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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 22, 2003 as doi:10.1096/fj.02-0967fje. |
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Department of Health Sciences, University of Genoa, Italy
3Correspondence: Department of Health Sciences, University of Genoa, Via A. Pastore 1, I-16132, Italy. E-mail: sdf{at}unige.it
SPECIFIC AIMS
Our aims were to understand the molecular mechanisms underlying the increased risk of developmental impairments, congenital diseases, and childhood cancer after transplacental exposure of the fetus to maternal cigarette smoke and to evaluate the protective effects of a chemopreventive agent given during pregnancy.
PRINCIPAL FINDINGS
1. Exposure of pregnant Swiss albino mice to smoke reduces parity, fetus body weight, and placenta weight
Whole-body exposure of dams to environmental cigarette smoke (ECS) throughout pregnancy resulted in a significant (P<0.01) reduction of parity (from 9.4±0.53 to 6.6±0.56 litters/pregnant dam, means ±SE), placenta weights (from 0.073±0.002 to 0.064±0.002 g), and body weights of fetuses (1.3±0.01 to 1.1±0.01 g).
2. Exposure of pregnant mice to smoke causes the formation of genomic alterations in fetal liver
Transplacental exposure to ECS resulted in a significant increase of bulky DNA adducts in fetus liver as detected by 32P postlabeling after enrichment with butanol (Table 1
). Figure 1
shows the autoradiographic patternsobserved in untreated mice (SHAM) and smoke-exposed mice (ECS). In parallel, there was a significant increase of oxidative DNA damage, measured in terms of 8-hydroxy-2'-deoxyguanosine (8-OH-dG). Transplacental exposure to ECS produced a significant toxicity in fetal liver, as shown by the decrease of the polychromatic-to-normochromatic erythrocyte (PCE/NCE) ratio, as well as a significant clastogenicity, as shown by the increased frequency of micronucleated (MN) PCE (Table 1)
.
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Administration of N-acetylcysteine-L-cysteine (NAC) with drinking water (1 g/kg body weight) throughout pregnancy did not affect per se levels of bulky DNA adducts, oxidative DNA damage, MN frequency in PCE, or PCE/NCE ratio in fetal liver, but significantly and strikingly inhibited all genomic alterations produced by ECS (Table 1)
.
3. Exposure of pregnant mice to smoke alters multigene expression in fetal liver
RNA was extracted from fetus liver and analyzed by cDNA array for the expression of 746 genes by using the AtlasTM mouse stress and mouse expression arrays. A list of these genes can be found in the web site www.atlasinfo.clontech.com. Within sham-exposed mice, 61 genes (8.2%) yielded a signal in fetus liver that was at least twofold higher than the background signal. The expression of 116 genes (15.5%) was increased at least twofold in the liver of fetuses from ECS-exposed mice compared with sham-exposed mice (see the scattergram reproduced in Fig. 1
). Only one gene, encoding for a growth factor, was down-regulated after exposure to ECS. There was a high correlation (r=0.934, P<0.05) between cDNA array and RT-PCR analyses in evaluating the effect of ECS on the expression of five selected genes.
4. Transplacental NAC does not substantially change the background gene expression in fetal mouse liver while attenuating smoke-related alterations
Oral administration of NAC to pregnant mice up-regulated 3 of the 746 tested genes in fetal liver. They included the genes that encode for two glutathione (GSH) S-transferase isoenzymes and for 
1-antitrypsin precursor. On the other hand, NAC attenuated the ECS-related induction of a gene (trypsin-related serine protease) that causes emphysema in hamsters. Administration of NAC to ECS-exposed pregnant mice normalized the expression of the majority of the 116 ECS up-regulated genes. In fact, 74 of these genes (63.8%) were no longer > twofold higher than sham-exposed mice; 26 (22.4%) were decreased > twofold compared with ECS-exposed mice.
5. Transplacental smoke induces hematopoietic cell proliferation in fetal liver
Transplacental ECS stimulated the expression of several genes that regulate leukocyte proliferation and differentiation, including c-myb, c-myc, L-myc, c-rel, c-fos, and Lfc proto-oncogenes, macrophage colony-stimulating factor (CSF) 1 receptor, mast/stem cell growth factor receptor, granulocyte CSF receptor precursor, and granulocyte-macrophage CSF receptor low-affinity subunit precursor. These effects were less evident after cotreatment with NAC.
6. Transplacental smoke induces fetal liver metabolism, oxidative mechanisms, and stress response
Exposure of pregnant mice to ECS increased, in fetus liver, the expression of 18 of 50 genes (36.0%) included in the category of "xenobiotic metabolism." This figure was reduced to 8 (16.0%) in fetuses from ECS-exposed dams treated with NAC during pregnancy. ECS induced the expression of genes encoding for cytochromes P450 (CYP) involved in metabolic activation of a variety of xenobiotics. At the same time, ECS induced the expression of genes involved in detoxification of xenobiotics and multidrug resistance (MDR) protein 2.
Neither ECS nor NAC affected the induction of 8-oxo-dGTPase, an enzyme that prevents 8-OH-dG incorporation into DNA, or of 8-oxoguanine DNA glycosylase 1 (OGG1), which removes 8-OH-dG. Therefore, the observed protective effect of NAC toward 8-OH-dG formation should not be ascribed to mechanisms involved in incorporation or repair of this altered base, but to the activity of NAC as a scavenger of reactive oxygen species (ROS) causing its formation. Exposure to ECS induced the expression of genes encoding for oxidative stress-induced protein, catalase, and precursors of two superoxide dismutases (SODs) in the cell cytosol and mitochondrial matrix.
As many as 120 of the 746 genes tested belong to the category "stress response." Eighteen (15.0%) were up-regulated by ECS in the absence of NAC treatment and nine (7.5%) were up-regulated by ECS in NAC-treated mice. Overexpression of some genes falling in this category has been associated with oxidative stress. Administration of NAC inhibited the ECS-related overexpression of MAP kinase 3 and heme oxygenase 1, which triggers the conversion of heme moieties to antioxidant derivatives.
The majority of the genes tested within the category "protein repair, removal, and folding" (15/21, 71.4%) were overexpressed in fetus liver after exposure of pregnant dams to ECS. Several genes in this category encode for proteins, such as heat shock proteins and T complex proteins, which function as chaperones in repair and control of damaged proteins. Heat shock proteins are involved in refolding processes of oxidatively damaged proteins. NAC prevented the overexpression of ECS-induced genes, with a sharp effect in the case of the rotamase gene.
7. Transplacental smoke down-regulates liver cell cycle and up-regulates apoptosis in fetal liver, whereas DNA repair is modest and poorly inducible
Fetal liver from untreated dams exhibited a poor propensity to repair DNA via different mechanisms, including base excision and nucleotide excision repair pathways involved in removal of modified nucleotides. Only a tiny proportion of genes involved in DNA repair (7 of the 115 genes analyzed: 6.4%) was induced by ECS, and none was induced more than twice after administration of NAC to ECS-exposed dams. GADD153 and GADD45 were among the genes whose induction by ECS was almost completely prevented by NAC. Up-regulation of GADD genes after DNA damage results in pleiotropic effects such as induction of DNA repair genes and apoptosis, control of genomic stability, and down-regulation of the cell cycle.
Within the category "cell cycle regulation," 19 of 96 genes (19.8%) were up-regulated by ECS. ECS induced the expression of five genes involved in the negative regulation of cell cycle. ECS induced 11 of 23 genes (47.8%) in the category of "apoptosis," all having proapoptotic functions via different mechanisms. NAC administration to pregnant mice resulted in an evident inhibition of the ECS-related induction of most proapoptotic genes.
Exposure of pregnant mice to ECS produced moderate effects on fetal liver genes in the category of "growth factors and cytoskeleton," "transcription factors," and "cell receptors." In these groups, ECS up-regulated 8 of 99 genes (8.1%), 6 of 100 genes (6.0%), and 5 of 99 genes (5.1%), respectively. Overall, only seven of the genes belonging to these categories were still up-regulated after treatment with NAC of ECS-exposed mice.
8. Transplacental ECS induces hypoxia-related genes
Another ECS-related effect detected by postgenomic analysis in fetal liver was the induction of tissue hypoxia. The gene encoding for rhodanese was strongly up-regulated in the liver of ECS-exposed fetuses. Multigene expression analyses provided evidence that the fetus counteracts CS-induced hypoxia by increasing erythrocyte production in liver, as suggested by an enhanced expression of genes encoding for 1) erythropoietin receptor precursors; 2) transferrin and low density lipoprotein receptors uptaking red blood cell components, such as iron and cholesterol; and 3) transcription of hematopoietic domains, such as CACCC box binding protein. Induction of two genes involved in blood vessel growth (i.e., vascular endothelial growth factor precursor and angiogenin) is likely to be related to ECS-induced hypoxia.
CONCLUSIONS AND SIGNIFICANCE
Exposure of mice to ECS throughout pregnancy caused genomic and transcriptional alterations in fetal liver that can be related mechanistically to noxious effects in the offspring. Because hepatocytes and blood cells or their precursors are in close contact in fetus liver, it is significant that liver erythrocytes were particularly susceptible to the toxic and cytogenetic damage produced by transplacental ECS and that ECS overexpressed several genes that regulate leukocyte proliferation and differentiation. Indeed, these findings may help explain why, in humans, the childhood leukemias/lymphomas are the tumors most often associated with maternal smoking during pregnancy or exposure of pregnant women to ECS. Multigene expression analyses provided evidence that the main mechanisms available to the liver to counteract DNA damage during developmental life are provided by down-regulation of cell replication and up-regulation of apoptosis rather than by modulation of DNA repair. These patterns are consistent with the impairment of parity, fetus body weight, and placenta weight observed after exposure of pregnant mice to ECS and with the notion that maternal smoking produces growth retardation in the offspring. Transplacental ECS modulated metabolism in fetal liver, with a prevalence of activating pathways over detoxifying mechanisms, as supported by the occurrence of multiple DNA adducts in liver cells. ECS induced hypoxia-related genes and genes involved in oxidative damage, known to play an important role in cigarette smoke carcinogenesis as shown in the present study by formation of 8-OH-dG in fetal liver. Stimulation by transplacental ECS of an emphysema-related gene is consistent with the fact that the prenatal exposure of human fetus to tobacco smoke has been linked to an enhanced susceptibility of children to respiratory diseases.
For the first time, evidence was provided that oral administration of a drug during pregnancy can significantly attenuate smoke-related molecular alterations. NAC, which is a nontoxic analog and precursor of GSH, has been investigated extensively as a cancer chemopreventive agent in experimental models and is known to protect mothers and fetuses when given during pregnancy as an antidote against acute intoxications. Transplacental NAC per se overexpressed three protective genes, prevented all monitored ECS-induced genotoxic effects in fetal liver, and normalized a large proportion of ECS-dysregulated genes.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0967fje; to cite this article, use FASEB J. (April 22, 2003) 10.1096/fj.02-0967fje 
2 Permanent address: National Center of Oncology, Sofia-1756, Bulgaria.
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