Evaluation of Toxicological Monitoring Markers Using Proteomic Analysis Donggeun Sul† Graduate School of Medicine and Environmental Toxico-Genomic and Proteomic Center, College of Medicine, Korea University, Anamdong 5, Sungbukku, Seoul 136-701, Korea Received July 28, 2006
In our study, we chose three different concentrations of FA (0, 5, and 10 ppm), and cytotoxic (lipid peroxidation and protein oxidation) and genotoxic assays (DNA damage) were carried out on plasma, blood, and liver cells of rats subjected to FA-inhalation treatment. The profiles of plasma protein changes determined using 2-DE analysis were also evaluated to identify potential toxicological monitoring markers in FA-exposed rats. Concern was raised that our genotoxic analyses did not follow previously published research data and that the results of our rat plasma proteomic studies were difficult to interpret because we did not directly determine the plasma concentration of FA. However, we had already determined the concentration of FA using HPLC in an exposure chamber to monitor FA inhalation concentrations. We suggest that our experimental design was suitable to determine the FA effects on rat using an inhalation chamber system. For the similarity of genotoxic effects in lymphocytes and liver cells, we chose to present our data on the general cytological toxic effects on lipid peroxidation and protein oxidation which revealed a similarity between plasma and liver cells of FA-exposed rats. We have shown strong correlations between genotoxicity and lipid peroxidation, and lipid peroxidation is known to mediate DNA damage in many in vitro, and in vivo studies. We are well aware of the ‘implausibility’ of leukemia induction by FA, but for precisely this reason, we feel the need for further study to prove the systemic genotoxic effects of FA. Keywords: DNA damage • formaldehyde • lipid peroxidation • protein oxidation
In our study,1 we chose three different concentrations of formaldehyde (FA) (0, 5, and 10 ppm), and cytotoxic (lipid peroxidation and protein oxidation) and genotoxic assays (DNA damage) were carried out on plasma, blood, and liver cells of rats subjected to FA-inhalation treatment. The profiles of plasma protein changes determined using 2-DE analysis were also evaluated with the aim of identifying potential toxicological monitoring markers in FA-exposed rats. In addition, we discussed the possible associations between cytotoxic and genotoxic assays with respect to plasma protein changes. Concern was raised that our genotoxic analyses did not follow previously published research data.2,3 I agree that the plausibility and biological significance of leukemia induction by FA has to be questioned4 and that many positive5-11 and negative12-16 data concerning cytogenic studies of FA-exposed lymphocytes have been discussed in human studies. In addition, animal studies have shown similar conflicts of cytogenic effects in bone marrow.17-19 Speit points out the similarity of genotoxic effects observed in lymphocytes and liver cells in rats exposed to FA, but we were also interested in DNA damage to lymphocytes and liver cells and chose to present our data on † To whom correspondence should be address: Dr. Donggeun Sul, Graduate School of Medicine and Environmental Toxico-Genomic and Proteomic Center, College of Medicine, Korea University, Anamdong 5, Sungbukku, Seoul 136-701, Korea. Tel., +82-2-920-6614; fax, +82-2-927-7220; e-mail,
[email protected].
10.1021/pr060384d CCC: $33.50
2006 American Chemical Society
the general cytological toxic effects on lipid peroxidation and protein oxidation which revealed a similarity between plasma and liver cells of FA-exposed rats. We have shown strong correlations between genotoxicity and lipid peroxidation, and the latter is known to mediate DNA damage in many in vitro and in vivo studies.20-24 Speit also postulates that it is uncertain whether the inhaled FA can cause a similar degree of DNA damage in lymphocytes and other organ cells because of the protective metabolism in the nasal mucosa and the lung. In other unpublished study, we have shown that FA-exposed mice exhibit a significant increase in genotoxicity and lipid peroxidation in lung tissue, bronchoalveolar lavage cells, lymphocytes, and lymph node. We have also shown increased level of reactive oxygen species (ROS) in the cells (5 × 105 cells/sample) isolated from the lung tissue. These data support the idea that FA can cross the alveolar-capillary barrier with deleterious effects on other organ tissues via blood circulation. Speit also suggests that the results of our rat plasma proteomic studies are difficult to interpret because we did not directly determine the plasma concentration of FA. However, we demonstrated the general toxic effects of FA on plasma, such as lipid peroxidation and protein oxidation, in our study. In related studies, we determined the concentration of isofluranes (in isoflurane-exposed rats) using GC/MS in different Journal of Proteome Research 2006, 5, 2525-2526
2525
Published on Web 08/30/2006
letters tissues, including rat lymphocytes, bone marrow, spleen, brain, livers, and lung, and analyzed the DNA damage, lipid peroxidation, and protein oxidation. The results showed very strong correlations between genotoxicity and lipid and protein oxidations.23 We did not determine the concentration FA in plasma and liver tissues because we had already determined the concentration of FA using HPLC in an exposure chamber to monitor FA inhalation concentrations. We suggest that our experimental design was suitable to determine the FA effects on rat using an inhalation chamber system. If we had added more information of ROS levels or direct FA concentration in plasma and liver cells, all doubt would be removed. However, such experiments are better performed in another general toxicological study in order to provide an exact quantitative determination of FA levels in different samples with the use of radiolabeled FA producers.25 We are well aware of the ‘implausibility’ of leukemia induction by FA, but for precisely this reason, we feel the need for further study to prove the systemic genotoxic effects of FA. We are currently investigating the effects of FA on induction of airway inflammation via regulation of the production of ROS in inhalation-exposed mice and hope that this study will provide deeper mechanistic insight into the effects of FA on complex biological systems.
Acknowledgment. This work was supported by the Medical Research Center for Environmental Toxico-Genomics & Proteomics of Korea University and by the Ministry of Environment as “The Eco-Technopia 21 project”. References (1) Im, H.; Oh, E.; Mun, J.; Khim, J.-Y.; Lee, E.; Kang, H.-S.; Kim, E.; Kim, H.; Won, N.-H.; Kim, Y.-H.; Jung, W.-W.; Sul, D. J. Proteome Res. 2006, 5, 1354-1366. (2) Speit, G.; Schmid, O. Mutat. Res. 2006, published online Apr 23, http://dx.doi.org/10.1016/j.mrrev.2006.02.002. (3) Merk, O.; Speit, G. Environ. Mol. Mutagen. 1999, 33, 167-172.
2526
Journal of Proteome Research • Vol. 5, No. 10, 2006
Sul (4) Heck, H.d’A.; Casanova, M. Regulat. Toxicol. Pharmacol. 2004, 40, 92-106. (5) Bauchinger, M.; Schmid, E. Mutat. Res. 1985, 158, 195-199. (6) He, J. L.; Jin, L. F.; Jin, H. Y. Biomed. Environ. Sci. 11, 1998, 8792. (7) Kitaeva, L. V.; Mikheeva, E. A.; Shelomova, L. F.; Shvartsman, P. Y. Genetika 1996, 32, 1287-1290. (8) Shaham, J.; Gurvich, R.; Kaufman, Z. Mutat. Res. 2000, 514, 115123. (9) Shaham, J.; Bomstein, Y.; Melzer, A.; Ribak, J. Int. J. Occup. Environ. Health 1997, 3, 95-104. (10) Suruda, A.; Schulte, P.; Boeniger, M.; Hayes, R. B.; Livingston, G. K.; Steenland, K.; Stewart, P.; Herrick, R.; Douthit, D.; Fingerhut, M. A. Cancer Epidemiol., Biomarkers Prev. 1993, 2, 453-460. (11) Yager, J. W.; Cohn, K. L.; Spear, R. C.; Fisher, J. M.; Morse, L. Mutat. Res. 1986, 174, 135-139. (12) Thomson, E. J.; Shackleton, S.; Harrington, J. M. Mutat. Res. 1984, 141, 89-93. (13) Vasudeva, N.; Anand, C. J. Am. Coll. Health 1996, 44, 177-179. (14) Ying, C. J.; Yan, W. S.; Zhao, M. Y.; Ye, X. L.; Xie, H.; Yin, S. Y.; Zhu, X. S. Biomed. Envrion. Sci. 1997, 10, 451-455. (15) Ying, C. J.; Ye, X. L.; Xie, H.; Yan, W. S.; Zhao, M. Y.; Xia, T.; Yin, S. Y. Biomed. Envrion. Sci. 1999, 12, 88-94. (16) Fleig, I.; Petri, N.; Stocker, W. G.; Thiess, A. M. J. Occup. Med. 1982, 24, 1009-1012. (17) Kitaeva, L. V.; Kitaev, E. M.; Pimenova, M. N. Tsitologiya 1990, 32, 1212-1216. (18) Dallas, C. E.; Scott, M. J.; Ward, J. B., Jr.; Thesis, J. C. J. Appl. Toxicol. 1992, 12, 199-203. (19) Natarajan, A. T.; Darroudi, F.; Bussman, C. J. M.; van Kesterenvan Leeuwen, A. C. Mutat. Res. 1983, 122, 355-360. (20) Wong, Y. T.; Ruan, R.; Tay, F. E. Free Radical Res. 2006, 40, 393402. (21) Park, J. E.; Yang, J. H.; Yoon, S. J.; Lee, J. H.; Yang, E. S.; Park, J. W. Biochimie 2002, 84, 1199-1205. (22) Box, H. C.; Maccubbin, A. E. Nutrition 1997, 13, 920-921. (23) Kim, H.; Oh, E.; Im, H.; Mun, J.; Yang, M.; Khim, J. Y.; Lee, E.; Lim, S. H.; Kong, M. H.; Lee, M.; Sul, D. Toxicology 2006, 220, 169-178. (24) Prasad, N. R.; Menon, V. P.; Vasudev, V.; Pugalendi, K. V. Toxicology 2005, 209 (3), 225-235. (25) Lengyel, J.; Kala´sz, H.; Szarva, T.; Peltz, C.; Szarka´ne´-Bolehovszky, A. J. Chromatogr. Sci. 2003, 41, 177-181.
PR060384D
