Abstract
Fetal growth restriction (FGR) is an abnormal developmental process associated with Fetal Basis of Adult Diseases (FeBAD) such as diabetes and hypertension. Maternal cigarette smoking is one of the factors correlated with FGR. Benzo[a]pyrene (BaP) is a major environmental toxin in cigarette smoke and has been shown to alter placental trophoblast cell functions; but, the mechanism is not understood. In this investigation, we utilized MALDI-TOF to analyze proteomic changes in term placental samples from smokers (=10/day) and non-smokers, and, in 48 hr-BaP-treated MCF7 breast cancer cells. Protein extracts from cells and tissues were subjected to SDS-PAGE followed by tandem mass spectrometric analysis. iTRAQ methodology using a set of four, isobaric reagents enabled multiplexed relative and absolute quantification of proteins. The SDA gels were compared and differentially-expressed proteins were collected via robotic sampler. Following trypsin digestion peptide sizes were determined using MALDI-TOF. The data were blasted against the Mascot proteomics database. There were major changes in the proteomic profiles of MCF7 cells treated with BaP compared to non-treated cells and between placentas from smokers compared to non-smokers. The affected proteins included heat shock protein 70 (Hsp70), which is a chaperone whose action promotes protein folding through repeated cycles of substrate binding and release in an ATP-dependent manner. The proteomic changes were extensive and included proteins such as HSP, indicating that smoking/BaP changes protein expressions that influence the fold-integrity of proteins. To test for genomic effects of BaP, chromosomal analysis was performed on placental- and bone marrow cells from treated and non-treated rats and showed an increase in chromosomal abnormalities in cultured and bone marrow cells from BaP-treated animals. We believe that BaP is toxic at both the proteomic and genomic levels. Apart from previously proposed genomic abnormalities the extensive list of proteins influenced by the BaP treatment indicates that there are several new potential mechanisms through which BaP affects placental cell function.
Introduction
The basis of cigarette smoking-induced fetal growth retardation is unknown, although proteomic changes seem most likely. In order to test this possibility, we have begun to adapt Matrix-Assisted Laser Desorption-Mass Spectrometry (MALDI-MS) applied in situ to tissue in order to characterize proteins within placental tissues as a means of assessing the effects of cigarette smoke on the proteins expressed in smoker and non-smoker placentas.
Materials and Methods
Cells: Placental trophoblast cell lines (Gift from Dr. S. Guller, Yale Univ., USA).
Tissues: Term placentas from non-smokers and smokers were obtained under an IRB-approved protocol.
MALDI-MS: MALDI-TOF was utilized to analyze proteomic changes in term placental samples from smokers (=10/day) and non-smokers and in BaP treated MCF7 breast cancer cells (positive control). Protein extracts from cells and tissues were subjected to SDS-PAGE followed by tandem mass spectrometric analysis.. iTRAQ methodology using a set of four, isobaric reagents enabled multiplexed relative and absolute quantification of proteins. The SDA gels were compared and differentially-expressed proteins were collected via robotic sampler. Following trypsin digestion peptide sizes were determined using MALDI-TOF. The data were blasted against the Mascot proteomics database.
In situ MALDI: Tissues were frozen, cut on a cryo-microtome, and the 1 micron sections applied to conductive metal plates for MALDI, with mirror-image sections applied to glass slides for histologic examination. MALDI MS, and MALDI MS/MS were applied using trypsin 1:10,000 w/w of protein and matrix alpha cyano 4 hydroxy cinnamic acid, 10mg/ml. 100:1 w/w of protein were applied directly to the tissue slices. Cellular integrity was maintained using the techniques of cryo-protection and instant cellular and tissue fixation prior to application of MALDI-MS that we have previously reported (14). This is a feasibility study to determine whether our method of MALDI-MS can identify proteins in human term placental tissue samples. Using a chemical printer, matrix was co-registered on cryo-preserved, trypsinized human placental sections, followed by MALDI with post-source decay (PSD) or collision-induced dissociation (CID). Mass-to-charge (m/z) data from the cells and control brain tissues were processed and the results used by Mascot# software to interrogate the National Centre for Biotechnology Information (NCBI) database.
Results
Chromosomal analysis indicated the presence of both numerical and structural abnormalities in BaP treated cells compared to control group. Differential proteomics indicated a clear proteomics chances in samples from non smokers compared to non smokers. There were major changes in the proteomic profiles of MCF7 cells treated with BaP compared to non-treated cells and between placentas from smokers compared and non-smokers. The affected proteins included heat shock protein 70 (Hsp70), which is a chaperone whose action promotes protein folding through repeated cycles of substrate binding and release in an ATP-dependent manner. The proteomic changes were extensive and included proteins such as HSP, indicating that smoking/BaP changes protein expressions that influence the folding-integrity of proteins. To test for genomic effects of BaP, chromosomal analysis was performed on placental- and bone marrow cells from treated and non-treated rats and showed an increase in chromosomal abnormalities in cultured and bone marrow
cells from BaP-treated animals. MALDI: Peptides were subjected to MALDI MS/MS in a collision chamber and dissociated with helium gas (CID). The mass/charge (m/z) spectra were subjected to bioinformatics via Mascot Ion software interrogation of the NCBI database. There were significant differences in the peptides identified from the trypsin digest of the tissue.
Conclusion
Cigarette smoking is associateds with ~10% of fetal growth retardation (Naftolin and Usher, 1975).
We have shown that smoke toxin BaP causes chromosomal abnormalities and reduction in Mitotic index of placental cells. We have found using differential proteomics that BaP influence proteins related to folding-integrity such as the heat shock protein 70 (Hsp70), which is a chaperone whose action promotes protein folding through repeated cycles of substrate binding and release in an ATP-dependent manner.
Metallothionine 2 has previously been shown to be induced in the placenta by smoking. Identifying it in a smoker placenta, but not in a non-smoker placentasupports out plan to monitor the proteomic in-situ using MALDI-MS.
Metallothionine is a protein that binds metals and may cause growth retardation of tissues, including the placenta. We are exploring the possibility that this may play a role in placental undergrowth in smoker's pregnancies marked by fetal growth restriction.
Related References
- Tanaka Y, et al., Proteomics 2006; 6: 4845.
- Jespersen S, et al., J. Anal. Chem. 1999; 71: 660.
- Caprioli RM, et al., J. Anal. Chem. 1997; 69: 4751.
- Naftolin F. (Ed). Berlin, Dahlem Konferenzen, 1978.
- Pevsner P, et al., Rapid Commun Mass Spectrom. 2007;21(3):429-36.
Support
NIH NICHD 047003 to FN
