Research in Dr. Smith's laboratory focuses on the biochemical and cellular mechanisms involved in the site-specific localization and production of toxicant-induced renal injury.  The compounds studied include therapeutic agents and environmental contaminants.  Research projects in her laboratory utilize a combination of in vitro  and in vivo  techniques to identify and understand the role structural and functional differences within the nephron play in the ultimate localization of toxic injury.  The experimental approach is designed such that mechanistic evaluations are conducted using in vitro  methods following characterization of the toxic events in an animal model.  The in vitro  models used include renal slices, renal tubule suspensions and renal cells in culture. 

Recent and current work has focused on delineating the mechanisms of vancomycin-induced nephrotoxicity. Dr. Smith's laboratory is currently trying to identify specific markers for distal tubular injury following vancomycin administration in rats.  These markers include the evaluation of vancomycin's effects on hormone-stimulated cAMP production in renal membrane preparations and kallikrein excretion in urine following in vivo  administration of vancomycin.  The utility of these markers for evaluating other distal tubular toxicants such as cyclodiene pesticides will also be studied.  Recent studies have characterized the time-course for accumulation of vancomycin within renal tissue with markers of proximal and distal tubular injury.  Her laboratory is now examining the mechanisms involved in vancomycin uptake in renal slices and in primary cultures of renal cortical cells.  These experiments will be expanded to examine the uptake and effects of vancomycin in primary cultures of proximal and distal cells specifically.  Studies evaluating the effects of vancomycin on mitochondrial function and the relationship of these effects on renal transport are also underway.  Additional research projects in Dr. Smith's laboratory include the study of the mechanisms of pentamidine-induced nephrotoxicity, cyclodiene pesticide-induced nephrotoxicity and the effects of radiocontrast agents in mesangial cells. 

Students in this laboratory receive training in both in vitro  and in vivo  toxicity evaluation.  The techniques available include:  whole animal nephrotoxicity assessment (urinary enzyme excretion and serum analyses), primary and secondary cell culture methods (proximal, distal and mesangial cells), cytotoxicity evaluation using microscopy (light & fluorescence), biochemical analyses of cell injury, HPLC analyses of toxicant and cellular constituent concentrations, as well as cell proliferation and phospholipid metabolism assays. 

Recent Publications

Smith HE and Smith MA. Pentamidine isethionate-induced nephrotoxicity I: in vivo effects in the female Sprague-Dawley rat. Fundamentals of Applied Toxicology (submitted) 

Kaplan DL, Mann PL, Zager PG and Smith MA. Increased contractility of mesangial cells isolated from galactose-fed rats in vitro. Cellular and Developmenal Biology (submitted) 

Smith MA, Swann J, and Acosta D. Isolation and primary culture of rat renal cortical epithelial cells. Journal of Tissue Culture Methods 11:207-210, 1988. 

Welder AA, Smith MA, Ramos K and Acosta D. Cocaine-induced cardiotxicity. In Vitro 2:205-13, 1988. 

Smith JH, Smith MA, Litterst CL, Copley MP, Uozumi J and Boyd MR. In vivo biochemical indices of nephrotoxicity of platinum analogs tetraplatin, CHIP & cisplatin at equimolar doses. Fundamntals of Applied Toxicology 10:45-61, 1988.

The focus of my research program is the understanding of the fundamental cellular and molecular processes that underlie malignant growth and therapeutic failure in human tumors, particularly those of the central nervous system.  My laboratory currently  is identifying and characterizing genes and gene products involved in these processes and we hope that this knowledge will provide novel molecular targets for therapeutic intervention, and novel diagnostic and prognostic parameters. 

One major focus of my research  is  aimed at understanding the mechanisms of DNA damage and its repair, both in the total cellular genome and in specific DNA sequences and the role they play in mediating therapeutic response in tumor cells.  We recently discovered that cellular glutathione (GSH) not only inhibits the DNA repair process, but also down-regulates  critical DNA repair-associated proteins,  both at the transcriptional and post-transcriptional levels.  Ongoing studies are directed at the nature and the mechanisms of the GSH involvement in the regulation of these  key DNA repair proteins. 

The second focus of my laboratory is on the role glutathione S-transferases (GSTs) in tumor progression and response to therapy.  We recently cloned two novel polymorphic variants of the GST-pi gene.  These findings represent the first evidence of allelic polymorphism in the locus of this gene.  Transfection studies with constructs of these variant GST-pi genes, are helping us determine their individual roles in mediating the malignant phenotype, therapeutic response, and clinical behavior of human gliomas. 

Other studies in my laboratory are directed at understanding the molecular basis of elevated GSH in brain tumors.  To this end, we have purified from human brain tumor cells a key protein, g-glutamylcysteinyl synthetase (g-GC-S ), involved in cellular GSH biosynthesis. Ongoing studies are defining how the regulation of g-GC-S may be involved in  the elevated GSH levels  observed in many human tumors. 

Recent Publications

Ali-Osman F, Berger MS, Rairkar A, and Stein DE. Enhanced repair of a cisplatin-damaged reporter chloramphenicol-O- acetyltransferase gene and altered activities of DNA polymerases alpha and beta, and DNA ligase in cells of a human malignant glioma following in vivo cisplatin therapy. Journal of Cellular Biochemistry 54:11-9, 1994. Abstract

Ali-Osman F, Berger MS, Rajagopal S, Spence A, and Livingston RB. Topoisomerase II inhibition and altered kinetics of formation and repair of nitrosourea and cisplatin-induced DNA interstrand cross-links and cytotoxicity in human glioblastoma cells. Cancer Research 53:5663-8, 1993. Abstract

Ali-Osman F, and Schofield D. Cellular and molecular studies in brain and nervous system oncology. Current Opinions in Oncology 2:655-65, 1990. 

Ishikawa T, and Ali-Osman F. Glutathione-associated cis-diamminedichloroplatinum(II) metabolism and ATP-dependent efflux from leukemia cells. Molecular characterization of glutathione-platinum complex and its biological significance. Journal of Biological Chemistry 268:20116-25, 1993. Abstract

Ali-Osman F. Prediction of clinical response to therapy of adult and pediatric brain tumor patients by chemosensitivity testing in the capillary brain tumor clonogenic cell assay. In: New Trends in Pediatric Neuro-Oncology, Vol.3, pp 220-230, Harwood Academic Publisher, Switzerland, 1991.

My laboratory is interested in the molecular mechanisms that underlie the regulation of programmed cell death (apoptosis). Many hormones, drugs and environmental intoxicants produce biological effects by altering the rate at which cells undergo apoptosis. We are applying the techniques of modern molecular biology (gene cloning, cellular transfections and transgenic mouse lineages) to investigations of the mechanisms that control gene expression during normal and drug-induced apoptosis. Studies in the laboratory encompass investigations in cultured cells, tumors and normal embryonic development.

The lab is fully equipped for molecular and cellular investigations that include molecular biological techniques, morphologic studies and digital image microscopy.

Recent Publications

Nagy L, Thomazy VA, Shipley GL, Fesus L, Lamph W, Heyman RA, Chandraratna RAS, and Davies PJA. Activation of retinoid X receptors (RXR) induces apoptosis in HL-60 cells. Molecular & Cellular Biology 7:3540-3551,1995.

Lu S, Chin K-V, Saydak M, Gentile V, Stein JP, and Davies PJA. Isolation and characterization of the human tissue transglutaminase gene promoter. Journal of Biological Chemistry 270:9748-9756, 1995.

Nagy L, Saydak M, Shipley N, Lu S, Basilion JP, Yan ZH, Syka P, Chandraratna RAS, Heyman RA, Stein JP, and Davies PJA. Identification and characterization of a versatile retinoid response element (RARE/RXRE) in the mouse tissue transglutaminase gene promoter. Journal of Biological Chemistry 271:4355-4365, 1996.

Davies PJA and Lippman SM. Biologic basis of retinoid pharmacology: implications for cancer prevention and therapy. Advances in Oncology 12:2-10,1996.

Research Interests

One of the primary focuses of Experimental Gynecology has been to investigate the effects of perinatal exposure of estrogen on the cervicovaginal (CV) tract of a mouse. Results from studies done our group indicates that 17§-estradiol both increases and decreases lactoferrin expression. That this outcome was dependent on animal age and the dose used. 17§-estradiol causes early expression of lactoferrin in the CV tract at all doses compared to controls in immature mice. In adult mice, increased lactoferrin expression was dependent on whether 17§- estradiol induced ovary-dependent or ovary-independent persistent cornification. The importance of this finding may lies in the possibility that thi may be correlated with disease progression similar to our findings with the protooncogene HER2/neu in both ovarian and endometrial cancers.

Although environmental estrogens have been implicated in the etiology of hormonally related cancers, i.e., breast and endometrial, questions still remain regarding their ability to play a significant role in the etiology of these diseases. A major finding in this area by our group has been to demonstrate the 17¶ estradiol, a relative biological inert compound in adult animals, when exposure occurs doing a critical period of development results in the formation of CV tumors. These results have begun to redefine how one characterizes what is a "weak estrogen. In addition, recent studies indicate that Polychlorinated Biphenyls (PCBs) such as 2',4',6'-Trichloro-4-biphenylol and 2',3',4',5'-Tetra-4-biphenylol are estrogenic both in neonates as well as adult animals. Results from these studies will help to clarify the potential of endocrine disruptors causing hormonal carcinogenesis when exposure occurs early in life.

Our research also focuses on determining the effects of perinatal exposure to natural estrogens on changes in mammary gland development. This research has demonstrated that neonatal estrogen treatment of female mice is associated with both the inhibition and promotion of postnatal mammary gland growth. Similar to the effect of neonatal estrogen exposure of the cervicovaginal tract, we propose that the inappropriate activation of Estrogen Receptor a (ER) during the same critical perinatal period is a key factor in mammary gland susceptibility to estrogen-induced alterations. Results from our studies indicate that mammary glands from 7 day old mice express low levels of ER. We presented the first report of a mammary gland epithelial cell line with a functional ER derived from immature mammary glands.

Selected Publications

Hajek RA, Van NT, Johnston DA, Jones LA: Exogenous Estradiol Increases Neonatal Estrogen-Induced Aneuploidy in Mouse Cervicovaginal Epithelium. Biology of Reproduction 49:908-917, 1993.

Jones LA, Hajek RA: Effects of Estrogenic Chemicals on Development, Environmental Health Perspectives 103(Suppl 7):63 Ð67, 1995.

Hajek RA, Robertson AD, Johnston DA, Van NT, Tcholakian RK, Wagner LA, Conti CJ, Meistrich ML, Contreas N, Edward CL, Jones LA. During Development, 17 a-estradiol is a potent estrogen and carcinogen. Environmental Health Perspectives 105-577-581, 1997.

DiPaolo D, Jones LA: Neonatal estrogen exposure in vivo alters mammary ER¶ expression in vitro. The Endocrine Society - 80th Annual Meeting, Endocrine Society Press, New Orleans, La., p. 308, 1998.

Martinez JM, Jones LA: Longterm Effects of Polychlorinated Biphenyls (PCBs) in the Neonatal BALB/c Mouse Model, Toxicological Sciences, vol. 48(1-S):21, 1999.

The research in my laboratory focuses on cytochrome P450 mediated metabolism of drugs, endogenous compounds and toxicants. We are interested in activation mechanism and in the interaction of activated metabolites with tissue targets. We are studying cytochrome P450 based activation in liver, colon and brain. We utilize physical characterization, enzyme purification and molecular biological techniques in pursuit of these goals. As an example, we have recently cloned six new cytochrome P450 forms from a brain cDNA expression library (1,2,3). We have expressed these forms in heterologous expression systems and isolated the purified recombinant protein from the expression system cells. With CYP4F5 we have shown that this P450 isoform catalyzes the hydroxylation of leukotriene B4, an important trigger for cellular inflammation (4). With CYP3A9 we have shown that the purified isoform catalyzes the actuation of estrogen to its putative carcinogenic form (5). These studies continue to examine the cytochrome P450-dependent mediation of toxicity. 

Recent Publications

Kawashima H and Strobel HW. cDNA cloning of a novel rat brain cytochrome P450 belonging to the Cyp 2D subfamily. Biochemistry and Biophysics Research Communications 209:535-540, 1995. 

Kawashima H and Strobel HW. cDNA cloning of three new forms of rat brain cytochrome P450 belonging to the CYP4F subfamily. Biochemistry and Biophysics Research Communications 217:1137-1144, 1995. 

Wang H, Kawashima H and Strobel HW. cDNA cloning of a novel CYP3A from rat brain. Biochemistry and Biophysics Research Communications 221:157-162, 1996. 

Wang H and Strobel HW. Regulation of CYP3A9 gene expression by estrogen and catalytic studies using cytochrome P450 3A9 expressed in E. coli. Archives of Biochemistry and Biophysics 344:365-372, 1997. 

Kawashima H, Kusunose, E, Thompson CM and Strobel HW. Protein expression, characterization and regulation of CYP4F4 and CYP4F5 cloned from rat brain. Archives of Biochemistry and Biophysics In Press.

The research efforts in my laboratory are concerned mainly with the immunotoxicology of ultraviolet radiation (UV) and JP-8 jet fuel. UV radiation is a ubiquitous environmental toxin. UV exposure is the major cause of non-melanoma skin cancer, the most prevalent form of human neoplasia. In addition UV radiation is immune suppressive and studies with experimental animals and biopsy proven skin cancer patients have shown that the immune suppression induced by UV exposure is a risk factor for skin cancer induction. We are interested, therefore, in determining the underlying mechanisms through which UV exposure induces immune suppression with the long-term goal of manipulating and preventing the immune suppressive effects in the hopes of preventing skin cancer induction. We find that UV irradiation of the skin activates a cytokine cascade (prostaglandin E2, interleukin (IL)-4, IL-10) that ultimately affects antigen-presenting cells and inhibits their function in vivo. Recent findings suggest that this cascade of events alters cytokine production by dendritic cells. Upon activation dendritic cells from UV-irradiated animals fail to secrete immune stimulatory IL-12 but rather secrete an immunosuppressive antagonist of IL-12, the IL-12p40 homodimer.

The second immunotoxicology project in my laboratory concerns the immune suppressive effects of JP-8 jet fuel. In the early 1990's the United States Air Force and the NATO allies switched to a new jet fuel, JP-8. Complaints of health problems in jet fuel handlers, engine mechanics and flight line personnel prompted the Air Force to start a systematic study of JP-8-induced immunotoxicity. We found that dermal application of JP-8 induces immune suppression. This occurs after one single large dose exposure to JP-8 or after multiple smaller exposures. It appears the T cell mediated immune reactions, such as contact and delayed-type hypersensitivity and T cell proliferation, are more susceptible to the effects of JP-8 than humoral immune responses such as antibody production. Recent findings indicating that injecting JP-8-treated mice with a selective inhibitor of the cyclooxygenase 2 enzyme prevents JP-8-induced immune suppression suggest the biological response modifier prostaglandin E2 is involved in JP-8 induced immune suppression. Moreover, it provides a potential mechanism to prevent immune suppression in vivo and ameliorate the immunotoxicology of jet fuel.

Selected Publications

Ullrich, S.E. Skin Immunology, in: Comprehensive Toxicology, I.G. Sipes, C.A. McQueen & A.J. Gandolfi, eds, Elsevier Science Ltd, Oxford, Vol 5. pp 153-174, 1997

Ullrich, S.E. T lymphocyte subpopulations and UVB-mediated Immunotoxicity, In: T lymphocyte subpopulations in Immunotoxicology, eds. I. Kimber & M. Selgrade, John Wiley & Sons, New York. p. 121-142, 1998.

Shreedhar, V., Giese, T., Sung, V.W. and Ullrich, S.E. A cytokine cascade including prostaglandin E2, interleukin-4 and interleukin-10 is responsible for UV-induced immune suppression. Journal of Immunology 160:3783-3789, 1998

Ullrich, S.E., Pride, M.W. and Moodycliffe, A.M. Antibodies to the costimulatory molecule CD86 interfere with ultraviolet radiation induced immune suppression. Immunology 94:417-423, 1998.

Ullrich, S.E. Dermal application of JP-8 jet fuel induces immune suppression. Toxicological Sciences 52:61-67, 1999.