Xenobiotic metabolism and its physiological consequences in high-Antarctic Notothenioid fishes

Abstract

The Antarctic ecosystem is progressively exposed to anthropogenic contaminants, such as polycyclic aromatic hydrocarbons (PAHs). So far, it is largely unknown if PAHs leave a mark in the physiology of high-Antarctic fish. We approached this issue via two avenues: first, we examined the functional response of the aryl hydrocarbon receptor (Ahr), which is a molecular initiating event of many toxic effects of PAHs in biota. Chionodraco hamatus and Trematomus loennbergii served as representatives for high-Antarctic Notothenioids, and Atlantic cod, Gadus morhua as non-polar reference species. We sequenced and cloned the Ahr ligand binding domain (LBD) of the Notothenioids and deployed a GAL4-based luciferase reporter gene assay expressing the Ahr LBD. Benzo[a]pyrene (BaP), beta-naphthoflavone and chrysene were used as ligands for the reporter gene assay. Second, we investigated the energetic costs of Ahr activation in isolated liver cells of the Notothenioids during acute, non-cytotoxic BaP exposure. In the reporter assay, the Ahr LBD of Atlantic cod and the Antarctic Notothenioids were activated by the ligands tested herein. In the in vitro assays with isolated liver cells of high-Antarctic Notothenioids, BaP exposure had no effect on overall respiration, but caused shifts in the respiration dedicated to protein synthesis. Thus, our study demonstrated that high-Antarctic fish possess a functional Ahr that can be ligand-activated in a concentration-dependent manner by environmental contaminants. This is associated with altered cost for cellular protein synthesis. Future studies have to show if the toxicant-induced activation of the Ahr pathway may lead to altered organism performance of Antarctic fish.

Supplementary information: The online version contains supplementary material available at 10.1007/s00300-021-02992-4.

Keywords: Aryl hydrocarbon receptor; Hepatocyte metabolism; Luciferase reporter gene assay; Notothenioids; Polycyclic aromatic hydrocarbons.

Fig. 1

Alignment of the ligand binding domain (LBD) and PAS-B amino acid sequences of rainbow trout, zebrafish, killifish, Atlantic cod and high-Antarctic Notothenioid Ahrs. The amino acid sequences were aligned using CLC Main Workbench. Differences in amino acid sequences are shaded in grey. The LBD is highlighted by black brackets, the PAS-B domain is depicted by a grey box. GenBank accession numbers are: rainbow trout (Oncorhynchus mykiss) Ahrβ #NP001117724.1, zebrafish (Danio rerio) Ahr2 #NP571339.1, killifish (Fundulus heteroclitus) Ahr2 #AAC59696.3, Gadus morhua Ahr2, Chionodraco hamatus Ahr, Trematomus loennbergii Ahr, Antarctic eelpout (Pachycara brachycephalum) Ahrβ #KY747527.1

Fig. 2

Phylogenetic analysis of fish Ahr amino acid sequences. The amino acid sequences were aligned using CLC Main Workbench. The phylogenetic tree was constructed by the neighbour-joining method using CLC Main Workbench, with the branch lengths corresponding to the evolutionary distance between sequence clusters. The GenBank accession of the sequences are as follows: seabream Ahr2 #AAN05089, killifish (Fundulus heteroclitus) Ahr1 #O57452, killifish (Fundulus heteroclitus) Ahr2 #AAC59696, goldfish (Carassius auratus) Ahr1 #ACT79400, goldfish Ahr2 #ACT79401, zebrafish (Danio rerio) Ahr1 #NP571103, zebrafish (Danio rerio) Ahr2 #NP571339, red seabream (Pagrus major) Ahr2 #BAE02825, Atlantic salmon (Salmo salar) Ahr1 #NP_001117158.1 Atlantic salmon (Salmo salar) Ahr2α #NP_001117156, Antarctic eelpout (Pachycara brachycephalum) Ahr2α #KY747528, Atlantic cod (Gadus morhua), Chionodraco (Chionodraco hamatus) Ahr2 and Trematomus (Trematomus loennbergii) Ahr2

Fig. 3

Ligand activation dose–response curves of Atlantic cod (Gadus morhua) (n = 3) Ahr2 ligand binding domain (Gm Ahr2), Chionodraco hamatus (n = 3) Ahr ligand binding domain (Ch Ahr LBD) and Trematomus Loennbergii (n = 3) Ahr ligand binding domain (Tl Ahr LBD). The ligand activation of Ahr by selected test compounds (a = beta-naphthoflavone (BNF), b = Benzo[a]pyrene (BaP), c = chrysene) is reported as fold-increase in luciferase activity in cells exposed to the test compound over cells exposed to solvent (dimethyl sulfoxide, not shown). Each data point represents the mean of triplicate wells measured in five independent experiments (± sem). The dose–response curves were fitted by non-linear regression (GraphPad Prism). * displays a statistically significant difference in luciferase activities in compound-exposed compared to dimethyl sulfoxide -treated cells (ANOVA, p ≤ 0.05. Detailed statistics are given in Table S3, supplementary). # shows a significant difference in luciferase activities compared to the luciferase activity of Chionodraco hamatus (ANOVA, p ≤ 0.05. Detailed statistics are given in Table S3, supplementary)

Fig. 4

Oxygen consumption (MO₂) of liver cells of Trematomus loennbergii (n = 8, plain bars) and Chionodraco hamatus (n = 6, striped bars). Hepatocyte MO₂ was measured under control and BaP-exposed conditions at 0 °C. Values are displayed as means ± sem

Fig. 5

Energy expenditure for protein biosynthesis in control (sham) and BaP-exposed liver cells of Trematomus loennbergii (n = 8, plain bars) and Chionodraco hamatus (n = 6, striped bars) assayed at 0 °C. Protein biosynthesis is displayed as % of control (sham) respiration. The # depicts a significant difference (unpaired t-test, t₆ = 3.536, p = 0.012) to Chionodraco hamatus at the respective assay conditions. The asterisk displays a significant difference (unpaired t-test, t₄ = 2.163, p = 0.048)) to the protein biosynthesis in BaP-exposed liver cells of Trematomus loennbergii. Values are means ± sem