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Sensitivity of Human Hepatocytes in Culture to Reactive Nitrogen Intermediates
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
233, 545– 549 (1997)
RC976472
Sensitivity of Human Hepatocytes in Culture to Reactive Nitrogen Intermediates Steven M. D’Ambrosio,1 Tatiana M. Oberyszyn,* Tonya Brady, Mary S. Ross,* and Fredika M. Robertson* Departments of Radiology and Pharmacology and *Department of Medical Microbiology and Immunology, The College of Medicine, The Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210
Received February 28, 1997
The cytotoxic effects of 3-morpholinosydnonimine (Sin-1) and S-nitroso-N-acetylpenicillamine-amine (SNAP) on replicatively active human hepatocyte cells in culture was determined as a function of oxidant type. Both Sin-1 which yields nitric oxide and peroxynitrite following the generation of superoxide anion plus nitric oxide, and SNAP which generates nitric oxide, induced dose dependent decreases in the colony forming capabilities of the human hepatocytes. Sin-1 was much more cytotoxic (LD50 Å 400 mM) than SNAP (LD50 Å 1250 mM). Comparatively, both compounds were much less cytotoxic than H2O2 (LD50 Å 96 mM). Sin-1 induced 4-fold higher levels of cellular nitrite than that generated by the chemical in cell free medium. Nitrotyrosine, a marker of peroxynitrite formation in cells, was immunohistochemically detected in hepatocytes treated with both Sin-1 and SNAP. The formation of 3-nitrotyrosine by hepatocytes incubated with SNAP, suggests that hepatocytes generate intracellular superoxide which reacts with the exogenous nitric oxide derived from SNAP to produce intracellular peroxynitrite, resulting in the SNAP cytotoxicity. The enhanced levels of Sin-1 cytotoxicity on the hepatocytes is suggested to be due both to the chemical generation of peroxynitrite and superoxide anion by Sin-1. These data indicate that peroxynitrite is formed in cultured human hepatocytes inhibiting their replication, and that peroxynitirite may play a significant role in the pathogenesis of liver disease. q 1997 Academic Press
The production of reactive nitrogen intermediates, including nitric oxide, nitronium ions, nitrogen dioxide 1
To whom correspondence should be addressed at 400 W. 12th Avenue, Rm. 103 Wiseman Hall, Columbus, OH 43210. Fax: 614292-7237. E-mail: [email protected]. Abbreviations used: Sin-1, 3-morpholinosydnonimine; SNAP, Snitroso-N-acetylpenicillamine-amine; CFE, colony-forming efficiency.
radical, peroxynitrite and peroxynitrous acid occurs in many pathological conditions associated with inflammatory processes (1-7). Nitric oxide is synthesized from L-arginine in an oxygen-dependent reaction, catalyzed by NADPH-dependent nitric oxide synthase (2,3,8-11). This activity is usually associated with activated monocytes, but has recently been identified in a wide variety of cell types within many different organs following toxic injury and inflammation. In the liver, the cell types that produce nitric oxide includes resident cells, such as activated hepatocytes and Kupffer cells, as well as, infiltrating polymorphonuclear leukocytes and monocytes (1-3,8-10,12-17). It has been calculated that activated cells can produce nitric oxide at the rate of 5 to 50 1 104 molecules per cell-sec (18). In addition to nitric oxide, there is increasing evidence that nitric oxide and superoxide anion can act together to form an even more toxic chemical, peroxynitrite (18-21). Peroxynitrite itself is a strong oxidizing agent and forms characteristic patterns of nitrosation in cellular proteins (22-24). Nitric oxide and its degradation products have been shown to be cytotoxic and mutagenic in a variety of bacterial and mammalian systems (18-20,2528), suggesting that nitric oxide and reactive nitrogen intermediates may be key mediators in the cellular and tissue responses to conditions which may produce large amounts of these reactive nitrogen intermediates. Chronic inflammation of the liver is usually associated with infections and/or chemical injury and is characteristic of many liver diseases (3,8,13,17,29). Following injury, the normally quiescent hepatocytes move from G0 to G1 and begin to repopulate the liver (3035). The combination of exposure to nitric oxide and its degradative products, as may occur during inflammation, and hepatocyte proliferation following injury, is thought to play an important role in the initiation and progression of liver diseases and cancer (3,14,17). However, the biological responses of human hepatocytes to reactive nitrogens has not yet been determined. In order to gain insights into the responses of human hepa-
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tocytes to reactive nitrogen, cultured human hepatocyte (36-38) were exposed with S-nitroso-N-acetylpenicillamine-amine (SNAP) which generates nitric oxide, and 3-morpholinosydnonimine (Sin-1) which yields peroxynitrite from the generation of superoxide plus nitric oxide. Cytotoxicity was determined by colony forming efficiency (CFE) following treatment and was correlated with levels of nitrite in the cellular medium. Immunohistochemical methods in combination with anti-nitrotyrosine antibodies were used to determine the extent of hepatocyte protein nitration, as an indicator of peroxynitrite. MATERIALS AND METHODS Cell culture. The human hepatocyte LI1029 cell line was established from a normal adult male liver from excess transplant tissue as previously described (36,37). For these experiments hepatocytes were used between subpassages five to seven, corresponding to cumulative population doublings between 11 and 16. The cell line exhibits a finite life-span of approximately 25 cumulative population doubling, and expresses many hepatic phenotypes including: epithelial morphology, expression of cytokeratin 18, and intracellular synthesis of both albumin and glycogen (37). The cultures were free of fibroblasts and other non-parenchymal cell types as determined by morphology and lack of immunostaining for cytokeratin 19, alpha fetoprotein, gamma glutamyl transpeptidase, and factor VIII antigens (37). Cell cultures were maintained and treated in the previously described modified alpha MEM specifically formulated to promote the growth of normal human hepatocytes in culture (36-38). Treatment of cells. Hydrogen peroxide (SIGMA, 30%) was diluted in water, Sin-1 (Alexis Corp., San Diego, CA) was dissolved in water and SNAP (Alexis Corp., San Diego, CA) was dissolved in DMSO. All solutions were filtered sterilized and added to the cells at the indicated concentrations in growth medium. Exponentially growing hepatocytes were treated at approximately 60% confluency and incubated at 377C for 30 min (H2O2) or 8 hr (Sin-1 and SNAP). The
FIG. 1. Effect of Sin-1, SNAP, and H2O2 on the cytotoxicity of normal human hepatocytes. Hepatocytes were incubated with 0 to 5 mM Sin-1 (l) or SNAP (s) for 8 hr, or with 0 to 200 mM H2O2 (n, inset) for 20 min. Following treatment, 400 to 1,000 cells were reseeded onto plates for determination of CFE, as described in Methods. Colonies of ú50 cells were allowed to form (approximately 15 days) after which the colonies were fixed and stained with crystal violet and counted. Each data point represents the mean { standard error of the mean of five replicates, from three independent cultures.
FIG. 2. Nitrite production in normal human hepatocytes treated with Sin-1 and SNAP. Normal human hepatocytes were incubated for 8 hr with 0, 0.25, 0.5 and 1.0 mM Sin-1 (l) or SNAP (s). Bar graphs indicate levels of nitrite generated by 1 mM Sin-1 (solid bar) and SNAP (open bar) incubated in cell free medium for 8 hr without hepatocytes. Supernatants were isolated and the levels of nitrite, as an indicator of nitric oxide production, was measured as described in Methods. Values are the means { SD for three independent measurements.
medium was then removed, filtered and stored at 0207C for analysis of nitrite/nitrate. The cells were washed with PBS and removed from the surface of the flasks with trypsin/EGTA. Cells (400 to 1,000) were seeded onto 60-mm2 plates in quintuplicates for determination of CFE (39). Colony formation was monitored under phase contrast microscopy. Colonies of ú50 cells were allowed to form (approximately 15 days) after which the colonies were fixed and stained with crystal violet. The mean CFE { s.e.m for each dose was determined (ratio of total colonies formed to the total number of cells plated) and cytotoxicity calculated relative to water and DMSO controls. Determination of nitrite levels in hepatocyte supernatants. Following the treatment of hepatocytes with Sin-1 and SNAP for 8 hr at 377C medium was collected, filtered and analyzed for nitrite and nitrate using Cayman’s Nitrate/Nitrite Assay Kit (Alexis Corp., San Diego, CA). This assay uses a two step process to first convert nitrate to nitrite utilizing nitrate reductase. The second step uses Griess Reagents and is based on the diazotization of an aryl amine by nitrite and the coupling of the product to form a deep purple azochromophore. Absorbance, at 540 nm, due to this chromophore was determined using a microtiter plate reader. A nitrite standard curve was generated at the time of assay and was used to quantitate the total nitrite concentration in the medium. The sensitivity of the assay is 2.0 mM nitrite. Two solvent controls (DMSO and media) were used to determine background values. Cellular production of nitrate/nitrite were determined by subtracting the values obtained from cell free medium from that obtained from the medium containing cells. Immunohistochemical determination of 3-nitrotyrosine. Immunochemical detection methods were as previously described (40) with the following modification for monolayer cell cultures. Hepatocytes were grown on LabTech 4-well plastic tissue culture slides (Nunc, Inc). Cells were treated with Sin-1 and SNAP for 8 hr as described above for the cytotoxicity experiments. Immediately following treatment, the cells were washed with PBS and immediately fixed in 10% neutral buffered formalin. The primary antibody used for these studies was a rabbit anti- nitrotyrosine (Upstate Biotechnology; Lake Placid, NY; 1:100 dilution). Slides were incubated with this antibody for 1.5 hr in a humidified chamber. A biotinylated secondary goat anti-rabbit (Vector Laboratories, Burlingame, CA; 1:200 dilution) was used as the secondary antibody. The slides were then incubated with horseradish peroxidase coupled to Strepavidin. Color was developed using DAB (diamino benzidine tetrahydrochloride) as the chromophore. Rabbit IgG antibody (Organon Teknika, Durham, NC) was used as the isotopic control to determine the extent of non-specific antibody binding.
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FIG. 3. Immunohistochemical detection of 3-nitrotyrosine in normal human hepatocytes treated with Sin-1 and SNAP. Photomicrographs show immunoreactive 3-nitrotyrosine immediately following treatment of hepatocytes with 0 and 1.0 mM Sin-1 (a,b) and SNAP (c,d) for 8 hr. Notice the lack of cellular staining of control cells, versus the intense staining around the nucleus diffusing into areas of the cytoplasm of cells treated with Sin-1 and SNAP.
Production of Nitrite in Hepatocytes Treated with Sin-1 and SNAP
RESULTS Cytotoxicity of Reactive Nitrogen in Human Hepatocytes The cytotoxic effects of SNAP which generates nitric oxide and Sin-1 which generates peroxynitrite from the reaction of superoxide with nitric oxide, were compared to the cytotoxic effects of H2 O2 (fig. 1). Exposure of human hepatocytes to increasing doses of Sin-1, SNAP, and H2O2 resulted in dose dependent decreases in the CFE of the hepatocytes (fig. 1). H2O2 was the most cytotoxic compound to the hepatocytes, exhibiting an LD50 of 96 mM. Comparatively, the reactive nitrogen generators, Sin-1 and SNAP, were 4- and 13-fold, respectively, less cytotoxic than the reactive oxygen generator, H2O2 . Sin1 which generates nitric oxide and superoxide anion was 3-fold more cytotoxic than SNAP, which primarily generates nitric oxide. The LD50 for Sin-1 was 400 mM, while the LD50 for SNAP was 1250 mM.
Studies were performed to quantitate and compare the levels of nitrite in the supernatants of the hepatocyte cultures incubated with Sin-1 and SNAP for 8 hr. Due to the high level of cytotoxicity induced by these compounds, nitrite was measured at doses between 0 and 1 mM Sin-1 and SNAP. Supernatants isolated from cell free medium, containing either the solvent (DMSO or water) or medium from cells treated with H2O2 , contained less than 5 mM nitrite (data not shown). As shown in figure 2, the levels of nitrite in the hepatocyte supernatants increased linearly as a function of Sin-1 and SNAP doses. The levels of nitrite in the supernatant of hepatocyte cultures incubated with Sin-1 were not significantly (pú0.05) different than those incubated with SNAP. Sin-1 appeared to induce the intracellular production of nitrite by hepatocytes, increasing the levels of nitrite in the supernants 4-fold from 82.6 mM (cell free) to 346.3 mM (with hepatocytes) (fig. 2).
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The levels of nitrite produced by SNAP increased only slightly, but not significantly, from 191.2 (cell free) to 244.3 mM (with hepatocytes). Nitration of Hepatocyte Protein Following Sin-1 and SNAP Treatments Immunohistochemistry was used to determine the formation of 3-nitrotyrosine in hepatocytes following treatment with Sin-1 and SNAP. 3-Nitrotyrosine has been used as a signature of the interaction of cellular proteins with peroxynitrite (7,22,24). A monoclonal antibody, specifically directed to this protein in cells, coupled with immunoperoxidase staining, was used to detect intracellular 3-nitrotyrosine protein. Cells treated with H2O2 or DMSO and medium controls contained only faint staining within the cytoplasm of hepatocytes (Fig. 3A,C). Following treatment with 1 mM of Sin1 or SNAP, all of the cells contained 3-nitrotyrosine localized around the nucleus diffusing into the cytoplasm and cellular extensions (Fig. 3B,D). There was not an discernable difference in the level of 3-nitrotyrosine or the number of hepatocytes stained following treatment of the hepatocytes with either Sin-1 (Fig. 3B) or SNAP (Fig. 3D). DISCUSSION The roles that nitric oxide and its highly reactive derivative peroxynitrite play in degenerative disease processes are somewhat controversial (5,41-43). Some studies have indicated that nitric oxide and peroxynitrite damage DNA, and are cytotoxic and mutagenic (18-20,25-28), while others have shown that nitric oxide protects against the cytotoxic effects of reactive oxygen (5,19,44-46). While liver parenchymal and nonparenchymal cell types are known to generate significant amounts of nitric oxide and peroxynitrite in response to injury and inflammation (1-3,8-10,12-17), very little is understood about the cellular effects of these reactive nitrogen intermediates in human hepatocytes. In this paper we demonstrate, for the first time, that human hepatocytes produce nitric oxide in response to oxidant stress and that peroxynitrite is formed in cultured human hepatocytes inhibiting their replication. The higher cytotoxicity induced by the exposure of human hepatocytes to Sin-1, compared to SNAP, is consistent with the generation of nitric oxide by Sin-1, combined with the highly reactive and cytotoxic agents superoxide and peroxynitrite (19,25). Incubation of the hepatocytes with Sin-1 stimulated the production of nitric oxide as evidenced by the 4-fold increase in nitrite in the medium. This induction of nitric oxide may be a protective response by the hepatocytes in response to oxidants generated by Sin-1, similar to other cellular systems which induce nitric oxide as a way of protecting cells from oxidant induced injury (2,4,8,14,47).
Our data demonstrate that SNAP, a generator of nitric oxide, induces a significant cytotoxicity to human hepatocytes in culture. The high amounts of nitrite measured in the supernatants of hepatocytes incubated with SNAP suggest that nitric oxide is cytotoxic to hepatocytes. However, there were immunodetectable levels of 3-nitrotyrosine in these cells following incubation with SNAP, suggesting that peroxynitrite may also be involved in the cytotoxic response of hepatocytes to SNAP. The presence of 3-nitrotyrosine (protein nitrotryosination) is consistent with other studies showing that peroxynitrite can be formed in vivo from the exogenous addition of nitric oxide with superoxide anion (19,20,48). In other systems, cells treated with nitric oxide have been found to accumulate superoxide due to the inhibition of mitochondrial superoxide dismutase and cytochrome-c oxidase by nitric oxide (19). Nitric oxide then reacts with superoxide anion to form peroxynitrite, supporting the hypothesis that the formation of peroxynitrite from nitric oxide may be responsible for a significant part of the ‘‘nitric oxide’’-dependent toxicity (19-21,49). Therefore, the cytotoxicity observed in the hepatocytes treated with SNAP, may be due more to the intracellular formation of peroxynitrite than to the presence of large quantities of nitric oxide. While immunodetection of 3-nitrotyrosine in the hepatocytes incubated with Sin-1 was expected from the generation of peroxynitrite, the level of staining was comparable to that observed with the hepatocytes incubated with SNAP. This was surprising, since Sin-1 is a direct generator of peroxynitrite, while peroxynitrite derived from SNAP requires reaction with cellular superoxide. This suggests that intracellular or chemical generation of peroxynitrite during the 8 hr incubation of hepatocytes with Sin-1 and SNAP, probably leads to saturating levels of immunodetectable 3-nitrotyrosine. Normally the adult liver, and the adult hepatocyte, are quiescent with minimal replicative activity (50). However, the liver, and its differentiated hepatocytes, is one of the few adult organs which can exhibit a physiological growth response in vivo following physical, infectious, and chemical injuries which stimulate cell loss (30,34,50-52). Such injuries also activate Kupffer cells and cause the influx of monocytes, lymphocytes and specific populations of polymorphonuclear leukocytes (13). Upon activation by soluble cytokine mediators such as g-interferon and lipopolysaccharide, these cells produce reactive oxygen and nitrogen intermediates including, superoxide anion, hydroperoxide, hydroxyl radicals, singlet oxygen, nitric oxide, and peroxynitrite (2,3,8-10,13,16,17). Hepatocytes also produce nitric oxide from the metabolism of arginine, albeit at low levels, which can be induced in response to inflammatory products excreted by Kupffer cells (2,8,14). While hepatocytes, like many other cellular systems, maintain a oxidant homeostasis, our studies indicate that the perturbation of this balance leads to cytotoxicity. Coupled
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with a toxic-proliferative response, as may occur in the liver, reactive nitrogen intermediates, as well as, the better characterized reactive oxygen intermediates, may be important risk factors in the induction and promotion of many degenerative disease processes in the liver. ACKNOWLEDGMENT Supported by Grant ES RO1-5727 from the National Institute of Environmental Sciences to S.M.D.
REFERENCES 1. Nussler, A. K., Heeckt, P. F., and Stadler, J. (1994) Z. Gastroenterol. 32, 24– 30. 2. Curran, R. D., Billiar, T. R., Stuehr, D. J., Hofmann, K., and Simmons, R. L. (1989) J. Exp. Med. 170, 1769 – 1774. 3. Ohshima, H., and Bartsch, H. (1994) Mutat. Res. 305, 253– 264. 4. Heck, D. E. (1996) Adv. Exp. Med. Biol. 387, 171 –176. 5. Rubbo, H., Darley-Usmar, V., and Freeman, B. A. (1996) Chem. Res. Toxicol. 9, 809 –820. 6. Tamir, S., Burney, S., and Tannenbaum, S. R. (1996) Chem. Res. Toxicol. 9, 821–827. 7. Beckman, J. S. (1996) Chem. Res. Toxicol. 9, 836–844. 8. Billiar, T. R., Curran, R. D., Ferrari, F. K., Williams, D. L., and Simmons, R. L. (1990) J. Surg. Res. 48, 349 –353. 9. Moncada, S., Palmer, R. M. J., and Higgs, E. A. (1991) Pharmacol. Rev. 43, 109–142. 10. Nathan, C. (1992) FASEB J. 6, 3051– 3064. 11. Moncada, S. (1992) Acta Physiol. Scand. 145, 201 –227. 12. Maher, J. J., and Friedman, S. L. (1993) Semin. Liver Dis. 13, 13–20. 13. Winwood, P. J., and Arthur, M. J. P. (1993) Sem. Liver Disease 13, 50–59. 14. Wagner, J. G., Ganey, P. E., and Roth, R. A. (1996) Hepatology 23, 803–810. 15. Fo¨rstermann, U., and Dun, N. J. (1996) Methods Enzymol. 268, 510–515. 16. Shimoda, R., Nagashima, M., Sakamoto, M., Yamaguchi, N., Hirohashi, S., Yokota, J., and Kasai, H. (1994) Cancer Res. 54, 3171 – 3172. 17. Haswell-Elkins, M. R., Satarug, S., Tsuda, M., Mairiang, E., Esumi, H., Sithithaworn, P., Mairiang, P., Saitoh, M., Yongvanit, P., and Elkins, D. B. (1994) Mutat. Res. 305, 241–252. 18. Tamir, S., DeRojas-Walker, T., Wishnok, J. S., and Tannenbaum, S. R. (1996) Methods Enzymol. 269, 230–243. 19. Beckman, J. S., and Koppenol, W. H. (1996) Am. J. Physiol. Cell Physiol. 271, C1424 –C1437. 20. Crow, J. P., and Beckman, J. S. (1996) Adv. Exp. Med. Biol. 387, 147–161. 21. Beckman, J. S., Beckman, T. W., Chen, J., Marshall, P. A., and Freeman, B. A. (1990) Proc. Natl. Acad. Sci. USA 87, 1620 –1624. 22. VanderVliet, A., O’Niell, C. A., Halliwell, B., Cross, C. E., and Kaur, H. (1994) FEBS Lett. 33, 89– 92. 23. Oshima, H., Friesen, M., Brouet, I., and Bartsch, H. (1990) Chem. Toxicol. 28, 647–652.
24. Ischiropoulos, H., Zhu, L., Chen, J., Tsai, M., Martin, J. C., Smith, C. D., and Beckman, J. S. (1992) Arch. Biochem. Biophys. 298, 431– 437. 25. Behar-Cohen, F. F., Heydolph, S., Faure, V., Droy-Lefaix, M. T., Courtois, Y., and Goureau, O. (1996) Biochem. Biophys. Res. Commun. 226, 842– 849. 26. Christen, S., Gee, P., and Ames, B. N. (1996) Methods Enzymol. 269, 267– 278. 27. Tamir, S., and Tannenbaum, S. R. (1996) Biochim. Biophys. Acta Rev. Cancer 1288, F31 –F36. 28. Juedes, M. J., and Wogan, G. N. (1996) Mutat. Res. Fundam. Mol. Mech. Mutagen. 349, 51 –61. 29. Biasi, F., Bosco, M., Chiappino, I., Chiarpotto, E., Lanfranco, G., Ottobrelli, A., Massano, G., Donadio, P. P., Vaj, M., Andorno, E., Rizzetto, M., Salizzoni, M., and Poli, G. (1995) Free Radic. Biol. Med. 19, 311 –317. 30. Shimbara, N., Atawa, R., Takashina, M., Tanaka, K., and Ichihara, A. (1996) Cytotechnology 21, 31– 43. 31. Bisgaard, H. C., Nagy, P., Santoni-Rugiu, E., and Thorgeirsson, S. S. (1996) Hepatology 23, 62– 70. 32. Columbano, A., and Shinozuka, H. (1996) FASEB J. 10, 1118 – 1128. 33. Farinati, F., Cardin, R., D’Errico, A., De Maria, N., Naccarato, R., Cecchetto, A., and Grigioni, W. (1996) Hepatology 23, 1468 – 1475. 34. Ohmura, T., Ledda-Columbano, G. M., Piga, R., Columbano, A., Glemba, J., Katyal, S. L., Locker, J., and Shinozuka, H. (1996) Am. J. Pathol. 148, 815–824. 35. Umemura, T., Sai-Kato, K., Takagi, A., Hasegawa, R., and Kurokawa, Y. (1996) Fundam. Appl. Toxicol. 30, 285– 289. 36. Gibson-D’Ambrosio, R. E., Crowe, D. L., Shuler, C. F., and D’Ambrosio, S. M. (1993) Cell Biol. Toxicol. 9, 385– 403. 37. Gibson-D’Ambrosio, R. E., and D’Ambrosio, S. M. (1996) in Cell and Tissue Culture: Laboratory Procedures (Doyle, A., Griffiths, J. B., and Newell, D. G., Eds.), pp. 12B:17.1–12B:17.5, Wiley, Salisbury, Wiltshire, UK. 38. Gibson-D’Ambrosio, R. E., Brady, T., and D’Ambrosio, S. M. (1995) Biotechniques 19, 784–790. 39. Tong, H. H., Park, J. H., Brady, T., Weghorst, C. M., and D’Ambrosio, S. M. (1997) Environ. Mol. Mutagen. 29, (in press). 40. Robertson, F. M., Long, B. W., Tober, K. L., Ross, M. S., and Oberyszyn, T. M. (1996) Carcinogenesis 17, 2053 –2059. 41. Marnett, L. J. (1996) Chem. Res. Toxicol. 9, 807–808. 42. Darley-Usmar, V., Wiseman, H., and Halliwell, B. (1995) FEBS Lett. 369, 131–135. 43. Williams, R. J. P. (1996) Chem. Soc. Rev. 25, 77– 83. 44. Granger, D. N., and Kubes, P. (1996) Methods Enzymol. 269, 434–442. 45. Wink, D. A., Hanbauer, I., Laval, F., Cook, J. A., Krishna, M. C., and Mitchell, J. B. (1994) Ann. NY Acad. Sci. 738, 265– 278. 46. Moncada, S., and Higgs, E. A. (1995) FASEB J. 9, 1319 –1330. 47. Duval, D. L., Miller, D. R., Collier, J., and Billings, R. E. (1996) Mol. Pharmacol. 50, 277–284. 48. Inoue, S., and Kawanishi, S. (1995) FEBS Lett. 371, 86–88. 49. Carreras, M. C., Pargament, G. A., Catz, S. D., Poderoso, J. J., and Boveris, A. (1994) FEBS Lett. 341, 65 –68. 50. Steer, C. J. (1995) FASEB J. 9, 1396 –1400. 51. Taub, R. (1996) FASEB J. 10, 413–427. 52. Diehl, A. M., and Rai, R. M. (1996) FASEB J. 10, 215 –227.
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RC976472
Sensitivity of Human Hepatocytes in Culture to Reactive Nitrogen Intermediates Steven M. D’Ambrosio,1 Tatiana M. Oberyszyn,* Tonya Brady, Mary S. Ross,* and Fredika M. Robertson* Departments of Radiology and Pharmacology and *Department of Medical Microbiology and Immunology, The College of Medicine, The Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43210
Received February 28, 1997
The cytotoxic effects of 3-morpholinosydnonimine (Sin-1) and S-nitroso-N-acetylpenicillamine-amine (SNAP) on replicatively active human hepatocyte cells in culture was determined as a function of oxidant type. Both Sin-1 which yields nitric oxide and peroxynitrite following the generation of superoxide anion plus nitric oxide, and SNAP which generates nitric oxide, induced dose dependent decreases in the colony forming capabilities of the human hepatocytes. Sin-1 was much more cytotoxic (LD50 Å 400 mM) than SNAP (LD50 Å 1250 mM). Comparatively, both compounds were much less cytotoxic than H2O2 (LD50 Å 96 mM). Sin-1 induced 4-fold higher levels of cellular nitrite than that generated by the chemical in cell free medium. Nitrotyrosine, a marker of peroxynitrite formation in cells, was immunohistochemically detected in hepatocytes treated with both Sin-1 and SNAP. The formation of 3-nitrotyrosine by hepatocytes incubated with SNAP, suggests that hepatocytes generate intracellular superoxide which reacts with the exogenous nitric oxide derived from SNAP to produce intracellular peroxynitrite, resulting in the SNAP cytotoxicity. The enhanced levels of Sin-1 cytotoxicity on the hepatocytes is suggested to be due both to the chemical generation of peroxynitrite and superoxide anion by Sin-1. These data indicate that peroxynitrite is formed in cultured human hepatocytes inhibiting their replication, and that peroxynitirite may play a significant role in the pathogenesis of liver disease. q 1997 Academic Press
The production of reactive nitrogen intermediates, including nitric oxide, nitronium ions, nitrogen dioxide 1
To whom correspondence should be addressed at 400 W. 12th Avenue, Rm. 103 Wiseman Hall, Columbus, OH 43210. Fax: 614292-7237. E-mail: [email protected]. Abbreviations used: Sin-1, 3-morpholinosydnonimine; SNAP, Snitroso-N-acetylpenicillamine-amine; CFE, colony-forming efficiency.
radical, peroxynitrite and peroxynitrous acid occurs in many pathological conditions associated with inflammatory processes (1-7). Nitric oxide is synthesized from L-arginine in an oxygen-dependent reaction, catalyzed by NADPH-dependent nitric oxide synthase (2,3,8-11). This activity is usually associated with activated monocytes, but has recently been identified in a wide variety of cell types within many different organs following toxic injury and inflammation. In the liver, the cell types that produce nitric oxide includes resident cells, such as activated hepatocytes and Kupffer cells, as well as, infiltrating polymorphonuclear leukocytes and monocytes (1-3,8-10,12-17). It has been calculated that activated cells can produce nitric oxide at the rate of 5 to 50 1 104 molecules per cell-sec (18). In addition to nitric oxide, there is increasing evidence that nitric oxide and superoxide anion can act together to form an even more toxic chemical, peroxynitrite (18-21). Peroxynitrite itself is a strong oxidizing agent and forms characteristic patterns of nitrosation in cellular proteins (22-24). Nitric oxide and its degradation products have been shown to be cytotoxic and mutagenic in a variety of bacterial and mammalian systems (18-20,2528), suggesting that nitric oxide and reactive nitrogen intermediates may be key mediators in the cellular and tissue responses to conditions which may produce large amounts of these reactive nitrogen intermediates. Chronic inflammation of the liver is usually associated with infections and/or chemical injury and is characteristic of many liver diseases (3,8,13,17,29). Following injury, the normally quiescent hepatocytes move from G0 to G1 and begin to repopulate the liver (3035). The combination of exposure to nitric oxide and its degradative products, as may occur during inflammation, and hepatocyte proliferation following injury, is thought to play an important role in the initiation and progression of liver diseases and cancer (3,14,17). However, the biological responses of human hepatocytes to reactive nitrogens has not yet been determined. In order to gain insights into the responses of human hepa-
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tocytes to reactive nitrogen, cultured human hepatocyte (36-38) were exposed with S-nitroso-N-acetylpenicillamine-amine (SNAP) which generates nitric oxide, and 3-morpholinosydnonimine (Sin-1) which yields peroxynitrite from the generation of superoxide plus nitric oxide. Cytotoxicity was determined by colony forming efficiency (CFE) following treatment and was correlated with levels of nitrite in the cellular medium. Immunohistochemical methods in combination with anti-nitrotyrosine antibodies were used to determine the extent of hepatocyte protein nitration, as an indicator of peroxynitrite. MATERIALS AND METHODS Cell culture. The human hepatocyte LI1029 cell line was established from a normal adult male liver from excess transplant tissue as previously described (36,37). For these experiments hepatocytes were used between subpassages five to seven, corresponding to cumulative population doublings between 11 and 16. The cell line exhibits a finite life-span of approximately 25 cumulative population doubling, and expresses many hepatic phenotypes including: epithelial morphology, expression of cytokeratin 18, and intracellular synthesis of both albumin and glycogen (37). The cultures were free of fibroblasts and other non-parenchymal cell types as determined by morphology and lack of immunostaining for cytokeratin 19, alpha fetoprotein, gamma glutamyl transpeptidase, and factor VIII antigens (37). Cell cultures were maintained and treated in the previously described modified alpha MEM specifically formulated to promote the growth of normal human hepatocytes in culture (36-38). Treatment of cells. Hydrogen peroxide (SIGMA, 30%) was diluted in water, Sin-1 (Alexis Corp., San Diego, CA) was dissolved in water and SNAP (Alexis Corp., San Diego, CA) was dissolved in DMSO. All solutions were filtered sterilized and added to the cells at the indicated concentrations in growth medium. Exponentially growing hepatocytes were treated at approximately 60% confluency and incubated at 377C for 30 min (H2O2) or 8 hr (Sin-1 and SNAP). The
FIG. 1. Effect of Sin-1, SNAP, and H2O2 on the cytotoxicity of normal human hepatocytes. Hepatocytes were incubated with 0 to 5 mM Sin-1 (l) or SNAP (s) for 8 hr, or with 0 to 200 mM H2O2 (n, inset) for 20 min. Following treatment, 400 to 1,000 cells were reseeded onto plates for determination of CFE, as described in Methods. Colonies of ú50 cells were allowed to form (approximately 15 days) after which the colonies were fixed and stained with crystal violet and counted. Each data point represents the mean { standard error of the mean of five replicates, from three independent cultures.
FIG. 2. Nitrite production in normal human hepatocytes treated with Sin-1 and SNAP. Normal human hepatocytes were incubated for 8 hr with 0, 0.25, 0.5 and 1.0 mM Sin-1 (l) or SNAP (s). Bar graphs indicate levels of nitrite generated by 1 mM Sin-1 (solid bar) and SNAP (open bar) incubated in cell free medium for 8 hr without hepatocytes. Supernatants were isolated and the levels of nitrite, as an indicator of nitric oxide production, was measured as described in Methods. Values are the means { SD for three independent measurements.
medium was then removed, filtered and stored at 0207C for analysis of nitrite/nitrate. The cells were washed with PBS and removed from the surface of the flasks with trypsin/EGTA. Cells (400 to 1,000) were seeded onto 60-mm2 plates in quintuplicates for determination of CFE (39). Colony formation was monitored under phase contrast microscopy. Colonies of ú50 cells were allowed to form (approximately 15 days) after which the colonies were fixed and stained with crystal violet. The mean CFE { s.e.m for each dose was determined (ratio of total colonies formed to the total number of cells plated) and cytotoxicity calculated relative to water and DMSO controls. Determination of nitrite levels in hepatocyte supernatants. Following the treatment of hepatocytes with Sin-1 and SNAP for 8 hr at 377C medium was collected, filtered and analyzed for nitrite and nitrate using Cayman’s Nitrate/Nitrite Assay Kit (Alexis Corp., San Diego, CA). This assay uses a two step process to first convert nitrate to nitrite utilizing nitrate reductase. The second step uses Griess Reagents and is based on the diazotization of an aryl amine by nitrite and the coupling of the product to form a deep purple azochromophore. Absorbance, at 540 nm, due to this chromophore was determined using a microtiter plate reader. A nitrite standard curve was generated at the time of assay and was used to quantitate the total nitrite concentration in the medium. The sensitivity of the assay is 2.0 mM nitrite. Two solvent controls (DMSO and media) were used to determine background values. Cellular production of nitrate/nitrite were determined by subtracting the values obtained from cell free medium from that obtained from the medium containing cells. Immunohistochemical determination of 3-nitrotyrosine. Immunochemical detection methods were as previously described (40) with the following modification for monolayer cell cultures. Hepatocytes were grown on LabTech 4-well plastic tissue culture slides (Nunc, Inc). Cells were treated with Sin-1 and SNAP for 8 hr as described above for the cytotoxicity experiments. Immediately following treatment, the cells were washed with PBS and immediately fixed in 10% neutral buffered formalin. The primary antibody used for these studies was a rabbit anti- nitrotyrosine (Upstate Biotechnology; Lake Placid, NY; 1:100 dilution). Slides were incubated with this antibody for 1.5 hr in a humidified chamber. A biotinylated secondary goat anti-rabbit (Vector Laboratories, Burlingame, CA; 1:200 dilution) was used as the secondary antibody. The slides were then incubated with horseradish peroxidase coupled to Strepavidin. Color was developed using DAB (diamino benzidine tetrahydrochloride) as the chromophore. Rabbit IgG antibody (Organon Teknika, Durham, NC) was used as the isotopic control to determine the extent of non-specific antibody binding.
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FIG. 3. Immunohistochemical detection of 3-nitrotyrosine in normal human hepatocytes treated with Sin-1 and SNAP. Photomicrographs show immunoreactive 3-nitrotyrosine immediately following treatment of hepatocytes with 0 and 1.0 mM Sin-1 (a,b) and SNAP (c,d) for 8 hr. Notice the lack of cellular staining of control cells, versus the intense staining around the nucleus diffusing into areas of the cytoplasm of cells treated with Sin-1 and SNAP.
Production of Nitrite in Hepatocytes Treated with Sin-1 and SNAP
RESULTS Cytotoxicity of Reactive Nitrogen in Human Hepatocytes The cytotoxic effects of SNAP which generates nitric oxide and Sin-1 which generates peroxynitrite from the reaction of superoxide with nitric oxide, were compared to the cytotoxic effects of H2 O2 (fig. 1). Exposure of human hepatocytes to increasing doses of Sin-1, SNAP, and H2O2 resulted in dose dependent decreases in the CFE of the hepatocytes (fig. 1). H2O2 was the most cytotoxic compound to the hepatocytes, exhibiting an LD50 of 96 mM. Comparatively, the reactive nitrogen generators, Sin-1 and SNAP, were 4- and 13-fold, respectively, less cytotoxic than the reactive oxygen generator, H2O2 . Sin1 which generates nitric oxide and superoxide anion was 3-fold more cytotoxic than SNAP, which primarily generates nitric oxide. The LD50 for Sin-1 was 400 mM, while the LD50 for SNAP was 1250 mM.
Studies were performed to quantitate and compare the levels of nitrite in the supernatants of the hepatocyte cultures incubated with Sin-1 and SNAP for 8 hr. Due to the high level of cytotoxicity induced by these compounds, nitrite was measured at doses between 0 and 1 mM Sin-1 and SNAP. Supernatants isolated from cell free medium, containing either the solvent (DMSO or water) or medium from cells treated with H2O2 , contained less than 5 mM nitrite (data not shown). As shown in figure 2, the levels of nitrite in the hepatocyte supernatants increased linearly as a function of Sin-1 and SNAP doses. The levels of nitrite in the supernatant of hepatocyte cultures incubated with Sin-1 were not significantly (pú0.05) different than those incubated with SNAP. Sin-1 appeared to induce the intracellular production of nitrite by hepatocytes, increasing the levels of nitrite in the supernants 4-fold from 82.6 mM (cell free) to 346.3 mM (with hepatocytes) (fig. 2).
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The levels of nitrite produced by SNAP increased only slightly, but not significantly, from 191.2 (cell free) to 244.3 mM (with hepatocytes). Nitration of Hepatocyte Protein Following Sin-1 and SNAP Treatments Immunohistochemistry was used to determine the formation of 3-nitrotyrosine in hepatocytes following treatment with Sin-1 and SNAP. 3-Nitrotyrosine has been used as a signature of the interaction of cellular proteins with peroxynitrite (7,22,24). A monoclonal antibody, specifically directed to this protein in cells, coupled with immunoperoxidase staining, was used to detect intracellular 3-nitrotyrosine protein. Cells treated with H2O2 or DMSO and medium controls contained only faint staining within the cytoplasm of hepatocytes (Fig. 3A,C). Following treatment with 1 mM of Sin1 or SNAP, all of the cells contained 3-nitrotyrosine localized around the nucleus diffusing into the cytoplasm and cellular extensions (Fig. 3B,D). There was not an discernable difference in the level of 3-nitrotyrosine or the number of hepatocytes stained following treatment of the hepatocytes with either Sin-1 (Fig. 3B) or SNAP (Fig. 3D). DISCUSSION The roles that nitric oxide and its highly reactive derivative peroxynitrite play in degenerative disease processes are somewhat controversial (5,41-43). Some studies have indicated that nitric oxide and peroxynitrite damage DNA, and are cytotoxic and mutagenic (18-20,25-28), while others have shown that nitric oxide protects against the cytotoxic effects of reactive oxygen (5,19,44-46). While liver parenchymal and nonparenchymal cell types are known to generate significant amounts of nitric oxide and peroxynitrite in response to injury and inflammation (1-3,8-10,12-17), very little is understood about the cellular effects of these reactive nitrogen intermediates in human hepatocytes. In this paper we demonstrate, for the first time, that human hepatocytes produce nitric oxide in response to oxidant stress and that peroxynitrite is formed in cultured human hepatocytes inhibiting their replication. The higher cytotoxicity induced by the exposure of human hepatocytes to Sin-1, compared to SNAP, is consistent with the generation of nitric oxide by Sin-1, combined with the highly reactive and cytotoxic agents superoxide and peroxynitrite (19,25). Incubation of the hepatocytes with Sin-1 stimulated the production of nitric oxide as evidenced by the 4-fold increase in nitrite in the medium. This induction of nitric oxide may be a protective response by the hepatocytes in response to oxidants generated by Sin-1, similar to other cellular systems which induce nitric oxide as a way of protecting cells from oxidant induced injury (2,4,8,14,47).
Our data demonstrate that SNAP, a generator of nitric oxide, induces a significant cytotoxicity to human hepatocytes in culture. The high amounts of nitrite measured in the supernatants of hepatocytes incubated with SNAP suggest that nitric oxide is cytotoxic to hepatocytes. However, there were immunodetectable levels of 3-nitrotyrosine in these cells following incubation with SNAP, suggesting that peroxynitrite may also be involved in the cytotoxic response of hepatocytes to SNAP. The presence of 3-nitrotyrosine (protein nitrotryosination) is consistent with other studies showing that peroxynitrite can be formed in vivo from the exogenous addition of nitric oxide with superoxide anion (19,20,48). In other systems, cells treated with nitric oxide have been found to accumulate superoxide due to the inhibition of mitochondrial superoxide dismutase and cytochrome-c oxidase by nitric oxide (19). Nitric oxide then reacts with superoxide anion to form peroxynitrite, supporting the hypothesis that the formation of peroxynitrite from nitric oxide may be responsible for a significant part of the ‘‘nitric oxide’’-dependent toxicity (19-21,49). Therefore, the cytotoxicity observed in the hepatocytes treated with SNAP, may be due more to the intracellular formation of peroxynitrite than to the presence of large quantities of nitric oxide. While immunodetection of 3-nitrotyrosine in the hepatocytes incubated with Sin-1 was expected from the generation of peroxynitrite, the level of staining was comparable to that observed with the hepatocytes incubated with SNAP. This was surprising, since Sin-1 is a direct generator of peroxynitrite, while peroxynitrite derived from SNAP requires reaction with cellular superoxide. This suggests that intracellular or chemical generation of peroxynitrite during the 8 hr incubation of hepatocytes with Sin-1 and SNAP, probably leads to saturating levels of immunodetectable 3-nitrotyrosine. Normally the adult liver, and the adult hepatocyte, are quiescent with minimal replicative activity (50). However, the liver, and its differentiated hepatocytes, is one of the few adult organs which can exhibit a physiological growth response in vivo following physical, infectious, and chemical injuries which stimulate cell loss (30,34,50-52). Such injuries also activate Kupffer cells and cause the influx of monocytes, lymphocytes and specific populations of polymorphonuclear leukocytes (13). Upon activation by soluble cytokine mediators such as g-interferon and lipopolysaccharide, these cells produce reactive oxygen and nitrogen intermediates including, superoxide anion, hydroperoxide, hydroxyl radicals, singlet oxygen, nitric oxide, and peroxynitrite (2,3,8-10,13,16,17). Hepatocytes also produce nitric oxide from the metabolism of arginine, albeit at low levels, which can be induced in response to inflammatory products excreted by Kupffer cells (2,8,14). While hepatocytes, like many other cellular systems, maintain a oxidant homeostasis, our studies indicate that the perturbation of this balance leads to cytotoxicity. Coupled
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with a toxic-proliferative response, as may occur in the liver, reactive nitrogen intermediates, as well as, the better characterized reactive oxygen intermediates, may be important risk factors in the induction and promotion of many degenerative disease processes in the liver. ACKNOWLEDGMENT Supported by Grant ES RO1-5727 from the National Institute of Environmental Sciences to S.M.D.
REFERENCES 1. Nussler, A. K., Heeckt, P. F., and Stadler, J. (1994) Z. Gastroenterol. 32, 24– 30. 2. Curran, R. D., Billiar, T. R., Stuehr, D. J., Hofmann, K., and Simmons, R. L. (1989) J. Exp. Med. 170, 1769 – 1774. 3. Ohshima, H., and Bartsch, H. (1994) Mutat. Res. 305, 253– 264. 4. Heck, D. E. (1996) Adv. Exp. Med. Biol. 387, 171 –176. 5. Rubbo, H., Darley-Usmar, V., and Freeman, B. A. (1996) Chem. Res. Toxicol. 9, 809 –820. 6. Tamir, S., Burney, S., and Tannenbaum, S. R. (1996) Chem. Res. Toxicol. 9, 821–827. 7. Beckman, J. S. (1996) Chem. Res. Toxicol. 9, 836–844. 8. Billiar, T. R., Curran, R. D., Ferrari, F. K., Williams, D. L., and Simmons, R. L. (1990) J. Surg. Res. 48, 349 –353. 9. Moncada, S., Palmer, R. M. J., and Higgs, E. A. (1991) Pharmacol. Rev. 43, 109–142. 10. Nathan, C. (1992) FASEB J. 6, 3051– 3064. 11. Moncada, S. (1992) Acta Physiol. Scand. 145, 201 –227. 12. Maher, J. J., and Friedman, S. L. (1993) Semin. Liver Dis. 13, 13–20. 13. Winwood, P. J., and Arthur, M. J. P. (1993) Sem. Liver Disease 13, 50–59. 14. Wagner, J. G., Ganey, P. E., and Roth, R. A. (1996) Hepatology 23, 803–810. 15. Fo¨rstermann, U., and Dun, N. J. (1996) Methods Enzymol. 268, 510–515. 16. Shimoda, R., Nagashima, M., Sakamoto, M., Yamaguchi, N., Hirohashi, S., Yokota, J., and Kasai, H. (1994) Cancer Res. 54, 3171 – 3172. 17. Haswell-Elkins, M. R., Satarug, S., Tsuda, M., Mairiang, E., Esumi, H., Sithithaworn, P., Mairiang, P., Saitoh, M., Yongvanit, P., and Elkins, D. B. (1994) Mutat. Res. 305, 241–252. 18. Tamir, S., DeRojas-Walker, T., Wishnok, J. S., and Tannenbaum, S. R. (1996) Methods Enzymol. 269, 230–243. 19. Beckman, J. S., and Koppenol, W. H. (1996) Am. J. Physiol. Cell Physiol. 271, C1424 –C1437. 20. Crow, J. P., and Beckman, J. S. (1996) Adv. Exp. Med. Biol. 387, 147–161. 21. Beckman, J. S., Beckman, T. W., Chen, J., Marshall, P. A., and Freeman, B. A. (1990) Proc. Natl. Acad. Sci. USA 87, 1620 –1624. 22. VanderVliet, A., O’Niell, C. A., Halliwell, B., Cross, C. E., and Kaur, H. (1994) FEBS Lett. 33, 89– 92. 23. Oshima, H., Friesen, M., Brouet, I., and Bartsch, H. (1990) Chem. Toxicol. 28, 647–652.
24. Ischiropoulos, H., Zhu, L., Chen, J., Tsai, M., Martin, J. C., Smith, C. D., and Beckman, J. S. (1992) Arch. Biochem. Biophys. 298, 431– 437. 25. Behar-Cohen, F. F., Heydolph, S., Faure, V., Droy-Lefaix, M. T., Courtois, Y., and Goureau, O. (1996) Biochem. Biophys. Res. Commun. 226, 842– 849. 26. Christen, S., Gee, P., and Ames, B. N. (1996) Methods Enzymol. 269, 267– 278. 27. Tamir, S., and Tannenbaum, S. R. (1996) Biochim. Biophys. Acta Rev. Cancer 1288, F31 –F36. 28. Juedes, M. J., and Wogan, G. N. (1996) Mutat. Res. Fundam. Mol. Mech. Mutagen. 349, 51 –61. 29. Biasi, F., Bosco, M., Chiappino, I., Chiarpotto, E., Lanfranco, G., Ottobrelli, A., Massano, G., Donadio, P. P., Vaj, M., Andorno, E., Rizzetto, M., Salizzoni, M., and Poli, G. (1995) Free Radic. Biol. Med. 19, 311 –317. 30. Shimbara, N., Atawa, R., Takashina, M., Tanaka, K., and Ichihara, A. (1996) Cytotechnology 21, 31– 43. 31. Bisgaard, H. C., Nagy, P., Santoni-Rugiu, E., and Thorgeirsson, S. S. (1996) Hepatology 23, 62– 70. 32. Columbano, A., and Shinozuka, H. (1996) FASEB J. 10, 1118 – 1128. 33. Farinati, F., Cardin, R., D’Errico, A., De Maria, N., Naccarato, R., Cecchetto, A., and Grigioni, W. (1996) Hepatology 23, 1468 – 1475. 34. Ohmura, T., Ledda-Columbano, G. M., Piga, R., Columbano, A., Glemba, J., Katyal, S. L., Locker, J., and Shinozuka, H. (1996) Am. J. Pathol. 148, 815–824. 35. Umemura, T., Sai-Kato, K., Takagi, A., Hasegawa, R., and Kurokawa, Y. (1996) Fundam. Appl. Toxicol. 30, 285– 289. 36. Gibson-D’Ambrosio, R. E., Crowe, D. L., Shuler, C. F., and D’Ambrosio, S. M. (1993) Cell Biol. Toxicol. 9, 385– 403. 37. Gibson-D’Ambrosio, R. E., and D’Ambrosio, S. M. (1996) in Cell and Tissue Culture: Laboratory Procedures (Doyle, A., Griffiths, J. B., and Newell, D. G., Eds.), pp. 12B:17.1–12B:17.5, Wiley, Salisbury, Wiltshire, UK. 38. Gibson-D’Ambrosio, R. E., Brady, T., and D’Ambrosio, S. M. (1995) Biotechniques 19, 784–790. 39. Tong, H. H., Park, J. H., Brady, T., Weghorst, C. M., and D’Ambrosio, S. M. (1997) Environ. Mol. Mutagen. 29, (in press). 40. Robertson, F. M., Long, B. W., Tober, K. L., Ross, M. S., and Oberyszyn, T. M. (1996) Carcinogenesis 17, 2053 –2059. 41. Marnett, L. J. (1996) Chem. Res. Toxicol. 9, 807–808. 42. Darley-Usmar, V., Wiseman, H., and Halliwell, B. (1995) FEBS Lett. 369, 131–135. 43. Williams, R. J. P. (1996) Chem. Soc. Rev. 25, 77– 83. 44. Granger, D. N., and Kubes, P. (1996) Methods Enzymol. 269, 434–442. 45. Wink, D. A., Hanbauer, I., Laval, F., Cook, J. A., Krishna, M. C., and Mitchell, J. B. (1994) Ann. NY Acad. Sci. 738, 265– 278. 46. Moncada, S., and Higgs, E. A. (1995) FASEB J. 9, 1319 –1330. 47. Duval, D. L., Miller, D. R., Collier, J., and Billings, R. E. (1996) Mol. Pharmacol. 50, 277–284. 48. Inoue, S., and Kawanishi, S. (1995) FEBS Lett. 371, 86–88. 49. Carreras, M. C., Pargament, G. A., Catz, S. D., Poderoso, J. J., and Boveris, A. (1994) FEBS Lett. 341, 65 –68. 50. Steer, C. J. (1995) FASEB J. 9, 1396 –1400. 51. Taub, R. (1996) FASEB J. 10, 413–427. 52. Diehl, A. M., and Rai, R. M. (1996) FASEB J. 10, 215 –227.
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