Hexachlorobenzene exerts genotoxic effects in a humpback whale cell line under stable exposure conditions

Abstract

Humpback whales, like other polar wildlife, accumulate persistent organic pollutants. In Southern hemisphere populations, hexachlorobenzene (HCB) dominates the contaminant profiles. HCB is linked to a variety of health effects and is classified as a group 2B carcinogen, but the mechanism of action is a matter of contention. Potential toxicological effects to humpback whales remain entirely unknown. The recently established humpback whale fibroblast cell line (HuWa) offers an in vitro model for toxicological investigations. We here combine this novel cell line with a passive dosing strategy to investigate whale-specific toxicity of HCB. The relevant partitioning coefficients were determined to produce stable and predictable exposure concentrations in small-scale bioassays. The system was used to assess acute toxicity as well as genotoxicity of HCB to the HuWa cell line. While we found some transient reductions in metabolic activity, measured with the indicator dye alamarBlue, no clear acute toxic effects were discernible. Yet, a significant increase in DNA damage, detected in the alkaline comet assay, was found in HuWa cells exposed to 10 μg L^-1 HCB during the sensitive phase of cell attachment. Collectively, this work provides a ready-to-use passive dosing system and delivers evidence that HCB elicits genotoxicity in humpback whale cells.

Fig. 1. Characterisation of the HCB passive dosing setup. (a) Loading of silicone O-rings. Top: time to steady-state by equilibrium partitioning from a loading buffer of methanol/water 60 : 40 (v/v) at room temperature and 250 rpm. Points represent the mean of three technical replicates (i.e. three independently prepared O-rings), error bars represent the standard deviation (SD). Bottom: resulting concentrations of HCB in silicone established from varying concentrations of HCB in LB. Means of three to twelve technical replicates with SD (note the differential scaling of the upper and lower segments of the axes). (b) Pre-equilibration of DMEM/F12 exposure medium. Top: time to steady-state in DMEM/F12 (1% FBS) with submerged HCB-loaded silicone O-rings in amber glass vials at 37 °C and 250 rpm. Means of three technical replicates with SD. Bottom: resulting concentrations of HCB in DMEM/F12 (solid symbols ●: with 1% FBS; open symbols ○: serum-free) established from varying concentrations of HCB in silicone. Means of three to six technical replicates with SD. (c) Concentration stability during exposure of cells. Concentration of HCB in exposure medium DMEM/F12 (1% FBS) immediately after pre-equilibration (at 0 hours, open symbol ○), and after transfer to a 24-well plate containing a confluent monolayer of cells (1 × 10⁴ cells per cm² seeded two days prior to exposure), with a loaded O-ring placed afloat, covered with aluminium foil, sealed and incubated at 37 °C and 30 rpm (solid symbols ●). Means of three technical replicates with SD. (d) Partitioning coefficients for HCB. Between LB and silicone (sil), and between sil and DMEM/F12.

Fig. 2. Impact of HCB exposure on cell viability. (a) Impact of HCB on established monolayers. Attached monolayers of HuWa_TERT were exposed to 1, 5, and 10 μg L⁻¹ HCB using the pre-equilibrated DMEM/F12 (1% FBS) exposure medium and silicone O-rings for passive dosing. Metabolic activity and membrane integrity were assessed relative to control after 3, 6, and 24 hours. Plots represent the median, 1^st and 3^rd quartile, and whiskers the 5^th and 95^th percentile of three biological replicates. (b) Impact of HCB on cells during attachment. HuWa_TERT cells in suspension were continuously exposed to 1, 5, and 10 μg L⁻¹ HCB using pre-equilibrated DMEM/F12 (1% FBS) exposure medium with passive dosing during the phase of attachment to well bottoms. Metabolic activity and membrane integrity were assessed relative to control after 3, 6, and 24 hours. Plots represent the median, 1^st and 3^rd quartile, and whiskers the 5^th and 95^th percentile of three biological replicates.

Fig. 3. Tail intensities of cells treated with HCB compared to HCB-free controls. (a) HCB-induced DNA damage. The comet assay was performed with HuWa_TERT cells after exposure to 10 μg L⁻¹ HCB for three hours during the phase of attachment to well bottoms using pre-equilibrated DMEM/F12 (1% FBS) exposure medium with passive dosing. Plots represent the mean and 95% CI of mean tail intensity of four biological replicates with 100 analysed cells each. (b) Apparent concentration dependency of HCB-induced DNA damage. The comet assay was performed after HuWa_TERT cells were exposed to 1, 5, and 10 μg L⁻¹ HCB for three hours during the phase of attachment to well bottoms using pre-equilibrated DMEM/F12 (1% FBS) exposure medium with passive dosing. Plots represent the median, 1^st and 3^rd quartile, and whiskers the 5^th and 95^th percentile, of one biological replicate with 100 analysed cells each in the control and the HCB-treated groups.