Results of the visual cue test.

<p>(A) Escape latency on d 7 of training and during the visual cue test expressed as a percentage of baseline escape latency (escape latency on the first training day). Additional parameters that influence performance in the MWM were assessed during the visual cue test including: (B) mean swim velocity and (C) rest time, both of which are presented as a function of training day. No statistically significant differences were identified using paired t-test (A) or repeated measures ANOVA (B,C).</p

MWM training increases transcription of genes associated with synaptic plasticity in multiple brain regions.

<p>Transcript levels of spinophilin (Spn), activity-regulated cytoskeleton-associated protein (ARC), neurogranin (RC3), Homer1a and Homer1b/c were analyzed in total RNA harvested from the cortex, cerebellum, and hippocampus of weanling mice after behavioral studies were completed. Data are presented as fold-change in transcript expression relative to non-maze-trained littermates as calculated by the Pfaffl equation, normalized to the housekeeping gene 18S rRNA (n = 9–12 animals per group). The dashed line represents a fold-change of 1, which indicates no difference in gene expression between MWM-trained animals and untrained littermate controls.</p

Weanling mice exhibit spatial memory after MWM training.

<p>Spatial memory was assessed in a probe trial administered on training day 8 with respect to: (A) percentage of time or (B) percentage of path length spent in the target quadrant relative to non-target quadrants. Data in panels A and B are presented as the mean ± SEM (n = 16 animals). Significantly different from target quadrant at ^a<i>p</i> < 0.05, ^c<i>p</i> < 0.001 as determined by repeated measures ANOVA with the Greenhouse-Geisser correction for LSD <i>post hoc</i> tests. Effect sizes: partial η² for % time = 0.66; partial η² for % path length = 0.68. Observed power: 100% for % time; 100% for % path length.</p

Weanling mice exhibit spatial learning in the Morris water maze (MWM).

<p>Spatial learning was assessed as a function of training day with respect to the following parameters: (A) escape latency, (B) percentage of time spent in the target quadrant, and (C) percentage of total path length spent in the target quadrant. Data are presented as the mean ± SEM (n = 16 animals). Since sex differences were not identified for any of the behavioral parameters shown in this Fig., data from males and females were combined to calculate mean values. Significantly different from training d 1 (A) or d 2 (B and C) at ^a<i>p</i> < 0.05, ^b<i>p</i> < 0.01, ^c<i>p</i> < 0.001 as determined using repeated measures ANOVA with LSD <i>post hoc</i> test. Effect sizes: partial η² for latency = 0.31, partial η² for % time = 0.21; partial η² for % path length = 0.17. Power: 99% for latency, 94% for % time, 84% for % path length. Note that escape latency was the only data collected on the first day of training because of a computer malfunction in collecting data on the first training day.</p

Influence of O₂ tension, 2-ME and serum on the metabolic activity of THP-1 cells.

<p>THP-1 cells were synchronized by serum deprivation for 48 h. (A) Undifferentiated THP-1 cells were plated in 96-well plates precoated with poly-D-lysine. (B) THP-1 cells were triggered to undergo macrophage differentiation by incubating with PMA at 20 ng/ml for 24 h. MTT was added to both undifferentiated and differentiated THP-1 cells for 3 h under varying O₂, 2-ME and serum conditions. MTT reduction, measured as the absorbance at 562 nm, was normalized to protein concentration. Data are presented as the mean ± SEM (n = 4 independent experiments. *Significantly different from +2-ME+FBS (standard culture conditions) under the same oxygen tension (one-way <i>ANOVA</i> and <i>post hoc</i> Tukey’s test); ^▴Significantly different from –2-ME+FBS under the same oxygen tension (one-way <i>ANOVA</i> and <i>post hoc</i> Tukey’s test); ^#Significantly different from the same culture condition in the 18% O₂ group (e.g., 18% O₂<i>versus</i> 5% O₂) by Student’s <i>t</i>-test. *, ^#, ^▴<i>p</i><0.05; **,^##,^▴▴<i>p</i><0.01; ***, ^###, ^▴▴▴<i>p</i><0.001.</p

Influence of oxygen tension on phagocytosis.

<p>THP-1 cells were cultured with PMA for 25 h to promote macrophage differentiation. In a subset of the cultures, the oxygen tension was switched from normoxic to hyperoxic or from hyperoxic to normoxic for the last hour of the incubation period. Phagocytosis was assessed as the uptake of <i>E.coli</i> BioParticles®. Data are presented as mean ± SEM (n = 3 per treatment group). *Significantly different from 25 h at 18% O₂ at <i>p</i><0.05; and ^ΔΔΔSignificantly different from 25 h at 5% O₂ and from 24 h at 18% O₂ → 1 h @ 5% O₂ at <i>p</i><0.001 (one-way <i>ANOVA</i> with <i>post hoc</i> Tukey’s analysis).</p

Influence of O₂ tension, 2-ME and serum on the rate of THP-1 differentiation.

<p>Differentiation of THP-1 cells from monocytic to macrophage cells is associated with transition from a non-adherent to an adherent cell type. To determine whether culture conditions affect PMA-stimulated differentiation of THP-1 cells to macrophages, cell adhesion was assessed at 3 and 24 h after addition of PMA (20 ng/ml) to the culture medium. Data are presented as the mean ± SEM of the protein concentration of adherent cells at 3 h as a percentage of the protein concentration of adherent cells at 24 h (n = 4 independent experiments). *Significantly different from +2-ME+FBS (standard culture conditions) under the same oxygen tension (one-way <i>ANOVA</i> and <i>post hoc</i> Tukey’s test); ^▴Significantly different from –2-ME+FBS under the same oxygen tension (one-way <i>ANOVA</i> and <i>post hoc</i> Tukey’s test); ^#Significantly different from the same culture condition in the 18% O₂ group (e.g., 18% O₂<i>versus</i> 5% O₂) by Student’s <i>t</i>-test. **, ^##,^▴▴<i>p</i><0.01; ***, ^###,^▴▴▴<i>p</i><0.001.</p

Oxygen tension influences LPS-induced NF-κB activation and release of cytokines in PMA-differentiated THP-1 cells.

<p>Undifferentiated THP-1 XBlue cells, which express an NF-κB reporter gene linked to secreted embryonic alkaline phosphatase (SEAP) were synchronized by serum deprivation for 48 h, and then differentiated with PMA (20 ng/ml) for 48 h in the absence of 2-ME and FBS. Differentiated THP-1 XBlue cells were then cultured in the absence (baseline) or presence of LPS (1 µg/ml) for an additional 24 h in either 18% (A, C) or 5% (B, D) O₂. SEAP activity was quantified by QuantiBlue at 630 nm (A,B). Conditioned media from these cultures were analyzed using a human Milliplex Kit® to simultaneously quantify multiple cytokines and chemokines released from differentiated THP-1 cell during the 24 h incubation. Each symbol in panels C and D represents the mean of duplicates from one of five wells run in a representative experiment. Data are presented as the mean ± SEM (n = 2 independent experiments). ***Significantly different from baseline under the same oxygen tension at <i>p</i><0.001,^###significantly different from 18% O₂ at <i>p</i><0.001 (Student’s <i>t</i>-test).</p

Oxygen tension significantly influences phagocytosis in PMA-differentiated THP-1 cells.

<p>Undifferentiated THP-1 cells were synchronized by serum deprivation for 48 h, plated at a density of 10⁵cells/well in a 96-well plate and differentiated with PMA (20 ng/ml) for 48 h in the absence of 2-ME and FBS. Differentiated THP-1 cells were washed and then incubated for 3 h with <i>E.coli</i> BioParticles®, which emit fluorescence upon acidification in lysosomes following phagocytosis. Phagocytosis, which was quantified by determining the fluorescence intensity at 600 nm, was blocked by pretreating cultures with cytochalasin D (2 µM) for 1 h prior to addition of <i>E. coli</i> BioParticles®. The mean fluorescence intensity was normalized to protein concentration as determined using the BCA protein assay. Data are presented as the mean ± SEM (n = 3 independent experiments). *Significantly different from control (– cytochalasin) treatment under the same oxygen tension; ^#significantly different from the same culture condition in the 18% O₂ treatment group (e.g., 18% O₂<i>versus</i> 5% O₂) by Student’s <i>t</i>-test. ***, ^###<i>p</i><0.001.</p

Effect of Pregnancy on the Disposition of 2,2′,3,5′,6-Pentachlorobiphenyl (PCB 95) Atropisomers and Their Hydroxylated Metabolites in Female Mice

Chiral PCBs, such as PCB 95, are developmental neurotoxicants that undergo atropisomeric enrichment in nonpregnant adult mice. Because pregnancy is associated with changes in hepatic cytochrome P450 enzyme activity as well as lipid disposition and metabolism, this study investigates the effect of pregnancy on the maternal disposition of chiral PCBs. Female C57BL/6 mice (8 weeks old) were dosed daily beginning 2 weeks prior to conception and continuing throughout gestation and lactation (56 days total) with racemic PCB 95 (0, 0.1, 1.0, or 6.0 mg/kg body wt/day) in peanut butter. Levels and chiral signatures of PCB 95 and its hydroxylated metabolites (OH-PCBs) were determined in adipose, blood, brain, and liver. Tissue levels of PCB 95 increased 4- to 12-fold with increasing dose, with considerable enrichment of the second eluting atropisomer in all tissues (EF range 0.11 to 0.26). OH-PCBs displayed atropisomeric enrichment in blood and liver but were not detected in adipose and brain. Levels of PCB 95 and its metabolites were 2- to 11-fold lower in pregnant dams relative to those previously reported in nonpregnant age-matched female mice; however, PCB 95 and OH-PCB profiles and chiral signatures were similar between both studies. In contrast, human brain samples contained racemic PCB 95 residues (EF = 0.50). These results demonstrate that changes in cytochrome P450 enzyme activity and lipid disposition during pregnancy reduce the PCB body burden in dams but do not affect metabolite profiles or chiral signatures. The differences in chiral signatures between mice and humans suggest species-specific differences in atropisomeric disposition, the toxicological significance of which remains to be determined