Stachybotrys chartarum Alters Surfactant-Related Phospholipid Synthesis and CTP:Cholinephosphate Cytidylyltransferase Activity in Isolated F
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《毒物学科学杂志》
Departments of Oral Biology and Human Anatomy and Cell Science, Faculties of Dentistry & Medicine, University of Manitoba, Winnipeg, Manitoba, Canada R3E0W2
Department of Biology, St. Mary's University, Halifax, Nova Scotia B3H3C3, Canada
National Research Council, Institute for Biodiagnostics, Winnipeg, Canada R3B1Y6
Health Canada, Health Products and Food Branch, Winnipeg, Manitoba, Canada A2J3Y1
Manitoba Institute of Child Health, Biology of Breathing Group, Children's Hospital Foundation, Winnipeg, Manitoba, Canada R3E3P4
ABSTRACT
Stachybotry chartarum, a fungal contaminant of water-damaged buildings commonly grows on damp cellulose-containing materials. It produces a complex array of mycotoxins. Their mechanisms of action on the pulmonary system are not entirely clear. Previous studies suggest spore products may depress formation of disaturated phosphatidylcholine (DSPC), the major surface-active component of pulmonary surfactant (PS). If S. chartarum can indeed affect formation of this phospholipid, then mold exposure may be a significant issue for pulmonary function in both mature lung and developing fetal lung. To address this possibility, fetal rat type II cells, the principal source of DSPC, were used to assess effects of S. chartarum extract on formation of DSPC. Isolated fetal rat lung type II cells prelabeled with 3H-choline and incubated with spore extract showed decreased incorporation of 3H-choline into DSPC. The activity of CTP:cholinephosphate cytidylyltransferase (CPCT), the rate-limiting enzyme in phosphatidylcholine synthesis was reduced by approximately 50% by a 1:10 dilution of spore extract. Two different S. chartarum extracts (isolates from S. chartarum (Cleveland) and S. chartarum (Hawaiian)) were used to compare activity of CPCT in the presence of phosphatidylglycerol (PG), a known activator. PG produced an approximate two-fold increase in CPCT activity. The spore isolate from Hawaii did not alter enzyme activity. S. chartarum (Cleveland) eliminated the PG-induced activation of CPCT. These results support previous observations that mold products alter PS metabolism and may pose a risk in developing lung, inhibiting surfactant synthesis. Different isolates of the same species of fungus are not equivalent in terms of potential exposure risks.
Key Words: lung; mold spores; cytidylyltransferase; surfactant.
INTRODUCTION
Fungal spores and their micellar components and products are common exposure factors in the everyday environment in virtually all parts of the world. Wheat dusts in Egypt (Abdel-Hafez et al., 1990), tobacco cigarettes (el Maghraby and Abdel Sater, 1993), soil and food products in France (Pieckova and Jesenska, 1999), and infant exposure in Cleveland (Etzel et al., 1998), although the latter has been questioned by a CD review (Centers for Disease Control and Prevention, 2000), all demonstrate the ubiquitous distribution and potentially hazardous effects of fungal products. These airborne contaminants represent a wide range of various fungal species (Cooley et al., 1998). Generally the bioaerosols generated from fungi are considered the main contributors to what has come to be known as sick-building syndrome (Lewis, 1994).
The materials from fungi appear to be associated with two main types of responses. The first is an allergenic response which may affect people to varying degrees, depending on a range of individual factors including such things as previous exposure levels, individual allergenic response potentials, or immunological status (Flannigan, 1994). The second type of response relates to the production of toxigenic mycotoxins, which, unlike allergenic materials, produce toxic responses in virtually all people (Jarvis, 1994). While exposure levels are dependent on many factors, and relatively low levels of these toxins and other volatile organic compounds may be released (Wilkins et al., 1998), not a great deal is known of the effects or modes of action of these materials. Some studies suggest that mycotoxin extracts of several different species of fungi may inhibit cellular replication potential or be cytotoxic as measured in an MTT-formazan assay using swine kidney cells (Gareis, 1994). However, as any given exposure, whether it induces an immunological reaction or represents a response to mycotoxin exposure, appears to originate through the respiratory system (Nikulin et al., 1997), lung cells would seem to be the model of choice. Our previous work suggests that S. chartarum reduces the synthesis of disaturated phosphatidylcholine (DSPC), the major pulmonary surfactant phospholipid. Furthermore intratracheal instillation of S. chartarum spores produces ultrastructural changes in mouse lung type II cells (Rand et al., 2002), suggesting that this cell type may be a target for S. chartarum mycotoxins. Consequently the objective of the present study was to determine the effects of S. chartarum on conversion of choline, the major biosynthetic precursor of disaturated phosphatidylcholine. As a means of establishing the effects of the toxin on surfactant synthesis, the model selected for study was the isolated fetal lung type II cell, as these late fetal lung cells closely mimic neonatal lung cells (Massaro et al., 1986).
MATERIALS AND METHODS
Animals. Pregnant Sprague-Dawley rats (virus antibody free, VAF, original breeding population, Charles River, Montreal, QC, Canada) were used for the experiments and were from the animal care facility at the University of Manitoba, in Winnipeg, MB. All procedures have been approved by the Canadian Council on Animal Care through the local protocol review committee at the University of Manitoba.
Preparation of spore extracts. Spores from Stachybotrys chartarum, strain 002 from Hawaii, (Department of Agriculture, Culture Collection, DAOM 225489) and from Cladosporium cladosporioides were isolated and cultured as described previously (Mason et al., 1998). Spores were suspended in physiological saline because this represents a more biologically relevant situation than that often used by methanol extraction (Andersson et al., 1997). Since Cladosporium cladosporioides does not produce known mycotoxins, it was used in some experiments as a negative control. The concentration of the spore samples was 2.0 x 106 spores per ml. Spores from American Type Culture Collection Stachybotrys chartarum, strain 58–17 (ATCC 201211) (Cleveland), is an isolate from a home in Cleveland (Jarvis et al., 1998).
The suspension was initially centrifuged at 2500 rpm for 15 min to collect the spores. The supernatant was decanted and discarded, and the pellet was resuspended in 10 ml of cold sterile Hank's Balanced Salt Solution (HBSS) and incubated for 3 hours. The spores were again collected by centrifugation and the supernate decanted for use in the experiments.
Fetal lung type II cell isolation and culture. Fetal lung type II cells were isolated as described previously (Batenburg et al., 1988; Scott, 1994). Pregnant rats were killed by an ip injection of Euthanyl on the 21st day of gestation (term is 23 days). The fetuses were removed, decapitated, and their bodies placed in cold sterile HBSS. Under sterile conditions, fetal lungs were removed, and mediastinal structures were dissected away. The lung tissue was washed twice with cold sterile HBSS and chopped on a Sorval tissue chopper (Sorval Instruments, Newtown, CT). Lung tissue was placed in a solution of HBSS with trypsin-EDTA (0.05%/0.01%) (Gibco, Missisauga, ON), in a water-jacketed trypsinization flask (Wheaton, Millville, NJ) and stirred on a magnetic plate for 45 min at 37°C. Trypsinization was arrested with the addition of Minimum Essential Medium, (MEM, Gibco, Missisauga, Ont.) with 10% carbon-stripped Newborn Calf Serum, (sNCS, Sigma, St. Louis, MO), and 1% each of antibiotic/antimycotic and Fungizone (Gibco, Missisauga, ON). The suspension was filtered through three layer of sterile Nitex gauze and centrifuged for 15 min at 250 x g. The supernatant was removed, and the pellet was resuspended in 50 ml of MEM with sNCS. The cells were plated in five sterile 75-cm2 culture flasks (Fisher Scientific, Mississauga, ON) and incubated for 1 h at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Since fibroblasts attach more quickly than type II cells, the fibroblasts were the first few cells to adhere to the plastic flasks. After the incubation period, the contents of the flasks were poured off into a second set of flasks and incubated for a further 30 min to allow for further separation of the fibroblasts. The contents of the flasks were pooled, and the cells were counted in a Coulter cell counter (Coulter Electronics, St Hialeah, FL). Cells were plated at a density of 8 x 105 cells/25-cm2 flask in MEM with 10% sNCS (v/v) and incubated under the conditions described above. The medium was changed after the first 24 h and every 48 h thereafter. This population of cells represents an 80–85% enrichment in perinatal type II alveolar cells (Batenburg et al., 1988).
In vitro incubation of cells with spore extracts. Enriched type II cell cultures were incubated with the spore extracts to investigate the effects of the fungal toxins on the radiolabeling of disaturated phosphatidylcholine (DSPC), the major surface-active phospholipid of pulmonary surfactant (Veldhuizen et al., 1998). The following approach was used to ensure similar cell numbers in each incubation. At confluence, the cells were removed with trypsin/EDTA. Trypsin action was terminated with the addition of MEM with sNCS. Cells were collected by centrifugation at 250 x g for 10 min. The supernatant was removed, and the pellet was resuspended in MEM with sNCS. The cells were centrifuged again at the same speed and for the same amount of time. The supernatant was removed, and the cells were resuspended in a known volume of MEM/sNCS. The cells were counted in a Coulter counter, and 5.00 x 105 cells per incubation were used.
To study the effects of the spore toxins, enriched cell cultures were incubated in microcentrifuge tubes with varying concentrations of Stachybotrys chartarum supernatant, HBSS, and [methyl-3H]choline chloride (specific activity, 58 Ci/mmole, New England Nuclear, Lachine, QC). Cells were incubated with varying concentrations of toxin, MEM with sNCS, and radioactive precursor at 37°C and at 5% CO2 with constant agitation for 18 h. The incubations were terminated by the addition of methanol.
Isolation of DSPC. Following the addition of methanol, the cells were transferred to test tubes. Lipids were extracted with a solution of chloroform/methanol/1% potassium chloride (1:1:0.9). The solvent phase containing the lipids was removed and concentrated. To isolate DSPC (Gilfillan et al., 1983), 0.5 ml of carbon tetrachloride containing 3.5 mg of osmium tetroxide was added to each sample, incubated for 15 min, and removed by evaporation. Samples were spotted onto thin layer chromatography plates (Whatman, LK5D) with authentic standards and developed in chloroform/methanol/water (65:25:4). Phospholipid spots were identified using iodine vapor, and radioactivity from the appropriate spots was measured by scraping the gel into scintillation vials and counting on a LS5801 scintillation counter. Quench was compensated using the method of H# based on the spectrum of 137Cs (Beckman Instruments, Palo Alto, CA).
Pulse Chase Experiment
In vitro incubation. Fetal type II cells were isolated as described above, and experiments were performed as described previously (Cherlet and Scott, 2002). Cells were prelabeled for 1 h in HBSS with 2 μCi of 3H-choline and removed to fresh MEM with sNCS with Cladosporium cladosporioides or S. chartarum spore supernates for 15, 30, 60, or 120 min. The medium was removed, and cells were scraped into test tubes. Cells that were incubated with MEM and sNCS alone served as controls.
Isolation of choline, choline-phosphate, and CDP-choline. Samples prelabeled with 3H-choline were extracted using chloroform/methanol/1% potassium chloride (1:1:0.9), and both the upper aqueous layer and the lower organic phase were saved. The aqueous samples were dried under air and resuspended in a known volume of water/ethanol (1:1). Intermediates were isolated by thin layer chromatography. Samples were spotted on plates and developed in methanol/0.78% NaCl/ammonium hydroxide (50:50:5) (Tokmakjian et al., 1981). Standards of choline, cholinephosphate, and CDP-choline were run on separate channels on each plate. Plates were dried thoroughly after development, and standards were visualized using iodine. Appropriate spots were scrapped into scintillation vials, and radioactivity was measured.
Separation of type II cell cytosolic fraction. Fetal rat type II cells were isolated as described above, centrifuged at 250 x g for 15 min to collect the cells, the supernatant was removed, and the pellet was resuspended in HBSS. Samples were frozen at –85°C to destroy cell integrity and were kept ice-cold at all times prior to the assay to preserve enzyme activity. The samples were centrifuged at 300 x g for 10 min to precipitate the nuclei. The pellet was discarded, and the supernatant was centrifuged at 10,000 x g to precipitate the mitochondria. The pellet was discarded, and the supernatant was centrifuged at 100,000 x g for 90 min. The supernatant containing the cytosolic fraction was retained. This sample was concentrated by centrifugation at 3000 rpm for 30 min at 4°C in a Millipore centrifuge Filter Device. Protein concentration was determined by the BioRad Protein Assay (BioRad, Missassauga, ON, Canada).
Assay of CTP:cholinephosphate cytidylyltransferase (CPCT, 2.7.7.15). The effects of Stachybotrys chartarum, strains 002 (Hawaiian) and 58–17 (Cleveland), on CPCT activity were measured. The enzyme was assayed as described (Cherlet and Scott, 2002; Sharma et al., 1993; Viscardi et al., 1989) in the presence or absence of undiluted or 1-in-10 diluted spore supernatant. The procedure was as follows. Stock solutions of magnesium acetate (100 mM, final concentration 10 mM), PIPES-maleate (pH 6.5, 1 M, final concentration 100 mM) were prepared. 14C-Cholinephosphate (specific activity, 50 mCi/mmol; final concentrations, 0.3 mM at 2 Ci/mol in 0.1 mM EGTA) was used in the assay. Cytidine triphosphate, (CTP, 50 mM, final concentration 5 mM) was made fresh each time for the assay. In some assays phosphatidylglycerol, a known activator of the enzyme, was used at concentrations of 0.25 mM. Distilled water was added to this stock solution according to the amount of spore supernatant required to ensure equal volumes of solution for each incubation. The enzyme was assayed in a total volume of 100 μl. Prior to starting the incubation the components, excluding the cytosolic protein, were aliquoted into microcentrifuge tubes, and the tubes were placed in a water bath of 37°C for 5 min. To start the incubations, 120 μg of cytosolic protein was added to each tube. Tubes were removed at various times (0–40 min) and immersed in boiling water to stop the reaction. This assay was repeated with the addition of phosphatidylglycerol with or without undiluted samples of the supernate from two Stachybotrys spore variants.
Tubes were centrifuged for 2 min at 13,500 rpm in a microcentrifuge to pellet protein. Fifty microliters (50% of the total volume) of the supernatant were spotted on thin layer chromatography plates (Whatman, LK5D) with CDPcholine authentic standard. Plates were developed in ethanol/water/20% ammonium hydroxide (100:50:2) (Cherlet and Scott, 2002). Plates were thoroughly dried, and CDPcholine spots were identified using iodine. Appropriate spots were scrapped into scintillation vials and radioactivity determined as indicated above.
Analysis of S. chartarum supernatants. Samples of the supernates collected as described above from Stachybotrys chartarum, strain 58–17 (ATC 201211) (Cleveland), Stachybotrys chartarum, strain 002 from Hawaii, (Department of Agriculture, Culture Collection, DAOM 225489), and Cladosporium cladosporioides were analyzed, and 1-ml samples were lyophilized and analyzed by high performance liquid chromatography. Spectra obtained from the samples were screened for aflatoxin B1, alternariol, alternariol methyl ether, altertoxin I, brefeldin A, cyclopenin, cyclopenol, deoxynivalenol, nivalenol, ochratoxin A, patulin, penicillic acid, penitrim A, roridin A, roridin E, satratoxin G, satratoxin H, verrucarin A, verrucarin J, and viridicatin. Detection of peaks was done using photodiode array spectral matching against spectra of known standards injected under identical conditions. Quantification was done by comparing peaks obtained from the samples against calibration curves using known quantities of toxin.
Statistical analysis. Where appropriate, statistical analysis was done by post hoc application of Duncan's New Multiple Range Test (Ott, 1977) assuming a significant difference at p < 0.05. Regression lines were fit by computer to the raw data using SigmaPlot (SPSS Incorporated, Chicago, IL).
RESULTS
The HPLC chromatogram of the supernate from Stachybotrys chartarum, strain 58–17 (Cleveland) is shown in Figure 1. Two peaks corresponding to satratoxin G and satratoxin H standards were detected. Quantification indicated that approximately 1 μg of each satrotoxin existed in the sample. The other spore sample from S. chartarum-002 (Hawaiian) did not show any identifiable toxins (results not shown).
The accumulation of radioactive precursor in water-soluble intermediates in the synthetic pathway for DSPC is shown in Figure 2. Intracellular levels of 3H-choline in control or S. chartarum (Cleveland)-exposed cells displayed a similar pattern, declining from high levels after 20–40 min to low levels by 120 min. Concurrently, levels of radiolabeled precursor appearing in cholinephosphate had peaked by 20 min, indicating that 3H-choline rapidly reaches cholinephosphate. Again, very little difference was observed in the pattern of labeling of this intermediate, and fitting of regression lines indicated a similar decline in appearance of label over 120 min. In contrast to the first two intermediates, CDPcholine labeling in cells exposed to C. cladosporioides or under control conditions displayed a slow increase or no change, respectively, over time. However, in cell samples exposed to S. chartarum, a different pattern of radiolabeling was observed. In this intermediate, radioactive precursor levels declined between 20 min and 60 min and remained low; radiolabeling of CDPcholine in control and S. chartarum-exposed cells was similar by the end of 120 min.
The radiolabeling of total phosphatidylcholine and DSPC at varying exposure levels to supernate from S. chartarum samples is shown in Figure 3. The lowest level of spore exposure (0.225 μl/ml) did not affect the label incorporation into DSPC. In contrast, the three higher dose levels all significantly reduced (p < 0.05) radiolabeling of DSPC by isolated fetal type II cells.
The in vitro activity of CTP:cholinephosphate cytidylyltransferase in the presence of supernate from S. chartarum strain 002 (Hawaiian) is shown in Figure 4. No differences were observed in the conversion of 14C-cholinephosphate to 14C-CDPcholine. In contrast, when the assay was conducted in the presence of supernate from S. chartarum strain 58–17 (Fig. 5A), a 1:10 dilution reduced the conversion of 14C-cholinephosphate to 14C-CDPcholine. Undiluted spore supernate reduced the enzyme activity to nearly undetectable levels. The rate of conversion of 14C-cholinephosphate to 14C-CDPcholine (Fig. 5B) showed clearly that the supernate from S. chartarum cultures dramatically reduced the activity of CTP:cholinephosphate cytidylyltransferase.
Since CTP:cholinephosphate cytidylyltransferase is activated by phosphatidylglycerol (PG) (Feldman and Weinhold, 1987; Sharma et al., 1993) the effects of this phospholipid on enzyme activity in the presence of extract from S. chartarum (Cleveland) or S. chartarum (Hawaiian) was measured (Fig. 6). PG increased the level of precursor conversion such that, by 40 min, more than twice as much 14C-cholinephosphate was converted to 14C-CDPcholine (Fig. 6A). Addition of extract from S. chartarum (Hawaiian) did not affect enzyme activity. In contrast, extract from S. chartarum (Cleveland) depressed enzyme activity to control levels. The rate of conversion is shown in Figure 6B. All rates declined initially and remained constant between 10 and 40 min. The rate of conversion of 14C-cholinephosphate to 14C-CDPcholine was reduced dramatically by extract from S. chartarum (Cleveland).
DISCUSSION
A wide range of toxigenic microbes are present in our environment (Johanning et al., 1998). These microbes produce a vast array of biologically active compounds, many of which are very complex (Jarvis and Mazzola, 1982). The toxic nature of these compounds has been related to a number of functional characteristics including, among other things, their ability to inhibit protein synthesis (Lee et al., 1999), induce apoptosis (Yang et al., 2000), inhibit replication (Lee et al., 1999), or induce an allergic response (Larsen et al., 1997). Specific respiratory symptoms including rhinitis, asthma, alveolitis, bronchiectasisfibrosis, and hemosiderosis have been reported in patients (Hossain et al., 2004). Given this wide range of potentially toxic effects, the presence of molds, fungi, and their spores and metabolic products within buildings may produce a variety of health problems. The respiratory system may be particularly susceptible to these effects and is therefore of particular concern (Hodgson et al., 1998). Among fungal species, S. chartarum, which produces a number of mycotoxins such as trichothecenes as well as some volatile toxic compounds (Gao and Martin, 2002), has recently been of interest due to its distribution in water-damaged buildings (Fung et al., 1998). Furthermore, some evidence suggests this mold may have been a factor in a number of recent infant deaths in Cleveland (2000), although caution must be exercised in attributing a causal relationship (Centers for Disease Control, 2000).
S. chartarum produces a range of potentially toxic compounds. These have documented effects on a number of physiological systems including the lung, since this is the primary route of exposure (Nikulin et al., 1997). In particular, extract from S. chartarum appears to alter cycling of pulmonary surfactant within the alveolar hypophase at the level of the air exchange tissues (Mason et al., 1998). Changes in morphology of surfactant-producing alveolar type II cells, including damage to lamellar bodies and nucleoli, have been documented in animals following intratracheal instillation of S. chartarum spores (Rand et al., 2002). Furthermore these spores may induce changes at the molecular level in phospholipid species within the surfactant (McCrae et al., 2001). Taken together, these results point to a direct effect on pulmonary surfactant phospholipid formation and/or secretion. Surfactant is synthesized and secreted by alveolar type II cells which, together with the type I cells, line the pulmonary side of the blood-air barrier. The surfactant is composed predominantly of phospholipids, although four surfactant proteins have been identified, and these appear to have a vital role in many phases of surfactant function (Hawgood and Poulain, 1995). However it remains that the phospholipids probably impart the surface-tension-lowering abilities to the surfactant. Within this complex mix, the disaturated fraction (DSPC), which is largely in the form of dipalmitoylphosphatidylcholine, represents a unique moiety which, due to the close packing allowed by the disaturated acyl chains, provides the ability to achieve very high surface pressure within the alveolus upon compression (Bi et al., 2001).
DSPC is formed de novo from intracellular choline, which undergoes phosphorylation to cholinephosphate (Batenburg and Haagsman, 1998). Reaction of cholinephosphate producing CDPcholine represents the rate-limiting step in phosphatidylcholine production. CDPcholine and diacylglycerol are subsequently enzymatically converted to phosphatidylcholine by choline-phosphotransferase. In the lung, the unsaturated fraction of this phospholipid destined for surfactant undergoes a deacylated-reacylation reaction to produce the disaturated species (Veldhuizen et al., 1998). We have previously shown that exposure to S. chartarum spores through intratracheal instillation in mice produces changes in lavageable surfactant subfractions and a reduction in formation of tissue DSPC in isolated fetal alveolar type II cells (Mason et al., 1998). In the present study, using a pulse-chase approach, we observed that extract from the spores reduced the level of radioactivity reaching CDPcholine. The production of this intermediate is generally considered to represent the rate-limiting step in synthesis of phosphatidylcholine (Post et al., 1984). Therefore it would seem that S. chartarum may inhibit enzyme activity, producing reduced levels of CDPcholine for subsequent synthesis of phosphatidylcholine and DSPC. Interestingly, the levels of total phosphatidylcholine that is both unsaturated and disaturated were not significantly altered after exposure to S. chartarum. Nevertheless DSPC labeling was significantly reduced. This observation suggests that the spore extract may affect CDPcholine destined for the surfactant-related subcellular pool for DSPC synthesis to a greater extent than the general pool related to constitutive functions of the cell. The significance of alterations in surfactant phospholipid composition are related to reduced pulmonary function observed in several disease states, for example, in asthma (Wright et al., 2000), and respiratory infections (Wright et al., 2000), as well as during mechanical ventilation (Veldhuizen et al., 2000).
The present study also demonstrated a major difference between spore isolates from different geographical locations. Phosphatidylglycerol, a known lipid activator of CTP:cholinephosphate cytidylyltransferase (Sharma et al., 1993) induced more than a two-fold increase in enzyme activity under normal assay conditions. The addition of extract from the Hawaiian isolate of S. chartarum did not alter the PG-induced lipid activation, which may relate to the low to undetectable levels of the satratoxin in supernate from this species. In contrast, addition of extract from S. chartarum (Cleveland) isolate, which did contain satratoxins G and H, completely reversed the PG activation of CPCT. It is not clear what other mycotoxins might be present in extract from S. chartarum (Cleveland) compared to S. chartarum (Hawaiian), and indeed the complex nature of the products from both of these spores makes this question difficult to answer. Nevertheless, lipid activation of CPCT is a well-documented phenomenon and appears to be dependent on protein intercalation into the membrane domain. In the case of the cytidylyltransferase, association between it and acyl chains of 12 or more carbon atoms appears to be particularly important (Cornell and Northwood, 2000). Furthermore changes in charge, specifically a negative membrane surface charge, are required for activation. Nevertheless, although the exact mechanisms for CPCT activation are not clear, association with membrane components may play a role. Specifically, activation may involve the removal of an inhibitory constraint in the catalytic domain by binding of the amphipathic alpha helical membrane binding domain. Given the wide range of mycotoxins produced by S. chartarum, and the complex activation mechanism(s) of CPCT, it is difficult to speculate as to which spore products might inhibit the activity of this enzyme.
In summary, the present investigation supports the supposition that mycotoxins produce toxins that may alter enzymatic activity related to de novo synthesis of pulmonary surfactant and its major phospholipids, although the mechanisms are not clear. Furthermore different strains of the same fungus are not equivalent in either their toxin profiles or ability to affect pulmonary tissues. As there remains some question as to the involvement of S. chartarum (Cleveland) in the incidence of lung hemosiderosis observed in Cleveland (Centers for Disease Control and Prevention, 2000), characterization of potential pulmonary effects of this and other fungal contaminants is important as more evidence accumulates supporting the reality of "sick-building syndromes" (Burge, 2004).
ACKNOWLEDGMENTS
Supported by the Natural Sciences and Engineering Research Council of Canada, the Biology of Breathing Group, Manitoba Institute of Child Health, and in part, by the Paul Thorlakson Foundation, University of Manitoba, Winnipeg, Canada. C. Hastings was supported by the Manitoba Medical Services Foundation through a student fellowship.
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Department of Biology, St. Mary's University, Halifax, Nova Scotia B3H3C3, Canada
National Research Council, Institute for Biodiagnostics, Winnipeg, Canada R3B1Y6
Health Canada, Health Products and Food Branch, Winnipeg, Manitoba, Canada A2J3Y1
Manitoba Institute of Child Health, Biology of Breathing Group, Children's Hospital Foundation, Winnipeg, Manitoba, Canada R3E3P4
ABSTRACT
Stachybotry chartarum, a fungal contaminant of water-damaged buildings commonly grows on damp cellulose-containing materials. It produces a complex array of mycotoxins. Their mechanisms of action on the pulmonary system are not entirely clear. Previous studies suggest spore products may depress formation of disaturated phosphatidylcholine (DSPC), the major surface-active component of pulmonary surfactant (PS). If S. chartarum can indeed affect formation of this phospholipid, then mold exposure may be a significant issue for pulmonary function in both mature lung and developing fetal lung. To address this possibility, fetal rat type II cells, the principal source of DSPC, were used to assess effects of S. chartarum extract on formation of DSPC. Isolated fetal rat lung type II cells prelabeled with 3H-choline and incubated with spore extract showed decreased incorporation of 3H-choline into DSPC. The activity of CTP:cholinephosphate cytidylyltransferase (CPCT), the rate-limiting enzyme in phosphatidylcholine synthesis was reduced by approximately 50% by a 1:10 dilution of spore extract. Two different S. chartarum extracts (isolates from S. chartarum (Cleveland) and S. chartarum (Hawaiian)) were used to compare activity of CPCT in the presence of phosphatidylglycerol (PG), a known activator. PG produced an approximate two-fold increase in CPCT activity. The spore isolate from Hawaii did not alter enzyme activity. S. chartarum (Cleveland) eliminated the PG-induced activation of CPCT. These results support previous observations that mold products alter PS metabolism and may pose a risk in developing lung, inhibiting surfactant synthesis. Different isolates of the same species of fungus are not equivalent in terms of potential exposure risks.
Key Words: lung; mold spores; cytidylyltransferase; surfactant.
INTRODUCTION
Fungal spores and their micellar components and products are common exposure factors in the everyday environment in virtually all parts of the world. Wheat dusts in Egypt (Abdel-Hafez et al., 1990), tobacco cigarettes (el Maghraby and Abdel Sater, 1993), soil and food products in France (Pieckova and Jesenska, 1999), and infant exposure in Cleveland (Etzel et al., 1998), although the latter has been questioned by a CD review (Centers for Disease Control and Prevention, 2000), all demonstrate the ubiquitous distribution and potentially hazardous effects of fungal products. These airborne contaminants represent a wide range of various fungal species (Cooley et al., 1998). Generally the bioaerosols generated from fungi are considered the main contributors to what has come to be known as sick-building syndrome (Lewis, 1994).
The materials from fungi appear to be associated with two main types of responses. The first is an allergenic response which may affect people to varying degrees, depending on a range of individual factors including such things as previous exposure levels, individual allergenic response potentials, or immunological status (Flannigan, 1994). The second type of response relates to the production of toxigenic mycotoxins, which, unlike allergenic materials, produce toxic responses in virtually all people (Jarvis, 1994). While exposure levels are dependent on many factors, and relatively low levels of these toxins and other volatile organic compounds may be released (Wilkins et al., 1998), not a great deal is known of the effects or modes of action of these materials. Some studies suggest that mycotoxin extracts of several different species of fungi may inhibit cellular replication potential or be cytotoxic as measured in an MTT-formazan assay using swine kidney cells (Gareis, 1994). However, as any given exposure, whether it induces an immunological reaction or represents a response to mycotoxin exposure, appears to originate through the respiratory system (Nikulin et al., 1997), lung cells would seem to be the model of choice. Our previous work suggests that S. chartarum reduces the synthesis of disaturated phosphatidylcholine (DSPC), the major pulmonary surfactant phospholipid. Furthermore intratracheal instillation of S. chartarum spores produces ultrastructural changes in mouse lung type II cells (Rand et al., 2002), suggesting that this cell type may be a target for S. chartarum mycotoxins. Consequently the objective of the present study was to determine the effects of S. chartarum on conversion of choline, the major biosynthetic precursor of disaturated phosphatidylcholine. As a means of establishing the effects of the toxin on surfactant synthesis, the model selected for study was the isolated fetal lung type II cell, as these late fetal lung cells closely mimic neonatal lung cells (Massaro et al., 1986).
MATERIALS AND METHODS
Animals. Pregnant Sprague-Dawley rats (virus antibody free, VAF, original breeding population, Charles River, Montreal, QC, Canada) were used for the experiments and were from the animal care facility at the University of Manitoba, in Winnipeg, MB. All procedures have been approved by the Canadian Council on Animal Care through the local protocol review committee at the University of Manitoba.
Preparation of spore extracts. Spores from Stachybotrys chartarum, strain 002 from Hawaii, (Department of Agriculture, Culture Collection, DAOM 225489) and from Cladosporium cladosporioides were isolated and cultured as described previously (Mason et al., 1998). Spores were suspended in physiological saline because this represents a more biologically relevant situation than that often used by methanol extraction (Andersson et al., 1997). Since Cladosporium cladosporioides does not produce known mycotoxins, it was used in some experiments as a negative control. The concentration of the spore samples was 2.0 x 106 spores per ml. Spores from American Type Culture Collection Stachybotrys chartarum, strain 58–17 (ATCC 201211) (Cleveland), is an isolate from a home in Cleveland (Jarvis et al., 1998).
The suspension was initially centrifuged at 2500 rpm for 15 min to collect the spores. The supernatant was decanted and discarded, and the pellet was resuspended in 10 ml of cold sterile Hank's Balanced Salt Solution (HBSS) and incubated for 3 hours. The spores were again collected by centrifugation and the supernate decanted for use in the experiments.
Fetal lung type II cell isolation and culture. Fetal lung type II cells were isolated as described previously (Batenburg et al., 1988; Scott, 1994). Pregnant rats were killed by an ip injection of Euthanyl on the 21st day of gestation (term is 23 days). The fetuses were removed, decapitated, and their bodies placed in cold sterile HBSS. Under sterile conditions, fetal lungs were removed, and mediastinal structures were dissected away. The lung tissue was washed twice with cold sterile HBSS and chopped on a Sorval tissue chopper (Sorval Instruments, Newtown, CT). Lung tissue was placed in a solution of HBSS with trypsin-EDTA (0.05%/0.01%) (Gibco, Missisauga, ON), in a water-jacketed trypsinization flask (Wheaton, Millville, NJ) and stirred on a magnetic plate for 45 min at 37°C. Trypsinization was arrested with the addition of Minimum Essential Medium, (MEM, Gibco, Missisauga, Ont.) with 10% carbon-stripped Newborn Calf Serum, (sNCS, Sigma, St. Louis, MO), and 1% each of antibiotic/antimycotic and Fungizone (Gibco, Missisauga, ON). The suspension was filtered through three layer of sterile Nitex gauze and centrifuged for 15 min at 250 x g. The supernatant was removed, and the pellet was resuspended in 50 ml of MEM with sNCS. The cells were plated in five sterile 75-cm2 culture flasks (Fisher Scientific, Mississauga, ON) and incubated for 1 h at 37°C in a humidified atmosphere of 5% CO2 and 95% air. Since fibroblasts attach more quickly than type II cells, the fibroblasts were the first few cells to adhere to the plastic flasks. After the incubation period, the contents of the flasks were poured off into a second set of flasks and incubated for a further 30 min to allow for further separation of the fibroblasts. The contents of the flasks were pooled, and the cells were counted in a Coulter cell counter (Coulter Electronics, St Hialeah, FL). Cells were plated at a density of 8 x 105 cells/25-cm2 flask in MEM with 10% sNCS (v/v) and incubated under the conditions described above. The medium was changed after the first 24 h and every 48 h thereafter. This population of cells represents an 80–85% enrichment in perinatal type II alveolar cells (Batenburg et al., 1988).
In vitro incubation of cells with spore extracts. Enriched type II cell cultures were incubated with the spore extracts to investigate the effects of the fungal toxins on the radiolabeling of disaturated phosphatidylcholine (DSPC), the major surface-active phospholipid of pulmonary surfactant (Veldhuizen et al., 1998). The following approach was used to ensure similar cell numbers in each incubation. At confluence, the cells were removed with trypsin/EDTA. Trypsin action was terminated with the addition of MEM with sNCS. Cells were collected by centrifugation at 250 x g for 10 min. The supernatant was removed, and the pellet was resuspended in MEM with sNCS. The cells were centrifuged again at the same speed and for the same amount of time. The supernatant was removed, and the cells were resuspended in a known volume of MEM/sNCS. The cells were counted in a Coulter counter, and 5.00 x 105 cells per incubation were used.
To study the effects of the spore toxins, enriched cell cultures were incubated in microcentrifuge tubes with varying concentrations of Stachybotrys chartarum supernatant, HBSS, and [methyl-3H]choline chloride (specific activity, 58 Ci/mmole, New England Nuclear, Lachine, QC). Cells were incubated with varying concentrations of toxin, MEM with sNCS, and radioactive precursor at 37°C and at 5% CO2 with constant agitation for 18 h. The incubations were terminated by the addition of methanol.
Isolation of DSPC. Following the addition of methanol, the cells were transferred to test tubes. Lipids were extracted with a solution of chloroform/methanol/1% potassium chloride (1:1:0.9). The solvent phase containing the lipids was removed and concentrated. To isolate DSPC (Gilfillan et al., 1983), 0.5 ml of carbon tetrachloride containing 3.5 mg of osmium tetroxide was added to each sample, incubated for 15 min, and removed by evaporation. Samples were spotted onto thin layer chromatography plates (Whatman, LK5D) with authentic standards and developed in chloroform/methanol/water (65:25:4). Phospholipid spots were identified using iodine vapor, and radioactivity from the appropriate spots was measured by scraping the gel into scintillation vials and counting on a LS5801 scintillation counter. Quench was compensated using the method of H# based on the spectrum of 137Cs (Beckman Instruments, Palo Alto, CA).
Pulse Chase Experiment
In vitro incubation. Fetal type II cells were isolated as described above, and experiments were performed as described previously (Cherlet and Scott, 2002). Cells were prelabeled for 1 h in HBSS with 2 μCi of 3H-choline and removed to fresh MEM with sNCS with Cladosporium cladosporioides or S. chartarum spore supernates for 15, 30, 60, or 120 min. The medium was removed, and cells were scraped into test tubes. Cells that were incubated with MEM and sNCS alone served as controls.
Isolation of choline, choline-phosphate, and CDP-choline. Samples prelabeled with 3H-choline were extracted using chloroform/methanol/1% potassium chloride (1:1:0.9), and both the upper aqueous layer and the lower organic phase were saved. The aqueous samples were dried under air and resuspended in a known volume of water/ethanol (1:1). Intermediates were isolated by thin layer chromatography. Samples were spotted on plates and developed in methanol/0.78% NaCl/ammonium hydroxide (50:50:5) (Tokmakjian et al., 1981). Standards of choline, cholinephosphate, and CDP-choline were run on separate channels on each plate. Plates were dried thoroughly after development, and standards were visualized using iodine. Appropriate spots were scrapped into scintillation vials, and radioactivity was measured.
Separation of type II cell cytosolic fraction. Fetal rat type II cells were isolated as described above, centrifuged at 250 x g for 15 min to collect the cells, the supernatant was removed, and the pellet was resuspended in HBSS. Samples were frozen at –85°C to destroy cell integrity and were kept ice-cold at all times prior to the assay to preserve enzyme activity. The samples were centrifuged at 300 x g for 10 min to precipitate the nuclei. The pellet was discarded, and the supernatant was centrifuged at 10,000 x g to precipitate the mitochondria. The pellet was discarded, and the supernatant was centrifuged at 100,000 x g for 90 min. The supernatant containing the cytosolic fraction was retained. This sample was concentrated by centrifugation at 3000 rpm for 30 min at 4°C in a Millipore centrifuge Filter Device. Protein concentration was determined by the BioRad Protein Assay (BioRad, Missassauga, ON, Canada).
Assay of CTP:cholinephosphate cytidylyltransferase (CPCT, 2.7.7.15). The effects of Stachybotrys chartarum, strains 002 (Hawaiian) and 58–17 (Cleveland), on CPCT activity were measured. The enzyme was assayed as described (Cherlet and Scott, 2002; Sharma et al., 1993; Viscardi et al., 1989) in the presence or absence of undiluted or 1-in-10 diluted spore supernatant. The procedure was as follows. Stock solutions of magnesium acetate (100 mM, final concentration 10 mM), PIPES-maleate (pH 6.5, 1 M, final concentration 100 mM) were prepared. 14C-Cholinephosphate (specific activity, 50 mCi/mmol; final concentrations, 0.3 mM at 2 Ci/mol in 0.1 mM EGTA) was used in the assay. Cytidine triphosphate, (CTP, 50 mM, final concentration 5 mM) was made fresh each time for the assay. In some assays phosphatidylglycerol, a known activator of the enzyme, was used at concentrations of 0.25 mM. Distilled water was added to this stock solution according to the amount of spore supernatant required to ensure equal volumes of solution for each incubation. The enzyme was assayed in a total volume of 100 μl. Prior to starting the incubation the components, excluding the cytosolic protein, were aliquoted into microcentrifuge tubes, and the tubes were placed in a water bath of 37°C for 5 min. To start the incubations, 120 μg of cytosolic protein was added to each tube. Tubes were removed at various times (0–40 min) and immersed in boiling water to stop the reaction. This assay was repeated with the addition of phosphatidylglycerol with or without undiluted samples of the supernate from two Stachybotrys spore variants.
Tubes were centrifuged for 2 min at 13,500 rpm in a microcentrifuge to pellet protein. Fifty microliters (50% of the total volume) of the supernatant were spotted on thin layer chromatography plates (Whatman, LK5D) with CDPcholine authentic standard. Plates were developed in ethanol/water/20% ammonium hydroxide (100:50:2) (Cherlet and Scott, 2002). Plates were thoroughly dried, and CDPcholine spots were identified using iodine. Appropriate spots were scrapped into scintillation vials and radioactivity determined as indicated above.
Analysis of S. chartarum supernatants. Samples of the supernates collected as described above from Stachybotrys chartarum, strain 58–17 (ATC 201211) (Cleveland), Stachybotrys chartarum, strain 002 from Hawaii, (Department of Agriculture, Culture Collection, DAOM 225489), and Cladosporium cladosporioides were analyzed, and 1-ml samples were lyophilized and analyzed by high performance liquid chromatography. Spectra obtained from the samples were screened for aflatoxin B1, alternariol, alternariol methyl ether, altertoxin I, brefeldin A, cyclopenin, cyclopenol, deoxynivalenol, nivalenol, ochratoxin A, patulin, penicillic acid, penitrim A, roridin A, roridin E, satratoxin G, satratoxin H, verrucarin A, verrucarin J, and viridicatin. Detection of peaks was done using photodiode array spectral matching against spectra of known standards injected under identical conditions. Quantification was done by comparing peaks obtained from the samples against calibration curves using known quantities of toxin.
Statistical analysis. Where appropriate, statistical analysis was done by post hoc application of Duncan's New Multiple Range Test (Ott, 1977) assuming a significant difference at p < 0.05. Regression lines were fit by computer to the raw data using SigmaPlot (SPSS Incorporated, Chicago, IL).
RESULTS
The HPLC chromatogram of the supernate from Stachybotrys chartarum, strain 58–17 (Cleveland) is shown in Figure 1. Two peaks corresponding to satratoxin G and satratoxin H standards were detected. Quantification indicated that approximately 1 μg of each satrotoxin existed in the sample. The other spore sample from S. chartarum-002 (Hawaiian) did not show any identifiable toxins (results not shown).
The accumulation of radioactive precursor in water-soluble intermediates in the synthetic pathway for DSPC is shown in Figure 2. Intracellular levels of 3H-choline in control or S. chartarum (Cleveland)-exposed cells displayed a similar pattern, declining from high levels after 20–40 min to low levels by 120 min. Concurrently, levels of radiolabeled precursor appearing in cholinephosphate had peaked by 20 min, indicating that 3H-choline rapidly reaches cholinephosphate. Again, very little difference was observed in the pattern of labeling of this intermediate, and fitting of regression lines indicated a similar decline in appearance of label over 120 min. In contrast to the first two intermediates, CDPcholine labeling in cells exposed to C. cladosporioides or under control conditions displayed a slow increase or no change, respectively, over time. However, in cell samples exposed to S. chartarum, a different pattern of radiolabeling was observed. In this intermediate, radioactive precursor levels declined between 20 min and 60 min and remained low; radiolabeling of CDPcholine in control and S. chartarum-exposed cells was similar by the end of 120 min.
The radiolabeling of total phosphatidylcholine and DSPC at varying exposure levels to supernate from S. chartarum samples is shown in Figure 3. The lowest level of spore exposure (0.225 μl/ml) did not affect the label incorporation into DSPC. In contrast, the three higher dose levels all significantly reduced (p < 0.05) radiolabeling of DSPC by isolated fetal type II cells.
The in vitro activity of CTP:cholinephosphate cytidylyltransferase in the presence of supernate from S. chartarum strain 002 (Hawaiian) is shown in Figure 4. No differences were observed in the conversion of 14C-cholinephosphate to 14C-CDPcholine. In contrast, when the assay was conducted in the presence of supernate from S. chartarum strain 58–17 (Fig. 5A), a 1:10 dilution reduced the conversion of 14C-cholinephosphate to 14C-CDPcholine. Undiluted spore supernate reduced the enzyme activity to nearly undetectable levels. The rate of conversion of 14C-cholinephosphate to 14C-CDPcholine (Fig. 5B) showed clearly that the supernate from S. chartarum cultures dramatically reduced the activity of CTP:cholinephosphate cytidylyltransferase.
Since CTP:cholinephosphate cytidylyltransferase is activated by phosphatidylglycerol (PG) (Feldman and Weinhold, 1987; Sharma et al., 1993) the effects of this phospholipid on enzyme activity in the presence of extract from S. chartarum (Cleveland) or S. chartarum (Hawaiian) was measured (Fig. 6). PG increased the level of precursor conversion such that, by 40 min, more than twice as much 14C-cholinephosphate was converted to 14C-CDPcholine (Fig. 6A). Addition of extract from S. chartarum (Hawaiian) did not affect enzyme activity. In contrast, extract from S. chartarum (Cleveland) depressed enzyme activity to control levels. The rate of conversion is shown in Figure 6B. All rates declined initially and remained constant between 10 and 40 min. The rate of conversion of 14C-cholinephosphate to 14C-CDPcholine was reduced dramatically by extract from S. chartarum (Cleveland).
DISCUSSION
A wide range of toxigenic microbes are present in our environment (Johanning et al., 1998). These microbes produce a vast array of biologically active compounds, many of which are very complex (Jarvis and Mazzola, 1982). The toxic nature of these compounds has been related to a number of functional characteristics including, among other things, their ability to inhibit protein synthesis (Lee et al., 1999), induce apoptosis (Yang et al., 2000), inhibit replication (Lee et al., 1999), or induce an allergic response (Larsen et al., 1997). Specific respiratory symptoms including rhinitis, asthma, alveolitis, bronchiectasisfibrosis, and hemosiderosis have been reported in patients (Hossain et al., 2004). Given this wide range of potentially toxic effects, the presence of molds, fungi, and their spores and metabolic products within buildings may produce a variety of health problems. The respiratory system may be particularly susceptible to these effects and is therefore of particular concern (Hodgson et al., 1998). Among fungal species, S. chartarum, which produces a number of mycotoxins such as trichothecenes as well as some volatile toxic compounds (Gao and Martin, 2002), has recently been of interest due to its distribution in water-damaged buildings (Fung et al., 1998). Furthermore, some evidence suggests this mold may have been a factor in a number of recent infant deaths in Cleveland (2000), although caution must be exercised in attributing a causal relationship (Centers for Disease Control, 2000).
S. chartarum produces a range of potentially toxic compounds. These have documented effects on a number of physiological systems including the lung, since this is the primary route of exposure (Nikulin et al., 1997). In particular, extract from S. chartarum appears to alter cycling of pulmonary surfactant within the alveolar hypophase at the level of the air exchange tissues (Mason et al., 1998). Changes in morphology of surfactant-producing alveolar type II cells, including damage to lamellar bodies and nucleoli, have been documented in animals following intratracheal instillation of S. chartarum spores (Rand et al., 2002). Furthermore these spores may induce changes at the molecular level in phospholipid species within the surfactant (McCrae et al., 2001). Taken together, these results point to a direct effect on pulmonary surfactant phospholipid formation and/or secretion. Surfactant is synthesized and secreted by alveolar type II cells which, together with the type I cells, line the pulmonary side of the blood-air barrier. The surfactant is composed predominantly of phospholipids, although four surfactant proteins have been identified, and these appear to have a vital role in many phases of surfactant function (Hawgood and Poulain, 1995). However it remains that the phospholipids probably impart the surface-tension-lowering abilities to the surfactant. Within this complex mix, the disaturated fraction (DSPC), which is largely in the form of dipalmitoylphosphatidylcholine, represents a unique moiety which, due to the close packing allowed by the disaturated acyl chains, provides the ability to achieve very high surface pressure within the alveolus upon compression (Bi et al., 2001).
DSPC is formed de novo from intracellular choline, which undergoes phosphorylation to cholinephosphate (Batenburg and Haagsman, 1998). Reaction of cholinephosphate producing CDPcholine represents the rate-limiting step in phosphatidylcholine production. CDPcholine and diacylglycerol are subsequently enzymatically converted to phosphatidylcholine by choline-phosphotransferase. In the lung, the unsaturated fraction of this phospholipid destined for surfactant undergoes a deacylated-reacylation reaction to produce the disaturated species (Veldhuizen et al., 1998). We have previously shown that exposure to S. chartarum spores through intratracheal instillation in mice produces changes in lavageable surfactant subfractions and a reduction in formation of tissue DSPC in isolated fetal alveolar type II cells (Mason et al., 1998). In the present study, using a pulse-chase approach, we observed that extract from the spores reduced the level of radioactivity reaching CDPcholine. The production of this intermediate is generally considered to represent the rate-limiting step in synthesis of phosphatidylcholine (Post et al., 1984). Therefore it would seem that S. chartarum may inhibit enzyme activity, producing reduced levels of CDPcholine for subsequent synthesis of phosphatidylcholine and DSPC. Interestingly, the levels of total phosphatidylcholine that is both unsaturated and disaturated were not significantly altered after exposure to S. chartarum. Nevertheless DSPC labeling was significantly reduced. This observation suggests that the spore extract may affect CDPcholine destined for the surfactant-related subcellular pool for DSPC synthesis to a greater extent than the general pool related to constitutive functions of the cell. The significance of alterations in surfactant phospholipid composition are related to reduced pulmonary function observed in several disease states, for example, in asthma (Wright et al., 2000), and respiratory infections (Wright et al., 2000), as well as during mechanical ventilation (Veldhuizen et al., 2000).
The present study also demonstrated a major difference between spore isolates from different geographical locations. Phosphatidylglycerol, a known lipid activator of CTP:cholinephosphate cytidylyltransferase (Sharma et al., 1993) induced more than a two-fold increase in enzyme activity under normal assay conditions. The addition of extract from the Hawaiian isolate of S. chartarum did not alter the PG-induced lipid activation, which may relate to the low to undetectable levels of the satratoxin in supernate from this species. In contrast, addition of extract from S. chartarum (Cleveland) isolate, which did contain satratoxins G and H, completely reversed the PG activation of CPCT. It is not clear what other mycotoxins might be present in extract from S. chartarum (Cleveland) compared to S. chartarum (Hawaiian), and indeed the complex nature of the products from both of these spores makes this question difficult to answer. Nevertheless, lipid activation of CPCT is a well-documented phenomenon and appears to be dependent on protein intercalation into the membrane domain. In the case of the cytidylyltransferase, association between it and acyl chains of 12 or more carbon atoms appears to be particularly important (Cornell and Northwood, 2000). Furthermore changes in charge, specifically a negative membrane surface charge, are required for activation. Nevertheless, although the exact mechanisms for CPCT activation are not clear, association with membrane components may play a role. Specifically, activation may involve the removal of an inhibitory constraint in the catalytic domain by binding of the amphipathic alpha helical membrane binding domain. Given the wide range of mycotoxins produced by S. chartarum, and the complex activation mechanism(s) of CPCT, it is difficult to speculate as to which spore products might inhibit the activity of this enzyme.
In summary, the present investigation supports the supposition that mycotoxins produce toxins that may alter enzymatic activity related to de novo synthesis of pulmonary surfactant and its major phospholipids, although the mechanisms are not clear. Furthermore different strains of the same fungus are not equivalent in either their toxin profiles or ability to affect pulmonary tissues. As there remains some question as to the involvement of S. chartarum (Cleveland) in the incidence of lung hemosiderosis observed in Cleveland (Centers for Disease Control and Prevention, 2000), characterization of potential pulmonary effects of this and other fungal contaminants is important as more evidence accumulates supporting the reality of "sick-building syndromes" (Burge, 2004).
ACKNOWLEDGMENTS
Supported by the Natural Sciences and Engineering Research Council of Canada, the Biology of Breathing Group, Manitoba Institute of Child Health, and in part, by the Paul Thorlakson Foundation, University of Manitoba, Winnipeg, Canada. C. Hastings was supported by the Manitoba Medical Services Foundation through a student fellowship.
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