Exposure protocol

Two identically sized chambers, each ~1 m3 in volume (1.2 × 0.8 × 1.2 m), were used in the present study. Each chamber could house a maximum of 9 rat cages (4 on the upper rack and 5 on the lower rack). These chambers were placed side by side for whole body inhalation exposure of rats in an air‐conditioned room in Beijing. The only difference between the 2 chambers was the presence or absence of a high‐efficiency particulate air (HEPA) filter placed in the inlet duct. The room was about 2 km away from northwestern fourth Ring Road, a major artery of the city with 8 main lanes and 6 auxiliary lanes that carries nearly 220,000 vehicles per day. Particulate matter concentrations were measured using 2 sets of single particle soot photometer and scanning mobility particle sizer and aerodynamic particle size, one for the chamber airstream and the other for ambient atmosphere. We calculated the removal efficiency of every particulate size. The removal efficiency of the HEPA filter was 98.99 ± 0.86% for particles larger than 2.5 μm in diameter and 70.61 ± 19.34% for PM 2.5 . PM 2.5 concentrations inside the chamber equipped with the HEPA filter were estimated using outdoor PM 2.5 concentrations and the filter removal efficiency for PM 2.5 . The temperature inside these chambers was controlled at 24 ± 1°C.

We started our experiments with pregnant Sprague Dawley rats. All the rats were kept in an animal care facility before the experiments commenced. We conducted the first set of experiments using 30 pregnant Sprague Dawley rats that were 12 wk old. On gestation d 4, rats were assigned to 2 groups: 18 entered the unfiltered chamber, and the remaining 12 entered the filtered chamber. The assignment was done by consideration of baseline body weight in such a way that the baseline weights were not significantly different between the two groups. In each chamber, no more than 2 rats were housed per cage to avoid overcrowding. One group was placed inside a chamber with the inlet air directly coming from outdoors; the other group was housed inside a same‐size chamber equipped with a HEPA filter at the air inlet. The chambers were placed side by side inside an air‐conditioned room in Beijing. Rats lived in these chambers naturally under a 12 h light/12 h dark cycle and were fed a normal chow diet. The animals were weighed when entering the chamber (d 0) and then every 3 d until d 14, when a series of end points was analyzed. Over this 14‐d period, (December 28, 2009, to January 10, 2010), PM 2.5 concentrations were 73.5 ± 61.3 μg/m3 in the unfiltered chamber and 19.8 ± 9.0 μg/m3 in the filtered chamber (means ± sd of hourly concentrations measured throughout the 14‐d period). The sd values indicated large variability in the hourly data. We observed large diurnal and day‐to‐day fluctuations in ambient PM 2.5 concentrations.

We found that 7 of 10 rats in the unfiltered air group and 4 of 6 in the filtered air group had produced pups. Among the rest of the pregnant rats not dissected, we found that 6 in the unfiltered group and 6 in the filtered group had delivered pups normally. The pups prenatally exposed to unfiltered air stayed in the unfiltered chamber, and the pups prenatally exposed to filtered air stayed in the filtered chamber continuously until they were analyzed either at 3 or 8 wk of age.