Nanoparticle-Based Radioconjugates for Targeted Imaging and Therapy of Prostate Cancer.

Prostate cancer is the second most frequent malignancy in men worldwide and the fifth leading cause of death by cancer. Although most patients initially benefit from therapy, many of them will progress to metastatic castration-resistant prostate cancer, which still remains incurable. The significant mortality and morbidity rate associated with the progression of the disease results mainly from a lack of specific and sensitive prostate cancer screening systems, identification of the disease at mature stages, and failure of anticancer therapy. To overcome the limitations of conventional imaging and therapeutic strategies for prostate cancer, various types of nanoparticles have been designed and synthesized to selectively target prostate cancer cells without causing toxic side effects to healthy organs. The purpose of this review is to briefly discuss the selection criteria of suitable nanoparticles, ligands, radionuclides, and radiolabelling strategies for the development of nanoparticle-based radioconjugates for targeted imaging and therapy of prostate cancer and to evaluate progress in the field, focusing attention on their design, specificity, and potential for detection and/or therapy.

Dietary Intervention with Blackcurrant Pomace Protects Rats from Testicular Oxidative Stress Induced by Exposition to Biodiesel Exhaust.

The exposure to diesel exhaust emissions (DEE) contributes to negative health outcomes and premature mortality. At the same time, the health effects of the exposure to biodiesel exhaust emission are still in scientific debate. The aim of presented study was to investigate in an animal study the effects of exposure to DEE from two types of biodiesel fuels, 1st generation B7 biodiesel containing 7% of fatty acid methyl esters (FAME) or 2nd generation biodiesel (SHB20) containing 7% of FAME and 13% of hydrotreated vegetable oil (HVO), on the oxidative stress in testes and possible protective effects of dietary intervention with blackcurrant pomace (BC). Adult Fisher344/DuCrl rats were exposed by inhalation (6 h/day, 5 days/week for 4 weeks) to 2% of DEE from B7 or SHB20 fuel mixed with air. The animals from B7 (n = 14) and SHB20 (n = 14) groups subjected to filtered by a diesel particulate filter (DPF) or unfiltered DEE were maintained on standard feed. The rats from B7+BC (n = 12) or SHB20+BC (n = 12), exposed to DEE in the same way, were fed with feed supplemented containing 2% (m/m) of BC. The exposure to exhaust emissions from 1st and 2nd generation biodiesel resulted in induction of oxidative stress in the testes. Higher concentration of the oxidative stress markers thiobarbituric acid-reactive substances (TBARS), lipid hydroperoxides (LOOHs), 25-dihydroxycholesterols (25(OH)2Ch), and 7-ketocholesterol (7-KCh) level), as well as decreased level of antioxidant defense systems such as reduced glutathione (GSH), GSH/GSSG ratio, and increased level of oxidized glutathione (GSSG)) were found. Dietary intervention reduced the concentration of TBARS, 7-KCh, LOOHs, and the GSSG level, and elevated the GSH level in testes. In conclusion, DEE-induced oxidative stress in the testes was related to the biodiesel feedstock and the application of DPF. The SHB20 DEE without DPF technology exerted the most pronounced toxic effects. Dietary intervention with BC in rats exposed to DEE reduced oxidative stress in testes and improved antioxidative defense parameters, however the redox balance in the testes was not completely restored.

The effect of surface modification of dendronized gold nanoparticles on activation and release of pyroptosis-inducing pro-inflammatory cytokines in presence of bacterial lipopolysaccharide in monocytes.

Biomedical applications of gold nanoparticles (AuNPs) may be limited by their toxicological effects. Although surface-modified AuNPs can induce apoptosis, less is known about whether they can induce other types of cell death. Pyroptosis, an inflammatory type of programmed cell death, can be induced in immune cells, especially macrophages, by bacterial endotoxins. Therefore, in this study, dendronized AuNPs were combined with bacterial lipopolysaccharides (LPSs) as the main stimulators of pro-inflammatory responses to test the induction of pyroptosis in THP-1 myeloid cell line. These AuNPs induced caspase-1 activity (3-4 times more compared to control) and enhanced the release of interleukin (IL)-18 and IL-1ß without inducing gasdermin D cleavage and related pore formation. The production of pro-inflammatory cytokines occurred mainly visible during LPS treatment, although their secretion was observed only after administration of dendronized AuNPs (release of IL-1ß to supernatant up to 80 pg/mL). These findings suggest that dendronized AuNPs can induce pyroptosis-like inflammatory mechanisms and that these mechanisms are enhanced in the presence of bacterial LPS. The intensity of this effect was dependent on AuNP surface modification. These results shed new light on the cytotoxicity of metal NPs, including immune responses, indicating that surface modifications play crucial roles in their nanotoxicological effects.

Hypothermia differentially modulates the formation and decay of NBS1, γH2AX and 53BP1 foci in U2OS cells exposed to gamma radiation.

In studies on the mechanism of DNA damage response where ionizing radiation is used as the DNA damaging agent, cells are often exposed to ionizing radiation on melting ice (corresponding to 0.8 °C). The purpose of this procedure is to inhibit cellular processes i.e. DNA repair. Low temperature at exposure has been shown to act in a radioprotective manner at the level of cytogenetic damage, but its mechanisms of action are poorly understood. The aim of the study was to analyze the effect of hypothermia at the level of formation and decay of NBS1, γH2AX, and 53BP1 foci, micronuclei, survival, cell cycle progression and oxidative stress in U2OS cells. The results show that hypothermia alone induced oxidative stress and foci. When applied in combination with radiation but only during the exposure time, it potentiated the formation of γH2AX and 53BP1 but not of NBS1 foci. When applied during irradiation and subsequent repair time, 53BP1 and NBS1 foci formed and decayed, but the levels were markedly lower than when repair was carried out at 37 °C. The frequency of micronuclei was elevated in cells irradiated at 0.8 °C, but only when analysed 20 h after irradiation which is likely due to a reduced G2 cell cycle block. Hypothermia reduced cell survival, both with and without radiation exposure. The temperature effect should be considered when cooling cells on melting ice to inhibit DNA repair in the induction of DNA damage.

Cisplatin Reduces the Frequencies of Radiotherapy-Induced Micronuclei in Peripheral Blood Lymphocytes of Patients with Gynaecological Cancer: Possible Implications for the Risk of Second Malignant Neoplasms.

Gynaecologic cancers are common among women and treatment includes surgery, radiotherapy or chemotherapy, where the last two methods induce DNA damage in non-targeted cells like peripheral blood lymphocytes (PBL). Damaged normal cells can transform leading to second malignant neoplasms (SMN) but the level of risk and impact of risk modifiers is not well defined. We investigated how radiotherapy alone or in combination with chemotherapy induce DNA damage in PBL of cervix and endometrial cancer patients during therapy. Blood samples were collected from nine endometrial cancer patients (treatment with radiotherapy + chemotherapy-RC) and nine cervical cancer patients (treatment with radiotherapy alone-R) before radiotherapy, 3 weeks after onset of radiotherapy and at the end of radiotherapy. Half of each blood sample was irradiated ex vivo with 2 Gy of gamma radiation in order to check how therapy influenced the sensitivity of PBL to radiation. Analysed endpoints were micronucleus (MN) frequencies, apoptosis frequencies and cell proliferation index. The results were characterised by strong individual variation, especially the MN frequencies and proliferation index. On average, despite higher total dose and larger fields, therapy alone induced the same level of MN in PBL of RC patients as compared to R. This result was accompanied by a higher level of apoptosis and stronger inhibition of cell proliferation in RC patients. The ex vivo dose induced fewer MN, more apoptosis and more strongly inhibited proliferation of PBL of RC as compared to R patients. These results are interpreted as evidence for a sensitizing effect of chemotherapy on radiation cytotoxicity. The possible implications for the risk of second malignant neoplasms are discussed.

Coralyne Radiosensitizes A549 Cells by Upregulation of CDKN1A Expression to Attenuate Radiation Induced G2/M Block of the Cell Cycle.

Coralyne is a synthetic analog of berberine related to protoberberine-isoquinoline alkaloids. Isoquinoline derivatives and analogs are renowned as potent radiosensitizers with potential medical application. In the present study, we investigated the effect of coralyne on the cell death, cytoskeletal changes and cell cycle progression of irradiated A549 cells. A clonogenic assay revealed that coralyne pretreatment decreased the viability of A549 cells in a time- and dose-dependent manner. Moreover, exposure to coralyne and ionizing radiation (IR) markedly altered the filamentous actin cytoskeletal architecture and integrin-ß binding sites of A549 cells. Treatment with 1-25 µM coralyne in combination with 2 Gy of IR significantly reduced the percentage of cells in G2/M phase compared with 2 Gy IR alone. These results indicate that coralyne is a potent radiosensitizing agent that may find an application in medicine.

Design and Evaluation of 223Ra-Labeled and Anti-PSMA Targeted NaA Nanozeolites for Prostate Cancer Therapy-Part II. Toxicity, Pharmacokinetics and Biodistribution.

Metastatic castration-resistant prostate cancer (mCRPC) is a progressive and incurable disease with poor prognosis for patients. Despite introduction of novel therapies, the mortality rate remains high. An attractive alternative for extension of the life of mCRPC patients is PSMA-based targeted radioimmunotherapy. In this paper, we extended our in vitro study of 223Ra-labeled and PSMA-targeted NaA nanozeolites [223RaA-silane-PEG-D2B] by undertaking comprehensive preclinical in vitro and in vivo research. The toxicity of the new compound was evaluated in LNCaP C4-2, DU-145, RWPE-1 and HPrEC prostate cells and in BALB/c mice. The tissue distribution of 133Ba- and 223Ra-labeled conjugates was studied at different time points after injection in BALB/c and LNCaP C4-2 tumor-bearing BALB/c Nude mice. No obvious symptoms of antibody-free and antibody-functionalized nanocarriers cytotoxicity and immunotoxicity was found, while exposure to 223Ra-labeled conjugates resulted in bone marrow fibrosis, decreased the number of WBC and platelets and elevated serum concentrations of ALT and AST enzymes. Biodistribution studies revealed high accumulation of 223Ra-labeled conjugates in the liver, lungs, spleen and bone tissue. Nontargeted and PSMA-targeted radioconjugates exhibited a similar, marginal uptake in tumour lesions. In conclusion, despite the fact that NaA nanozeolites are safe carriers, the intravenous administration of NaA nanozeolite-based radioconjugates is dubious due to its high accumulation in the lungs, liver, spleen and bones.

Design and Evaluation of 223Ra-Labeled and Anti-PSMA Targeted NaA Nanozeolites for Prostate Cancer Therapy-Part I.

Prostate cancer is the second most frequent malignancy in men worldwide. Unfortunately, current therapies often lead to the onset of metastatic castration-resistant prostate cancer (mCRPC), causing significant mortality. Therefore, there is an urgent need for new and targeted therapies that are advantageous over the current ones. Recently, the PSMA-targeted radioligand therapy of mCRPC has shown very promising results. In line with this, we described the synthesis of a new radioimmunoconjugate, 223RaA-silane-PEG-D2B, for targeted mCRPC therapy. The new compound consists of a NaA zeolite nanocarrier loaded with the α-particle emitting Ra-223 radionuclide, functionalized with the anti-PSMA D2B antibody. Physicochemical properties of the synthesized compound were characterized by standard methods (HR-SEM, TEM, XRD, FTIR, EDS, NTA, DLS, BET, TGA). The targeting selectivity, the extent of internalization, and cytotoxicity were determined in LNCaP C4-2 (PSMA+) and DU-145 (PSMA-) cells. Our results supported the 223RaA-silane-PEG-D2B synthesis and revealed that the final product had a diameter ca. 120 nm and specific activity 0.65 MBq/1mg. The product was characterized by a high yield of stability (>95% up to 12 days). The conjugation reaction resulted in approximately 50 antibodies/nanoparticle. The obtained radioimmunoconjugate bound specifically and internalized into PSMA-expressing LNCaP C4-2 cells, but not into PSMA-negative DU-145 cells. 223RaA-silane-PEG-D2B demonstrated also potent cytotoxicity in LNCaP C4-2 cells. These promising results require further in vivo evaluation of 223RaA-silane-PEG-D2B with regard to its toxicity and therapeutic efficacy.

Targeted Radionuclide Therapy of Prostate Cancer-From Basic Research to Clinical Perspectives.

Prostate cancer is the most commonly diagnosed malignancy in men and the second leading cause of cancer-related deaths in Western civilization. Although localized prostate cancer can be treated effectively in different ways, almost all patients progress to the incurable metastatic castration-resistant prostate cancer. Due to the significant mortality and morbidity rate associated with the progression of this disease, there is an urgent need for new and targeted treatments. In this review, we summarize the recent advances in research on identification of prostate tissue-specific antigens for targeted therapy, generation of highly specific and selective molecules targeting these antigens, availability of therapeutic radionuclides for widespread medical applications, and recent achievements in the development of new-generation small-molecule inhibitors and antibody-based strategies for targeted prostate cancer therapy with alpha-, beta-, and Auger electron-emitting radionuclides.

Selective cytotoxicity and antifungal properties of copper(II) and cobalt(II) complexes with imidazole-4-acetate anion or 1-allylimidazole.

The physicochemical properties of metal complexes determine their potential applications as antitumor agents. In this study, the antitumor properties of mononuclear cobalt(II) and copper(II) coordination compounds (stoichiometry: [Co(iaa)2(H2O)2]·H2O (iaa = imidazole-4-acetate anion), [Co(1-allim)6](NO3)2 (1-allim = 1-allylimidazole), [Cu(iaa)2H2O] and [Cu(1-allim)4(NO3)2]) and their ligands have been evaluated on human lung carcinoma A549 cells and normal bronchial BEAS-2B cells. Designing the chemical structure of new antitumor agents the possible interactions with macromolecules such as DNA or proteins should be take into account. PCR gene tlr4 product served as DNA model, whereas lysozyme and phage-derived endolysin (both peptidoglycan degrading enzymes) were applied as protein/enzyme model. The interactions were analysed using PCR-HRM and circular dichroism, FT-IR, spectrophotometry, respectively. Additionally, the antimicrobial properties of the complexes at a non-cytotoxic concentration were analyzed against S. aureus, E. coli, P. aeruginosa and C. albicans strains. The results obtained in this study showed the selective cytotoxicity of metal complexes, mainly [Cu(1-allim)4(NO3)2] towards tumor cells. From all tested compounds, only [Co(iaa)2(H2O)2].H2O non-covalently interacts with DNA. Cu(II) and Co(II) complexes did not affect the secondary conformation of tested proteins but modified the hydrolytic activity of enzymes (lysozyme and endolysin). Moreover, only [Co(iaa)2(H2O)2].H2O exhibited the antifungal properties. In conclusion, Co(II) and Cu(II) metal complexes bearing two imidazole-4-acetate ligands seemed to be promising antitumor and antifungal agents for future drug design and application.

Lung effects of 7- and 28-day inhalation exposure of rats to emissions from 1st and 2nd generation biodiesel fuels with and without particle filter - The FuelHealth project.

Increased use of 1st and 2nd generation biofuels raises concerns about health effects of new emissions. We analyzed cellular and molecular lung effects in Fisher 344 rats exposed to diesel engine exhaust emissions (DEE) from a Euro 5-classified diesel engine running on B7: petrodiesel fuel containing 7% fatty acid methyl esters (FAME), or SHB20 (synthetic hydrocarbon biofuel): petrodiesel fuel containing 7% FAME and 13% hydrogenated vegetable oil. The Fisher 344 rats were exposed for 7 consecutive days (6 h/day) or 28 days (6 h/day, 5 days/week), both with and without diesel particle filter (DPF) treatment of the exhaust in whole body exposure chambers (n = 7/treatment). Histological analysis and analysis of cytokines and immune cell numbers in bronchoalveolar lavage fluid (BALF) did not reveal adverse pulmonary effects after exposure to DEE from B7 or SHB20 fuel. Significantly different gene expression levels for B7 compared to SHB20 indicate disturbed redox signaling (Cat, Hmox1), beta-adrenergic signaling (Adrb2) and xenobiotic metabolism (Cyp1a1). Exhaust filtration induced higher expression of redox genes (Cat, Gpx2) and the chemokine gene Cxcl7 compared to non-filtered exhaust. Exposure time (7 versus 28 days) also resulted in different patterns of lung gene expression. No genotoxic effects in the lungs were observed. Overall, exposure to B7 or SHB20 emissions suggests only minor effects in the lungs.

Biological effects of mixed-ion beams. Part 2: The relative biological effectiveness of CHO-K1 cells irradiated by mixed- and single-ion beams.

The relative biological effectiveness (RBE) values were determined for single- and mixed-ion beams containing carbon and oxygen ions. The CHO-K1 cells were irradiated with beams with the linear energy transfer (LET) values of 236-300 and 461-470 keV/µm for 12C and 16O ions, respectively. The RBE was estimated as a function of dose, survival fraction (SF) and LET. The SF was not affected by varying contributions of the constituent ions to the total mixed dose. The RBE has the same value for single-ion exposures with ions with LET 300 (12C) and 470 keV/µm (16O).

Gene expression changes in rat brain regions after 7- and 28 days inhalation exposure to exhaust emissions from 1st and 2nd generation biodiesel fuels - The FuelHealth project.

While the impact of emissions from combustion of fossil fuel on human health has been extensively studied, current knowledge of exhaust exposure from combustion of biofuels provides limited and inconsistent information about its neurotoxicity. The objective of the present work was to compare the gene expression patterns in rat frontal cortex and hippocampus after exposure to diesel exhaust emissions (DEE) from combustion of two 1st generation fuels, 7% fatty acid methyl esters (FAME) (B7) and 20% FAME (B20), and a 2nd generation 20% FAME/hydrotreated vegetable oil (SHB20: synthetic hydrocarbon biofuel), with and without diesel particulate filter (DPF). The Fisher 344 rats (n = 7/treatment) were exposed to DEE for 7 days (6h/day), and for 28 days (6h/day, 5 days/week) in whole body exposure chambers. The controls were breathing room air. Brain histological examinations did not reveal any adverse exposure-related effects of DEE in frontal cortex or in hippocampus. Gene expression analysis showed that several genes associated with antioxidant defenses and inflammation were statistically differently expressed in DEE exposed animals versus control. In addition, the gene expression changes between the exposure groups were compared, where the observed rank order in frontal cortex was B7 > B20 > SHB20 after 7 days of exposure, and SHB20 > B7 = B20 after 28 days of exposure. In the hippocampus, the rank order was B7 > SHB20 > B20. Effect of DPF treatment was observed for Tnf only. Overall, moderate increases in bio-components in diesel blends do not appear to result in dramatic alterations in gene expression or adverse histopathological effects.

Biological effects of mixed-ion beams. Part 1: Effect of irradiation of the CHO-K1 cells with a mixed-ion beam containing the carbon and oxygen ions.

Carbon and oxygen ions were accelerated simultaneously to estimate the effect of irradiation of living cells with the two different ions. This mixed ion beam was used to irradiate the CHO-K1 cells, and a survival test was performed. The type of the effect of the mixed ion beam on the cells was determined with the isobologram method, whereby survival curves for irradiations with individual ion beams were also used. An additive effect of irradiation with the two ions was found.

Fullerenol C60(OH)36 protects human erythrocyte membrane against high-energy electrons.

Fullerenols (polyhydroxylated fullerene C60) are nanomaterial with potentially broad applicability in biomedical sciences with high antioxidant ability, thus, we investigated the radioprotecting potential of fullerenol C60(OH)36 on human erythrocytes irradiated by high-energy electrons of 6 MeV. The results demonstrate that C60(OH)36 at concentration of 150 µg/mL protects the erythrocytes against the radiation-induced hemolysis (comparing to non-protected cells, we observed 30% and 39% protection for 0.65 and 1.3 kGy irradiation doses, respectively). The protecting effect was confirmed by 32% decreased release of potassium cations comparing to the cells irradiated without C60(OH)36. Measurements of the amount of lactate dehydrogenase (LDH) released from the irradiated erythrocytes showed that the size of the pores formed by irradiation was not sufficient to release LDH across the erythrocyte membranes. We also report a significant decrease of the affinity of acetylcholinesterase (AChE) for the substrate in the presence of fullerenol, indicating the relatively strong adsorption of C60(OH)36 to components of plasma membrane. Changes in membrane fluidity detected by fluorescence spectroscopy and conformational changes in membrane proteins detected by spin labeling suggest the dose-dependent formation of disulfide groups as an effect of oxidation and this process was inhibited by C60(OH)36. We suppose that scavenging the ROS as well as adsorption of fullerenol to membrane proteins and steric protection of -SH groups against oxidation are responsible for the observed effects.

Hypothermia modulates the DNA damage response to ionizing radiation in human peripheral blood lymphocytes.

PURPOSE: Low temperature at exposure has been shown to act in a radioprotective manner at the level of cytogenetic damage. It was suggested to be due to an effective transformation of DNA damage to chromosomal damage at low temperature. The purpose of the study was to analyze the kinetics of aberration formation during the first hours after exposing human peripheral blood lymphocytes to ionizing radiation at 0.8 °C and 37 °C. MATERIALS AND METHODS: To this end, we applied the technique of premature chromosome condensation. In addition, DNA damage response was analyzed by measuring the levels of phosphorylated DNA damage responsive proteins ATM, DNA-PK and p53 and mRNA levels of the radiation-responsive genes BBC3, FDXR, GADD45A, XPC, MDM2 and CDKN1A. RESULTS: A consistently lower frequency of chromosomal breaks was observed in cells exposed at 0.8 °C as compared to 37 °C already after 30 minutes postexposure. This effect was accompanied by elevated levels of phosphorylated ATM and DNA-PK proteins and a reduced immediate level of phosphorylated p53 and of the responsive genes. CONCLUSIONS: Low temperature at exposure appears to promote DNA repair leading to reduced transformation of DNA damage to chromosomal aberrations.

Genotoxic potential of diesel exhaust particles from the combustion of first- and second-generation biodiesel fuels-the FuelHealth project.

Epidemiological data indicate that exposure to diesel exhaust particles (DEPs) from traffic emissions is associated with higher risk of morbidity and mortality related to cardiovascular and pulmonary diseases, accelerated progression of atherosclerotic plaques, and possible lung cancer. While the impact of DEPs from combustion of fossil diesel fuel on human health has been extensively studied, current knowledge of DEPs from combustion of biofuels provides limited and inconsistent information about its mutagenicity and genotoxicity, as well as possible adverse health risks. The objective of the present work was to compare the genotoxicity of DEPs from combustion of two first-generation fuels, 7% fatty acid methyl esters (FAME) (B7) and 20% FAME (B20), and a second-generation 20% FAME/hydrotreated vegetable oil (SHB: synthetic hydrocarbon biofuel) fuel. Our results revealed that particulate engine emissions from each type of biodiesel fuel induced genotoxic effects in BEAS-2B and A549 cells, manifested as the increased levels of single-strand breaks, the increased frequencies of micronuclei, or the deregulated expression of genes involved in DNA damage signaling pathways. We also found that none of the tested DEPs showed the induction of oxidative DNA damage and the gamma-H2AX-detectable double-strand breaks. The most pronounced differences concerning the tested particles were observed for the induction of single-strand breaks, with the greatest genotoxicity being associated with the B7-derived DEPs. The differences in other effects between DEPs from the different biodiesel blend percentage and biodiesel feedstock were also observed, but the magnitude of these variations was limited.

A comparative analysis of in vitro toxicity of diesel exhaust particles from combustion of 1st- and 2nd-generation biodiesel fuels in relation to their physicochemical properties-the FuelHealth project.

Biodiesels represent more carbon-neutral fuels and are introduced at an increasing extent to reduce emission of greenhouse gases. However, the potential impact of different types and blend concentrations of biodiesel on the toxicity of diesel engine emissions are still relatively scarce and to some extent contradictory. The objective of the present work was to compare the toxicity of diesel exhaust particles (DEP) from combustion of two 1st-generation fuels: 7% fatty acid methyl esters (FAME; B7) and 20% FAME (B20) and a 2nd-generation 20% FAME/HVO (synthetic hydrocarbon biofuel (SHB)) fuel. Our findings indicate that particulate emissions of each type of biodiesel fuel induce cytotoxic effects in BEAS-2B and A549 cells, manifested as cell death (apoptosis or necrosis), decreased protein concentrations, intracellular ROS production, as well as increased expression of antioxidant genes and genes coding for DNA damage-response proteins. The different biodiesel blend percentages and biodiesel feedstocks led to marked differences in chemical composition of the emitted DEP. The different DEPs also displayed statistically significant differences in cytotoxicity in A549 and BEAS-2B cells, but the magnitude of these variations was limited. Overall, it seems that increasing biodiesel blend concentrations from the current 7 to 20% FAME, or substituting 1st-generation FAME biodiesel with 2nd-generation HVO biodiesel (at least below 20% blends), affects the in vitro toxicity of the emitted DEP to some extent, but the biological significance of this may be moderate.

No adverse lung effects of 7- and 28-day inhalation exposure of rats to emissions from petrodiesel fuel containing 20% rapeseed methyl esters (B20) with and without particulate filter - the FuelHealth project.

Increased use of biofuels raises concerns about health effects of new emissions. We analyzed relative lung health effects, on Fisher 344 rats, of diesel engine exhausts emissions (DEE) from a Euro 5-classified diesel engine running on petrodiesel fuel containing 20% rapeseed methyl esters (B20) with and without diesel particulate filter (DPF). One group of animals was exposed to DEE for 7 days (6 h/day), and another group for 28 days (6 h/day, 5 days/week), both with and without DPF. The animals (n = 7/treatment) were exposed in whole body exposure chambers. Animals breathing clean air were used as controls. Genotoxic effects of the lungs by the Comet assay, histological examination of lung tissue, bronchoalveolar lavage fluid (BALF) markers of pulmonary injury, and mRNA markers of inflammation and oxidative stress were analyzed. Our results showed that a minor number of genes related to inflammation were slightly differently expressed in the exposed animals compared to control. Histological analysis also revealed only minor effects on inflammatory tissue markers in the lungs, and this was supported by flow cytometry and ELISA analysis of cytokines in BALF. No exposure-related indications of genotoxicity were observed. Overall, exposure to DEE with or without DPF technology produced no adverse effects in the endpoints analyzed in the rat lung tissue or the BALF. Overall, exposure to DEE from a modern Euro 5 light vehicle engine run on B20 fuel with or without DPF technology produced no adverse effects in the endpoints analyzed in the rat lung tissue or the BALF.

Proinflammatory effects of diesel exhaust particles from moderate blend concentrations of 1st and 2nd generation biodiesel in BEAS-2B bronchial epithelial cells-The FuelHealth project.

Biodiesel fuel fuels are introduced at an increasing extent as a more carbon-neutral alternative to reduce CO2-emissions, compared to conventional diesel fuel. In the present study we have investigated the impact of increasing the use of 1st generation fatty acid methyl ester (FAME) biodiesel from current 7% blend (B7) to 20% blend (B20), or by increasing the biodiesel content by adding 2nd generation hydrotreated vegetable oil (HVO) based biodiesel (SHB; Synthetic Hydrocarbon Biofuel) on toxicity of diesel exhaust particles (DEP) in an in vitro system. Human bronchial epithelial BEAS-2B cells were exposed for 4 and 20h to DEP from B7, B20 and SHB at different concentrations, and examined for effects on gene expression of interleukin 6 (IL-6), CXCL8 (IL-8), CYP1A1 and heme oxygenase-1 (HO-1). The results show that both B20 and SHB were more potent inducers of IL-6 expression compared to B7. Only B20 induced statistically significant increases in CXCL8 expression. By comparison the rank order of potency to induce CYP1A1 was SHB>B7>B20. No statistically significant difference were observed form HO-1 expression, suggesting that the differences in cytokine responses were not due to oxidative stress. The results show that even moderate increases in biodiesel blends, from 7% to 20%, may increase the proinflammatory potential of emitted DEP in BEAS-2B cells. This effect was observed for both addition of 1st generation FAME and 2nd generation HVO biodiesel.