Jürgen Bünger

Mutagenic and cytotoxic effects of exhaust particulate matter of biodiesel compared to fossil diesel fuel

The mutagenic and cytotoxic effects of diesel engine exhaust (DEE) from a modern passenger car using rapeseed oil methyl esters (RME, biodiesel) as fuel were directly compared to DEE of diesel fuel (DF) derived from petroleum. Combustion particulate matter was collected on glass fiber filters coated with polytetrafluoroethylene (PTFE) from an exhaust dilution tunnel using three different engine test cycles on a chassis dynamometer. Filters were extracted with dichloromethane in a soxhlet apparatus for 12 h. The mutagenicity of the extracts was tested in the Salmonella typhimurium/mammalian microsome plate-incorporation assay using strains TA97a, TA98, TA100, and TA102. The toxicity to the established cell line L929 (mouse lung fibroblasts) was investigated in the neutral red assay. In the tester strains TA98 and TA100 a significant increase of mutations resulted for the particle extracts of both fuels, but for DF the revertants were significantly higher compared to RME. The highest levels of revertants were observed in tests including a cold start phase. This was probably due to incomplete combustion in the cold engine and a lower conversion rate of the cold catalytic converter. Testing with activated liver S9 fraction induced a slightly lower increase of revertants in most experiments. TA97a and TA102 showed no significant enhancement of spontaneous mutations. In the FTP-75 test cycle RME extracts showed slightly higher toxic effects to the L929 cells than DF, whereas in the other tests no significant differences were observable. These results indicate a higher mutagenic potency of DEE of DF compared to RME. This is probably due to the lower content of polycyclic aromatic compounds (PAC) in RME exhaust, although the emitted masses of RME were higher in most test procedures applied in this study.

Influence of Biodiesel and Different Designed Diesel Fuels on the Exhaust Gas Emissions and Health Effects

The more stringent regulations for diesel engine emissions lead to the requirement that both fuels and engines must be developed jointly. In the future, so-called designer fuels will help to achieve the stringent limits.In our research, conventional diesel fuel, biodiesel, Swedish low sulfur diesel fuel MK1 and a specially designed diesel fuel were compared using a DaimlerChrysler diesel engine, running the modes of the ECE 49 test cycle. The results for regulated and non-regulated gaseous emissions, particulate matter size distributions as well as mutagenic effects of particle extracts are reported.

Cyto- and genotoxic effects of coordination complexes of platinum, palladium and rhodium in vitro

The growing industrial use of platinum group elements as catalysts, especially in automobile exhaust detoxification (trimetal catalytic converters), is causing increasing occupational and environmental pollution. The cytotoxic and mutagenic properties of industrially used coordination complexes of platinum, palladium and rhodium were investigated using the neutral red cytotoxicity assay on two established cell lines and theSalmonella typhimurium/microsome test system (Ames test). Cytotoxic effects of the platinum complexes, measured as ED50, occurred at test concentrations of 0.2 mM. The analogous palladium salts tested were 3 times less toxic with ED50 being 0.6 mM, while the rhodium salts proved to be 30 times less toxic (ED50=6 mM). Levels of toxicity of the different complexes of a particular metal did not differ significantly from each other, which indicates that the metal itself is responsible for the toxic effects. In the Ames test, the spontaneous mutation rates increased by factors of 3 to 20 when the four tester strains were exposed to the platinum complexes. The analogous rhodium compounds proved to be considerably less mutagenic, and palladium demonstrated no mutagenic potential. As all of the four tester strains contain different mutations, the mutagenic potential of platinum and rhodium complexes appears to be based on a variety of mechanisms that damage DNA. From these in vitro experiments, it can be concluded that water-soluble complex salts of rhodium are less toxic and have a smaller mutagenic potential than the analogous platinum complexes. For palladium there is no evidence of any mutagenic property. From this point of view, the development of a catalytic converter containing predominantly palladium may be a possible means of minimizing potential health risks from this exhaust detoxification technique.

Increased risk for therapy-associated hematologic malignancies in patients with carcinoma of the breast and combined homozygous gene deletions of glutathione transferases M1 and T1.

The most serious long-term complications of anti-tumor therapy are secondary malignancies. Parameters which might allow an estimation of the individual risk to develop a therapy-induced neoplasia are urgently needed. We examined whether the genotypes of the glutathione S-transferases (GST) M1 and T1, which metabolize various cytostatic drugs, as well as reactive oxygen species, influence the risk for secondary neoplasia. In a retrospective study, we analyzed peripheral blood lymphocyte or bone marrow DNA samples from 213 patients with acute myeloid leukemia (AML) and 128 with myelodysplastic syndromes (MDS) 44 of whom suffered from therapy-associated AML/MDS. The control group consisted of 239 healthy individuals with comparable composition as to race and sex. GSTM1 and GSTT1 were analyzed by multiplex PCR. Comparison between patients and control group revealed a significant (P=0.0003) overrepresentation of combined deletions of both GSTM1 and GSTT1 (double null genotype) in the group of patients with AML/MDS secondary to chemo- and/or radiotherapy of a carcinoma of the breast. In this group, 55% of the patients displayed the double null genotype as compared with 8.8% in the control group. We conclude that patients with carcinoma of the breast and inheritance of a combined gene deletion of GSTM1 and GSTT1 might bear an increased risk to develop a secondary therapy-induced hematologic neoplasia. An insufficient detoxification of cytostatic drugs such as cyclophosphamide is suggested to represent the underlying pathomechanism.

Mutagenicity of the glutathione and cysteine S-conjugates of the haloalkenes 1,1,2-trichloro-3,3,3-trifluoro-1-propene and trichlorofluoroethene in the Ames test in comparison with the tetrachloroethene-analogues

The nephrotoxic and nephrocarcinogenic potential of the haloalkenes is associated with the conjugation of the chemicals to L-glutathione. Subsequent processing of the haloalkene glutathione S-conjugates via the cysteine conjugate beta-lyase pathway in the mammalian kidney yields nephrotoxic and mutagenic species. To investigate whether S-conjugates of the model chlorofluoroalkenes 1,1,2-trichloro-3,3,3-trifluoro-1-propene (CAS # 431-52-7) and trichlorofluoroethene (CAS # 359-29-5) show comparable effects, we have synthesised the respective cysteine and glutathione S-conjugates and subjected them to the Ames test. The cysteine and glutathione S-conjugates of tetrachloroethene (CAS # 127-18-4), S-(1,2,2-trichlorovinyl)-L-cysteine (TCVC) and S-(1,2,2-trichlorovinyl)glutathione (TCVG) were used as positive controls and reference substances. S-(1,2-dichloro-3,3,3-trifluoro-1-propenyl)-L-cysteine (DCTFPC) and S-(2,2-dichloro-1-fluorovinyl)-L-cysteine (DCFVC) showed clear dose-dependent mutagenic effects with the Salmonella typhimurium tester strains TA100 and TA98. Using TCVC as a reference substance the following ranking in mutagenic response was established: TCVC>DCTFPC>DCFVC. S-(1,2-dichloro-3,3,3-trifluoro-1-propenyl)glutathione (DCTFPG) and S-(2,2-dichloro-1-fluorovinyl)glutathione (DCFVG) showed potent dose-dependent mutagenic effects with the S. typhimurium tester strain TA100 in the presence of a rat kidney S9-protein fraction; tests carried out in the absence of the bioactivation system resulted only in background rates of revertants. Using TCVG as a reference substance the following ranking in mutagenic response was established: TCVG=DCTFPG>DCFVG. The data obtained provide a basis for further studies on the mutagenic and presumable carcinogenic potential of the substances.

Monitoring and Analysis of Occupational Exposure to Chain Saw Exhausts

The extent of inhalation exposure to loggers from two-stroke chain saws was measured and evaluated under various conditions. Carbon monoxide, measured by personal air monitoring and determination of carboxyhemoglobin levels of the loggers, was used as an indicator of exhaust exposure. Video recordings were made to analyze the influence of varying working conditions and the individual handling of the chain saw on the amount of pollutants inhaled. The American Conference of Governmental Industrial Hygienists biological exposure index (BEI) for carboxyhemoglobin (3.5%) was exceeded during logging in heavy timber stands. When workers were paid on a piecework basis, carboxyhemoglobinemia increased to its maximum level in the first 2-3 hours of the shift and then declined. After 8 hours carboxyhemoglobin levels were 20-30% lower compared with the maximum. Increased exhaust inhalation with short-term exposures to carbon monoxide up to 400 ppm was observed in the following conditions: (1) felling operations, (2) other operations performed in a leaning of squatting position, (3) limbing in thick tops of coniferous trees, (4) working at low wind velocity, and (5) working in thick forest stands. Maximum allowable blood concentrations for carboxyhemoglobin are exceeded in chain saw operators in logging operations. Blood sampling at the end of the workday is not always suitable for determining the highest carboxyhemoglobin levels in loggers during the shift. The exposure of chain saw operators to exhaust increases under certain conditions.

Exhaust Gas Emissions and Health Effects from Biodiesel, Fossil Diesel Fuel, and Swedish Low Sulfur Diesel Fuel MKI

In Germany more than 500.000 tons of biodiesel (rape seed oil methylester, RME) were produced in 2001. More than 600.000 tons are expected in 2002. In the U.S.A. biodiesel derives from both, rape seed oil and soy bean oil. It is necessary to judge the environmental and health effects that derive from the use of biodiesel in combustion engines. A comparison of regulated and non regulated emissions and of the mutagenic potency of particulate matter has been carried out at the example of a state of the art diesel engine. As fuels European diesel fuel (DIN EN 590), Swedish low sulfur diesel fuel (MKI) and rapeseed oil methylester were tested.

Detection of unknown toxic mycotoxins inAspergillus nidulans using a structure-activity approach

Mycotoxins are secondary metabolites of filamentous fungi that can cause various acute and chronic toxic effects in humans. Previous work by Büngeret al. exhibited that the cytotoxicityof Aspergillus nidulans, one of the most frequent toxigenic moulds in composting plants, could not be explained by its content of identified mycotoxins. The presence of additional mycotoxins or other toxic prinpiples was assumed, which may be detected by a structure-activity approach.An HPLC-diode array detector method was used to separate and characterize the components of theA. nidulans-extract within 50 minutes/analysis. Aliquots of the extract were chromatographed and nine 5-minutes-fractions were collected and lyophilized. Rechromatography of aliquots of the residues confirmed the accuracy of the 5-minutes-cuts.The cytotoxicity of these fractions was estimated in three cell lines (A-549, L-929 and Hep-G2) using the neutral red assay (NRU assay). Ethanol/dichloromethane (1:1, v/v) was proven to be a suitable solvent mixture with a low cytotoxicity. HPLC-fractions were dissolved in this mixture prior to the NRU assay. Three 5-minutes-fractions exhibited a strong cytotoxicity in this screening system and will be further analysed to identify the underlying unknown toxic principles.