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Biotransformation Research Highlights
Title: Protection against X-irradiation Toxicity
Background and Advances
Implications & Public Health Impact
Center Contribution
Key Researchers
Publication(s)
Grant Support
Title: Structure and Function of Mammalian Cytochromes P450
Background and Advances
Implications & Public Health Impact
Center Contribution
Key Researchers
Publication(s)
Grant Support
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Title: Protection against X-irradiation Toxicity
Background and Advances: A thrust of the Biotransformation Core is to
understand the pharmacological and physiological significance of
detoxification Phase II enzymes including glutathione S-transferases (GSTs).
The overall objective of these studies is to elucidate the protective
role of glutathione (GSH) -linked detoxification mechanisms against
environmental toxins and oxidative stress. Regulation of the expression
of GSTs and their induction by suitable non-toxic inducers of synthetic
and plant origin is being pursued to identify effective inhibitors of
chemical carcinogenesis.
Exposure to xenobiotics usually results in oxidative stress due to the
generation of reactive oxygen species (ROS) formed during P450-mediated
metabolism of xenobiotics. Lipid peroxidation initiated by ROS has been
shown to be involved in the etiology of cataractogenesis and other age
related degenerative diseases. 4-hydroxynonenal (4HNE), the toxic
end-product lipid peroxidation that is involved in the mechanisms of
cataractogenesis, is an endogenous substrate of GSTs. Dr. Awasthi’s
laboratory is currently studying the mechanisms of catalysis and
expression of novel sub-group of alpha-class GST isoenzymes (mGSTA4-4,
rGSTA4-4 and hGSTA 5.8) in rodents and humans. These enzymes show
substrate preferences for 4-HNE and other products of lipid peroxidation.
The goal is to determine the role of these enzymes in protection against
oxidative stress-induced lipid peroxidation and subsequent age related
degenerative diseases such as cataractogenesis, macular degeneration and
atherosclerosis. The physiological significance of these groups of GSTs
has been demonstrated by Dr. Awasthi’s studies showing that transfection
with mGSTA4-4 protects cells from oxidative stress, and that the
induction of rGSTA4-4 in lens epithelium protects rat lens from
cataractogenesis caused by oxidative stress. They have shown that
glutathione peroxidase (GPx) activity of GSTA1-1 and GSTA2-2 plays a
major role in defending against ROS toxicity and that GSTs protection
against oxidative stress, heat- and UV-induced damage, and apoptosis are
particularly important. Over-expression of GST1-1 or GST2-2 protects
cells of various origins from apoptosis caused by UV irradiation,
oxidative stress and environment toxins/drugs such as naphthalene and
doxorubicin. GSTA1-1 and GSTA2-2 provide this protection by attenuating
lipid peroxidation through their GPx activity towards fatty acid
hydroperoxides and phospholipids hydroperoxides generated during ROS-induced
lipid peroxidation. These studies indicate that GSTs can modulate
stress-mediated signaling by regulating the levels of lipid peroxidation
products. These novel findings on physiological roles of GSTs provide
new concepts in the field of signaling and open up possibilities for
future collaborative research with other Center Investigators.
Studies are also underway for structural and functional characterization
of a novel non-ABC GSH-conjugate transporter RLIP76, which is
ubiquitously expressed in human tissues. Through functional
reconstitution of purified RLIP76 in proteoliposomes, it has been
established that this versatile ATP-dependent transporter can mediate
ATP-dependent transport of not only the anionic GSH-conjugates, but also
of the cationic amphilic compounds, such as doxorubin. Recent work from
Dr. Awasthi’s laboratory has shown that over expression of RLIP76
confers resistance to the cancer cells against the cationic as well as
anionic chemical therapeutic agents. These studies suggest that this
novel transporter plays as important role in multi-drug resistance of
cancer cells. Non-small lung cancer cells (NSCLC) are highly resistant
to some of the choice cancer drugs such as doxorubicin, which are
effective in killing small cell lung cancer cells (SCLC) but not NSCLC.
Dr. Awasthi’s studies have shown that higher resistance of NSCLC or to
doxorubicin correlates with enhanced efflux of doxorubicin by NSCLC due
to increased transport activity of RLIP76. Furthermore, his studies
suggest that RLIP76 provide protection against radiation by transporting
the glutathione-conjugates of toxic electrophilic endogenous compounds
generated during irradiation. These findings may have important clinical
implications, and collaborative studies with oncologists are being
pursued to explore clinical potential of these findings. Current studies
are delineating the mechanisms by which this multifunctional protein
mediates ATP-dependent transport of drugs/xenobiotics with diverse
structures and its role in the mechanisms of MDR.
High Impact Research Findings: Studies from Dr. Awasthi’s laboratory had
earlier shown that he novel GSH-conjugate transporter RLIP76 provides
protection against the toxicity of chemotherapeutic agents and UV- and
X-irradiations in cell cultures. His recent in vivo studies (Cancer
Research 65:2, 2005) have shown that RLIP76 (-/-) mice are highly
sensitive to X-irradiation as compared to the RLIP76 (+/+) wild-type
mice, and that intraperitoneal injection of RLIP76 liposomes to the
RLIP76 (-/-) restores their inherent resistance to X-irradiation
comparable to that of the wild-type (RLIP76 +/+) mice. More importantly,
these studies show that RLIP76 liposomes administered wild type mice
acquire several fold increased resistance to X-irradiation as indicated
by substantially increased survival time as compared to X-irradiated
control wild-type mice. These studies suggest that: 1) inhibiting RLIP76
could be used for sensitization to radiation, 2) RLIP76 liposomes could
be used to minimize X-irradiation toxicity to tissues during radiation
therapy, and 3) RLIP76 liposomes could be used as radioprotective
agents. These studies were conducted in collaboration with Dr. Paul
Boor, Ph.D. who is an Oxidative Stress Core member and Dr. Sanjay
Awasthi, M.D. an oncologist at UT Arlington.
Implications and Public Health Impact: In vivo studies using RLIP76 null
mice highlighted above strongly suggest potential clinical use of RLIP76 liposomes. Since RLIP76 inhibition potentiates the toxicity of
X-irradiation, the inhibition or depletion of RLIP76 could be an
important strategy to enhance efficacy of radiation therapy. RLIP76
mediated protection against X-irradiation also opens up the possibility
of RLIP76 proteoliposomes as effective radioprotective agents for
patients during radiation therapy. The ability of RLIP76 proteoliposomes
to provide remarkable protection against X-irradiation toxicity could
particularly be important to strategies for negating radiation toxicity
to populations exposed to radiation during accidents or other
catastrophic events such as an unforeseen “dirty bomb” attack.
Center Contribution: The expertise of Center Investigators, Drs. Ansari
and Boor, are utilized to pursue this research. The Molecular Genetics
and Biomolecular Resource Facility cores have been utilized.
Key Researchers:
G.A. Shakeel Ansari, Biotransformation Research Core, Department of
Biochemistry and Molecular Biology and Department of Pathology
Yogesh C. Awasthi, Biotransformation Research Core, Department of
Biochemistry and Molecular Biology
Paul J. Boor, Oxidative Stress and Signaling Research Core, Department
of Pathology
Publication(s):
Awasthi S, Singhal SS, Sushma Y, Singhal Y, Drake K, Nadkar A, Zajac E,
Wickramarachchi D, Row N, Yacoub A, Boor P, Dwivedi S, Dent P, Jarman
WE, Johns B, Awasthi YC 2005; RLIP76 is a major determinant of radiation
sensitivity. Cancer Research 65(14):1-7.
Awasthi YC, Ansari GAS, Awasthi S, 2005; Phase II: Conjugation enzymes,
glutathione transferases and transport systems. Methods in Enzymology.
Volume 401:379-409.
Liang FQ, Assadi R, Morehead P, Awasthi YC, Godley BF, 2005; Enhanced
expression of glutathione S-transferase A1-1 protects against oxidative
stress in human retinal pigment epithelial cells. Exp. Eye Res.
8:113-119.
Patrick B, Li J, Jeyabal PV, Reddy PM, Yang y, Sharma R, Sinha M, Luxon
B, Zimniak P, Awasthi S, Awasthi YC. Depeltion of 4-hydroxynonenal
hGSTA4-transfected HLB B-3 cells results in profound changes in gene
expression. Biochem. Biophy. Res. Commun. 334(2):425-432.
Stuckler D, Singhal J, Singhal SS, Yadav S, Awasthi YC, Awasthi S, 2005;
RLIP76 transports vinorelbine and mediates rrug resistance in non-small
cell lung cancer. Cancer Res.65 (3).
Yadav S, Zaja CE, Singhal SS, Singhal J, Drake K, Awasthi YC, and
Awasthi S, 2005; POB1 over-expression inhibits RLIP76-mediated transport
of glutathione-conjugates, drugs and promotes apoptosis. Biochem.
Biophys. Res. Commun. 328: 1003-9.
Grant Support:
NIH/NIEHS
5 RO1 ES 12171 Protection of Oxidant Toxicity by GSTs
NIH/NEI
5 RO1 EY 04396 Detoxification of Xenobiotics in Ocular Tissue
Title: Structure and Function of Mammalian Cytochromes P450
Background and Advances: Another major thrust of research in the Biotransformation Core is to understand the structural determinants of specificity of individual cytochrome P450 enzymes (CYP). They constitute a superfamily of hemoproteins that play a pivotal role in the metabolism of a wide variety of foreign (xenobiotics) and endogenous compounds. Three gene families (CYP1, CYP2, and CYP3) are thought to be responsible for the majority of xenobiotic oxidation in human liver. Heterogeneity in the expression levels and/or activities of these enzymes is a major determinant of individual response to medications and likely contributes to individual susceptibility to environmental toxicants as well. 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.
The ultimate goal is to predict ligand interactions with wild-type and variant forms of the enzymes in order to improve drug discovery and therapy, safety assessment of chemicals, and individual risk assessment upon exposure to xenobiotics. Substrate interactions with cytochromes P450 have been proposed to occur in three stages: 1) recognition by surface residues; 2) entry into the buried active site through a hydrophobic access channel; and 3) orientation in the active site to allow catalysis. Research during the past year utilizing x-ray crystallography and isothermal titration calorimetry has elucidated how ligands of different size and shape may induce very different conformations of cytochrome P450 2B4. 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 allow us to model the membrane-associated state of P450 and provide insight into how lipophilic substrates access the buried active site.
The ultimate goal of the research involving cholesterol-metabolizing P450s is to understand whether these important enzymes could serve as targets for regulation of serum cholesterol levels via post-translational modulation of their activities by exogenous compounds. Previous studies in Dr. Pikuleva’s laboratory showed the importance of the membrane-protein interactions for catalytic efficiency of cholesterol-metabolizing P450s. The focus during the past year was on the active site of cytochromes P450 7A1 and 27A1, the two key enzyme in cholesterol degradation. Investigation of P450 27A1 was carried out in collaboration with Dr. Halpert. Computer modeling supported by site-directed mutagenesis suggests that there is a very tight complementary fit between cholesterol and the P450 7A1 substrate pocket, and this “’goodness of fit” seems to be the feature that contributes in part to strict substrate specificity and high catalytic efficiency of this enzyme. The substrate fit in the P450 27A1 active site is not as good as in P450 7A1, therefore different physiological substrates occupy different sub-regions within the P450 27A1 substrate pocket and bind in different orientations. The data provide insight into how cholesterol-metabolizing P450s have adapted to fit their specific roles in cholesterol elimination.
Implications and Public Health Impact: The major causes of variation in xenobiotic metabolism are genetic polymorphisms, induction or inhibition due to concomitant drug therapies or environmental factors, physiological status, and disease states. P450 genetic polymorphisms in particular are increasingly being recognized as major contributors to therapeutic failure or adverse effects of drugs and environmental chemicals. 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. Accordingly, detailed knowledge of the structural basis of substrate specificities of cytochromes P450 is crucial for predicting and/or rationalizing individual and species differences in xenobiotic metabolism and response, as well as metabolic interactions between compounds. Understanding of how cholesterol-metabolizing P450s function provides a basis for development of new therapeutic strategies aimed at lowering serum cholesterol levels. Furthermore, distinct biding of the P450 27A1 substrates may provide insight into why phenotypic manifestations of the disease associated with the deficiency of P450 27A1 activity which are so diverse.
Center Contribution: The Center brought together the biochemical and biophysical expertise of Dr. Halpert’s and Dr. Pikuleva’s laboratories with the computational expertise of Dr. Braun. In addition, the expertise of Dr. Ansari in lipid chemistry proved essential for Dr. Pikuleva’s progress in purifying and characterizing cholesterol-metabolizing P450 enzymes. Dr. Ansari will continue to be a key collaborator in Dr. Pikuleva’s future studies evaluating polyunsaturated fatty acids as post-translational modulators of the activity of cholesterol-metabolizing P450s. With the recent evidence that membrane interactions may be very important for substrate recognition and access, the involvement of Dr. Ansari may also be of value in future Dr. Halpert’s studies.
Key Researchers:
Shakeel Ansari, Biotransformation Research Core, Department of Biochemistry and Molecular Biology and Department of Pathology.
James R. Halpert, Biotransformation Research Core, Department of Pharmacology and Toxicology.
Werner Braun, DNA Repair and Mutagenesis Research Core, Department of Biochemistry and Molecular Biology.
Irina Pikuleva, Biotransformation Research Core, Department of Pharmacology and Toxicology.
Publication(s):
Zhao, Y., White, M.A., Muralidhara, B.K., Sun, L., Halpert, J.R., and Stout, C.D. (2005). Structure of microsomal cytochrome P450 2B4 complexed with the antifungal drug bifonazole: Insight into P450 conformational plasticity and membrane interaction. J. Biol. Chem.[Epub ahead of print, Dec. 21].
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., [Epub ahead of print, Jan. 26].
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., Pikuleva, I.A. A simple and rapid method to measure cholesterol binding to P450s and other proteins. (2005). J. Lip. Res. 46,1561-1568.
Mast, N., Murtazina, D., Liu, H., Graham, S.E., Bjorkhem, I., Halpert, J.R., Peterson, J., and
Pikuleva, I.A. (2005) Distinct binding of cholesterol and 5b-cholestan-3a,7a,12a-triol to cytochrome P450 27A1: evidence from modeling and site-directed mutagenesis studies. Biochemistry (submitted).
Grant Support:
NIH/NIEHS R01 ES03619
Molecular Basis of Selective P450 2B Function
NIH/NIGMS R01 GM62882
Cholesterol Metabolizing P450s: Structure and Function
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