect of PBN on the development ofSTZ-induced diabetes, PBN (150 mg/kg body weight)dissolved in saline was administered 30 min before STZinjections for 5 consecutive days. Fasting blood glucoselevels were monitored over 25 days.

Determination of NFkB activation In Vivo

For NFkB activation analysis, mice were injectedwith 50 mg/kg alloxan or 50 mg/kg STZ (iv) and killedat 30 min after injection (n 5 5 in each time interval).Pancreas was immediately removed. NFkB activationwas determined using an electrophoretic mobility shiftassay (EMSA). To determine the effect of PBN on al-loxan- or STZ-induced NFkB activation, animals wereinjected with PBN (150 mg/kg) 30 min before alloxan orSTZ administration.

Crude nuclear extracts were prepared from pancreatictissue as described by Deryckere and Gannon [25]. Dou-ble stranded synthetic oligonucleotides probes for NFkB(59-AGTGAGGGGACTTTCCCAGGC-39) (Promega,Madison, WI, USA) were end-labeled using [g-32P](Amersham, Arlington Heights, IL, USA) and T4polynucleotide kinase (Boehringer Manneheim, India-napolis, IN, USA). Binding reactions containing equalamounts of protein (;7 mg) and labeled oligonucleotideprobes were performed for 20 min at room temperaturein binding buffer (4% glycerol, 1 mM MgCl2, 0.5 mMethylenediaminetetraacetate, pH 8.0, 0.5 mM dithiothre-tol, 50 mM NaCl, 10 mM Tris, 50mg/ml poly [dI-dC].Specific binding was confirmed using 100–400 fold ex-cess unlabeled NFkB oligonucleotide as a specific com-petitor. Protein-DNA complexes were separated using

605PBN, NFkB, and IDDM

6% nondenaturing polyacrylamide gel electrophoresisfollowed by radiography to detect the level of retardationproduced by binding to NFkB probe.

Nitrite analysis

Pancreatic tissue was homogenized in 100 mM N-[2-hydroxyethyl]piperazine-N•-[2-ethanesulfonic acid] buf-fere and then centrifuged at100,0003 g for 60 min.Nitrite formation was used as an indirect measure ofnitric oxide (NO) production. Nitrite concentration wasdetermined using the Griess reaction as described byHevel and Marletta [26]. Protein concentration was de-termined using the Lowry method [27].

Ability of PBN to stabilize alloxan- and STZ-inducedradicals

Pancreatic or liver tissue (0.5 g) was homogenized in150 mM phosphate buffer. Homogenates were then in-cubated with 20 mg/ml alloxan or streptozotoein (STZ)and 14 mg of spin trap, PBN (Sigma, St. Louis, MO,USA) at 37°C for 90 min. Control samples containedPBN only. After the incubation period, PBN spin adductswere extracted with 6 ml of benzene. These adducts wereconcentrated to a 0.2 ml sample and degassed withnitrogen gas before EPR analysis. Samples were placedin quartz round cell and analyzed at room temperaturewith a Bruker X band EPR (Brucker, Milton, ON, Can-ada) spectrometer operating at 9.78 GHz. Extractionsand concentration was performed as previous describedPBN adducts were analyzed by a Bruker X band spec-trometer. For identification of PBN-adduct, solutionswere transferred to hexane for normal phase high-perfor-mance liquid chromatography (HPLC) or to acetonitrile/water (70/30) for reverse phase HPLC.

Identification of PBN-adduct by HPLC

HPLC analyses were performed on a Liquid Chromato-graphic Series 1050 (Hewlett Packard, Palo Alto, CA,USA). Two HPLC systems were used for analyses.

Normal phase systemA variable UV absorbance detector set at 289 nm, a

bondclone 10 silica column (3003 3.9 mm; Phenome-nex, Torrance, CA, USA) were used. The mobile phasewas a mixture of hexane/isopropanol (100/3). The flowrate was 1.0 ml/min and the UV spectrum was recordedat a chart speed 0.25 cm/min.

Reverse phase systemAn ODS Spheri-5 (Waters, Milford, MA, USA) (4.6

mm3 22 cm) column was utilized and acetonitrile-water(70/30) containing 0.02 M phosphate buffer (pH 7.4) wasselected as the mobile phase. A flow rate of 0.7 ml/minand chart speed 0.5 cm/min was used. An electrochem-ical detector with a pyrolytic graphite-working electrodeat 0.7 V vs. Ag/AgCl reference electrode was connectedfor simultaneous monitoring.

Synthesis of alloxan free radical adduct by photolysis

PBN (50 mg) and alloxan (20 mg) were added to 2 mlbenzene and degassed with nitrogen for 10 min. Thesolution was illuminated by UV light under stirring andnitrogen degassing for 12 min. The UV light was gen-erated from 200 W Hg, using 5.2 amperes by an Arclamp power supply. The adduct produced was transferredto hexane for normal phase HPLC or to acetonitrile/water (70/30) for reverse phase HPLC.

Subcellular distribution of alloxan free radical adduct

Mouse tissue (3 g) was homogenized in 3 ml of 150mM phosphate buffer containing 100 mM EDTA. Ho-mogenates were incubated with 60 mg/ml alloxan and 14mg/ml PBN for 90 min at 37°C. Following incubationperiod, differential centrifugation was performed to sep-arate subcellular components. To remove the nuclearfraction, samples were centrifuged at 6003 g, the pelletwas removed, washed and resuspended in phosphatebuffer. The first supernatant was again centrifuged at10,0003 g to separate the mitochondrial fraction. Themitochondrial pellet was removed, washed, and resus-pended in phosphate buffer. The second supernatant wascentrifuged at100,000 3 g for 60 min. The pelletcontaining the microsomal fraction was removed,washed and resuspended in phosphate buffer. The cyto-solic supernatant was also removed. PBN spin adductswere extracted with 6 ml of benzene. These adducts wereconcentrated to a 0.2 ml sample and degassed withnitrogen gas before EPR analysis. Samples were placedin quartz round cell and analyzed at room temperaturewith a Bruker X band EPR spectrometer operating at9.78 GHz.

Statistics

One way analysis of variance (ANOVA) was per-formed to assess the differences between diabetic, PBNtreated, and control groups at various endpoints. Differ-ence in means among treatments was tested by using

606 E. HO et al.

Duncan’s test. Level of significance was evaluated atp , .05.

RESULTS

Hyperglycemia, a commonly used marker for theprevalence of diabetes was used to assess the severity ofIDDM. Mice injected intraperitoneally (ip) with alloxanat the level or higher than 150 mg/kg BW display symp-toms characteristic of diabetes including hyperglycemia,weight loss, and polyuria. Animals injected with alloxanalone showed significantly elevated fasting blood glu-cose levels compared with controls (Figs. 1A, 1B and1C). A dose-dependent increase in the severity of hyper-glycemic after injection (ip) of increasing doses of al-loxan was observed. Pretreatment of PBN attenuated the

severity of alloxan-induced hyperglycemia at all threedoses (Fig. 1). Treatment with PBN significantly reducedfasting blood glucose levels in mice injected with 200and 300 mg/kg alloxan (Figs. 1A and B). At a dose of150 mg/kg alloxan (Fig. 1C), fasting blood glucose lev-els were brought back to that of control with PBNtreatment.

Fig. 2. Pretreatment with PBN reduced STZ-induced hyperglycemia.Mice (n 5 5) were injected with 40 mg/kg STZ for 5 consecutive days(-F-, STZ 1 Sham). PBN (150 mg/kg ip) was injected 30 min beforeSTZ injections (-■-, STZ-injected1 PBN). Controls (-Œ-) were in-jected with saline only. Mean values were compared at each day usingone-way ANOVA. Level of significance was evaluated using Duncan’stest atp , .05.

Fig. 3. Administration of PBN inhibits alloxan-induced NFkB activa-tion in pancreas. Control group received only sham saline injections.Alloxan group received alloxan only (50 mg/kg iv). ALX1 PBN groupwas injected with 150 mg/kg PBN ip 30 min before alloxan (50 mg/kgBWiv). N-acetylcysteine (NAC) is known to inhibit NFkB activation.ALX 1 NAC group was used for external comparison. Pancreatictissue was removed from mice 30 min after alloxan injection. Nuclearextracts were prepared from five pooled pancreas samples from eachgroup. This figure is a representative of two individual experiments.

Fig. 1. Pretreatment with PBN reduces alloxan-induced hyperglycemiaat several three doses of alloxan. Mice (n 5 5 at each dose) wereinjected with (A) 300 mg/kg alloxan, (B) 200 mg/kg alloxan, or (C) 150mg/kg alloxan (-}- Alloxan 1 Sham). PBN (150 mg/kg ip) wasinjected 30 min before alloxan injections (ip) (-■- Alloxan-injected1PBN). Controls (-Œ-) were injected with saline only. Mean values werecompared at each day using one-way ANOVA. Level of significancewas evaluated using Duncan’s test atp , .05.

607PBN, NFkB, and IDDM

Multiple low-dose STZ model was used to inducehyperglycemia. In mice injected with multiple doses ofSTZ, fasting blood glucose levels begin to rise shortlyafter last injection on day 5 and continues to stay ele-vated through day 25 (Fig. 2). Similar to alloxan-induceddiabetes, pretreatment of PBN before STZ injectionsattenuated the hyperglycemic response.

Figure 3 illustrates that the pretreatment of PBN in-hibits alloxan-induced NFkB activation in pancreas invivo. Similar to previous studies, maximal NFkB activa-tion was observed 30 min after alloxan administration(lane 2). However, pretreatment of PBN (30 min beforealloxan administration) fully inhibited the activation ofNFkB (lane 4). N-acetylcysteine (NAC) is known to

Fig. 4. (A) Time course of NFkB activation following STZ injection. Mice were injected with 50 mg/kg STZ i.v. A total of fivepancreas samples were pooled together for nuclear extract preparation at each time point. Lanes 2, 4, 6, 8 are specific competitorreactions (unlabelled NFkB oligo) to confirm the specificity of NFkB binding. These figures are representative of two individualexperiments. (B) Pretreatment with PBN inhibits alloxan-induced NFkB activation in pancreas. Control group received only shamsaline injections. STZ group received STZ only (50 mg/kg iv). STZ1 PBN group was injected with 150 mg/kg PBN i.p. 30 min priorto STZ (50 mg/kg BW iv). Lanes 2, 4, and 6 are specific competitor reactions (unlabeled NFkB oligo) to confirm specificity of NFkBbinding. Pancreatic tissue was removed from mice 30 min following STZ injection. Nuclear extracts were prepared from 5 pooledpancreas samples from each group. This figure is a representative of 2 individual experiments.

608 E. HO et al.

inhibit NFkB activation. Thus NAC treatment (500mg/kg ip) with alloxan acts as an external control forcomparison. This result clearly demonstrates that pre-treatment with PBN effectively inhibited alloxan-in-duced NFkB activation in the pancreas compared withnontreated animals.

Figure 4A illustrates the time course of NFkB activa-tion after administration of STZ. STZ-induced activationof NFkB in vivo has not been previously demonstrated.Lanes 2, 4, 6, and 8 confirm the specificity of NFkBbinding using a cold specific competitor reaction (seeMaterials and Methods). At time 0, no NFkB activationis observed, and maximal activation of NFkB is evident30 min after injection of STZ. Figure 4B demonstratesthat similar to alloxan, PBN pretreatment inhibits STZ-induced NFkB activation in the pancreas. Saline-injectedanimals show no activation (lane 1) and STZ-injectedanimals show significant activation at 30 min followinginjection (lane 3). However, PBN-treated animals showmarkedly lower levels of activation with STZ (lane 5).Thus, PBN is also effective inhibiting STZ-inducedNFkB activation.

One downstream target of NFkB activation is theinducible form of nitric oxide synthase (iNOS). Induc-tion of this protein can be indirectly assessed through themeasurement of changes in NO production. To deter-mine the effect of alloxan and PBN on NO production,pancreatic nitrite levels were determined in the pancreasfrom animals on day 9 after alloxan injection. Figure 5demonstrates that animals injected with alloxan (Allox-an) shows a significant elevation in pancreatic nitritelevels compared with saline-injected controls (Control).Animals that were pretreated with PBN and injected with

alloxan (ALX 1 PBN) showed no increase in nitritelevels compared with controls (Control). Thus, the re-sults suggest indirectly that iNOS, a down stream targetof NFkB, is induced after alloxan exposure. Pretreatmentwith PBN can inhibit this induction, possibly throughinhibition of NFkB.

EPR spin trapping studies were performed to assessthe ability of PBN to trap and stabilize alloxan-inducedfree radicals. Figure 6 shows the EPR spectra of PBNspin adducts of alloxan-induced free radicals in pancre-atic and liver homogenate. A more intense signal can bedetected in pancreas compared with liver homogenateswhen identical amounts of tissue are used. Hyperfinesplitting constants are aN 5 14.46 and ab

H 5 2.38. Thesesplitting constants suggest that the radical detected is acarbon centered radical.

To identify the primary radical trapped with PBN, wecompared the hyperfine splitting constants of the EPRspectra obtained from tissue homogenate (aN 5 14.46,ab

H 5 2.38) and from a photolysis reaction (aN 5 14.50,ab

H 5 2.31). These similar splitting demonstrates thatPBN-alloxan adduct generated from tissue homogenatesand photolysis reactions are most likely identical (Table1 and Fig. 7). HPLC studies demonstrate that retentiontimes of the PBN adduct both in the homogenate andphotolysis reaction, are also identical (Table 1). Thisconfirms that the primary free radical produced is mostlikely an alloxan radical.

Subcellular distribution of alloxan adduct was inves-tigated in order to find the possible site of metabolism ofthe alloxan or target of alloxan radical in the cells. Thestrongest signal of alloxan radical was detected in thenuclear fraction (Fig. 8). Significantly smaller EPR sig-nals are also present in the mitochondrial, microsomal,and cytosolic fractions. This suggests that the alloxanradical is either being produced primarily in the nuclearregion of the cell or the nuclear region is the main targetof the alloxan radical.

DISCUSSION

The results clearly demonstrate that pretreatment ofthe spin trapping agent, PBN is capable of reducing theseverity of hyperglycemia induced by two structurallydiverse diabetogenic compounds in CD-1 mice (Figs. 1and 2). To determine the mechanism that PBN protectsagainst alloxan- or STZ-induced diabetes, we examinedthe ability of alloxan and STZ to induce the activation ofNFkB and determined if PBN could inhibit NFkB acti-vation and its downstream target. There is no doubt thatboth alloxan and STZ induce NFkB activation in vivo(Figs. 3 and 4). This is the first time that STZ adminis-tration has been shown to induce NFkB activation inpancreas in an in vivo model. It is also interesting to note

Fig. 5. Pretreatment of PBN inhibits alloxan-induced NO formation inthe pancreas. Nitrite production was used as an indirect measure of NOproduction. Nitrite levels were assessed in saline-injected (Control),alloxan-injected (Alloxan) and alloxan-injected/PBN supplemented(150 mg/kg PBN) (ALX1 PBN) animals. Pancreas was removed onDay 14 following saline or alloxan injection. Values are expressed asmean6 SE (n 5 6). Comparison of means was performed usingone-way ANOVA. Level of significance was evaluated using Duncan’stest atp , .05.

609PBN, NFkB, and IDDM

that both alloxan [28] and STZ (Fig. 4A) rapidlyinduce NFkB activation in pancreas within 30 minafter iv injection. In another commonly used model forIDDM using IL-1 in culture, NFkB activation is ap-parent in isolated islets after IL-1 exposure in a similartime frame [29].

Pretreatment with PBN 30 min before alloxan or STZinjection effectively inhibits NFkB activation in the pan-creas in both models (Figs. 3 and 4B). Previous work hasshown that PBN is maximally distributed to tissueswithin 30 min [30]. The mechanism by which PBN

inhibits alloxan-induced diabetes and NFkB activationmay be through its ability to trap and stabilize freeradicals. We have previously demonstrated that anotherantioxidant, N-acteylcysteine (NAC), can also inhibitalloxan-induced NFkB activation and significantly re-duce the severity of hyperglycemia and weight loss as-sociated with IDDM development. EPR spin trappingstudies suggest that PBN can stabilize alloxan-inducedfree radicals (Fig. 6) and the radical species trapped is analloxan radical (Table 1). In the metabolism of alloxan,both an alloxan radical and ROS can be produced. Theability of alloxan to produce free radicals has been de-termined by several investigators [8,31–33]. For exam-ple, production of an alloxan radical has been demon-strated by EPR experiments [34]. Although we did notdetect ROS-PBN adducts, it is possible that these adductsare simply not stable enough to be detected. The detec-tion of various EPR signals largely depends on the rate ofproduction and decay. Carbon-centered radicals such asCCl4 radical, are highly stable PBN adducts [35]. Thusthe prevalence of the carbon-centered alloxan radicalmay be due to an increased stability compared to ROS

Fig. 6. EPR spectra of PBN spin adducts of alloxan-induced free radicals trapped in pancreatic and liver homogenates. Alloxanmonohydrate (10 mg/ml) and PBN (14 mg/ml) were incubated in 0.5 g pancreatic or liver tissue for 90 min at 37°C. PBN spin adductswere extracted and analyzed by EPR. Instrument settings were: microwave power, 20 mW; modulation amplitude, 1 G; receiver gain,5 3 105; time constant 100 ms, scan range, 100 G and sweep time, 50s.

Table 1. Comparison of Products Obtained From Tissue Homogenateor Photolysis

HPLC retention time (min)

EPRsplittingconstants

(G)

Normal phase Reverse phase aN abH

Product from homogenate 15.79 4.71 14.46 2.38Product from photolysis 15.76 4.69 14.50 2.31

610 E. HO et al.

adducts. There also exist several lines of indirect evi-dence for ROS production during alloxan metabolism.For example, addition of ROS scavengers such as super-oxide dismutase, catalase and hydroxyl scavengers [36,37] protect against alloxan cytotoxicity in vitro, whichsuggests that ROS are in fact produced with alloxan. Asecond possibility is that although PBN does not trapROS, PBN may indirectly reduce ROS production bytrapping the alloxan radical and subsequently blockingdialuric acid cycling that produces ROS.

In attempts to understand the sites of alloxan metab-olism or cytotoxicity, we performed EPR experimentsfollowing subcellular fractionation in an in vitro system.We expected to find the alloxan radical in the microso-mal fraction, the site of mixed function oxidase (MFO)systems. However, subcellular distribution studies re-vealed that the alloxan radical resides predominantly inthe nucleus. Peak height is an indicator of free radicalconcentration. Figure 8 clearly shows that signal inten-

sity (peak height) is much greater in the nuclear frac-tions. It is unlikely such a significant difference can bedue to contamination or solubility differences. Thus, itappears the alloxan radical either is largely produced inthe nucleus, or the nuclear region is the primary site ofradical attack. This lead us to believe that alloxan me-tabolism may not be through classic xenobiotic metabo-lism pathways. It is currently unknown why the alloxanradical is concentrated in the nucleus and what precisefunctional consequence it has. It has been shown thatalloxan induces islet DNA strand breaks [38,39]. In vitro,there is evidence for increased 8-oxodG formation, amarker for oxidative DNA damage, in calf thymus DNAwith alloxan [34]. Future studies that precisely identifysubcellular distribution and localization using radiola-beled alloxan are needed. The functional consequencesof the nuclear distribution of the alloxan radical, includ-ing the determination DNA adduct formation, should bealso examined in our IDDM models.

Fig. 7. Identification of PBN adducts generated from alloxan. (Spectra A) PBN-alloxan adducts generated in tissue homogenate: alloxanmonohydrate (20 mg/ml) and PBN (14 mg/ml) were incubated in tissue homogenate for 90 min at 37°C. PBN spin adducts wereextracted and analyzed by HPLC and EPR. (Spectra B) PBN-alloxan radical produced by photolysis: alloxan monohydrate (20 mg/ml)and PBN (50 mg/ml) were incubated in 2 ml benzene exposed to UV light for 12 min. PBN spin adducts were extracted and analyzedby HPLC and EPR. Spectra A1 and B1: normal phase HPLC of products from tissue homogenate or photolysis reaction. Spectra A2

and B2: EPR spectra of products from tissue homogenate or photolysis reaction. Spectra A3 and B3: reverse phase HPLC of productsfrom tissue homogenate and photolysis reaction. EPR instrument settings were: microwave power, 20 mW; modulation amplitude, 1G; receiver gain, 53 105; time constant 100 ms, scan range, 100G and sweep time, 50s.

611PBN, NFkB, and IDDM

The mechanism by which PBN inhibits STZ-inducedhyperglycemia is not clear. It is well known that STZreleases NO in its metabolism [10]. We were, however,unable to detect the presence of PBN-STZ or NO adductsin the pancreas. Other investigators, however, shownusing N-methyl-D-glucamine dithiocarbamatehave(MGD) as a spin trap, that PBN can reduce MGD-NOproduction by STZ [40]. The peak height of NO radicalstrapped with MGD is significantly reduced when PBN iscoinjected with STZ compared with STZ alone. Themechanism by which PBN inhibits NO production maybe through the direct inhibition of NFkB activation (Fig.4B). This concept is supported by other disease modelsthat are known to produce NO. In a model for septicshock, PBN has shown to inhibit NFkB activation andboth mRNA and protein expression of iNOS inductionafter LPS treatment [21,41]. Thus, the effect of PBN onSTZ-induced hyperglycemia may primarily be throughinhibition of NFkB activation and the downstream reg-

ulation of iNOS transcription. These observations areconsistent with our hypothesis that NFkB activation is acentral signal in the diabetogenic process.

The role of ROS and NFkB in the pathogenesis ofmany human inflammatory and autoimmune diseases hasbeen gaining widespread attention [42]. The use of phar-macological agents that target NFkB activation has be-come a new therapeutic strategy for many of these dis-eases including inflammatory bowel disease [43],atherosclerosis [44] and rheumatoid arthritis [45], andseptic shock [46]. Although IDDM is largely consideredto be an immune disorder, this link between ROS andcellular response is a relatively unexplored area of re-search, leaving a large gap in the understanding of themechanisms that signalb-cell death. NFkB is a ubiqui-tous rapid response transcription factor in cells involvedin immune and inflammatory reactions. In our labora-tory, we have confirmed that alloxan and STZ specifi-cally activates NFkB in the pancreas within 30 min of

Fig. 8. Subcellular distribution of alloxan-induced free radical adducts. Tissue (3 g) was homogenized in 0.15 M phosphate buffer andincubated with 60 mg/ml alloxan and 14 mg/ml PBN for 90 min at 37°C. Samples were then sequentially centrifuged at 6003 g,10,0003 g and 100,0003 g to retrieve nuclear (A), mitochondrial (B), microsomal (C), and cytosolic (D) fractions. In each fraction,PBN spin adducts were extracted and analyzed by EPR. EPR instrument settings were: microwave power, 20 mW; modulationamplitude, 1 G; receiver gain, 53 105; time constant 100 ms, scan range, 100G and sweep time, 50s.

612 E. HO et al.

injection. No activation is seen in other tissues such asliver [28]. It is speculated that NFkB contributes toimmunologically mediated diseases by exerting its effecton the regulation of the expression of genes that partic-ipate in pathways involving inflammation or susceptibil-ity to apoptosis. We have not determined any specificexpression of cytokines, chemokines, cell adhesion mol-ecules, and immunoreceptors after NFkB activation in-duced by alloxan or STZ in this study. However, it hasbeen shown that NO production in the pancreas is ele-vated by alloxan [28] and STZ [10]. An increase in NOproduction is most likely through the induction of iNOS,another downstream target of NFkB. Although iNOS isknown to be stimulated during inflammatory and im-mune responses, we currently are unable to determine ifiNOS is being induced in the pancreatic islets them-selves, or from infiltrated immune cells. To determinethe source of NO, in situ experiments that cross-stain foriNOS and infiltrated immune cells can be performed.Future studies investigating the expression of variouscytokines, chemokines and adhesion molecules to di-rectly confirm the link between free radicals, NFkB,inflammation and IDDM also need to be pursued.

The NFkB activation process can be inhibited byantioxidants or pharmacologic agents at several differentpoints of the activation pathway. For example, glucocor-ticoids inhibit NFkB by directly upregulating IkB ex-pression, thereby trapping NFkB in inactive cytoplasmiccomplexes or by interfering with NFkB transactivation[47]. Cyclosporine and protease inhibitors prevent NFkBactivation by inhibiting IkB degradation [48]. Deox-yspergualin inhibits NFkB activation by blocking itsnuclear translocation [49]. Aspirin and salicylates inhibitupstream events inducing IkB phosphorylation [50].Some antioxidants inhibit NFkB activation by influenc-ing the redox state of the cell. We have previously shownthat N-acetylcysteine (NAC) supplementation increasedpancreatic glutathione levels, inhibited alloxan-inducedNFkB activation and concurrently reduced the severityof diabetes [28]. In this study, PBN appears to have morethan one possible mechanism to inhibit NFkB activation,e.g., through a direct inhibitory effect on the activationcascade or indirectly through a reduction of oxidativestress or both. Regardless of the mechanisms, the inhi-bition of alloxan- and STZ-induced NFkB activation inpancreas and concurrently reduced the severity of hyper-glycemia with PBN treatment is consistent with ourhypothesis that NFkB activation is a critical signal in thedevelopment of IDDM.

If NFkB activation is a critical step in the diabetogenicprocess, this knowledge will act as a useful tool for screen-ing different classes of compounds, such as antioxidants, fortheir value in IDDM therapy or prevention. Selective mod-ulation of NFkB activation or its downstream effect could

lead to the attenuation of the disease outcomes. A focus onthe control of cellular response pathways will bring the fieldto a level of understanding that is needed to develop effec-tive preventative strategies. This research is the first step inunifying the many factors to ROS signaling in the diabeto-genic processes in a relevant in vivo system and will helpmove us closer to finding effective treatment or preventionstrategies for IDDM.

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ABBREVIATIONS

BW—body weightEMSA—electromobility shift assayEPR—electron paramagnetic resonanceHPLC—high-performance liquid chromatographyip—intraperitonealiv—intravenousIDDM—insulin-dependent diabetes mellitusikB—inhibitory subunitkBiNOS—inducible nitric oxide synthaseNAC—N-acetylcysteineNFkB—nuclear transcription factorkBNO—nitric oxidePBN—a-phenyl-tert-butylnitroneROS—reactive oxygen speciesSTZ—streptozotocin

614 E. HO et al.