Effects of microbial cocultivation on inflammatory and cytotoxic potential of spores

Microbial growth on moisture-damaged building materials is commonly associated with adverse health effects in the occupants. In moisture damage situations, the environmental conditions as well as the dominant microbial species will vary, leading to a diversity of microbes and continual changes in the different microbial populations. Currently, very little is known about the effects of microbial cocultures on the potential harmfulness of the microbial population. In this study we have investigated the effects of cocultivation of certain indoor air microbes on the inflammatory and cytotoxic potential of their spores. We grew various microbial combinations made from strains of Streptomyces californicus, Stachybotrys chartarum, Aspergillus versicolor, and Penicillium spinulosum on wetted plasterboard. After 5 or 10 wk of growth, the spores were collected from the plasterboards, mouse RAW264.7 macrophages were exposed to the spores, and after 24 h the induced inflammatory and cytotoxic responses were analyzed. Among all the tested microbes and their combinations, the spores of Str. californicus proved to be the most potent inducer of cytotoxicity and inflammatory responses. These results indicate also that microbial coculture may support the growth of certain microbes with high immunotoxic potency such as Str.californicus. Furthermore, coculture containing S. chartarum and A. versicolor caused a synergistic increase in cytotoxicity compared to the sum response induced by the pure cultures, but no effect on inflammatory responses was detected. Generally, spore-induced cytotoxicity and production of inflammatory markers increased during the growth period from 5 to 10 wk, suggesting that the immunotoxic potency of spores increases with time.     

Induction of Cytotoxicity and Production of Inflammatory Mediators in RAW264.7 Macrophages by Spores Grown on Six Different Plasterboards

Dampness and microbial growth in buildings are associated with respiratory symptoms in the occupants, but details of the phenomenon are not sufficiently understood. The current study examined the effects of growth conditions provided by six plasterboards on cytotoxicity and inflammatory potential of the spores of Streptomyces californicus, Penicillium spinulosum, Aspergillus versicolor, and Stachybotrys chartarum. The microbes were isolated from mold problem buildings and thereafter grown on six different plasterboards. The spores were harvested, applied to RAW264.7 macrophages (10⁴, 10⁵, 10⁶ spores/10⁶     

The Proportions of Streptomyces californicus and Stachybotrys chartarum in Simultaneous Exposure Affect Inflammatory Responses in Mouse RAW264.7 Macrophages

Abstract

Adverse health outcomes associated with moisture-damaged buildings originate from an exposure consisting of complex interactions between various microbial species and other indoor pollutants. The concentrations and proportions of microbial components in such environments can vary greatly with the growth conditions. In this study, we aimed to evaluate the effects of simultaneous exposure with modified proportions of actinobacteria Streptomyces californicus and fungi Stachybotrys chartarum on inflammatory responses (cytokines macrophage inflammatory protein 2 [MIP2], interleukin 6 [IL-6] and tumor necrosis factor α [TNFα]; nitric oxide) and cytotoxicity (MTT-test and DNA content analysis) in mouse RAW264.7 macrophage cell line. Five different proportions of microbial spores were studied (Str. californicus: S. chartarum 10:1; 5:1; 1:1; 1:5; 1:10). RAW264.7 cells were coexposed to the total dose of 3 × 10⁵ spores/ml for 24 h and also both of these microbial spores on their own at the respective doses. At least the 1.5-fold synergistic increase in cytokine production of RAW264.7 macrophages was detected when coexposure contained an equal amount or more fungal spores (S. chartarum) than bacterial spores (Str. californicus) compared to the sum response caused by these microbial spores separately. On the contrary, NO production after coexposure was nearly 40% less than the sum response induced by the microbial spores separately, when coexposure contains 5 times more bacterial than fungal spores. In addition, coexposure slightly changed the cytotoxic potency of the spores. The present results revealed that mutual proportions of fungal and bacterial spores in simultaneous exposure affect the nature of their interactions leading to increased or suppressed production of inflammatory mediators in RAW264.7 macrophages.     

Effect of liner and core materials of plasterboard on microbial growth,spore-induced inflammatory responses,and cytotoxicity in macrophages

Microorganisms, when grown on wetted plasterboards, can produce bioactive compounds capable of inducing inflammatory and toxic reactions in mammalian cells. The paper liner of plasterboard is commonly regarded as the major substrate for microbial growth. In this study, we cultured Stachybotrys chartarum, Aspergillus versicolor, Penicillium spinulosum, and Streptomyces californicus on liners and cores of plasterboards in order to examine the role of these main plasterboard components on microbial growth and the resulting bioactivity, which was assessed as the ability of microbial spores to induce inflammatory responses and to evoke cytotoxicity in mouse macrophages. The microbes, isolated from mold problem buildings, were grown under saturated humidity conditions on wetted liners and cores of six different plasterboards. The spores were collected, applied to RAW264.7 macrophages at different doses, and evaluated 24 h after exposure for their ability to evoke cytotoxicity and to stimulate production of nitric oxide (NO), tumor necrosis factor alpha (TNFalpha), and interleukin-6 (IL-6). In general, microbial growth was better on the cores than on the liners. All of the studied microbes collected from cores induced a dose-dependent production of TNFalpha in macrophages. The TNFalpha production stimulated by spores of Stachybotrys, Aspergillus, and Streptomyces paralleled their cytotoxicity. Spores of Streptomyces and Aspergillus collected from liners were among the most potent inducers of NO and IL-6. Good growth of Stachybotrys on cores was associated with high cytotoxicity. Penicillium grew only on cores, but it did not induce major inflammatory mediator productions, nor was it significantly cytotoxic. These results indicate that previously reported microbial growth on plasterboards and spore-induced production of important inflammatory mediators and cell death in macrophages is not only due to the paper liner of plasterboard, but the core material also has a crucial role.     

EFFECT OF LINER AND CORE MATERIALS OF PLASTERBOARD ON MICROBIAL GROWTH,SPORE-INDUCED INFLAMMATORY RESPONSES,AND CYTOTOXICITY IN MACROPHAGES

Microorganisms, when grown on wetted plasterboards, can produce bioactive compounds capable of inducing inflammatory and toxic reactions in mammalian cells. The paper liner of plasterboard is commonly regarded as the major substrate for microbial growth. In this study, we cultured Stachybotrys chartarum, Aspergillus versicolor, Penicillium spinulosum, and Streptomyces californicus on liners and cores of plasterboards in order to examine the role of these main plasterboard components on microbial growth and the resulting bioactivity, which was assessed as the ability of microbial spores to induce inflammatory responses and to evoke cytotoxicity in mouse macrophages. The microbes, isolated from mold problem buildings, were grown under saturated humidity conditions on wetted liners and cores of six different plasterboards. The spores were collected, applied to RAW264.7 macrophages at different doses, and evaluated 24 h after exposure for their ability to evoke cytotoxicity and to stimulate production of nitric oxide (NO), tumor necrosis factor alpha (TNFα), and interleukin-6 (IL-6). In general, microbial growth was better on the cores than on the liners. All of the studied microbes collected from cores induced a dose-dependent production of TNFα in macrophages. The TNFα production stimulated by spores of Stachybotrys, Aspergillus, and Streptomyces paralleled their cytotoxicity. Spores of Streptomyces and Aspergillus collected from liners were among the most potent inducers of NO and IL-6. Good growth of Stachybotrys on cores was associated with high cytotoxicity. Penicillium grew only on cores, but it did not induce major inflammatory mediator productions, nor was it significantly cytotoxic. These results indicate that previously reported microbial growth on plasterboards and spore-induced production of important inflammatory mediators and cell death in macrophages is not only due to the paper liner of plasterboard, but the core material also has a crucial role.     

Induction of cytotoxicity and production of inflammatory mediators in raw264.7 macrophages by spores grown on six different plasterboards

Dampness and microbial growth in buildings are associated with respiratory symptoms in the occupants, but details of the phenomenon are not sufficiently understood. The current study examined the effects of growth conditions provided by six plasterboards on cytotoxicity and inflammatory potential of the spores of Streptomyces californicus, Penicillium spinulosum, Aspergillus versicolor, and Stachybotrys chartarum. The microbes were isolated from mold problem buildings and thereafter grown on six different plasterboards. The spores were harvested, applied to RAW264.7 macrophages (10(4), 10(5), 10(6) spores/10(6) cells), and evaluated 24 h after exposure for the ability to cause cytotoxicity and to stimulate production of nitric oxide (NO), interleukin-1 beta (IL-1beta), tumor necrosis factor alpha (TNFalpha) and interleukin-6 (IL-6). The data indicate clear differences between spores of different microbes in their ability to induce the production of these inflammatory mediators and to cause cell death in macrophages. Also, for each microbe, the induction ability specifically depended on the brand of plasterboard. The spores of Streptomyces californicus collected from all plasterboards were the most potent at inducing NO and cytokine production. Cytotoxicity caused by P. spinulosum and Streptomyces californicus spores was consistent with NO, IL-1beta and IL-6 production induced by those microbes. However, the production of these inflammatory mediators by the spores of Stachybotrys chartarum was not parallel to their ability to cause cell death. The low productions of NO and cytokines were associated with high cytotoxicity caused by the spores of the A. versicolor. These data suggest that growth condition of microbes on different plasterboards affect the ability of microbial spores to induce inflammatory responses and cytotoxicity in macrophages.     

DNA damage and p53 in RAW264.7 cells induced by the spores of co-cultivated Streptomyces californicus and Stachybotrys chartarum

Our recent studies have revealed that the co-cultivation of environmental microbes, Streptomyces californicus and Stachybotrys chartarum, potentiates the immunotoxic properties of the spores. In the present study, the spore-induced genotoxic potential of these microbes was investigated. Dose related differences in genotoxic and cytotoxic effects and in p53 level in mouse RAW264.7 macrophages were studied after 24h exposure to the spores of separately cultivated Streptomyces californicus or Stachybotrys chartarum alone, a simple spore-mixture of these microbes as well as to the spores of co-cultivated microbes. The genotoxic effect of the exposures was determined by the Comet assay and p53 level was analyzed by immunoblotting. Cytotoxicity was assessed by using flow cytometric analysis and also by the MTT test. The results revealed that the spores of co-cultivated microbes evoked DNA damage, p53 accumulation and cytotoxicity at a lower dose than the other exposures, and at the highest dose there was a 2.5-fold increase in DNA damage compared to control. In addition, the spores of Streptomyces californicus alone induced a 1.5-fold increase in DNA damage compared to control, dose dependent p53 accumulation and also extensive cytotoxicity. In contrast, the mixture of separately cultivated spores or the spores of Stachybotrys chartarum alone did not induce DNA damage with any tested dose although they triggered significant cytotoxicity and a slightly increased p53 level. Our results suggest that the detected genotoxic responses are the result of DNA damage in RAW264.7 cells by some genotoxically active metabolite(s) and the production of this compound was stimulated in Streptomyces californicus when it was co-cultivated with Stachybotrys chartarum.     

Biological responses of Raw 264.7 macrophage exposed to two strains of Stachybotrys chartarum spores grown on four different wallboard types

The many benefits of building “green” have motivated the use of sustainable products in the design and execution of the built environment. However, the use of these natural or recycled materials, some of which have been treated with antimicrobials, provides a growth opportunity for microorganisms with the potential to elicit adverse health effects especially in the presence of an antimicrobial. The focus of this research was to determine the effects of Stachybotrys chartarum (strains Houston and 51–11) grown under different conditions on a macrophage cell line (Raw 264.7) using endpoints, including cytotoxicity, and those associated with immunity specifically inflammation and MHC class II expression. The fungi were grown on four different gypsum products, and macrophages were exposed to whole spores of both strains and fragmented spores of strain 51–11. Whole spores of the Houston strain elicited no cytotoxicity with some level of inflammation, while exposure to whole spores of 51–11 caused variable responses depending on the wallboard type supporting the fungal growth. High concentrations of fragmented 51–11 spores primarily resulted in the apoptosis of macrophage with no inflammation. None of the fungal strains caused elevated levels of major histocompatibility complex (MHC) class II expression on the surface of Raw cells. Mycotoxin levels of 51–11 spores from all of the wallboard types measured >250?ng/μL of T2 equivalent toxin based on activity. Collectively, the data demonstrated that all of the wallboard types supported growth of fungi with the ability to elicit harmful biological responses with the potential to negatively impact human health.     

The proportions of Streptomyces californicus and Stachybotrys chartarum in simultaneous exposure affect inflammatory responses in mouse RAW264.7 macrophages

Adverse health outcomes associated with moisture-damaged buildings originate from an exposure consisting of complex interactions between various microbial species and other indoor pollutants. The concentrations and proportions of microbial components in such environments can vary greatly with the growth conditions. In this study, we aimed to evaluate the effects of simultaneous exposure with modified proportions of actinobacteria Streptomyces californicus and fungi Stachybotrys chartarum on inflammatory responses (cytokines macrophage inflammatory protein 2 [MIP2], interleukin 6 [IL-6] and tumor necrosis factor a [TNFa]; nitric oxide) and cytotoxicity (MTT-test and DNA content analysis) in mouse RAW264.7 macrophage cell line. Five different proportions of microbial spores were studied (Str. californicus: S. chartarum 10:1; 5:1; 1:1; 1:5; 1:10). RAW264.7 cells were coexposed to the total dose of 3x10(5) spores/ml for 24 h and also both of these microbial spores on their own at the respective doses. At least the 1.5-fold synergistic increase in cytokine production of RAW264.7 macrophages was detected when coexposure contained an equal amount or more fungal spores (S. chartarum) than bacterial spores (Str. californicus) compared to the sum response caused by these microbial spores separately. On the contrary, NO production after coexposure was nearly 40% less than the sum response induced by the microbial spores separately, when coexposure contains 5 times more bacterial than fungal spores. In addition, coexposure slightly changed the cytotoxic potency of the spores. The present results revealed that mutual proportions of fungal and bacterial spores in simultaneous exposure affect the nature of their interactions leading to increased or suppressed production of inflammatory mediators in RAW264.7 macrophages.     

Seasonal variation in the toxicological properties of size-segregated indoor and outdoor air particulate matter

Ambient air particulate matter (PM) as well as microbial contaminants in the indoor air are known to cause severe adverse health effects. It has been shown that there is a clear seasonal variation in the potency of outdoor air particles to evoke inflammation and cytotoxicity. However, the role of outdoor sources in the indoor air quality, especially on its toxicological properties, remains largely unknown. In this study, we collected size segregated (PM_10–2.5, PM_2.5–0.2 and PM_0.2) particulate samples with a high volume cascade impactor (HVCI) on polyurethane foam and fluoropore membrane filters. The samples were collected during four different seasons simultaneously from indoor and outdoor air. Thereafter, the samples were weighed and extracted with methanol from the filters before undergoing toxicological analyses. Mouse macrophages (RAW264.7) were exposed to particulate sample doses of 50, 150 and 300 μg/ml for 24 h. Thereafter, the levels of the proinflammatory cytokine (TNF-α), NO-production, cytotoxicity (MTT-test) and changes in the cell cycle (SubG₁, G₁, S and G₂/M phases) were investigated. PM_10–2.5 particles evoked the highest inflammatory and cytotoxic responses. Instead, PM_2.5–0.2 samples exerted the greatest effect on apoptotic activity in the macrophages. With respect to the outdoor air samples, particles collected during warm seasons had a stronger potency to induce inflammatory and cytotoxic responses, whereas no such clear effect was seen with the corresponding indoor air samples. Outdoor air samples were associated with higher inflammatory potential, whereas indoor air samples had overall higher cytotoxic properties. This indicates that the outdoor air has a limited influence on the indoor air quality in a modern house. Thus, the indoor sources dominate the toxicological responses obtained from samples collected inside house.     

Inflammatory cytokine gene expression in THP-1 cells exposed to Stachybotrys chartarum and Aspergillus versicolor

Very little is known about the mechanisms that occur in human cells upon exposure to fungi as well as their mycotoxins. A better understanding of toxin‐regulated gene expression would be helpful to identify safe levels of exposure and could eventually be the basis for establishing guidelines for remediation scenarios following a water intrusion event. In this research, cytokine mRNA expression patterns were investigated in the human monocytic THP‐1 cell line exposed to fungal extracts of various fragment sizes obtained from Stachybotrys chartarum RTI 5802 and/or Aspergillus versicolor RTI 3843, two common and well‐studied mycotoxin producing fungi. Cytokine mRNA expression was generally upregulated 2–10 times following a 24 h exposure to fungal extracts. Expression of the proinflammatory interleukin‐1β, interleukin‐8, and tumor necrosis factor‐α genes increased while the anti‐inflammatory gene interleukin‐10 also increased albeit at very low level, suggesting that negative feedback regulation mechanism of production of proinflammatory cytokines initiated upon 24 h of incubation. In addition, submicron size extracts of A. versicolor caused significant death of THP‐1 cells, whereas extracts of S. chartarum caused no cell death while the mixture of the two fungi had an intermediate effect. There was no general correlation between gene expression and fragment sizes, which suggests that all submicron fragments may contribute to inflammatory response. © 2011 Wiley Periodicals, Inc. Environ Toxicol, 2013.     

Interactions between Streptomyces californicus and Stachybotrys chartarum can induce apoptosis and cell cycle arrest in mouse RAW264.7 macrophages

Exposure to complex mixtures of bacteria and fungi in moisture-damaged buildings is a potential cause of inflammatory related symptoms among occupants. The present study assessed interactions between two characteristic moldy house microbes Streptomyces californicus and Stachybotrys chartarum. Differences in cytotoxic and inflammatory responses in mouse (RAW264.7) macrophages were studied after exposure to the spores of co-cultivated microbes, the mixture of separately cultivated spores, and the spores of either of these microbes cultivated alone. The RAW264.7 cells were exposed to six doses (1 x 10(4) to 3 x 10(6) spores/ml) for 24 h, and the time course of the induced responses was evaluated after 4, 8, 16, and 24 h of exposure (1 x 10(6) spores/ml). The cytotoxic potential of the spores was characterized by the MTT test, DNA content analysis, and enzyme assay for caspase-3 activity. The production of cytokines (IL-1beta, IL-6, IL-10, TNFalpha, and MIP2) was measured immunochemically and nitric oxide by the Griess method. Co-cultivation increased the ability of the spores to cause apoptosis by more than 4-fold and the proportion of RAW264.7 cells at the G2/M stage increased nearly 2-fold when compared to the response induced by the mixture of spores. In contrast, co-cultivation decreased significantly the ability of the spores to trigger the production of NO and IL-6 in RAW264.7 cells. In conclusion, these data suggest that co-culture of S. californicus and S. chartarum can result in microbial interactions that significantly potentiate the ability of the spores to cause apoptosis and cell cycle arrest in mammalian cells.     

Co-cultivation of Streptomyces californicus and Stachybotrys chartarum stimulates the production of cytostatic compound(s) with immunotoxic properties

We have recently shown that the actinobacterium Streptomyces californicus and the fungus Stachybotrys chartarum originating from moisture damaged buildings possess both immunotoxic and immunostimulatory characteristics, which are synergistically potentiated by microbial interaction. In the search for the causative agent(s) behind the immunotoxicity, the cytostatic effects of the co-cultivated spores of S. californicus and S. chartarum were compared to those caused by widely used cytostatic agents produced by streptomycetes. The RAW264.7 macrophages were exposed to four doses of doxorubicin (DOX), actinomycin D (AMD), mitomycin C (MMC) or phleomycin (PHLEO) for 24 h. Kinetics of the spores of the co-cultivated and the separately cultivated microbes (1x10(6) spores/ml) was compared to DOX (0.15 muM). Apoptotic responses were analyzed by measuring DNA content and mitochondria membrane depolarization with flow cytometer, and by the fluorometric caspase-3 assay. The present data indicate that interactions during co-cultivation of S. californicus and S. chartarum stimulate the production of an unidentified cytostatic compound(s) capable of inducing mitochondria mediated apoptosis and cell cycle arrest at S-G(2)/M. The spores of co-cultivated microbes caused a 4-fold collapse of mitochondrial membrane potential and an almost 6-fold caspase-3 activation and DNA fragmentation when compared to control. Similar responses were induced by DNA cleaving compounds, especially DOX and AMD, at the relatively low concentrations, but not the spores of the same microbes when they were grown separately. These data suggest that when growing in the same habitat, interactions between S. californicus and S. chartarum stimulates the production of an unknown cytostatic compound(s) which evoke immunotoxic effects similar to those by chemotherapeutic drugs.     

Effect of growth medium on potential of Streptomyces Anulatus spores to induce inflammatory responses and cytotoxicity in RAW264.7 macrophages

Epidemiological studies have shown an association between microbial growth in buildings and increased risk of respiratory symptoms and disease related to inflammatory reactions in the inhabitants96. The current study examined the affects of growth conditions of Streptomyces anulatus, isolated from indoor air of a moldy building, on the inflammatory potential of spores of this microbe. Spores were harvested from 15 growth media formulations, applied to RAW264.7 macrophages (10(5), 10(6), or 10(7) spores/million cells), and evaluated for the ability to stimulate production of inflammatory mediators and cytotoxicity in these cells 24 h after exposure. Streptomyces anulatus spores induced dose-dependent production of nitric oxide (NO) in macrophages, reaching a level from 4.2 microM to 39.2 microM depending on the composition of the growth medium of the microbe. Expression of inducible NO synthase (iNOS) was detected in macrophages after exposure to spores collected from all growth media. Production of reactive oxygen species (ROS) was significantly increased only by the highest dose of S. anulatus spores grown on glycerol-arginine agar. Furthermore production of cytokines was affected by growth medium; the highest dose-dependent levels of interleukin 6 (IL-6) ranged from 900 to 7800 pg/ml, and the levels of tumor necrosis factor alpha (TNFalpha) varied from 490 to 3200 pg/ml. The amount of dead macrophages after the exposure varied from 11% to 96%, depending also on the growth media of the microbe. Altogether, our results suggest that the growth medium of S. anulatus has a fundamental role in the ability of the spores to induce inflammatory responses and cytotoxicity in mammalian cells.     

EFFECT OF GROWTH MEDIUM ON POTENTIAL OF Streptomyces Anulatus SPORES TO INDUCE INFLAMMATORY RESPONSES AND CYTOTOXICITY IN RAW264.7 MACROPHAGES

Epidemiological studies have shown an association between microbial growth in buildings and increased risk of respiratory symptoms and disease related to inflammatory reactions in the inhabitants. The current study examined the affects of growth conditions of Streptomyces anulatus, isolated from indoor air of a moldy building, on the inflammatory potential of spores of this microbe. Spores were harvested from 15 growth media formulations, applied to RAW264.7 macrophages (10⁵, 10⁶, or 10⁷     

Aspartate semialdehyde dehydrogenase inhibition suppresses the growth of the pathogenic fungus Candida albicans

Potent inhibitors of an essential microbial enzyme have been shown to be effective growth inhibitors of Candida albicans, a pathogenic fungus. C. albicans is the main cause of oropharyngeal candidiasis, and also causes invasive fungal infections, including systemic sepsis, leading to serious complications in immunocompromised patients. As the rates of drug-resistant fungal infections continue to rise novel antifungal treatments are desperately needed. The enzyme aspartate semialdehyde dehydrogenase (ASADH) is critical for the functioning of the aspartate biosynthetic pathway in microbes and plants. Because the aspartate pathway is absent in humans, ASADH has the potential to be a promising new target for antifungal research. Deleting the asd gene encoding for ASADH significantly decreases the survival of C. albicans, establishing this enzyme as essential for this organism. Previously developed ASADH inhibitors were tested against several strains of C. albicans to measure their possible therapeutic impact. The more potent inhibitors show a good correlation between enzyme inhibitor potency and fungal growth inhibition. Growth curves generated by incubating different C. albicans strains with varying enzyme inhibitor levels show significant slowing of fungal growth by these inhibitors against each of these strains, similar to the effect observed with a clinical antifungal drug. The most effective inhibitors also demonstrated relatively low cytotoxicity against a human epithelial cell line. Taken together, these results establish that the ASADH enzyme is a promising new target for further development as a novel antifungal treatment against C. albicans and related fungal species.     

Evaluation of Cytotoxic and Mutagenic Effects of Coriolus versicolor and Funalia trogii Extracts on Mammalian Cells

This study examined the in vitro cytotoxic activities of standardized aqueous bioactive extracts prepared from Coriolus versicolor and Funalia trogiiATCC 200800 on HeLa and fibroblast cell lines using a MTT (3-[4,5-dimetiltiazol-2-]-2–5-difeniltetrazolium bromide) cytotoxicity assay. F. trogii and C. versicolor extracts were cytotoxic to both cell lines. At 10 μL treatment level, F. trogii and C. versicolor extracts inhibited proliferation of HeLa cancer cells by 71.5% and 45%, respectively, compared with controls. Toxicity was lower toward normal fibroblasts. In the latter case, treatment at 10 μL level with F. trogii and C. versicolor extracts reduced cell proliferation by 51.3% and 38.7%, respectively. In separate experiments, the mitotic index (MI) obtained with 3 μL treatment level of unheated extracts of the two fungi was comparable to the MI value obtained by treatment with 4 μg/mL MMC (anticancer agent mitomycin-C). A significant induction of sister chromatid exchange (SCE) was observed in normal cultured lymphocytes treated with MMC (4 μg/mL). MMC treatment reduced replication index compared with treatment with unheated F. trogii extract and negative controls (p < 0.001). In contrast to MMC, F. trogii extracts did not affect the proliferation of human lymphocytes compared with controls (p > 0.05). Laccase and peroxidase enzyme activities in F. trogii extract were implicated in their inhibitory effect on cancer cells. F. trogii extract was concluded to have antitumor activity.     

Ability of Doxorubicin-Loaded Nanoparticles to Overcome Multidrug Resistance of Tumor Cells After Their Capture by Macrophages

Purpose. Investigation of the ability of doxorubicin-loaded nanoparticles (NP/Dox) to overcome multidrug resistance (MDR) when they have first been taken up by macrophages. Methods. The growth inhibition of P388 sensitive (P388) and resistant (P388/ADR) tumor cells was evaluated in a coculture system consisting of wells with two compartments. The tumor cells were seeded into the lower compartment, the macrophages were introduced into the upper part in which the drug preparations were also added. Results. Doxorubicin exerted lower cytotoxicity on tumor cells in coculture compared with direct contact. In P388/ADR, NP/Dox cytotoxicity was far higher than that of free doxorubicin (Dox). Three different formulations of cyclosporin A (either free (CyA), loaded to nanoparticles (NP/CyA) or in a combined formulation with doxorubicin (NP/Dox-CyA)), were added to modulate doxorubicin efficacy. The addition of cyclosporin A to Dox increased drug cytotoxicity. Both CyA added to NP/Dox and NP/Dox-CyA were able to bypass drug resistance. Conclusions. Despite the barrier role of macrophages, NP/Dox remained far more cytotoxic than Dox against P388/ADR. Both NP/ Dox + CyA and NP/Dox-CyA allowed to overcome MDR, but the last one should present greater advantagein vivo by confining both drugs in the same compartment, hence reducing the adverse effects.     

Inflammatory responses in mice after intratracheal instillation of spores of Streptomyces californicus isolated from indoor air of a moldy building

Microbial growth in buildings is associated with respiratory symptoms in the occupants. However, the specific effects of the microbes and the way they provoke clinical manifestations are poorly understood. In the current study, mice were exposed via intratracheal instillation to single doses of the spores of Streptomyces californicus, isolated from indoor air of a moisture-damaged building (2.2 x 10(7), 1.1 x 10(8), and 3.3 x 10(8) spores), or lipopolysaccharide (50 microg). Inflammation and toxicity in lungs were evaluated 24 h later. The time course of the effects was explored with the dose of 1.1 x 10(8) spores for up to 7 days. The microbial spores elevated proinflammatory cytokine (i.e., TNFalpha and IL-6) levels in bronchoalveolar lavage fluid (BALF) and in serum in a dose- and time-dependent manner and evoked expression of inducible nitric oxide synthase in BAL cells. Both TNFalpha and IL-6 responses peaked at 6 h after instillation, but TNFalpha leveled off more quickly than IL-6. The cytokine surge was followed by inflammatory cell recruitment into airways. Moreover, the spores increased dose- and time-dependently total protein, albumin, hemoglobin, and lactate dehydrogenase concentrations in BALF during the first 24 h. Histopathological examination of lungs confirmed the inflammatory changes. With the exception of macrophage and lymphocyte numbers, all parameters returned to control level at 7 days. In summary, these observations indicate that the spores of S. californicus are capable of provoking an acute inflammation in mouse lungs and can cause cytotoxicity. Thus, S. californicus can be considered as a species with potential to cause adverse health effects in occupants of moisture-damaged buildings.     

Efficiency of log wood combustion affects the toxicological and chemical properties of emission particles

Context: Particulate matter (PM) has been identified as a major environmental pollutant causing severe health problems. Large amounts of the harmful particulate matter (PM) are emitted from residential wood combustion, but the toxicological properties of wood combustion particles are poorly known.

Objective: To investigate chemical and consequent toxicological characteristics of PM₁ emitted from different phases of batch combustion in four heating appliances.

Materials and methods: Mouse RAW264.7 macrophages and human BEAS-2B bronchial epithelial cells were exposed for 24 h to different doses (15–300 µg/mL) of wood combustion particles. After the exposure, cytotoxicity, genotoxicity, production of the inflammatory mediators (TNF-α and MIP-2) and effects on the cell cycle were assessed. Furthermore, the detected toxicological responses were compared with the chemical composition of PM₁ samples including PAHs, metals and ions.

Results: All the wood combustion samples exerted high cytotoxicity, but only moderate inflammatory activity. The particles emitted from the inefficient phase of batch combustion in the sauna stove (SS) induced the most extensive cytotoxic and genotoxic responses in mammalian cells. Polycyclic aromatic hydrocarbons (PAHs) and other organic compounds in PM₁ samples might have contributed to these effects. Instead, water-soluble metals seemed to participate in the cytotoxic responses triggered by the particles from more efficient batch combustion in the masonry heaters. Overall, the toxicological responses were decreased when the combustion phase was more efficient.

Conclusion: Efficiency of batch combustion plays a significant role in the harmfulness of PM even under incomplete wood combustion processes.