Multi-walled carbon nanotubes induce stronger migration of inflammatory cells in vitro than asbestos or granular particles but a similar pattern of inflammatory mediators.

Biopersistent pro-inflammatory fibers are suspected human carcinogens. Cytotoxicity and transcription of pro- and anti-inflammatory mediators of different fibers were investigated in functional relationship to chemotaxis in vitro as a model for fiber-induced inflammation of the lung. We challenged NR8383 rat macrophages with multi-walled carbon nanotubes (MWCNT) and various asbestos fibers. The resulting cell supernatants were than studied using the Particle-induced Cell Migration Assay (PICMA) and cytotoxicity was determined using the LDH test. Expression of inflammatory mediators was analyzed with qPCR and verified by ELISA. Chrysotile A and the rigid, needle-shaped NM-401 caused the strongest cytotoxic effects and the largest number of migrated cells. In contrast, the MWCNT NM-400, NM-402, and NM403 were apparently non-cytotoxic but induced pronounced cell migration showing a very steep dose response. However, the strength of cell migration and cytotoxicity of the asbestos fibers were correlated. The expression profile of inflammatory mediators was comparable, although cytotoxicity of the MWCNT NM-401 and NM-403 differed strongly. Induction of the corresponding proteins was confirmed for CCL2, CCL3, CXCL1, CXCL3, IL1RA (IL1RN), CSF1, GDF15 and TNFa. Chrysotile A and NM-401 induced much stronger chemotaxis than the non-fibrous particles reported in our previous study. Cytotoxic and chemotactic effects correspond to the induction of inflammatory mediators.

Barium sulfate micro- and nanoparticles as bioinert reference material in particle toxicology.

The inhalation of particles and their exposure to the bronchi and alveoli constitute a major public health risk. Chemical as well as particle-related properties are important factors for the biological response but are difficult to separate from each other. Barium sulfate is a completely inert chemical compound, therefore it is ideally suited to separate these two factors. The biological response of rat alveolar macrophages (NR8383) was analyzed after exposure to barium sulfate particles with three different diameters (40 nm, 270 nm, and 1.3 µm, respectively) for 24 h in vitro (particle concentrations from 12.5 to 200 µg mL-1). The particles were colloidally stabilized as well as fluorescently-labeled by carboxymethylcellulose, conjugated with 6-aminofluorescein. All kinds of barium sulfate particles were efficiently taken up by NR8383 cells and found inside endo-lysosomes, but never in the cell nucleus. Neither an inflammatory nor a cytotoxic response was detected by the ability of dHL-60 and NR8383 cells to migrate towards a chemotactic gradient (conditioned media of NR8383 cells) and by the release of inflammatory mediators (CCL2, TNF-α, IL-6). The particles neither caused apoptosis (up to 200 µg mL-1) nor necrosis (up to 100 µg mL-1). As only adverse reaction, necrosis was found at a concentration of 200 µg mL-1 of the largest barium sulfate particles (1.3 µm). Barium sulfate particles are ideally suited as bioinert control to study size-dependent effects such as uptake mechanisms of intracellular distributions of pure particles, especially in nanotoxicology.

A variant of the CXCL11 gene may influence susceptibility to contact allergy, particularly in polysensitized patients.

BACKGROUND: Hereditary factors may influence individual susceptibility to contact allergy. OBJECTIVES: To investigate genetic variants with impacts on early inflammatory reactions and T cell functions that possibly increase the risk of contact allergy. PATIENTS AND METHODS: Three hundred and seventy two patients undergoing patch testing were recruited from the Information Network of Departments of Dermatology (IVDK). Of these, 133 were monosensitized and 239 were polysensitized, defined as reacting to three or more unrelated sensitizers. Within the polysensitized individuals, a subgroup with at least one particularly strong patch test reaction (strong reactors; n = 194) was considered. Three hundred and forty-seven blood bank donors served as controls. Fifteen genetic variants in 13 genes were analysed. RESULTS: The homozygous variant CXCL11 AA genotype (rs6817952) was significantly more frequent among polysensitized patients (10 of 239 = 4.2%; p = 0.0048; odds ratio 7.49; 95%CI: 1.7-36.1) than among monosensitized patients (2.2%) and in the control group (0.6%). None of the remaining genetic variants investigated were characterized by similarly strong associations. However, the significance was lost after correction for multiple comparisons. CONCLUSIONS: The homozygous variant CXCL11 genotype is associated with an increased risk of contact allergy. To confirm this exploratory finding, further independent studies are needed.

Combusting vegetable oils in diesel engines: the impact of unsaturated fatty acids on particle emissions and mutagenic effects of the exhaust.

High particle emissions and strong mutagenic effects were observed after combustion of vegetable oil in diesel engines. This study tested the hypothesis that these results are affected by the amount of unsaturated or polyunsaturated fatty acids of vegetable oils. Four different vegetable oils (coconut oil, CO; linseed oil, LO; palm tree oil, PO; and rapeseed oil, RO) and common diesel fuel (DF) were combusted in a heavy-duty diesel engine. The exhausts were investigated for particle emissions and mutagenic effects in direct comparison with emissions of DF. The engine was operated using the European Stationary Cycle. Particle masses were measured gravimetrically while mutagenicity was determined using the bacterial reverse mutation assay with tester strains TA98 and TA100. Combustion of LO caused the largest amount of total particulate matter (TPM). In comparison with DF, it particularly raised the soluble organic fraction (SOF). RO presented second highest TPM and SOF, followed by CO and PO, which were scarcely above DF. RO revealed the highest number of mutations of the vegetable oils closely followed by LO. PO was less mutagenic, but still induced stronger effects than DF. While TPM and SOF were strongly correlated with the content of polyunsaturated fatty acids in the vegetable oils, mutagenicity had a significant correlation with the amount of total unsaturated fatty acids. This study supports the hypothesis that numbers of double bounds in unsaturated fatty acids of vegetable oils combusted in diesel engines influence the amount of emitted particles and the mutagenicity of the exhaust. Further investigations have to elucidate the causal relationship.

Particle-induced cell migration assay (PICMA): A new in vitro assay for inflammatory particle effects based on permanent cell lines.

Inflammation is a decisive pathophysiologic mechanism of particle toxicity and accumulation of neutrophils in the lung is believed to be a crucial step in this process. This study describes an in vitro model for investigations of the chemotactic attraction of neutrophils in response to particles using permanent cell lines. We challenged NR8383 rat macrophages with particles that were characterized concerning chemical nature, crystallinity, and size distribution in the dry state and in the culture medium. The cell supernatants were used to investigate migration of differentiated human leukemia cells (dHL-60 cells). The dose range for the tests was determined using an impedance-based Real-Time Cell Analyzer. The challenge of NR8383 cells with 32-96 µg cm(-2) coarse and nanosized particles resulted in cell supernatants which induced strong and dose-dependent migration of dHL-60 cells. Quartz caused the strongest effects - exceeding the positive control "fetal calf serum" (FCS) several-fold, followed by silica, rutile, carbon black, and anatase. BaSO4 served as inert control and induced no cell migration. Particles caused NR8383 cells to secrete chemotactic compounds. The assay clearly distinguished between the particles of different inflammatory potential in a highly reproducible way. Specificity of the test is suggested by negative results with BaSO4.

Amylenes do not lead to bacterial mutagenicity in contrast to structurally related epoxides.

Amylenes are unsaturated hydrocarbons (C5H10), such as 1-pentene, 2-pentene, 2-methyl-but-1-en (3-methyl-1-butene), 2-methyl-but-2-en (isopentene), and 3-methyl-but-1-en. We investigated bacterial mutagenicity of 1-pentene, 2-pentene, and 3-methyl-but-1-en in the Ames test. 2-Pentene was investigated as racemate and as pure diastereomers. We included the methyltransferase deficient Salmonella Typhimurium strain YG7108 and the application of a gas-tight preincubation to reduce the risk of false negative results. 1,2-Epoxypentane which may arise from 1-pentene was used as positive control. None of the investigated amylenes showed mutagenic effects, whereas 1,2-epoxypentane was mutagenic exceeding 100 µ g per plate. An exceptional high reverse mutation in the negative control plates in the experiments with 1,2-epoxypentane was obviously caused by evaporation into the incubator which was shown by placing the control plates in a separate apparatus. No differences were seen upon use of YG7108 and its parent strain TA1535. In conclusion, 1,2-epoxypentane is most probably not a substrate of the deleted bacterial methyltransferases. The comparison of the bacterial mutagenicity of the investigated amylenes and 1,2-epoxipentane suggests that epoxidation of amylenes in the S9-mix does not proceed effectively or is counterbalanced by detoxifying reactions. The assessment of mutagenic effects of short chained aliphatic epoxides can be underestimated due to the evaporation of these compounds.

Combustion of hydrotreated vegetable oil and jatropha methyl ester in a heavy duty engine: emissions and bacterial mutagenicity.

Research on renewable fuels has to assess possible adverse health and ecological risks as well as conflicts with global food supply. This investigation compares the two newly developed biogenic diesel fuels hydrotreated vegetable oil (HVO) and jatropha methyl ester (JME) with fossil diesel fuel (DF) and rapeseed methyl ester (RME) for their emissions and bacterial mutagenic effects. Samples of exhaust constituents were compared after combustion in a Euro III heavy duty diesel engine. Regulated emissions were analyzed as well as particle size and number distributions, carbonyls, polycyclic aromatic hydrocarbons (PAHs), and bacterial mutagenicity of the exhausts. Combustion of RME and JME resulted in lower particulate matter (PM) compared to DF and HVO. Particle numbers were about 1 order of magnitude lower for RME and JME. However, nitrogen oxides (NOX) of RME and JME exceeded the Euro III limit value of 5.0 g/kWh, while HVO combustion produced the smallest amount of NOX. RME produced the lowest emissions of hydrocarbons (HC) and carbon monoxide (CO) followed by JME. Formaldehyde, acetaldehyde, acrolein, and several other carbonyls were found in the emissions of all investigated fuels. PAH emissions and mutagenicity of the exhausts were generally low, with HVO revealing the smallest number of mutations and lowest PAH emissions. Each fuel showed certain advantages or disadvantages. As proven before, both biodiesel fuels produced increased NOX emissions compared to DF. HVO showed significant toxicological advantages over all other fuels. Since jatropha oil is nonedible and grows in arid regions, JME may help to avoid conflicts with the food supply worldwide. Hydrogenated jatropha oil should now be investigated if it combines the benefits of both new fuels.

Potential hazards associated with combustion of bio-derived versus petroleum-derived diesel fuel.

Fuels from renewable resources have gained worldwide interest due to limited fossil oil sources and the possible reduction of atmospheric greenhouse gas. One of these fuels is so called biodiesel produced from vegetable oil by transesterification into fatty acid methyl esters (FAME). To get a first insight into changes of health hazards from diesel engine emissions (DEE) by use of biodiesel scientific studies were reviewed which compared the combustion of FAME with common diesel fuel (DF) for legally regulated and non-regulated emissions as well as for toxic effects. A total number of 62 publications on chemical analyses of DEE and 18 toxicological in vitro studies were identified meeting the criteria. In addition, a very small number of human studies and animal experiments were available. In most studies, combustion of biodiesel reduces legally regulated emissions of carbon monoxide, hydrocarbons, and particulate matter. Nitrogen oxides are regularly increased. Among the non-regulated emissions aldehydes are increased, while polycyclic aromatic hydrocarbons are lowered. Most biological in vitro assays show a stronger cytotoxicity of biodiesel exhaust and the animal experiments reveal stronger irritant effects. Both findings are possibly caused by the higher content of nitrogen oxides and aldehydes in biodiesel exhaust. The lower content of PAH is reflected by a weaker mutagenicity compared to DF exhaust. However, recent studies show a very low mutagenicity of DF exhaust as well, probably caused by elimination of sulfur in present DF qualities and the use of new technology diesel engines. Combustion of vegetable oil (VO) in common diesel engines causes a strongly enhanced mutagenicity of the exhaust despite nearly unchanged regulated emissions. The newly developed fuel "hydrotreated vegetable oil" (HVO) seems to be promising. HVO has physical and chemical advantages compared to FAME. Preliminary results show lower regulated and non-regulated emissions and a decreased mutagenicity.

Mutagenicity of diesel engine exhaust is eliminated in the gas phase by an oxidation catalyst but only slightly reduced in the particle phase.

Concerns about adverse health effects of diesel engine emissions prompted strong efforts to minimize this hazard, including exhaust treatment by diesel oxidation catalysts (DOC). The effectiveness of such measures is usually assessed by the analysis of the legally regulated exhaust components. In recent years additional analytical and toxicological tests were included in the test panel with the aim to fill possible analytical gaps, for example, mutagenic potency of polycyclic aromatic hydrocarbons (PAH) and their nitrated derivatives (nPAH). This investigation focuses on the effect of a DOC on health hazards from combustion of four different fuels: rapeseed methyl ester (RME), common mineral diesel fuel (DF), SHELL V-Power Diesel (V-Power), and ARAL Ultimate Diesel containing 5% RME (B5ULT). We applied the European Stationary Cycle (ESC) to a 6.4 L turbo-charged heavy load engine fulfilling the EURO III standard. The engine was operated with and without DOC. Besides regulated emissions we measured particle size and number distributions, determined the soluble and solid fractions of the particles and characterized the bacterial mutagenicity in the gas phase and the particles of the exhaust. The effectiveness of the DOC differed strongly in regard to the different exhaust constituents: Total hydrocarbons were reduced up to 90% and carbon monoxide up to 98%, whereas nitrogen oxides (NO(X)) remained almost unaffected. Total particle mass (TPM) was reduced by 50% with DOC in common petrol diesel fuel and by 30% in the other fuels. This effect was mainly due to a reduction of the soluble organic particle fraction. The DOC caused an increase of the water-soluble fraction in the exhaust of RME, V-Power, and B5ULT, as well as a pronounced increase of nitrate in all exhausts. A high proportion of ultrafine particles (10-30 nm) in RME exhaust could be ascribed to vaporizable particles. Mutagenicity of the exhaust was low compared to previous investigations. The DOC reduced mutagenic effects most effectively in the gas phase. Mutagenicity of particle extracts was less efficiently diminished. No significant differences of mutagenic effects were observed among the tested fuels. In conclusion, the benefits of the DOC concern regulated emissions except NO(X) as well as nonregulated emissions such as the mutagenicity of the exhaust. The reduction of mutagenicity was particularly observed in the condensates of the gas phase. This is probably due to better accessibility of gaseous mutagenic compounds during the passage of the DOC in contrast to the particle-bound mutagens. Concerning the particulate emissions DOC especially decreased ultrafine particles.

Variations of the melanocortin-1 receptor and the glutathione-S transferase T1 and M1 genes in cutaneous malignant melanoma.

Variations in the melanocortin-1 receptor (MC1R) and in the glutathione-S transferase genes mu1 (GSTM1) and theta 1 (GSTT1) have been reported to influence UV sensitivity and melanoma risk. MC1R is one of the major genes that determine skin pigmentation because the melanocortin-1 receptor regulates eumelanin synthesis. GSTT1 and GSTM1 are enzymes expressed in the skin that detoxify products of oxidative stress reactions caused by UV irradiation. In this study variations in the MC1R, GSTM1 and T1 genes were analyzed in 347 healthy subjects and 322 patients with cutaneous malignant melanoma by direct cycle sequencing, RFLP and multiplex PCR. Important phenotypic characteristics of the study participants were obtained to assess whether genetic associations occurred independently of phenotypic risk factors for melanoma. We found an association of the MC1R D84E and R151C polymorphisms with melanoma (odds ratios for carriage of the rare allele 4.96, 95% CI [1.06-23.13], P = 0.032, and 1.69, 95% CI [1.12-2.55], P = 0.013, respectively). Melanoma risk increased with the number of variant MC1R alleles carried by an individual (P = 0.003). In a multivariate model, however, only the D84E polymorphism influenced melanoma risk independently of the risk factors fair skin type, high nevus count and high age (P = 0.047). There was no effect of homozygous GST M1 or T1 deletions on melanoma risk. In contrast to previous data, there was no evidence that GSTM1 deficiency influences melanoma risk in the subgroup of individuals with red or blond hair.

Mutagenicity of cytochrome P450 2E1 substrates in the Ames test with the metabolic competent S. typhimurium strain YG7108pin3ERb5.

UNLABELLED: Poor metabolic competence of in vitro systems was proposed to be one of their major shortcomings accounting for false negative results in genotoxicity testing. For several "low molecular weight cancer suspects" this was specifically attributed to the lack of cytochrome P450 2E1 (CYP2E1) in conventional in vitro metabolising systems. One promising attempt to overcome this problem is the transfection of "methyltransferase-deficient"S. typhimurium strains with the plasmid pin3ERb5. This plasmid contains DNA encoding for a complete electron transport chain, comprising P450 reductase, cytochrome b5 and cytochrome P450 2E1. In order to answer the question if CYP2E1 substrates that yield negative or inconclusive results in the Ames test can be activated by metabolic competent bacterial strains, we used YG7108pin3ERb5 to investigate the following compounds: acetamide, acrylamide, acrylonitrile, allyl chloride, ethyl acrylate, ethyl carbamate, methyl-methacrylate, vinyl acetate, N-nitrosopyrrolidine, trichloroethylene and tetrachloroethylene. N-Nitrosodiethylamine served as a positive control. In addition to these known or proposed CYP2E1 substrates, we investigated the polycyclic aromatic hydrocarbon benzo[alpha]pyrene and the heterocyclic aromatic amines 2-aminofluorene and 2-aminoanthracene. RESULTS: The extensive metabolic competence of the transformed strain is underlined by results showing strong mutagenicity between 10 and 500 micro g N-nitrosopyrrolidine per plate. Unexpectedly, 2-aminoanthracene was mutagenic at a concentration range between 25 and 250 micro g per plate using YG7108pin3ERb5. Moreover, we demonstrate for the first time a clear response of sufficiently characterised allyl chloride in the Ames test at a reasonably low concentration range between 300 and 1500 micro g per plate. We achieved similar results in the parent strain YG7108 with conventional metabolic activation. Without metabolic activation less pronounced mutagenicity occurred, suggesting a contribution of a direct alkylating effect. Propylene oxide is usually contained in allyl chloride as stabilizer at amounts up to 0.09%. Though YG7108 revealed to be very sensitive towards propylene oxide, allyl chloride dissolved in water was not mutagenic, showing that no water soluble compounds contribute to its mutagenicity. None of the remaining compounds showed mutagenic effects using YG7108pin3ERb5. CONCLUSION: YG7108pin3ERb5 and its parent strain YG7108 are sensitive for compounds which are negative in conventional tester strains including N-nitrosodiethylamine, N-nitrosopyrrolidine, propylene oxide and allyl chloride.

Variations in the genes encoding the peroxisome proliferator-activated receptors alpha and gamma in psoriasis.

The three peroxisome proliferator-activated receptor (PPAR) subtypes alpha, delta (beta), and gamma belong to the group of nuclear receptors that act as ligand-activated transcription factors. Recently, expression of PPAR alpha and gamma in keratinocytes has been demonstrated, and ligands of PPAR alpha and gamma have been found to enhance epidermal maturation and protect against cutaneous inflammation. There is evidence for a possible role of PPARs in psoriasis, as the expression of PPAR alpha and gamma is decreased in lesional skin and treatment with PPAR gamma agonists improves psoriatic keratinocyte pathology in vitro and in vivo. We performed a case-control study to search for possible associations between variations in the genes encoding PPAR alpha and gamma and psoriasis. Seven variations in these genes were analyzed in 192 patients with chronic plaque-type psoriasis and 330 healthy controls by PCR-based methods. No association between any of the investigated PPAR variants and psoriasis was found. Our findings argue against a significant contribution of the investigated PPAR variations to the genetic basis of psoriasis.

Cytokine gene polymorphisms in allergic contact dermatitis.

Susceptibility to contact allergy may be influenced by genetically determined alterations in the production of pro- and anti-inflammatory cytokines. This report focuses on functional polymorphisms in the genes encoding for several cytokines involved in the pathogenesis of contact allergic responses, including tumour necrosis factor (TNF)-alpha (G-238 A, G-308 A), interleukin (IL)-1beta (C-511G, T+ 3953C), its natural antagonist, the IL-1 receptor antagonist (VNTR intron 2), and IL-6 (G-174C). Polymorphisms were investigated by PCR techniques among polysensitized individuals, defined as individuals with confirmed contact sensitization to para-substituted aryl compounds and at least one other structurally unrelated allergen (n = 86), and healthy control individuals without a history of eczema (n = 310). The distribution of TNFA-308 genotypes was significantly different in these groups (Padjusted= 0.0378). Compared with carriers of 2 wild-type alleles (TNFA-308*1/1 (*G/G)), carriers of the TNFA-308*1/2 (*G/A) and TNFA-308*2/2 (*A/A) genotypes tended to be more common among polysensitized individuals [OR = 1.54, 95% CI (0.92-2.55) and OR = 2.36 (0.84-6.51), respectively]. No significantly different distribution of genotypes was detected at any other polymorphic loci among control individuals without eczema and polysensitized subjects. These findings suggest a possible relationship between the TNFA-308 polymorphism and contact allergy. The results need to be confirmed in future studies.

Thimerosal induces micronuclei in the cytochalasin B block micronucleus test with human lymphocytes.

Thimerosal is a widely used preservative in health care products, especially in vaccines. Due to possible adverse health effects, investigations on its metabolism and toxicity are urgently needed. An in vivo study on chronic toxicity of thimerosal in rats was inconclusive and reports on genotoxic effects in various in vitro systems were contradictory. Therefore, we reinvestigated thimerosal in the cytochalasin B block micronucleus test. Glutathione S-transferases were proposed to be involved in the detoxification of thimerosal or its decomposition products. Since the outcome of genotoxicity studies can be dependent on the metabolic competence of the cells used, we were additionally interested whether polymorphisms of glutathione S-transferases (GSTM1, GSTT1, or GSTP1) may influence the results of the micronucleus test with primary human lymphocytes. Blood samples of six healthy donors of different glutathione S-transferase genotypes were included in the study. At least two independent experiments were performed for each blood donor. Significant induction of micronuclei was seen at concentrations between 0.05-0.5 micro g/ml in 14 out of 16 experiments. Thus, genotoxic effects were seen even at concentrations which can occur at the injection site. Toxicity and toxicity-related elevation of micronuclei was seen at and above 0.6 micro g/ml thimerosal. Marked individual and intraindividual variations in the in vitro response to thimerosal among the different blood donors occurred. However, there was no association observed with any of the glutathione S-transferase polymorphism investigated. In conclusion, thimerosal is genotoxic in the cytochalasin B block micronucleus test with human lymphocytes. These data raise some concern on the widespread use of thimerosal.