Arsenic transport by zebrafish aquaglyceroporins

Abstract

Background: Arsenic is one of the most ubiquitous toxins and endangers the health of tens of millions of humans worldwide. It is a mainly a water-borne contaminant. Inorganic trivalent arsenic (AsIII) is one of the major species that exists environmentally. The transport of AsIII has been studied in microbes, plants and mammals. Members of the aquaglyceroporin family have been shown to actively conduct AsIII and its organic metabolite, monomethylarsenite (MAsIII). However, the transport of AsIII and MAsIII in in any fish species has not been characterized.

Results: In this study, five members of the aquaglyceroporin family from zebrafish (Danio rerio) were cloned, and their ability to transport water, glycerol, and trivalent arsenicals (AsIII and MAsIII) and antimonite (SbIII) was investigated. Genes for at least seven aquaglyceroporins have been annotated in the zebrafish genome project. Here, five genes which are close homologues to human AQP3, AQP9 and AQP10 were cloned from a zebrafish cDNA preparation. These genes were named aqp3, aqp3l, aqp9a, aqp9b and aqp10 according to their similarities to the corresponding human AQPs. Expression of aqp9a, aqp9b, aqp3, aqp3l and aqp10 in multiple zebrafish organs were examined by RT-PCR. Our results demonstrated that these aquaglyceroporins exhibited different tissue expression. They are all detected in more than one tissue. The ability of these five aquaglyceroporins to transport water, glycerol and the metalloids arsenic and antimony was examined following expression in oocytes from Xenopus leavis. Each of these channels showed substantial glycerol transport at equivalent rates. These aquaglyceroporins also facilitate uptake of inorganic AsIII, MAsIII and SbIII. Arsenic accumulation in fish larvae and in different tissues from adult zebrafish was studied following short-term arsenic exposure. The results showed that liver is the major organ of arsenic accumulation; other tissues such as gill, eye, heart, intestine muscle and skin also exhibited significant ability to accumulate arsenic. The zebrafish larvae also accumulate considerable amounts of arsenic.

Conclusion: This is the first molecular identification of fish arsenite transport systems and we propose that the extensive expression of the fish aquaglyceroporins and their ability to transport metalloids suggests that aquaglyceroporins are the major pathways for arsenic accumulation in a variety of zebrafish tissues. Uptake is one important step of arsenic metabolism. Our results will contribute to a new understanding of aquatic arsenic metabolism and will support the use of zebrafish as a new model system to study arsenic associated human diseases.

Figure 1

Phylogenetic tree of the zebrafish aquaporin and aquaglyceroporin family. Zebrafish sequences were obtained by blasting the protein database with human AQP9 and AQP1. Proteins with different sequences were chosen to be aligned.

Figure 2

Sequence alignment of five zebrafish aquaglyceroporins. Black background represents the identical residues while grey background represents the similar residues.

Figure 3

Expression of mRNA of zebrafish aquaglyceroporins.The expression of mRNA of aqp9a, aqp9b, aqp3, aqp3l, and aqp10 in isolated zebrafish tissues. Tissues were isolated from adult zebrafish, homogenized, and total cDNA was obtained. A zebrafish β-actin gene was used to be a positive control.

Figure 4

Water permeation and glycerol transport by zebrafish AQP-expression Xenopus oocytes. A. Water osmolarity (P_f) is determined in oocytes that have expressed zebrafish aquaglyceroporin genes. (n = 5 oocytes were used. P < 0.01 is calculated from water control and Aqp injected samples). B. Glycerol transport is determined by applying 0.5 mM [³H] glycerol for 30 min. The standard deviation was calculated using Sigma Plot 10 (n = 7 oocytes were used from same frog, P < 0.01 is calculated from water control and Aqp injected samples).

Figure 5

Uptake of As^III, MAs^III and Sb^III by zebrafish AQP-expression Xenopus oocytes. Various metalloids were added at 1 mM final concentration to oocytes that were injected by cRNA. Oocytes were incubated for 30 min and digested for metalloid quantification. The standard deviation was calculated from three replicates and plotted using Sigma Plot 10 (n = 7 oocytes were used, P < 0.01 calculated from water control and Aqp injected samples). A. Uptake of As^III. B. Uptake of MAs^III. C. Uptake of Sb^III.

Figure 6

Effect of short-term arsenic exposure on accumulation of arsenic in zebrafish. After arsenic treatment, organs from adult fish were isolated (n = 6) and homogenized. Totally 10 zebrafish larvae were pooled as one sample (n = 4). Standard deviation is determined by Sigma plot 10.