Ingvar Eide

Diesel exhaust particles and carbon black have adjuvant activity on the local lymph node response and systemic IgE production to ovalbumin

The possible adjuvant effect of diesel exhaust particles (DEP) on the response to the model allergen ovalbumin (OA) was studied in BALB/c mice using the popliteal lymph node (PLN) assay. In addition to changes in PLN weight, cell numbers and cell proliferation, specific serum IgE anti-OA antibody levels were measured. OA inoculated together with DEP into one hind footpad gave a significantly augmented response (increase in weight, cell numbers and cell proliferation) in the draining popliteal lymph node as compared to DEP or OA alone. Also, the local lymph node response was of longer duration when DEP were given with the allergen. Experiments in thymus-deficient nu/nu mice indicated that the lymph node response observed in BALB/c mice was of a specific immunologic character and not an unspecific inflammatory reaction. The OA-specific IgE response was increased in mice receiving OA together with DEP as compared to the response in mice receiving OA without DEP. Carbon black (CB) was given with and without OA in some experiments, as a surrogate for the non-extractable core of DEP. CB was found to resemble DEP in its capacity to increase the local lymph node response and serum specific IgE response to OA, but CB appeared to be slightly less potent than DEP. Thus, both DEP and CB had a significant adjuvant effect on the local immune-mediated inflammatory response and on the systemic specific IgE response to allergen. The results indicate that the non-extractable particle core contributes substantially to the adjuvant activity of DEP.

Relationship between composition and toxicity of motor vehicle emission samples.

In this study we investigated the statistical relationship between particle and semivolatile organic chemical constituents in gasoline and diesel vehicle exhaust samples, and toxicity as measured by inflammation and tissue damage in rat lungs and mutagenicity in bacteria. Exhaust samples were collected from “normal” and “high-emitting” gasoline and diesel light-duty vehicles. We employed a combination of principal component analysis (PCA) and partial least-squares regression (PLS; also known as projection to latent structures) to evaluate the relationships between chemical composition of vehicle exhaust and toxicity. The PLS analysis revealed the chemical constituents covarying most strongly with toxicity and produced models predicting the relative toxicity of the samples with good accuracy. The specific nitro-polycyclic aromatic hydrocarbons important for mutagenicity were the same chemicals that have been implicated by decades of bioassay-directed fractionation. These chemicals were not related to lung toxicity, which was associated with organic carbon and select organic compounds that are present in lubricating oil. The results demonstrate the utility of the PCA/PLS approach for evaluating composition–response relationships in complex mixture exposures and also provide a starting point for confirming causality and determining the mechanisms of the lung effects.

Endogenous and background DNA adducts by methylating and 2-hydroxyethylating agents

Detection of 7-alkylguanine DNA adducts is useful to assess human exposure to and the resulting DNA damage caused by simple alkylating agents. The background 7-methylguanine (7-MG) and 7-hydroxyethylguanine (7-HEG) adduct levels were determined in human and rat tissues, using thin-layer chromatography (TLC) combined with high pressure liquid chromatography (HPLC). In addition, these two adduct levels were also compared in various tissues between smokers and non-smokers. The results demonstrated that the background level of 7-alkylguanine adducts in WBC and lung tissues of non-smokers was 2.9 and 4.0 adducts/107 nucleotides, respectively. In smokers with lung cancers 7-MG adduct level in lung samples (6.3+/-1.9 adducts/107 nucleotides) and in bronchus samples (6.1+/-1.5 adducts/107 nucleotides) was significantly higher than that in WBC samples (3.3+/-0.9 adducts/107 nucleotides). 7-HEG adduct levels obtained from the same individuals were 0.8+/-0.3 in lung, 1.0+/-0.8 in bronchus and 0.6+/-0.2 adducts/107 nucleotides in WBC, respectively. Animal studies showed that background levels of 7-MG (2.1-2.5 adducts/107 nucleotides) in control rats were approximately 2-4-fold higher than 7-HEG levels (0.6-0.9 adducts/107 nucleotides). After a 3-day exposure to 300 ppm ethene, 7-HEG adducts accumulated to a similar extent in different tissues of rats, with the mean adduct level of 5.6-7.0 in liver, 7.4 in lymphocytes and 5.5 adducts/107 nucleotides in kidney.

The adjuvant activity of diesel exhaust particles and carbon black on systemic IgE production to ovalbumin in mice after intranasal instillation

The adjuvant activity of diesel exhaust particles (DEP) on systemic IgE production to ovalbumin (OA) was studied in mice after intranasal administration. The main purpose was to elucidate which part of the particles was responsible for the effect, the carbon core and/or the adsorbed organic substances. Female Balb/cA mice were immunized with OA either alone or in combination with DEP or carbon black particles (CB), the latter used as a surrogate for the non-extractable carbon core of DEP. Controls were given DEP, CB or buffer alone. The animals were immunized four times. 1 and 2 weeks after the last immunization anti OA IgE antibody in serum was analysed by enzyme linked immunosorbent assay (ELISA). An increased response to the antigen was observed in animals receiving OA together with DEP or CB, compared with animals receiving OA alone. The increased response was seen as both increased number of responding animals and increased serum anti OA IgE antibody. For OA + DEP 37% of the animals showed a serum anti OA IgE response, whereas 22% of the OA + CB animals and 10% of the OA animals responded. In conclusion, this work shows that not only DEP, but also CB have an adjuvant activity for specific IgE production after intranasal instillation. However, the activity of DEP may be more pronounced than that of CB. The results imply that both the organic matter adsorbed to DEP and the non-extractable carbon core are responsible for the observed adjuvant effect.

Automated curve resolution applied to data from multi-detection instruments

Abstract This work presents a method for automated resolution of multicomponent data. The proposed procedures corrects background, cuts the data into smaller sub matrices, and resolves complex overlapping systems without human intervention. Basic constraints on concentration and spectral profiles are defined and used for determining the number of components and for cutting complex peak clusters into smaller sub matrices. The procedures are applied to multicomponent data from high performance liquid chromatography with diode array detection (HPLC-DAD), flow injection analysis with diode array detection (FIA-DAD), and gas chromatography–mass spectrometry (GC–MS).

Mutagenicity testing of organic extracts of diesel exhaust particles after fractionation and recombination

Abstract A new strategy for the evaluation of mixtures is presented. The mixture used was the organic extract of diesel exhaust particles (DEP). After extraction with dichloromethane (DCM), the crude extract was fractionated according to polarity into five fractions: aliphatic hydrocarbons, polycyclic aromatic hydrocarbons (PAHs), nitro-PAHs, dinitro-PAHs, and polar compounds. After dissolving in dimethylsulphoxide␣(DMSO), the three fractions containing the primary mutagens (fractions 3–5) were recombined in different combinations to create new extracts. The blend matrix was obtained using a mixture design at three dose levels to support an empirical model with linear, interaction, and quadratic terms (Taylor polynome). The recombined extracts were tested in the Ames Salmonella assay using strain TA100. Multivariate data analysis was performed with projections to latent structures (PLS). The best model describing the relation between the mutagenicity (response) and the three fractions (variables) contained two interaction terms. The model showed high correlation (r2) and prediction properties (Q2), the latter obtained after cross validation. Interaction terms are only indications of possible synergism or antagonism and have to be evaluated with respect to dose-additivity and response-additivity. The incorporation of dose in the design reduced the number of samples (recombined extracts) significantly, compared to determining dose-response curves on each sample (i.e. the recombined extracts in different dilutions). Furthermore, instead of running two independent experiments as required in the standard procedure for the Ames test, predictions and verifications of a few new samples were used. The principle of fractionation and recombination, and the use of mixture design may in principle be extended to an unlimited number of variables. An adaptation of mixture design to the isobole method is discussed.

Inhalation experiments with mixtures of hydrocarbons Experimental design, statistics and interpretation of kinetics and possible interactions

Abstract The paper describes experimental and statistical methods for toxicokinetic evaluation of mixtures in inhalation experiments. Synthetic mixtures of three C9 n-paraffinic, naphthenic and aromatic hydrocarbons (n-nonane, trimethylcyclohexane and trimethylbenzene, respectively) were studied in the rat after inhalation for 12 h. The hydrocarbons were mixed according to principles for statistical experimental design using mixture design at four vapour levels (75, 150, 300 and 450 ppm) to support an empirical model with linear, interaction and quadratic terms (Taylor polynome). Immediately after exposure, concentrations of hydrocarbons were measured by head space gas chromatography in blood, brain, liver, kidneys and perirenal fat. Multivariate data analysis and modelling were performed with PLS (projections to latent structures). The best models were obtained after removing all interaction terms, suggesting that there were no interactions between the hydrocarbons with respect to absorption and distribution. Uptake of paraffins and particularly aromatics is best described by quadratic models, whereas the uptake of the naphthenic hydrocarbons is nearly linear. All models are good, with high correlation (r2) and prediction properties (Q2), the latter after cross validation. The concentrations of aromates in blood were high compared to the other hydrocarbons. At concentrations below 250 ppm, the naphthene reached higher concentrations in the brain compared to the paraffin and the aromate. Statistical experimental design, multivariate data analysis and modelling have proved useful for the evaluation of synthetic mixtures. The principles may also be used in the design of liquid mixtures, which may be evaporated partially or completely.

Supplemental role of the Ames mutation assay and gap junction intercellular communication in studies of possible carcinogenic compounds from diesel exhaust particles

This study presents a new strategy for the carcinogenic evaluation of complex chemical mixtures based on genotoxic and nongenotoxic assays. We studied the ability of organic extracts of diesel exhaust particles (DEP) to induce point mutations in five different Salmonella typhimurium strains (Ames test) and to inhibit gap junction intercellular communication (GJIC) in rat liver epithelial cell lines. A crude extract of DEP was prepared by extraction with dichloromethane (DCM), and fractionated according to polarity into five fractions: aliphatic hydrocarbons, polycyclic aromatic hydrocarbons (PAH), nitro-PAH, dinitro-PAH, and polar compounds. Statistical experimental design, multivariate data analysis, and modeling were used to quantify the mutagenicity of individual and combined DEP fractions in the Ames assay. Quantitative determination of GJIC was carried out using a recently described combination of scrape loading and digital image analysis. Both assays responded to the DEP extract, but the responses were due to different fractions. The nitro-PAH fraction showed the strongest mutagenic potential, followed by the dinitro-PAH fraction. The effect on GJIC was due to the fraction containing the polar components, followed by the dinitro-PAH fraction. The extract was found to induce both basepair substitutions and frameshift mutations, through activation by bacterial nitroreductases. Hyperphosphorylation of connexin43, the major connexin in the epithelial cell lines, was less evident for DEP extract than for other communication inhibitors such as phorbol esters and growth factors, and consequently inhibitors of the protein kinase C (PKC) and mitogen-activated protein (MAP) kinase pathway were unable to counteract the inhibition by DEP extract. Since the Ames test is a well accepted method to screen for substances with genotoxic activity while inhibition of GJIC is associated with effect of tumor promoters and nongenotoxic carcinogens, it is not surprising but encouraging and interesting that the present data indicate that the two endpoints supplement each other as screening tests and in the evaluation of hazardous compounds in complex mixtures.

Mutagenicity testing of organic extracts of diesel exhaust particles after spiking with polycyclic aromatic hydrocarbons (PAH).

Abstract In the present study, spiking was used as a strategy to evaluate the mutagenicity of individual compounds in a mixture. Mutagenicity of individual polycyclic aromatic hydrocarbons (PAH) was evaluated in an organic extract of diesel exhaust particles (DEP). The particles were extracted with dichloromethane (DCM). After replacing DCM with dimethylsulphoxide (DMSO), the extract was spiked with four individual PAH: benzo(a)pyrene, benzo(a)anthracene, pyrene and fluoroanthene. The PAH were added separately and in various combinations to the extract to determine the effects of each variable and to identify possible interactions between the individual PAH and between the PAH and the extract. The study was designed as a fractional factorial experiment with the five variables (the DEP extract and the four PAH), giving 16 (instead of 32) mixtures plus a triplicate centrepoint and background, i.e. a total of 20. The fractionated factorial design used in the present work supports a model with linear and interaction terms. The mixtures were tested for mutagenicity in the Ames assay using four strains of Salmonella typhimurium in the presence of rat liver xenobiotic enzymes (S9-mix). Projections to Latent Structures (PLS) was used to quantify the mutagenicity of each compound and possible interactions. The four individual PAH and the DEP extract acted additively in the Ames test with 10% S9-mix.

Persistence of 7-(2-hydroxyethyl)guanine± DNA adducts in rats exposed to ethene by inhalation

Quantification of 7 2 hydroxyethyl guanine 7 HEG adduct in DNA of livers and lymphocytes of male Sprague-Dawley rats exposed to 300 ppm ethene by inhalation 12 h a day for three consecutive days was performed to evaluate the potential of ethene to produce DNA adducts in these tissues. The persistence of 7 HEG in livers and lymphocytes was studied in rats sacrificed 0, 1, 5, and 20 days after the last exposure. DNA samples from control and treated animals were analysed for 7 HEG and 7 methylguanine 7 MG adducts by thin layer chromatography TLC combined with a high pressure liquid chromatography HPLC assay. After a 3 day exposure to ethene, 7 HEG accumulated to a similar extent in liver and lymphocytes, with the mean adduct level of 7.0 0.7 adducts per 107 nucleotides in liver and 7.4 0.7 adducts per 107 nucleotides in lymphocytes of rats sacrificed immediately after cessation of exposure. The approximate half life of 7 HEG was 5 days in liver and 3 days in lymphocytes, which is consistent with the loss of adduct primarily by spontaneous depurination. In addition, the background levels of 7 HEG and 7 MG were determined in the liver and lymphocytes from the control rats.Quantification of 7 2 hydroxyethyl guanine 7 HEG adduct in DNA of livers and lymphocytes of male Sprague-Dawley rats exposed to 300 ppm ethene by inhalation 12 h a day for three consecutive days was performed to evaluate the potential of ethene to produce DNA adducts in these tissues. The persistence of 7 HEG in livers and lymphocytes was studied in rats sacrificed 0, 1, 5, and 20 days after the last exposure. DNA samples from control and treated animals were analysed for 7 HEG and 7 methylguanine 7 MG adducts by thin layer chromatography TLC combined with a high pressure liquid chromatography HPLC assay. After a 3 day exposure to ethene, 7 HEG accumulated to a similar extent in liver and lymphocytes, with the mean adduct level of 7.0 0.7 adducts per 107 nucleotides in liver and 7.4 0.7 adducts per 107 nucleotides in lymphocytes of rats sacrificed immediately after cessation of exposure. The approximate half life of 7 HEG was 5 days in liver and 3 days in lymphocytes, which is consistent with the loss of ...