NONYLPHENOL INDUCES EXPRESSION OF THE T-BOX6 GENE IN ZEBRAFISH EMBRYOS: MATERIALS AND METHODS

MATERIALS AND METHODS

Chemicals

The synthesized NP used in this study (Table 1) were synthesized and purified as described in Preuss et al (2006)18. All isomers had a purity >99%. Technical nonylphenol (t-NP) and dimethyl sulfoxide (DMSO 99,9%) was purchased from Sigma-Aldrich/Fluka (Deisenhofen, Germany). Substances used for RNA extraction were purchased from AppliChem (Darmstadt, Germany), enzymes used for cDNA synthesis and real time PCR were supplied by MBI Fermentas (St. LeonRot, Germany).

Table 1 : Characterization of the nonyphenol isomers including induced spinal malformations with effective concentration (EC50)

Abbreviation Name Structure Spinalmalformation EC50 ± SEM spinalmalformation[^mol/L]
p22 NP 4-(2’methyl-2’-octyl)phenol „XT1 very short tail n.d.
p252 NP 4-(2’,5’-dimetyl-2’heptyl)phenol HorrW^ bended tail 7.9 ± 16.2
p262 NP 4-(2’,5’-dimetyl-2’hexyl)phenol 4ОX bended tail 7.4 ± 0.8
p33 NP 4-(3’methyl-3’-octyl)phenol bended tail n.d.
p353 NP 4-(3’,5’-dimetyl-3 ’heptyl)phenol „oO^ broadened tail tip 14.5 ± 0.45
p363 NP 4-(3’,5’-dimetyl-3 ’hexyl)phenol swollen tail tip 10.0 ± 0.25
t- NP technical mixture bended tail 9.5 ± 0.58

Breeding of zebrafish

250 zebrafishes were held in breeding groups of 20 females and 30 males. Fish were kept at 26°C and a light/dark period of 14h/10h in tap water. For collection of eggs translucent plastic spawning boxes (12 x 20 x 12 cm) with a stainless steel mesh insert (3 – 4 mm mesh size) were placed in the aquaria. On top of the mesh some green plastic net material and green glass marbles animated the fish to spawn into the box. Although not all fish spawned into the boxes, up to 3000 eggs/day could be collected from the stock using 2 boxes for each breeding group. Eggs are 1.0 – 1.2 mm in diameter and have a transparent chorion. Fertilized and well developed eggs in the 4 to 8 cell stage were selected for the test and placed in 24-well dishes (Nunc GmbH, Wiesbaden, Germany). The mean spontaneous lethality rate was 2%. Fertilization rates were > 80%.

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