Nitric Oxide Induces Oxidative Damage in Addition to Deamination in

Nov 4, 1994 - combination with oxygen radicals in DNA damage was investigated. DNA deamination. (xanthine) and oxidation [5-(hydroxymethyl)uracil ...
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Chem. Res. Toxicol. 1995,8, 473-477

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Nitric Oxide Induces Oxidative Damage in Addition to Deamination in Macrophage DNA Teresa deRojas-Walker,? Snait Tamir,? Hong Ji,t John S. Wishnok,? and Steven R. Tannenbaum*>t>s Division of Toxicology and Department of Chemistry, Massachusetts Znstitute of Technology, 77 Massachusetts Avenue, 56-311, Cambridge, Massachusetts 02139-4307 Received November 4, 1994@ Inflammatory cells such as phagocytes, neutrophils, and macrophages have been implicated in the pathogenesis of several forms of clinical and experimental tumor development. It is hypothesized that this process is mediated by the production of reactive species including NO', 0 2 * - , H202, and ONOO- which inflict DNA damage. In this study, the role of NO' in combination with oxygen radicals in DNA damage was investigated. DNA deamination (xanthine) and oxidation [5-(hydroxymethyl)uracil (5HMU), 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FAPY-GI, and 8-oxoguanine (8oxoG)I products were identified in the DNA activated with Escherichia coli lipopolysaccharide (LPS)and mouse of macrophages (RAW264.7) y-interferon (INF-y). The formation of these products was inhibited by N-methyl-L-arginine (NMA), a nitric oxide synthase inhibitor. NMA inhibited only the production of nitric oxide and had no effect on superoxide production. These results demonstrate that NO' plays a dual role in damaging the DNA of activated macrophages. Autoxidation of NO' leads to nitrosating species which cause deamination of bases. Reaction of NO' with 0 2 ' - leads to DNA oxidative damage due to the formation of peroxynitrite which may have HO'-like oxidizing potential. Another possible mechanism of oxidative damage by NO' could be the mobilization of free iron by NO' which could ultimately cause Fenton-type reactions. Therefore, nitric oxide not only leads to deamination of DNA bases but is also a n obligatory factor in oxidative damage to DNA.

Introduction In the past several years there has been an explosion of interest in NO', a free radical, gaseous molecule that occupies a central role in mammalian physiology (1,2). Nitric oxide is formed in many different cell types through a common biochemical pathway which involves the oxidation of a guanido nitrogen of L-arginine via nitric oxide synthase (NOS)l (3). The NO' thus generated in vivo undergoes subsequent oxidation and is ultimately excreted as urinary nitrate (4). Numerous other investigations concerning problems of cardiovascular physiology, neuronal signaling, endotoxic shock, sexual function, etc., have resulted in the discovery of a family of NOS (EC 1.14.23).These NOS include both constitutive and inducible forms, all of which are cytochrome P450 enzymes utilizing L-arginine as the sole substrate (5). Although NO' produced in this manner has critical functions in homeostasis and host defense, there may also be collateral reactions which lead to DNA damage and/ or cell death in the generator cells or neighboring cells

* To whom correspondence and requests for reprints should be addressed. +Division of Toxicology. Department of Chemistry. Abstract published in Advance ACS Abstracts, February 15, 1995. Abbreviations: BSTFA, bis(trimethylsily1)trifluoroacetamide; DMEM, Dulbecco's modified Eagle's medium; FAF'Y-G, 2,6-diamino4-hydroxy-5-formamidopyrimidine;5HMU, 5-(hydroxymethyl)uracil; HEPES, N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonicacid; HBSS, Hank's balanced salt solution; HICS, heat inactivated calf serum; HRPO, horseradish peroxidase; INF-y, ,y-interferon; LPS, lipopolysaccharide; MEM, minimal Eagle's medium; NMA, N-methyl-L-arginine; NOS, nitric oxide synthase(s); 8oxoG, 8-oxoguanine; PBS, phosphate-buffered saline; PMA, phorbol 12-myristate 13-acetate; SOD, superoxide dismutase.

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of the host organism (I,6-91. Damage to DNA in cells exposed to NO' could be caused by a t least two major pathways: one arising from reaction of NO' with molecular oxygen, and one arising from the reaction of NO' with superoxide. Reaction with 0 2 yields Nz03 which can either (a) nitrosate secondary amines to form carcinogenic or mutagenic N-nitrosoamines or (b) nitrosate primary amines on nucleic acid bases (6). The latter reaction leads to deamination of purines and pyrimidines in various forms of RNA and DNA, thereby leading to mutations (10).Reaction with superoxide leads rapidly to the formation of peroxynitrite anion (ONOO-) which decomposes via reactive intermediates that may oxidize cellular constituents (11). In addition to point mutations at the site of the modified bases there are parallel changes that might also be important for toxicity or mutagenesis, such as DNA strand breaks, and DNADNA or DNA-protein cross-links. The inflammatory phagocytes, macrophages and neutrophils, produce copious quantities of reactive species (i.e., NO', OZ'-, HzOz, and ONOO-) and as such are considered to have a higher cytotoxic potential than other cell types (1,6). In situations where infection and/or inflammation may continue over months or longer, target cells will be exposed to substantial quantities of these highly reactive chemical species. It follows that similar DNA damage will occur to the phagocytic cells themselves. To this end, DNA oxidation and deamination were studied in activated macrophages. Although these are terminally differentiated cells, the type of DNA damage found following activation should be representative of the type of damage that could occur in target cells.

0893-228x/95/2708-0473$09.00/0 0 1995 American Chemical Society

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Experimental Procedures Materials. High-glucose Dulbecco's modified Eagle's medium (DMEM) and heat inactivated calf serum (HICS) were obtained from BioWhittaker, Inc. (Walkersville, MD). Escherichia coli LPS (serotype 0127:B), Trypan blue, N-(a-hydroxyethyl)piperazine-N'-2-ethanesulfonicacid (HEPES), cglutamine, sodium pyruvate, glucose, NaHC03, EDTA, Hank's balanced salt solution without phenol red (HBSS), arginine, ferricytochrome c, horseradish peroxidase (HRPO), phenol red, superoxide dismutase (SOD), sodium azide, phosphate-buffered saline, p€J 7.4 (PBS), phorbol 12-myristate 13-acetate (PMA), 54hydroxymethylluracil (BHMU), and xanthine were purchased from Sigma Chemical Co. (St. Louis, MO). Auto-POW supplemented minimal Eagle's medium without phenol red, L-glutamine, or N d C 0 3 (MEM) was purchased from ICN Biomedical, Inc. (Costa Mesa, CAI. INF-y was purchased from Genzyme (Cambridge, MA). lOO-mm, 6-well, and 96-well tissue culture plates were obtained from Costar (Cambridge, MA). Qiagen-Plasmid Kit was purchased from Qiagen Inc. (Chatsworth, CA). SepPak C-18 was purchased from Millipore Co. (Milford, MA). Bis(trimethylsily1)trifluoroacetamide(BSTFA)was purchased from Supelco (Bellefonte, PA). [1,3-15N~lXanthine was obtained from Cambridge Isotope Laboratory. 80xoG was purchased from Chemical Dynamics Corp. (South Planfield, NJ). N-Methyl-L-arginine (NMA) was a gift from Dr. Michael A. Marletta (The University of Michigan, Ann Arbor, MI). FAPY-G was a gift from Robert Turesky (Nestle, Lausanne, Switzerland), and [13C,3-15N]80~oG and [13C,3J5N]FAPY-G were gifts from Dr. Miral Dizdaroglu (National Institute of Standards and Technology, Gaithersburg, MD). 5HMU-2-l3C,5-dz,6-dwas synthesized from uracil-2J3C,6-d and paraf~rmaldehyde-~~C,dz using the method of Cline et al. (12). Macrophages. An immortalized macrophage cell line, RAW264.7, was originally obtained from the American Type Tissue Culture Collection (Camden, NJ) and has been passaged and kept frozen a t MIT since 1989. The cell line was certified prior to use to be free of mycoplasma. Macrophages were grown in 100-mm tissue culture dishes in DMEM supplemented with 10% HICS and kept in a humid 5% cOz-95% air atmosphere a t 37 "C. After removing the cells from the culture dishes by vigorous pipetting, live cell numbers were determined via Trypan blue exclusion, after which the cells were diluted and plated at the appropriate cell densities. Cells were allowed to adhere for 2 h. Induction of Nitric Oxide Production. Cell sheets were washed and then covered with MEM supplemented with 15 mM HEPES, 4 mM L-glutamine, 1 mM sodium pyruvate, 19 mM glucose, 23.8 mM NaHC03, and 10% HICS (SMEM). Macrophages were activated with LPS at 1pg/mL and INF-y a t 500 units/mL for the desired time period. To some plates was added the NO' synthase inhibitor, NMA (6 mM), 5 h after the LPS/ INF-y. Nitric Oxide Concentration. The total production or delivery of NO' under these conditions was determined by analyzing the media for the presence of nitrate and nitrite using an automated procedure previously described (13). Superoxide Assay. Superoxide production was measured using an automated procedure previously described by Pick and Mizel (14). Briefly, macrophages were plated at 5 x 105 cells/ well in 96-well tissue culture dishes. Cells were activated (iNMA), and the superoxide produced was measured at specified time intervals (Table 1)via the oxidation of ferricytochrome c. Cells activated with PMA were used as a positive control. At each time point, cells were washed five times with PBS and covered with 100 pL of a ferricytochrome c solution (160 mM) in phenol red-free HBSS supplemented with arginine (0.6 mM). Unstimulated cells were used as reference. A duplicate plate without ferricytochrome c was utilized for nitrate and nitrite analyses. Hydrogen Peroxide Assay. The measurement of HzOz production by macrophages is based on the HRPO-dependent conversion of phenol red by HzOz into a compound with increased absorbance a t 610 nm as previously described (14,

deRojas-Walker et al.

Table 1. Summary of the Amount of Superoxide (a= 12), Nitrite, and Nitrate (n = 4) Produced by Activated RAW264.7 Macrophates over 2 h, Measured at Different Time Points following Activation with LPS and I " - y r"Y5 x lo5 macrophages time after stimulation (h) superoxide nitrate nitrite 0.6 f 0.1 Of0 Of0 0-2 6-8 1.5 i 0.4 1.0 i 0.1 0.6 f 0.3 1.2 i 0.3 1.2 f 0.5 1.5 i 0.1 12-14 0.7 i 0.3 0.9 f 0.2 1.0 i 0.4 18-20 24-26 0.8 f 0.3 0.7 f 0.2 1.0 i 0.5 15). Macrophages were plated at 5 x lo6 cells/well in 6-well tissue culture plates, activated with LPS and INF-y for 12 h, washed five times with PBS, and covered with 1mL of a phenol red solution (supplemented SMEM without the HEPES or serum, 0.2 g/L phenol red, and 19 units/mL of HRPO). Cells activated with PMA were used as a positive control. At the completion of incubation the cell-free supernatant was transferred to a microcentrifuge tube and centrifuged for 5 min (4000 rpm) a t 5 "C. The reaction was terminated by removing 900 pL of the supernatant and adding 100 pL of a 1 N NaOH solution to bring the pH above 12.5. HzOz production was monitored over 2-6 h in cells incubated with and without SOD (300 UniWmL) and sodium azide (1mM). A standard curve (116 pM HzOz) was generated for data calculations. DNA Deamination and Oxidation Products. Macrophages were plated a t 1 x lo6 celldmL in several 100-mmtissue culture plates. Cells were activated with LPS and INF-y for 24 h; activated cells with NMA and inactivated cells with or without NMA were used as controls. Cells f INF-y were used as controls. The cell sheets were washed twice with PBS and removed as a suspension in a 0.02% EDTA solution. Cells for each test condition were combined, pelleted at 400g, washed, resuspended in 10% DMSO a t 5 x lo7 celldml, and kept frozen at -125 "C until analysis. Cells were quickly thawed, washed with ice-cold PBS, and incubated at 37 "C, first with 100 pg/mL RNase for 1 h and then with 1 mg/mL proteinase K for 1 h. The DNA was isolated using a QiagenPlasmid column. The DNA was precipitated with 2-propanol and washed with ethanol. The sample was air-dried and then dissolved in water and mixed by shaking a t 5 "C overnight. The absorbance of the sample was measured at 260, 280, and 230 nm to check the purity and estimate the DNA concentration of the sample. Acid Hydrolysis. Sample DNA (50 pg) was added t o the isotopically-labeled internal standards (5Hhfu-2-13C,5-d2,6-d, [1,3J5Nz] xanthine, [l3C,3J5N1 8oxoG, and [l3C,3J5N1FAPYG) and treated with 0.5 mL of 60% formic acid in 1 mL ReactiVials a t 100 "C for 1h. The vials were silanized before use and were sealed under Nz. After cooling briefly, the samples were dried under a stream of Nz and then further dried by Speed-Vac. The samples were diluted in HzO and applied to a Sep-Pak C-18 and eluted with 1mL of H2O followed by 1mL of 2.5% MeOH and 3 mL of MeOH. The water and MeOH eluates were combined and dried in a Speed-Vac. Derivatization. The hydrolyzed samples were derivatized at 130 "C for 20 min with 25 pL of BSTFA and 15 pL of acetonitrile. Pyridine (10pL) was added as catalyst. GC/MS Analysis. The derivatized samples and standards were analyzed on a Hewlett-Packard (HP) Model 5971A mass selective detector interfaced with an HP Model 5890 gas chromatograph. A n HP Vectra 486 data system was used for data acquisition and processing. The electron energy was 70 eV. Dwell times for selected-ion monitoring runs were 50 ms/ ion. GC separation was done on an HP-1 fused-silica capillary column (12.5 m x 0.2 mm i.d., phase thickness 0.33 pm). The injector and detector temperatures were 240 and 280 "C, respectively. The oven temperature program was 100 "C for 30 s, increasing to 300 "C at a rate of 20 Wmin. Helium was the carrier gas a t a flow rate of 2 mumin. "he electron multiplier voltage was approximately 1900 eV (16). Typically, 1-2 pL of each sample was injected for analysis, and mlz = 353,

Nitric Oxide Induces Oxidative DNA Damage

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Table 2. Summary of the Levels of DNA Deamination and Oxidation Products Formed in RAW264.7 Macrophages after Activation' per

lo5 basesb

modified bases xanthine 5HMU 8oxoG FAPY-G control ( n = 6) 5 f 0.9 0.3 f 0.1 20 f 8.0 2 f 0.4 INF-)I( n = 2) 7 f4.2 0.3 f 0.1 26 f 6.6 2 f 0.0 LPS + INF-./ (TZ = 4) 29 f 7.4' 1.4 f 0.4' 48 f 6.2' 3 f 0.6 LPS + INF-./ NMA 7 f 1.5 0.4 f 0.2 22 f 5.1 2 f 0.6 ( n = 4)

guanine increased 2.5-fold above the levels found in inactivated macrophages (Table 2). FAPY-G was not significantly elevated. The production of all modified bases was inhibited by NMA, the competitive inhibitor of nitric oxide synthase. Macrophages preincubated with INF-y and then stimulated with PMA did not exhibit DNA oxidation or deamination above background.

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Cells were activated for 24 h with LPS INF-y with or without the NOS inhibitor NMA. * Mean f SD. p 50.005. a

355, 358, 362, 440, 442, 444, and 446 were monitored in the selected-ion mode. The peak area ratios of the DNA base products to their respective isotopically-labeled internal standards were determined using 3581362 for BHMU, 3531355 for xanthine, 4421446 for FAPY-G, and 4401444 for 8oxoG, and calculations were made by comparisons to standard curves. Statistics. Data were analyzed using a one-way analysis of variance. Statistical significance was assigned at p (0.01.

Results Production of Reactive Oxygen Species. The amount of NO' produced by macrophages activated with LPS and INF-y can be estimated by measuring the levels of NO' reaction products, nitrite and nitrate, in the media. Activated macrophages produced 110 pM NO' reaction products over 24 h. The nitric oxide synthase inhibitor NMA inhibited NO' production by 91% as observed by a decrease in nitrite and nitrate levels. The production of superoxide above background under these conditions was continuous from the beginning of activation, whereas NO' production was not detectable until 6 h after activation (see Table 1). The amount of superoxide produced was not affected by NMA (data not shown). Hydrogen peroxide formation could not be measured in RAW264.7 cells stimulated with either PMA INF-y or LPS INF-y, conditions which produced 02-(Table 11, even after 6 h of incubation with SOD. In the presence of SOD and the catalase inhibitor sodium azide, low levels of H2Oz were detected; however, the quantity of HzO2 measured was one-tenth the superoxide produced (0.066 f 0.009 nmol of H20& x lo5cells stimulated with LPS INF-y). These suggest that the low levels of HzOz measured were due to the high efficiency of catalase in these cells. In order to determine the capacity of the endogenous catalase to break down HzO2, additional hydrogen peroxide (final concentration 4 pM) was added to macrophages with and without sodium azide. No HzOz was found in the absence of sodium azide. Only 1pM of the 4 pM HzOz added was observed in the presence of 1 mM sodium azide. This suggests that the endogenous catalase was not completely inhibited at this concentration of sodium azide. Higher sodium azide concentrations were not utilized due to concerns about toxicity. Oxidation and Deamination of DNA. DNA deamination and oxidation products (xanthine, 5-HMU, FAPYG, and 8oxoG) were measured in the DNA of macrophages after activation with LPS and INF-y. The data in Table 2 represent an average of different experiments each analyzed in triplicates. There was a statistically significant increase in the number of modified bases found in activated macrophages when compared to NMAinhibited and control cells. Following 24 h of activation with LPS + INF-y xanthine levels increased 6-fold, 5-(hydroxymethyl)uracil increased &fold, and 8-oxo-

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Discussion Nitrosative deamination is a well-known consequence of the reaction of primary amines with N203 via exposure to acidic nitrite. Therefore, this reaction should also occur under nonacidic conditions when N203 is generated by the reaction of nitric oxide with molecular oxygen. Nitrosation of primary amines results in rapid deamination via the reaction of diazonium ions with HzO. In these studies the deamination product of guanine, xanthine, was significantly increased above background in macrophages activated with LPS and INF-y (see Table 2). Xanthine formation by activated macrophages was inhibited by NMA. The deamination of nucleic acids by NO' can lead to mispairing, as well as DNA-DNA and/ or DNA-protein cross-linking; also the relative instability of xanthine and hypoxanthine in DNA can lead to depurination and subsequent strand breakage (17-20). Subsequent mutation following the deamination of DNA bases has been shown in previous studies: NO' induced mutations a t the tk and hprt gene loci in a human lymphoblastoid cell line (71, C T mutations in E. coli (81, and AT GC transitions in the pSP189 supF gene (21). Thus, the deamination of nucleic acid bases observed in this study may contribute to the molecular mechanisms of mutagenesis in cells exposed to NO'. A recent published study reveals the complexity of the mutagenic process that results from base deamination, and the potential importance of xanthine formation (36). The formation of DNA oxidation products (5HMU and 8oxoG) produced by macrophages activated with LPS INFy was inhibited by NMA, and NO' synthase inhibitor (see Table 2); however, NMA did not affect the levels of superoxide produced by activated macrophages. These data suggest that DNA oxidation products, produced by macrophages are NO'-dependent. A major 02'-generating system in macrophages is the NADPH oxidase complex which is arranged vectorially in the cell membrane so that electrons pass across it from the NADPH-oxidizing site on the inside of the cell membrane to the molecular oxygen-reducing site outside the cell membrane (22, 23). In this manner, 0 2 ' - is generated outside the cell where it has little chance to interact with DNA. The major internal sources of 02'in macrophages are the electron transport chains of mitochondria and the cytochromes in the endoplasmic reticulum; however, cellular systems have evolved so that most of the radical intermediates of oxygen reduction are rapidly processed by superoxide dismutase in combination with glutathione peroxidase and/or catalase to innocuous products (23). In cells competent for SOD, the steady-state concentration of 0 2 ' - is in the subnanomolar range (241, but even these low levels might produce a background level of oxidation of DNA bases. When produced in conjunction with cytosolic NO', an additional reactive oxidative species is formed, possibly ONOO-, which can further oxidize DNA bases. We have shown that calf thymus DNA treated with ONOO- has increased levels of 8oxoG (unpublished data). The reaction of 02'and NO' to yield peroxynitrite has been observed in

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aqueous solutions (251,and the formation of peroxynitrite by rat alveolar macrophages has been reported (26). Under these conditions, peroxynitrite subsequently decomposes to give a species with hydroxyl radical-like reactivity. The major final product of this reaction is nitrate. The levels of nitrate and superoxide produced by macrophages in this study (see Table 1) support the hypothesis that ONOO- was produced. Furthermore, the relatively higher bimolecular reaction rate constant of NO’ with 0 2 ’ - compared to that of SOD with 02.- also suggests that ONOO- will be formed (27). A second explanation for NO’-induced DNA oxidation is the induction of iron release from iron-sulfur proteins. NO’ reacts with the 4Fe-4S cluster of iron regulatory factor, which is a cytoplasmic aconitase, to yield an ironnitrosyl complex. This iron-nitrosyl complex could cause the disassembly of the iron-sulfur cluster (28). This free iron can be subsequently translocated to the nucleus where it could participate in Fenton chemistry. A third possible explanation relates to the possible inhibition effect of NO’ on repair enzymes (29)which are involved in the repair of oxidative damage. The DNA oxidation products formed in macrophages activated with LPS INF-y, 8oxoG, and BHMU have numerous consequences in cells. 80xoG has been shown in vitro to have a significant DNA miscoding potential which can give rise to G T transversions, ultimately leading to mutagenesis and genetic instability (30-32). In addition, BHMU in mammalian DNA can be cytotoxic (33, 34). Numerous other oxidized bases isolated from animal tissues have yet to be evaluated for genotoxic potential (35). In tissues undergoing an inflammatory reaction, both the infiltrating and resident cell populations produce a time-dependent mixture of nitrogen oxide radicals and oxygen radicals. These different radical species interact to form new reactive intermediates which may contribute to DNA damage. Although the flux of radicals per unit time is low, an inflammatory condition that continues for years becomes a significant risk factor for carcinogenic cell transformation. The chemistry of DNA damage by NO’, although potentially complex with respect to the overall mechanisms and the actual alterations of the nucleic acid, might arise from two fundamental processes, Le., reaction with molecular oxygen to form nitrosating species and reactions with superoxide to form peroxynitrite. It remains to be seen whether the type of DNA damage found in activated macrophages will also occur in target cells.

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Acknowledgment. This work was supported by National Institutes of Health Grants PO1 CA26731 (National Cancer Institute), ES02109 and T32 ES07020 (National Institute of Environmental Health Sciences) and the Rothschild Foundation. We thank Carl Nathan for suggestions on the oxygen radical experiments. References Nathan, C. (1992)Nitric oxide as a secretory product of mammalian cells. FASEB J. 6,3051-3064. Lancaster, J. R.,Jr. (1992)Nitric oxide in cells. A m . Scient. 80, 248-259. Marletta, M. A., Yoon, P. S., Iyengar, R., Leaf, C. D., and Wishnok, J . S. (1988)Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate. Biochemistry 27,87068711. Leaf, C. D., Wishnok, J. S., and Tannenbaum, S. R. (1989) L-arginine is a precursor for nitrate biosynthesis in humans. Biochem. Biophys. Res. Commun. 163,1032-1037.

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Nitric Oxide Induces Oxidative DNA Damage (30) Wood, M. L., Dizdaroglu, M., Gajewski, E., and Essigmann, J. M. (1990) Mechanistic studies of ionization radiation and oxidative mutagenesis: genetic effects of a single 8-hydroxyguanine (7-hydro-8-oxoguanine) residue inserted at a unique site in a viral genome. Biochemistry 29, 7024-7032. (31) Reid, T. M., and Loeb, L. A. (1992)Mutagenic specificity of oxygen radicals produced by human leukemia cells. Cancer Res. 52, 1082-1086. (32) Cheng, K. C., Cahill, D. S., Kasai, H., Nishimura, S., and Loeb, L. A. (1992) 8-Hydroxyguanine, a n abundant form of oxidative DNA damage, causes G-T and A-C substitutions. J. Biol. Chem. 267,166-172. (33) Boorstein, R. J., Chiu, L. N., and Teebor, G. W. (1992) A mammalian cell line deficient in activity of the DNA repair enzyme 5-hydroxymethyluracii is resistant to the toxic effects of

Chem. Res. Toxicol., Vol. 8, No. 3, 1995 477 the thymidine analog 5-hydroxymethyl-2’-deoxyuridine. Mol. Cell. Biol. 12, 5536-5540. (34) Kasai, H., Iida, A., Yamaizumi, Z., Nishimura, S., and Tanooka, H. (1990) 5-Formyldeoxyuridine: a new type of DNA damage induced by ionizing radiation and its mutagenicity to Salmonella strain TA102. Mutat. Res. 243, 249-253. (35) Wagner, J. R., Hu, C., and Ames, B. N. (1992) Endogenous oxidative damage of deoxycytidine in DNA. Proc. Natl. Acad. Sci. U S A . 89,3380-3384. (36) Schumutte, C., Rideout, W. M., 111, Shen, J.-C., and Jones, P. A. (1994) Mutagenicity of nitric oxide is not caused by deamination of cytosine or 5-methylcytosine in double-stranded DNA. Carcinogenesis 15, 2899-2903.

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Announcements International Society for the Study of Xenobiotics: Fourth International ISSX Meeting The Fourth International ISSX Meeting will be held August 27-31, 1995, a t the Westin Hotel in Seattle, Washington. The meeting will consist of two optional short courses, poster sessions, regular sessions, and commercial exhibits. Additionally, there will be a graduate student/ postdoctoral fellow “Best Paper Competition”. Abstract deadline is April 20, 1995. Short courses to be offered on Sunday, August 27th, are as follows: “Immunological and Kinetic Methods for Identifying and Characterizing Specific Drug Metabolizing Enzymes”; and “Regulatory Issues which Impact on the Role of Drug Metabolism in Drug Development”. Topics for the August 28-31 sessions are as follows: “In VitroAn Vivo Correlations”; “Agricultural Chemicals”; “Immune Responses to Xenobiotics”;“Sulfotransferases”; “Horizonsin Toxicology: Mechanisms of Acute Lethal Cell Injury”; “Mechanisms of Teratogenesis”; “Cytochrome P450 Active-Site Modeling”; “Species Correlation and Scaling”; “Regulation of Enzyme Expression”; “New Methodology”;“Biotechnology and Proteins as Drugs”; “New Aspects”; “Metabolism of Xenobiotics in the Brain”; and “Debate: Natural Carcinogens-Fact or Fancy?”. For more information, pleast contact: Convention Services Northwest, 1809 7th Avenue, Suite 1414, Seattle, WA (telephone: 206-292-9198; fax: 206-292-0559). TX9504772

Meeting Calendar June 17-20, 1995

Benzene ’95: An International Conference on Toxicity-CarcinogenesisEpidemiology-Risk [Chem. Res. Toxicol. 8 (21, 321, 19951.

July 23-27, 1995

9th International Conference on Cytochrome P450 [Chem. Res. Toxciol. 7 (51, 701, 19941.

October 11- 13, 1995

European Conference on Combination Toxicology [Chem. Res. Toxicol. 8 (21, 321, 19951. TX950476+