ToxSci Advance Access originally published online on May 4, 2007
Toxicological Sciences 2007 98(2):526-541; doi:10.1093/toxsci/kfm102
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Neurotoxicity and Inflammation in the Nasal Airways of Mice Exposed to the Macrocyclic Trichothecene Mycotoxin Roridin A: Kinetics and Potentiation by Bacterial Lipopolysaccharide Coexposure
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,1
* Department of Food Science and Human Nutrition
Center for Integrative Toxicology
Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, Michigan 48824
1 To whom correspondence should be addressed at 234 G.M. Trout Building, Michigan State University, East Lansing, MI 48824-1224. Fax: (517) 353-8963. E-mail: pestka{at}msu.edu.
Received February 7, 2007; accepted April 27, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Macrocyclic trichothecene mycotoxins produced by indoor air molds potentially contribute to symptoms associated with damp building illnesses. The purpose of this investigation was to determine (1) the kinetics of nasal inflammation and neurotoxicity after a single intranasal instillation of roridin A (RA), a representative macrocyclic trichothecene; and (2) the capacity of lipopolysaccharide (LPS) to modulate RA's effects. C57Bl/6 female mice were intranasally instilled once with 50 µl of RA (500 µg/kg body weight [bw]) in saline or saline only and then nose and brain tissues were collected over 72 h and processed for histopathologic and messenger RNA (mRNA) analysis. RA-induced apoptosis specifically in olfactory sensory neurons (OSNs) after 24 h postinstillation (PI) causing marked atrophy of olfactory epithelium (OE) that was maximal at 72 h PI. Concurrently, there was marked bilateral atrophy of olfactory nerve layer of the olfactory bulbs (OBs) of the brain. In the ethmoid turbinates, upregulated messenger RNA (mRNA) expression of the proapoptotic gene FAS and the proinflammatory cytokines tumor necrosis factor-
, interleukin (IL)-6, IL-1, and macrophage inhibitory protein-2 was observed from 6 to 24 h PI, whereas expression of several other proapoptotic genes (PKR, p53, Bax, and caspase-activated DNAse) was detectable only at 24 h PI. Simultaneous exposure to LPS (500 ng/kg bw) and a lower dose of RA (250 µg/kg bw) magnified RA-induced proinflammatory gene expression, apoptosis, and inflammation in the nasal tract. Taken together, the results suggest that RA markedly induced FAS and proinflammatory cytokine expression prior to evoking OSN apoptosis and OE atrophy and that RA's effects were augmented by LPS. Key Words: trichothecene; mycotoxin; apoptosis; neurotoxicity; inflammation; endotoxin.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Adverse human health effects are often attributed to damp indoor air environments generated by excessive condensation, failure of water-use devices, or building envelope breach during flooding (Institute of Medicine, 2004). Symptoms typically reported by affected occupants of water-damaged buildings include cough, rhinosinusitis, exacerbation of asthma, and increased susceptibility to respiratory infections. For example, a recent National Institute of Occupational Safety and Health (NIOSH) study (Cox-Ganser et al., 2005
) reported that excessive respiratory symptoms and physician-diagnosed asthma occurred among occupants in an office building that was subject to water incursions. These effects contributed to overall burden of illness delineated in terms of absences, use of breathing medications, and health-related quality of life.
Damp building–related illnesses are frequently linked to aberrant growth of mold (Andersson et al., 1997; Fog Nielsen, 2003). In 2004, an Institute of Medicine (IOM) expert committee (Institute of Medicine, 2004) concluded that there are sufficient scientific data for an association between the exposure to moldy damp indoor environments and (1) upper respiratory tract symptoms (nasal congestion, sneezing, runny or itchy nose, throat irritation) as well as (2) some lower respiratory tract symptoms/syndromes (cough, wheeze, asthma exacerbation, hypersensitivity pneumonitis in susceptible people), but that insufficient evidence exists to date for other sometimes disparate health conditions (airflow obstruction, mucous membrane irritation, chronic obstructive pulmonary disease, pulmonary hemorrhage, neurologic effects, and cancer).
The black mold Stachybotrys chartarum is a saprophytic fungus that grows on cellulosic building materials including wallboard, ceiling tiles, and cardboard (Andersson et al., 1997; Boutin-Forzano et al., 2004; Tuomi et al., 1998, 2000). It has been postulated that building-related S. chartarum, its trichothecene mycotoxins, or other byproducts are possible etiologic contributors to debilitating respiratory illnesses (Fung et al., 1998; Hossain et al., 2004; Jarvis et al., 1986, 1998; Kilburn, 2004) as well as immune dysfunction (Johanning et al., 1996) and cognitive impairment (Gordon, 1999). The aforementioned IOM report (Institute of Medicine, 2004) suggested, however, that while in vitro and in vivo research on S. chartarum suggest that adverse effects in humans are indeed "biologically plausible," establishing a clear association with building-related illnesses will require rigorous study from the perspectives of mechanisms, dose–response and exposure assessment.
The trichothecene mycotoxins comprise more than 180 structurally related sesquiterpenoid metabolites and are produced by Fusarium, Stachybotrys, Myrothecium, and other molds (Grove, 1988, 1993, 2000). Trichothecenes are potent translational inhibitors that have in common a 9, 10 double bond, and a 12, 13 epoxide group, but extensive variation exists relative to ring oxygenation patterns. The Type D or "macrocyclic" trichothecenes, which have a cyclic diester or triester ring linking C-4 to C-15 (e.g., satratoxins or roridins), are produced by Stachybotrys and Myrothecium. Macrocyclic trichothecenes occur in the outer plasmalemma surface and the inner wall layers of fungal conidiospores (Gregory et al., 2004). Dry spores are readily aerosolized and have a respirable mean aerodynamic diameter of approximately 5 µm (Sorenson et al., 1987; Yike and Dearborn, 2004). In addition, nonviable, fine (
1 µm in diameter) airborne particulates can also contain trichothecenes (Brasel et al., 2005). Thus, human exposure to these toxins is indeed possible.
Several experimental studies of S. chartarum spores have focused on the contributions of trichothecenes to airway pathology. Rand et al. (2002) determined, in direct intratracheal instillation studies of mice, that respiratory pathology caused by fungal spores could be reproduced with pure isosatratoxin F. After investigating the effects of a single tracheal instillation of fungal spores on survival, growth, histopathology of the lung, and respiration in the rat pup model, Yike et al. (2002) concluded that mycotoxins were critical for the hemorrhagic and inflammatory responses. In subsequent work, this group demonstrated that toxic S. chartarum spores or associated macrocyclic trichothecenes initiated acute inflammatory responses in the rodent lung (Yike et al., 2005; Yike and Dearborn, 2004). Recently, our laboratory has observed that acute intranasal exposure to satratoxin G and isosatratoxin F, macrocyclic trichothecenes produced by S. chartarum, causes inflammation and apoptosis in the murine nose 24 h after instillation (Islam et al., 2006). Both olfactory sensory neurons (OSNs) and the olfactory bulb (OB) of the brain are targets of satratoxin G–induced toxicity. Elevated proinflammatory cytokine gene expression is also observed at 24 h PI, suggesting that these mediators might contribute to OSN apoptosis as well as accompanying rhinitis and mild focal encephalitis observed in toxin-exposed mice.
Occupants of damp buildings are likely to be exposed to other microbial products besides fungi (Institute of Medicine, 2004). Notably, endotoxin often co-occurs with molds in damp buildings (Bornehag et al., 2004; Foto et al., 2005; Heinrich et al., 2003; Kovesi et al., 2006; Sebastian and Larsson, 2003; Sebastian et al., 2005) as was dramatically evidenced in New Orleans following Hurricane Katrina (Solomon et al., 2006). A recent NIOSH study suggested that occupant respiratory problems might result from the combined exposure to fungi and endotoxin and that both agents should be assessed in epidemiological investigations of indoor environments (Park et al., 2006). Oral trichothecene toxicity is amplified by coexposure to lipopolysaccharide (LPS), the major bioactive component of endotoxin (Chung et al., 2003a,b; Islam et al., 2002; Mbandi and Pestka, 2006; Tai and Pestka, 1988b; Zhou et al., 1999, 2000). LPS might similarly enhance the nasal toxicity of macrocyclic trichothecenes.
Since satratoxins are not commercially available and they are extremely difficult to purify from fungal culture, characterization of their toxic effects and mechanisms whether alone or in the context of co-occurring contaminants presents a technical challenge. One possible alternative would be to employ another macrocyclic trichothecene as a surrogate for the satratoxins. Roridin A (RA) is one such structurally related trichothecene that is produced in copious amounts by the fungus Myrothecium and that is commercially available. In preliminary studies, we have observed that, like the satratoxins, RA can induce inflammation and OSN loss in mice following intranasal instillation. The purpose of this study was two fold. First, we determined the relative kinetics of proinflammatory cytokine and proapoptotic gene expression following intranasal RA exposure and further related these effects to OSN apoptosis and OE atrophy. Second, we assessed the capacity of LPS to modulate RA-induced gene expression and toxicity in the nasal airway. This investigation (1) confirmed that RA mimicked satratoxin G's toxic effects in the nose, (2) identified from expression kinetics those genes likely to contribute to apoptosis, and (3) demonstrated the capacity of LPS to exacerbate RA's adverse effects.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Experimental design.
RA was purchased from Sigma Chemical Co. (St Louis, MO). Toxin purity was > 99% as determined by a single high performance liquid chromatography peak at 260 nm (Hinkley and Jarvis, 2001
). LPS derived from Escherichia coli serotype 0111:B4 with an activity of 1.5 x 106 EU/mg was purchased from Sigma. Animal studies were conducted in accordance with National Institutes of Health guidelines as overseen by the All University Committee on Animal Use and Care at Michigan State University. Pathogen-free female C57Bl/6 mice (7–8 weeks, Charles River, Portage, MI) were randomly assigned to experimental groups (n = 6) and housed in polycarbonate cages containing Cell-Sorb plus bedding (A & W Products, Cincinnati, OH) and covered with filter bonnets. Mice were provided free access to food and water. Room lights were set on a 12-h light/dark cycle, and temperature and relative humidity were maintained between 21°C–24°C and 40–55% humidity, respectively. For each experiment, mice were anesthetized with 4% halothane and 96% oxygen and then instilled intranasally at 50 µl per mouse with RA (250 or 500 µg/kg body weight [bw]) and/or LPS (500 ng/kg bw) dissolved in a vehicle of pyrogen-free saline (Abbott Laboratories, IL) or with vehicle(s) alone. Selection of RA doses was based on capacity to mimic satratoxin G olfactory effects in preliminary experiments. Mice were euthanized after various time intervals and selected tissues from the nose and brain were subjected to histopathologic and messenger RNA (mRNA) analysis as described below.
Animal necropsies and tissue processing for light microscopic examination.
Mice were sacrificed at 6, 12, 24, or 72 h PI. For light morphologic examination and morphometric analyses of nasal cavity, mice were deeply anesthetized via ip injection of 0.1 ml of 12% (wt/vol) sodium pentobarbital and killed via exsanguination by cutting the abdominal aorta. Heads from each mouse were immediately removed and 1 ml of 10% (vol/vol) neutral buffered formalin (Fisher Scientific Co., Fairlawn, NJ) was flushed retrograde through the nasopharyngeal meatus. After the lower jaw, skin, muscles, and dorsal cranium were removed, the head with the intact brain was immersed and stored in a large volume of the fixative for at least 24 h prior to further tissue processing.
After fixation, transverse tissue blocks from the head of these mice were selected for light microscopy as previously described (Islam et al., 2006). Prior to sectioning, the heads were decalcified in 13% (vol/vol) formic acid for 7 days and then rinsed in tap water for at least 4 h. The nasal cavity of each mouse was transversely sectioned at four specific anatomic locations (Islam et al., 2006; Mery et al., 1994) (Fig. 1A). The most proximal nasal section was taken immediately posterior to the upper incisor teeth (proximal, T1); the middle section was taken at the level of the incisive papilla of the hard palate (middle, T2); the third nasal section was taken at the level of the second palatal ridge (T3); and the most distal nasal section (T4) was taken at the level of the intersection of the hard and soft palate and through the proximal portion of the OB of the brain (Fig. 1B). Tissue blocks were embedded in paraffin and the anterior face of each block was sectioned at a thickness of 5 µm, and stained with hematoxylin and eosin.
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Immunohistochemistry.
Unstained and hydrated paraffin sections from the same nasal blocks (T1–T4) were incubated first with a nonspecific protein blocking solution containing normal sera (Vector Laboratories Inc., Burlingame, CA) and then with specific dilutions of primary polyclonal antibodies directed against caspase-3 (1:100; rabbit anti-caspase-3 antibody; Abcam, Inc., Cambridge, MA), olfactory marker protein (OMP) (1:4000; goat anti-OMP antibody provided by Dr. Frank Margolis, University of Maryland), or neutrophils infiltrating the nasal mucosa (1:600; rabbit anti-rat neutrophil antibody provided by Dr Robert Roth, Michigan State University). Tissue sections used for caspase-3 or OMP detection were pretreated prior to the blocking solution with 3% (vol/vol) H2O2 in methanol to destroy endogenous peroxidase. Tissue sections were then incubated with biotinylated antispecies IgG. Immunoreactivity of caspase-3 and OMP was visualized with Vector R.T.U. Elite ABC-Peroxidase Reagent followed by Nova Red chromagen. Anti-neutrophil antibody treatment was sequentially followed with biotinylated anti-rabbit IgG, Streptavidin–Phosphatase complex (KPL laboratories, Gaithersburg, MD) and Vector Red chromagen. After immunohistochemistry, slides were lightly counterstained with hematoxylin.
Light microscopic morphometry.
Thickness of the olfactory epithelium (OE) lining the medial surface of the second ethmoid turbinates (2E) in T3 (Fig. 1) was morphometrically evaluated as previously described (Islam et al., 2006). Briefly, measurements were conducted at a final magnification of x1920 using a light microscope (Olympus BX40; Olympus America Inc., Melville, NY), coupled to a 3.3 megapixel digital color camera (Q-Color 3 Camera, Quantitative Imaging Corp., Burnaby, BC, Canada), and a PC (Dell Dimension 8200, Austin, TX). The morphometric analyses were performed using a cycloid grid overlay and software for counting points and intercepts (Stereology Toolbox, Davis, CA) (Hyde et al., 1990, 1991). The percentage volume density, Vv, the proportion of the epithelium composed of cytoplasm, nuclei, or apoptotic nuclear fragments was determined by point counting and calculated using the following formula:
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Other standard morphometric and image analysis techniques were used to determine the numeric cell density of mature OSNs in OE. Morphometric estimates of the numeric cell density of OSNs immunohistochemically reactive for OMP (protein indicator of mature OSNs) were determined via light microscopy (x790 final magnification) by counting the number of the nuclear profiles of these immunoreactive neuroepithelial cells in the OE lining the medial surface of the second ethmoid turbinates (2E) in T3 (Fig. 1) and dividing by the length of the underlying basal lamina. The length of the basal lamina was determined from the contour length of a computerized digital image of the basal lamina using Scion Image program (Scion Corporation, Fredrick, MD). All numeric cell density data were expressed as the number of OSN nuclei/mm basal lamina.
Necropsy and tissue processing for PCR.
Mice used for real-time PCR analyses of nasal and brain tissues were anesthetized and killed at designated times after RA instillation as described above. Immediately upon death, the head of each mouse was removed from the carcass after the skin, muscles, and lower jaw were removed from the head, the nasal airways were opened by splitting the nose in a sagittal plane adjacent to the midline. The nasal septum was removed thereby exposing the nasal turbinates projecting from the lateral wall of each nasal passage (Fig. 1). Using a dissecting microscope and ophthalmic surgical instruments, all ethmoid turbinates and OB were dissected from both nasal passages and brain, respectively. These excised tissues were immediately stored in RNAlater (Ambion Inc., Austin, TX). Total RNA was isolated using RNeasy Protect Mini kit (Qiagen Inc. Valencia, CA) within 7 days after removal.
Real-time PCR.
Real-time PCR for proapoptotic genes (Fas, FasL, PKR, p53, Bax, Bcl-2, caspase-activated DNAse [CAD]) and proinflammatory cytokine genes (interleukin [IL]-1, IL-1ß, tumor necrosis factor [TNF]-, IL-6, MIP-2) were performed on an ABI PRISM 7900HT using Taqman One-Step RT-PCR (reverse transcription–polymerase chain reaction) Master Mix and Assays-on-Demand primer/probe gene expression products according to the manufacturer's protocols (Applied Biosystems, Foster City, NY). Relative quantification of apoptotic and cytokine gene expression was carried out using an 18S RNA control and an arithmetic formula method (Audige et al., 2003; Islam et al., 2006).
Statistics.
All data were analyzed with SigmaStat v 3.1 (Jandel Scientific; San Rafael, CA) with the criterion for significance set at p < 0.05. Morphometric and RT-PCR data were subjected to one-way analysis of variance with Student–Newman–Keuls post hoc test. The capacity of LPS to magnify RA effects in a nonadditive manner was evaluated by comparing the response of cotreated mice to the predicted mean additive response (Islam and Pestka, 2006).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Intranasal RA Exposure Induces Apoptosis in OE
Light microscopic examination of the nasal tissues (T1–4) revealed that all mice (6/6) exposed to RA (500 µg/kg bw) and sacrificed at 1 and 3 days PI had conspicuous nasal epithelial and inflammatory lesions in the dorsocaudal half of the nasal passages that is normally lined by OE (Fig. 1). These lesions were not apparent in the nasal cavity of saline vehicle–treated controls nor were they observed in RA-instilled mice that were sacrificed after only 6 and 12 h. RA-induced alterations were not present in regions of the nasal airways lined by other nasal epithelial types including respiratory, transitional, or squamous epithelium.
RA-induced OE lesions at 1 day PI consisted of numerous individual epithelial cells with morphologic features characteristic of apoptosis in the OE lining all the ethmoid turbinates and the adjacent lateral walls that border the lateral meatus in the distal regions of both nasal passages (T3 and T4), with the most dorsolateral ethmoid turbinates (1E and 2E) being most severely affected (Fig. 1B). RA-induced apoptosis was also present in the OE of the mid and ventral septum lining the middle medial meatus in T3, but not in T4. In the middle of the nasal passages (T2), prior to the distal regions containing the ethmoid turbinates (T3 and T4), RA-induced apoptotic lesions in the OE were only detected in a small mucosal region of the lateral walls and septum lining the middle medial meatus where the OE meets the respiratory epithelium. RA-induced nasal epithelial lesions were undetectable in the most proximal regions of the nasal passages (T1). Interestingly, the OE lining the dorsal medial meatus throughout the nasal passages (T1–T4) had no microscopic evidence of RA-induced apoptosis of OE or any other epithelial alterations.
RA Induces OE Atrophy and Loss of OSNs
When effects of RA on OE effects (Fig. 2A–J) were assessed over time, RA-induced OE lesions and OE atrophy were first detectable at 24 h PI (Fig. 2 G,H) and to a much greater extent at 72 h PI (Figs. 2I and 2J). Morphometry of OE lining ethmoid turbinate 2 (region 2E, Fig. 1B) confirmed that greater atrophy occurred in mice sacrificed 3 days PI than in mice sacrificed 1 day PI, with an approximately 33% reduction in epithelial thickness compared to control mice (Fig. 2K).
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OMP, a specific peptide found only in mature OSNs (Kream and Margolis, 1984
), was dramatically reduced in the OE of RA-instilled mice compared to saline-instilled control mice (Figs. 3A and 3B). Consistent with OSN loss, immunohistochemical examination demonstrated a marked reduction of the normally dense mat of cilia projecting from the dendritic knobs and lining the surface of the nasal airway lumen (Figs. 3C and 3D). There was also noticeable atrophy of the OMP-positive olfactory nerve bundles located in the lamina propria underlying the atrophic OE. Morphometric analysis revealed approximately a 90% loss in OSNs per mm of OE in the nasal mucosa lining the dorsolateral meatus at 3 days PI when compared to the identical region in vehicle-instilled mice (Fig. 4).
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Concurrent with the loss of OMP-positive OSNs in the OE and lamina propria in the nasal mucosa, there was also marked bilateral atrophy and vacuolation of the outer tissue layer (olfactory nerve layer) of the OBs (Fig. 5A) in the brains of these RA-treated mice at 72 h PI. Loss of OMP-stained axons of the OSNs in the brain was most evident in the lateral and medial aspects of each OB (Figs. 5B and 5C). Loss of OMP-staining was also evident in the adjacent glomerular layer of the OB where the axons of OSNs first synapse with other neurons in the brain.
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RA Induces Apoptosis of OSN in OE at 24 h PI
RA-induced conspicuous apoptotic lesions in the OE at 24 h. Toxin-induced apoptosis was defined by light morphologic features of condensation and shrinkage of individual epithelial cells; clumping, fragmentation, and margination of nuclear chromatin; and numerous widely scattered cellular fragments (apoptotic bodies) (Figs. 2G, 2H, and 6A, 6B). Morphologic detection of RA-induced apoptosis in the OE corresponded with the immunohistochemical detection of the activated caspase-3 in many of the apoptotic cells (Figs. 6A and 6B). RA-induced apoptosis was restricted to OSNs whose cell bodies and nuclei reside in the middle nuclear layers of the OE below the distinct apical row of sustentacular (support) cell nuclei and above the basal cell nuclei near the basal lamina. Prominent anti-caspase-3 staining was also present in olfactory nerve bundles (axons of OSNs) in the underlying lamina propria of the mice instilled with RA. Therefore, the marked RA-induced atrophy of OE, that was most prominent at 72 h PI, was likely a result of widespread apoptosis of OSN at 24 h with subsequent loss of these neuronal cells. Very few apoptotic cells or cellular fragments remained in the atrophic OE at 72 h PI, and there was no increase in immunohistochemical detectable activated caspase-3, above that of saline-instilled controls, in these nasal tissues at this later time point.
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RA Upregulates Proapoptotic gene Expression in the Ethmoid Turbinates
Real-time PCR analysis of microdissected OE-lined ethmoid turbinates from RA-treated mice demonstrated a marked upregulation of the proapoptotic genes Fas, PKR, p53, Bax, and CAD (Fig. 7). Fas mRNA expression was elevated as early as 6 h PI, remained significantly elevated up to 24 h PI, but returned to baseline control levels at 72 h PI. Although there was also a trend toward higher mRNA expression for PKR, p53, Bax, and CAD at 12 h, these genes were significantly elevated only at 24 h PI and returned to control values at 72 h PI. Thus, apoptotic death of OSNs in the OE was preceded by Fas expression and concurrent with expression of these other proapoptotic genes.
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RA Induces Neutrophil Infiltration in OE
Along with the marked atrophy of OE at 72 h PI, RA-instilled mice also had prominent accumulations of mucoproteinaceous material with exfoliated and degenerating cellular debris from the dendritic portions of the apoptotic OSNs in the nasal airways adjacent to the luminal surfaces of the atrophying OE. With secondary degeneration of these exfoliated dendritic fragments, there was accompanying infiltration of numerous phagocytic cells consisting mainly of polymorphonuclear leukocytes (neutrophils) and only occasional mononuclear cells (monocytes and macrophages) (Figs. 2I, 3D, and 8A–D). Infiltrating neutrophils were also widely scattered in the atrophic OE and lamina propria of the RA-altered olfactory mucosa (Figs. 8C and 8D).
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RA Upregulates Proinflammatory Gene Expression in the Ethmoid Turbinates and the OB
In the previous study with satratoxin G, there was a robust infiltration of mucosubstances filled with neutrophils in the nasal airways and sporadic incidences of neutrophils in the OB (Islam et al., 2006
). Therefore, proinflammatory cytokines and the neutrophil chemoattractive protein MIP-2 were measured in the tissues from ethmoid turbinate (ET) and OB. The results showed that there was markedly increased expression of mRNAs for the proinflammatory cytokines TNF-, IL-1, IL-1ß, IL-6, and MIP-2 in the ethmoid turbinates of mice intranasally instilled with RA (Fig. 9A). Except for IL-1, upregulation of these mRNAs was evident as early as 6 h PI. Expression of all genes remained elevated at 12 and 24 h PI and returned to control levels at 72 h PI. As observed for the ethmoid turbinates, IL-6 and MIP-2 mRNA levels were also transiently elevated in the OB from 6 to 12 h PI (Fig. 9B), whereas the other proinflammatory cytokines were not affected (data not shown). Thus, proinflammatory gene expression in the ethmoid turbinate and OE generally occurred much earlier (6 h PI) than either apoptotic gene expression or onset of apoptosis OSN apoptosis (24 h PI).
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LPS Enhances RA-Induced Apoptosis of OSNs, OE Atrophy, and Neutrophilic Rhinitis
The effects of intranasal coexposure to RA (250 µg/kg bw) and LPS (500 ng/kg bw) on OE apoptosis were also assessed at 24 h PI. Mice intranasally instilled with vehicle or with LPS alone did not exhibit any exposure-related epithelial or inflammatory lesions in the nasal airways (Figs. 10A and 10C). Mice instilled with RA alone exhibited moderate increases in apoptotic cells in the OE lining similar regions of the nasal airways that were altered with the higher RA dose (500 µg/kg bw) in the previously described experiment (Fig. 10B). In contrast, mice coinstilled with LPS and RA, had a marked neutrophilic rhinitis with degeneration, exfoliation and atrophy of turbinate and septal OE, especially in the nasal mucosa lining the dorsolateral meatus and middle medial meatus, respectively (Fig. 10D). Neutrophilic influx in the nasal mucosa and adjacent nasal airways were most prominent in the regions with the greatest OE lesions.
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Increased inmmunohistochemical staining for anti-caspase-3 staining in the OE, compared to that of the saline-instilled control mice (Fig. 11A), was evident in mice intranasally instilled with RA alone (Fig. 11B), but not LPS alone (Fig. 11C). There were markedly more anti-caspase-3–stained apoptotic cells and cellular fragments in the degenerating OE of mice coexposed to RA and LPS (Fig. 11D), compared to those of mice exposed only to RA.
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Proapoptotic gene expression was also compared in microdissected OE-lined ethmoid turbinates from mice treated with vehicle, RA only, LPS only, or RA plus LPS at 24 h PI. LPS alone induced mild Fas, FasL, and PKR expression while RA-induced PKR only. Marked mRNA upregulation of Fas, FasL, and PKR was observed in the cotreated mice (Fig. 12). Thus, coadministration of RA and LPS caused greater proapoptotic gene expression and apoptosis in the OE than either toxin alone.
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Based on the prior kinetic study with RA, 12 h PI was chosen to compare proinflammatory cytokine gene expression in mice instilled with LPS and/or RA (Fig. 13). LPS but not RA alone induced TNF-
, IL-1, and IL-1ß in ethmoid turbinates (Fig. 13A) and OB (Fig. 13B). There was a trend toward elevated expression of all three cytokines following cotreatment with RA and LPS, but these changes were not significant compared to LPS alone. IL-6 and MIP-2 mRNAs were marginally induced by either LPS or RA alone in both ethmoid turbinates (Fig. 13A) and OB (Fig. 13B). However, coexposure to both agents dramatically elevated these genes as compared to either toxin alone. Thus, LPS could magnify OSN apoptosis and inflammation following a single acute coexposure to RA.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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As a portal of entry of inhaled air, the nose is a prime target for airborne toxic and infectious agents. Surface epithelial cell populations lining the nasal passages are often the initial targets for inhaled toxicants. Morphologically and functionally distinct epithelia line the mammalian nasal passages that include (1) squamous epithelium, (2) ciliated, pseudostratified, respiratory epithelium, (3) nonciliated transitional epithelium, and (4) OE, located in the dorsal or dorsoposterior aspect of the nasal cavity (Harkema et al., 2006
). Very low satratoxin G doses (as little as 25 µg/kg bw or 0.5 µg/mouse) cause apoptosis of OSNs of the OE in the mouse as evidenced by morphologic examination at the light and electron microscopic levels, immunohistochemical detection of activated caspase-3, and proapoptotic gene expression (Islam et al., 2006). Concurrently, satratoxin G causes rhinitis and a mild focal encephalitis in the OB of the brain. Yike and Dearborn (2004) estimated that a single S. chartarum spore contains 1 pg equivalent of satratoxin G, suggesting that a single exposure to 5 x 105 spores could deliver sufficient toxin in the mouse to evoke olfactory toxicity. The results presented herein indicate that the related macrocyclic trichothecene RA similarly targets OSNs and can also initiate inflammatory responses in the nose and OB. It should be noted that approximately 10 times more RA than satratoxin G was required to achieve equivalent effects. Nevertheless, RA should be a suitable surrogate to uncover underlying mechanisms of macrocyclic trichothecene toxicity in the nasal airway and to investigate interactive effects with endotoxin, a co-occurring airborne toxicant.
The OE and OSNs are vulnerable to injury and death from direct exposure to inhaled toxins and infectious agents. OE undergoes continuous neuronal cell renewal throughout the life span of an individual. Its regenerative ability following injury enables the OE to maintain olfactory sensory function. Compared to other neurons, OSNs are unique in that they have relatively short life spans (30–40 days or longer) and are continuously being replaced through basal cell proliferation and differentiation (neuronal regeneration) (Graziadei and Monti-Graziadei, 1978). Similar to other epithelial cells, but unlike most neuronal cells, OSNs undergo continuous apoptosis and genesis throughout life as part of the normal turnover of mature OE. Experimentally, OSNs in laboratory animals can be induced to die via manipulative methods such as olfactory bulbectomy, transection of the olfactory nerve at the cribiform plate, and intranasal exposure to chemicals known to be toxic to the OE, such as zinc sulfate and methyl bromide (Cowan and Roskams, 2002, 2004). While bulbectomy and nerve transection evoke selective apoptosis in death of OSNs, most chemical toxins cause necrosis (oncosis) of the OSNs along with other epithelial cells (sustentacular and basal cells) in the OE. Thus, it is quite remarkable that RA, as described here, and the satratoxins, as reported previously (Islam et al., 2006), are exquisitely specific in initiating OSN apoptosis and OE atrophy in the nose and in the OB.
RA might induce OSN apoptosis in two ways. One plausible mechanism is via death receptors. OSN apoptosis has been previously linked to specific genes associated extrinsic death receptor pathways involving TNF- and Fas (Cowan and Roskams, 2002). Both TNF- and Fas directly induce apoptosis in in vitro OE organ cultures (Farbman et al., 1999; Suzuki and Farbman, 2000). Consistent with this mechanism, the results presented here indicated that RA-induced TNF- and Fas mRNA expression from 6 to 24 h PI which preceded or was concurrent with OSN apoptosis, induction of caspase-3 mRNA and caspase-3 activation at 24 h PI. The origins of induced TNF- might be OSNs or adjacent cells in the OE which would promote autocrine or paracrine responses, respectively. For Fas upregulation to have an effect it would have to occur within the susceptible OSN population. A second potential mechanism is that RA directly induces apoptosis in the OSNs by initiating mitochondrial cell death via an intrinsic pathway involving p53 and Bax. Previously, we have demonstrated in macrophages that the trichothecene deoxynivalenol induced p38-dependent apoptosis involving p53, Bax, and caspase-3 (Zhou et al., 2005) and furthermore, that PKR plays a critical role in p38 activation as well as apoptosis (Zhou et al., 2003). Here, it was shown, at 24 h PI, that RA markedly upregulated mRNA expression for p53 and Bax as well as PKR; furthermore, increased expression of these genes at 24 h PI was concurrent with OSN apoptosis, induction of caspase-3 mRNA, and caspase-3 activation. Consistent with the latter findings, satratoxin H–induced caspase-3 activation and apoptosis in PC12 neural cells are p38 dependent (Nusuetrong et al., 2005). It should be emphasized that induction of the aforementioned extrinsic and intrinsic pathways by RA is not mutually exclusive and might involve extensive cross-talk. Future studies should clarify the specific cell types within the OE that express TNF-, Fas and proapoptotic mRNAs and further relate these mRNA data to expression of proteins encoded by these genes.
It is not readily apparent why RA and the satratoxins specifically target OSNs while nasal respiratory epithelium and other cell types in the OE remain unaffected. One possibility is that OSN sensitivity results from longer regional exposure to epithelial cells in OE compared with the exposure to cells within the respiratory epithelium. Prolonged regional exposure could be caused by the much slower rate of mucociliary clearance of inhaled agents from OE-lined ethmoid turbinates, which are covered by immotile cilia, as compared to other regions of the nasal cavity that are lined by respiratory epithelium containing motile cilia with high ciliary beat frequencies. Such movement facilitates rapid regional flows of mucus from of the nasal cavity, through the nasopharynx and ultimately into the upper digestive tract (Morgan et al., 1984). A second possibility is that slower RA clearance from OE compared with respiratory epithelium might result from differences in factors that impact clearance of chemicals from the nasal airway, such as mucosal metabolism or blood flow. Finally, Yike et al. (2005) reported that satratoxin G can form covalent adducts with proteins. Formation of such adducts with OSN receptors which cover a wide surface area on the OE might provide a signal that drives apoptosis.
It was also notable that the dorsomedial meatus, a region of the nasal cavity that is populated by OE, was consistently spared from toxin-induced injury. This was similarly observed for the satratoxins (Islam et al., 2006). The potential exists that macrocyclic trichothecenes or their metabolites might bind only specific OSN receptors, thereby enhancing uptake and resultant toxicity. In support of this contention, populations of distinct odorant receptors can be divided into four specific topographical regions of the OE, one of which lines the dorsomedial meatus (Ressler et al., 1993). Thus, odorant receptors might be involved in OSN toxicity caused by these mycotoxins.
While we are unaware of inhaled toxicants other than the macrocyclic trichothecenes that specifically target OSNs, it has been reported that exposure of mice to high intravenous doses of some tubulin-targeting antitumor drugs, like vincristine, induces specific apoptosis of OSNs with subsequent OE atrophy in the mouse (Kai et al., 2002, 2004). Subsequent studies by that group suggest that the initial event of vincristine-induced apoptosis in the mouse OE was mitotic arrest with high drug retention (Kai et al., 2005), and, that mice, particularly females, were more susceptible to vincristine-induced OE apoptosis than rats or monkeys (Kai et al., 2006). Interestingly, vincristine and other microtubule-interfering agents induce mitogen activated protein kinase–mediated activation of activator protein-1 and the transcription of COX-2 (Subbaramaiah et al., 2000). Since vincristine interferes with protein synthesis by disrupting the cytoplasmic microtubule network and function of membrane-associated ribosomes (Walker and Whitfield, 1985), it is tempting to speculate that tubulin-targeting antitumor drugs might induce a ribotoxic stress response in OSN and adjacent cells thereby causing similar outcomes to those of the macrocyclic trichothecenes.
Instillation of endotoxin into the airways of laboratory rodents causes a similar inflammatory response to those observed in humans, including cytokine production and neutrophil infiltration. Both structural and cellular changes in the airways of laboratory rodents are elicited by intranasal instillation (Harkema and Hotchkiss, 1991, 1992; Steiger et al., 1995) and aerosolized endotoxin (Gordon and Harkema, 1994). Among these are epithelial cytotoxicity, hyperplasia, and increased synthesis, storage, and secretion of mucosubstances by airway mucous cells. In this study, we observed that LPS markedly amplifies OSN apoptosis and inflammation following a single acute coexposure to RA. These findings are significant because the effects of Stachybotrys and other molds in water-damaged buildings might be amplified by cocontaminating inflammagenic particulates containing LPS, most notably Gram-negative bacteria (e.g., E. coli, P. aeruginosa) (Andersson et al., 1997; Park et al., 2006). Potentiation of apoptosis by LPS appeared to correlate with increased Fas, FasL, and PKR, but not with TNF- or IL-1 mRNAs, in the OE. The increased neutrophilic rhinitis that occurred with RA and LPS coexposure also correlated with increased IL-6 and MIP-2 expression.
Consistent with the findings of this study, the trichothecenes T-2 and deoxynivalenol become more toxic in the presence of endotoxin, thereby causing elevated tissue injury and mortality (Tai and Pestka, 1988a,b; Taylor et al., 1989, 1991; Zhou et al., 1999, 2000). Coexposure of mice to subtoxic doses of LPS and deoxynivalenol markedly upregulates proinflammatory cytokine expression and subsequently induces apoptosis in thymus, Peyer's patches, bone marrow, and spleen (Islam et al., 2002). Remarkably, a single LPS exposure can sensitize the innate immune system to trichothecene-induced lymphoid apoptosis for at least 24 h (Islam and Pestka, 2006).
Taken together, our observations here and previously that the OE and OB are specific targets of macrocyclic trichothecenes should be a critical consideration in future studies of damp building–related illnesses and the potential etiologic role of S. chartarum and other molds. Acute inflammation of nose (allergic rhinitis) and brain (encephalitis) are significant health problems that can affect many aspects of daily life because of physical discomfort and impairment along with emotional distress (Baiardini et al., 2006; Olgar et al., 2006). The genes upregulated here could likely contribute to OSN apoptosis as well as accompanying rhinitis and even mild encephalitis. The observation that LPS exacerbated RA-induced proinflammatory gene expression, apoptosis, and inflammation in the OE suggests that co-occurring inflammagens have the potential to interact with macrocylic trichothecenes in evoking even more severe pathologic effects in the nasal cavity. While the data presented herein and previously (Islam et al., 2006) provide important insight into targets and mechanisms, future perspectives must include assessment of dose dependency and latency of recovery in nasal tissue after chronic exposure to macrocyclic trichothecenes alone, as well as the contributions of spore matrix and co-occurring inflammagens found in indoor air. It will also be critically important to understand the extent to which toxicant-induced inflammation and neuronal injury occur in other parts of the brain along the olfactory pathway and whether such injury could contribute to neurocognitive dysfunction. Finally, all mechanistic findings must be considered in the context of accurate quantitative assessments of human exposure to trichothecenes using both state-of-the-art sampling and analytical methods and relevant biomarkers.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONSUPPLEMENTARY DATAREFERENCES |
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Supplementary data are available online at http://toxsci.oxfordjournals.org/.
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ACKNOWLEDGMENTS |
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This research was funded by a Michigan State University Foundation Strategic Partnership Grant and Public Health Service Grant ES03358 (J.J.P.) from the National Institute for Environmental Health Sciences. We thank Lori Bramble, Sarah Godbehere, Amy Porter, Rick Rosebury, Kathleen Campbell, and Dr Annette Thelen for technical assistance.
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