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Spores of Aspergillus versicolor Isolated from Indoor Air of a Moisture-damaged Building Provoke Acute Inflammation in Mouse Lungs

Spores of Aspergillus versicolor Isolated from Indoor Air of a Moisture-damaged Building Provoke Acute Inflammation in Mouse Lungs. Juha Jussila, H. Komulainen, V. Kosma, A. Nevalainen, J. Pelkonen, M. Hirvonen. Inhalation Toxicology, 14:1261-1277, 2002.

Aspergillis versicolor and increased concentrations of its spores are frequently found in buildings damaged by water. One of the mycotoxins produced is a carcinogen, sterigmatocytin.  Penetration of spores to the deep lung is allowed by small aerodynamic diameters of 2.0 to 3.6 um. These spores may carry various mycotoxins into the deep lung. Although asthma, chronic bronchitis, cough, chronic respiratory tract infections and rhinitis in humans are associated with moisture-damaged buildings, cause and effect has not yet been shown. The purpose of this study was to describe cytotoxicity and inflammation induced by Aspergillis versicolor in mouse lungs. A single intratracheal (i.t.) exposure to mold spores followed by serial sacrifices up to 28 d. as well as exposure to 4 different doses of spores followed by a single sacrifice time were performed.   A dose-response relationship between number of spores and changes indicating inflammatory damage in mouse lungs was demonstrated.

An earlier study from this laboratory showed increased production of such proinflammatory mediators as TNF-alpha, IL-6 and NO when Aspergillis versicolor spores were exposed to mouse macrophages in vitro. These spores, isolated from a moisture-damaged building, were grown on plasterboard. The current study analyzes the same mediators in addition to changes in mouse lung histopathology.

Study Design:

The Aspergillis versicolor spores used for i.t. instillation were in a prepared homogenous suspension.  This suspension was originally isolated from mold within a moisture-damaged building.

Dose-response was studied with 4 doses of spores (1 x 105, 1 x 106, 1 x 107, 1 x 108) or HBSS control. Mice were killed 24 hr after the instillation for analysis. The time courses of the responses were studied with a single dose (5 x 106 spores per animal) of spores or HBSS control. Animals were killed at 6 h, 24 h, 3 d, 7 d, 14 d, 21 d, and 28 d after instillation.

The mice at each sacrifice time were killed, blood collected and serum separated for cytokine anaylsis. The lungs, liver, spleen, and lymph nodes of 3 to 4 mice per group were prepared for standard H and E 5 um paraffin sections. The lungs of 6 to 7 mice per group were lavaged with HBSS. BALF was collected and cell differentials performed. TNF-alpha, IL-6, albumin, total protein and LDH concentrations were measured in the lavage supernatant. RBC’s were hemolyzed and hemoglobin measured. Lavaged cells were also used to measure iNO synthase by Western blot.

Results:

Cytokine levels were both spore dose magnitude and time post-dose dependent.  Both TNF alpha (TNF) and IL-6 in the BALF indicate proinflammatory cytokine production. They were maximal 6 hrs after instillation of the spores. The magnitude of IL-6 was twice that of TNF although the TNF increase lasted longer. When the systemic cytokine levels were studied in the blood serum, only the highest dose of spores (1 x 108) caused an increase in IL-6. There was no increase in IL-6 or TNF at lower doses of spores.  BALF cells did not exhibit an increase in iNOS synthase.

The total cell number increased in a dose dependent fashion when inflammatory cells differentials were performed on BALF,. The first increase was in neutrophils, then macrophages followed by a small increase in lymphocytes between 3 to 14 days. No changes in eosinophil numbers were observed. Hemoglobin was elevated only at the highest spore density at 24 hr.

LDH, albumin and protein all increased in the BALF in a dose dependent fashion.

Increased neutrophil numbers in the alveoli and bronchiolar lumens indicated inflammation with light microscope microscopy at 24 hr after the spore dose. The quantity of inflammation was dose dependent. Macrophages and lymphocytes were most common 3 days after exposure. These changes were graded as minimal, mild or severe.  No histological changes were observed in the spleen, liver, or lymph nodes.

Discussion:

The investigators have shown that Aspergillis versicolor spores originally isolated from a moisture-damaged building cause acute inflammation in the lungs of mice after a single intratracheal dose. Both biochemical markers in BALF and pathological changes in the lungs documented the inflammatory changes after a single dose of spore suspension. TNF and IL6 proinflammatory cytokines increased dose and time dependently after exposure. The influx of inflammatory cells was typical of a standard inflammatory response. Changes in the lung histology observed in 5 µm sections were typical of inflammation as well.

The authors conclude that only the highest spore dose (1 x 108) was cytotoxic because this was the only dose that caused increased serum IL-6, vascular leakage, and increased levels of albumin, protein, LDH, and hemoglobin in the airways. Longer exposures to Aspergillis versicolor will be necessary before more complete information on adverse effects of these spores can be obtained.

The only unexpected finding was the continued presence of macrophages present in the BALF at the end of the 28 d observation period. The authors speculate that this may be due to the presence of uncleared spores or pieces of spores inhibiting phagocytosis. They also introduce the possibility of the macrophage overloading phenomenon described by Morrow (1992) as being responsible for the high inflammation at the highest spore dose.  This is considered  a non-specific effect occurring only with high particle concentrations.  Macrophages are overloaded as a result.  However this phenomenon would not be relevant to realistic indoor concentrations of particles.

In addition the Aspergillis versicolor spores did not induce expression of NO producing iNOS synthase protein  this experiment. Other studies using the same mold do increase iNOS synthase. This is a seeming contradiction requiring more study. However the authors do not recommend using NO measurements to measure airway inflammation in humans exposed to moisture-damaged buildings with microbe contamination.  The companion paper to this article describes the reasoning for this last conclusion more completely (Purokivi et al, 2002).

The authors conclude that Aspergillis versicolor spores might cause adverse health effects in humans who spend time in moisture-damaged buildings.

References:

Morrow, P. E. 1992.  Dust overloading of the lungs:  Update and appraisal.  Toxicol Appl. Pharmacol.  113: 1 - 12.

Purokivi, M., M.-R. Hirvonen, J. Randell, M. Roponen, H. Tukiainen.  2002.  Nitric oxide alone is an insufficient biomarker of exposure to microbes in a moisture-damaged building.  Inhalation Toxicology, 14: 1279 - 1290, 2002.

Editor’s comment:

Toxicologists have recently begun to study the potential toxicity of mold to humans. This is one of the first studies to define some of the mechanisms by which mold spore toxicity in the respiratory tract may occur. Early studies of mold involved measuring concentrations of mold in various areas of water-exposed buildings. The most toxic of the spore effects come from microbes isolated from moisture-damaged buildings. This is the third study involving Aspergilla in this newsletter series of reviews.  See: Aspergillis in the Environment and Aerosolized Antibiotic Therapy of Aspergillosis.  

A definition of "moisture-damaged" in reference to buildings from which mold spores were collected would have been helpful in this manuscript because the conclusion depends upon exposure to spores from mold in moisture-damaged buildings.  Apparently mold in buildings that are not damaged by moisture does not cause these toxic effects.

The emerging study of mold spores within the field of toxicology is similar to that of the study of ultrafine particles because both areas are of recent interest. Similarly both areas of research show promise in explaining mechanisms of respiratory disease development and, possibly, how interactions between different inhaled agents may act in causing or altering respiratory tract illnesses.

 

By: Susan G. Shami, ScD, Editor