Division of Toxicology, Department of Pharmacology and Toxicology, Indiana University School of Medicine, 635 Barnhill Dr., MS 1021, Indianapolis, Indiana 46202
Received February 2, 2002; accepted April 5, 2002
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: catalase; cyanide; morphological transformation; oxidative stress; SHE cells; superoxide dismutase.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Several short-term in vitro and in vivo test systems have been used to assess the mutagenicity and carcinogenicity of chemicals. Cell transformation in Syrian hamster embryo (SHE) cells measures the carcinogenic potential of xenobiotics by assessing transformed colonies based on morphological criterion (Berwald and Sachs, 1963). This in vitro system exhibits a multistage transformation process similar to that seen in carcinogenesis in vivo (Barrett and Tso, 1978
; Isfort et al, 1994
; LeBoeuf et al., 1990
). Morphological transformation is the first identifiable stage during the transformation process and shows a > 85% concordance rate with the 2-year rodent bioassays (Isfort et al., 1996
; LeBoeuf et al., 1996
).
Oxidative stress resulting from either overproduction of prooxidants or deficiency of antioxidants has been suggested as an important factor in many degenerative diseases, such as rheumatoid (Mapp et al., 1995), cardiovascular, and neurodegenerative diseases (Giacosa and Filiberti, 1996
; Simonian and Coyle, 1996
). Several studies have shown that the induction of oxidative stress is involved in cyanide-induced neurodegenerative disease (Akira et al., 1994
; Ardelt et al., 1989
; Gunasekar et al., 1996
). Resulting from oxidative stress, cyanide induces lipid peroxidation in vitro that is reversible by coincubation with N-acetyl cysteine and curcumin (Bhattacharya et al., 1999). The induction of oxidative stress by cyanide may involve increases in reactive oxygen species and nitric oxide (Gunasekar et al., 1996
; Mills et al., 1996
), inhibition of antioxidant systems (Ardelt et al., 1989
), and inhibition of mitochondrial function (Way, 1984
). Inhibition of mitochondrial function can result in the production of additional reactive oxygen species, principally the superoxide anion. In addition, the basal ganglia are affected by cyanide exposure, resulting in neurological sequelae similar to those seen in individuals with Parkinsons disease (Carella et al., 1988
).
Oxidative stress has been shown to participate in the carcinogenesis process (Guyton and Kensler, 1993; Trush and Kensler, 1991
; Vuillaume, 1987
). Although no direct evidence exists to link chronic exposure to low doses of cyanide with cancer development, cyanide was shown to induce aneuploidy in Drosophila. Several groups have identified a causal link between the induction of aneuploidy and cancer development (Dellarco et al., 1986
; Holliday, 1989
; Li et al., 2000
; Osgood and Sterling, 1991
). Cyanide has also tested positive in the Salmonella typhimurium mutagenicity assay (Kushi et al., 1983
).
In the present study, cyanide was evaluated for its ability to induce morphological transformation in the SHE cell system. In addition, the examination of oxidative stress end points (evaluation of oxidative damage and antioxidant status) following cyanide exposure were examined in SHE cells in an attempt to elucidate the potential mechanism(s) by which cyanide induces cellular transformation.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SHE cell transformation assay.
SHE cell transformation was conducted as described previously (Kerckaert et al., 1996). Briefly, feeder cells were prepared by reconstituting cryopreserved SHE cells in DMEM-L and culturing at 37°C, 10% CO2, 95% humidity for 72 h. The cells were then rinsed with CMF-HBSS, detached (0.05% trypsin-EDTA, 5 ml), resuspended in 30 ml culture medium, and exposed to X-ray (
5000 rad) such that the cells remained viable but not capable of replication. The cells were plated at a density of 2 x 104/ml in 60-mm culture dishes (2 ml/dish) and incubated for 24 h. Target cells (cells to be used for clonal growth) were prepared as described above for feeder cells. After 24 h, target cells were plated on feeder cells at a density of 85 cells/dish in 2 ml complete medium. Following an additional 24-h incubation, cells were treated and allowed to grow for 7 days. Colonies were then fixed with methanol, stained with Giemsa, and scored for morphological transformation as described (Kerckaert et al., 1996
).
Cell culture and treatment.
For assays other than the examination of morphological transformation, cells were cultured as follows. Cells were isolated from the embryos of Syrian golden hamsters at day 13 of gestation (Charles River, Portage, MI) and cryopreserved as described (Kerckaert et al., 1996). For each experiment, cryopreserved SHE cells (2 x 106) were thawed and cultured in DMEM-L at 37°C, 10% CO2, and 95% humidity. At 90% confluency, cells were trypsinized and plated at a density of 1 x 106 cells per 60-mm culture dish. Cell cultures were then treated when approximately 75% confluent.
Analysis of 8-hydroxydeoxyguanosine (OH8dG).
OH8dG was measured as described previously (Shigenaga et al., 1994). Briefly, DNA was isolated from nuclei using a sodium iodide choatropic method (Wang et al., 1994
). DNA was dissolved in Tris-HCl buffer (10 mM, pH 7.0) containing 2 mM butylated hydroxytoluene (BHT) and 0.1 mM desferral. DNA (100200 µg) was digested sequentially with nuclease P1 (10 units, 37°C, 30 min) followed by alkaline phosphatase (14 units, 37°C, 60 min). After centrifugation (10,000 x g, 10 min, 4°C), the supernatant was removed for analysis. OH8dG was measured by HPLC and detected electrochemically (set at E1: 100 mV, 1 µA; E2: 400 mV, 5 µA; esa, Inc., Chelmsford, MA). 2-Deoxyguanosine (2-dG) was detected using an ultraviolet detector (Waters PDA, 260 nm; Waters Inc., Milford, MA). OH8dG and 2-dG were quantified from standard curves and expressed as the ratio of OH8dG to 2-dG.
Detection of hydroxyl radicals.
Hydroxyl radical formation in SHE cells was determined by monitoring the nonenzymatic aromatic hydroxylation of salicylic acid, a reaction that reflects hydroxyl radical production (Floyd et al., 1984). Four hours prior to harvest, salicylic acid (10 mM) was added to SHE cell cultures. After treatment, trichloroacetic acid was added (5% v/v final concentration) and the cell cultures were incubated on ice for 15 min prior to collection. The cell extracts were centrifuged (10,000 x g, 10 min). The resultant supernatant was injected into HPLC and 2,3-DHBA resolved on a Nova-Pak C18 reversed-phase analytical column and eluted with sodium citrate buffer (30 mM, pH to 4.65) at a flow rate of 1.3 ml/min. 2,3-DHBA was detected electrochemically (ESA Colouchem II, set at 0.5 µA, 350 mV; esa, Inc., Chelmsford, MA). Salicylic acid was measured at 296 nm (Waters PDA, Waters, Inc. Milford, MA). 2,3-DHBA and salicylic acid were quantitated from standard curves, and the results expressed as the ratio of 2,3-DHBA relative to salicylic acid.
Enzymatic antioxidant activity assays.
After treatment, SHE cells were washed twice with PBS (0°C) and collected. Cell pellets were resuspended in lysis buffer (0.1% triton X-100, 10 mM potassium phosphate buffer, pH 7.2). Cell membranes were disrupted by sonication (10 s; Fisher model 550 sonic dismembranetor; Fisher Scientific, Pittsburgh, PA). Cell lysates were centrifuged (13,000 x g; 15 min), and the supernatants were removed for enzyme measurement. Protein content in supernatants was determined using the Bio-Rad DC protein assay, based on the method of Lowry et al. (1951), with bovine serum albumin serving as a standard.
Superoxide dismutase activity was measured using the inhibition of the autooxidation of pyrogallol (Marklund and Marklund, 1974). Briefly, an aliquot of cell extract was mixed with buffer (50 mM Tris HCl, 50 mM cacodylic acid, 1 mM diethylenetriamine pentaacetic acid, pH 8.2) containing 2 mM pyrogallol. The autooxidation of pyrogallol and the inhibition of this reaction were monitored spectrophotometrically. Under the conditions of this assay, one unit of superoxide dismutase activity was equivalent to the amount of enzyme that resulted in a 50% inhibition of pyrogallol autooxidation.
Catalase activity was determined by monitoring the enzyme-catalyzed decomposition of hydrogen peroxide by potassium permanganate (Cohen et al., 1970). Samples (50 µl) were added to test tubes, followed by the addition of H2O2, then allowed to incubate on ice for 3 min. The reaction was stopped by the addition of H2SO4. KMnO4 was then added, and absorbance was recorded at 480 nm. Under the conditions of this assay, one unit of enzyme activity equals k/(0.00693) (Aebi, 1974
), where k = log (S0/S2) x (2.3/t), S0 = absorbance of standard absorbance of blank, S2 = absorbance of standard absorbance of sample, and t = time interval. The measured activities were normalized to the protein content of each sample.
Statistics.
For studies examining morphological transformation, data were analyzed using the Fisher exact test (Kerckaert et al., 1996). For all other studies, the data were analyzed by one-way ANOVA followed by Duncans test. For all studies, treatment groups were considered statistically different from controls at p < 0.05.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cyanide has been previously shown to induce oxidative stress and damage in a number of biological systems. Administration of sublethal doses of potassium cyanide to CF1 mice induced lipid peroxidation in brain (Johnson et al., 1987). This action has been ascribed to the activation of xanthine oxidase (and subsequent increase in reactive oxygen species) as well as altered regulation of neuronal calcium homeostasis. The induction of lipid peroxidation by cyanide was also observed in cultured neurons from chick embryo telencephalon (Muller and Krieglstein, 1995
) and in mouse brain cortical slices incubated with potassium cyanide in vitro (Ardelt et al., 1994
).
Several studies document an increase in reactive oxygen species following cyanide exposure. Through the inhibition of the mitochondrial respiratory chain at the ubiquinone-cytochrome b site, cyanide produces the superoxide anion. In addition, the generation of hydroxyperoxide was also seen in cyanide-treated PC12 cells (Ardelt et al., 1994; Mills et al., 1996
), rat leukemia 2H3 cells (Arai et al., 1999
), and cerebellar granule cells (Gunasekar et al., 1996
). The formation of the superoxide anion was also shown to be produced nonenzymatically via interaction of cyanide with flavonoids (Hodnick et al., 1994
). In addition to the generation of reactive oxygen species, cyanide is a potent inhibitor of the enzymatic antioxidants catalase, superoxide dismutase, and glutathione peroxidase (Ardelt et al., 1989
; Kanthasamy et al., 1997
). These mechanisms may function in concert to produce the oxidative stress and damage seen after cyanide exposure.
The induction of oxidative stress by cyanide in SHE cells appears to involve both the production of reactive oxygen species and the depletion of the cellular antioxidants catalase and superoxide dismutase. Inhibition of these antioxidants was seen within a short duration after cyanide exposure (4-h and 1-day treatments). The inhibition of these enzymatic antioxidants may have accounted for or contributed to the increase in reactive oxygen species seen in SHE cells treated with cyanide at the early time points (4 h and 1 day).
Although the cellular activity of superoxide dismutase had returned to control level and catalase activity was increased significantly after 2 days and 7 days of treatment with cyanide, the level of reactive oxygen species continued to be elevated over control throughout 7 days of study. This suggests that additional mechanisms must be functioning to maintain the level of reactive oxygen species seen in these studies. One such possibility is the inhibition of mitochondrial respiration and the subsequent generation of superoxide anion by cyanide. It is not clear whether cyanide remains bioavailable at the later time points or if, because of irreversible binding to the mitochondria, cyanide produces a sustained production of the superoxide anion through the time points examined in the present study. Additional studies are being performed to address this issue.
The induction of oxidative stress by cyanide in SHE cells was further evidenced by the formation of the oxidative DNA adduct OH8dG. OH8dG is the most prevalent form of oxidative DNA damage and is commonly used as a biomarker for oxidative stress (Kasai, 1997; Grollman and Moriya, 1993
; Shigenaga et al., 1994
). The results of the present study showed that 500 µM cyanide induced significant increase of OH8dG in SHE cells after 1 and 2 days of exposure, whereas no increase in oxidative DNA damage was seen after 4 h of treatment. Thus, it appears that the induction of oxidative DNA damage occurs subsequent to an increase in hydroxyl radical formation by cyanide. Additionally, treatment with a lower nontransforming concentration of cyanide also failed to induce an increase in either hydroxyl radical formation or oxidative DNA damage, further supporting a link between the induction of oxidative stress by cyanide and cellular transformation.
The formation of OH8dG has been associated with cancer development (Floyd, 1990). OH8dG is mutagenic in several in vitro systems (Kamiya et al., 1992
; Moriya et al., 1991
; Shibutani et al., 1991
; Wood et al., 1990
) and is known to inhibit human DNA methyltransferase in vitro (Turk et al., 1995
). This suggests that OH8dG could be involved in carcinogenesis through both genotoxic and nongenotoxic mechanisms. Recent results from our laboratory have demonstrated a causal relationship between the level of OH8dG in SHE cells and the induction of morphological transformation (Zhang et al., 2000a
). Collectively, the results from the present studies showing the inhibition of cyanide-induced morphological transformation by antioxidant cotreatment and the formation of OH8dG in SHE cells by cyanide provide direct evidence linking the induction of oxidative stress to cyanide-induced cellular transformation.
The finding of increased catalase activity in SHE cells may be a result of gene regulation by reactive oxygen species. Low levels of reactive oxygen intermediates have been shown to modulate gene expression (Schulze-Osthoff et al., 1995). Several groups have demonstrated that the catalase and superoxide dismutase gene expression levels were upregulated by hydrogen peroxide and other agents that produce active oxygen species (Cornelissen et al., 1997
; Diez-Fernandez et al., 1998
; Rohrdanz and Kahl, 1998
). Thus, the increase in hydroxyl radicals produced by cyanide at the early time points in the present study may serve as second messengers to regulate the expression of catalase and/or superoxide dismutase genes, resulting in the observed increases in the activity of these antioxidants seen at the later treatment points. Besides gene upregulation, catalase is regulated at both the posttranscriptional and posttranslational levels (Clerch, 1995
; Reimer et al., 1994
) as well as by gene amplification (Yamada et al., 1991
). Further studies are needed to determine whether the increased catalase and superoxide dismutase activity is regulated at the transcriptional level, posttranscriptional level, or posttranslational level, as well as the signaling pathways involved in their regulation.
In summary, the present study showed that cyanide induced morphological transformation in SHE cells after 7 days of continuous treatment. These results also demonstrated that oxidative stress (both the inhibition of antioxidant enzyme activity and production of reactive oxygen species) is involved in the induction of morphological transformation by cyanide. Results of the present study, together with findings demonstrating the induction aneuploidy in Drosophila and mutagenicity in Salmonella typhimurium by cyanide, point to the need for chronic bioassays to evaluate the carcinogenic potential of cyanide.
![]() |
NOTES |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Akira, T., Henry, D., Baldwin, R. A., and Wasterlain, D. G. (1994). Nitric oxide participates in excitotoxic mechanisms induced by chemical hypoxia. Brain Res. 645, 285290.[ISI][Medline]
Arai, M., Imai, H., Koumura, T., Yoshida, M., Emoto, K., Umeda, M., Chiba, N., and Nakagawa, Y. (1999). Mitochondrial phospholipid hydroperoxide glutathione peroxidase plays a major role in preventing oxidative injury to cells. J. Biol. Chem. 274, 49244933.
Ardelt, B. K., Borowitz., J. L., and Isom, E. G. (1989). Brain lipid peroxidation and antioxidant protectant mechanisms following acute cyanide intoxication. Toxicology 56, 147154.[ISI][Medline]
Ardelt, B. K., Borowitz, J. L., Maduh, E. U., Swain, S. L., and Isom, G. E. (1994). Cyanide-induced lipid peroxidation in different organs: Subcellular distribution and hydroperoxide generation in neuronal cells. Toxicology 89, 127137.[ISI][Medline]
Barrett, J. C., and Tso, P. O. P. (1978). Evidence for the progressive nature of neoplastic transformation in vitro. Proc. Natl. Acad. Sci. U.S.A. 75, 37613765.[Abstract]
Berwald, Y., and Sachs, L. (1963). In vitro transformation with chemical carcinogens. Nature 200, 11821184.[ISI]
Bhattacharya, R., and Lakshmana Rao, P. V. (2001). Pharmacological interventions of cyanide-induced cytotoxicity and DNA damage in isolated rat thymocytes and their protective efficacy in vivo. Toxicol. Lett. 119, 5970.[ISI][Medline]
Blanc, P., Hogan, M., Mallin, K., Hryhorczuk, S., Hessl, S., and Bernard, B. (1985). Cyanide intoxication among silver-reclaiming workers. JAMA 253, 367371.[Abstract]
Carella, F., Grassi, M. P., Savoiardo, M., Contri, P., Rapuzzi, B., and Mangoni, A. (1988). Dystonic-Parkinsonian syndrome after cyanide poisoning: Clinical and MRI findings. J. Neurol. Neurosurg. Psychiatry 51, 13451348.[Abstract]
Chandra, H., Gupta, G. N., Bhargava, S. K., Clerk, S. H., and Mahendra, P. N. (1980). Chronic cyanide exposurea biochemical and industrial hygiene study. J. Anal. Toxicol. 4, 161165.[ISI][Medline]
Clerch, L. B. (1995). A 3 untranslated region of catalase mRNA composed of a stem-loop and dinucleotide repeat elements binds a 69-kDa redox-sensitive protein. Arch. Biochem. Biophys. 317, 267274.[ISI][Medline]
Cohen, G., Dembiec, D., and Marcus, J. (1970). Measurement of catalase activity in tissue extracts. Anal. Biochem. 34, 3038.[ISI][Medline]
Cornelissen, J., Van Kuilenburg, A. B., Voute, P. A., and Van Gennip, A. H. (1997). MIBG causes oxidative stress and up-regulation of anti-oxidant enzymes in the human neuroblastoma cell line SK-N-BE(2c). Int. J. Cancer 72, 486490.[ISI][Medline]
Dellarco, V. L., Mavournin, K. H., and Waters, W. D. (1986). An introduction of a series of U.S. Environmental Protection Agency special committee reports on testing approaches for the detection of chemically induced aneuploidy. Mutat. Res. 167, 37.[ISI][Medline]
Diez-Fernandez, C., Sanz, N., Alvarez, A. M., Wolf, A., and Cascales, M. (1998). The effect of non-genotoxic carcinogens, phenobarbital and clofibrate, on the relationship between reactive oxygen species, antioxidant enzyme expression and apoptosis. Carcinogenesis 19, 17151722.[Abstract]
Floyd, R. A. (1990). The role of 8-hydroxyguanine in carcinogenesis. Carcinogenesis 11, 14471450.[ISI][Medline]
Floyd, R. A., Watson, J. J., and Wong, P. K. (1984). Sensitive assay of hydroxyl free radical formation utilizing high detection of phenol and salicylate hydroxylation products. J. Biochem. Biophys. Methods 10, 221235.[ISI][Medline]
Funata, N., Song, S.-Y., Okeda, R., Funata, M., and Higashino, F. (1984). A study of experimental cyanide encephalopathy in the acute phasephysiological and neuropathological correlation. Acta Neuropathol. (Berl.) 64, 99107.[ISI][Medline]
Giacosa, A., and Filiberti, R. (1996). Free radicals, oxidative damage and degenerative diseases. Eur. J. Cancer Prev. 5, 307312.[ISI][Medline]
Grollman, A. P., and Moriya, M. (1993). Mutagenesis by 8-oxoguanine: An enemy within. Trends Genet. 9, 246249.[ISI][Medline]
Gunasekar, P. G., Sun, P. W., Kanthasamy, A. G., Borowitz, J. L., and Isom, G. E. (1996). Cyanide-induced neurotoxicity involves nitric oxide and reactive oxygen species generation after N-methyl-D-aspartate receptor activation. J. Pharmacol. Exp. Ther. 277, 150155.[Abstract]
Guyton, K. Z, and Kensler, T. W. (1993). Oxidative mechanisms in carcinogenesis. Br. Med. Bull. 49, 523544.[Abstract]
Hodnick, W. F., Duval, D. L., and Pardini, R. S. (1994). Inhibition of mitochondrial respiration and cyanide-stimulated generation of reactive oxygen species by selected flavonoids. Biochem. Pharmacol. 47, 573580.[ISI][Medline]
Holliday, R. (1989). Chromosome error propagation and cancer. Trends Genet. 5, 4245.[ISI][Medline]
Isfort, R. J., Cody, D. B., Doersen, C., Kerckaert, G. A., and LeBoeuf, R. A. (1994). Alterations in cellular differentiation, mitogenesis, cytoskeleton and growth characteristics during Syrian hamster embryo cell multistep in vitro transformation. Int. J. Cancer 59, 114125.[ISI][Medline]
Isfort, R. J., Kerckaert, G. A., and LeBoeuf, R. A. (1996). Comparison of the standard and reduced pH Syrian hamster embryo (SHE) cell in vitro transformation assays in predicting the carcinogenic potential of chemicals. Mutat. Res. 356, 1163.[ISI][Medline]
Isom, G. E, Gunasekar, P. G., and Borowitz, J. L. (1999). Cyanide and neurodegenerative disease. In Chemicals and Neurodegenerative Disease (S. C. Bondy, Ed.), pp. 101129. Prominent Press, Scottsdale, AZ.
Isom, G. E., and Way, J. L. (1984). Effects of oxygen on the antagonism of cyanide intoxication: Cytochrome oxidase in vitro. Toxicol. Appl. Pharmacol. 74, 5762.[ISI][Medline]
Johnson, J. D., Conroy, W. G., Burris, K. D., and Isom, G. E. (1987). Peroxidation of brain lipids following cyanide intoxication in mice. Toxicology 46, 2128.[ISI][Medline]
Kales, S. N., Dinklage, D., Dickey, J., and Goldman, R. H. (1997). Paranoid psychosis after exposure to cyanide. Arch. Environ. Health. 52, 245246.[ISI][Medline]
Kamiya, H., Miura, K., Ishikawa, H., Inoue, H., Nishimura, S., and Ohtsuka, E. (1992). C-Ha-ras containing 8-hydroxyguanine at codon 12 induces point mutations at the modified and adjacent positions. Cancer Res. 52, 34833485.[Abstract]
Kanthasamy, A. G., Ardelt, B., Malave, A., Mills, E. M., Powley, T. L., Borowitz, J. L., and Isom, G. E. (1997). Reactive oxygen species generated by cyanide mediate toxicity in rat pheochromocytoma cells. Toxicol. Lett. 93, 4754.[ISI][Medline]
Kasai, H. (1997). Analysis of a form of oxidative DNA damage, 8-hydroxy-2-deoxiguanosine, as a marker of cellular oxidative stress during carcinogenesis. Mutat. Res. 387, 147163.[ISI][Medline]
Kerckaert, G. A., Isfort, R. J., Carr, G. J., Aardema, M. J., and LeBoeuf, R. A. (1996). A comprehensive protocol for conducting the Syrian hamster embryo cell transformation assay at pH 6.70. Mutat. Res. 356, 6584.[ISI][Medline]
Kushi, A., Matsumoto, T., and Yoshida, D. (1983). Mutagen from the gaseous phase of protein pyrolyzate. Agric. Biol. Chem. 47, 19791982.[ISI]
LeBoeuf, R. A., Kerckaert, G. A., Aardema, M. J., and Gibson, D. P. (1990). Multistage neoplastic transformation of Syrian hamster embryo cells cultured at pH 6.70. Cancer Res. 50, 37223729.[Abstract]
LeBoeuf, R. A., Kerckaert, G. A., Aardema, M. J., Gibson, D. P., Brauninger, R., and Isfort, R. (1996). The pH 6.7 Syrian hamster embryo cell transformation assay for assessing the carcinogenic potential of chemicals. Mutat. Res. 356, 85127.[ISI][Medline]
Li, R., Sonik, A., Stindl, R., Rasnick, D., and Duesberg, P. (2000). Aneuploidy vs. gene mutation hypothesis of cancer: Recent study claims mutation but is found to support aneuploidy. Proc. Natl. Acad. Sci. U.S.A. 97, 32363241.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. 64, 95102.
Mapp, P. I, Grootveld, M. C, and Blake, D. R. (1995). Hypoxia, oxidative stress and rheumatoid arthritis. Br. Med. Bull. 5, 419436.
Marklund, S., and Marklund, G. (1974). Involvement of the superoxide anion radical in the autooxidation of pyrogallol and a convenient assay for superoxide dismutase. J. Biochem. 47, 469474.
Mills, E. M., Gunasekar, P. G., Pavlakovic, G., and Isom, G. E. (1996). Cyanide-induced apoptosis and oxidative stress in differentiated PC12 cells. J. Neurochem. 67, 10391046.[ISI][Medline]
Moriya, M., Ou, C., Bodepudi, V., Johnson, F., Takeshita, M., and Grollman, A. P. (1991). Site-specific mutagenesis using a gapped duplex vector: A study of translesion synthesis past 8-oxodeoxyguanosine in E. coli. Mutat. Res. 254, 281288.[ISI][Medline]
Muller, U., and Krieglstein, J. (1995). Inhibitors of lipid peroxidation protect cultured neurons against cyanide-induced injury. Brain Res. 678, 265268.[ISI][Medline]
Osgood, C., and Sterling, D. (1991). Dichloroacetonitrile, a by-product of water chlorination, induces aneuploidy in Drosophila. Mutat. Res. 261, 8591.[ISI][Medline]
Patel, M. N., Peoples, R. W., Yim, G. K., and Isom, G. E. (1994). Enhancement of NMDA-mediated responses by cyanide. Neurochem. Res. 19, 13191323.[ISI][Medline]
Reimer, D. L., Bailley, J., and Singh, S. M. (1994). Complete cDNA and 5 genomic sequences and multilevel regulation of the mouse catalase gene. Genomics 21, 325336.[ISI][Medline]
Rohrdanz, E., and Kahl, R. (1998). Alterations of antioxidant enzyme expression in response to hydrogen peroxide. Free Radic. Biol. Med. 24, 2738.[ISI][Medline]
Schulze-Osthoff, K., Los, M., and Baeuerle, P. A. (1995). Redox signaling by transcription factors NF-B and AP-1 in lymphocytes. Biochem. Pharmacol. 50, 735741.[ISI][Medline]
Shibutani, S., Takeshita, M., and Grollman, A. P. (1991). Insertion of specific bases during DNA synthesis past the oxidation-damaged base 8-oxodG. Nature 349, 431434.[ISI][Medline]
Shigenaga, M. K., Aboujaoude, E. N., Chen, Q., and Ames, B. N. (1994). Assays of oxidative DNA damage biomarkers 8-oxo-2-deoxyguanosine and 8-oxyguanine in nuclear DNA and biological fluids by high-performance liquid chromatography with electrochemical detection. Methods Enzymol. 234, 1733.
Simonian, N. A., and Coyle, J. T. (1996). Oxidative stress in neurodegenerative diseases. Annu. Rev. Pharmacol. Toxicol. 36, 83106.[ISI][Medline]
Trush, M. A., and Kensler, T. W. (1991). An overview of the relationship between oxidative stress and chemical carcinogenesis. Free Radic. Biol. Med. 10, 201209.[ISI][Medline]
Turk, P. W., Laayoun, A., Smith, S. S., and Weitzman, S. A. (1995). DNA adduct 8-hydroxy-2-deoxyguanosine (8-hydroxyguanine) affects function of human DNA methyltransferase. Carcinogenesis 16, 12531255.[Abstract]
Vuillaume, M. (1987). Reduced oxygen species, mutation, induction and cancer initiation. Mutat. Res. 186, 4372.[ISI][Medline]
Wang, L., Hirayasu, K., Ishizawa, M., and Kobayashi, Y. (1994). Purification of genomic DNA from human whole blood by isopropanol-fractionation with concentrated Na1 and SDS. Nucleic Acids Res. 22, 17741775.[ISI][Medline]
Way, J. L. (1984). Cyanide intoxication and its mechanism of antagonism. Annu. Rev. Pharmacol. Toxicol. 24, 451481.[ISI][Medline]
Wood, M. L., Diadaroglu, M., Gajewski, E., and Essigmann, J. M. (1990). Mechanistic studies of ionizing radiation and oxidative mutagenesis: Genetic effects of single 8-hydroxyguanine (7-hydro-8-oxoguanine) residue inserted at a unique site in a viral genome. Biochemistry 29, 70247032.[ISI][Medline]
Yamada, M., Hashinaka, K., Inazawa, J., and Abe, T. (1991). Expression of catalase and myeloperoxidase genes in hydrogen peroxide-resistant HL-60 cells. DNA Cell Biol. 10, 735742.[ISI][Medline]
Zhang, H., Xu, Y., Kamendulis, L. M., and Klaunig, J. E. (2000a). Morphological transformation by 8-hydroxy-2-deoxyguanosine in Syrian hamster embryo (SHE) cells. Toxicol. Sci. 56, 303312.