Genes involved in testicular development and functions affected in fetal testis explants exposed to MEHP.

<p>Fold-change values expressed relative to time-matched untreated controls, with reciprocal transformation from expression ratios. NS: no significant change in gene expressions. Values in italics indicate a down-regulation and values in bold indicate an upregulation.</p><p>*p value<0.01 and</p><p>**p<0.001.</p

17-hydroxyprogesterone (17-OHP) production is increased whereas androstenedione production is decreased by exposure of the fetal testes to MEHP.

<p>Effects of 10 µM MEHP on 17-OHP and androstenedione secretion by fetal rat testes cultured for 72 h, beginning at GD14.5. We determined 17-OHP and androstenedione concentrations with specific RIAs. Values are means +/−SEM of 8 (17-OHP) and 7 (androstenedione) testes from fetuses of 2 different litters in 2 independent experiments. The numbers indicate the percentage decrease or increase relative to the corresponding control. <i>* p<0.05 and ** p<0.01</i> in <i>Wilcoxon Mann-Whitney tests comparing treated and control testes</i>.</p

MEHP inhibits the production of testosterone and 5α-DHT by the fetal testis.

<p>Effects of MEHP on the production of testosterone and 5α-DHT by fetal rat testes cultured for 72 h beginning on GD14.5. Testosterone and 5α-DHT were determined with specific RIAs. Control testes were cultured in medium containing DMSO, whereas the treated testes were cultured in medium containing 1, 10 or 100 µM MEHP diluted in DMSO. The testes of different fetuses from different dams were selected at random. Values are means +/− SEM of 10 testes from the fetuses of 2 litters in 3 independent experiments. The number indicates the percentage decrease relative to the corresponding control. <i>* p<0.05</i> in <i>Wilcoxon Mann-Whitney tests comparing treated and control testes in the 5alpha-DHT assay * p<0.05</i> in non parametric <i>ANOVA for the MEHP dose-response testosterone assay</i>.</p

Supplementation of the medium with testosterone and testosterone precursors reveals that only the 17,20 lyase activity of CYP17 is affected by MEHP treatment.

<p>Effect of 3.5×10⁻⁷ M pregnenolone (+Pregnenolone), 17-OHP (+17-OHP) androstenedione (+Androstenedione) or 10⁻⁷ M testosterone (+Testosterone) in the presence or absence of 10 µM MEHP on 17-OHP, androstenedione, testosterone and 5α-DHT secretion <i>in vitro</i>. Data are presented relative to the corresponding control (Basal). Values are means +/−SEM of 7 testes from fetuses taken from 2 litters in 3 independent experiments. * <i>p<0.05 and ** p<0.01</i> in <i>Wilcoxon Mann-Whitney tests comparing treated and control testes; NS: not significant</i>.</p

Expression of a number of genes involved in cholesterol transport and steroidogenesis, as determined by real-time quantitative PCR.

<p><i>18 S</i> transcript levels were used as a reference, and relative transcript levels were calculated as a fold-change relative to control. The names of the genes investigated are cytochrome P450, family 17, subfamily A, polypepetide 1 (<i>Cyp17a1</i>), cytochrome P450, family 11, subfamily A, polypepetide 1 (<i>Cyp11a1</i>), 3 beta- and steroid delta-isomerase 1 (<i>Hsd3b1</i>), Steroidogenic acute regulatory protein (<i>Star</i>). Data are presented as means ± SEM for various cDNAs synthesized from the mRNA of 6 to 12 testes from the fetuses of 2 litters in 3 or 4 independent experiments. The number indicates the percentage decrease relative to the control. <i>^* p<0.05</i> in <i>Wilcoxon Mann-Whitney tests comparing treated and control to testes; NS: not significant</i>.</p

Western blot analysis of P450c17 and Cytochrome b5 protein levels after MEHP exposure.

<p>A) Representative Western blot of protein extracts (20 µg) from a pool of 8 control testes or testes exposed to 10 µM MEHP. Blots were incubated with anti-p450c17, anti-Cytochrome b5 and anti-GAPDH antibodies, to control for equal loading. The apparent molecular masses (kDa) are indicated on the left. B) Quantification of band intensity on three blots, carried out with Quantity One software (Biorad). Data are expressed in arbitrary units relative to the corresponding control. The number indicates the percentage decrease relative to control. * <i>p<0.05 in Wilcoxon Mann-Whitney tests comparing treated and control testes. </i><i>NS: not significant</i>.</p

Adult CYP20-exposed offspring displayed maladaptive behavior in response to highly stressful conditions.

<p><b>A</b>: The openfield was used as a weakly challenging task. Total distance travelled in the apparatus (1–2), time spent and the mean speed in different parts of the openfield were used to assess emotional reactivity (3). <b>B</b>: Emotional reactivity in response to highly challenging environments was evaluated by confronting mice to a novel cage with no sawdust. Total distance travelled was monitored over the total duration of the test (1). Data were also analyzed on a minute-by minute basis (2–3). <b>C-D</b>: Emotional reactivity in response to highly challenging environments was evaluated in the forced swimming task and the tail suspension task. In each condition, total time spent immobile was scored over the total duration of the test (1) and on a minute-by-minute basis (2). <b>E</b>: Habituation to a second exposure to stressful environments was assessed by confronting mice to the forced swimming task 24h later the first trial. Performance on day 2 is depicted on (1) and (2), and habituation between day 1 and day 2 is depicted on (3) and (4). Data are mean +/- sem; n = 20–21 /group. *** p < 0.001, ** p < 0.01, *p < 0.05 and # p < 0.09.</p

Putative Adverse Outcome Pathway for CYP-induced developmental neurotoxicity.

<p>Brown boxes show molecular initiating events (MIE), key events (KEs) and adverse outcomes (AOs) related to cypermethrin exposure at the highest dose (20mg/kg; CYP20). Orange boxes show MIE, KEs and AOs related to cypermethrin exposure at the lower dose (5mg/kg; CYP5). Common mechanisms / commonly observed changes are in double-lined boxes.</p

Behavioral tests used in the two experiments.

<p>Control and CYP-exposed offspring were exposed to both weakly and highly emotionally challenging environments. Conditions were defined as weakly challenging when the animals could escape from the stress generated by the apparatus and luminosity was low (no more than 30 lux). On the contrary, conditions were defined as highly challenging when the animals were not able to escape from the stress generated by the apparatus and luminosity was high (200 lux). The orange circles show the conditions for the CYP5-exposed offspring while the brown circles show the conditions for the CYP20-exposed offspring.</p

Adult CYP5-exposed offspring displayed maladaptive behavior in response to social contexts.

<p><b>A</b>: In the three-chambered sociability test, CYP5-exposed offspring displayed no preference for the chamber containing the social partner (mouse 1), unlike the controls (1). When the object was replaced by a novel social partner (mouse 2) in a subsequent trial, CYP5-exposed offspring, as well as the controls, preferred interacting with the novel partner compared to the already known partner (2). * <i>p</i> < 0.05, ** <i>p</i> < 0.01, *** <i>p</i> < 0.001 compared to time spent in contact with wire cup containing mouse 1. <b>B</b>: Social skills were also assessed in more realistic conditions: in a territorial context <i>i</i>.<i>e</i>. a male—male interaction task and in a <b>C:</b> reproductive context <i>i</i>.<i>e</i>. male—female interaction task. In these tasks, both social and non-social behaviors were scored over the total duration of the test (1). The microstructure of social / non-social behaviors was also monitored (2). And each parameter was measured on a minute-by-minute basis (3). Data are mean +/- sem; n = 13–14 / group. *p < 0.05, ** p < 0.01, *** p < 0.001, # p < 0.09 compared to the controls.</p