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Recent epidemiological studies have reported significantly increasing hospital admission rates for mental disorders such as anxiety and depression, not only in adults but also in children and adolescents, indicating more research is needed for evaluation of the etiology and possible reduction and prevention of these disorders. The aim of the present study was to examine the associations between perinatal exposure to traffic-related air pollutants and anxiety-like behaviors and alterations in neurological and immunological markers in adulthood using a rat model. Sprague Dawley pregnant rats were exposed to clean air (control), diesel exhaust (DE) 101 ± 9 μg/m3 or diesel exhaust origin secondary organic aerosol (DE-SOA) 118 ± 23 μg/m3 from gestational day 14 to postnatal day 21. Anxiety-related behavioral tests including open field tests, elevated plus maze, light/dark transition tests and novelty-induced hypophagia were performed on 10-week-old rats. The hippocampal expression of neurotransmitters, neurotrophic factors, and inflammatory molecular markers was examined by real-time RT-PCR. Anxiety-like behaviors were observed in both male and female rat offspring exposed to DE or DE-SOA. Moreover, serotonin receptor (5HT1A), dopamine receptor (Drd2), brain-derived neurotrophic factor and vascular endothelial growth factor A mRNAs were significantly decreased, whereas interleukin-1β, cyclooxygenase-2, heme oxygenase-1 mRNAs and microglial activation were significantly increased in both male and female rats. These findings indicate that brain developmental period exposure to traffic-related air pollutants may induce anxiety-like behaviors via modulation of neurotransmitters, neurotrophic factors, and immunological molecular markers, triggering neuroinflammation and microglia activation in rats.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impaired social communication, poor social interactions, and repetitive behaviors. We aimed to examine autism-like behaviors and related gene expressions in rats exposed to diesel exhaust (DE)-origin secondary organic aerosol (DE-SOA) perinatally. Sprague&ndash, Dawley pregnant rats were exposed to clean air (control), DE, and DE-SOA in the exposure chamber from gestational day 14 to postnatal day 21. Behavioral phenotypes of ASD were investigated in 10~13-week-old offspring using a three-chambered social behavior test, social dominance tube test, and marble burying test. Prefrontal cortex was collected to examine molecular analyses including neurological and immunological markers and glutamate concentration, using RT-PCR and ELISA methods. DE-SOA-exposed male and female rats showed poor sociability and social novelty preference, socially dominant behavior, and increased repetitive behavior. Serotonin receptor (5-HT(5B)) and brain-derived neurotrophic factor (BDNF) mRNAs were downregulated whereas interleukin 1 &beta, (IL-&beta, ) and heme oxygenase 1 (HO-1) mRNAs were upregulated in the prefrontal cortex of male and female rats exposed to DE-SOA. Glutamate concentration was also increased significantly in DE-SOA-exposed male and female rats. Our results indicate that perinatal exposure to DE-SOA may induce autism-like behavior by modulating molecules such as neurological and immunological markers in rats.

In Japan, people using water from the well contaminated with high-level arsenic developed neurological, mostly cerebellar, symptoms, where diphenylarsinic acid (DPAA) was a major compound. Here, we investigated the adverse effects of developmental exposure to 20mg/l DPAA in drinking water (early period [0-6 weeks of age] and/or late period [7-12]) on behavior and cerebellar development in male rats. In the open field test at 6 weeks of age, early exposure to DPAA significantly increased exploratory behaviors. At 12 weeks of age, late exposure to DPAA similarly increased exploratory behavior independent of the early exposure although a 6-week recovery from DPAA could reverse that change. In the passive avoidance test at 6 weeks of age, early exposure to DPAA significantly decreased the avoidance performance. Even at 12 weeks of age, early exposure to DPAA significantly decreased the test performance, which was independent of the late exposure to DPAA. These results suggest that the DPAA-induced increase in exploratory behavior is transient, whereas the DPAA-induced impairment of passive avoidance is long lasting. At 6 weeks of age, early exposure to DPAA significantly reduced the concentration of cerebellar total glutathione. At 12 weeks of age, late, but not early, exposure to DPAA also significantly reduced the concentration of cerebellar glutathione, which might be a primary cause of oxidative stress. Early exposure to DPAA induced late-onset suppressed expression of NMDAR1 and PSD95 protein at 12 weeks of age, indicating impaired glutamatergic system in the cerebellum of rats developmentally exposed to DPAA.

An incident of poisoning occurred in Japan in 2003 when high-level contamination with arsenic, mainly diphenylarsinic acid (DPAA), was found in well water. People using this water particularly experienced cerebellar symptoms. In the present study, we investigated the adverse effects of DPAA on the cerebellum in vitro and in vivo to understand the biological mechanisms that cause cerebellar symptoms. Comprehensive gene expression analyses in primary cultured ratcerebellar cells exposed to 10 μM DPAA for 24 hours indicated significant alterations in the mRNA expression of genes encoding antioxidative stress proteins (heme oxigenase 1 and heat shock protein72) and neuroactive and vasoactive peptides (neuropeptide Y, adrenomedullin, monocyte chemoattractant protein 1, and fibroblast growth factor 2). Further analyses of proteins revealed that cultured cerebellar astrocytes expressed these antioxidative stress proteins and peptides in response to exposure to DPAA. In addition, these adverseeffects were also observed in the cerebellum exposed in vivo to DPAA (100 mg/L) for 21 days. These results suggested that cerebellarastrocytes irregularly secrete neuroactive and vasoactive peptidesagainst DPAA-induced oxidative stress, which leads to abnormal neural functions and disrupted cerebellar autoregulation dynamics and results in the onset of cerebellar symptoms.

In the present study, in order to reveal novel adverse effects of ultrafine particles (UFP) on the central nervous system, the effects of nanoparticle-rich diesel exhaust particles (NRDEP; count mode diameter, 21.45 nm) on emotional behavior, learning capability and brain neurotransmitter levels were studied in rats by intranasal instillation (iNI). NRDEP (10 and 50 µg/rat) was instilled into 2-week old infant, male rats once a week for 4 weeks. Spontaneous motor activity measured was observed to be inverse to the dose level. In active avoidance tests using a shuttle box, NRDEP-treated animals showed a lower avoidance performance than control animals given air-instillation. The levels of dopamine and its metabolite (DOPAC) in the medial mammillary nucleus of the brain tended to be lower in the NRDEP-treated animals. From these results, although the effects of NRDEP by iNI on the emotionality and the brain neurotransmitter levels were not fully clear, the results obtained by avoidance testing suggested involvement of UFP in learning capability.

The tissue distribution and chemical forms of arsenic were compared in two animal species with different metabolic capacity and toxicity to arsenic. Hamsters and rats were given a single oral dose of arsenite (iAsIII) at 5.0 mg As/kg body weight, and then the concentrations of arsenic were determined; more than 75% of the dose accumulated in rat red blood cells (RBCs) in the form of dimethylarsinous acid (DMAIII), whereas less than 0.8% of the dose accumulated in hamster RBCs, mostly in the form of monomethylarsonous acid (MMAIII). Reflecting the low accumulation in RBCs, more than 63% of the dose was recovered in hamster urine within one week (7.8-fold higher than that in rat urine). The quantity of arsenic distributed in the liver and kidneys was significantly higher in hamsters than in rats, and arsenic in livers stayed much longer in hamsters than in rats. Arsenic accumulated more and was retained longer in the kidneys than in the livers in both animals, and in hamster kidneys, it accumulated at levels higher than those in rat kidneys in the form of MMAIII bound to proteins. In the first 24 h urine, dimethylmonothioarsinic (DMMTAV) and dimethyldithioarsinic (DMDTAV) acids were detected in hamsters, but only DMMTAV was found in rats, together with an unknown arsenic metabolite in both animals. The unknown urinary arsenic metabolite was identified as monomethylmonothioarsonic acid (MMMTAV; CH3As(=S)(OH)2). The present results indicate that in hamsters, arsenic does not accumulate in RBCs, and therefore, hamsters exhibit a more uniform tissue distribution and faster urinary excretion of arsenic than rats. In addition, arsenic was thiolated more in hamsters than in rats excreting mono and dimethylated thioarsenicals in urine.

Glutathione (GSH) plays an important role in the metabolism of arsenite and arsenate by generating arsenic-glutathione complexes. Although dimethylarsinic acid (DMA(V)) is the major metabolite of inorganic arsenicals (iAs) in urine, it is not clear how DMA(V) is produced from iAs. In the present study we report that S-(dimethylarsino)-glutathione (DMA(III)(SG)), a putative precursor of dimethylarsinic acid DMA(V), was unstable in the culture medium without excess GSH and generated volatile substances which were highly cytotoxic for both rat heart microvascular endothelial cells and HL60, a human leukemia cell line. Cytotoxicity of DMA(III)(SG) was higher than that of iAs and its LC(50) value was calculated to be 7.8 microM in the endothelial cells. To our surprise DMA(III)(SG) effectively killed cells in the neighbor wells of the same multi-well dish, indicating that volatile toxic compounds generated from DMA(III)(SG) in the culture medium. High performance lipid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICPMS) analyses suggested that the freshly generated volatile compounds dissolved into aqueous solution and formed an unstable arsenic compound and the unstable compound was further converted to DMA(V). These results suggested that DMA(III)(SG) exerts its cytotoxicity by generating volatile arsenicals and is implicated in the metabolic conversion of inorganic arsenicals into DMA(V), a major final metabolite of inorganic arsenicals in most mammals.

We analyzed the health risk of particulate matters in the air to humans using bioassay data and a mathematical model. We designed an original dosimetry model to estimate the particle concentration in human respiratory organs, and the concentration of the inhaled particles at the target organ was used for interspecies extrapolation from rat to human. Our model is based on the conventional dosimetry model and deposition model in the previous literature, but clearance parameters have been newly introduced for the simulation of long-term exposure. Lung cancer was set as the risk endpoint in our risk study, and the dose-response relationship at the target organ (lung) was quantitatively analyzed by the benchmark dose (BMD) method. For interspecies extrapolation based on target organ concentration, we assumed benchmark concentration (BMC) related to 1% excess cancer in rats and humans, and the human equivalent concentration (HEC) was searched by back-estimation using our model. The obtained HEC was 948 to 1098 mg/m3, and the unit risk to humans was 9.11 to 10.5 x 10(-9) per 1 microg/m3 of particulate matter. The estimated cancer risk for Japanese people in general was estimated as approximately 9-10 persons per 100,000,000 when the particle concentration in the air is 10 microg/m3.

Diesel exhaust particles (DEP) induce pulmonary diseases including asthma and chronic bronchitis. Comprehensive evaluation is required to know the mechanisms underlying the effects of air pollutants including DEP on lung diseases. Using a cDNA microarray, we examined changes in gene expression in SV40T2 cells, a rat alveolar type II epithelial cell line, following exposure to an organic extract of DEP. We identified candidate sensitive genes that were up- or down-regulated in response to DEP. The cDNA microarray analysis revealed that a 6-h exposure to the DEP extract (30 microg/ml) increased (2-fold) the expression of 51 genes associated with drug metabolism, antioxidation, cell cycle/proliferation/apoptosis, coagulation/fibrinolysis, and expressed sequence tags (ESTs), and decreased (0.5-fold) that of 20 genes. In the present study, heme oxygenase (HO)-1, an antioxidative enzyme, showed the maximum increase in gene expression; and type II transglutaminase (TGM-2), a regulator of coagulation, showed the most prominent decrease among the genes. We confirmed the change in the HO-1 protein level by Western blot analysis and that in the enzyme activity of TGM-2. The organic extract of DEP increased the expression of HO-1 protein and decreased the enzyme activity of TGM-2. Furthermore, these effects of DEP on either HO-1 or TGM-2 were reduced by N-acetyl-l-cysteine (NAC), thus suggesting that oxidative stress caused by this organic fraction of DEP may have induced these cellular responses. Therefore, an increase in HO-1 and a decrease in TGM-2 might be good markers of the biological response to organic compounds of airborne particulate substances.

Although inorganic arsenate (iAsV) and arsenite (iAsIII) are metabolized in liver and excreted into bile and urine, the metabolites in the bile after the oral intake of iAs remain unclear. Male Sprague-Dawley rats were orally (po) or intravenously (iv) exposed to iAs and methylated arsenics, and the arsenic speciation in the urine and bile was analyzed by high performance liquid chromatography-inductively coupled argon plasma mass spectrometry. Arsenic caused induction of multidrug resistance-associated protein 2 (MRP2), and changes of glutathione (GSH) levels in the liver and bile were also determined. The metabolic speciation studies revealed that arsenic was excreted into bile in the methylarsenic-diglutathione (MADG) and/or dimethylarsenic acid (DMAV) forms in iAsIII- or iAsV-po rats, but that MADG and arsenic-triglutathione (ATG) are the main forms excreted into bile both in iAsIII- and iAsV-iv rats. In MADG-po rats, the MADG was excreted into bile in the MADG and DMAV forms. Monomethylarsonic acid (MMAV)- and DMAV-iv rats did not excrete significant amounts of either MMAV or DMAV into bile and mostly excreted into urine in the unchanged chemical forms. Taken together, the DMAV detected in the bile is mostly supposed to be the dissociation of dimethylarsenic-glutathione (DMAG). Urinary arsenic speciation showed that arsenic metabolized to 43% methylated DMAV, 47% unmethylated iAsIII, and 10% iAsV in iAsIII-iv rats, whereas only 3% methylated DMAV, 87% unmethylated iAsV, and 10% iAsIII were detected in iAsV-iv rats. Arsenic was accumulated dose dependently, and arsenic concentration was significantly higher in the iAsIII-po rat liver than in the iAsV-po rat liver. GSH levels in the bile were decreased by relatively higher doses of iAsV-po, but significantly increased by iAsIII- or iAsV-iv. iAs-exposure increased the expression of MRP2 in the liver. Pretreatment with buthionine sulfoximine predominantly inhibited arsenic excretion into bile in iAs-iv rats. In conclusion, our data demonstrated that biliary and urinary arsenic excretion and speciation are affected by the route, dose, and chemical forms of arsenical administration, and GSH plays a key role in arsenic metabolism. We are also first to show that DMAV that probably originated from DMAG is excreted into the bile in iAs-po rats.

Clark 1 (diphenylarsine chloride) and Clark 2 ( diphenylarsine cyanide) were used as chemical weapon agents (CWA), and the soil contamination by these CWA and their degraded products, diphenyl and phenyl arsenicals, has been one of the most serious environmental issues. In a series of comparisons in toxicity between trivalent and pentavalent arsenicals we investigated differences in the accumulation and toxicity of phenylarsine oxide (PAO(3+)) and phenylarsonic acid (PAA(5+)) in rat heart microvascular endothelial cells. Both the cellular association and toxicity of PAO(3+) were much higher than those of PAA(5+), and LC50 values of PAO(3+) and PAA(5+) were calculated to be 0.295 muM and 1.93 mM, respectively. Buthionine sulfoximine, a glutathione depleter, enhanced the cytotoxicity of both PAO(3+) and PAA(5+). N-Acetyl-L-cysteine (NAC) reduced the cytotoxicity and induction of heme oxygenase-1 (HO-1) mRNA in PAO(3+)-exposed cells, while NAC affected neither the cytotoxicity nor the HO-1 mRNA level in PAA(5+)-exposed cells. The effect of NAC may be due to a strong affinity of PAO(3+) to thiol groups because both NAC and GSH inhibited the cellular accumulation of PAO(3+), but PAA(3+) increased tyrosine phosphorylation levels of cellular proteins. These results indicate that the inhibition of protein phosphatases as well as the high affinity to cellular components may confer PAO(3+) the high toxicity.

The aim of this study was to gain insight into early events in the lung epithelial cells following acute Cd exposure. We adopted the polymerase chain reaction (PCR)-based subtraction technique and found several genes that were upregulated in immortalized rat lung type 2 epithelial cells (SV40T2). The upregulation of those genes was confirmed by Northern blot analysis and categorized into three groups (highly, moderately, and weakly inducible genes). Heme oxygenase-1 (HO-1), HSP 72, hepatic steroid hydroxylase/CYPIIA2, and Cd-inducible gene 1 (cdig1, a new gene, Accession Nos. AB086233 and AB086234 ) were highly inducible genes, testosterone-repressed prostate message 2 mRNA was moderately inducible, and collagen-binding protein and cdig2 (another new gene, Accession No. AB086193 ) mRNAs were weakly inducible. The expression of cdig1 increased linearly with time up to 9 h, while that of HO-1 reached the maximum value at 4 h in response to 10 μM Cd.

Intake of inorganic arsenic is known to cause vascular diseases as well as skin lesions and cancer in humans. We investigated the differences in cytotoxicity, uptake rate of arsenic, and gene expression of antioxidative enzymes between arsenite (As(3+))- and arsenate (As(5+))-exposed rat heart microvessel endothelial cells. As(3+) was more cytotoxic than As(5+), and LC(50) values were calculated to be 36 and 220 micro M, respectively. As(3+) (1-25 micro M) increased mRNA levels of antioxidant enzymes such as heme oxygenase-1 (HO-1), thioredoxin peroxidase 2, NADPH dehydrogenase, and glutathione S-transferase P subunit. HO-1 mRNA levels showed the most remarkable increase in response to As(3+). cDNA microarray analysis indicated that there was no prominent difference in arsenic-induced transcriptional changes between As(3+)- and As(5+)-exposed cells, when the cells were exposed to one-fourth the LC(50) concentration of arsenic (9 and 55 micro M for As(3+) and As(5+), respectively). N-acetyl- l-cysteine (NAC) reduced both the cytotoxicity of inorganic arsenic and the HO-1 mRNA level, and buthionine sulfoximine enhanced cytotoxicity of inorganic arsenic. As(3+) was taken up by the endothelial cells 6-7 times faster than As(5+), and the presence of NAC in the culture medium did not change the uptake rate of As(3+). These results suggest that the effects of NAC on arsenic-induced cytotoxicity and oxidative stress were due to the antioxidative role of non-protein thiols and not to chelation of arsenic in the culture medium. The difference in cellular uptake of arsenic between As(3+) and As(5+) appeared not to be due to the ionic charge on arsenic (at physiological pH, trivalent arsenic is neutral whereas pentavalent arsenic is negatively charged). These results suggest that the higher toxicity of As(3+) compared with that of As(5+) is probably due to the faster uptake of As(3+) by endothelial cells, and inorganic arsenic exerts its toxicity at least in part via intracellular oxidative stress.

Background Previous studies have shown that lipopolysaccharide-induced inflammation in the lung results in tyrosine nitration. The objective of this study was to evaluate the contribution of myeloperoxidase and peroxynitrite pathway to the tyrosine nitration in lipopolysaccharide-administered lungs of rats that were otherwise untreated or leukocyte-depleted by cyclophosphamide or received inhaled nitric oxide (NO). Methods The authors analyzed the immunoreactivity of inducible nitric oxide synthase (iNOS), nitrotyrosine (a product of the myeloperoxidase or peroxynitrite pathway), and chlorotyrosine (a byproduct of the myeloperoxidase pathway) by use of specific antibodies. The number of neutrophils in bronchoalveolar lavage fluid (BALF) and levels of myeloperoxidase activity in lung homogenates were also measured. Results Lipopolysaccharide enhanced the immunoreactivity of iNOS, nitrotyrosine, and chlorotyrosine in alveolar wall cells, alveolar macrophages, and neutrophils. Leukocyte depletion by cyclophosphamide and inhibition of leukocyte accumulation in the lungs by NO inhalation did not eliminate the increase in iNOS immunoreactivity in alveolar macrophages after lipopolysaccharide treatment, but nitrotyrosine and chlorotyrosine were not produced in these cells. Tyrosine nitration in response to lipopolysaccharide was associated with increases in neutrophil count in BALF and in myeloperoxidase activity in lung homogenates, whereas NO inhalation suppressed the neutrophil count in BALF and reduced tyrosine nitration and chlorination. Conclusions These findings suggest that myeloperoxidase pathway has a role in tyrosine nitration in the lungs of lipopolysaccharide-treated rats, and that NO inhalation during early phase of inflammation does not increase but rather decreases tyrosine nitration and chlorination, possibly by reducing neutrophil sequestration.

Diesel exhaust particles (DEP) induce pulmonary diseases including asthma and chronic bronchitis. Comprehensive evaluation is required to know the effects of pollutants including DEP on these and other lung diseases. Alveolar macrophages (AM) and epithelial cells are important cellular targets for pollutants such as DEP in the lung. Alveolar macrophages encounter and phagocytose DEP in the alveolar space, and their biological responses have been implicated in DEP-induced pulmonary diseases. Expression profiles of genes induced by DEP in AM will lead to better understanding of the mechanisms involved in pulmonary diseases. To characterize the effect of the DEP extract on AM systematically, we analyzed the gene expression in AM exposed to DEP extract using the Atlas Rat Toxicology Array II. The finding in cDNA microarray was further confirmed by Northern blot analysis. AM were exposed to 10 microg/ml of DEP extract for 6 h in order to elucidate early response to DEP extract in AM. Early response to DEP extract in AM may affect the alteration of gene expression in subsequent responses so that it is important to identify the alteration in early response. In this study, the transcription of 6 genes in the cDNA microarray was significantly elevated by exposure of the AM to DEP extract. These genes were heme oxygenase (HO)-1 and -2, thioredoxin peroxidase 2 (TDPX-2), glutathione S-transferase P subunit (GST-P), NAD(P)H dehydrogenase, and proliferating cell nuclear antigen (PCNA). The antioxidative enzymes such as HO, TDPX-2, GST-P, and NAD(P)H dehydrogenase may play a role in the pulmonary defense against oxidative stress caused by various pollutants including DEP. PCNA may have contributed to the repair of DNA damage and to cell proliferation caused by exposure to these pollutants. Our results suggest that cDNA microarray analysis is a useful tool to investigate the biological responses to pulmonary toxicants.

The present study was designed to investigate the incorporation of zinc (Zn) into cultured UMR-106 osteoblasts in response to mineralization caused by the addition of beta-glycerophosphate. As a result of the induced mineralization, the contents of calcium (Ca), phosphorus (P), and Zn in the monolayer increased, whereas the magnesium (Mg) content did not change. The activity of alkaline phosphatase (ALP) also increased during the process. The zinc distribution in the cell monolayer was studied using Zinquin, a fluorescent zinc ion chelator. The double fluorescent labeling with Zinquin and calcein revealed that zinc was localized both as intracellular vesicles and extracellular clusters, whereas calcium was colocalized with extracellular zinc. These results suggest that zinc is involved in the mineralization process of UMR-106 cells.

Alveolar macrophages meet various types of particulate substances deposited deep in the lung. We report differences in biologic responses of alveolar macrophages between phagocytosis of fine spherical and fibrous particles. Although titanium dioxide (TiO(2)) is thought to be biologically inert, the cytotoxicity of fibrous TiO(2) (F-TiO(2)) was much higher than spherical TiO(2) (S-TiO(2)). Differential display and the subsequent Northern blot analysis indicated that transcription of krox-20/egr-2 gene was slightly and greatly upregulated in S- and F-TiO(2)-exposed alveolar macrophages, respectively. The messenger RNA (mRNA) level of krox-20/egr-2 increased up to 8 h in F-TiO(2)-exposed alveolar macrophages, whereas krox-20/egr-2 mRNA level was transiently increased in response to adhesion to the culture dish. Stimulation with lipopolysaccharide also increased krox-20/egr-2 mRNA level transiently, although the mRNA level rebounded after 8 h. The analysis with 5' rapid amplification of complementary DNA ends suggested that there is a heterogeneity in the upstream region of this gene (krox-20/egr-2 and krox-20H1; accession numbers AB032420 and AB032419, respectively). The polymerase chain reaction analysis with specific primers for krox-20/egr-2 and krox-20H1 indicated that both genes were almost equally upregulated after either adhesion to the plastic dish or phagocytosis of F-TiO(2). These results suggest that both krox-20/egr-2 and krox-20H1 are implicated in adhesion and phagocytosis, and that the expression of krox-20 may reflect interaction with foreign substances and adhesion in alveolar macrophages.

Inhalation exposure to fluoride compounds has been associated with respiratory failure. We have addressed effects of fluoride on alveolar macrophages and lung responses to intratracheally (i.t.) instilled fluoride in rats. I.t. instillation of fluoride at doses of 200 and 400 microg F/rat caused significant polymorphonuclear leukocyte (PMN) infiltration in the rat lung at 20 h post-administration, while 100 microg fluoride did not recruit a significant number of PMNs in the alveolar space. Total RNA was extracted from the lung lavage cells obtained from 5 h post i.t. instillation and mRNA levels of chemokines and proinflammatory cytokines were semi-quantitatively evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). I.t. instillation of fluoride significantly enhanced mRNA expression of cytokines such as interleukin-1beta (IL-1beta), tumor necrosis factor-alpha, cytokine-induced neutrophil chemoattractant, and macrophage inflammatory proteins-1alpha and -2. Fluoride-induced augmentation in IL-1beta mRNA expression was also examined by Northern hybridization following in vitro exposure of alveolar macrophages to fluoride. However, the enhancement of IL-1beta mRNA expression following in vitro exposure to fluoride was observed only at 500 microM, a dose higher than the 50% lethal concentration (LC(50)). Non-specific adhesion of alveolar macrophages to the plastic dish was significantly increased following in vitro exposure to fluoride. The fluoride-induced non-specific adhesion was significantly reduced by anti-CD18, suggesting that beta(2) integrin played a role in the increase of adherence. Those results suggest that fluoride activates alveolar macrophages, enhances the production of chemokines and proinflammatory cytokines, and causes PMN infiltration in the lung.

Nitric oxide (NO) may either protect against or contribute to inflammatory lung injury. In this study we investigated whether inhalation of 20 ppm NO alters tyrosine nitration, and we assessed the degree of lung inflammation and edema in rats after lipopolysaccharide (LPS) instillation. The amount of nitrotyrosine relative to the total amount of tyrosine was measured in lung homogenates, and lung tissue sections were stained for nitrotyrosine and aminotyrosine (a reduced form of nitrotyrosine). Leukocytes in bronchoalveolar lavage fluid (BALF) were counted, and myeloperoxidase activity was measured in lung homogenate. Lung edema and inflammatory cell accumulation in lung tissue were estimated by extravascular lung water weight (EVLW) and extravascular dry lung weight (EVDW), respectively. LPS instillation caused increases in nitrotyrosine concentration and immunohistochemical staining of nitrotyrosine and aminotyrosine in the lungs. LPS instillation increased the BALF leukocyte count, myeloperoxidase activity in lung tissue, and both EVLW and EVDW. Inhalational exposure to 20 ppm NO induced nitrotyrosine and aminotyrosine formation only in bronchial epithelial cell surface of the lungs not instilled with LPS. NO inhalation reduced the increases in nitrotyrosine and aminotyrosine in LPS-instilled lung tissue as well as the leukocyte count in BALF and myeloperoxidase activity in lung tissue, but it did not significantly change EVLW or EVDW. Leukocyte depletion in LPS-instilled rats reduced interstitial inflammatory cells, which were stained with nitrotyrosine and aminotyrosine, and attenuated the nitrotyrosine staining of alveolar capillaries. These results suggest that inhalation of 20 ppm NO reduces leukocyte accumulation in the lungs and inhibits tyrosine nitration caused by LPS instillation.

Although an excess intake of fluoride has been reported to cause skeletal fluorosis, very little is known about the mechanism of adverse effects of fluoride on bone. In the present study cytotoxic effects of fluoride were studied using the osteosarcoma cell line, UMR 106. The DNA ladder formation upon agarose electrophoresis and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) staining revealed that UMR 106 underwent apoptosis following exposure to 5 mM fluoride for 8 h. On the other hand exposure to A23187, a calcium ionophore, caused necrosis while co-exposure to fluoride and A23187 inhibited fluoride-mediated apoptosis in UMR 106. The proliferation of UMR 106 cells cultured for 6 days in the presence of 0.5 mM fluoride was significantly decreased compared to the control culture. The cytotoxic effects of fluoride were modulated by both the cell density and the pH of the culture medium. The fluoride-induced viability loss in UMR 106 was enhanced in culture of high cell-density and inversely correlated with pH of the culture medium. Enhancement of fluoride cytotoxicity at acidic pH was also observed in rat alveolar macrophages and RAW 264, a macrophage cell line. The results suggest that fluoride-mediated apoptosis and culture conditions, including pH of the medium, should be taken into consideration to evaluate toxicity of fluoride in vitro.

Diphenylarsinic acid (DPAA(V)) is a chemical precursor as well as a degradation product of arsenic-containing chemical weapons such as Clark 1 (diphenylarsine chloride) and Clark 2 (diphenylarsine cyanide). Compared to inorganic arsenicals, toxicological findings on DPAA(V) are limited. To elucidate the mechanism of DPAA(V) toxicity, we investigated the metabolic behavior of DPAA(V) in the body. Specifically, we examined the distribution and biliary excretion of DPAA(V) and its metabolites in rats orally administered DPAA(V) at a dose of 1.0 mg As kg(-1) body weight. DPAA(V) was excreted in bile, either as the original DPAA(V) or as a DPAA-GSH complex (DPAG(III)), as determined by HPLC-ICP-MS and HPLC-ESI-MS, with DPAG(III) being the main chemical form. Approximately 1.7% and 2.4% of the dose was accumulated in erythrocytes three hours and three days after administration, respectively. Approximately 91% of the dose was excreted in urine and feces as DPAA(V) in three days, mostly in the urine. Accumulation of arsenic in erythrocytes was investigated in vitro by incubating (at 37 °C for up to three hours) DPAA(V) or DPAG(III) with a suspension of rat erythrocytes (10% in Tris-HCl-buffered saline). Approximately 80% of the DPAG(III) dose was taken up by erythrocytes within thirty seconds, while approximately 35% of the DPAA(V) dose was taken up by erythrocytes after three hours of incubation. DPAG(III) was possibly hydrolyzed and converted to trivalent unconjugated arsenical (diphenylarsinous acid, DPA(III)), which also was accumulated in erythrocytes. As expected, DPA(III) exhibited greater affinity for erythrocyte proteins than did DPAA(V).

The present study was undertaken primarily to investigate accumulation of polymorphonuclear leukocytes (PMN) within the lung vasculature after intraperitoneal injection of lipopolysaccharide (LPS) and migratory responses of those intravascular PMN to intratracheally instilled LPS in mice. Intraperitoneally injected LPS was absorbed into the blood rapidly, and the concentration of circulating LPS peaked at 1 h postinjection. Tumor necrosis factor concentration in blood began to increase at 0.5 h and also peaked at 1 h postinjection. The number of lung vascular-associated PMN was increased 7.5-fold at 0.5 h post-intraperitoneal injection. However, neither diapedesis of PMN nor injury was observed in the lung, while mixed cell infiltration was observed in the liver. Although intraperitoneally injected LPS caused a significant PMN accumulation within the lung vasculature, pre-intraperitoneal injection of LPS dramatically and dose dependently abolished both intratracheal LPS-inducible PMN infiltration and apparent plasma protein leakage into the alveolar space. On the other hand, pre-intratracheal instillation of LPS enhanced rather than reduced intraperitoneal LPS-inducible PMN infiltration into the peritoneal cavity. In rats, a sublethal dose of intraperitoneally injected LPS caused a modest increase in alveolar PMN and yet reduced intratracheal LPS-inducible robust transpulmonary PMN infiltration. Although mechanisms of different migratory responses of the lung vasculature-associated PMN are unknown, the inhibitory effects of intraperitoneally injected LPS on transpulmonary PMN infiltration may be applicable to treatments for septic lung diseases.

Because both alveolar macrophages and pulmonary epithelial cells are primary target cells of inhaled endotoxin, it is of interest to study the interaction of alveolar macrophages with epithelial cells following exposure to lipopolysaccharide (LPS). Repeated bronchoalveolar lavage suggested that rat alveolar macrophages became adhesive to epithelial cells in response to intratracheally instilled LPS at 0.5 h post-exposure in vivo. Adherence of alveolar macrophages to SV40T2 (rat type II pulmonary epithelial cells transformed by SV40) was also increased following 0.5-h stimulation with LPS in vitro. The adherence was increased with dose and reached a plateau at 2 micrograms/ml LPS. The transepithelial resistance of the SV40T2 monolayer was decreased by coculture with macrophages in the presence of LPS for 4 h. The transepithelial resistance was not changed by exposure of SV40T2 alone to LPS or conditioned medium obtained from LPS-stimulated macrophages, suggesting that the intercellular interaction between alveolar macrophages and epithelial cells or unstable products of LPS-exposed alveolar macrophages was associated with the decrease in transepithelial resistance. Following the decrease in transepithelial resistance, lysis of SV40T2 was observed in the LPS-exposed coculture system. However, the lysis of SV40T2 did not occur until 12 h after the addition of LPS, indicating that the junctional change of the monolayer preceded cell death in SV40T2. Cytotoxicity of LPS-stimulated macrophages was significantly reduced by NG-monomethyl-L-arginine (NMMA), an inhibitor of nitric oxide synthase, but NMMA did not reduce the decrease in transepithelial resistance. These results suggest that the greater adherence of alveolar macrophages to epithelial cells, the junctional change of the epithelial monolayer and the lysis of epithelial cells occur in this order in the LPS-exposed alveolar macrophage-SV40T2 coculture system, and the greater adherence of alveolar macrophages may play a role in LPS-induced inflammation in the rat lung.

Diphenylarsinic acid (DPAA) was a major compound found in the arsenic poisoning incident that occurred in Kamisu, Ibaraki, Japan in 2003. People exposed to DPAA via contaminated well water suffered from several neurological disorders, including cerebellar symptoms. We previously reported that DPAA induces cellular activation in cultured rat cerebellar astrocytes, dose-dependent promotion of cell growth (low DPAA), cell death (high DPAA), and increased phosphorylation of mitogen-activated protein (MAP) kinases (p38MAPK, SAPK/JNK, and ERK1/2). Moreover, DPAA induces up-regulation of oxidative stress-counteracting proteins, activation of CREB phosphorylation, increased protein expression of c-Jun and c-Fos, and aberrant secretion of brain-active cytokines (MCP-1, adrenomedullin, FGF2, CXCL1, and IL-6). Here, we explored the role of MAP kinases in DPAA-induced activation of astrocytes using specific MAP kinase signaling inhibitors [SB203580 (p38MAPK), SP600125 (SAPK/JNK), SCH772984 (ERK1/2), and U0126 (MEK1/2, a kinase for ERK1/2)]. DPAA-induced activation of MAP kinases had little contribution to DPAA-induced cell growth and death. On the other hand, a power relationship among MAP kinases was also observed, in which p38MAPK suppressed DPAA-induced SAPK/JNK and ERK1/2 activation, whereas ERK1/2 and MEK1/2 facilitated p38MAPK and SAPK/JNK activation. In addition, SAPK/JNK had minimal effects on the activation of other MAP kinases. DPAA-induced activation of transcription factors and secretion of brain-active cytokines were submissively but intricately dominated by MAP kinases. Collectively, our results indicate that DPAA-induced activation of MAP kinases is neither a cell growth-promoting response nor a cytoprotective one but leads to transcriptional disruption and aberrant secretion of brain-active cytokines in cerebellar astrocytes.

Exposure to asbestos is a risk for malignant mesothelioma (MM) in humans. Among the commercially used types of asbestos (chrysotile, crocidolite, and amosite), the carcinogenicity of chrysotile is not fully appreciated. Here, we show that all three asbestos types similarly induced MM in the rat peritoneal cavity and that chrysotile caused the earliest mesothelioma development with a high fraction of sarcomatoid histology. The pathogenesis of chrysotile-induced mesothelial carcinogenesis was closely associated with iron overload: repeated administration of an iron chelator, nitrilotriacetic acid, which promotes the Fenton reaction, significantly reduced the period required for carcinogenesis; massive iron deposition was found in the peritoneal organs with high serum ferritin; and homozygous deletion of the CDKN2A/2B/ARF tumour suppressor genes, the most frequent genomic alteration in human MM and in iron-induced rodent carcinogenesis, was observed in 92.6% of the cases studied with array-based comparative genomic hybridization. The induced rat MM cells revealed high expression of mesoderm-specific transcription factors, Dlx5 and Hand1, and showed an iron regulatory profile of active iron uptake and utilization. These data indicate that chrysotile is a strong carcinogen when exposed to mesothelia, acting through the induction of local iron overload. Therefore, an intervention to remove local excess iron might be a strategy to prevent MM after asbestos exposure.

Speciation analysis using high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP MS) is now commonly used to investigate metabolic and toxicological aspects of some metals and metalloids. We have developed a rapid method for simultaneous identification and quantification of metabolites of selenium (Se) compounds using multiple standards labelled with different isotopes. A mixture of the labelled standards was spiked in a selenised garlic extract and the sample was subjected to speciation analysis by HPLC-ICP MS. The selenised garlic contains γ-glutamyl-methylselenocysteine, methylselenocysteine, and selenomethionine and the concentrations of those Se compounds were 723.8, 414.8, and 310.7 ng Se ml(-1), respectively. The isotopically labelled standards were also applied to the speciation of Se in rat urine. Selenate, methylselenonic acid, selenosugar, and trimethyselenium ions were found to be excreted by the present speciation procedure. Multiple standards labelled with different stable isotopes enable high-throughput identification and quantitative measurements of Se metabolites.

Although radioactive yttrium (Y) has been used for medical treatment, little attention has been directed toward the toxicity of Y. We report time-course and dose-related changes in tissue distribution, subcellular localization, clearance, and acute toxicity of iv-injected yttrium chloride (YCl3) in rats. Intravenously injected Y was predominantly distributed to plasma in the blood. At doses more than 0.2 mg Y/rat, most plasma Y appears to be in colloidal material which was composed of proteins and some minerals. Electron microscopic analyses revealed that the colloidal material was taken up by phagocytic cells in the liver and spleen. The liver Y was slowly cleared with a half-time of 144 days at a dose of 1 mg Y/rat. Glutamic-oxaloacetic and glutamic-pyruvate transaminase activities in blood plasma were increased with a peak at 20 hr postinjection at a dose of 1 mg Y/rat and returned to their control values at 170 hr postinjection, indicating that iv-injected YCl3 caused acute hepatic injury. Some of the plasma Ca was translocated to the colloidal material and plasma Ca concentration was increased transiently following injection of YCl3, probably because of resorption of bone. At a dose of 1 mg Y/rat, a significant and tremendous amount of Ca was deposited in the liver (over 10-fold) and spleen (over 100-fold), while Ca concentration was only slightly increased in the lung and kidney (less than 1.5-fold). These results indicate that the liver and spleen are primary target organs of iv-injected YCl3.

Pulmonary clearance and toxicity of cupric oxide (CuO) dusts, which are probably formed in refining and smelting factories, were investigated. Groups of three rats received intratracheal (i.t.) instillation of CuO at a dose of 20 micrograms Cu/rat in time-course experiments (up to 7 days post-instillation). Other groups of three rats received i.t. instillation of CuO at doses of 2.5, 5, 10, 30, 50 and 100 micrograms Cu/rat and were killed at 2 days post-instillation in dose-effect experiments. Intratracheally instilled CuO particles were cleared from the lung with a half-time of 37 h. Copper binding metallothionein (MT) was induced in a dose-dependent manner and detected at 12 h to 3 days post-instillation. Rapid clearance of CuO from the lung and induction of MT at 12 h post-instillation suggest that CuO particles were solubilized and then cleared from the lung. The acute pulmonary toxicity of CuO was evaluated by cytological (numbers of macrophages and polymorphonuclear leukocytes), biochemical and elemental inflammatory indices (lactate dehydrogenase and beta-glucuronidase activities and protein, sulfur, phosphorus and calcium contents) in the bronchoalveolar lavage (BAL) fluid. These inflammatory indices peaked at 12 h to 3 days post-instillation, and increased with dose over the dose range, except for phosphorus content. Dose-effect relationships in BAL inflammatory indicators of CuO-injected (i.t.) groups were compared to those of CuSO4-injected (i.t.) groups. The results of the comparison indicated that there was no significant difference in acute inflammatory potency between CuSO4 (soluble form of Cu) and CuO (insoluble form of Cu) in the rat lung.

Selenium is an essential trace element found in vegetables as selenomethionine (SeMet) and methylselenocysteine (MeSeCys). In the present study, we used stable isotopes of Se to investigate differences between how SeMet and MeSeCys are metabolized, using methylseleninic acid (MSA) as a reference methylselenol source. A mixture containing (76)Se-SeMet, (77)Se-MeSeCys, and (82)Se-MSA (each 25 microg Se/kg b.w.) was orally administered to rats, and then, speciation analyses of Se in urine and exhaled gas were conducted using HPLC-inductively coupled plasma (ICP)-MS and GC-ICP-MS, respectively. The proportions of (76)Se-, (77)Se-, and (82)Se-selenosugar (Se-sugar) to total urinary Se metabolites originating from each tracer were very similar, while the proportion of (77)Se-tirmethylselenonium (TMSe) was much less than that of(76)Se- and (82)Se-TMSe in urine, suggesting that(77)Se-SeMet is less efficiently metabolized to TMSe. Similarly, there was significantly less (77)Se-dimethylselenide (DMSe) originating from (77)Se-SeMet than(76)Se- and (82)Se-DMSe originating from (76)Se-MeSeCys and (82)Se-MSA in exhaled gas. It is generally accepted that DMSe and TMSe are metabolites of methylselenol, a putative metabolic intermediate in Se metabolism. Methylselenol is believed to be responsible for the cancer chemoprevention effects of Se. These results suggest that MeSeCys is converted to methylselenol more efficiently than is SeMet and that urinary TMSe and exhaled DMSe might be useful biomarkers for the generation of cancer chemopreventive forms of Se.

Respiratory exposure to asbestos has been linked with mesothelioma in humans. However, its carcinogenic mechanism is still unclear. Here we studied the ability of chrysotile, crocidolite and amosite fibers to induce oxidative DNA damage and the modifying factors using four distinct approaches. Electron spin resonance analyses revealed that crocidolite and amosite containing high amounts of iron, but not chrysotile, catalyzed hydroxyl radical generation in the presence of H(2)O(2), which was enhanced by an iron chelator, nitrilotriacetic acid, and suppressed by desferal. Natural iron chelators, such as citrate, adenosine 5'-triphosphate and guanosine 5'-triphosphate, did not inhibit this reaction. Second, we used time-lapse video microscopy to evaluate how cells cope with asbestos fibers. RAW264.7 cells, MeT-5 A and HeLa cells engulfed asbestos fibers, which reached not only cytoplasm but also the nucleus. Third, we utilized supercoiled plasmid DNA to evaluate the ability of each asbestos to induce DNA double strand breaks (DSB). Crocidolite and amosite, but not chrysotile, induced DNA DSB in the presence of iron chelators. We cloned the fragments to identify break sites. DSB occurred preferentially within repeat sequences and between two G:C sequences. Finally, i.p. administration of each asbestos to rats induced not only formation of nuclear 8-hydroxy-2'-deoxyguanosine in the mesothelia, spleen, liver and kidney but also significant iron deposits in the spleen. Together with the established carcinogenicity of i.p. chrysotile, our data suggest that asbestos-associated catalytic iron, whether constitutional or induced by other mechanisms, plays an important role in asbestos-induced carcinogenesis and that chemoprevention may be possible through targeting the catalytic iron.

Trivalent arsenicals such as arsenite (iAs(III)), monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III)) are more toxic than analogous pentavalent compounds such as arsenate (iAs(V)), monomethylarsonic acid (MMA(V)) and dimethylarsinic acid (DMA(V)). It has been reported that arsenic-glutathione (As-GSH) complexes such as arsenic triglutathione (ATG) and methylarsenic diglutathione (MADG) are major metabolites in rat bile following intravenous administration of iAs(III). Recently, we have shown that both ATG and MADG are unstable and easily hydrolyzed to iAs(III) and MMA(III), respectively, and that MMA(III) is oxidized to MMA(V) in bile. In the present study we report the effects of H(2)O(2) and GSH on the stability of As-GSH complexes in rat bile. Male SD rats were injected intravenously with saline or iAs(III) at a dose of 0.2 or 2.0 mg As/kg body weight, and bile fluid was collected on ice for 30 min. To estimate the stability of As-GSH complexes in bile, ATG or MADG was added to untreated, heat-treated, catalase-treated, or dialyzed bile, and then incubated at 37 degrees C for 10 min. Concentrations of biliary H(2)O(2) and GSH in the higher dose group were 12.6- and 4.5-times higher than the control value, respectively. Exogenously added trivalent arsenicals were oxidized to pentavalent arsenicals in the bile depending on the biliary concentration of H(2)O(2). Both catalase and dialysis prevented oxidation of trivalent arsenicals to the corresponding pentavalent compounds. Exogenously added GSH stabilized As-GSH complexes in bile. These results suggest that H(2)O(2) converts trivalent arsenicals to less toxic pentavalent arsenicals, whereas GSH prevents hydrolysis of As-GSH complexes and the generation of unconjugated toxic trivalent arsenicals.

Though copper (Cu)-handling workers are known to be at risk to develop metal fume fever and acute respiratory failure, very little is known about the metabolic fate and pulmonary toxicity of Cu compounds deposited in the lung. We have investigated time-course and dose-related changes in Cu contents, the production of metallothionein (MT) and inflammatory responses in the rat lung following intratracheal instillation of cupric sulfate (CuSO4). Intratracheally instilled Cu was cleared rapidly from the lung with a half-time of 7.5 h. Copper-binding MT was induced in the lung tissue following Cu instillation and the amount of MT increased with the dose of CuSO4. However, the production of MT contributed little to the accumulation of Cu in the lung. The pulmonary toxicity of CuSO4 was evaluated by examining time-course and dose-effect profiles of cytological and biochemical inflammatory indices (enzymes, protein and elements) retrieved in bronchoalveolar lavage fluids. These results revealed that the inflammatory indices reached maximum values in their activities and contents at 12 h to 3 days after instillation and a dose of 5 μg Cu/rat was sufficient to produce acute inflammatory responses in the rat lung.

Acute biochemical changes in the rat lung and liver following intratracheal instillation of cadmium oxide (CdO) were observed at a dose of 5 μ Cd/rat to investigate the defense mechanism to Cd intoxication via airway. In the lung metallothionein (MT) was induced, reaching a maximum at 2 days. A slight increase in reduced glutathione (GSH) concentration was observed at 4 days. The activity of glucose-6-phosphate dehydrogenase (G6PDH) was increased and Superoxide dismutase (SOD) activity was slightly decreased, but glutathione peroxidase (GPx) and glutathione reductase (GR) activities were not changed. These observations suggested that MT played a key role in detoxification of instilled CdO, but that the antioxidant enzymes had a minimal role. In the liver MT and GSH concentrations were diminished 7 h after instillation and returned to their control levels. Hepatic GPx activity was increased 1 day after instillation and the significantly elevated level lasted up to 7 days, while hepatic GR activity was decreased. These hepatic biochemical changes are suggested to be due to the secondary effects of the lung injury.

Endothelial cells play important roles in anticoagulant and fibrinolytic systems. Recent studies suggest that increases in ambient particulate matter (PM) levels have been associated with an increase in mortality rate from cardiovascular diseases. We examined the production of heme oxygenase-1 (HO-1) and factors related to the fibrinolytic function by rat heart microvessel endothelial cells exposed to organic extracts of diesel exhaust particles (OE-DEP) and urban fine particles (OE-UFP) to investigate the direct effects of these soluble organic fractions in these PM on the fibrinolytic function of endothelial cells. The cell monolayer exposed to 10 microg/ml OE-DEP produced a larger amount of HO-1 than cells exposed to 10 microg/ml OE-UFP. OE-DEP and OE-UFP exposure reduced plasminogen activator inhibitor-1 (PAI-1) production by the cells but did not affect the production of thrombomodulin, tissue-type plasminogen activator, or urokinase-type plasminogen activator. Increased PAI-1 synthesis in response to treatment with 1.0 ng/ml tumor necrosis factor-alpha or 0.5 ng/ml transforming growth factor-beta1 was reduced by OE-DEP exposure. Suppression of PAI-1 production by OE-DEP exposure was mediated through oxidative stress and was independent of HO-1 activity. These results suggest that exposure to the soluble organic fraction of PM and DEP induced oxidative stress and reduced the PAI-1 production of endothelial cells.

Inorganic arsenicals such as arsenite (iAs(III)) and arsenate (iAs(V)) are well-known human carcinogens. Arsenic is metabolized by repetitive reduction and oxidative methylation, and is excreted mainly in urine as monomethylated arsenicals (MMAs) and dimethylated arsenicals (DMAs). Recently, it has been shown that iAs(III) administered intravenously or orally is excreted into bile as arsenic-glutathione (As-GSH) complexes such as arsenic triglutathione [As(GS)(3)] and methylarsenic diglutathione [CH(3)As(GS)(2)]. In order to carry out the speciation of As-GSH complexes, it is important to understand their stability. The present study was designed to clarify the stability of As-GSH complexes in rat bile, and the role of GSH in stabilizing these complexes. Arsenic species were separated on an anion-exchange column and were analyzed by high-performance liquid chromatography-inductively coupled argon plasma mass spectrometry (HPLC-ICP MS). As(GS)(3) and CH(3)As(GS)(2) were unstable in bile and were hydrolyzed to iAs(III) and monomethylarsonous acid (MMA(III)) in the absence of GSH. As(GS)(3) appeared to be stable in the presence of 10mM GSH. Exogenously added GSH also stabilized CH(3)As(GS)(2) in bile at the concentrations of 5mM or higher. It has been suggested that trivalent arsenicals, especially MMA(III), are more toxic than corresponding pentavalent ones. These results suggest that GSH plays an important role in preventing hydrolysis of As-GSH complexes and the generation of well-known toxic trivalent arsenicals.

Since inhaled fluoride is implicated in the acute respiratory failure, cytotoxic effects of fluoride on alveolar macrophages, primary target cells of inhaled toxicants, were investigated. The LC50 of sodium fluoride was estimated to be 0.41 mM, while 1 mM sodium chloride, bromide and iodide had virtually no effects on the viability of alveolar macrophages. Photomicroscopic observation revealed that nuclei of the fluoride-exposed alveolar macrophages were fragmented. The ladder formation was observed when DNA isolated from fluoride-exposed alveolar macrophages was electrophoresed in agarose gel. These results suggest that cytotoxicity of fluoride is associated with apoptosis in rat alveolar macrophages.

The metabolism of arsenic is generally accepted to proceed by repetitive reduction and oxidative methylation; the latter is mediated by arsenic methyltransferase (Cyt19). In human urine, the major metabolites of inorganic arsenicals such as arsenite (iAsIII) and arsenate (iAsV) are monomethylarsonic acid (MMA(V)) and dimethylarsinic acid (DMA(V)). On the other hand, in rat bile, the major metabolites of iAsIII have been reported to be arsenic-glutathione (As-GSH) complexes. In the present study we investigate whether these As-GSH complexes are substrates for arsenic methyltransferase by using human recombinant Cyt19. Analyses by high-performance liquid chromatography-inductively coupled plasma mass spectrometry suggested that arsenic triglutathione (ATG) was generated nonenzymatically from iAsIII when GSH was present at concentrations 2 mM or higher. Human recombinant Cyt19 catalyzed transfer of a methyl group from S-adenosyl-L-methionine to arsenic and produced monomethyl and dimethyl arsenicals. The methylation of arsenic was catalyzed by Cyt19 only when ATG was present in the reaction mixture. Moreover, monomethylarsonic diglutathione (MADG) was a substrate of Cyt19 for further methylation to dimethylarsinic glutathione (DMAG). On the other hand, monomethylarsonous acid (MMA(III)), a hydrolysis product of MADG, was not methylated to dimethyl arsenical by Cyt19. These results suggest that As-GSH complexes such as ATG and MADG were converted by Cyt19 to MADG and DMAG, respectively. Both MADG and DMAG were unstable in solution when the GSH concentration was lower than 1 mM, and were hydrolyzed and oxidized to MMA(V) and DMA(V), respectively. Metabolism of iAsIII to methylated arsenicals by Cyt19 was via ATG and MADG rather than by oxidative methylation of iAsIII and MMA(III).

Although arsenic exposure causes liver disease and/or hepatoma, little is known about molecular mechanisms of arsenic-induced liver toxicity or carcinogenesis. We investigated the effects of arsenic on expression of cancer-related genes in a rat liver following subchronic exposure to sodium arsenate (1, 10, 100 ppm in drinking water), by using real-time quantitative RT-PCR and immunohistochemical analyses. Arsenic accumulated in the rat liver dose-dependently and caused hepatic histopathological changes, such as disruption of hepatic cords, sinusoidal dilation, and fatty infiltration. A 1-month exposure to arsenic significantly increased hepatic mRNA levels of cyclin D1 (10 ppm), ILK (1 ppm), and p27Kip1 (10 ppm), whereas it reduced mRNA levels of PTEN (1 ppm) and β-catenin (100 ppm). In contrast, a 4-month arsenic exposure showed increased mRNA expression of cyclin D1 (100 ppm), ILK (1 ppm), and p27Kip1 (1 and 10 ppm), and decreased expression of both PTEN and β-catenin at all 3 doses. An immunohistochemical study revealed that each protein expression accords closely with each gene expression of mRNA level. In conclusion, subchronic exposure to inorganic arsenate caused pathological changes and altered expression of cyclin D1, p27Kip1, ILK, PTEN, and β-catenin in the liver. This implies that arsenic liver toxicity involves disturbances of some cancer-related molecules.

Excess intake of arsenic is known to cause vascular diseases as well as skin lesions and cancer in humans. Recent reports suggest that trivalent methylated arsenicals, which are intermediate metabolites in the methylation process of inorganic arsenic, are responsible for the toxicity and carcinogenicity of environmental arsenic. We investigated acute toxicity and accumulation of monomethylarsonic acid (MMA(V)), dimethylarsinic acid (DMA(V)), trimethylarsine oxide (TMAO), and monomethylarsonous acid diglutathione (MMA(III) (GS)(2)) in rat heart microvessel endothelial (RHMVE) cells. MMA(V) (LC(50) = 36.6 mM) and DMA(V) (LC(50) = 2.54 mM) were less toxic than inorganic arsenicals (cf. LC(50) values for inorganic arsenite (iAs(III)), and inorganic arsenate (iAs(V)) was reported to be 36 and 220 microM, respectively, in RHMVE cells. TMAO was essentially not toxic. However, MMA(III) (GS)(2) was highly toxic (LC(50) = 4.1 microM). The order of cellular arsenic accumulation of those four organic arsenic compounds was MMA(III) (GS)(2) >> MMA(V) > DMA(V) > TMAO. MMA(III) (GS)(2) was efficiently taken up by the cells and cellular arsenic content increased with the concentration of MMA(III) (GS)(2) in culture medium. N-acetyl-l-cysteine (NAC) reduced cellular arsenic content in DMA(V)-exposed cells and also decreased the cytotoxicity of DMA(V), whereas it changed neither cellular arsenic content nor the viability in MMA(V)-exposed cells. mRNA levels of heme oxygenase-1 (HO-1) were decreased by NAC in DMA(V)-exposed, but MMA(V)-exposed cells. Buthionine sulfoximine (BSO), a cellular glutathione (GSH) depleting agent, enhanced the cytotoxicity of MMA(V). However, BSO reduced, rather than enhanced, the cytotoxicity of DMA(V). These results suggest that intracellular GSH modulated the toxic effects of arsenic in opposite ways for MMA(V) and DMA(V). Even though intracellular GSH decreased the cytotoxicity of MMA(V), extracellularly added GSH enhanced the cytotoxicity of MMA(V). The use of high-performance liquid chromatography (HPLC)-inductively coupled plasma mass spectrometric analyses suggested that a small amount of MMA(V) was converted to MMA(III) (GS)(2) in the presence of GSH. These results suggest that MMA(III) (GS)(2) is highly toxic compared to other arsenic compounds because of faster accumulation of this species by cells, in addition to having the toxic nature of methylated trivalent organic arsenics.

Exposure to fine particulate materials is associated with an increase in mortality rate of cardiovascular diseases. Particles deposited in the lung may affect the vascular system both directly (leaching of soluble components from particles) and indirectly (via cytokines and mediators). The present study addressed cytotoxicity and oxidative stress potency of organic extracts of diesel exhaust particles (OE-DEP) and urban fine particles (OE-UFP) in rat heart microvessel endothelial (RHMVE) cells. The LC(50) values of OE-DEP and OE-UFP were calculated to be 17 and 34 microg/ml, respectively, suggesting that OE-DEP was more cytotoxic than OE-UFP. The viability of OE-DEP- and OE-UFP-exposed cells was ameliorated by N-acetyl-L-cysteine (NAC). The cell monolayer was exposed to 0 (control), 1, 3, and 10 microg/ml OE-DEP for 6 h and mRNA levels of antioxidant enzymes such as heme oxygenase-1 (HO-1), thioredoxin peroxidase 2 (TRPO), glutathione S-transferase P subunit (GST-P), and NADPH dehydrogenase (NADPHD) were quantitated by northern analysis. All those mRNA levels increased dose-dependently with OE-DEP and HO-1 mRNA showed the most marked response to OE-DEP. mRNA levels of those antioxidant enzymes and heat shock protein 72 (HSP72) in OE-DEP-exposed cells were higher than those of OE-UFP-exposed cells as compared at the same concentration. The transcription levels of HO-1 and HSP72 in OE-DEP- and OE-UFP-exposed cells were also reduced by NAC. Those results suggest that the organic fraction of particulate materials in the urban air has a potency to cause oxidative stress to endothelial cells and may be implicated in cardiovascular diseases through functional changes of endothelial cells.

Macrophages are known to adhere to a plastic dish via beta2 integrin (CR3) and scavenger receptors. Although their functions such as phagocytosis, endocytosis, and nitric oxide production have been investigated on adherent macrophages in vitro, very little is known about intracellular signals triggered by adhesion to a plastic dish. Recently we reported that the mRNA level of krox-20/egr-2 was significantly increased in rat alveolar macrophages following exposure to fibrous titanium dioxide particles. In the present study we report that up-regulation of krox-20/egr-2 gene expression following adhesion to a plastic dish and homophilic adhesion in rat alveolar macrophages and rat macrophage cell line, NR8383. The mRNA level of krox-20/egr-2 increased with a peak 1 hr after adhesion to a plastic dish in both cell types. Piceatannol inhibited tyrosine-phosphorylation of Syk and decreased both adhesion and krox-20/egr-2 mRNA level. In contrast staurosporine, a serine/threonine kinase inhibitor, increased adherence of macrophages and yet prohibited the adhesion-dependent increase in krox-20/egr-2 gene expression. When NR8383 cells are cultured in suspension, the cells aggregated naturally and produced cell clumps. The mRNA level of krox-20/egr-2 also increased in response to the homophilic intercellular adhesion. The increased mRNA level of krox-20/egr-2 was not caused by inflammatory stimuli, because lipopolysaccharide did not affect the aggregation-dependent up-regulation of krox-20/egr-2 gene. The up-regulation of krox-20/egr-2 gene due to the homophilic cell aggregation was also inhibited either by piceatannol or staurosporine. Those results suggest that krox-20/egr-2 gene expression is triggered by sensing non-specific and homophilic cellular adhesion and the following phosphorylation of signal transducing proteins including Syk and staurosporine-inhibitable kinases.

Inhalation exposure to cadmium (Cd) is associated with inflammatory lesion in the lung. In the present study we have investigated cytotoxic effects of Cd on immortalized rat lung epithelial cells (SV40-T2) and gene expression in those cells following in vitro exposure to sublethal concentrations of cadmium chloride (CdCl(2)). The polymerase chain reaction (PCR)-based subtraction method was used to find differentially expressed genes between control and Cd-exposed SV40-T2 cells. The most prominent cDNA on an agarose gel was identical to a fragment of rat heme oxygenase (HO) gene. Northern blot analysis indicated that the level of HO mRNA expression in SV40-T2 cells was increased to 38-fold of the control value following exposure to 2.5 microM CdCl(2) for 4 h. These results suggest that HO gene expression is one of the most sensitive biomarkers for acute exposure to Cd in the lung.

We investigated pulmonary clearance of yttrium (Y) and acute lung injury following intratracheal instillation (i.t.) of yttrium chloride (YCl3) in saline- or YCl3-pretreated rats (30 days before the second challenge). About 67% of the initial dose of Y remained in the lung even 31 days after the i.t. treatment. The pretreatment with YCl3 significantly reduced i.t.-YCl3-induced increases in biochemical inflammatory indicators in bronchoalveolar lavage fluid (BALF), such as lactate dehydrogenase, beta-glucuronidase, and alkaline phosphatase activities and protein concentration, while the pretreatment increased the number of polymorphonuclear leukocyte (PMN) in BALF. These results suggest that the augmentation of PMN infiltration does not play an important role, if any, in i.t. YCl3-induced increases in biochemical indicators in BALF. The reduction of the increases in those biochemical inflammatory indicators may be due, at least in part, to the increase of manganese-superoxide dismutase (Mn-SOD) activity in the lung tissue, because the lung Mn-SOD activity in the YCl3-pretreated group was two times higher than that of the saline-pretreated group.

It is known that murine macrophages produce nitric oxide (NO) when stimulated with lipopolysaccharide (LPS) or interferon-gamma (IFN-gamma), and NO mediates the tumoricidal activity of activated macrophages. The present study was designed to investigate whether the intercellular adhesion is necessary for activated rat alveolar macrophages to exert the full cytotoxic effects. Rat alveolar macrophages produced NO dose dependently in response to either LPS or IFN-gamma, and caused DNA fragmentation in rat type II pneumocytes transformed with SV40 (SV40T2). Chemically produced NO also caused the DNA fragmentation and viability loss in SV40T2, and both of them were inhibited by a NO radical scavenger. The cytotoxicity of activated macrophages was reduced by NG-monomethyl-L-arginine, a competitive nitric synthase inhibitor, and neither superoxide dismutase nor catalase modulated the cytotoxicity. Although alveolar macrophages stimulated with either LPS or IFN-gamma caused DNA fragmentation of SV40T2, only LPS increased the intercellular adherence between macrophages and SV40T2. The intercellular adhesion was reduced by both anti-CD18 and anti-CD11a. However, those antibodies did not affect the cytotoxicity of LPS-stimulated macrophages. These results clearly indicate that NO-mediated cytotoxicity is caused predominantly by diffusion of NO, and the beta 2 integrin-mediated intercellular adhesion does not play an important role, if any, in activated macrophage-mediated cytotoxic effects on SV40T2.

Rats were exposed to nickel sulfate (NiSO4) either by intratracheal (IT) instillation or by acute aerosol inhalation, and pulmonary clearance of Ni and pulmonary inflammatory responses were studied. The half-time of Ni in the lung (initial lung burden = 50 micrograms Ni/rat) was about 32 h in both the IT instillation and inhalation groups. Ni retention in the lung tissue following IT instillation of NiSO4 was saturable with reference to dose, suggesting that clearance rate of Ni from the rat lung depends on lung burden of Ni. Lung inflammatory responses were evaluated by biochemical, elemental and cytological indicators in bronchoalveolar lavage fluid (BALF) following IT instillation of NiSO4. Activities of lactate dehydrogenase and beta-glucuronidase, contents of lysozyme, protein, sulfur and calcium, and the number of polymorphonuclear leukocytes were increased with a peak at 2-3 days post-instillation, while BALF alkaline phosphatase (ALP) activity was significantly decreased after IT instillation of NiSO4. Lung tissue ALP activity was also decreased by NiSO4. Because Ni does not inhibit ALP directly, the decrease in ALP activity is probably due to functional changes of type II cells (a major source of BALF ALP). Thiobarbituric acid reacting substances in the lung tissue were not changed by NiSO4, suggesting that lipid peroxidation plays a minimal, if any role, in the Ni-induced inflammation in the rat lung.

In a preceding study, we reported that the numbers of macrophages and polymorphonuclear leukocytes (PMN) were increased in bronchoalveolar lavage fluid (BALF) following the intratracheal instillation of nickel sulfate (NiSO4) in rats. In the present study, BALF chemotactic activities for both macrophages and PMN were measured to investigate if the increases of these inflammatory cells in BALF depend on increases in chemotactic activities in epithelial lining fluid (ELF) of the lung. Both the number of PMN and the PMN chemotactic activity peaked at 2 days post-instillation and they were significantly correlated. However, the PMN chemotactic activity was inversely correlated with concentration of leukotriene B4 (LTB4), a well-known chemotaxin. Although PMN were not observed in control BALF, LTB4 concentration in the control ELF (ca. 5×10−7 M) was estimated to have a potential to attract PMN chemotactically through a membrane in in vitro migration assay. These results suggest that the presence of LTB4 in ELF itself does not trigger transpulmonary PMN infiltration. The rat BALF were fractionated by high performance liquid chromatography (HPLC), and PMN chemotactic activity of each fraction was measured. The elution profiles of PMN chemotactic activity showed that there were at least two different chemotaxins in BALF obtained from the NiSO4-exposed rats. Macrophage chemotactic activity in BALF also peaked at 2 days post-instillation. However, the number of macrophages was not significantly correlated with the chemotactic activity for macrophage in BALF. The HPLC study showed that the macrophage chemotactic substance in the BALF obtained from NiSO4-exposed rats was different from complement fragment (C5a) and its MW was estimated to be 10–12 kD.

When 2-chloro[14C]dibenzofuran was intravenously administered to the rats, about 86% of radioactivity was found in the urine, the large intestinal contents and the feces within 24 h. Approximately 3% of 2-chlorodibenzofuran (2CDF) radioactivity was present in the adipose tissue 48 h after an intravenous administration. A similar excretion pattern of the compound was observed in orally administered rats. From experiments in vitro, approximately 80% of 2CDF was present in the red blood cell fraction and the remainder in the plasma fraction. Bile cannulation studies revealed involvement of enterohepatic circulation in the metabolism of the compound. Bile specimens that were subjected to enzymatic hydrolysis by arylsulfatase and/or beta-glucuronidase showed the presence of various yet unidentified conjugated substances. A comparative study using 3-chlorodibenzofuran (3CDF) showed a similar distribution pattern in the 2CDF-treated rats, but 2CDF appeared to accumulate more in the adipose tissue and red blood cells. The present study demonstrates that most of 2CDF is quickly metabolized to give rise to a number of metabolites and excreted from the body, but suggests that the compound once distributed in the adipose tissue may remain there for a relatively long period of time.

The hepatic metabolism of 2-chlorodibenzofuran was investigated in the rat. When 2-chloro[14C]dibenzofuran was intravenously administered to bile duct-cannulated rats, about 90% of radioactivity was excreted in bile and urine within 6 hr, the bile being the preferential route. Another group of rats received oral administration of cold 2-chlorodibenzofuran and bile fluid was collected by surgical bile duct cannulation for qualitative analysis. Three hydroxylated metabolites were isolated by high performance liquid chromatography from the bile fluid after hydrolytic digestion with sulfatase and beta-glucuronidase and were identified by mass and nuclear magnetic resonance spectrometries to be 2-chloro-3,7-dihydroxydibenzofuran, 2-chloro-3-hydroxydibenzofuran and 2-chloro-7-hydroxydibenzofuran, respectively. Analyses of the radioactive bile fluid by thin layer chromatography revealed that there was no detectable amounts of unmetabolized 2-chlorodibenzofuran in the bile fluid and the hydroxylated metabolites were present not as aglycons but as conjugated substances. The results suggest that 2-chlorodibenzofuran is rapidly metabolized in the rat, and the 3 and/or 7 positions play an important role in the metabolism of 2-chlorodibenzofuran.

Pulmonary toxicity of zinc oxide (ZnO) was evaluated by investigating the metabolic fate and inflammatory potency of ZnO instilled into the rat lung. Groups of three rats received single intratracheal instillations of ZnO suspension at a dose of 100 micrograms Zn/rat in the time-course experiment or received 20, 50, 100, 200, 500 and 1000 micrograms Zn/rat and were killed 2 days after treatment in the dose-response experiment. It was suggested that ZnO particles were solubilized in the bronchoalveolar milieu and cleared from the lung with a half-life of 14 h. Metallothionein (MT) was induced with a peak at 2 days. The content of MT was proportional to the dose of ZnO, but contributed little for the accumulation of Zn in the lung. A dose of 20 micrograms Zn/rat was sufficient to develop maximum responses for beta-glucuronidase activity and surfactant content in the bronchoalveolar lavage fluid. Moreover, the activity of lactate dehydrogenase and protein content in the lavage fluid increased significantly at 20 micrograms Zn/rat. These results suggest that the recommended ZnO concentration in the work-place atmosphere of 5 mg/m3 might not be adequate.

Groups of rats received a single intratracheal instillation of saline (control), 1.6 mg crocidolite, and 1.6 mg chrysotile. At 1, 3, 6, 9, and 12 mo after instillation, respiratory functional tests were performed and the lungs were biochemically analyzed. Throughout 12 mo, obstructive small-airway abnormalities with air trapping were observed in the asbestos-treated rats. The degree of airway obstruction and air trapping was higher in the chrysotile-treated group than in the crocidolite-treated group. Lung weight increased significantly in both asbestos-treated groups. Collagen and elastin content of unit lung weight significantly increased in the chrysotile-treated group compared with the control and crocidolite-treated group throughout the experimental period up to 12 mo. However, the collagen and elastin content of unit lung weight in the crocidolite-treated group was not as different from the control group. These results indicate that chrysotile is more fibrogenic than crocidolite, as has been suggested in histopathological studies.