Phenyl-N-tert-butylnitrone Demonstrates Broad-Spectrum Inhibition of Apoptosis-Associated Gene Expression in Endotoxin-Treated Rats

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Archives of Biochemistry and Biophysics Vol. 365, No. 1, May 1, pp. 71–74, 1999 Article ID abbi.1999.1159, available online at on

Phenyl-N-tert-butylnitrone Demonstrates Broad-Spectrum Inhibition of Apoptosis-Associated Gene Expression in Endotoxin-Treated Rats Charles A. Stewart, Katrina Hyam, Gemma Wallis, Hong Sang, Kent A. Robinson, Robert A. Floyd, Yashige Kotake, and Kenneth Hensley 1 Free Radical Biology and Aging Research Program, Oklahoma Medical Research Foundation, 825 Northeast 13th Street, Oklahoma City, Oklahoma 73104

Received December 28, 1998, and in revised form February 10, 1999

Systemic exposure to gram-negative bacterial substances such as lipopolysaccharide (LPS, or endotoxin) induces an uncontrolled, massive inflammatory reaction which culminates in multiple system organ failure and death. Septic shock often does not respond to corticosteroids; however, certain low-molecularweight antioxidant compounds have been discovered to possess potent anti-inflammatory action, and some of these novel compounds can rescue animals from experimentally induced septic shock. Phenyl-N-tertbutylnitrone (PBN) is the archetype of the nitrone class of antioxidants which we have previously shown to suppress LPS-induced cytokine biosynthesis in vivo. Using a multiprobe ribonuclease protection assay, we now demonstrate the ability of PBN to suppress proapoptotic gene expression in the LPSinduced model of endotoxic shock. The broad-spectrum gene-suppressive affects of PBN are discussed in the context of inflammatory signal transduction and models are proposed to explain why certain antioxidants may also possess anti-inflammatory and antiapoptotic properties. © 1999 Academic Press Key Words: inflammation; cytokines; apoptosis; nitrone; septicemia.

Apoptosis, or programmed cell death, occurs as a regulated function of tissue development that requires de novo biosynthesis of specific apoptosis-related proteins (1). Pathological initiation of apoptosis in mature tissue can be elicited by exposure to inflammatory stimuli such as bacterial endotoxin (lipopolysaccha1 To whom correspondence should be addressed. Fax: (405) 2711795. E-mail: [email protected]

0003-9861/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.

ride, LPS) 2 or by certain cytokines (2, 3). Apoptosis of irreplaceable neurons is thought to occur in neurodegenerative conditions, including Alzheimer’s disease (4). Accordingly, there is a pressing need to understand cellular mechanisms underlying initiation of the apoptotic program and to identify strategies of inhibiting apoptosis in vivo. One possible means of regulating apoptosis is through the inhibition of oxidation-sensitive signal transduction pathways which regulate expression of proapoptotic genes. Various chemical oxidants, including hydrogen peroxide (H 2O 2), are biosynthesized in cells exposed to inflammatory cytokines (5–10). The purpose of generating intracellular oxidants is unclear, but seems to involve stimulation of protein kinase cascades linked to transcription factor activation (5, 7– 8, 10 –21). It is therefore likely that agents which antagonize oxidative stress may inhibit signal transduction pathways linked to proapoptotic gene expression. The nitrone class of antioxidants has received particular attention as a means of suppressing inflammation and correlated oxidative stress in vivo (22–26). The archetypical representative of this antioxidant family is a-phenyl-N-tert-butylnitrone (PBN), which was originally developed as a means of trapping and detecting free radical intermediates (27). PBN and related nitrones are documented to abrogate postischemic tissue damage (28 –30), rescue animals from septic shock (23–25, 31), and suppress chemically induced thymocyte apoptosis (32) and spontaneous apoptosis of 2 Abbreviations used: LPS, lipopolysaccharide; PBN, a-phenyl-Ntert-butylnitrone: NFkB, nuclear factor kB; RPA, ribonuclease protection assay; GAPDH, glyceraldehyde phosphate dehydrogenase; L32, L32 ribosomal RNA.




FIG. 1. Autoradiogram of the multiprobe RPA demonstrating transcriptional upregulation of several proapoptotic messages in the liver of LPS-treated adult rats. Acronyms: fas-A, fas antigen (CD95 receptor); fas-L, fas ligand; ICE, interleukin-converting enzyme (caspase 1); YAMA, caspase 3 (apopain, CPP32); ICH L/S, caspase 2 (long and short forms).

Down’s syndrome neurons (33). The precise mechanism(s) of nitrone action is unknown; however, we have shown PBN to suppress activation of specific transcription factors such as nuclear factor kB (NFkB) and AP-1 (32). We have previously demonstrated that PBN inhibits transcription of nine distinct proinflammatory cytokines in endotoxin-treated rats and that this suppression correlates with decreased activation of the NFkB and AP-1 transcription factors (34). The present study extends this previous work by determining whether PBN also inhibits transcription of apoptosis-associated genes during LPS-induced septicemia. Using a multiprobe ribonuclease protection assay (RPA), eight apoptosis-associated mRNA species and two constitutively expressed “housekeeping” mRNA species were detected and quantified. PBN administration immediately following an LPS challenge strongly suppressed transcription of all apoptosis-associated mRNA species which were investigated. These results clarify the mechanism of nitrone action and suggest that large suites of genes may be coregulated during inflammation by related oxidant-sensitive signal transduction mechanisms. MATERIALS AND METHODS Animals and experimental manipulations. Male Wistar rats were purchased from Charles River Laboratories (Wilmington, MA) and housed in the Oklahoma Medical Research Foundation laboratory animal care facility. PBN was synthesized and purified in this laboratory. LPS (Escherichia coli 055:B5) was purchased from Sigma Chemical Co. (St. Louis MO). All compounds were dissolved in phosphate-buffered 0.1 N saline prior to administration. Animals were injected intraperitoneally with either saline or LPS (4 mg/kg). Thirty minutes later, half of the saline group and half of the LPS-treated group were injected intraperitoneally with PBN (150 mg/kg). Thirty

minutes or 2.5 h after this second injection, all animals were asphyxiated under CO 2 and liver tissues dissected free. RPA for apoptosis-associated mRNA. Liver sections (approx 100 mg) were frozen on dry ice at necropsy. This tissue was homogenized in TRIzol reagent (Life Technologies, Gaithersburg, MD) with a Dounce-type homogenizer. Total RNA in the resulting extract was quantified spectrophotometrically at 260 nm. A panel of apoptosisassociated RNA species was detected using a multiprobe ribonuclease protection assay system (Riboquant; Pharmingen, San Diego, CA). Radiolabeled probes were synthesized from DNA templates containing a T7 RNA polymerase promoter (Pharmingen). Templates were transcribed in the presence of 100 mCi [a- 32P]UTP to yield radioactive probes of defined size for each mRNA. Probes were hybridized with 5–10 mg total hippocampal RNA, and then samples were treated with RNase A and T1 to digest single-stranded RNA. Intact double-stranded RNA hybrids were resolved on 5% polyacrylamide/8 M urea gels. Dried gels were developed using a phosphorimager (Molecular Dynamics, Sunnyvale, CA), and bands were quantified using instrument-resident densitometry software (ImageQuant, Molecular Dynamics). Within each sample, the density of each apoptosis-associated mRNA band was divided by the sum of the L32 ribosomal RNA (L32) 1 glyceraldehyde phosphate dehydrogenase (GAPDH) bands. The resulting value for each mRNA species was then expressed as a percentage of the average of the same parameter within control samples (taken from animals which had been injected only with saline). Finally, the dried gel was then redeveloped by traditional autoradiography. The data in this paper describe analysis of eight apoptosis-associated mRNA species by RPA; a similar analysis of nine proinflammatory cytokine mRNA species was performed on the same livers and has previously been described (34). Statistical analysis. The nitrone effect on gene expression was assessed by analysis of variance. For each specific mRNA species quantified, differences between treatment groups were statistically assessed by two-tailed Student’s t test.


The results of the multiprobe RPA analysis are depicted in Figs. 1 and 2. While the mRNA levels for the housekeeping genes L32 and GAPDH were similar




FIG. 2. Densitometric quantitation of the data illustrated in Fig. 1. Each bar represents the mean 6 SE of all animals in each group as depicted in Fig. 1. Quantitation was based on data collected by an automated phosphorimager. *Indicates significant inhibition of the LPS-induced gene expression; P , 0.01 by Student’s t test.

across all treatment groups, differential expression of apoptosis-related mRNA products was clearly evident. All eight apoptosis-associated gene products increased within 3 h of LPS treatment, though the magnitude of expression varied considerably (from 1.5-fold upregulation in the case of bcl-2 to 22-fold increased expression for the fas antigen). YAMA (caspase 3) was also strongly induced by LPS (approximately 3-fold above baseline), although a considerable amount of this mRNA species was present in the liver of control animals (Figs. 1 and 2). All apoptosis-associated gene products which were induced by LPS were induced to a much lesser extent in those animals which had also received PBN. In most cases, PBN suppressed mRNA transcription by 50 – 60%; however, fas-A and bax induction were almost completely suppressed by PBN (80 and 90% inhibition, respectively). No clear relationship was observed, however, between the magnitude of LPS-induced gene expression and the sensitivity of the induction to PBN. Likewise, no relationship was observed between the basal levels of the various mRNA species and their inducibility. Interestingly, in those rats which received PBN alone, there was little change among the several proapoptotic mRNA species which were present at detectable quantities in normal animals. The mRNA levels for fas antigen and YAMA, however, decreased by approximately 30% after PBN treatment (Figs. 1 and 2). The PBN suppression of apoptosis-associated mRNA species was highly significant (P , 0.01 by analysis of variance). No proapoptotic gene transcription was evident within 30 min of the LPS challenge (data not shown). The time course of proapoptotic gene transcription following LPS challenge therefore correlates closely with the time course of proinflammatory cytokine expression in the same animals (34).

Several previous studies have documented the ability of PBN to suppress gene transcription in LPSchallenged rats. Most notably, PBN has previously been shown to inhibit LPS-induced expression of inducible nitric oxide synthase (31), tumor necrosis factor-a, and interferon-g (23–25, 34). PBN rescues endotoxin-treated animals from septicemic death (24), implying that the inflammatory processes which are inhibited by PBN are significant features in the endotoxic pathology. Nitrone suppression of proapoptotic gene induction is less commonly reported, though PBN has been shown to inhibit expression of the proapoptotic, immediate early gene product c-Fos in ischemiadamaged liver (29). The broad-spectrum gene suppressive effects of PBN are remarkable and necessitate careful consideration. The broad-spectrum transcriptional suppression seen in PBN-treated animals may result from the ability of the nitrone to inhibit transcription factor activation. We have previously shown PBN to antagonize LPSinduced activation of NFkB and AP-1 transcription factors in vivo (34). In separate work, we have found PBN to inhibit phosphoactivation of the p38 MAPK in astrocytes exposed to H 2O 2 or the inflammatory cytokine IL1-b (10). Therefore, several discrete (although probably interdependent) proinflammatory and proapoptotic signaling pathways are responsive to nitrone presence. At least two plausible models for nitrone action can be proposed. In the first model, nitrones act simultaneously in multiple cell types at some central regulator which is responsible for integrating inflammatory signaling pathways. In this model, the nitrone may act globally by suppressing biosynthesis of H 2O 2 or other oxidizing agents which normally stimulate stress-responsive transcription factor activation. In the second model, PBN could interfere locally with a specific event early in the inflammatory cascade initiated by LPS challenge. If the second case is true, then widespread transcription factor activation and coupled gene induction never occurs because the necessary inflammatory ligands are never biosynthesized. These two models are not mutually exclusive. For instance, PBN could preferentially interfere with a specific signal transduction pathway in certain immune system cells, thereby preventing complete endotoxic shock, while simultaneously inhibiting signal transduction processes in nonimmune system cells. Proposed cellular targets for nitrone action include cyclooxygenase (COX), which PBN inhibits weakly (35), and mitochondria, wherein PBN weakly inhibits NADHlinked H 2O 2 biosynthesis (36). In cell culture, PBN and other antioxidants either increase phosphatase activity or prevent oxidant-induced loss of phosphatase activity



(10). Some action of PBN is therefore possible with respect to antagonizing oxidation-sensitive protein phosphorylation cascades linked to inflammatory and apoptotic gene regulation (10). The precise molecular targets of PBN and related nitrones remain to be discerned. Furthermore, it remains to be determined whether specific signal transduction systems of immune-responsive cells may be preferentially affected by nitrones. These issues are currently being pursued. ACKNOWLEDGMENTS This work was supported in part by a grant (GM54878) from the National Institute of General Medical Sciences, National Institutes of Health.

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