Synergistic proinflammatory interactions of microbial toxins and structural components characteristic to moisture-damaged buildings.

Indoor exposure to microbes and their structural and metabolic compounds is notoriously complex. To study proinflammatory interactions between the multiple microbial agents, macrophages derived from human THP-1 monocytic cells were exposed to several concentrations of microbial toxins alone (emodin, enniatin B, physcion, sterigmatocystin, valinomycin) and in combination with microbial structural components (bacterial lipopolysaccharide [LPS] or fungal ß-glucan). While the expression of proinflammatory cytokines TNFα and IL-1ß to single toxins alone was modest, low-dose co-exposure with structural components increased the responses of emodin, enniatin B, and valinomycin synergistically, both at the mRNA and protein level, as measured by RT-qPCR and ELISA, respectively. Co-exposure of toxins and ß-glucan resulted in consistent synergistically increased expression of several inflammation-related genes, while some of the responses with LPS were also inhibitory. Co-exposure of toxins with either ß-glucan or LPS induced also mitochondrial damage and autophagocytosis. The results demonstrate that microbial toxins together with bacterial and fungal structural components characteristic to moisture-damaged buildings can have drastic synergistic proinflammatory interactions at low exposure levels.

Urine, hair, and nails as indicators for ingestion of uranium in drinking water.

The concentration of uranium in urine, hair, and nails due to continuous exposure through ingestion of drinking water was studied. The study population consisted of 205 individuals living in 134 different households in southern Finland where drinking water is supplied from private drilled wells. The population was selected to include a broad range of uranium daily intake from drinking water (0.03-2,775 microg d). The uranium content in drinking water, urine (overnight collection), hair and nails was determined by ICPMS. Uranium in urine was corrected for the matrix effects by use of thallium as an internal standard and adjusted by creatinine normalization. Hair and toenail samples were rinsed to remove external contamination prior to acid digestion and analysis. The uranium content in all excretion pathways was correlated with the uranium intake, particularly at elevated levels (> or =10 microg d) where drinking water was the major source of exposure to uranium. The median of the individual uranium absorption factors for urine, hair, and toenails were fu=0.003, fh=0.003, and fn=4 x 10, respectively. The association between the different bioassays was examined. The absorption factor, f1, was calculated for the population with an intake above 10 microg d and was below 0.01 for 72% of the study persons (range 0.0002 to 0.070). No statistically significant difference in f1 values was found between women and men. However, the absorption factor was higher among younger (< 60 y) than older (> or =60 y) subjects and among people with a lower exposure (below 100 microg d) than among those that ingest over 100 microg d.

Induction of oxidative stress in murine cell lines by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX).

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), the potent bacterial mutagen produced during chlorination of drinking water, was tested for the induction of oxidative stress in two murine cell lines: NIH 3T3 (fibroblasts) and L929 (fibrosarcoma cells). Following 1 h MX treatment at concentrations between 100 and 1000 microM, cellular stress conditions were monitored by measuring reactive oxygen species formation (ROS) and reduced glutathione levels (GSH). The kinetics of ROS formation and GSH depletion was investigated from 10 min to 1 h. MX caused detachment of cells at 1000 microM in L929 cells and at 300 microM in NIH 3T3 cells but the viability of the cells, measured by the trypan blue assay, decreased only by 20 and 7%, respectively, in 1h. MX increased ROS production in L929 cells in a dose-dependent manner, by 120% at 500 microM of MX in 1 h. The maximum ROS production was attained already in 10min. In NIH 3T3 cells, the ROS production was slightly, but not statistically significantly stimulated at 200 microM between 20 and 60 min. Concomitantly, MX decreased the intracellular content of GSH dose-dependently in both cell lines, by 48% in L929 cells at 500 microM of MX and 32% in NIH 3T3 cells at 200 microM of MX in one hour. The majority of this GSH depletion had occurred in 10 min. These findings indicate that MX induces oxidative stress in mammalian cells in vitro though the sensitivity of cells may differ for this effect.

Effects of mobile phone radiation on UV-induced skin tumourigenesis in ornithine decarboxylase transgenic and non-transgenic mice.

PURPOSE: The effects of low-level radiofrequency radiation (RFR) on ultraviolet (UV)-induced skin tumorigenesis were evaluated in ornithine decarboxylase (ODC) and non-transgenic mice. MATERIALS AND METHODS: Transgenic female mice over-expressing the human ODC gene and their non-transgenic littermates (20 animals in the cage control group, and 45-49 animals in the other groups) were exposed for 52 weeks to UV radiation or a combination of UV radiation and pulsed RFR. The UV dose was 240 Jm(-2) (1.2 x human minimum erythemal dose) delivered three times a week. One group of animals was exposed to Digital Advanced Mobile Phone System (DAMPS)-type RFR, the other group to Global System for Mobile (GSM)-type RFR at a nominal average specific absorption rate of 0.5 W kg(-1), 1.5 h day(-1), for 5 days a week. The skin was carefully palpated weekly for macroscopic tumours. Histopathological analyses of all skin lesions and of a specified dorsal skin area were performed on all animals. RESULTS: UV exposure resulted in development of macroscopic skin tumours in 11.5 and 36.8% of non-transgenic and transgenic animals, respectively. The RFR exposures did not give a statistically significant effect on the development of skin tumours in either transgenic or non-transgenic animals, or in combined analysis, but tumour development appeared slightly accelerated especially in non-transgenic animals. No effects of RFR exposures were found on excretion of 6-hydroxymelatonin sulphate into urine or on polyamine levels in dorsal skin. CONCLUSION: RFR exposures did not significantly enhance skin tumourigenesis. However, the slightly accelerated tumour development may warrant further evaluation.

Systemic immunoresponses in mice after repeated exposure of lungs to spores of Streptomyces californicus.

Microbial growth in moisture-damaged buildings is associated with respiratory and other symptoms in the occupants. Streptomyces spp. are frequently isolated from such buildings. In the present study, we evaluated the responses of mice after repeated exposure to spores of Streptomyces californicus. Mice were exposed via intratracheal instillation to six doses (at 7-day intervals) of the spores of S. californicus, originally isolated from the indoor air of a moisture-damaged building, at three dose levels (2 x 10(3), 2 x 10(5), and 2 x 10(7) spores). Inflammation and toxicity, including changes in cell populations in the lungs, lymph nodes, and spleen, were evaluated 24 h after the last dosage. The exposure provoked a dose-dependent inflammatory cell response, as detected by the intense recruitment of neutrophils, but the numbers of macrophages and lymphocytes in the airways also increased. The cellular responses corresponded to the dose-dependent increases in inflammation- and cytotoxicity-associated biochemical markers (i.e., levels of albumin, total protein, and lactate dehydrogenase) in bronchoalveolar lavage fluid. The spore exposure increased the number of both activated and nonactivated T lymphocytes. Also, the amounts of CD3(-) CD4(-) and unconventional CD3(-) CD4(+) lymphocytes in the lung tissue were augmented. Interestingly, the spore exposure decreased cells in the spleen. This effect was strongest at the dose of 2 x 10(5) spores. These results indicate that the spores of S. californicus are capable of provoking both immunostimulation in lungs (inflammation) and systemic immunotoxicity, especially in the spleen. The immunotoxic effect resembled that caused by chemotherapeutic agents, originally isolated from Streptomyces spp. Thus, S. californicus must be considered a microbial species with potential to cause systemic adverse health effects in occupants of moisture-damaged buildings.

Effects of mobile phone radiation on X-ray-induced tumorigenesis in mice.

The increased use of mobile phones has raised the question of possible health effects of such devices, particularly the risk of cancer. It seems unlikely that the low-level radiofrequency (RF) radiation emitted by them would damage DNA directly, but its ability to act as a tumor promoter is less well characterized. In the current study, we evaluated the effect of low-level RF radiation on the development of cancer initiated in mice by ionizing radiation. Two hundred female CBA/S mice were randomized into four equal groups at the age of 3 to 5 weeks. The mice in all groups except the cage-control group were exposed to ionizing radiation at the beginning of the study and then to RF radiation for 1.5 h per day, 5 days a week for 78 weeks. One group was exposed to continuous NMT (Nordic Mobile Telephones)-type frequency-modulated RF radiation at a frequency of 902.5 MHz and a nominal average specific absorption rate (SAR) of 1.5 W/kg. Another group was exposed to pulsed GSM (Global System for Mobile)-type RF radiation (carrier-wave frequency 902.4 MHz, pulse frequency 217 Hz) at a nominal average SAR of 0.35 W/kg. The control animals were sham-exposed. Body weight, clinical signs, and food and water consumption were recorded regularly. Hematological examinations and histopathological analyses of all lesions and major tissues were performed on all animals. The RF-radiation exposures did not increase the incidence of any neoplastic lesion significantly. We conclude that the results do not provide evidence for cancer promotion by RF radiation emitted by mobile phones.

Differences in inflammatory responses and cytotoxicity in RAW264.7 macrophages induced by Streptomyces Anulatus grown on different building materials.

Streptomyces anulatus, an indicator microbe of mold in buildings, was grown on different building materials in order to study the impact of growth conditions on the ability of the spores of this microbe to induce toxicity and inflammatory responses. The microbes were grown for 2 months on sterilized and unsterilized wood, chipboard, concrete, plaster board and mineral wool in tight glass vessels under humid conditions. The highest microbial spore concentration was detected on the sterilized mineral wool followed by the sterilized plaster board and the unsterilized mineral wool. Mouse RAW264.7 macrophages were exposed in vitro for 24 h to the spores of S. anulatus and the production of the inflammatory mediators, nitric oxide (NO), tumor necrosis factor alpha (TNF alpha), interleukin-6 (IL-6), interleukin-10 (IL-10) and cytotoxicity, were measured. The dose equivalent to 5 x 10(5) spores/ml of medium was used to compare the different materials. The most intense production of NO (11.6 microM), TNF alpha (560 pg/ml) and IL-6 (2800 pg/ml) in macrophages was induced by the spores grown on sterilized plaster board. They also caused the greatest loss of cell viability (39%). The spores grown on sterilized concrete induced significant production of NO (1.5 microM) and decreased cell viability (22%), and the spores grown on unsterilized and sterilized mineral wool increased production of NO (4.1 microM and 0.8 microM, respectively). The spores did not stimulate production of the anti-inflammatory cytokine IL-10. These results indicate that the ability of S. anulatus to induce inflammatory responses and cytotoxicity in macrophages is dependent on the growth conditions provided by different building materials.

Development of preimplantation mouse embryos after exposure to a 50 Hz magnetic field in vitro.

Effect of sinusoidal 50 Hz magnetic field (MF) on development of preimplantation CBA/S mouse embryos in vitro was studied. Superovulated and in vivo fertilized preimplantation embryos were collected at one cell stage and divided to control and MF-exposed groups. Sinusoidal 50 Hz MF with field strength of 10 A/m r.m.s., corresponding a flux density of 13 microT r.m.s., was used to expose the embryos in culture at 37 degrees C in a CO2-incubator. The developmental stage and abnormalities were recorded twice daily except once daily during weekends. The vitality and developmental stages of the embryos were similar in both groups although slightly more dead embryos were found during the 1st day in MF-exposed group (P<0.05) and the development of MF-exposed embryos was slightly impaired. In conclusion, the exposure to sinusoidal 50 Hz MF at field strength of 10 A/m did not significantly disturb the development of the mouse embryos in vitro up to the blastocyst stage.

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.

Effects of 50 Hz magnetic fields on cancer induced by ionizing radiation in mice.

PURPOSE: To evaluate the effects of 50 Hz magnetic fields (MF) on the development of cancer induced by ionizing radiation. MATERIALS AND METHODS: A total of 150 female CBA/S mice were randomized into three equal groups at the age of 3-5 weeks. One of the groups served as a 'cage-control group'. The two other groups were exposed to ionizing radiation in the beginning of the study. One of these two groups was exposed 24 h per day, for 1.5 years, to a 50Hz vertical MF, the intensity of which varied regularly between 1.3, 13 and 130 muT. The other served as a control group and was sham-exposed to MF in similar, but unenergized, exposure racks. Body weights, clinical signs, and food and water consumption were recorded regularly. Haematological examination, and the histopathological analysis of all lesions and major tissues were performed on all animals. RESULTS: MF exposure did not increase the incidence of any primary neoplasms. However, the incidence of basophilic liver foci, a probable pre-neoplastic change in liver, was increased. The incidence of hepatocellular adenomas was unchanged, whereas the incidence of hepatocellular carcinomas was slightly, but not statistically significantly, elevated. CONCLUSIONS: It is concluded that overall the results of this study do not support a role for MF as a tumour promoter.

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.

Effects of low-frequency magnetic fields on implantation in rats.

Effects of 50-Hz sinusoidal magnetic fields (MFs) on embryo implantation, serum 17beta-estradiol, progesterone, testosterone, and melatonin levels, and on estrogen receptor (ER) and progesterone receptor (PgR) densities in the uterus were studied during the preimplantation and implantation periods in rats. Pregnant Wistar rats were exposed to magnetic r.m.s. field strengths of 10 or 100 A/m (13 or 130 microT) or sham-exposed (controls) from day 0 of pregnancy for 24 h/day and killed during light and dark periods between 70 h and 176 h after ovulation. MFs did not influence the mean total number of implantations. The nocturnal mean serum melatonin concentration decreased by 34 and 38% at 10 and 100 A/m, respectively. At the same time, the first embryos, at an early developmental stage, arrived in the uterus in the MF-exposed groups. Serum estradiol and progesterone levels did not significantly change. Nuclear PgR and ER densities in the uterus decreased before implantation and there was an increased incidence of early stage embryos and fewer hatched embryos were found in the uterus at 100 A/m. During the early implantation period, the uterine cytosolic ER/PgR-ratio was increased at 100 A/m and no implants were concomitantly found in uterus. The nuclear ER/PgR-ratio decreased during implantation in both MF-groups due to decreased nuclear ER density. At the same time, 19% and 15% of the embryos (calculated from the corpora luteae) at 10 and 100 A/m, respectively, were yet morulae and not implanted. In summary, the results show that MFs do not impair implantation in rats although there may be some borderline changes in the transport and development of embryos and associated endocrinologic parameters.

Enhancement of 3-methylcholanthrene-induced neoplastic transformation by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone in the two-stage transformation assay in C3H 10T1/2 cells.

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)furanone (MX) is a mutagenic by-product found in chlorinated drinking water. It is a multi-site carcinogen in Wistar rats although the mechanisms of action of the carcinogenesis remain unresolved. We evaluated the ability of MX to promote development of transformation foci in a two-stage cell transformation assay in vitro. C3H 10T1/2 mouse embryonic fibroblasts were exposed to 3-methylcholanthrene (MC, 5 microg/ml) in the initiation phase and to MX (0.5, 1 or 2 microg/ml) or the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, the positive control, 0.3 microg/ml) during the promotion phase of the assay on dishes. In other experiments, the cells were exposed to MX (0.5, 5 or 10 microg/ml) only in the initiation phase. At the end of the assay (6 weeks from the start of the assay), the transformation foci were counted and scored after fixation and staining of the cells. MC increased the total number of transformation foci per dish and the number of malignant type III foci, and TPA further promoted this phenomenon. When MX was added during the promotion phase in the MC-initiated cells, it promoted the development of the transformation foci in a dose-dependent manner. MX alone (added as an initiator) also slightly increased the development of the foci, including the malignant forms (type II and III), but the effect was not dose-dependent. In contrast to MC-induced foci, TPA did not promote the development of MX-initiated foci, it even decreased their number. The results suggest that MX may also have potential to promote tumor development.

Comparable DNA and chromosome damage in Chinese hamster ovary cells by chlorohydroxyfuranones.

Chlorinated drinking water contains several chlorohydroxyfuranone (CHF) by-products whose contribution to cancer risk is not presently known. 3,4-Dichloro-5-hydroxy-2(5H)-furanone (MCA), 3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone (CMCF), and 3- chloro-4-methyl-5-hydroxy-2(5H)-furanone (MCF) were studied for the induction of DNA damage, using the alkaline single-cell gel (SCG)/comet assay, and for chromosome damage, using sister-chromatid exchange (SCE) and chromosome aberration (CA) tests, in Chinese hamster ovary (CHO) cells. 3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), the known genotoxic chlorination by-product and a rat carcinogen, was used as a reference chemical. The SCG analyses were done using concentrations that caused little or no cytotoxicity compared to that of the concurrent control cultures. In the cytogenetic tests, the CHFs were tested up to maximum cytotoxicity. MX and MCA were the most cytotoxic of the compounds in CHO cells followed by CMCF and MCF. All of the CHFs induced DNA damage, SCEs and CAs (mainly chromatid-type breaks and exchanges) in a concentration-related manner, with the exception that MCA was a weak inducer of SCEs. There were no significant differences between the lowest concentration of MX, MCA, and CMCF to cause DNA damage (SCG assay). Based on comparisons of the slopes of regression lines, MX was somewhat more potent than either MCA or CMCF, and MCF was clearly less potent than the other three compounds in the assay. The order of potency was MX > CMCF > MCA > MCF in inducing SCEs and MX > MCA > CMCF > MCF in inducing CAs. The data show that there are differences in the potency of genotoxicity among the CHFs tested. In many cases, however, the extent of maximum effect observed was comparable between the compounds. The results suggest that besides MX other CHFs should be considered in the evaluation of genotoxic risks associated with the consumption of chlorinated drinking water.

Effects of 2-ethylhexanoic acid on the production of reactive oxygen species in human polymorphonuclear leukocytes in vitro.

2-Ethylhexanoic acid (2-EHA), is an industrial chemical and a toxic biotransformation product of the plasticizer di(2-ethylhexyl)phthalate. Its immunological effects are unknown. 2-EHA resembles structurally C18 fatty acids, which are known activators of respiratory burst in human polymorphonuclear leukocytes (PMNL). Therefore, we exposed PMNL to 2-EHA in vitro and measured the production of reactive oxygen species (ROS) and explored the associated cellular mechanisms. 2-EHA (10-2000 microM) inhibited dose-dependently formyl-methionyl-leucyl-phenylalanine (FMLP)-induced respiratory burst in PMNL. Moreover, 2-EHA decreased oxidative burst evoked by the protein kinase C (PKC) activators, phorbol myristate acetate (PMA) and dioctanoyl-s,n-glycerol (DIC(8)). 2-EHA affected neither the levels of free intracellular calcium nor inhibited PKC. The results indicate that 2-EHA inhibits activation of PMNL to produce ROS, i.e. has an immunosuppressive effect in vitro. The site of action in the PKC is after activation of this enzyme.

3-Cloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a rat thyroid gland carcinogen, does not affect serum levels of TSH and thyroid hormones.

3-Chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a chlorine disinfection by-product in drinking water, causes follicular adenomas and carcinomas in thyroid glands of Wistar rats with an unknown mechanism. We evaluated effects of MX on blood thyroid stimulating hormone (TSH), thyroxine (T(4)), triiodothyronine (T(3)), prolactin (PRL) and growth hormone (GH) levels in male and female Wistar rats to assess their role in the tumorigenesis. The levels of TSH, PRL and GH in serum of male rats were not significantly affected by a single dose of 1, 10 or 60 mg/kg of MX administered by gavage 2 h before sampling. In repeated dose experiments MX was administered at dose levels of 1, 10 or 60 mg/kg of MX (40 mg/kg for females) in water by gavage daily for 1 or 3 weeks. Thyroid glands, adrenal glands and the liver were evaluated for morphological changes and cell proliferation activity after staining with proliferating cell nuclear antigen (PCNA). The dose of 60 mg/kg MX was toxic upon repeated administration. Nevertheless, MX did not affect blood TSH and T(4) levels at any time point in either sex. T(3) concentration increased transiently in males (by 37% after week 1) at the highest MX dose but not in females. MX did not change the weights of thyroid glands, their morphology and cell proliferation activity by the end of the week 3. MX did not affect blood PRL levels but decreased GH levels in males at all doses after the first week of MX treatment. The results indicate that MX does not alter blood TSH and thyroid hormone levels in rats, and imply that MX may not cause thyroid follicular cell tumors by TSH-mediated hormonal promotion.

No consistent pattern of mutations in p53 and ras genes in liver tumors of rat treated with the drinking water mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX).

The frequency of point mutations in p53 (exons 4-7) and in Ki-ras, Ha-ras, and N-ras (exons 1 and 2) and the expression of p53 protein were evaluated in the liver tumors of Wistar rats of a 104-week carcinogenicity study on 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), a chlorine disinfection by-product in drinking water. Mutations were analyzed in 16 hepatocellular adenomas, 7 hepatocellular carcinomas, 23 cholangiomas, and 2 cholangiocarcinomas of the MX-treated animals and one hepatocellular carcinoma and cholangiocarcinoma in control animals using PCR-SSCP (polymerase chain reaction-single-strand conformation polymorphism) or PCR-TGGE (temperature gradient gel electrophoresis) and direct sequencing. The expression of the p53 protein (wild-type and mutated protein) was detected by immunohistochemistry (CM5 antibody). The expression of p53 and that of the proliferating cell nuclear antigen (PCNA, 19 A2) were also evaluated in livers of female animals exposed to MX for 1 week, 3 weeks, or 18 weeks. Altogether, four mutations were found in p53 in three tumors, in two hepatocellular adenomas, and one cholangiocarcinoma, all in females receiving the highest MX dose (6. 6 mg/kg/day) of the study. Three of the mutations were G:C --> A:T transitions and one was an A:T --> T:A transversion. The mutations were scattered at different codons and positions of the codon. One hepatocellular adenoma contained two p53 mutations. All cholangiomas and cholangiocarcinomas, but no hepatocellular adenomas and carcinomas, overexpressed the p53 protein. MX treatment did not induce p53 expression at any age in the liver or alter the expression of the PCNA in the liver of younger animals. The p53 protein was overexpressed in hyperplastic bile ducts in aged rats but not in bile ducts of younger rats (up to 24 weeks). No mutations were observed in either Ki-ras, Ha-ras, or N-ras of the liver tumors. These data suggest that point mutations in p53, Ki-ras, Ha-ras, and N-ras are not involved in the MX-induced liver carcinogenesis in rats.

Nitric oxide and proinflammatory cytokines in nasal lavage fluid associated with symptoms and exposure to moldy building microbes.

Epidemiological data indicate that living or working in a moldy building is associated with increased risk of respiratory symptoms and disease related to inflammatory reactions, but biochemical evidence linking cause and effect is still scarce. The staff working in a mold-contaminated school, and a reference group without such exposure, were studied. Nasal lavage was performed and health data were collected with a questionnaire at the end of the spring term, after a 2.5-mo summer vacation, and at the end of the fall term. Here we show that concentrations of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and nitric oxide (NO) in nasal lavage fluid were significantly higher in the exposed than in the control subjects at the end of the first exposure period. These inflammatory mediators decreased to reference group concentrations during the period when there was no exposure and the production of NO and IL-6 increased again during the reexposure in the fall term. Reports of cough, phlegm, rhinitis, eye irritation, and fatigue paralleled the changes in the measured inflammatory markers. These results point to an association between inflammatory markers in the nasal lavage fluid, the high prevalence of respiratory symptoms among the occupants, and chronic exposure to molds in the indoor environment.

Diesel particles induce nitric oxide production in murine alveolar macrophages and rat airways.

The acute adverse health effects among respiratory and cardiovascular patients have been associated with particulate air pollution, containing diesel particles (DP). The mechanisms of these effects are unknown, but they may involve inflammation. We investigated the effects of DP (30-3000 µg/10(6) cells) on cell viability and production of nitric oxide (NO), interleukin-1 (IL-1), interleukin-6 (IL-6), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in murine RAW 264.7 macrophage cultures in vitro. DP caused a dose- and time-dependent NO-production and was cytotoxic in murine RAW 264.7 macrophages. Cytotoxicity preceded the increases in NO production. DP had minimal effects on cytokine production. A single intratracheal instillation of DP 1 and 5 mg/rat increased NO production and protein concentration in bronchoalveolar lavage fluid, and caused pulmonary edema and hemorrhage. The present results indicate that DP can induce both NO production and cytotoxicity in the lower respiratory tract, which may contribute to the short-term adverse respiratory effects of these particles.

Streptomyces anulatus from indoor air of moldy houses induce NO and IL-6 production in a human alveolar epithelial cell-line.

Moisture associated microbial growth in buildings may cause respiratory symptoms such as pulmonary inflammation. We studied the effects of spores of Streptomyces anulatus, commonly found in moldy buildings, on the production of nitric oxide (NO), interleukin-6 (IL-6), interleukin-5 (IL-5), interleukin-4 (IL-4), and tumour necrosis factor alpha (TNFα), as well as cell viability in human alveolar II type epithelial cell line (A549). Cells were exposed in vitro to S. anulatus spores with and without interferon-γ (IFNγ) in vitro. Lipopolysaccharide (LPS) was used as a reference substance. S. anulatus alone, and in combination with IFNγ induced NO and IL-6 production and decreased cell viability whereas IL-4, IL-5 or TNFα production were not affected. IFNγ alone had a weaker but otherwise similar effect as S. anulatus on NO and IL-6 production and it potentiated the effects of S. anulatus. LPS did not induce NO or cytokine production, or affect cell viability in A549 cells. These data indicate that spores of S. anulatus induce the excretion of inflammatory mediators in respiratory epithelial cells, which may partly explain the adverse respiratory health effects experienced by individuals exposed to the indoor air of moldy houses.