Heavy Metals-Induced Expression of ABCB 10 Gene in Zebrafish Danio rerio

ABCB10 is a mitochondrial ATP-binding cassette (ABC) transporter. It is involved in multi-drug resistance and it has a potential role in the regulation of oxidative stress induced by toxic substances, such as heavy metals. Little is known about the role of ABCB10 gene in metal detoxification in fishes generally and Zebrafish particularly. Also the effect of Tubifex worms on the gene expression has not been discussed before, despite the importance of these worms in metals transfer and bioavailability. Therefore, the impact of contaminated sediment with Cu and Cu on ABCB10 gene expression was investigated in this study. In addition, the effect of Tubifex worms on the expression of Zebrafish ABCB10 gene was also studied. Zebrafish was exposed for 7 days to contaminated sediment with 315 mg/kg and 84.8 mg/kg of Cu and Cd, respectively. Relative gene expression was recorded in Zebrafish different organs (brain, gills, muscles and digestive tract), in 4 experimental groups in presence and absence of Tubifex worms. The highest level of ABCB10 expression was recorded in digestive tract samples in all tested groups followed by muscles then gills, while brain samples recorded the least induction level for ABCB10 expression. Tubifex worms showed an effective influence on Zebrafish ABCB10 expression with higher up-regulation level compared with those recorded for Cu and Cd contamination.


ABCB10
is a multi-drug resistance protein, it is also called Mitochondrial ATP-binding cassette, sub-family B (MDR/TAP), member 10 (Thiriet, 2012).This protein encoded by ABCB10 gene which is localized in the mitochondrial inner membrane.
The ABCB10 gene is conserved in Chimpanzee, Rhesus monkey, dog, cow, mouse, rat, chicken, Zebrafish, fruit fly, mosquito and C.elegans.It is highly expressed during erythroid differentiation and hemoglobin synthesis (Liesa et al., 2012).ABCB10 gene may regulate oxidative stress by its transport activities.Also it has a potential therapeutic role against diseases involved in increasing mitochondrial ROS production and oxidative stress (Liesa et al., 2012).Therefore, it participates in mitochondrial antioxidant response in eukaryotic cells (Hyde et al., 2012).
Heavy metal is a serious source of contamination threatening all aquatic systems specially lakes.Due to lake's closed system, toxic materials can rapidly accumulate in water, sediment and aquatic organisms (Gurcu et al., 2010).
One of the most heavily polluted lakes in Egypt is Lake Manzala (Abdel-Gawad & El-Sayed, 1998).
Heavy metals contamination has a genotoxic effects on the aquatic organisms (Myers et al., 2008;Valko et al., 2006), by activating the production of reactive oxygen species (ROS), leading to oxidative stress and DNA damage (Farombi et al., 2007), that could be transferred to the next generations (Bolognesi & Hayashi, 2011).As a defense mechanism against heavy metal toxicity, aquatic organisms activate the cellular detoxification mechanisms.Different studies have illustrated the role of some genes in multi-xenobiotic resistance process to extrude toxins outside the cell.Such genes are ABC transporters (Aleo et al., 2005;Kingtong et al., 2007;Morgan et al., 2007).
Zebrafish, Danio rerio is one of the perfect vertebrate models for toxicology, pharmacology, developmental biology, and genetics studies.Its importance as a model organism is due to its small size, short life cycle, rapid growth and its completed genome sequence (http://www.ensembl.org/Danio_rerio)(Broughton et al., 2001;Gilbert, 2006).
Danio rerio has been extensively used in toxicological studies to investigate the mutagenic effects of water pollution on the integrity of DNA (Gonzalez et al., 2006;Scholz et al., 2008;Popovic et al., 2010).
Another macrobenthic organism is used as a bioindicator to sediment contamination , Tubifex tubifex worm is a conveyor belt feeder that has a great role in metal transfer and bioavailability between sediment and water column during its feeding process (Anschutz et al., 2012;Ciutat & Boudou, 2003).Moreover, it is used as a favourable food for Zebrafish.
Few previous studies investigated the role of ABCB10 gene in cellular detoxification and toxins secretion in humans (Zhang et al., 2000), but no recorded reference about the role of Zebrafish ABCB10 in heavy metals detoxificatoion.In addition, it is not previously studied the impact of Tubifex worms on the gene expression of ABCB10 gene in Zebrafish.
Therefore, the aim of this work was to investigate the impact of contaminated sediment (with relevant concentrations of Cu and Cd found in Lake Manzala) on Zebrafish ABCB10 expression.The effect of Tubifex worms on Zebrafish ABCB10 gene expression was also investigated.

MATERIALS AND METHODS Zebrafish Danio rerio maintenance
Adult males D. rerio were purchased from a local pet farm in Bordeaux-France, and transferred to the acclimation tank at 21 º C. Fishes were fed twice a day (50mg diet /fish/day) for acclimation period of 30 days.

Tubifex tubifex maintenance
Sixty batches of Tubifex worms, 200 worms each were purchased from natural pond (SARL GREBIL père & fils, Paris -France).Worms were acclimated on clean sediment from Garonne River, Bordeaux-France at 20º C for 15 days.

Heavy metals treatment
Sediment from Garonne River, Bordeaux-France was sampled and sieved.Mixture of Copper (Copper standard, Cu-Titrisol, Merck) and Cadmium (Cadmium standard, CdCl 2 -Titrisol, Merck) stock solutions were added and homogenized with the sediment to have a final concentration of 315 mg/kg and 84.8 mg/kg of Cu and Cd respectively.Sediment was stored at 4ºC for 7 days before the beginning of the experimental protocol.
Four Zebrafishes were distributed in each unit, while worms were distributed as 56000 worms/m 2 in D and G conditions only.All experimental unites were arranged in a thermostatic chamber in Arcachon marine station, Arcachon-France.
Water temperature was set at 21º C (optimum temperature for Zebrafish and Tubifex worms) (Schaefer & Ryan, 2006), with source of aeration and 12 hr photoperiod of florescent light for 7 days of exposure.

Total RNA Isolation
After 7 days of exposure, 5 fish samples from each condition were dissected to collect skeletal muscles, digestive tract, gills and brain samples.All tissue samples were stored in RNA later solution (Qiagene) at -80 ºC.Total RNAs were isolated from tissues samples using absolutely RNA miniprep kit (Agilant), then first strand of cDNA was synthesized using Affinity Script cDNA synthesis kit (Agilent).

Primers design
Primers for Zebrafish ABCB10 gene (XM_001343182) and the housekeeping gene β-Actin gene (NM_131031) were designed according to their sequence in the GenBank.

Statistical analysis
The deferential gene expression was tested for significance using non parametric Mann-Whitney test (P<0.05),after applying the Shapiro-Wilk normality test (1% risk) using SigmaStat 3.5 program.

Relative expression of Zebrafish ABCB10 gene
After 7 days of exposure to contaminated sediment with Cu and Cd, the relative gene expression of ABCB10 gene to β-actin was evaluated in Zebrafish different organs in presence and absence of Tubifex worms.
Among the 4 different tissues, the highest level of Zebrafish ABCB10 gene expression was recorded in digestive tract samples in all tested groups.Samples from control group D recorded the highest induction in the gene expression compared to samples from all other groups, while gill samples showed very low level of gene expression in all treatments.
Generally, D. rerio different organs almost recorded same trend in gene expression levels in the 4 experimental groups, as digestive tract samples showed the highest level of gene expression followed by muscles samples then gills and brain samples (Fig. 1).Transcriptional response of Zebrafish ABCB10 gene to Cu and Cd contamination Impact of Cu and Cd contamination on ABCB10 gene expression was evaluated by comparing the relative gene expression of ABCB10 gene in contaminated group F against control group C, and contaminated group G against control group D. Significant up-regulation by 6.4-times was recorded in muscles samples of the contaminated group F compared to C group.In addition to upregulation in the gene expression by 2.2-, 4.1-and 3.4-times in brain, gills and digestive tract samples respectively.The presence of Tubifex in both control group D and the contaminated group G had a significant effect on Zebrafish ABCB10 expression.Tubifex worms caused downregulation in gene expression in the 4 tested organs of the contaminated group G compared to D group samples (Table1and Fig. 2).Transcriptional response of Zebrafish ABCB10 gene to Tubifex worms Zebrafish ABCB10 expression was highly up-regulated in presence of Tubifex worms in control group D compared to control group C; brain, gills, muscles and digestive tract samples recorded 3.8-, 14.3-, 13.1-and 28.4-times up-regulation respectively.
The presence of worms in contaminated environment showed down-regulation in Zebrafish ABCB10 gene in brain, gills, and muscles samples of G group compared to F group.In addition to 2.3-times up-regulation in the gene expression in digestive tract samples of G group compared to F group samples (Table 1and Fig. 3).DISCUSSION ABCB10 gene expression was used in this study to invistigate Zebrafish defense mechanism against polymetallic contamination with Cu and Cd.Tubifex worms were used for its effective role in metal transfer and bioavailabilty between sediment-water column as bioturbator organism (Ciutat & Boudou, 2003).Therefore, its influence on Zebrafish ABCB10 expression was also investigated in this study.
ABCB10 is a mitochondrial ATPbinding cassette transporter, sub family B (MDR/TAP) member 10.It is involved in multidrug resistance, heme biosynthesis in liver and spleen (Nilsson et al., 2009), heme transport (Lee et al., 2012), and it is highly expressed in erythrocytes (Chambers et al., 2007).
Results of relative gene expression showed high induction in gene expression in digestive tract samples in all the experimental groups, with the highest expression level in the control group D (+fish+worms), followed by samples from group G, then samples from group F and group C. ABCB10, as a member of ABCB transporters which are located in bloodbrain barrier, liver, mitochondria and participates in peptides and bile transports (Annilo et al., 2006), and it has a serious role in cellular detoxification through lipophilic anion extrusion (Bellamy, 1996) .It was clear from the differential gene expression results that ABCB10 gene was upregulated in 4 tested organs as a result of metallic contamination with Cu and Cd, as it has a serious role in heavy metals detoxification (Bellamy, 1996).
The comparison of relative gene expression in tissue samples in the contaminated group G (+fish+worms) against control group D (+fish+worms) showed significant down-regulation in gene expression in all tissues samples, * * Heavy Metals-Induced Expression of ABCB10 Gene in Zebrafish Danio rerio 103 this is due to the high level of gene expression in control group D in all the tested organs.
It was surprising to notice that Tubifex worms were more effective on Zebrafish ABCB10 gene expression than Cu and Cd contamination.The significant high level of ABCB10 gene expression in presence of Tubifex in uncontaminated sediment in group D compared to group C could be explained due to the stimulatory effect of these worms on fishes attraction and exploration behaviour (Saglio et al., 1990).More exploration behaviour may cause more consumption of Tubifex as food, and more activity in the digestive tract to store many substances and to produce bile in the liver, then more expression of ABCB10 gene to extrude bile and to participate in blood detoxification (Field et al., 2003).
While the effect of Tubifex worms was significantly decreased in contaminated sediment, heavy metals contamination of the sediment might have caused inhibition in worms activities (Arrate et al., 2004) , less consumption of Tubifex as food, which could have less effect on the exploration and attraction activities of Zebrafish with less expression for ABCB10 gene.
The final results confirmed that there are two main organs that are associated in dissolved metal uptake inside aquatic animal's body, gills and digestive tract, while muscles are the final storage site for metals (Amiard et al., 1989;Lagadic et al., 1997).Therefore, it was very clear that digestive tract recorded the highest expression level followed by muscles then gills which act as a short time storage organ for heavy metals (Amiard et al., 1989;Lagadic et al., 1997).The least affected organ with heavy metals contamination in the presence of Tubifex worms was brain.
The combined effect of Cu and Cd contamination and Tubifex worms was evaluated by comparing the relative gene expression of group G samples with the gene expression of group C samples.Results showed up-regulation in the gene expression in all tested organs with significant increase in brain and muscles samples in group G compared to group C.That increase in gene expression was less than the effect of the metallic contamination or Tubifex worms alone.It might be due to the fact that Tubifex can be additional source of contamination because of its high ability to bioaccumulate different metals such as Cd in its body (Arrate et al., 2004;Ciutat & Boudou, 2003).Subsequently, it acts as a source of contaminated diet for Zebrafish leading to serious levels of Reactive Oxygen Species (ROS), causing oxidative stress that might cause DNA damage, inhibition in cells control on apoptotic death and by the end all the functions inside the cells could be affected (Lee & Shacter, 1999;Turpaev, 2002).

Fig. 4 :
Fig. 4: Differential gene expression of Zebrafish ABCB10 in relation to both heavy metals contamination and Tubifex worms *Statistically Significant difference (p<0.05)

Heavy Metals-Induced Expression of ABCB10 Gene in Zebrafish Danio rerio 101Table 1 :
The differential expression of ABCB10 gene in Zebrafish Danio rerio