Effects of first-generation in utero exposure to diesel engine exhaust on second-generation placental function, fatty acid profiles and foetal metabolism in rabbits: preliminary results

Atmospheric pollution has major health effects on directly exposed subjects but intergenerational consequences are poorly characterized. We previously reported that diesel engine exhaust (DE) could lead to structural changes in the placenta of in utero exposed rabbits (first generation, F1). The effects of maternal exposure to DE were further studied on second-generation (F2) rabbits. Pregnant F0 females were exposed to filtered, diluted DE (1 mg/m3, median particle diameter: 69 nm) or clean filtered air (controls) for 2 h/day, 5 days/week by nose-only exposure during days 3–27 post-conception (dpc). Adult female offspring (F1) were mated to control males: F1 tissues and F2 foeto-placental units were collected at 28 dpc and placental structure and gene expression (microarray) analysed. Fatty acid profiles were determined in foetal and maternal plasma, maternal liver and placenta. In F1, compared to controls, hepatic neutral lipid contents were increased in exposed animals without change in the blood biochemistry. In F2, the placental lipid contents were higher, with higher monounsaturated fatty acids and reduced pro-inflammatory arachidonic acid (AA), without placental structural changes. Conversely, the proportion of anti-inflammatory n-3 polyunsaturated fatty acids in F2 plasma was increased while that of AA was decreased. Gene set enrichment analyses (GSEA) of F2 placenta transcriptomic data identified that the proteasome complex and ubiquitin pathways genes were over-represented and ion channel function and inflammation pathways genes were under-represented in exposed animals. These preliminary results demonstrate that diesel engine exhaust exposure and in utero indirect exposure should be considered as a programming factor within the context of the DOHaD (Developmental Origins of Health and Disease) with a probable intergenerational transmission.

5µm thick sections of liver slices from in utero exposed F1 pregnant rabbits (n=6) on the left side or control F1 pregnant rabbits (n=3) on the right side. These liver sections were colored with (a and b) Hematoxyline Eosine Safran (HES) coloration to reveal the different cellular components, (c and d) a  (Microm Microtech, France) to visualize collagen fibers, (e and f) a PAS (Periodic Acid Schiff) staining to highlight the presence of glycogen, and (g and h) a Prussian blue coloration (Perl's coloration) to highlight possible ferric deposits and activation of Kupffer cells (KC). All colored sections were analyzed under light microscopy and scanned using a Nanozoomer Digital Pathology System (NDP Scan U10074-01, Hamamatsu, Japan). At this x40 magnification, we can observe hepatic parenchyma and the arrangement of hepatocytes (H). They form flattened, anastomosing blades, the thickness of which is of a single cell, and between which the blood circulates slowly towards the centrilobular vein (CLV). The sinusoids (S) are bordered by a discontinuous layer of endothelial cells, which do not rest on any basement membrane and which are separated from the hepatocytes by a small space (Disse space); it drains into the lymphatic portals. The cytoplasm of hepatocytes is highly eosinophilic, due to the presence of numerous mitochondria, with very fine basophilic granulations linked to numerous free ribosomes and granular endoplasmic reticulum. The stained section with PAS shows the presence of glycogen grains, which, being polysaccharides, are PAS-positive (colored in magenta), in control sections mostly.
Liver sections from in utero exposed (E) F1 dams, once pregnant at 28 dpc, showed marked microvacuolar steatosis, with micro and macro lipid vacuoles inside hepatocyts and heterogeneous dilated sinusoids, without fibrosis (a, c, e and g); Liver slice from control (C) F1 dams, once pregnant at 28 dpc, showed moderated microvacuolar steatosis (b, d and h) and mostly glycogen overload using PAS coloration (f).
At 28dpc, immunodetection of vimentin to label fetal capillaries was performed on labyrinthine area sections from the control group (a) and the exposed group (b). Black immunostaining represents fetal vessels (FV), blue cells are trophoblasts cells (T) and the white spaces with erythrocytes are maternal space (MS). profile in plasma of F1 pregnant female rabbits at 28dpc.
The PCA, plotting individual factor scores, provides a representation in a space of reduced dimensions, thus providing data structure and highlighting groups of homogeneous individuals. For this, we have looked for sub-areas in which projection of the cloud deforms as little as possible the initial data summarized in Table 2: Fatty acid profiles of F1 maternal plasma (with FA expressed as % of total FA). The first principal axis is the one that maximizes the variance when data are projected onto a line and the second one is orthogonal to it, and still maximizes the remaining variance. The variables, here the fatty acids of the plasma profile, are plotted into a so-called Variables factor map (or "correlation circle" of the PCA, (a)), where the angle formed by any two variables represented as vectors, reflects their actual pairwise correlation. The Variables factor map allows identifying the variables that contribute "positively" vs. "negatively" to the PCA axes. The main plan, designed by the dimensions 1 and 2, represents 53.7% of the inertia of the data table, summarized in Table 2. The individual factor map (b) of this PCA shows that, as illustrated by the confidence ellipses drawn around the individuals, no specific FA signature characterized each group according to their in utero exposure, exposed (black) or control (red), since the dimension 1 does not correlate with the in utero treatment of the F1 female (v.test < 2). The main contribution of FA to dimension 1 concern a positive correlation (only the correlations >0.80 are reported here) with C22:5n-6 (0.91, p=4.4e-05), SFA (0.89, p=9.88e-05), C16:0 (0.83, p=8.38e-04), n-6/n-3 ratio (0.81, p=1.57e-03) and C18:0 (0.80, p=1.63e-03), and a negative correlation with MUFA (-0.96, p=1.32e-06). The main contributions of FA to dimension 2 concern a positive correlation with n-6 PUFA (0.0.90, p=5.31e-05), C15:0 (0.89, p=1.09e-04), C22:6n-3 (0.79, p=1.97e-03) and a negative correlation with C20:5n-3 (-0.63, p=2.82e-02). Using the v.test from FactomineR, the statistical outputs performed on this PCA (Chi2) show nevertheless that the FA profile of plasma of the F1 Exposed female rabbits is significantly different of the control one (p=0.034), and that these differences are attributed to a positive association with C16:0 (v.test=2.057, p=0.039), and a negative association with C16:1n-7 (v.test=-2.426, p=0.015), C22:1n-9 (v.test=-2.502, p=0.012) and C18:3n-3 (v.test=-2.738, p=0.006), which confirm the statistical results obtained on data Table 2.
Supplementary Figure S4 online: Principal Component Analyses (PCA) of the fatty acid profile of F2 placenta phospholipids (PL) at 28dpc.
Supplemental Figure S7 online. Hierarchical clustering of transcriptomes of F2 EC (noted P in blue on the graphs) and CC (noted T in red on the graphs) placentas (n=8 per condition), in rabbit.
A. Hierarchical clustering was based on the Pearson correlation coefficient of F2 EC and CC placentas transcriptome. B. Correlation circles around the groups of the Principal Component Analyses (PCA) in the plane constituted by the dimensions 4 and 5 presented here for example. Foetal sex among groups is indicated with different colours. C. Histogram of raw pValues (justifying the use of GSEA analyses). Sixteen F0 females (N=7 Controls, N=9 Exposed dams) gave birth to F1 offspring, which were raised under control conditions. Altogether, 72 F1 offspring survived until adulthood, including 18 control females, 40 exposed females, 11 control males and 16 exposed males. All animals were weaned at 5 weeks of age. After becoming sexually mature adults at 6.5 months of age, a limited number of F1 nulliparous females (11 F1 control and 11 F1 exposed females) were dedicated to the production of the F2 generation. These females were mated with unexposed males. In the controls, only 5 out 11 rabbits were diagnosed as pregnant, among which one rabbit died at mid-pregnancy and another aborted; in the exposed group 10 out 11 rabbits were pregnant, including 1 abortion at mid-pregnancy. All females that were still pregnant (N=3 controls and N=9 exposed) were euthanized at 28 dpc to collect second generation (F2) foeto-placental units. The litter sizes were 6 to 13 fetuses without difference in sex ratio or change in fetal weight between groups.