Mixture genotoxicity of 2,4-dichlorophenoxyacetic acid, acrylamide, and maleic hydrazide on human Caco-2 cells assessed with comet assay.

Assessment of genotoxic properties of chemicals is mainly conducted only for single chemicals, without taking mixture genotoxic effects into consideration. The current study assessed mixture effects of the three known genotoxic chemicals, 2,4-dichlorophenoxyacetic acid (2,4-D), acrylamide (AA), and maleic hydrazide (MH), in an experiment with a fixed ratio design setup. The genotoxic effects were assessed with the single-cell gel electrophoresis assay (comet assay) for both single chemicals and the ternary mixture. The concentration ranges used were 0-1.4, 0-20, and 0-37.7 mM for 2,4-D, AA, and MH, respectively. Mixture toxicity was tested with a fixed ratio design at a 10:23:77% ratio for 2.4-D:AA:MH. Results indicated that the three chemicals yielded a synergistic mixture effect. It is not clear which mechanisms are responsible for this interaction. A few possible interactions are discussed, but further investigations including in vivo studies are needed to clarify how important these more-than-additive effects are for risk assessment.

Genotoxic potential of two herbicides and their active ingredients assessed with comet assay on a fish cell line, epithelioma papillosum cyprini (EPC).

The aim of this study was to optimize the epithelioma papillosum cyprini (EPC) cell line handling procedure for the comet assay to investigate the genotoxic potential of widely used pesticides. The effects of various media and handling of the EPC cell line were examined. Results indicated that avoiding trypsin to detach cells led to lower level of DNA damage in the negative control. Further, two commonly used herbicides (Dezormon and Optica trio) and their four active ingredients (4-chloro-o-tolyloxyacetic acid, 2,4-dichlorophenoxyacetic acid, 2-(4-chloro-2-methylphenoxy)propionic acid, 2-(2,4-dichlorophenoxy)propionic acid) individually and in a ternary mixture were examined with the comet assay. Data showed that among the active ingredients only 2,4-D and MCPA induced DNA damage, while both herbicides were genotoxic at high concentrations.

Comet assay on tetraploid yeast cells.

Tetraploid yeast cells (Saccharomyces cerevisiae) were used in the comet assay with the intention of developing a new, fast and easy assay for detecting environmental genotoxic agents without using higher organisms. Two DNA-damaging chemicals, H(2)O(2) and acrylamide, together with wastewater from three municipal treatment plants were tested for their effect on the yeast-cell DNA. The main problem with using yeast in the comet assay is the necessity to degrade the cell wall. This was achieved by using Zymolase 100 T twice during the procedure, since Zymolase 20 T did not open the cell wall. Analytical problems that arose due to the small amount of DNA in the yeast nuclei in haploid and diploid cells, which contain 13 Mbp and 26 Mbp DNA per cell, respectively, were solved by using tetraploid yeast cells (52 Mbp) instead. DNA damage was shown after exposure to H(2)O(2) and acrylamide. The lowest dose causing significant DNA damage was 20 microM for H(2)O(2) and 200mg/l for acrylamide. Tertiary-treated wastewater from the outlets of three municipal wastewater-treatment plants was tested, but did not cause DNA damage. Even though it is possible to produce comets with tetraploid yeast cells, the amount of DNA is likely too small for a proper comet assay.

Intersex in Littorina littorea and DNA damage in Mytilus edulis as indicators of harbour pollution.

Intersex in snails (Littorina littorea) and DNA damage in blue mussels (Mytilus edulis) were analysed to assess how these bio-indicators reflected the level of chemical contamination at two sites in a highly contaminated harbour in Denmark. The comet assay using mussel gill cells was an indicator of exposure to genotoxic chemicals, and the intersex index (ISI) observed in snails was an indicator of exposure to butyltin (BT) compounds. Biota and sediments were analysed for heavy metals (Cd, Cu, Pb and Zn), butyltin compounds (TBT, DBT and MBT), nine PCB congeners and 19 PAH compounds. The biological effects were found to reflect the levels of the chemicals, and it was concluded that intersex in L. littorea and DNA damage in M. edulis can be used as bio-indicators of harbour pollution.

Genotoxicity, inflammation and physico-chemical properties of fine particle samples from an incineration energy plant and urban air.

Airborne particulate matter (PM) was sampled by use of an electrostatic sampler in an oven hall and a receiving hall in a waste-incineration energy plant, and from urban air in a heavy-traffic street and from background air in Copenhagen. PM was sampled for 1-2 weeks, four samples at each site. The samples were extracted and examined for mutagenicity in Salmonella typhimurium strains TA98, YG1041 and YG5161, for content of inorganic elements and for the presence of eight polycyclic aromatic hydrocarbons. The induction of IL-6 and IL-8 mRNA expression and the presence of DNA damage - tested by the comet assay - were determined after 24-h incubations with human A549 lung epithelial cells. The PM(2.5) concentration was about twofold greater in the oven hall than in the receiving hall. The particle size distribution in the receiving hall was similar to that in street air (maximum mode at about 25nm), but the distribution was completely different in the oven hall (maximum mode at about 150nm). Also chemically, the samples from the oven hall were highly different from the other samples. PM extracts from the receiving hall, street and background air were more mutagenic than the PM extracts from the oven hall. PM from all four sites caused similar levels of DNA damage in A549 cells; only the oven hall samples gave results that were statistically significantly different from those obtained with street-air samples. The receiving hall and the urban air samples were similarly inflammatory (relative IL-8 mRNA expression), whereas the oven hall did not cause a statistically significant increase in IL-8 mRNA expression. A principal component analysis separated the oven hall and the receiving hall by the first principal component. These two sites were separated from street and background air with the second principal component. Several clusters of constituents were identified. One cluster consisted of all the polycyclic aromatic hydrocarbons (PAH), several groups of metals and one group of the biological endpoints (DNA damage, IL-6 and IL-8 mRNA expression). The PAH and the inorganic content of the air in the receiving hall may be due to vehicle emissions and suspended waste particles. The inorganic content in the street and background air may have been influenced by break wear, road emissions and long-range transport. The results from a partial least-square regression analysis predicted that both PAHs and a group of metals including Fe and Mn contributed to IL-6 and IL-8 induction. Only Mn and Sr were predicted to influence DNA damage statistically significantly. Among the PAHs only chrysene had influence on DNA damage. The PM from the oven hall was markedly different from the PM at other locations in particle size distribution, chemical composition and the resulting biological effects when A549 cells were incubated with the PM. These characteristics and observations in the oven hall indicated that the PM source was oven exhaust, which was well combusted.

DNA damage, acetylcholinesterase activity and lysosomal stability in native and transplanted mussels (Mytilus edulis) in areas close to coastal chemical dumping sites in Denmark.

Biomarkers of genotoxicity (DNA damage, measured as tail moment in the Comet assay), neurotoxicity (acetylcholinesterase inhibition, AChE) and general stress (lysosomal membrane stability, LMS) were studied in native and transplanted blue mussels (Mytilus edulis) in coastal areas of western Denmark potentially affected by anthropogenic pollution originating from chemical dumping sites. The results indicate responses to pollution in all the biomarkers applied at the suspected areas, but the results were not consistent. Seasonal fluctuations in exposure situations at the study sites make interpretation of chemical pollution complex, as seen especially in the variability in results on DNA damage, and also in regard to AChE activity. These investigations further stress the importance of understanding the effects of natural factors (salinity, temperature, water levels, rain and storm events) in correct interpretation of the biomarker data obtained. In addition, adaptation of populations to local contamination may play a role in some of the response patterns observed.

Monitoring DNA damage in indigenous blue mussels (Mytilus edulis) sampled from coastal sites in Denmark.

Damage to DNA detected by use of the single-cell gel electrophoresis (comet) assay was monitored in blue mussels, Mytilus edulis, sampled from coastal waters in Denmark. Mussels from five locations in Køge Bay, an area receiving wastewater from many industries and municipalities, were collected five times during 1999 and six times in 2001. In 1999, both gill cells and haemolymph cells were examined, and sediments were sampled on three dates from the same five locations. In the autumn of 1999, mussels were also collected at six reference sites without known pollution. Results showed a significantly higher level of DNA damage in gill cells compared with haemolymph cells. Because of this, only gill cells were sampled for the monitoring in 2001. Levels of DNA damage, expressed as tail moments, were significantly higher for the mussels in Køge Bay when compared with levels of DNA damage in mussels from the non-polluted coastal areas. No clear seasonal variation was demonstrated. Analysis of the correlation between chromium, nickel, cadmium and mercury in sediments and tail moments in haemolymph and gill cells from the five sites showed a statistically significant positive correlation between tail moments and chromium, nickel and cadmium (P<0.01). The overall conclusion was that the comet assay on blue mussels could be useful for screening of genotoxic pollution in marine waters.

Comet assay on gill cells and hemocytes from the blue mussel Mytilus edulis.

Gill cells and hemocytes from the blue mussel Mytilus edulis were examined for DNA damage using the comet assay after laboratory exposure in vitro and in vivo to methyl methansulfonate (MMS). Hydrogen peroxide and UV radiation were used as positive control. Comet assay was also carried out on hemocytes from blue mussels sampled at polluted and unpolluted coastal areas. After 60 min in vitro exposure of gill cells to MMS, the highest response, a tail moment of 6.70+/-4.25, was obtained at 1.0mg/L. At higher doses the response decreased. After 2 days in vivo exposure a dose response was seen at concentrations between 1.0 and 33.0mg/L MMS for both gill cells and hemocytes. However, after 4 days in vivo exposure using the same concentrations of MMS, a maximum effect was seen at a 10 times lower concentration of 3.3mg/L. At the higher doses, the effect decreased. Hemocytes from blue mussels sampled at four polluted sites in Køge Bay had a great variation in tail moments with the highest value of 5.38+/-4.39. The average of all samples from Køge Bay had tail moments of 2.75+/-1.00(n=19), which was significantly higher (P<0.05) than the average, 1.72+/-1.16(n=10), of samples from unpolluted coastal waters.