A gene to organism approach--assessing the impact of environmental pollution in eelpout (Zoarces viviparus) females and larvae

Abstract

A broad biomarker approach was applied to study the effects of marine pollution along the Swedish west coast using the teleost eelpout (Zoarces viviparus) as the sentinel species. Measurements were performed on different biological levels, from the molecular to the organismal, including measurements of messenger RNA (mRNA), proteins, cellular and tissue changes, and reproductive success. Results revealed that eelpout captured in Stenungsund had significantly higher hepatic ethoxyresorufin O-deethylase activity, high levels of both cytochrome P4501A and diablo homolog mRNA, and high prevalence of dead larvae and nuclear damage in erythrocytes. Eelpout collected in Göteborg harbor displayed extensive macrovesicular steatosis, whereby the majority of hepatocytes were affected throughout the liver, which could indicate an effect on lipid metabolism. Results also indicate that eelpouts collected at polluted sites might have an affected immune system, with lower mRNA expression of genes involved in the innate immune system and a higher number of lymphocytes. Biomarker assessment also was performed on livers dissected from unborn eelpout larvae collected from the ovary of the females. No significant differences were noted, which might indicate that the larvae to some extent are protected from effects of environmental pollutants. In conclusion, usage of the selected set of biological markers, covering responses from gene to organism, has demonstrated site-specific biomarker patterns that provided a broad and comprehensive picture of the impact of environmental stressors.

Keywords: Biomarker; Eelpout; Gene expression; Genotoxicity; Histopathology.

Figure 1

Eelpout sampling sites on the Swedish west coast in November 2011. Brofjorden is located close to a big oil refinery, Stenungsund is in the outlet of an area with chemical industries, and Göteborg harbor is Scandinavia's largest general oil port. Fjällbacka was used as a northern reference site. Billdal was used as a reference site for the genotoxic assays.

Figure 2

Erythrocytic nuclear abnormalities assay in eelpout erythrocytes: Images of different cell nucleus types. Erythrocyte with (A) micronucleus, (B) bleb, (C) bud, (D) lobed nucleus, (E) notched nucleus, (F) binucleated with bridge, (G) circular nucleus, and (H) binucleated erythrocyte.

Figure 3

Frequency of (A) micronuclei, (B) blebbed nuclei, (C) budded nuclei, (D) lobed nuclei, (E) notched nuclei, (F) binucleated nuclei with bridges, (G) circular nuclei, and (H) binucleated nuclei (mean ± standard error) in erythrocytes from eelpouts collected in Fjällbacka, Stenungsund, Göteborg, and Billdal (14–15 individuals per site). All fish used were sexually mature females with the exception of fish from Fjällbacka, which were a mix of males and juveniles. Fjällbacka and Billdal are considered as reference sites. Lower‐case letters indicate significant differences between sites (p < 0.05). nd = nuclear abnormalities not detected.

Figure 4

Quantitative polymerase chain reaction results for (A) complement component C7, (B) hepcidin, (C) lysozyme C, (D) CYP1A, (E) diablo homolog, and (F) DDIT4. Gene expression levels in Fjällbacka were set to 1, and levels in polluted sites were set accordingly (mean ± standard error). Lower‐case letters indicate significant differences between sites (p < 0.05). CYP1A = cytochrome P4501A; DDIT4 = DNA damage transcript 4.

Figure 5

(A) Reference liver section demonstrating mild macrovesicular steatosis (arrow). Scale bar = 100 µm. (B) Liver section demonstrating extensive macrovesicular steatosis characterized by the presence of elevated numbers of singular large vacuoles within hepatocytes. Vacuoles were often seen displacing nuclei within affected hepatocytes. Scale bar = 50 µm.

Figure 6

Dendrogram showing unsupervised hierarchical clustering (Euclidean distance) of 31 biological markers. The clustering was based on mean values for the markers measured in each of the 4 sites: Fjällbacka (reference), Brofjorden, Stenungsund, and Göteborg harbor. The biological markers were divided into 7 clusters, marked by color. DIABLO = diablo homolog; EROD = ethoxyresorufin O‐deethylase (CYP1A activity); CYP1A = cytochrome P4501A; DDIT4 = DNA damage transcript 4; tot. abnorm. fry = total abnormal fry; LysC = lysozyme C; GST = glutathione S‐transferase; GR = glutathione reductase; HN Pleomorph = hepatocellular and nuclear pleomorphism; Hb = hemoglobin; GSI = gonad somatic index; MA = macrophage aggregates; iRBC = immature red blood cells; MCHC = mean corpuscular hemoglobin concentration (ratio of Hb/Ht); CF = condition factor; regeneration = hepatocellular regeneration; C7 = complement component C7; Ht = hematocrit; LSI = liver somatic index; WBC = white blood cells.