Chemical and toxicological evaluation of transformation products during advanced oxidation processes.

The entry of pharmaceuticals into the water cycle from sewage treatment plants is of growing concern because environmental effects are evident at trace levels. Ozonation, UV- and UV/H(2)O(2)-treatment were tested as an additional step in waste water treatment because they have been proven to be effective in eliminating aqueous organic contaminants. The pharmaceuticals carbamazepine, ciprofloxacin, diclofenac, metoprolol and sulfamethoxazole as well as the personal care products galaxolide and tonalide were investigated in terms of degradation efficiency and by-product formation in consideration of toxic effects. The substances were largely removed from treatment plant effluent by ozonation, UV- and UV/H(2)O(2)-treatment. Transformation products were detected in all tested treatment processes. Accompanying analysis showed no genotoxic, cytotoxic or estrogenic potential for the investigated compounds after oxidative treatment of real waste waters. The results indicate that by-product formation from ozonation and advanced oxidation processes does not have any negative environmental impact.

Toxicity of volatile methylated species of bismuth, arsenic, tin, and mercury in Mammalian cells in vitro.

The biochemical transformation of mercury, tin, arsenic and bismuth through formation of volatile alkylated species performs a fundamental role in determining the environmental processing of these elements. While the toxicity of inorganic forms of most of these compounds are well documented (e.g., arsenic, mercury) and some of them are of relatively low toxicity (e.g., tin, bismuth), the more lipid-soluble organometals can be highly toxic. In the present study we investigated the cyto- and genotoxicity of five volatile metal(loid) compounds: trimethylbismuth, dimethylarsenic iodide, trimethylarsine, tetramethyltin, and dimethylmercury. As far as we know, this is the first study investigating the toxicity of volatile metal(loid) compounds in vitro. Our results showed that dimethylmercury was most toxic to all three used cell lines (CHO-9 cells, CaCo, Hep-G2) followed by dimethylarsenic iodide. Tetramethyltin was the least toxic compound; however, the toxicity was also dependend upon the cell type. Human colon cells (CaCo) were most susceptible to the toxicity of the volatile compounds compared to the other cell lines. We conclude from our study that volatile metal(loid) compounds can be toxic to mammalian cells already at very low concentrations but the toxicity depends upon the metal(loid) species and the exposed cell type.

Cellular uptake, subcellular distribution and toxicity of arsenic compounds in methylating and non-methylating cells.

Arsenic is a known human carcinogen, inducing tumors of the skin, urinary bladder, liver and lung. Inorganic arsenic, existing in highly toxic trivalent and significantly less toxic pentavalent forms, is methylated to mono- and di-methylated species mainly in the liver. Due to the low toxicity of pentavalent methylated species, methylation has been regarded as a detoxification process for many years; however, recent findings of a high toxicity of trivalent methylated species have indicated the contrary. In order to elucidate the role of speciation and methylation for the toxicity and carcinogenicity of arsenic, systematic studies were conducted comparing cellular uptake, subcellular distribution as well as toxic and genotoxic effects of organic and inorganic pentavalent and trivalent arsenic species in both non-methylating (urothelial cells and fibroblasts) and methylating cells (hepatocytes). The membrane permeability was found to be dependent upon both the arsenic species and the cell type. Uptake rates of trivalent methylated species were highest and exceeded those of their pentavalent counterparts by several orders of magnitude. Non-methylating cells (urothelial cells and fibroblasts) seem to accumulate higher amounts of arsenic within the cell than the methylating hepatocytes. Cellular uptake and extrusion seem to be faster in hepatocytes than in urothelial cells. The correlation of uptake with toxicity indicates a significant role of membrane permeability towards toxicity. Furthermore, cytotoxic effects are more distinct in hepatocytes. Differential centrifugation studies revealed that elevated concentrations of arsenic are present in the ribosomal fraction of urothelial cells and in nucleic and mitochondrial fractions of hepatic cells. Further studies are needed to define the implications of the observed enrichment of arsenic in specific cellular organelles for its carcinogenic activity. This review summarizes our recent research on cellular uptake, distribution and toxicity of arsenic compounds in methylating and non-methylating cells.

Methylated bismuth, but not bismuth citrate or bismuth glutathione, induces cyto- and genotoxic effects in human cells in vitro.

Bismuth compounds are widely used in industrial processes and products. In medicine, bismuth salts have been applied in combination with antibiotics for the treatment of Helicobacter pylori infections, for the prevention of diarrhea, and in radioimmunotherapy. In the environment, bismuth ions can be biotransformed to the volatile bismuth compound trimethylbismuth (Me3Bi) by methanobacteria. Preliminary in-house studies have indicated that bismuth ions are methylated in the human colon by intestinal microflora following ingestion of bismuth-containing salts. Information concerning cyto- and genotoxicity of these biomethylated products is limited. In the present study, we investigated the cellular uptake of an organic bismuth compound [monomethylbismuth(III), MeBi(III)] and two other bismuth compounds [bismuth citrate (Bi-Cit) and bismuth glutathione (Bi-GS)] in human hepatocytes, lymphocytes, and erythrocytes using ICP-MS. We also analyzed the cyto- and genotoxic effects of these compounds to investigate their toxic potential. Our results show that the methylbismuth compound was better taken up by the cells than Bi-Cit and Bi-GS. All intracellularly detected bismuth compounds were located in the cytosol of the cells. MeBi(III) was best taken up by erythrocytes (36%), followed by lymphocytes (17%) and hepatocytes (0.04%). Erythrocytes and hepatocytes were more susceptible to MeBi(III) exposure than lymphocytes. Cytotoxic effects of MeBi(III) were detectable in erythrocytes at concentrations >4 microM, in hepatocytes at >130 microM, and in lymphocytes at >430 microM after 24 h of exposure. Cytotoxic effects for Bi-Cit and Bi-GS were much lower or not detectable in the used cell lines up to a tested concentration of 500 microM. Exposure of lymphocytes to MeBi(III) (250 microM for 1 h and 25 microM/50 microM for 24 h) resulted in significantly increased frequencies of chromosomal aberrations (CA) and sister chromatid exchanges (SCE), whereas Bi-Cit and Bi-GS induced neither CA nor SCE. Our study also showed an intracellular production of free radicals caused by MeBi(III) in hepatocytes but not in lymphocytes. These data suggest that biomethylation of bismuth ions by the intestinal microflora of the human colon leads to an increase in the toxicity of the primary bismuth salt.

Vanadium pentoxide-coated ultrafine titanium dioxide particles induce cellular damage and micronucleus formation in V79 cells.

Surface-treated titanium dioxide (TiO(2)) particles coated with vanadium pentoxide (V(2)O(5)) are used industrially for selective catalytic reactions such as the removal of nitrous oxide from exhaust gases of combustion power plants (SCR process) and in biomaterials for increasing the strength of implants. In the present study, untreated ultrafine TiO(2) particles (anatase, diameter: 30-50 nm) and vanadium pentoxide (V(2)O(5))-treated anatase particles were tested for their cyto- and genotoxic effects in V79 cells (hamster lung fibroblasts). Cytotoxic effects of the particles were assessed by trypan blue exclusion, while genotoxic effects were investigated by micronucleus (MN) assay. In addition, the generation of reactive oxygen species (ROS) was determined by the acellular method of electron spin resonance technique (ESR) and by the cellular technique of determination of thiobarbituric acid-reactive substances (TBARS). Our results demonstrate that V(2)O(5)-treated TiO(2) particles induce more potent cyto- and genotoxic effects than untreated particles. Further, acellular and cellular radical formation was more pronounced with V(2)O(5)-anatase than untreated anatase. Thus, data indicate that V(2)O(5)-treated TiO(2) particles were more reactive than natural anatase and capable of inducing DNA damage in mammalian cells through production of free radicals.

The cyto- and genotoxicity of organotin compounds is dependent on the cellular uptake capability.

Organotin compounds have been widely used as stabilizers and anti-fouling agents with the result that they are ubiquitously distributed in the environment. Organotins accumulate in the food chain and potential effects on human health are disquieting. It is not known as yet whether cell surface adsorption or accumulation within the cell, or indeed both is a prerequisite for the toxicity of organotin compounds. In this study, the alkylated tin derivatives monomethyltin trichloride (MMT), dimethyltin dichloride (DMT), trimethyltin chloride (TMT) and tetramethyltin (TetraMT) were investigated for cyto- and genotoxic effects in CHO-9 cells in relation to the cellular uptake. To identify genotoxic effects, induction of micronuclei (MN), chromosome aberrations (CA) and sister chromatid exchanges (SCE) were analyzed and the nuclear division index (NDI) was calculated. The cellular uptake was assessed using ICP-MS analysis. The toxicity of the tin compounds was also evaluated after forced uptake by electroporation. Our results show that uptake of the organotin compounds was generally low but dose-dependent. Only weak genotoxic effects were observed after exposure of cells to DMT and TMT. MMT and TetraMT were negative in the test systems. After forced uptake by electroporation MMT, DMT and TMT induced significant DNA damage at non-cytotoxic concentrations. The results presented here indicate a considerable toxicological potential of some organotin species but demonstrate clearly that the toxicity is modulated by the cellular uptake capability.

Trimethylantimony dichloride causes genotoxic effects in Chinese hamster ovary cells after forced uptake.

In our study, we demonstrate that trimethylantimony dichloride (TMSb) does not induce micronucleus (MN) formation, chromosome aberrations (CA) or sister chromatid exchanges (SCE) under normal conditions in Chinese hamster ovary (CHO-9) cells in vitro up to an applied concentration of 1 mM, nor is it significantly cytotoxic. TMSb is taken up by the cells in a dose-dependent manner, but the percentage uptake of incubation substrate is low (max 0.05%). Intracellular TMSb concentration is two-fold increased after electroporation and under these forced uptake conditions MN formation is also significantly elevated. These data indicate that resistance to TMSb in CHO-9 cells occurs at the uptake and not at the intracellular level.

Forced uptake of trivalent and pentavalent methylated and inorganic arsenic and its cyto-/genotoxicity in fibroblasts and hepatoma cells.

Mammals are able to convert inorganic arsenic to mono-, di-, and trimethylated metabolites. In previous studies we have shown that the trivalent organoarsenic compounds are more toxic than their inorganic counterparts and that the toxicity is associated with the cellular uptake of the arsenicals. In the present study, we investigated cyto-/genotoxic effects of the arsenic compounds arsenate [As(i)(V)], arsenite [As(i)(III)], monomethylarsonic acid [MMA(V)], monomethylarsonous acid [MMA(III)], dimethylarsinic acid [DMA(V)], dimethylarsinous acid [DMA(III)], and trimethylarsine oxide [TMAO(V)] after an extended exposure time (24 h) and compared the uptake capabilities of fibroblasts (CHO-9 cells: Chinese hamster ovary) used for genotoxicity studies, with those of hepatic cells (Hep G2: hepatoma cell-line). To find out whether the arsenic compounds are bound to membranes or if they are present in the cytosol, the amount of arsenic was measured in whole-cell extracts and in membrane-removed cell extracts by inductively coupled plasma-mass spectrometry (ICP-MS). In addition, we forced the cellular uptake of the arsenic compounds into CHO-9 cells by electroporation and measured the intracellular arsenic concentrations before and after this procedure. Our results show that organic and inorganic arsenicals are taken up to a higher degree by fibroblasts compared to hepatoma cells. The arsenic metabolite DMA(III) was the most membrane permeable species in both cell lines and induced strong genotoxic effects in CHO-9 cells after an exposure time of 24 h. The uptake of all other arsenic species was relatively low (<1% by Hep G2 and <4% by CHO cells), but was dose-dependent. Electroporation increased the intracellular arsenic levels as well as the number of induced MN in CHO-9 cells. With the exception of As(i)(III) and DMA(III) in CHO-9 cells, the tested arsenic compounds were not bound to cell membranes, but were present in the cytosol. This may indicate the existence of DMA(III)-specific exporter proteins as are known for As(i)(III). Our results indicate that the uptake capabilities of arsenic compounds are highly dependent upon the cell type. It may be hypothesized that the arsenic-induced genotoxic effects observed in fibroblasts are due to the high uptake of arsenicals into this cell type. This may explain the high susceptibility of skin fibroblasts to arsenic exposure.

Uptake of inorganic and organic derivatives of arsenic associated with induced cytotoxic and genotoxic effects in Chinese hamster ovary (CHO) cells.

Humans are exposed to arsenic and their organic derivatives, which are widely distributed in the environment, via food, water, and to a lesser extent, via air. Following uptake, inorganic arsenic undergoes biotransformation to mono- and dimethylated metabolites. Recent findings suggest that the methylation reactions represent a toxification rather than a detoxification pathway. In the present study, the genotoxic effects and the cellular uptake of inorganic arsenic [arsenate, As(i)(V); arsenite, As(i)(III)] and the methylated arsenic species monomethylarsonic acid [MMA(V)], monomethylarsonous acid [MMA(III)], dimethylarsinic acid [DMA(V)], dimethylarsinous acid [DMA(III)], trimethylarsenic oxide [TMAO(V)] were investigated in Chinese hamster ovary (CHO-9) cells. The chemicals were applied at different concentrations (0.1 microM to 10 mM) for 30 min and 1 h, respectively. Cytotoxic effects were investigated by the trypan blue extrusion test and genotoxic effects by the assessment of micronucleus (MN) induction, chromosome aberrations (CA), and sister chromatid exchanges (SCE). Intracellular arsenic concentrations were determined by ICP-MS techniques. Our results show that MMA(III) and DMA(III) induce cytotoxic and genotoxic effects to a greater extent than MMA(V) or DMA(V). Viability was significantly decreased after incubation (1 h) of the cells with > or = 1 microM As(i)(III), > or = 1 microM As(i)(V), > or = 500 microM MMA(III), > or = 100 microM MMA(V), and 500 microM DMA(V) and > or = 0.1 microM DMA(III). TMAO(V) was not cytotoxic at concentrations up to 10 mM. A significant increase of the number of MN, CA and SCE was found for DMA(III) and MMA(III). As(i)(III + V) induced CA and SCE but no MN. TMAO(V), MMA(V) and DMA(V) were not genotoxic in the concentration range tested (up to 5 mM). The nuclear division index (NDI) was not affected by any of the tested arsenic compounds after a recovery period of 14 to 35 h. When the uptake of the chemicals was measured by ICP-MS analysis, it was found that only 0.03% MMA(V) and DMA(V), and 2% MMA(III), As(i)(III) and (V) were taken up by the cells. In comparison, 10% of the DMA(III) dose was taken up. The total intracellular concentration of all arsenic compounds increased with increasing arsenic concentrations in the culture medium. Taken together, these data demonstrate that arsenic compounds in the trivalent oxidation state exhibit the strongest genotoxic effects. Trivalent organoarsenic compounds are more membrane permeable than the pentavalent species. The potency of the DNA damage decreases in the order DMA(III) > MMA(III) > As(i)(III and V) > MMA(V) > DMA(V) > TMAO(V). We postulate that the induction of genotoxic effects caused by the methylated arsenic species is primarily dependent upon their ability to penetrate cell membranes.

Environmental distribution, analysis, and toxicity of organometal(loid) compounds.

The biochemical modification of the metals and metalloids mercury, tin, arsenic, antimony, bismuth, selenium, and tellurium via formation of volatile metal hydrides and alkylated species (volatile and involatile) performs a fundamental role in determining the environmental processing of these elements. In most instances, the formation of such species increases the environmental mobility of the element, and can result in bioaccumulation in lipophilic environments. While inorganic forms of most of these compounds are well characterized (e.g., arsenic, mercury) and some of them exhibit low toxicity (e.g., tin, bismuth), the more lipid-soluble organometals can be highly toxic. Methylmercury poisoning (e.g., Minamata disease) and tumor development in rats after exposure to dimethylarsinic acid or tributyltin oxide are just some examples. Data on the genotoxicity (and the neurotoxicity) as well as the mechanisms of cellular action of organometal(loid) compounds are, however, scarce. Many studies have shown that the production of such organometal(loid) species is possible and likely whenever anaerobic conditions (at least on a microscale) are combined with available metal(loid)s and methyl donors in the presence of suitable organisms. Such anaerobic conditions can exist within natural environments (e.g., wetlands, pond sediments) as well as within anthropogenic environmental systems (e.g., waste disposal sites and sewage treatments plants). Some methylation can also take place under aerobic conditions. This article gives an overview about the environmental distribution of organometal(loid) compounds and the potential hazardous effects on animal and human health. Genotoxic effects in vivo and in vitro in particular are discussed.

Effects of asbestos on initiation of DNA damage, induction of DNA-strand breaks, P53-expression and apoptosis in primary, SV40-transformed and malignant human mesothelial cells.

Human mesothelial cells (HMC), the progenitor cells of asbestos-induced mesothelioma, are particularly sensitive to the genotoxic effects of asbestos, although the molecular mechanisms by which asbestos induces injury in HMC are not well known. The high susceptibility of HMC to simian virus 40 (SV40)-mediated transformation is assumed to play a causative role in the pathogenesis of mesothelioma. The aim of this study was to investigate the asbestos-induced DNA damage in cultured HMC and SV40-transformed HMC (MeT-5A) compared with their malignant counterparts, i.e. human mesothelioma cells (MSTO). The time-dependent initiation of DNA-strand breaks as well as the induction of oxidative DNA base modifications were key factors for investigation. HMC, MeT-5A and MSTO cells were exposed to chrysotile and crocidolite asbestos (3 microg/cm2) during different time periods (1-72 h). DNA damage was investigated by use of the Comet assay and alkaline unwinding, the latter in combination with the Fpg protein. The P53 level was analyzed by immunofluorescence, and measurement of apoptosis was conducted by flow cytometry. We found a significant induction of DNA damage in asbestos-treated HMC already after an exposure time of 1.5 h. This effect could not be observed in treated MeT-5A and MSTO cells. Also, a time-dependent significant increase in DNA-strand breaks was observed by alkaline unwinding in asbestos-treated HMC, but not in treated MeT-5A and MSTO cells. In none of the three cell lines we could detect oxidative DNA damage recognized by the Fpg protein (e.g. 8-oxo-guanine), up to 24 h after exposure to asbestos. In contrast to what was found in HMC, P53 was over-expressed in untreated MeT-5A and MSTO. The induction of apoptosis by asbestos fibers was suppressed in MeT-5A and MSTO cells. Crocidolite fibers induced the higher genotoxic effects and chrysotile the more pronounced apoptotic effects. We conclude that asbestos induces DNA damage in HMC already after a very short exposure time in the absence of 8-oxo-guanine formation. The presence of SV40-Tag in MeT-5A and MSTO cells results in an increased expression of P53, but not in additive genotoxic effects after exposure to asbestos. The deregulation of the apoptotic pathway may lead to proliferation of genomically damaged cells and finally to the development of mesothelioma.

Blood-brain barrier permeability and monocyte infiltration in experimental allergic encephalomyelitis: a quantitative MRI study.

Enhanced cerebrovascular permeability and cellular infiltration mark the onset of early multiple sclerosis lesions. So far, the precise sequence of these events and their role in lesion formation and disease progression remain unknown. Here we provide quantitative evidence that blood-brain barrier leakage is an early event and precedes massive cellular infiltration in the development of acute experimental allergic encephalomyelitis (EAE), the animal correlate of multiple sclerosis. Cerebrovascular leakage and monocytes infiltrates were separately monitored by quantitative in vivo MRI during the course of the disease. Magnetic resonance enhancement of the contrast agent gadolinium diethylenetriaminepentaacetate (Gd-DTPA), reflecting vascular leakage, occurred concomitantly with the onset of neurological signs and was already at a maximal level at this stage of the disease. Immunohistochemical analysis also confirmed the presence of the serum-derived proteins such as fibrinogen around the brain vessels early in the disease, whereas no cellular infiltrates could be detected. MRI further demonstrated that Gd-DTPA leakage clearly preceded monocyte infiltration as imaged by the contrast agent based on ultra small particles of iron oxide (USPIO), which was maximal only during full-blown EAE. Ultrastructural and immunohistochemical investigation revealed that USPIOs were present in newly infiltrated macrophages within the inflammatory lesions. To validate the use of USPIOs as a non-invasive tool to evaluate therapeutic strategies, EAE animals were treated with the immunomodulator 3-hydroxy-3-methylglutaryl Coenzyme A reductase inhibitor, lovastatin, which ameliorated clinical scores. MRI showed that the USPIO load in the brain was significantly diminished in lovastatin-treated animals. Data indicate that cerebrovascular leakage and monocytic trafficking into the brain are two distinct processes in the development of inflammatory lesions during multiple sclerosis, which can be monitored on-line with MRI using USPIOs and Gd-DTPA as contrast agents. These studies also implicate that USPIOs are a valuable tool to visualize monocyte infiltration in vivo and quantitatively assess the efficacy of new therapeutics like lovastatin.

Smoking enhances asbestos-induced genotoxicity, relative involvement of chromosome 1: a study using multicolor FISH with tandem labeling.

Several experimental and epidermological studies have indicated augmentation of asbestos induced diseases by cigarette smoke by the mechanisms, which are still unknown. To determine whether smoking affects genetic system of the cells and further modifies asbestos induced genotoxicity, whole blood from non-smokers and smokers was exposed to asbestos fibres separately in vitro and micronucleus test was performed. The number of micronuclei was found to be significantly higher (P<0 05) in cases of smoker's lymphocytes, asbestos exposed non-smokers lymphocytes as well as asbestos exposed smokers lymphocytes, as compared with unexposed non-smokers lymphocytes. Further we investigated involvement of chromosome 1 in the damaging process using multicolor FISH technique. FISH is fast and reliable method, distinguishing both structural and numerical alterations. The centric/pericentric regions of chromosome 1 (cen-q12) were labeled, as the pericentric heterochromatin region 1 (q12) is quite large, highly repetitive and prone to breakage. Multicolor FISH assay suggested that the genetic damage by asbestos fibres mainly involve chromosome 1 but in case of cigarette smoking the damage is not strictly connected to chromosome 1 only, but also involves damage to other chromosomes. Further the study suggested that smoking makes genetic system of the cells more vulnerable to the deleterious effects of asbestos.

Meningeal and perivascular macrophages of the central nervous system play a protective role during bacterial meningitis.

Meningeal (MM) and perivascular macrophages (PVM) constitute major populations of resident macrophages in the CNS that can be distinguished from microglial cells. So far, there is no direct evidence that demonstrates a possible role of MM and PVM in the CNS during normal or pathologic conditions. To elucidate the role of the MM and PVM during CNS inflammation, we have developed a strategy using a single intraventricular injection of mannosylated clodronate liposomes, which results in a complete and selective depletion of the PVM and MM from the CNS. Depletion of the MM and PVM during experimental pneumococcal meningitis resulted in increased illness, which correlated with higher bacteria counts in the cerebrospinal fluid and blood. This was associated with a decreased influx of leukocytes into the cerebrospinal fluid, which occurred despite an elevated production of relevant chemokines (e.g., macrophage-inflammatory protein-2) and a higher expression of vascular adhesion molecules (e.g., VCAM-1). In contrast, the higher bacterial counts correlated with elevated production of local and systemic inflammatory mediators (e.g., IL-6) indicating enhanced local leukocyte and systemic immune activation, and this may explain the worsening of the clinical signs. These findings show that the PVM and MM play a protective role during bacterial meningitis and suggest that a primary action of these macrophages is to facilitate the influx of leukocytes at the blood-brain barrier. More in general, we demonstrate for the first time that the PVM and MM play a crucial role during inflammation in the CNS.

Mutational analysis of N-ras, p53, p16INK4a, p14ARF and CDK4 genes in primary human malignant mesotheliomas.

Nineteen specimens from primary human malignant mesotheliomas obtained from 19 patients were screened for activating point mutations in the oncogenes N-ras and CDK4 by combined RFLP-PCR/SSCP analysis. In addition, all tumours were screened for deletions and point mutations in the tumour suppressor genes p53, p16INK4a (CDKN2A) and p14ARF (exon-1beta) by combined multiplex-PCR/SSCP analysis. No mutations were found in N-ras, p53 and CDK4. Three tumours displayed homozygous deletion (co-deletion of exons 1, 2 and 3) of p16INK4a. One of them displayed additional homozygous deletion of p14ARF (exon-1beta). Two silent point mutations and 2 polymorphisms were found in p16INK4a in 3 tumours. Our preliminary data indicate that disarrangement of the Rb1 pathway may be involved in mesothelioma formation.

Rat macrophages: membrane glycoproteins in differentiation and function.

Macrophages (mphi) play a crucial role in the immune system. The rat offers unique advantages for studying the biology of mphi. Firstly, monoclonal antibodies (mAb) against many rat mphi surface glycoproteins have become available. These have not only demonstrated a considerable heterogeneity among mphi, but have also allowed the characterization of various mphi surface molecules in terms of structure, expression regulation and function. Furthermore, substantial numbers of rat mphi can be isolated from various sites (e.g. blood, peritoneal cavity, lung and other tissues), enabling proper molecular and functional analysis of these mphi populations. Finally, a number of (unique) experimental models for human diseases have been developed in the rat, making possible the evaluation of the involvement of mphi in pathogenesis. For this, a method for the selective elimination of mphi from various tissues in vivo has proven very useful. Here, we will review the contribution that the rat has made to understanding the immunobiology of mphi. In particular, we will discuss the surface (glyco)proteins on rat mphi in differentiation and function, and the involvement of mphi in rat models of disease.

Kerosene soot genotoxicity: enhanced effect upon co-exposure with chrysotile asbestos in Syrian hamster embryo fibroblasts.

Epidemiological and experimental studies have suggested an enhancement of asbestos-induced bronchogenic carcinoma by cigarette smoke. Further, our recent experimental and epidemiological studies have indicated that besides smoking, several other compounds including kerosene soot may accelerate disease processes in asbestos-exposed animals as well as in the humans. Incomplete combustion of kerosene oil generates large volumes of soot, which contains various polycyclic aromatic hydrocarbons and aliphatic compounds. As reported earlier, exposure to kerosene soot is known to cause biochemical and pathological changes in the pulmonary tissue, which may cause cardiopulmonary disorders. In this study we investigated genotoxic effects caused by kerosene soot and chrysotile asbestos as well as co-exposure of kerosene soot and chrysotile using Syrian hamster embryo fibroblasts (SHE). The micronucleus assay revealed a significant increase of induced micronuclei (MN), (P

Dielectric changes in membrane properties and cell interiors of human mesothelial cells in vitro after crocidolite asbestos exposure.

Asbestos induces cytogenetic and genotoxic effects in cultured cell lines in vitro. For further investigations of the fiber-induced cellular changes, electrorotation (ROT) measurements can be used to determine early changes of surface properties and dielectric cellular changes. In the present study, human mesothelial cells (HMC) were exposed to nontoxic concentrations of crocidolite asbestos (1 microg/cm(2)) for 12, 24, 30, 50, and 72 hr, and were investigated for changes in dielectric properties, morphologic and biochemical changes using ROT measurements, electron microscopy, and flow cytometry, respectively. The results of ROT measurements revealed slightly increased internal conductivity and decreased membrane conductance of HMC during the first 12 hr of exposure to crocidolite. This may be due to functional changes of ion channels of the cellular membrane. However, after exposures of >= 30 hr, reduced internal conductivity and increased membrane conductance of HMC occurred. These effects may be caused by permeabilization of the cell membrane and the leakage of ions into the surrounding medium. The membrane capacitance of HMC is always decreased during exposure of cells to crocidolite fibers. This decreased membrane capacitance may result from the observed reduction in the number of microvilli and from the shrinkage of cells as observed by electron microscopy and flow cytometry. Changes in composition of the plasma membrane were also observed after the labeling of phosphatidylserines (PS) on the cell surface. These observed changes can be related to apoptotic events. Whereas during the first 50 hr of exposure only a small number of HMC with increased exposure of PS on the cell surface was detected by flow cytometry, the dielectric properties of HMC showed marked changes during this time. Our results show that surface property changes of the cellular membrane of HMC as well as interior dielectric changes occur after the exposure of cells to crocidolite fibers. The observed changes are discussed in terms of complex combined cellular effects after amphibole asbestos exposure.

Occupational and Environmental Factors Enhancing the Genotoxicity of Asbestos.

Epidemiological and experimental studies have suggested the enhancement of asbestos-induced disease processes by simultaneous exposure to kerosene, its soot, and cigarette smoke in asbestos-exposed animals as well as in humans. To determine the influence of these factors on the genotoxic potential of asbestos, a micronucleus test was performed in Syrian hamster embryo fibroblasts (SHE) and human lymphocytes. To observe the specific chromosomal damages, multicolor fluorescence in situ hybridization (FISH) was done in the lymphocytes from smokers and nonsmokers exposed in vitro to asbestos. Significantly higher numbers of micronuclei were observed in SHE cells after combined treatment with chrysotile and kerosene soot (111 micronuclei/1000 cells) in comparison to chrysotile and kerosene soot separately. Kinetochore staining revealed mainly clastogenic effects in all the cases. In human lymphocytes exposed in cultures to chrysotile and crocidolite the numbers of micronuclei were found higher in smokers than nonsmokers. Multicolor FISH assay suggested that asbestos fibers inflict high damage within 1q12 and in the region between 1cen and 1q12 of chromosome 1. In the exposed population of an asbestos cement factory, the highest genetic damage was found in the blood lymphocytes of exposed smokers. The study suggests that smokers occupationally exposed to asbestos and domestically to kerosene soot are at higher risk for the early development of asbestos-induced diseases.

Regulation of CD 163 on human macrophages: cross-linking of CD163 induces signaling and activation.

CD163 is a member of the group B scavenger receptor cysteine-rich (SRCR) superfamily. This study describes aspects of the tissue distribution, the regulation of expression, and signal transduction after cross-linking of this receptor at the cell surface of macrophages. CD163 showed an exclusive expression on resident macrophages (e.g., red pulp macrophages, alveolar macrophages). The expression was inducible on monocyte-derived macrophages by glucocorticoids but not by interleukin-4 (IL-4), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interferon-gamma. The combination of IL-4 or GM-CSF with glucocorticoids resulted in a further increase. Subcellular analysis of alveolar macrophages by immunoelectron microscopy showed a plasma membrane localization of the antigen. Cross-linking of CD163 with monoclonal antibody induced a protein tyrosine kinase-dependent signal that resulted in (1) slow-type calcium mobilization, (2) inositol triphosphate production, and (3) secretion of IL-6 and GM-CSF. The data suggest a function for the SRCR-superfamily receptor CD163 in the regulation of inflammatory processes by macrophages.