Chem. Res. Toxicol. 2007, 20, 3-5
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PerspectiVe Future of ToxicologysLipid Peroxidation in the Future: From Biomarker to Etiology Koji Uchida* Graduate School of Bioagricultural Sciences, Nagoya UniVersity, Nagoya 464-8601, Japan ReceiVed NoVember 1, 2006
On the basis of a large number of reports concerning the detection of lipid peroxidation products as biomarkers in human diseases, there is no doubt that their steady-state levels increase under pathophysiological states associated with oxidative stress. The key question is whether they play any causative roles. This may be a matter of primary concern, which represents an important direction to pursue in the future of toxicology. My latest approach from autoimmune response induced by lipid peroxidation products will be illustrated. Lipid Peroxidation-Specific Epitopes as Biomarkers
Contents Introduction Lipid Peroxidation-Specific Epitopes as Biomarkers Lipid Peroxidation-Specific Epitopes as Self-Antigens HNE-Specific Epitopes as Sources of Anti-DNA Antibodies Is Lipid Peroxidation an Etiology of SLE? Conclusion
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Introduction As most of the readers of this journal well know, lipid peroxidation plays a role in the pathogenesis of many types of tissue injuries and especially in the tissue damage induced by several toxic substances. In addition, lipid peroxidation has been implicated in the pathogenesis of numerous diseases including atherosclerosis, diabetes, cancer, and rheumatoid arthritis, as well as in drug-associated toxicity, postischemic reoxygenation injury, and aging (1). However, current evidence for the involvement of lipid peroxidation in the pathogenesis is mostly indirect. Therefore, a natural interest is whether lipid peroxidation is directly involved in human diseases. This may be a matter of primary concern, which represents an important direction to pursue in the future of toxicology. In this perspective, after a brief summary of recent approaches that have been explored for protein-bound lipid peroxidation products as biomarkers, I will illustrate our latest approach to establish the involvement of the modified proteins with lipid peroxidation products in the etiology of a human disease. This short perspective is therefore going to be based on my personal view and interest in the future of toxicology. * To whom correspondence should be addressed. Tel: 81-52-789-4127. Fax: 81-52-789-5741. E-mail:
[email protected].
Lipid peroxidation generates a large number of toxic products, such as 2-alkenals, 4-hydroxy-2-alkenals, and ketoaldehydes (2). A number of chemists in the toxicology and free radical biology fields have significantly contributed to the identification of these aldehydes and characterized their in vitro effects. In addition, they have established the utility of these reactive aldehydes as the biomarkers for oxidative stress. A colorimetric assay using 2-thiobarbituric acid, for example, is probably the easiest way of evaluating lipid peroxidation. Highly sensitive and selective gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry assays have also been utilized to accurately estimate the biological levels of lipid peroxidation products. An alternative and probably more popular approach for the detection of lipid peroxidation products in biological samples is the use of antibodies. Immunological detection is a powerful tool that can be used to evaluate the presence of a desired target and its subcellular localization. The major advantages of this technique over the chemical approaches are the evaluation of small numbers of cells or archival tissues that may otherwise not be subject to analysis. Antibodies are usually raised by immunizing animals with protein-bound lipid peroxidation products and targeting specific antigenic structures (“lipid peroxidation-specific epitopes”) generated on the amino acid side chains of proteins. The antibodies developed through this procedure bind not only to the modified protein used as the immunogen but also to a variety of other proteins on which the same epitope is found. Thus, antibodies generated against a modified protein with lipid peroxidation-specific products recognize a variety of similarly modified proteins. Figure 1 represents a crystal structure of the Fab fragment of a monoclonal antibody with a lipid peroxidation-specific epitope, showing how specifically the antibody recognizes the epitope. Such properties make the antibodies directed to the lipid peroxidation-specific epitopes useful and reliable for the immunological evaluation of lipid peroxidation in vitro and in vivo. Taking advantage of the fact that the lipid peroxidation-specific epitopes are excellent immunogens that are capable of stimulat-
10.1021/tx600304n CCC: $37.00 © 2007 American Chemical Society Published on Web 12/05/2006
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ing adaptive immune response, a number of monoclonal antibodies against these epitopes have been developed. The table in Supporting Information lists the lipid peroxidation-specific monoclonal antibodies that have been established by my group at Nagoya University in collaboration with NOF Co., Ltd. (Tokyo, Japan). The development of these specific antibodies has enabled us to obtain very important evidence for the presence of lipid peroxidation-specific epitopes in vivo.
Lipid Peroxidation-Specific Epitopes as Self-Antigens What, then, is the physiological and pathophysiological role of the epitope production? Does it have anything to do with chemical toxicology? Answering such questions seems important when it comes to considering the fact that lipid peroxidation is one of the most important sources of endogenous toxic metabolites. Some insights have come from innate responses. Binder et al. proposed that the oxidation-derived epitopes, including the lipid peroxidation-specific epitopes generated on self-antigens, are important immunodominant targets of natural antibodies and suggested that these antibodies play an important function in the host response to the consequences of oxidative stress to the oxidative events that occur when cells undergo apoptosis (3). The lipid peroxidation-specific epitopes have also been shown to stimulate an autoimmune response. They are the targets of the B-cell-mediated immune responses and induce T-cell responses and add the potential of certain aldehydes to induce autoimmunity by breaking the B-cell tolerance to nonmodified proteins (4). The modification of self-proteins by lipid peroxidation-specific aldehyde species, such as 4-hydroxy2-nonenal (HNE),1 indeed results in breaking the tolerance to self-proteins (4). In addition, the immunization of animals with oxidized low-density lipoproteins (LDLs) has been shown to mediate the enhanced production of autoantibodies directed against the HNE-modified LDL (5, 6). More recently, modification of a lupus-associated protein with HNE has been shown to increase the antigenicity and to facilitate epitope spreading (7). These observations and the fact that the HNE adduction within proteins is enhanced in aging and stressed cells and occurs under physiological conditions (2, 8) suggest that the post-translational protein modification with lipid peroxidation products, HNE in particular, may serve as a trigger for the autoimmune response.
HNE-Specific Epitopes as Sources of Anti-DNA Antibodies HNE is one of the major products generated during the peroxidation of ω6 polyunsaturated fatty acids, such as linoleic acid and arachidonic acid, and is believed to be largely responsible for the cytopathological effects observed during oxidative stress. The natural form of HNE is a racemic mixture of the 4R- and 4S-enantiomers. HNE specifically reacts with nucleophilic amino acids, such as cysteine, histidine, and lysine, and uniquely forms stable cyclic hemiacetal adducts possessing three chiral centers in the cyclic hemiacetal structure (9). Because of this chirality, the HNE adduct is composed of eight configurational isomers. Several different types of monoclonal antibodies have been raised against the protein-bound HNE and have attested to be specific for the HNE-histidine adduct (9). On the basis of a large number of reports on the detection of proteinbound HNE in human diseases, there is no doubt that the steadystate levels of HNE increase under pathophysiological states 1Abbreviations: HNE, 4-hydroxy-2-nonenal; LDL, low-density lipoprotein; SLE, systemic lupus erythematosus.
Figure 1. Crystal structure of the Fab fragment of a monoclonal antibody with a lipid peroxidation-specific epitope. The hydrophobic domains at the antigen-binding site of the antibody are colored red. The antigen (R-HNE-histidine adduct) is shown in the wireframe model. This figure is courtesy of Dr. Sohei Ito (University of Shizuoka).
associated with oxidative stress. Considerable progress has also recently been made toward understanding the mechanisms of action of HNE. Some years ago, when we analyzed the variable genes and primary structure of the antibodies against the HNEspecific epitopes, we accidentally found that the sequence of a monoclonal antibody was highly homologous to the anti-DNA autoantibodies, the hallmark of systemic lupus erythematosus (SLE) (10). In addition, we carried out crystallographic and molecular modeling studies and demonstrated that the combining sites of high-affinity anti-HNE monoclonal antibodies possessed a stereoelectronic complementarity to DNA ligands (10, 11). These findings suggested that the HNE-specific epitopes could be the source of the anti-DNA antibodies. A more attractive hypothesis is that the HNE-specific epitopes may represent immunologic triggers for human autoimmune diseases and allergies.
Is Lipid Peroxidation an Etiology of SLE? Antibodies against nuclear antigens are a common manifestation of the human SLE. SLE is an autoimmune disease in which the body’s own immune system is directed against the body’s own tissues. Of the multiple antinuclear antibodies described in this disease, antibodies against DNA are among the most characteristic; yet, the triggering antigen for the disease is still unknown. Several lines of experimental evidence suggest that lipid peroxidation plays a role in the SLE. (i) Patients with SLE have been shown to have an enhanced urinary excretion of isoprostanes, consistent with enhanced lipid peroxidation (12). (ii) The levels of the lipid peroxidation products, such as HNE and malondialdehyde, are significantly elevated in children with a high SLE disease activity (13). (iii) Elevated levels of oxidized LDL together with elevated levels of autoantibodies related to the oxidized LDL in female patients with SLE have also been realized (14). Consistent with these observations, our preliminary experiments have shown that an anti-DNA autoantibody obtained from a spontaneous murine model of SLE significantly cross-reacts with the HNE-modified protein as well as the native DNA and that there is a significant correlation between the serum anti-DNA and the anti-HNE titers in both the control and the patients with SLE (Toyoda, K., Akagawa, M., Matsuda,
PerspectiVe
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T., and Uchida, K. Unpublished data). These findings suggest that the lipid peroxidation modification of cellular components, generating a variety of antigenic structures, may give rise to the production of multispecific antibodies that simultaneously recognize different epitope structures. Clearly, it is important to establish whether this lipid peroxidation-based multiple crossreactivity occurs in vivo. Furthermore, the characterization of the biological consequences of the production of such antibodies also merits immediate attention.
Conclusion On the basis of a large number of reports concerning the detection of lipid peroxidation-specific epitopes as biomarkers in human diseases, there is no doubt that the steady-state levels of lipid peroxidation products increase under pathophysiological states associated with oxidative stress. Considerable progress has also recently been made toward understanding the mechanisms of action of lipid peroxidation products (15). However, there are a number of interesting questions still remaining to be answered. Particularly, as raised by Daniel C. Liebler in the first Future of Toxicology perspective in this series (16), the key question is whether lipid peroxidation products and other biological reactive intermediates play causative roles, and if so, how do they act? Currently, I am trying to answer this question from the autoimmune response induced by lipid peroxidationspecific epitopes. Somebody may be approaching this same subject from a different direction. There is no doubt that the task ahead of us is difficult but cannot be avoided for the future of lipid peroxidation in toxicology. To comment on this and other Future of Toxicology perspectives, please visit our Perspectives Open Forum at http:// pubs.acs.org/journals/crtoec/openforum. Acknowledgment. I thank Dr. Sohei Ito for providing the X-ray crystallographic data (Figure 1). Supporting Information Available: Table of lipid peroxidation-specific monoclonal antibodies. This material is available free of charge via the Internet at http://pubs.acs.org.
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(3) Binder, C. J., Shaw, P. X., Chang, M. K., Boullier, A., Hartvigsen, K., Horkko, S., Miller, Y. I., Woelkers, D. A., Corr, M., and Witztum, J. L. (2005) The role of natural antibodies in atherogenesis. J. Lipid Res. 46, 1353-1363. (4) Wuttge, D. M., Bruzelius, M., and Stemme, S. (1999) T-cell recognition of lipid peroxidation products breaks tolerance to self proteins. Immunology 98, 273-279. (5) Palinski, W., and Witztum, J. L. (2000) Immune responses to oxidative neoepitopes on LDL and phospholipids modulate the development of atherosclerosis. J. Intern. Med. 247, 371-380. (6) Palinski, W., Yla-Herttuala, S., Rosenfeld, M. E., Butler, S., Socher, S. A., Parthasarathy, S., Curtiss, L. K., and Witztum, J. L. (1990) Antisera and monoclonal antibodies specific for epitopes generated during oxidative modification of low density lipoprotein. Atherosclerosis 10, 325-335. (7) Scofield, R. H., Kurien, B. T., Ganick, S., McClain, M. T., Pye, Q., James, J. A., Schneider, R. I., Broyles, R. H., Bachmann, M., and Hensley, K. (2005) Modification of lupus-associated 60-kDa Ro protein with the lipid oxidation product 4-hydroxy-2-nonenal increases antigenicity and facilitates epitope spreading. Free Radical Biol. Med. 38, 719-728. (8) Uchida, K. (2003) 4-Hydroxy-2-nonenal: A product and mediator of oxidative stress. Prog. Lipid Res. 42, 318-343. (9) Uchida, K. (2000) Role of reactive aldehyde in cardiovascular diseases. Free Radical Biol. Med. 28, 1685-1696. (10) Akagawa, M., Ito, S., Toyoda, K., Ishii, Y., Tatsuda, E., Yamaguchi, S., Shibata, T., Ishino, K., Kishi, Y., Adachi, T., Tsubata, T., Takasaki, Y., Hattori, N., Matsuda, T., and Uchida, K. (2006) Bispecific antibodies against modified protein and DNA with oxidized lipids. Proc. Natl. Acad. Sci. U.S.A. 103, 6160-6165. (11) Ito, S., Tatsuda, E., Ishino, K., Suzuki, K., Sakai, H., and Uchida, K. (2006) Crystallization and molecular-replacement studies of the monoclonal antibody mAbR310 specific for the (R)-HNE-modified protein. Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 62, 562-564. (12) Iuliano, L., Pratico, D., Ferro, D., Pittoni, V., Valesini, G., Lawson, J., Fitzgerald, G. A., and Violi, F. (1997) Enhanced lipid peroxidation in patients positive for antiphospholipid antibodies. Blood 90, 39313935. (13) Grune, T., Michel, P., Sitte, N., Eggert, W., Albrecht-Nebe, H., Esterbauer, H., and Siems, W. G. (1997) Increased levels of 4-hydroxynonenal modified proteins in plasma of children with autoimmune diseases. Free Radical Biol. Med. 23, 357-360. (14) Frostegard, J., Svenungsson, E., Wu, R., Gunnarsson, I., Lundberg, I. E., Klareskog, L., Horkko, S., and Witztum, J. L. (2005) Lipid peroxidation is enhanced in patients with systemic lupus erythematosus and is associated with arterial and renal disease manifestations. Arthritis Rheum. 52, 192-200. (15) West, J. D., and Marnett, L. J. (2006) Endogenous reactive intermediates as modulators of cell signaling and cell death. Chem. Res. Toxicol. 19, 173-194. (16) Liebler, D. C. (2006) The poisons within. Application of toxicology mechanisms to fundamental disease processes. Chem. Res. Toxicol. 19, 610-613.
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