To order to identify genes which show changes in the brain in response to MeHg exposure and may play a role in causing the decreased swimming activity in zebrafish embryos, the toxicogenomic screen is performed in parallel to the study of the marker genes of the CNS or the neurotransmitter system. Through the toxicogenomic screen, I identified 24 genes which were specially regulated in the brain under MeHg exposure. These genes are involved in diverse biological processes and molecular functions.
In response to MeHg exposure, the expression of ADP-ribosylation factor-like (arfl) was up-regulated in scattered cells in the brain (Fig. 3.8 A, A’) and such an expression was also observed in other parts of the body (supplementary disc). arf are members of the Arf family of the Ras superfamily of GTPase enzymes which can bind and hydrolyse GTP (Gillingham and Munro et al., 2007; Wennerberg et al., 2005). They are involved in the regulation of membrane traffic and organisation of the cytoskeleton (Donaldson and Honda, 2005; D'Souza-Schorey and Chavrier, 2008).
activating transcription factor 3 (atf3) was induced in patches of cells in the brain in
implicated in immune responses and oncogenesis (Hai et al., 2010; Thompson et al., 2009).
The expression of angiotensinogen (agt) was slightly increased in the boundaries between forebrain and midbrain and between midbrain and hindbrain and in the outer region of the tectum in embryos exposed to MeHg (Fig. 3.8 C, C’). agt is a part of the renin-angiotensin system that plays an important role in the regulation of blood pressure and water balance. In both human and rat brains, Agt was shown to be expressed by astrocytes (Intebi et al., 1990; Stornetta et al., 1988; Wosik et al., 2007). In the human brain, AGT was required for blood-brain barrier maintenance (Wosik et
al., 2007).
The expression of CCAAT/enhancer binding protein (C/EBP), gamma (cebpg) was up-regulated in the outer region of the tectum after MeHg treatment (Fig. 3.8 D, D’).
cebpg is a transcription factor of the C/EBP family which regulates viral and cellular
CCAAT/enhancer element-mediated transcription. A pervious study showed that the levels of expressions of cebpg correlated with those of antioxidant and DNA repair genes in normal bronchial epithelial cells (Mullins et al., 2005).
chromobox protein homolog 7-like (cbx7l) was induced in patches of cells in the brain
in MeHg-treated embryos ((Fig. 3.8 E, E’, arrows). It belongs to the polycomb group proteins which repress transcription epigenetically through the remodelling of chromatin and are involved in the control of cell cycle and oncogenesis (Morey and Helin, 2010; Schwartz, Pirrotta, 2008). Studies using human cancer cell lines showed that CBX7 was highly expressed in cells of urothelial carcinomas (Hinz et al., 2008), prostate cancer (Bernard et al., 2005), follicular lymphomas (Scott et al., 2007) and gastric cancer (Zhang et al., 2010), but down-regulated in those of thyroid carcinoma (Pallante et al., 2008) and pancreatic cancer (Karamitopoulou et al., 2010).
The expression of cocaine and amphetamine regulated transcript protein type I-like
(cartIl) in small groups of cells located in the brain was slightly increased after MeHg
exposure (Fig. 3.8 F, F’, arrows). Cart has been shown to be involved in multiple physiological functions, including feeding, endocrine regulation, and mediation of the
expressed and the expression was noted in neurons in the hypothalamus (Elias et al., 2001).
The expression of v-fos FBJ murine osteosarcoma viral oncogene homolog (fos) was strongly induced in the dorsal part of the brain after MeHg exposure (Fig. 3.8 G, G’).
Fos is a transcription factor with a leucine-zipper DNA binding domain. It is
important in normal and pathological conditions, and its deregulation has been shown to be linked with a variety of pathological conditions including oncogenesis, hypoxia, and neurological, immunological and skeletal defects (Herdegen and Waetzig, 2001; Milde-Langosch, 2005; Prabhakar and Kumar, 2004; Saitoh et al., 1993; Wagner and Eferl, 2005). In rodents, medium- or long-term dietary intake of Hg-contaminated rice were also shown to increase Fos expression levels in the brain (Cheng et al., 2005; 2009).
glutamate-cysteine ligase modifier subunit (gclm) showed a general up-regulation in
the brain in MeHg-treated embryos (Fig. 3.8 H, H’). It encodes a part of the enzyme glutamate cysteine ligase which is critical for the synthesis of the GSH antioxidant (Lu, 2009). Increased Gclm expression levels were observed in conditions of oxidative stress caused by various substances (Lavoie et al., 2009; Thompson et al., 2009; Woo et la., 2008). In human patients, polymorphism of GCLM has been reported to be associated with coronary endothelial vasomotor dysfunction and myocardial infarction (Koide et al., 2003; Nakamura et al., 2003).
homeodomain leucine zipper gene (homez) was strongly up-regulated in the brain,
especially in the dorsal hindbrain region after MeHg treatment (Fig. 3.8 I, I’). It is a transcription factor containing leucine-zipper like motifs (Bayarsaihan et al., 2003). In mouse, it showed restricted expressions in the brain, the optic vesicle and the otic placode during later stages of embryonic development (Bayarsaihan et al., 2003).
interferon regulatory factor (irf9) showed a ubiquitous up-regulation in the brain in
MeHg-treated embryos (Fig. 3.8 J, J’). Irf are transcription factors that involve in the protective defences of the immune system through the regulation of the transcription
the ability of uninfected host cells to resist new infection by virus (Gastl and Huber, 1988; Pestka et al., 1987; Taniguchi et al., 2001).
The expression of lysosomal-associated membrane glycoprotein 1-like (lamp1l) was up-regulated in the brain, especially in the epithelium lining the brain ventricle (Fig. 3.8 K, K’). Lamp1 together with Lamp2 account for about 50% of all the proteins of the lysosome membranes (Hunziker et al., 1996). In mouse embryonic fibroblasts, lacking both Lamp1 and Lamp2 resulted in an accumulation of autophagic vacuoles and disturbed cholesterol metabolism (Eskelinen et al., 2004).
opn1lw1 showed a general decrease in expression levels in the epiphysis in addition to
in the eye after MeHg exposure (Fig. 3.8 L, L’). opn1lw1 encodes a long-wavelength sensitive cone opsin having maximum light absorption at the red light region. Opsins are light-responsive membrane-bound G protein-coupled receptors of the retinylidene protein family (chromophore, vitamin A derivatives) found in photoreceptor cells (Nickle and Robinson, 2007).
6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4-like (pfkfb4l) showed an
increase in expression in the whole brain after MeHg treatment (Fig. 3.8 M, M’).
pfkfb4 is a member of the Pfkfb enzymes which induce glycolysis through activating
6-phosphofructo-1-kinase and blocking gluconeogenesis by inhibiting fructose-1,6- bisphosphatase (Pilkis et al., 1995). The expressions of PFKFBs have been shown to be induced in cancer cells and by hypoxia (Minchenko et al., 2005a, 2005b, 2005c).
ppp1r15a was up-regulated in the tegmentum and rhombomeres after MeHg exposure
(Fig. 3.8 N, N’). Ppp1r15a encodes the growth arrest and DNA damage 34 (GADD34) protein which dephosphorylates the alpha subunit of translation initiation factor 2 (eIF2α), promoting the recovery of protein synthesis in the unfolded protein response (a response to the accumulation of unfolded or misfolded proteins in the lumen of endoplasmic reticulum) (Novoa et al., 2001). Ppp1r15a mutant cells exhibit impaired recovery of protein synthesis while Ppp1r15a knockout mice are phenotypically indistinguishable from wild type (Kojima et al., 2003; Novoa et al., 2003).
The expression of peroxiredoxin1 (prdx1) was slightly up-regulated in the hypothalamus after MeHg exposure (Fig. 3.8 O, O’). Prdx1 is a member of the antioxidant thioredoxin-dependent peroxidase family (Immenschuh and Baumgart- Vogt, 2005; Rhee et al., 2005). It protects cellular components by removing the low levels of ROS produced as a result of normal cellular metabolism and is a stress- inducible antioxidant (Ishii and Yanagawa, 1997; Rhee et al., 2005). Prdx1 is also known as natural killer enhancing factor A (NkefA) and in human cell lines, it has been shown to be implicated in enhancing natural killer cell activity and antivirual activity (Geiben-Lynn, et al., 2003; Shau et al., 1993).
SAR1a gene homolog (sara2) showed mild up-regulation in the brain in embryos
exposed to MeHg (Fig. 3.8 P, P’). sara2 is a zebrafish homologue of the yeast S.
cerevisiae SAR1, which is also a small GTPase of the Arf family as arfl (Gillingham
and Munro et al., 2007). It is found in COPII vesicles (a type of vesicle which transports proteins from the rough endoplasmic reticulum (ER) to the Golgi apparatus) (Bi et al., 2002) and it regulates the membrane constriction, assembly and fission of the vesicles and the transport of ER (Bielli et al., 2005; Long et al., 2010). The expression of sine oculis homeobox homolog 3a (six3a) in the forebrain was up- regulated after MeHg exposure (Fig. 3.8 Q, Q’). six3a is a member of the Six gene family which was first identified in Drosophila for compound-eye formation (Kawakami et al., 2000). In zebrafish, six3a plays important roles in both eye and rostal forebrain developments (Kobayashi et al., 1998; Seo et al., 1998).
solute carrier family 16 (monocarboxylic acid transporters), member 9a (slc16a9a)
showed a general decrease in the expression in the whole brain after MeHg treatment (Fig. 3.8 R, R’). slc16a9a is a member of Slc16 gene family (Meredith and Christian, 2008). Some members of this family catalyse the transport of monocarboxylates such as lactate, pyruvate and ketone or bodies aromatic amino acids (Halestrap and Meredith, 2004; Meredith and Christian, 2008). However, the substrate(s) of slc16a9a is still unknown.
belongs to the UDP-Glucuronosyltransferase (Ugt) superfamily. Members of this superfamily encode enzymes responsible for the glucuronidation of the xenobiotics, producing the more water soluble glucuronic acid-bound xenobiotics, and thus, aid the excretion of the xenobiotics from the body (King et al., 2000; Miners et al., 2002). The Ugt5 family exist only in teleosts and amphibians, but not in reptiles, birds or mammals (Huang and Wu, 2010).
Eight other genes with unknown identity including zgc:101661, AW115990,
BE016163, BI474700 and BM036361 were also ectopically expressed in the brain
after MeHg exposure (Fig. 3.8 T, T’ – X, X’). The changes in the expression patterns of these 24 genes in the brain might suggest the induction of the behavioural changes reported by Samson et al. (2001) after MeHg exposure.
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Fig. 3.8 Changes in gene expression in the zebrafish brain after MeHg treatment.
Lateral (A, A’- E, E’, G, G’- K, K’, M, M’ – N, N’, P, P’- V, V’) and dorsal (F, F’, L L’, O, O’, W, W’, X, X’) views of 72-hpf-old control (A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P, Q, R, S, T, U, V, W, X) and MeHg-treated (A’, B’, C’, D’, E’, F’, G’, H’, I’, J’, K’, L’, M’, N’, O’, P’, Q’, R’, S’, T’, U’, V’, W’, X’) embryos performed with in situ hybridisation are shown. The dotted line in A indicates the position of the eye; the arrows in L, L’, X, X’ mark the epiphysis. Up-regulations of arfl (A, A’), atf3 (B, B’), agt (C, C’), cebpg (D, D’), cbx7l (E, E’), cartIl (F, F’, the patches of cells with increased expression are indicated by the arrows), fos (G, G’), gclm (H, H’), homez (I, I’), irf9 (J, J’), lamp1l (K, K’), pfkfb4l (M, M’), ppp1r15a (N, N’), prdx1 (O, O’, the asterisks indicate the hypothalamus), sara2 (P, P’), six3a (Q, Q’, eyes removed for better observation of the expression pattern in the brain), utg5a2 (S, S’), zgc:101661 (T, T’), AW115990 (U, U’), BE016163 (V, V’), BI474700 (W, W’) and BM036361 (X, X’), and down-regulations of opn1lw1 (L, L’) and slc16a9a (R, R’) were observed in the brain of