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Bile and liver metallothionein behavior in copper-exposed fish

https://doi.org/10.1016/j.jtemb.2013.09.003Get rights and content

Abstract

The present study analyzed metallothionein (MT) excretion from liver to bile in Nile Tilapia (Oreochromis niloticus) exposed to sub-lethal copper concentrations (2 mg L−1) in a laboratory setting. MTs in liver and bile were quantified by spectrophotometry after thermal incubation and MT metal-binding profiles were characterized by size exclusion high performance liquid chromatography coupled to ICP-MS (SEC-HPLC–ICP-MS). Results show that liver MT is present in approximately 250-fold higher concentrations than bile MT in non-exposed fish. Differences between the MT profiles from the control and exposed group were observed for both matrices, indicating differential metal-binding behavior when comparing liver and bile MT. This is novel data regarding intra-organ MT comparisons, since differences between organs are usually present only with regard to quantification, not metal-binding behavior. Bile MT showed statistically significant differences between the control and exposed group, while the same did not occur with liver MT. This indicates that MTs synthesized in the liver accumulate more slowly than MTs excreted from liver to bile, since the same fish presented significantly higher MT levels in liver when compared to bile. We postulate that bile, although excreted in the intestine and partially reabsorbed by the same returning to the liver, may also release MT-bound metals more rapidly and efficiently, which may indicate an efficient detoxification route. Thus, we propose that the analysis of bile MTs to observe recent metal exposure may be more adequate than the analysis of liver MTs, since organism responses to metals are more quickly observed in bile, although further studies are necessary.

Introduction

Metallothioneins (MTs) are considered good biomarkers for metal exposure. Liver is expected to accumulate the highest metallothionein levels in the body, since it is the major detoxifying organ [1]. Previous research has indicated that metallothioneins are excreted in varying degrees in mammals. For example, Bremner et al. [2] verified that the metallothionein isoform MT-I is excreted in varying amounts into blood, urine and bile in rats, while Jaw and Jeffery [3] observed that, in rats administered zinc, mercury or cadmium, cadmium was excreted into the bile in inverse proportion to the hepatic metallothionein content, while metallothionein content did not appear to bear any relationship to biliary excretion of mercury or zinc. Mohan et al. [4], on the other hand, analyzing biliary copper excretion in the neonatal rat verified that biliary copper excretion per day in young rats is relatively low in the first week of life and is independent of MT secretion.

No studies regarding biliary MT in fish, however, were available until recently, when Hauser-Davis et al. [5] reported that MTs are excreted from the liver into bile in fish as an alternative detoxification route, and that they follow the same trend as hepatic MT in situations of environmental metal exposure. Previous studies show that sub-lethal copper concentrations directly affect hepatic MT [6], however no studies regarding bile MT in these situations are available.

Nile tilapia (Oreochromis niloticus) are good sentinel organisms for metal exposure, as they are particularly exposed to sediment-associated contamination such as metals [7], since metals in general adsorb to sediments and particulate matter and they are limno-benthofagous, feeding on suspended particles.

The biliary excretion of several compounds of environmental concern has been validated as an alternative indicator of environmental exposure in fish, since this matrix also excretes substances from blood and liver that have not been excreted by the kidneys, such as metals and organic compounds [8], [9]. In between feeding bile accumulates in the gallbladder and, to accommodate the continual influx of fresh bile, water is continuously extracted from the gallbladder, concentrating bile and its constituents (i.e., biliary proteins, xenobiotics). This, allied to the fact that the rapid metabolism and elimination of several contaminants by vertebrates usually results in low concentrations in muscle and liver, indicating limited usefulness of the chemical analysis of fish tissues in environmental contamination situations [10]. Bile analysis in these cases, therefore, is more advantageous, since it is a less complex matrix than liver, concentrates and rapidly excretes xenobiotics and other compounds and, there may be no need to sacrifice animals, since bile can be obtained by chronic cannulation of the hepatic bile duct [6].

The aim of the present study, therefore, was to analyze hepatic and bile metallothionein in tilapia exposed to sub-lethal copper levels compared to a control group and investigate biliary and hepatic MT accumulation and metal binding behavior. Chronic copper contamination is of great concern in freshwater bodies in general, and more so in Brazil, where high levels of this environmental contaminant are present in several regions, justifying the choice of this metal in the present study [11].

Section snippets

Sample collection and laboratory exposure experiment

Nile Tilapia specimens (O. niloticus) (n = 20) were obtained from a commercial aquaculture facility in southeastern Brazil, in the municipality of Silva Jardim, Rio de Janeiro, in which fish are raised specifically for human consumption in tanks under controlled conditions. The fish were taken to the laboratory and acclimated in two 500 L tanks containing dechlorinated tap water at pH 7.0. Copper, in the form of copper sulfate (CuSO4·5H2O) was dissolved to obtain 2 mg L−1 of Cu in each tank. This

SDS-PAGE analyses

After the purification process, qualitative Laemmli 1D electrophoresis showed the presence of 14 kDa protein bands in liver and bile samples (fig. 1), as described previously for Nile tilapia, for both liver and bile, respectively [5], [21]. Other faint bands when present in the gels were significantly above or below this molecular weight, excluding their identity as MT. Therefore, the purification procedure was considered adequate for the present study. The objective of the SDS-PAGE analysis

Conclusions

The comparison between biliary and liver metallothionein in Nile tilapia in the present study indicates that metal exposure causes increased MT expression in both liver and bile, although this difference was non-significant in liver, and that bile MT seems to be a better indicator for Cu exposure than hepatic MT, since biliary MT excretion showed significantly different MT expression than liver MT. Because of this, bile MT analysis would be more adequate than liver MT if the aim were to observe

Conflict of interest

The authors report no conflict of interest.

Acknowledgment

The main author was granted a post-doctorate fellowship from CNPQ – National Research Council, Brazil. MAZA is also thankful to the Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP, São Paulo, Brazil.

References (31)

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