Genotoxicity of Heterocyclic PAHs in the Micronucleus Assay with the Fish Liver Cell Line RTL-W1

Heterocyclic aromatic hydrocarbons are, together with their un-substituted analogues, widely distributed throughout all environmental compartments. While fate and effects of homocyclic PAHs are well-understood, there are still data gaps concerning the ecotoxicology of heterocyclic PAHs: Only few publications are available investigating these substances using in vitro bioassays. Here, we present a study focusing on the identification and quantification of clastogenic and aneugenic effects in the micronucleus assay with the fish liver cell line RTL-W1 that was originally derived from rainbow trout (Oncorhynchus mykiss). Real concentrations of the test items after incubation without cells were determined to assess chemical losses due to, e.g., sorption or volatilization, by means of gas chromatography-mass spectrometry. We were able to show genotoxic effects for six compounds that have not been reported in vertebrate systems before. Out of the tested substances, 2,3-dimethylbenzofuran, benzothiophene, quinoline and 6-methylquinoline did not cause substantial induction of micronuclei in the cell line. Acridine caused the highest absolute induction. Carbazole, acridine and dibenzothiophene were the most potent substances compared with 4-nitroquinoline oxide, a well characterized genotoxicant with high potency used as standard. Dibenzofuran was positive in our investigation and tested negative before in a mammalian system. Chemical losses during incubation ranged from 29.3% (acridine) to 91.7% (benzofuran) and may be a confounding factor in studies without chemical analyses, leading to an underestimation of the real potency. The relative potency of the investigated substances was high compared with their un-substituted PAH analogues, only the latter being typically monitored as priority or indicator pollutants. Hetero-PAHs are widely distributed in the environment and even more mobile, e.g. in ground water, than homocyclic PAHs due to the higher water solubility. We conclude that this substance class poses a high risk to water quality and should be included in international monitoring programs

Corrected and uncorrected REPs relative to NQO, as well as respective corrected and uncorrected EC₂₅ values in the micronucleus assay with RTL-W1 cells.

<p>Chemical losses in the micronucleus assay used for calculation of corrected EC₂₅ (i.e. by multiplying the residual compound fraction with the EC₂₅) and REP values were derived from GC-MS measurements in 6-well microplates without cells.</p><p><i>n.d.: Inactive in assay system, i.e. substances which did not reach 25% induction of the NQO standard; n.a.: not available; STD: standard substance; LOQ: limit of quantification.</i></p><p>¹<i>Maximun tested concentrations based on cytotoxicity data from Hinger & Brinkmann et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085692#pone.0085692-Hinger1" target="_blank">[<i>13</i>]</a>.</p

Exemplary concentration-response curve (carbazole) measured in the micronucleus assay with RTL-W1 cells (closed circles).

<p>The concentration-response curve for NQO (open circles) and the blanks, i.e. control cells without treatment (open square and lower dashed line), are given for reference. Induction factors are fold-changes relative to blanks. EC₂₅ values relative to the maximum induction of NQO (upper dashed line) were calculated to derive fixed-effect-level-based REPs. Concentration values on the x-axis refer to nominal medium concentrations of the substances. Circles represent mean values measured in duplicate experiments, error bars the standard deviation. The red line depicts 25% of the maximum induction caused by the standard NQO. Asterisks denote statistically significant differences compared to blanks (one-way ANOVA with Dunnet’s test, <i>p</i>≤0.05).</p

Uncorrected and corrected relative potency (REP) values of hetero-PAHs.

<p>Uncorrected values refer to nominal concentrations and corrected to measured concentrations in the exposure medium after incubation without cells. <i>N.d.: inactive in assay system, i.e. substances which did not reach the stipulated level of 25% induction of the NQO standard.</i></p

Dose-response curves in the micronucleus assay with RTL-W1 cells for all investigated heterocyclic compounds (closed circles).

<p>Standard curves for NQO (open circles) and blanks, i.e. control cells without treatment (open squares, grey line), are given for reference. Induction factors are fold-changes relative to blanks. Concentration values on the x-axis refer to nominal medium concentrations of the substances. Dots represent mean values measured in duplicate experiments, error bars the standard deviation. Asterisks denote statistically significant differences compared to blanks (one-way ANOVA with Dunnet’s test, <i>p</i>≤0.05).</p