Experimental exposure regimen to second-hand smoke (SHS).

<p>apoE−/− mice on a C57BL/6J background were developmentally exposed to either filtered air or 1 mg/m³ total suspended particulate (TSP) SHS. The <i>in utero</i> group were exposed to 1 mg/m³ TSP from gestation days 1–19, followed by filtered air exposure until sacrifice at 12–14 weeks of age. The neonatal group were exposed to 1 mg/m³ TSP within 24 hours of birth until weaning at 3 weeks of age, followed by filtered air exposure until sacrifice at 12–14 weeks of age. Control mice were exposed exclusively to filtered air, until sacrifice at 12–14 weeks of age.</p

Aconitase activity from aortas harvested from mice exposed to SHS during development.

<p>Aconitase is specifically inactivated by superoxide (O₂^.−) and peroxynitrate (ONOO⁻) and therefore provides an indirect measure of O₂^−. associated oxidant stress. A) Aconitase activity was measured in aorta homogenates from mice exposed to filtered air, neonatal SHS, or <i>in utero</i> SHS by following the production of cis-aconitate from isocitrate at 240 nm. An asterisk (*) indicates a significant difference exists compared to filtered air control (p≤0.001; n = 8/group). B) Immunoblot showing aconitase protein levels; the bar graph below depicts the quantification of relative aconitase protein levels to control (n = 8/group).</p

Oil red-O staining of aortas from developmental SHS exposed mice.

<p>Atherosclerotic lesion levels were assessed by quantifying the percent of oil red-O staining of whole aortas from mice exposed either to <i>in utero</i> or neonatal SHS (1 mg/m³ TSP). A) Bar graph demonstrating the percent levels of oil red-O positive staining aortic area relative to total aortic area from control, neonatal, and <i>in ute</i>ro exposed apoE −/− mice. An asterisk (*) indicates a significant difference exists compared to filtered air control (p≤0.001; n = 5, 6, and 8 for control, <i>in utero</i>, and neonatal groups, respectively). B) <i>En face</i> images of oil red-O stained aortas; arrows indicate presence of positively stained area.</p

Adult weights from apoE−/− exposed to developmental SHS.

<p>Bar graph depicting average weight of mice at 12–14 weeks of age, prior to sacrifice. An asterisk (*) indicates a significant difference (p<0.05) exists compared to control (n = 39, 30, and 19 for control, <i>in utero</i>, and neonatal groups, respectively).</p

Mitochondrial DNA (mtDNA) copy number and deletion levels associated with developmental SHS exposure.

<p>Genomic DNA was extracted from whole aortas and mtDNA copy number and mtDNA deletion levels and frequency were estimated by QPCR. A) MtDNA copy number in control, <i>in utero</i> or neonatal exposure apoE−/− mice. Values are expressed relative to control. An asterisk indicates a significant difference (p<0.01; n = 10, 8, and 7 for control, <i>in utero</i>, and neonatal groups, respectively) exists from control. B) QPCR results showing mtDNA deletions in the control, neonatal, and <i>in utero</i> groups (indicated); lanes 1–3 are from control, <i>in utero</i>, and neonatal samples, respectively. Lanes 4–7 are from <i>in utero</i> exposed, lanes 8–11 are from control, and lanes 12–15 are from neonatal exposed samples. C) Mean number of different mtDNA deletions per group (represented by a different band per mtDNA deletion), as assessed by QPCR. An asterisk indicates a significant difference (p<0.05; n = 5/per group) exists from control. D) Mean amount of mtDNA deletion levels in aortic tissues from control, neonatal, and <i>in utero</i> exposed apoE−/− mice quantified by utilizing QPCR. An asterisk indicates a significant difference (p<0.01; n = 5/per group) exists from control and neonatal.</p