methimazole-induced cell death in rat olfactory receptor neurons occurs via apoptosis triggered...

Methimazole-Induced Cell Deathin Rat Olfactory Receptor NeuronsOccurs Via Apoptosis Triggered ThroughMitochondrial Cytochrome c-MediatedCaspase-3 Activation Pathway

Takashi Sakamoto,1 Kenji Kondo,2 Akinori Kashio,2 Keigo Suzukawa,2

and Tatsuya Yamasoba2*1Department of Otolaryngology, Mutual Aid Association for Tokyo Metropolitan Teachers and Officials,Sanraku Hospital, Tokyo, Japan2Department of Otolaryngology and Head and Neck Surgery, Graduate School of Medicine,Faculty of Medicine, The University of Tokyo, Tokyo, Japan

The administration of methimazole is known to inducecell death in rat olfactory receptor neurons (ORNs). Weinvestigated whether this injury occurs via apoptosis orthrough necrosis and whether it involves the extrinsicor intrinsic pathway. Rats were intraperitoneally injectedwith vehicle (control) or 300 mg/kg methimazole. Theexperimental animals were also administered vehicle ora caspase-3 or caspase-9 inhibitor 30 min earlier. Theadministration of methimazole induced cell death pre-dominantly in the mature ORNs and partially reducedolfactory sensitivity in the rats; the injured cells wereTUNEL-positive and showed a nuclear staining pattern.This insult induced cytochrome c release from the mito-chondria and a significant increase in the immunoreac-tivity of activated caspase-3 and caspase-9 as well asthat of cleaved poly-ADP-ribose-polymerase; in addi-tion, it caused a significant increase in the fluorogenicactivity of caspase-3 and caspase-9. However, it didnot affect the immunoreactivity of activated caspase-8or the fluorogenic activity of caspase-8. Pretreatmentwith a caspase-3 or caspase-9 inhibitor nearly com-pletely prevented the morphologic, biochemical, andfunctional changes induced by methimazole. These find-ings suggest strongly that methimazole-induced celldeath in rat ORNs is predominantly apoptosis; more-over, the majority of this apoptotic cell death is triggeredthrough mitochondrial cytochrome c-mediated caspase-3 activation pathway, and both caspase-3 and cas-pase-9 inhibitors can prevent methimazole-induced celldeath in the ORNs. VVC 2006 Wiley-Liss, Inc.

Key words: apoptosis; caspase; methimazole; olfact-ory receptor neuron; cytochrome c

Persistent olfactory disturbance is a serious problemin humans because it diminishes quality of life and theability to carry out normal activities of daily living

(Miwa et al., 2001). The olfactory receptor neurons(ORNs), which play a crucial role in olfaction, are bipo-lar neurons with their dendritic processes projecting intothe nasal lumen and their axons extending up to thecentral nervous system without synapsing. Because theORNs are exposed directly to the external environment,they are vulnerable to injuries from a variety of externalstimuli. The ORNs have a characteristic adaptive pro-perty of turnover and replacement of themselves fromthe basal progenitor cells throughout life to compensatefor the losses induced by external injuries (Farbman,1990). Despite this property, olfactory disturbance oftenpersists in humans after insults, such as viral infection,head trauma, congenital deficiency, rhinosinusitis, aller-gic rhinitis, and nasotoxicants (Kallmann et al., 1944;Sumner, 1964; Fein et al., 1966; Henkin and Smith,1971; Henkin et al., 1975; Schiffman, 1983; Yamagishiet al., 1989). The mechanisms of pathologic cell death ofthe ORNs in such situations are not well known.

Several in vivo experimental models have beenestablished for inducing mass degeneration of the ORNs.These include olfactory bulbectomy, olfactory nervetransection, topical application of chemicals, such as zincsulfate, Triton X-100, and methyl bromide into the nasalcavity and systemic injection of nasotoxic drugs, such as

Contract grant sponsor: Ministry of Education, Culture, Sports, Science

and Technology of Japan; Contract grant number: 18591901.

*Correspondence to: Dr. Tatsuya Yamasoba, Department of Otolaryn-

gology and Head and Neck Surgery, Faculty of Medicine, The Univer-

sity of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-8655, Japan.

E-mail: [email protected]

Received 19 August 2006; Revised 22 October 2006; Accepted 23

October 2006

Published online 14 December 2006 in Wiley InterScience (www.

interscience.wiley.com). DOI: 10.1002/jnr.21155

Journal of Neuroscience Research 85:548–557 (2007)

' 2006 Wiley-Liss, Inc.

3-methylindole, dichlobenil, and methimazole. The modeof cell death in the ORNs differs among these experi-mental models. Olfactory bulbectomy and olfactorynerve transection, which mimic olfactory dysfunctioncaused by head injuries, have been reported to inducemassive apoptosis in the mature ORN population (Hol-comb et al., 1995; Deckner et al., 1997). In contrast,most chemicals applied to the ORNs, which potentiallymimic the toxic environmental stimuli, have been reportedto induce necrosis. It remains controversial whether intra-peritoneal (i.p.) injection of nasotoxic agents, a model ofacquired drug-induced hyposmia or anosmia in humans,induces apoptosis or necrosis. Understanding the mecha-nism of such nasotoxic drug-induced cell death in theORNs is important to establish new pharmacotherapeuticinterventions to prevent/treat drug-induced olfactory dys-function in humans.

Methimazole belongs to the thioureylene class ofantithyroid drugs. In humans, a wide range of methima-zole-associated adverse effects have been reported, includ-ing hyposmia and anosmia (Hallman, 1953). In themetabolic process, the flavin-containing monooxygenaseoxidizes methimazole to intermediate metabolites ofsulfenic (R-SOH) and sulfinic (R-SO2H) acids, whichare considered to be responsible for the toxic effects ofmethimazole (Kedderis and Rickert, 1985).

Caspase-3 plays a pivotal role in apoptotic celldeath; it is one of the effector caspases and is activatedthrough at least two major pathways, caspase-8-mediatedextrinsic and caspase-9-mediated intrinsic pathways. Arecent study has shown that apoptosis in mouse ORNsinduced by bulbectomy is associated with caspase-9 andcaspase-3 activation (Cowan et al., 2001).

In the present study, we examined whethermethimazole-induced cell death in rat ORNs wasapoptotic or necrotic and assessed whether it involvedthe extrinsic or intrinsic apoptotic pathway.

MATERIALS AND METHODS

Animals and Experimental Design

The current study was carried out using 72 male Sprague-Dawley rats (Saitama Experimental Animals Supply Co. Ltd.,Saitama, Japan) weighing approximately 100 g. Rats werehoused in a light-controlled room with a 12-hr light-darkcycle and were allowed ad lib access to food and water. Allprocedures were conducted according to the guidelines ofThe University of Tokyo for the handling and care of labora-tory animals. Rats were assigned randomly to the followingfour study groups (n ¼ 18 each).

Group 1 (vehicle 1 vehicle). Rats were injected i.p.ab initio as well as 30 min later with 2 ml of 0.01 M phos-phate-buffered saline (PBS) containing 10% dimethyl sulfoxide(DMSO).

Group 2 (vehicle 1 methimazole). Rats were in-jected i.p. with 2 ml of vehicle initially and 30 min later with300 mg/kg methimazole dissolved in 2 ml of vehicle.

Group 3 (caspase-3 inhibitor 1 methimazole). Ratsin this group were injected i.p. with 9 mg/kg cell-permeable

caspase-3 inhibitor (Ac-DEVD-CHO; Sigma-Aldrich Corp.,St. Louis, MO) dissolved in 2 ml of vehicle and 30 min later with300 mg/kg methimazole dissolved in 2 ml of vehicle.

Group 4 (caspase-9 inhibitor 1 methimazole). Allrats from this group received an initial i.p. injection of 9.6mg/kg cell-permeable caspase-9 inhibitor (Ac-LEHD-CHO;Alexis Biochemicals Corp., San Diego, CA) dissolved in 2 mlof vehicle followed 30 min later by another i.p. injection of300 mg/kg methimazole dissolved in 2 ml of vehicle.

On Day 1, 48 rats were anesthetized deeply with a mix-ture of ketamine hydrochloride (40 mg/kg, intramuscular[i.m.]) and xylazine hydrochloride (10 mg/kg, i.m.) andeuthanized. In each group, 6 euthanized rats were subjectedto histologic evaluation and another 6 to Western blottingand fluorogenic caspase activity assay. Another six rats in eachgroup underwent behavioral testing from Day 1–7 for theevaluation of olfactory sensitivity; these rats were euthanizedunder deep anesthesia on Day 7 for histologic evaluation.

Immunohistochemistry

Rats were perfused intracardially with 4% paraformalde-hyde in 0.1 M phosphate buffer, decapitated, and post-fixedfor 24 hr in the same fixative. The nasal tissues, including theolfactory epithelium (OE), were decalcified with 10% ethyl-enediaminetetraacetic acid (EDTA) solution (pH 7.0) andembedded in paraffin. Coronal sections (4 lm thick) on Level4 (Young, 1981) were cut and mounted on silane-coatedslides. Deparaffinized sections were autoclaved for 10 min inthe Target Retrieval Solution (S1700; Dako Japan Inc., Kyoto,Japan) for antigen retrieval. Immunohistochemistry was carriedout using either of the following antibodies: anti-olfactorymarker protein (OMP) antibody (goat polyclonal; WakoChemicals, Dallas, TX; 1:2,000 dilution), anti-activatedcaspase-3 antibody (rabbit polyclonal, Cell Signaling Technol-ogy Inc., Beverly, MA; 1:5,000 dilution), anti-activatedcaspase-8 antibody (rabbit polyclonal; generously provided byDr. D. Nicholson, Merck Frosst Canada Ltd., Dorval, QC,Canada; 1:2,000 dilution), anti-activated caspase-9 antibody(rabbit polyclonal; Cell Signaling Technology Inc.; 1:1,000dilution) and anti-cleaved poly-ADP-ribose-polymerase (PARP)antibody (rabbit polyclonal; Cell Signaling Technology Inc.;1:500 dilution). Immunoreaction was detected using the fol-lowing secondary antibody systems: Histofine Simple StainMAX-PO (G) (Nichirei Corp., Tokyo, Japan) and CSA II(Dako Japan Inc., Tokyo, Japan), a biotin-free tyramide signalamplification system, according to the manufacturer’s instruc-tions. The tissue sections were counterstained with hematoxy-lin except for those subjected to OMP immunostaining. Tenhigh-power fields (3400) were selected randomly in a blindfashion from each of three sections prepared from a rat andexamined under the light microscope. The labeling index ofeach antibody was obtained by a modified Photoshop-basedimage analysis. The original method was depicted by Lehret al (1997). In brief, an image was digitized on magneticoptical disks. Using the Magic Wand tool in the Select menuof Photoshop, a cursor was placed on a portion of the immu-nostained area. The tolerance level of the Magic Wand toolwas adjusted so that the entire immunostained area was

Apoptosis in Olfactory Receptor Neurons 549

Journal of Neuroscience Research DOI 10.1002/jnr

selected. Using the Similar command in the Select menu, allthe immunostained areas were selected automatically.Subsequently, the image was transformed to an 8-bit grayscaleformat. An optical density plot of the selected areas was gen-erated using the Histogram tool in the Image menu. The meanstaining intensity and the number of pixels in the selectedareas were quantified. Next, the background was selected byusing the Inverse tool in the Select menu. The mean back-ground intensity was quantified using the Histogram tool asmentioned above. The immunostaining intensity was calcu-lated as the difference between the mean staining intensityand the mean background intensity. The immunostained ratiowas calculated as the ratio of the number of pixels in all theimmunostained areas to that in the entire image. The labelingindex was defined as the product of the immunostained ratioand the immunostaining intensity.

TUNEL Assay

Deparaffinized coronal sections were also processed forTUNEL staining using an In Situ Cell Death Detection Kit,POD (Roche Diagnostics K. K., Tokyo, Japan) according tothe manufacturer’s instructions. Tissue sections were counter-stained with 5% methyl green solution. Ten high-power fields(3400) were selected randomly in a blind fashion from threesections prepared from each rat and examined under the lightmicroscope. The apoptotic labeling index was estimated bythe same method as described above.

Western Blotting Analysis

On Day 1, after euthanasia under deep anesthesia, theOE was dissected from the nasal bones; it was then snap-frozen in liquid nitrogen and stored at �808C until analysis.The cytosolic fraction of the OE was prepared as describedpreviously (Rajgopal et al., 2003) and was used for Westernblotting analysis of cytochrome c and b-actin. The proteinconcentration was quantified by using the RC DC proteinassay kit (Medical and Biological Laboratories Inc., Nagoya,Japan). Protein samples (25 lg) were separated by sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis and trans-ferred onto a polyvinylidene difluoride (PVDF) membrane.After blotting, the PVDF membrane was blocked with 5%(w/v) skim milk dissolved in PBS for 2 hr. The blots wereprobed overnight at 48C with either a mouse monoclonalanti-cytochrome c antibody (Phar-Mingen Inc., San Diego,CA; 1:2,000 dilution) or a mouse monoclonal anti-b-actinantibody (Abcom Ltd., Cambridge, UK; 1:2,000 dilution).The blots were further processed for enhanced chemilumines-cence by the ECL Plus (GE Healthcare Biosciences K. K.,Tokyo, Japan) detection kit. Optical density (OD) of cyto-chrome c and b-actin were measured using a densitometer.The OD of b-actin was used as the internal control for thecytosolic fraction. Data were expressed as OD of cytochromec/OD of b-actin.

Fluorogenic Caspase-3, -9, and -8 Activity Assay

Caspase activities were measured using a fluorometricprotease assay kit (Medical and Biological Laboratories Inc.).Aliquots of cytosolic fraction containing 25 lg of protein

obtained from the four groups on Day 1 were incubated withfluorogenic substrates Ac-DEVD-7-amino-4-trifluoromethyl-coumarin (AFC) for caspase-3 activity assay, Ac-LEHD-AFCfor caspase-9 activity assay, and Ac-IETD-AFC for caspase-8activity assay in a reaction buffer at 378C for 1 hr. Fluores-cence (excitation at 400 nm and emission at 505 nm) wasmeasured every 15 min using the VersaFluor FluorometerSystem (Bio-Rad Laboratories Inc., Tokyo, Japan). Thespecific activity was calculated from a linear increase of therelative fluorescence unit (RFU) as a function of time andexpressed as the difference in relative fluorescence unit perminute per mg of protein (DRFU/min/mg protein).

Behavioral Test for Evaluating Olfactory Function

To evaluate olfactory sensitivity, a 2-bottle preferencetest using vanillin was carried out as described previously(Kanemura, 2003). Briefly, a bottle containing only distilledwater and another containing 0.1% vanillin (Wako PureChemicals Industries Ltd., Tokyo, Japan) dissolved in distilledwater were inserted at different sides into a cage; the sideswere interchanged every day. Intake of the content of thebottles was measured from Day 1–7. The intake ratio wascalculated as follows: intake of 0.1% vanillin in distilled water/intake of 0.1% vanillin in distilled water þ intake of simpledistilled water. Using an electrophysiologic method, Kane-mura (2003) showed that 0.1% vanillin solution did notinduce the gustatory response. Because rats tend to dislike theodor of vanillin, an increase in the intake ratio indicates thedeterioration of olfactory function.

Statistical Analysis

All values described in the text and figures are expressedas mean 6 standard deviation (SD) for n observations. Datawere analyzed using the one-way analysis of variance(ANOVA) followed by the Bonferroni post-hoc test forpair-wise comparisons among the groups in case of a signifi-cant F-test; statistical significance was inferred at a value ofP < 0.05.

RESULTS

Expression of OMP

OMP is expressed in mature ORNs and the olfac-tory bulb. The anti-OMP antibody is used as a markerof mature ORNs. In Group 2, the injured cells in theORNs were stained with OMP on Day 1 (Fig. 1Ab);the immunoreactivity of OMP was reduced on Day 1and more significantly on Day 7 (Fig. 1Af), indicatingthat most of the injured cells were mature ORNs. Incontrast, the expression of OMP was not changed eitheron Day 1 or Day 7 in Groups 3 (Fig. 1Ac,g) and 4(Fig. 1Ad,h) when compared to that in Group 1 (con-trol); however, the structure of the OE was preservedsimilarly to that observed in the control (Fig. 1Aa,e).The labeling index of OMP was significantly (P < 0.01)smaller in Group 2 compared to those in the othergroups both on Day 1 and Day 7 (Fig. 1B). These find-ings indicate that the mature ORNs were mainly dam-

550 Sakamoto et al.


aged by methimazole and that both caspase-3 and -9inhibitors prevented the degree of damage similarly.

Expression of Activated Caspase-3, Caspase-9, andCaspase-8

On Day 1, substantial amounts of cells positive foranti-activated caspase-3 and -9 antibodies were observedin the injured cells in Group 2 (Fig. 2Ab,f). In the othergroups, such positive cells were observed only sparsely(Fig. 2Aa,c-e,g,h). The labeling index of activated cas-pase-3 and -9 was significantly (P < 0.01) greater inGroup 2 compared to that in the other groups (Fig.2B,C). Positive cells for anti-activated caspase-8 antibodywere observed only sparsely in all the groups (Fig. 2Ai-l). The labeling index of activated caspase-8 on Day 1did not differ significantly amongst the groups (Fig. 2D).These findings indicate that methimazole-induced cellinjury involves the activation of caspase-3 and -9, but

not caspase-8. It is intriguing that the labeling index ofactivated caspase-3 did not increase in animals pretreatedwith a caspase-3 inhibitor (Group 3) whereas that ofactivated caspase-9 did not do so in animals pretreatedwith a caspase-3 inhibitor (Group 3) or a caspase-9 in-hibitor (Group 4).

Expression of Cleaved PARP

PARP is an enzyme involved in DNA repair andone of caspase-3-specific cleavage substrates. The exis-tence of cleaved PARP shows that the activity of acti-vated caspase-3 remains intact. On Day 1, numerouscells labeled with anti-cleaved PARP antibody wereobserved in injured cells in Group 2 (Fig. 3Ab), whereassuch cells were evident only sparsely in the other groups(Fig. 3Aa,c,d). The labeling index of cleaved PARP wassignificantly (P < 0.01) greater in Group 2 compared tothat in the other groups (Fig. 3B); it did not differ sig-

Fig. 1. Representative photographs of immunostaining with anti-OMP antibody (A) and the labeling index of OMP (B). On Day 1,the expression of OMP in Group 2 (vehicle (Veh) þ methimazole(Mz) group) (Ab) decreases compared to that in Group 1 (Veh þVeh group) (Aa), Group 3 (caspase-3 inhibitor (C-3i) þ Mz group)(Ac), and Group 4 (caspase-9 inhibitor (C-9i) þ Mz group) (Ad).

On Day 7, the expression of OMP in Group 2 (Af) decreases com-pared to that in Group 1 (Ae), Group 3 (Ag), and Group 4 (Ah).The labeling indices of OMP on Day 1 and Day 7 were significantlysmaller in Group 2 (Veh þ Mz group) compared to those in theother groups (B). Scale bar ¼ 50 lm. *P < 0.01 vs. the othergroups.



nificantly amongst the other groups. These findings indi-cate that methimazole-induced cell injury involved theactivation of caspase-3, and both caspase-3 and -9 inhib-itors suppressed the activation of pro-caspase-3 to thecontrol level.

TUNEL Assay

On Day 1, numerous TUNEL-positive cells show-ing a nuclear staining pattern were observed in theinjured cells in Group 2 (Fig. 3Af); however, cellsdisplaying strong cytoplasmic staining, which indicatesnecrosis, were not observed. In the other groups,

TUNEL-positive cells were observed only sparsely(Fig. 3Ae,g,h). The labeling index of TUNEL stainingon Day 1 was significantly (P < 0.01) greater in Group2 compared to that in the other groups (Fig. 3C). Thesefindings indicate that methimazole-induced cell injuryinvolved DNA fragmentation in an apoptotic fashionand that both caspase-3 and -9 inhibitors suppressedDNA fragmentation to the control level.

Fluorogenic Caspase-3, -9, and -8 Activity Assays

The activity of caspase-3 in Group 2 was signifi-cantly (P < 0.01) greater compared to that in the other

Fig. 2. Representative photographs of immunostaining with anti-activated caspase-3, -9, and -8 antibody on Day 1 (A). The expres-sion of activated caspase-3 in Group 2 (vehicle (Veh) þ methimazole(Mz) group) (Ab) increases in the injured cells compared to thatin Group 1 (Veh þ Veh group) (Aa), Group 3 (caspase-3 inhibitor(C-3 i) þ Mz group) (Ac), and Group 4 (caspase-9 inhibitor (C-9 i)(Ad). Similarly, the expression of activated caspase-9 in Group 2 (Af)increases in the injured cells compared to that in Groups 1 (Ae), 3

(Ag), and 4 (Ah). The expression of activated caspase-8 is not differ-ent among the groups (Ai-l). Scale bar ¼ 50 lm. The labeling indexof activated caspase-3 and -9 in Group 2 is significantly greater com-pared to that in the other groups (B,C); however, that of activatedcaspase-8 shows no significant difference among the groups (D).*P < 0.01 vs. the other groups. Abbreviations of a-C-3, a-C-9, anda-C-8 stand for activated caspase-3, activated caspase-9, and activatedcaspase-8, respectively.

552 Sakamoto et al.


groups (Fig. 4A). The activity of caspase-9 in Group 2was significantly (P < 0.05) greater compared to that inGroups 1 and 4 (Fig. 4B). The activity of caspase-8showed no significant difference among the groups (Fig.4C). An increase in caspase-3 activity induced by methi-mazole was suppressed by caspase-9 inhibitor.

Western Blotting Analysis

Cytochrome c was detected as a single band of mo-lecular mass (15 kDa) in the cytosolic fraction of theOE. On Day 1, the levels of cytochrome c was increasedin Groups 2–4 compared to that in the control (Group1) (Fig. 4D). b-Actin, which was used as the internalcontrol and detected as a single band of molecular mass(42 kDa) in the cytosolic fraction, was present similarlyin all groups (Fig. 4D). The OD ratio was significantly(P < 0.01) greater in Groups 2–4 compared to that in

Group 1; however, Groups 2–4 did not differ signifi-cantly in terms of the OD ratio (Fig. 4E). These findingsindicate that methimazole induced the release of cyto-chrome c from mitochondria into the cytosol in the OEand that caspase inhibitors did not affect the extent ofthe release of cytochrome c.

Behavioral Test for Evaluating OlfactoryFunction

The intake ratio from Day 1 to Day 7 was signifi-cantly (P < 0.01) greater in Group 2 compared to thatin the other groups; however, it did not differ amongthe other groups (Fig. 4F). These results indicate thatthe administration of methimazole partially reduced ol-factory sensitivity, and pretreatment with a caspase-3 orcaspase-9 inhibitor prevented this reduction.

Fig. 3. Representative photographs of immunostaining with anti-cleaved PARP and TUNEL assay on Day 1 (A). The expression ofcleaved PARP in the injured cells is increased in Group 2 (vehicle(Veh) þ methimazole (Mz) group) (Ab) compared to that in Group1 (Veh þ Veh group) (Aa), Group 3 (caspase-3 inhibitor (C-3 i) þMz group) (Ac), and Group 4 (caspase-9 inhibitor (C-9 i) (Ad).Similarly, the number of TUNEL-positive cells in the injured olfac-

tory epithelium is increased in Group 2 (Af) compared to that in theother groups (Ae,g,h). Scale bar ¼ 50 lm. The labeling index ofcleaved PARP is significantly greater in Group 2 compared to that inthe other groups (B). Similarly, the labeling index of TUNEL assayis significantly greater in Group 2 compared to that in the othergroups (C). *P < 0.01 vs. the other groups. Abbreviation of cPARPstands for cleaved PARP.



DISCUSSION

The present study showed the following findings:1) the administration of methimazole predominantlydamaged the mature ORNs in the OE and partiallyreduced olfactory sensitivity in the rats; 2) the injuredcells showed TUNEL labeling in a nuclear staining

pattern; 3) the administration of methimazole inducedthe release of cytosolic cytochrome c from the mito-chondria; it also induced an increase in the immunoreac-tivity of activated caspase-3, caspase-9, and cleavedPARP and that in the fluorogenic activity of caspase-3and caspase-9, but did not affect the immunoreactivity

Fig. 4. Fluorogenic caspase activity assay on Day 1 (A–C). The ac-tivity of caspase-3 is significantly greater in Group 2 (vehicle (Veh)þ methimazole (Mz) group) compared to that in the other groups(A). The activity of caspase-9 is significantly greater in Group 2 thanthat in Groups 1 and 4 (B). The activity of caspase-8 shows no sig-nificant difference among the groups (C). *P < 0.01 vs. the othergroups. þP < 0.05 vs. the Veh þ Veh and caspase-9 inhibitor (C-9i) þ methimazole (Mz) groups. Western blotting analysis for cyto-chrome c on Day 1 shows that the level of the band in Groups 2–4(animals treated with methimazole and either vehicle or caspase

inhibitors) is denser than that in controls untreated with methimazole(Group 1) (D). Densitometric semiquantitative analysis shows thatthe OD ratio of cytochrome c/b-actin in Groups 2–4 is significantlygreater than that in Group 2 (E). #P < 0.05 vs. Group 1 (Veh þVeh group). The results of a behavioral test evaluating olfactory sen-sitivity (F). The intake ratio is calculated using intake of content ofbottles from Day 1 to Day 7. The intake ratio is significantly greaterin Group 2 compared to that in the other groups; however, it is notdifferent among Groups 1, 3, and 4. *P < 0.01 vs. the other groups.

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or fluorogenic activity of caspase-8; and 4) pretreatmentwith a caspase-3 or caspase-9 inhibitor nearly completelyprevented these methimazole-induced morphologic,biochemical, and functional changes. These findingssuggest strongly that methimazole-induced cell deathobserved in the rat ORNs is predominantly apoptosis;moreover, this apoptotic cell death is triggered primarilythrough mitochondrial cytochrome c-mediated caspase-3activation pathway, and both caspase-3 and caspase-9inhibitors can prevent methimazole-induced cell deathin the OE.

Thyroid hormones are known to play a criticalrole in olfactory neurogenesis during development and apersistent thyroid hormone deficiency can lead to areduced olfactory surface area in developing rats (Pater-nostro and Meisami, 1993, 1994). Moreover, in hypo-thyroid adult mice, a decreased thickness of the OE anda loss of the sense of smell have been observed (Beardand Mackay-Sim, 1987; Mackay-Sim and Beard, 1987).However, pretreatment with thyroid hormones, i.e.,thyroxine, did not prevent methimazole-induced cellinjury in the olfactory epithelium 14 days after theadministration of methimazole (Bergman and Brittebo,1999). Moreover, it takes 2 or 3 weeks for the storageof thyroid hormones in thyroid to be exhausted. There-fore, hypothyroidism induced by the administration ofmethimazole is unlikely to cause massive cell death inthe ORNs.

Apoptosis and necrosis have long been consideredas 2 distinct mechanisms of cell death. These two typesof cell death, however, can occur simultaneously in tis-sues or cell cultures following exposure to the sameinsult (Ankarcrona et al., 1995; Leist et al., 1995; Shi-mizu et al., 1996). Recent studies have shown that acontinuum exists between apoptosis and necrosis (Martinet al., 1998) and that both types of cell death can becaused simultaneously depending on the intensity of theinsult as well as the intracellular level of ATP (Leistet al., 1997; Formigli et al., 2000). TUNEL assay hasbeen widely used as a specific marker for apoptosis(Gavrieli et al., 1992); however, both apoptotic and ne-crotic cells are stained with this assay. Two types ofTUNEL-positive cells are differentiated in a traumaticbrain injury model in rats (Rink et al., 1995). Type Icells with morphologic features of necrosis display strongcytoplasmic staining, and Type II cells with morphologicfeatures of apoptosis display strong nuclear and weakcytoplasmic staining. The cytoplasmic staining could beexplained by leakage of small molecular DNA (Russell,1983). In the present study, the injured ORNs did notshow a cytoplasmic pattern in TUNEL assay. Moreover,they expressed activated caspase-3 and cleaved PARP.Thus, the majority of cell death in rat ORNs inducedby the administration of methimazole is considered tooccur via apoptosis.

Many reports have, in addition, showed that whennecrosis and apoptosis occur simultaneously, blockingapoptotic cell death can shift the mode of cell deathfrom apoptosis to necrosis (Xiang et al., 1996; Hirsch

et al., 1997). Inhibition of methimazole-induced celldeath by pretreatment with a caspase-3 or caspase-9 in-hibitor induced no evidence of necrotic morphologicalchanges in the current study. This finding is also sup-portive of the notion that methimazole-induced celldeath in the ORNs is predominantly apoptotic.

Caspase-3 plays a pivotal role in chromatin con-densation and DNA fragmentation during the disman-tling of a cell with the ultimate formation of apoptoticbodies. Activation of caspase-3 is induced by at least twomajor initiator pathways, namely, the extrinsic andintrinsic pathways. In the extrinsic pathway, cell surfacedeath receptors such as Fas and TNF are oligomerizedby binding specific ligands that recruit the adaptor pro-teins to the active receptor complex. Pro-caspase-8 isactivated through adaptor proteins by autoproteolysis(Muzio et al., 1996; Boldin et al., 1996). In the intrinsicpathway, cytochrome c is released into the cytosol fromthe mitochondria and associates with Apaf-1, an adaptermolecule that can bind to and activate pro-caspase-9 inthe presence of dATP (Zou et al., 1997; Li et al., 1997;Srinivasula et al., 1998). Both pathways converge finallyat the sequential activation of pro-caspase-3. A linkbetween the 2 pathways by Bid has been reported (Scar-abelli et al., 2002). Bid is cleaved by activated caspase-8enabling it to translocate to the mitochondria and pro-mote cytochrome c release (Li et al., 1998). Recent stud-ies have also reported the presence of caspase-independ-ent apoptosis (Carmody and Cotter, 2000; Cande et al.,2004). The present study showed that the administrationof methimazole resulted in an increase in the levels ofcaspase-3 and caspase-9 and in the release of cytochromec from the mitochondria in the OE; these suggest thatthe majority of methimazole-induced cell death in theORNs is induced through the intrinsic pathway.Because pretreatment with a caspase-9 inhibitor nearlycompletely prevented methimazole-induced morphologicchanges and increase in activated caspase-3, caspase-9,and cleaved PARP in the OE, and reduction of olfac-tory sensitivity, similarly to a caspase-3 inhibitor, it isunlikely that translocated Bid enhances the activation ofpro-caspase-9 or that apoptosis via caspase-independentpathway is involved.

Caspase inhibitors such as Ac-DEVD-CHO andAc-LEHD-CHO are cell-permeable peptide aldehydes(Nicholson et al., 1995; Thornberry et al., 1997) thatmerely competitively inhibit each caspase activity, anddo not affect the activation process of each zymogen.Theoretically, the inhibition of activated caspase-3 by acaspase-3 inhibitor is considered to suppress the fluoro-genic activity of caspase-3 but not that of caspase-9 orthe immunoreactivity of active caspase-3 or caspase-9.The inhibition of activated caspase-9 by a caspase-9 in-hibitor is considered to suppress the fluorogenic activityof caspase-3 and caspase-9 in addition to the immuno-reactivity of active caspase-3; however, it does not sup-press the immunoreactivity of active caspase-9. Thepresent study, nevertheless, showed that pretreatmentwith a caspase-3 inhibitor prevented the methimazole-



induced increase in the immunoreactivity of activatedcaspase-3 and -9 as well as the fluorogenic activity ofboth caspase-3 and caspase-9. Pretreatment with a cas-pase-9 inhibitor also unexpectedly prevented the methi-mazole-induced increase in the immunoreactivity ofboth activated caspase-3 and caspase-9 and the fluoro-genic activity of both caspase-3 and caspase-9. Directfeed back activation of caspase-9 by caspase-3 has beenreported (Srinivasula et al., 1996) and is considered toact as a feed-forward cycle that, once initiated, mayresult rapidly in uncontrolled neuronal apoptosis. Thiscomplex interaction between caspase 3 and caspase 9may explain why a caspase-3 inhibitor prevented themethimazole-induced increase in the fluorogenic activityof caspase-9 and a caspase-9 inhibitor prevented themethimazole-induced increase in the immunoreactivityof activated caspase-9.

After bulbectomy, the proapoptotic signals by cas-pase-3 and caspase-9 were propagated from the axonalend to the cell body of the ORNs (Cowan et al., 2001).Contrastingly, in the present study, it was difficult todetermine the direction of propagation of the proapop-totic signals because it is believed that the proapoptoticstimuli are derived from the vicinity, i.e., Bowman’sglands and the supporting cells, of the cell body of theORNs. It is remarkable that apoptosis was induced simi-larly through the intrinsic pathway by these two differentstimuli. In the study by Cowan et al. (2001), a chief pre-dictive factor determining the timing of apoptosis wasreported to be the length of the ORN axon from theolfactory bulb receiving the insult. In the present study,apoptosis was induced throughout the OE to a similarextent regardless of the distance from the olfactory bulb.Methimazole is taken up into the Bowman’s glands,which are located diffusely in the lamia propria of theolfactory mucosa and, to a lower extent, into the sup-porting cells within 30 min after i.p. injection (Bergmanand Brittebo, 1999). Thus, the stimulus inducing celldeath in the present study is considered to be independ-ent of the distance from the olfactory bulb.

The accurate optimal period for the administrationof caspase inhibitors needs to be clarified. We observedpreviously that the number of TUNEL-positive cellsbegan to increase at 12 hr after methimazole injection(unpublished data). Methimazole is taken up in the vi-cinity of the ORNs within 30 min after the i.p. injec-tion. Because olfactory epithelium contains abundant fla-vin-containing monooxygenase, it has the outstandingability to oxidize methimazole to intermediate sulfenicand sulfinic acid metabolites (Genter et al., 1995). Themolecular weights of the olfactory-toxic sulfenic and sul-finic acids are 130.2 and 146.2, respectively; thus, thesemetabolites are extremely small and can spread easilyfrom Bowman’s glands and the supporting cells to thecell body of the ORNs. Moreover, caspase inhibitors arenot long-acting because of their reversibility. As a resultof these circumstances, we consider it reasonable toadminister caspase inhibitors 30 min before methimazoleinjection.

The results presented indicate that the majority ofmethimazole-induced cell death in rat ORNs occurs viaapoptosis through the mitochondrial cytochrome c-medi-ated caspase-3 activation pathway and can be preventedby pretreatment with a caspase-3 inhibitor or a caspase-9inhibitor. These findings are useful in establishing newtherapeutic strategies for preventing/rescuing pathologiccell death in the acute phase of acquired drug-inducedhyposmia or anosmia in humans.

ACKNOWLEDGMENTS

The authors thank Dr. D. Nicholson for his gener-ous gift of anti-activated caspase-8 antibody and Ms. Y.Kurasawa and Ms. A. Tsuyuzaki for providing technicalassistance. This research was supported by a grant(18591901) from the Ministry of Education, Culture,Sports, Science and Technology of Japan to T.S.

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