Additional file 1 of Biotechnological metabolization system has the potential to improve the predictive ability of the fish embryo acute toxicity (FET) test with the zebrafish (Danio rerio)
Creators: name: Reichstein, Inska S.
name: Becker, Alischa H.
name: Johann, Sarah
name: Braunbeck, Thomas
name: Schiwy, Sabrina
name: Hollert, Henner
name: Schiwy, Andreas
Publisher: name: figshare
Year: 2024
Subjects: subject: Biochemistry
subject: Neuroscience
subject: Physiology
subject: FOS: Biological sciences
subject: Chemical Sciences not elsewhere classified
subject: Ecology
subject: Inorganic Chemistry
subject: FOS: Chemical sciences
subject: Mental Health

description: Additional file 1: Table S1. List of chemicals. Table S2. List of materials. Table S3. List of lethal-equivalent and sublethal endpoints in the Fish Embryo Acute Toxicity Test (FET). Table S4. EC50, EC10 and hill slope values derived from FET data in zebrafish (Danio rerio) embryos at 120 h post fertilization (hpf) for the five model pro-toxicants. EC50, EC10 and hill slope values were calculated with (w/) or without (w/o) addition of S9 homogenates (0.01 mg/ml rat S9 or 0.05 mg/ml ewoS9R combined with 0.5 mM NADPH); 95% profile likelihood confidence limits in brackets. n.d. = not determinable. Figure S1. Range-finding experiments for the identification of optimal pre-metabolization conditions. Effects [%] of two S9 homogenates and NADPH zebrafish (Danio rerio) embryos at 120 h post fertilization (hpf) in the FET. Model for nonlinear regression (GraphPad Prism 9): [agonist] vs. response, variable slope (four parameters), bottom = 0%, top = 100%. Dotted lines indicate 10% effect (considered the non-effect threshold according to OECD TG 236). Data given as mean values ± standard deviation from independent biological replicates (n = 3 – 4). Figure S2. EROD activity of S9 homogenates respresented as nmol formed resorufin/mg proteinS9-homogenate in presence of 0.5 mM NADPH and 100 µM dicoumarol. The amount of resorufin formed was calculated by additionally recording the fluoresence of resorufin at excitation/emission wavelenghts of 588 nm/635 nm (concentration range 0—4 µM) in the presence of 0.5 mM NADPH and 100 µM dicoumarol (see Formula S1). The mean and error bars show the calculated nmol resorufin formed/mg protein (n = 3) with standard deviation. Figure S3. Concentration–response curve (concentration [μM] vs. effects [%] in zebrafish (Danio rerio) embryos at 120 h post fertilization (hpf) after expsoure to bisphenol A without NADPH and with or without addition of S9 homogenates. S9 homogenates were used at the concentrations of 0.01 mg/ml (rat S9) or 0.05 mg/ml (ewoS9R). Process controls contained S9 homogenates only and NADPH at the same concentrations, but no test substance. Data given as mean values ± standard deviatiom from n = 3—4 independent biological replicates. Model for nonlinear regression (GraphPad Prism 9): [agonist] vs. response, variable slope (four parameters), bottom = 0%, top = 100%. * Significant differences of the best-fit calues (hill slope, EC50) with S9 homogenate from runs without addition of metabolization. Figure S4. Overview on the type and quantity ratios of the effects after exposure to model substances at 120 h post fertilization (hpf)) in the FET with Danio rerio. For this, the observed effects of all tested concentrations were summed up to determine the percentage frequency of the individual effects. Blood congestion, no/reduced heartbeat, no/slow blood flow and heart deformations are all considered as “cardio-vascular effects”. The S9-homogenates were used at the concentrations of 0.01 mg/ml (rat S9) or 0.05 mg/ml (ewoS9R) together with or without 0.5 mM NADPH. Figure S5. Biotransformation potential of two metabolization systems within the FET using Danio rerio embroys at 48 h post fertilization (hpf). Allyl alcohol (A), TDCPP (B), bisphenol A (C), benzo[a]pyrene (D), chlorpyrifos (E) were tested with or without the addition of 0.01 mg/ml rat S9 or 0.05 mg/ml ewoS9R together with 0.5 mM NADPH. Process controls contained only S9 homogenates and NADPH at the same concentrations w/o chemicals. Data are given as means ± standard deviation (n = 3–4). Model for nonlinear regression (GraphPad Prism 9): [agonist] vs. response, variable slope (four parameters), bottom = 0%, top = 100%. *Significant differences of the best-fit values (hillslope, EC50) with S9 homogenate compared to no addition of an external metabolization system.