BIOLOGICAL ACTIVITIES OF THE METABOLITES OF MICROBES PRESENT IN THE INDOOR AIR

Project leader: Atte von Wright, University of Kuopio, Department of Pharmaceutical Chemistry P.O.Box 1627, FIN-70211 Kuopio, Finland, tel. +358-17-162 087, e-mail: Atte.vonWright@uku.fi
 
 

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TIIVISTELMÄ SUOMEKSI

Researchers:
Susanna Nieminen, Post-graduate student, University of Kuopio, Department of Pharmaceutical Chemistry, P.O. Box 1627, 70211 Kuopio, Finland, tel. +358-17-163573),
Riikka Kärki (former Saarelainen), University of Kuopio, Department of Pharmaceutical Chemistry, P.O. Box 1627, 70211 Kuopio, Finland

Consortium: Moisture, mould and health
Financing SYTTY organization: The Academy of Finland
Funding from SYTTY / Total funding of project (€): 127250 / 149586
Person-months of work funded by SYTTY / Total person-months of work: 40 / 50

KEY WORDS: Actinomycetes, fungi, DNA-repair,  genotoxins, indoor air
 

EXTENDED ABSTRACT

1 Introduction

Using a screening method based on the sensitivity of a DNA-repair deficient Escherichia coli strain to DNA-damaging agents, it was found that several actinomycetes and fungi isolated from the indoor air of houses with a moisture problem produce unidentified DNA-reacting compounds. As DNA-reactivity or genotoxicity has been considered an indicator of carcinogenicity, this finding is of potential concern. Consequently, the aim of this project is to characterise both chemically and toxicologically the most potent genotoxins produced by the indoor air microbes. The strains studied were selected on the basis of their apparent toxicity to the DNA-repair-deficient E. coli, the genotoxic compounds were extracted using organic solvents, and purified with different chromatographic methods. The chemical characterisation is based on the NMR-data. The range of biological activities of the purified compounds is further studied  using different methods briefly described below. Further, the production of genotoxins in different mbuilding mnaterials intentionally contaminated with a genotoxic mold, has been monitored.

2 Methods

2.1. The tested indoor air microbial isolates
Altogether 26 actinomycete isolates and 16 fungal strains, originally isolated from the indoor air of problem houses and stored in the culture collection of The National Public Health Institute were used for secreening tests. The strains had been taxonomically characterised to species or at least to genus level.

2.2. Detection of genotoxic activity
The screening of the genotoxic metabolites is based on the differential killing of Escherichia coli strains having different DNA-repair capacities. If a DNA-repair-deficient strain is consistently more sensitive than a repair-proficient isogenic strain to the culture medium or extracts of a certain Actinomycete or fungal isolate, the production of some genotoxic compound(s) can be assumed. The repair-deficient strain used was CM871, defective both in the excision and recombination repair, and the repair proficient isogenic control was WP2. In practice the screening was done by placing agar discs from confluent actinomycete or fungal plates on the lawn of test strains. A zone of inhibition around the agar plug on the CM871 plate together with no or little growth inhibition of WP2 was interpreted as an indication of DNA-damaging metabolites.

2.3. Extraction of genotoxic metabolites
Active organic extraction from both acidified and alkalised growth medium and chloroform was used to extract the active compounds from liquid actinomycete or fungal growth medium. The extraction was performed when the growth medium started to show activity in the DNA-repair assay (performed on CM871 and WP2 plates with 100 µl aliquotes of growth medium pipetted into wells made in the agar)

2.4. Purification procedure
A combination of chromatographic methods (TLC, silica gel column chromatography, HPLC) has been applied to the purification of the active compounds, the eluents, chromatography conditions and other parameters adjusted individually according to the compound(s) in question. The purification process was monitored by testing the different fractions obtained from each chromatographic step for genotoxic activity.

2.4. Identification of the compounds
Purified compounds obtained from HPLC were subjected to NMR spectrometry (both proton and 13C spectra) and mass spectrometry (University of Kuopio, Department of Chemistry). The spectra were analysed by Professor Hartmut Laatsch (Institute of Organic Chemistry,University of Gölttingen, Germany).

2.5. Tests with contaminated building materials
The production of genotoxins in different building materials was studied by artificially contaminating gypsum board, chipboard, mineral wool and spruce wood with a spore suspension of a genotoxic mold and incubating the materials in humid atmosphere untill a visible growth was observed. The genotoxic metabolites were analysed both from the fungal mycelium and from the infested building material.

2.5. Genotoxicity testing
The potential mutagenicities of the purified fractions were tested using the Ames test, and SOS-chromotest (bacterial tests). The cytogenetic effects are studied in different mammalian cell systems [Sister chromatid exchange (SCE) and Comet assay] by the National Public Health Institute.

2.6. Cytotoxicity testing
The cytotoxicities of the purified fractions were tested using a mouse hepatoma cell line cpapble for xenobiotic metabolism. The effects of the purified toxins on the cytokine induction will be studied by the National Public Health Institute.

3 Results and discussion

So far genotoxins produced by three indoor air molds and actinomycetes have been extracted for the purification process described above. The genotoxins produced by one particular Aspergillus fumigatus isolate HT 30 have been purified and chemically characterised. The compound, strongly cytotoxic  gliotoxin, although known before, has not been extensively screend for genotoxic activity.

In different genotoxicity tests (Ames test, SOS-chromotest, SCE-test and Comet assay) gliotoxin has been shown to fdamage both bacterial and mammalian DNA. However, the damage does not seem to induce point mutations.

In tests with infested building materials detectable amounts (1 - 20 µg per sample) of giliotoxins were detected in gypsum board, chipboard and spruce wood. No gliotoxin was associated with tha ctual fungal  mycelium.

4 Conclusions and future plans

The isolation and purification process suggested in the original research plan has proved sound. The characterisation of the genotoxins produced by the indoor air microbes is possible, at least with the most potent ones. When the chemical nature of the toxins is known, their direct analysis from environmental samples will also become possible.

The purification of other genotoxins from the alrready obtained crude extracts will continue. As soon as the chemical identities are known, growth and production tests on building materials in simulated conditions will be performed. It should be noted that the project has been granted an extension untill the end of 2002.

5 Co-operation

In Finland the main collaborators are the research group of Doc. Maija-Riitta Hirvonen and Dr.Jorma Mäki-Paakkanen  from the national Public Health Institute. Dr. Olavi Raatikainen (University of Kuopio, Centre for Training and Development) has acted as an advisor in the project. Professor Reino Laatikainen  (department of chemistry in University of Kuopio) has been very helpful with the NMR-analysis. The main foreign collaborator is professor Hartmut Laatsch, (Institute of Organic Chemistry, University of Göttingen, Germany), who has kindly put his expertise on the chemistry of natural compounds to the disposal of the project.
 

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