University of Texas Medical Branch Galveston

Cellular Response Mechanisms to Environmental Challenge

James R Halpert, Ph.D.
jhalpert@utmb.edu
http://www.utmb.edu/niehs/

Project Description

The mission of this Center is to integrate, coordinate and foster interactions and collaborations among a group of established investigators pursuing research pertinent to the effects of environmental factors on human health. The proximity to sources of many significant environmental problems, such as ozone pollution, emissions of fine particulates, hazardous chemical releases, hazardous waste sites, and pediatric lead exposure, makes UTMB a compelling site for a multidisciplinary environmental health sciences center. During the nine years of its existence, this Center has emerged as a national leader in elucidating cellular response mechanisms to environmental challenge and in working with the community to enhance awareness of environmental health issues and elaborate prevention and intervention strategies. The overarching theme of the UTMB NIEHS Center is the role of oxidative stress in mediating the health effects of exposure to environmental factors. Center investigators are aided by four service cores (Molecular Genetics, Biomolecular Resource Facility, Cell Biology, and Synthetic Organic Chemistry), which provide advanced technologies, unique reagents, and specialized expertise, as well as cost-effective and efficient access to more routine services or research materials. Scientific findings from the Center are communicated to the public through a vibrant COEP with advice from a Community Outreach Board.

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Program Highlights

Prevention of Colorectal Cancer and Sepsis by Aldose Reductase Inhibition

It is well known that increased expression of cytokines and chemokines elicits oxidative stress and cytotoxicity in many inflammatory diseases. Over 100,000 deaths in the U.S. each year can be attributed to an excessive inflammatory response alone. Moreover, inflammation plays a critical role in the pathophysiology of a number diseases; hence, elucidating the mechanisms that regulate inflammatory signals has profound importance for understanding and managing a very large array of disease processes, including cancer. Dr. Satish K. Srivastava and his associates have recently demonstrated that aldose reductase (AR), an enzyme that was known to reduce mainly aldosugars, efficiently reduces lipid aldehydes and their glutathione conjugates, and mediates cytotoxic signals of cytokines, chemokines and endotoxins. Studies to delineate the mechanism(s) of AR’s mediation in the inflammatory signals will be important in understanding the therapeutic significance of AR inhibitors in the prevention of inflammatory disorders such as colon cancer, sepsis, arthritis and asthma.

Using cellular and mouse models of colon cancer and sepsis, Dr. Srivastava’s group has shown that AR is an obligatory mediator of cytokine, growth factor and bacterial endotoxin signals that cause increased expression of NF-κB dependent inflammatory markers and tissue damage. Inhibition of AR prevents growth factor and cytokine–induced expression of Cox-2 and PGE2, and growth in human colon cancer cells. Further, siRNA based ablation of AR completely prevented the tumor growth in nude mice xenografts bearing SW480 human colon adenocarcinoma cells. These results suggest that AR inhibitors could be effectively used as chemopreventive drugs in colorectal cancer. In another study, inhibition of AR prevented bacterial endotoxin, lipopolysaccharide–induced expression of inflammatory cytokines, chemokines, Cox-2 and iNOS in murine macrophages as well as mouse serum, liver, kidney and heart. Further, inhibition of AR prevents septic shock–induced myocardial fractional shortening and contractile functions in mouse heart. Also, inhibition of AR increases survival in mice injected with lethal doses of lipopolysaccharide. These results suggest that inhibition of AR could prevent sepsis-induced Cardiomyopathy, and that AR inhibitors could be excellent anti-inflammatory drugs. Thus this innovative research has assigned a novel physiological role to AR in mediation of inflammatory signals and has laid the foundation for potential translation of these studies to prevent/treat colorectal cancer and septic shock in humans.

Tammali, R., Ramana, K.V., Singhal, S.S., Awathi, S. and Srivastava, S.K. (2006) Aldose reductase regulates growth factor-induced cyclooxygenase-2 expression and prostaglandin E2 production in human colon cancer cells. Cancer Res. 66:9705-9713.

Ramana, K.V., Willis, M.S., White, M.D., Horton, J.W., DiMaio, M., Srivastava, D., Bhatnagar, and Srivastava, S.K. (2006) Endotoxin-induced cardio myopathy and systemic inflammation in mice is prevented by aldose reductase inhibition. Circulation 114:1838-1846.

Ramana, K.V., Fadl, A.A., Reddy, A.B.M., Tammali, R., Chopra, A.K. and Srivastava, S.K. (2006) Aldose reductase mediates the lipopolysaccharides-induced release of inflammatory mediators in RAW264.7 murine macrophages. J. Biol. Chem. 281:33019-33029.

Ramana, K.V., Bhatnagar, A., Srivastava, S., Yadav, U.C., Awasthi, S., Awasthi, Y.C., Srivastava S.K. (2006) Mitogenic responses of vascular smooth muscle cells to lipid peroxidation-derived aldehyde 4-hydroxy-trans-2-nonenal (HNE): role of aldose reductase-catalyzed reduction of the HNE-glutathione conjugates in regulating cell growth. J Biol Chem. 281:17652-17660.

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Structure and Function of Mammalian Cytochromes P450

Three gene families (CYP1, CYP2, and CYP3) are thought to be responsible for the majority of xenobiotic oxidation in human liver. The major causes of individual variation are genetic polymorphisms, induction or inhibition due to concomitant drug therapies or environmental factors, physiological status, and disease states. In addition to their crucial role in individual differences in xenobiotic response in humans, cytochromes P450 are also major contributors to species differences in metabolism. Such species differences are very important in the proper choice of animal species as surrogates for humans in safety evaluation of drugs and other chemicals. Other gene families, including CYP7, CYP11, CYP27, and CYP46, are key determinants of metabolism of endogenous compounds such as cholesterol. Deficiencies in these enzymes can lead to abnormal accumulation of cholesterol and a variety of disease phenotypes that may be exacerbated by environmental exposures. In addition, understanding of how cholesterol-metabolizing P450s function provides a basis for development of new therapeutic strategies aimed at lowering serum cholesterol levels.

Recent research utilizing x-ray crystallography and isothermal titration calorimetry has elucidated how ligands of different size and shape induce very different conformations of cytochrome CYP2B4. Structurally plastic regions were identified that undergo correlated conformational changes in response to ligand binding. Molecular modeling has been used to understand the results. The most plastic regions are putative membrane binding motifs involved in substrate access or substrate binding. The results provide insight into how lipophilic substrates access the buried active site and have led to the realization that P450 2B enzymes, and presumably other mammalian xenobiotic-metabolizing cytochromes P450, operate by an induced-fit mechanism. Recent investigation by site-directed mutagenesis and molecular modeling of the active sites of cytochromes CYP7A1 and 27A1 provide insight into how the two key enzymes in cholesterol degradation have adapted to fit their specific physiological roles. There is a very tight complementary fit between cholesterol and the CYP7A1 substrate pocket, which appears to be the feature that contributes in part to strict substrate specificity and high catalytic efficiency of this enzyme. The substrate fit in the CYP27A1 active site is less good, suggesting that different physiological substrates occupy different sub-regions within substrate pocket and bind in different orientations. The most recent breakthrough has been the solution of X-ray crystal structures of substrate-bound and ligand-free CYP46A1, a key enzyme in cholesterol elimination from the brain that may play a role in Alzheimer’s disease. Substrate binding induces an opening of a channel, presumably water channel, that is not seen in any other structurally characterized mammalian P450.

Zhao, Y., White, M.A., Muralidhara, B.K., Sun, L., Halpert, J.R., and Stout, C.D. (2006). Structure of microsomal cytochrome P450 2B4 complexed with the antifungal drug bifonazole: Insight into P450 conformational plasticity and membrane interaction. J. Biol. Chem. 281:5973-5981.

Muralidhara, B.K., Negi, S., Chin, C.C., Braun, W., and Halpert, J.R. (2006) Conformational flexibility of mammalian cytochrome P450 2B4 in binding imidazole inhibitors of different ring chemistry and side chains: Solution thermodynamics and molecular modeling. J. Biol. Chem. 281:8051-8061.

Mast, N., Graham, S.E., Andersson, U., Bjorkhem, I., Hill, C., Peterson, J., Pikuleva. I.A. (2005). Cholesterol binding to cytochrome P450 7A1, a key enzyme in bile acid biosynthesis. Biochemistry 44, 3259-3271.

Mast, N., Murtazina, D., Liu, H., Graham, S.E., Bjorkhem, I., Halpert, J.R., Peterson, J., and Pikuleva, I.A. (2006) Distinct binding of cholesterol and 5β-cholestan-3α,7α,12α-triol to cytochrome P450 27A1: Evidence from modeling and site-directed mutagenesis studies. Biochemistry 45:4396-4404.

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