A rainbow trout liver cell line, RTL-W1, was used to evaluate the toxic potential of a heavy fuel oil (HFO) HFO 7102, and its fractions, which together with the HFO are referred to as the oil samples. The fractions were F2, F3, F3-1, F3-2 and F4 and had been prepared by low-temperature vacuum distillation by collaborators at Queen's University. For presentation to the cells, HFO 7102 and its fractions were made into High Energy-Chemically Enhanced Water Accommodated Fractions (HE-CEWAFs). The procedure for this involved adding Corexit 9500 to the oil samples, mixing them on a vortex, and letting the phases settle. The HE-CEWAFs were added to RTL-W1 cell cultures, and at various times afterwards cell viability and CYP1A induction were monitored. Cell viability was evaluated with two dyes, Alamar Blue, which monitors energy metabolism, and 5-carboxfluorescein diacetate acetoxymethyl ester (CFDA AM), which measures plasma membrane integrity. With both indicator dyes, Corexit 9500 was cytotoxic but the concentrations eliciting cytotoxicity varied with the cell culture media. In Leibovitz's L-15 with fetal bovine serum (FBS), which was the medium used for studying CYP1A induction, Corexit 9500 was only cytotoxic at concentrations of 0.1 % (v/v) and greater. For the oil samples, F3-2 at 1 mg/ml and F4 at 10 mg/ml, which were the highest testable concentrations for each, no loss of cell viability was observed over 24 h. The other oil samples were cytotoxic only at their highest testable concentrations, which ended being between 1 and 10 mg/ml. CYP1A induction was monitored in RTL-W1 as catalytic activity and as the level of CYP1A (P4501A) protein. The catalytic activity was assayed as 7-ethoxyresorufin o-deethylase (EROD) activity; the CYP1A protein level, by western blotting. The positive control was 2, 3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which strongly induced both EROD activity and CYP1A protein. Corexit 9500 by itself neither induced EROD activity nor CYP1A protein. All the oil samples induced both EROD activity and CYP1A protein. With both endpoints, the most potent fraction was F3; the least potent, F3-2. As the induction of CYP1A is associated with the development of blue sac disease (BSD) and mortality in early life stages of fish, the results suggest that HFO 7102 and its fractions have the potential to reduce recruitment of young into adult fish populations. CYP1A induction by F3 was studied further, again through EROD activity and western blotting. As the F3 concentration was increased, EROD activity increased but declined at high concentrations, whereas CYP1A protein continued to increase. This suggests the presence of compounds in F3 that at high concentrations inhibit the catalytic activity of CYP1A. When F3 was presented to RTL-W1 cultures together with TCDD, CYP1A protein was induced but not EROD activity. Again this suggests that F3 contains inhibitor(s) of CYP1A as well as inducers. When cultures were exposed to either F3 or TCDD for 24 h and then followed by western blotting for up to 6 days after F3 or TCDD removal, CYP1A levels declined in F3 cultures but not in TCDD cultures. This suggests that RTL-W1 were able to inactivate CYP1A inducer(s) in F3 through metabolism. Overall the results suggest that the pattern of CYP1A induction by F3, and by extension, HFO involves complex interactions between the many chemical components in these mixtures. Likely the most important chemicals are the polycyclic aromatic hydrocarbons (PAHs).

A rainbow trout liver cell line, RTL-W1, was used to evaluate the toxic potential of a heavy fuel oil (HFO) HFO 7102, and its fractions, which together with the HFO are referred to as the oil samples. The fractions were F2, F3, F3-1, F3-2 and F4 and had been prepared by low-temperature vacuum distillation by collaborators at Queen's University. For presentation to the cells, HFO 7102 and its fractions were made into High Energy-Chemically Enhanced Water Accommodated Fractions (HE-CEWAFs). The procedure for this involved adding Corexit 9500 to the oil samples, mixing them on a vortex, and letting the phases settle. The HE-CEWAFs were added to RTL-W1 cell cultures, and at various times afterwards cell viability and CYP1A induction were monitored. Cell viability was evaluated with two dyes, Alamar Blue, which monitors energy metabolism, and 5-carboxfluorescein diacetate acetoxymethyl ester (CFDA AM), which measures plasma membrane integrity. With both indicator dyes, Corexit 9500 was cytotoxic but the concentrations eliciting cytotoxicity varied with the cell culture media. In Leibovitz's L-15 with fetal bovine serum (FBS), which was the medium used for studying CYP1A induction, Corexit 9500 was only cytotoxic at concentrations of 0.1 % (v/v) and greater. For the oil samples, F3-2 at 1 mg/ml and F4 at 10 mg/ml, which were the highest testable concentrations for each, no loss of cell viability was observed over 24 h. The other oil samples were cytotoxic only at their highest testable concentrations, which ended being between 1 and 10 mg/ml. CYP1A induction was monitored in RTL-W1 as catalytic activity and as the level of CYP1A (P4501A) protein. The catalytic activity was assayed as 7-ethoxyresorufin o-deethylase (EROD) activity; the CYP1A protein level, by western blotting. The positive control was 2, 3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which strongly induced both EROD activity and CYP1A protein. Corexit 9500 by itself neither induced EROD activity nor CYP1A protein. All the oil samples induced both EROD activity and CYP1A protein. With both endpoints, the most potent fraction was F3; the least potent, F3-2. As the induction of CYP1A is associated with the development of blue sac disease (BSD) and mortality in early life stages of fish, the results suggest that HFO 7102 and its fractions have the potential to reduce recruitment of young into adult fish populations. CYP1A induction by F3 was studied further, again through EROD activity and western blotting. As the F3 concentration was increased, EROD activity increased but declined at high concentrations, whereas CYP1A protein continued to increase. This suggests the presence of compounds in F3 that at high concentrations inhibit the catalytic activity of CYP1A. When F3 was presented to RTL-W1 cultures together with TCDD, CYP1A protein was induced but not EROD activity. Again this suggests that F3 contains inhibitor(s) of CYP1A as well as inducers. When cultures were exposed to either F3 or TCDD for 24 h and then followed by western blotting for up to 6 days after F3 or TCDD removal, CYP1A levels declined in F3 cultures but not in TCDD cultures. This suggests that RTL-W1 were able to inactivate CYP1A inducer(s) in F3 through metabolism. Overall the results suggest that the pattern of CYP1A induction by F3, and by extension, HFO involves complex interactions between the many chemical components in these mixtures. Likely the most important chemicals are the polycyclic aromatic hydrocarbons (PAHs).

Gewässer sind Lebensgrundlage, jedoch gleichzeitig Schadstoffsenken für eine Vielzahl von Kontaminanten. Biologische Wirkungstests und das Biomonitoring aquatischer Proben sind daher besonders wichtig, um Umwelt-Gefahrenpotenziale erkennen zu können. Der "Comet Assay" (Einzelzell-Gelelektrophorese) ist ein Indikator von DNA-Strangbrüchen und wurde hier als Test auf gentoxische Wirkungen erprobt und angewandt. Mit bekannten, gentoxischen Substanzen wurden Nachweisgrenzen und Dosis-Wirkungs-Beziehungen für die Zelllinien RTG-2 und RTL-W1 (aus der Regenbogenforelle, Oncorhynchus mykiss) in vitro ermittelt und methodische Parameter an die Zellen angepasst. Der Test reagierte sehr sensitiv auf 4-Nitrochinolin-1-oxid. Die Substanz war daher geeignet, um in weiteren Versuchen als Positivkontrolle zu dienen. Zur Bewertung der Messdaten wurde ein geeignetes statistisches Verfahren gefunden, das auch historische Kontrollen mit einbezog. Der zeitliche Verlauf der DNA-Schädigung des Testsystems mit RTG-2-Zellen wurde ermittelt, und durch Inhibition der DNA-Reparatur mit Aphidicolin wurden Zusammenhänge zwischen der Entstehung von DNA-Strangbrüchen, der DNA-Reparaturkapazität sowie der Metabolisierungskapazität untersucht. In einer zweiten Phase wurden unbehandelte Wasserproben aus Rhein, Elbe sowie weitere Oberflächenwasserproben mit dem Comet Assay an RTG-2-Zellen getestet. Bei 15 von 49 Proben zeigten sich gentoxische Effekte. In einer dritten Phase wurden Erythrozyten von freilebenden Döbeln, Leuciscus cephalus, aus der Mosel mit dem Comet Assay untersucht. Die Fische von drei Messstellen zeigten erhöhte Werte von DNA-Schädigungen, gegenüber einer vierten, stromabwärts gelegenen Messstation. Korrelationen mit den Ergebnissen zusätzlicher Biomarker ergaben sich nur teilweise. Chemische Analysen von Wasser- oder Gewebeproben ließen keine Rückschlüsse auf verursachende Kontaminanten zu - gerade dies unterstreicht jedoch die Wichtigkeit biologischer Tests bei komplexen Proben. / Bodies of Water are both vital resources and pollutant sinks for a multitude of contaminants. Therefore, biological effect tests and biomonitoring of aquatic samples are of particular importance to detect potential environmental hazards. The "comet assay" (single cell gel electrophoresis) is an indicator for DNA strand breaks and was explored and applied as a genotoxicity test in the present study. Known genotoxic substances were used to determine the detection limits and dose-response relationships for the cell lines RTG-2 and RTL-W1 (from rainbow trout, Oncorhynchus mykiss) in vitro, and to adapt methodological parameters to the cells. The test was very sensitive to 4-Nitroquinoline-1-oxide. This substance was therefore well-suited to serve as positive control in further experiments. In order to evaluate the measurement data, an appropriate statistical procedure was developed, which also took "historical" controls into account. The time course of DNA damage in the test system using RTG-2 cells was determined, and relationships between the origin of DNA strand breaks, DNA repair capacity and the metabolizing capacity of the cells was investigated by means of inhibition of DNA repair with Aphidicoline. In the second stage, native water samples from the rivers Rhine and Elbe and further surface waters were tested with the comet assay, using RTG-2 cells. 15 out of 49 samples showed genotoxic effects. In a third stage, erythrocytes of feral chub, Leuciscus cephalus, from the Moselle river were examined with the comet assay. The fish from three measuring stations showed elevated values of DNA damage compared to fish sampled from a downstream station. There were only partly correlations with the results from additional biomarkers. Chemical analyses of water and tissue samples did not permit conclusions on effect-causing substances.However, this emphasizes the importance of biological tests in dealing with complex environmental samples.