Teratogenic effects of solvents between 24 and 48 hours post-fertilization.

<p>Embryos at 2 dpf (<b>a, b, c, d</b>) treated for the previous 24 hours with <b>a</b>, embryo medium, <b>b</b>, 1% DMF, <b>c</b>, 1% solketal or <b>d</b>, 2% glycerol. Embryos in <b>b</b>, <b>c</b>, and <b>d</b> display no or very slow blood circulation as well as no touch response (all were immotile). Treatment with 1% DMF or 1% solketal also resulted in brain hemorrhaging (red arrows), pericardial edemas, yolk necrosis (black arrows) and shortening along the AP axis. <b>d</b>, Other than the observed blood flow defects, embryos treated with 2% glycerol appeared morphologically normal. All embryos are depicted with anterior to the left, dorsal to the top.</p

Teratogenic effects of solvents within the first 24 hours post-fertilization.

<p>Typical results are shown. Embryos at 1 dpf treated for the previous 24 hours with <b>a</b>, embryo medium (control) or <b>b,</b> 1% DMF (from 4-cell stage), <b>c</b>, 1% DMF (from 4 hpf), <b>d</b>, 0.5% albumin (from 4-cell stage), <b>e</b>, 1.5% acetonitrile (from 4 hpf), and <b>f</b>, 2.5% glycerol (from 4-cell stage). <b>b</b>, embryos treated from 4-cell stage with 1% DMF display severe epiboly defects. The eyes (red arrow) and some somites are still visible (blue arrow), but most other structures are no longer clearly discernable. Massive cell death is also present surrounding the yolk (black asterisk). <b>c</b>, 1% DMF applied from 4 hpf onwards no longer inhibits epiboly. However, embryos display body curvature and shortening, loss of ventral tail fin (green arrow), no clear brain segmentation (black arrow), pericardial edema and a widened choroid fissure (orange arrow). <b>d</b>, embryos treated with 0.5% albumin are morphologically normal but display a significant delay in development (up to 9 hours) relative to control. <b>e</b>, 1.5% acetonitrile-treated embryos display anterior-posterior axis truncation, body curvature, microcephaly, lack of a midbrain-hindbrain boundary, incomplete pigmentation in the eyes, trunk and tail, ill-defined somitic boundaries, a bumpy notochord and ‘roughened’ epidermis and fin structures. <b>f</b>, embryos treated with 2.5% glycerol display anterior-posterior axis truncation with no extended tail, u-shaped somites, and <i>cardia bifida</i> (purple arrows). With the exception of embryo in panel b, which is oriented with anterior to the top, dorsal to the right, all other embryos are depicted with anterior to the left, dorsal to the top.</p

Phenotypic effects of solvents and carriers above maximum tolerated concentrations after 24 hours.^*

*<p>Phenotypes with an average penetrance of at least 50% (in comparison to dead or apparently normal embryos or larvae) are indicated by the following abbreviations: AH, atrial hemorrhage; AP, anterior-posterior axis truncation; BC, body curvature; BH, brain hemorrhage; BS, brain segmentation defects; CB, <i>cardia bifida</i>; CF, widened choroid fissure; CY, cyclopia; DD, developmental delay; DH, delayed hatching; EH, elongated heart; EP, epiboly defects; GG, bright green gall bladders; HJ, hypomorphic jaw; HO, hypoactivity; HY, hyperactivity; IC, impaired circulation; IM, impaired motility (lack of touch response); IP, incomplete pigmentation; LP, loss of posture; MC, microcephaly; NB, necrosis in body cavity; NH, necrosis in head; NO, bumpy notochord; PE, pericardial edema; SB, incompletely inflated or uninflated swim bladder; SB+, enlarged swim bladder; SO, somite defects; YE, yolk sac edema. Phenotypes were not recorded for penetrance levels below 50% (i.e. where the majority of embryos or larvae were dead or apparently normal); these cases are denoted by “-”. The lowest concentration (in % of solvent or carrier) at which an average penetrance of at least 50% was observed for the shown phenotypic effects is given in parentheses.</p

Teratogenic effects of solvents between 72 and 96 hours post-fertilization.

<p>Zebrafish embryos at 4 dpf, treated for the previous 24 hours with <b>a</b>, embryo medium, or <b>b </b><b>and </b><b>c</b>, 2.5% DMSO. DMSO-treated larvae display either smaller or no swim bladders (red arrows) and necrosis in the yolk area surrounding the internal organs (white asterisk). All embryos are depicted with anterior to the left, dorsal to the top.</p

Maximum-tolerated concentrations of solvents and carriers in zebrafish embryos and larvae.

<p>This table depicts the highest concentrations for each solvent and carrier, at each developmental stage tested, at which no phenotypic abnormalities could be observed. Embryos and larvae were incubated in water containing each solvent or carrier at the concentration indicated, for 24 hours beginning at the developmental stage indicated. In determining the maximum tolerated concentration (MTC) for each solvent and carrier, all embryos and larvae were allowed to develop to 9 dpf, so as to detect any deleterious effects appearing after this 24-hour exposure window; none were detected at the MTCs shown.</p

Concentration-dependent toxicities of solvents and carriers in zebrafish embryos and larvae.

<p>Effects of various solvent or carrier concentrations on zebrafish embryos and larvae at different developmental stages. Green bars indicate a normal phenotype, blue bars indicate an abnormal phenotype, and red bars indicate embryos or larvae that were dead at the timepoint of analysis. All dysmorphologies and other aberrant phenotypes (e.g. slow heart rate, lack of circulation) are classified as “abnormal” phenotypes in this summary. Each bar represents collated data of 3 separate experiments, for a total of 30 embryos or larvae. The depicted developmental stage (e.g. 4 hpf or 3 dpf) represents the timepoint at which the solvent was added to the medium. Phenotypes were analyzed 24 hours later. Values that are significantly different from the control group are indicated by asterisks: *, p<0.05; **, p<0.01; ***, p<0.001.</p

Representative HPLC chromatograms of mouse brain homogenate analysis for ar-turmerone determination.

<p>A) blank mouse brain extract, B) blank mouse brain extract spiked with ar-turmerone (AT) and internal standard (IS) and C) brain extract sample of a dosed mouse. </p

Evaluation of the effect of ar-turmerone on mouse motor function and balance.

<p>An intravenous dose of 50 mg/kg of ar-turmerone (AT) does not cause any alteration of motor skills in mice. Sensitivity of this model was confirmed by detection of motor and balance deficits induced by diazepam (DPZ) in mice at 1 mg/kg. Compared to control group (VHC), DZP-treated mice displayed a significant increase in number of footslips (A), falls (B) and time to reach goal box (C). Statistically significant differences between control and sample groups are labeled as ** for p < 0.001 (one-way ANOVA test).</p

Evaluation of the anticonvulsant activity of ar-turmerone (AT) as determined by the 6-Hz model.

<p>A) After 30 min of i.p. administration, complete protection was observed for ar-turmerone at a dose range of 0.1 - 50 mg/kg. A group of vehicle-treated mice (VHC) was included as a negative control. Sodium valproate at 300 mg/kg (VAL300) and levetiracetam at 50 mg/kg (LEV50) were used as positive controls. B) After 24 h i.p. administration (50 mg/kg), protection was observed after electrical induction. Statistically significant differences between control (VHC) and sample groups are labeled as * for p < 0.05 (Fisher’s exact test).</p

Behavioral profile of zebrafish larvae during a one-hour exposure to vehicle or PTZ.

<p>The average movement (y-axis) of vehicle- and 20 mM PTZ-treated larvae are denoted by closed triangles (grey) and closed circles (black) respectively. The mean movement ± SD of 12 larvae is depicted per minute (x-axis) of the tracking session. By 15 min, the frequency of convulsion-like episodes in all larvae reached a rate of one or more per minute, which is reflected in the decrease in SD after this time point.</p