- Email: [email protected]
The role of oxygen-free radicals and their secondary production by polymorphonuclear neutrophils — a comment
Mutation Research, 139 (1984) 211-212
211
Elsevier
MRLett 0547
Letter to the Editor
The role of oxygen-free radicals and their secondary production by polymorphonuclear neutrophils - a comment (Accepted 17 January 1984)
There has been a great deal of interest in the role of oxygen-free radicals and their secondary production by polymorphonuclear neutrophils (PMN) in the process of mutagenesis. Because of its reactive nature, the biological activity of one such free radical, the superoxide radical, is often misunderstood. Two papers on this subject which appeared recently in Mutation Research Letters, deserve critical comment. The first paper (Phagocytosis-induced mutagenesis in bacteria, Barak et al., Mutation Res., 121 (1983) 7-16) reported mutational events with dark mutants of the luminous bacterium Phytobacteriumfischeri when incubated with PMN cells. Reversion to the luminous phenotype was taken as a mutational event and was measured in a scintillation counter. The authors reported that PMN cells were able to cause reversion of the bacterial cells to the luminescent phenotype with maximum mutagenic effect during the first 15 min of phagocytosis. The difficulty with this paper is that the authors fail to report the degree of luminescence generated by stimulated P M N cells. Without this control, one cannot distinguish the degree of chemiluminescence caused by oxygen-free radicals, from luminescence generated by bacteria which have undergone a mutational event. The experimental design does not appear to take into consideration the fact that the presence of bacteria alone will act as a stimulant for neutrophils to undergo an oxidative burst to release oxygen-free radicals. Such a phenomenon was first reported by Allen et al., Biochem. Biophys. Res. Commun., 47 (1972) 679-684. Thus it is quite probable that the 0165-7992/84/$ 03.00 © 1984 Elsevier Science Publishers B.V.
luminescence observed is merely the response of PMN cells to bacterial stimulation. The control data reported in this article fail to account for this problem. Addition of superoxide dismutase and other free-radical scavengers may only be decreasing levels of chemiluminescence generated by the oxidative burst of P M N cells. Heat-killed and disintegrated phagocytes would be unable to undergo the oxidative reaction and therefore would not illicit a luminescent response. These controls do not conclusively demonstrate mutagenic activity in the bacteria themselves. The authors' statement that 'the presence of oxygen is essential for the mutagenic activity' may be more telling than they realize. The second paper (Superoxide anion generated by potassium superoxide is cytotoxic and mutagenic to Chinese hamster ovary cells, Cunningham and Lokesh, Mutation Res., 121 (1983) 299-304) made use of potassium superoxide as a source of superoxide free radicals in a 1-h treatment of C H O cells. The amount of superoxide free radical generated was ascertained by the reduction of cytochrome c. Fig. 1 of this paper reports a correlation of the concentration of potassium superoxide in #g/ml with the amount of cytochrome c reduced in nmoles/ml. This correlation may occur but the nature of this reaction is such that the reduction of cytochrome c is a rate-dependent process, and reduced cytochrome c cannot be reported as a defined quantity in nmoles/ml (McCord and Fridovich, J. Biol. Chem., 243 (1968) 5735-5760). Accordingly, this assay does not quantitate levels of potassium superoxide, but the production of free radicals
212
capable of causing reduction of cytochrome c. The authors should therefore either have expressed the rates in moles of ferricytochrome c reduced/rain or reported the rate of production of reduced ferricytochrome c in saturated solution in moles/min (Fridovich, J. Biol. Chem., 245 (1970)4053-4057). Conversion to production of Oi- may then be made using the molar extinction coefficient of ferricytochrome c (reduced minus oxidized forms). Thus, quantities of Oi- would be expressed in terms of rate of production, i.e. nmoles/min/mg of KO2 used. The authors imply that the superox-
ide free radical is a stable entity in itself, while in fact it is an extremely transient molecule which dismutates spontaneously. It is not possible under the experimental conditions outlined to state that '0.985 _+ 0.01 nmoles Oi- was generated with 0.1 /~g/ml KO2'. This figure does not provide an accurate estimate of the dose received by CHO cells during their 1-h period with potassium superoxide. Ann Hanham Mutagenesis Section, Environmental Health Centre, Tunney's Pasture, Ottawa, Ont. KIA OL2 (Canada)
211
Elsevier
MRLett 0547
Letter to the Editor
The role of oxygen-free radicals and their secondary production by polymorphonuclear neutrophils - a comment (Accepted 17 January 1984)
There has been a great deal of interest in the role of oxygen-free radicals and their secondary production by polymorphonuclear neutrophils (PMN) in the process of mutagenesis. Because of its reactive nature, the biological activity of one such free radical, the superoxide radical, is often misunderstood. Two papers on this subject which appeared recently in Mutation Research Letters, deserve critical comment. The first paper (Phagocytosis-induced mutagenesis in bacteria, Barak et al., Mutation Res., 121 (1983) 7-16) reported mutational events with dark mutants of the luminous bacterium Phytobacteriumfischeri when incubated with PMN cells. Reversion to the luminous phenotype was taken as a mutational event and was measured in a scintillation counter. The authors reported that PMN cells were able to cause reversion of the bacterial cells to the luminescent phenotype with maximum mutagenic effect during the first 15 min of phagocytosis. The difficulty with this paper is that the authors fail to report the degree of luminescence generated by stimulated P M N cells. Without this control, one cannot distinguish the degree of chemiluminescence caused by oxygen-free radicals, from luminescence generated by bacteria which have undergone a mutational event. The experimental design does not appear to take into consideration the fact that the presence of bacteria alone will act as a stimulant for neutrophils to undergo an oxidative burst to release oxygen-free radicals. Such a phenomenon was first reported by Allen et al., Biochem. Biophys. Res. Commun., 47 (1972) 679-684. Thus it is quite probable that the 0165-7992/84/$ 03.00 © 1984 Elsevier Science Publishers B.V.
luminescence observed is merely the response of PMN cells to bacterial stimulation. The control data reported in this article fail to account for this problem. Addition of superoxide dismutase and other free-radical scavengers may only be decreasing levels of chemiluminescence generated by the oxidative burst of P M N cells. Heat-killed and disintegrated phagocytes would be unable to undergo the oxidative reaction and therefore would not illicit a luminescent response. These controls do not conclusively demonstrate mutagenic activity in the bacteria themselves. The authors' statement that 'the presence of oxygen is essential for the mutagenic activity' may be more telling than they realize. The second paper (Superoxide anion generated by potassium superoxide is cytotoxic and mutagenic to Chinese hamster ovary cells, Cunningham and Lokesh, Mutation Res., 121 (1983) 299-304) made use of potassium superoxide as a source of superoxide free radicals in a 1-h treatment of C H O cells. The amount of superoxide free radical generated was ascertained by the reduction of cytochrome c. Fig. 1 of this paper reports a correlation of the concentration of potassium superoxide in #g/ml with the amount of cytochrome c reduced in nmoles/ml. This correlation may occur but the nature of this reaction is such that the reduction of cytochrome c is a rate-dependent process, and reduced cytochrome c cannot be reported as a defined quantity in nmoles/ml (McCord and Fridovich, J. Biol. Chem., 243 (1968) 5735-5760). Accordingly, this assay does not quantitate levels of potassium superoxide, but the production of free radicals
212
capable of causing reduction of cytochrome c. The authors should therefore either have expressed the rates in moles of ferricytochrome c reduced/rain or reported the rate of production of reduced ferricytochrome c in saturated solution in moles/min (Fridovich, J. Biol. Chem., 245 (1970)4053-4057). Conversion to production of Oi- may then be made using the molar extinction coefficient of ferricytochrome c (reduced minus oxidized forms). Thus, quantities of Oi- would be expressed in terms of rate of production, i.e. nmoles/min/mg of KO2 used. The authors imply that the superox-
ide free radical is a stable entity in itself, while in fact it is an extremely transient molecule which dismutates spontaneously. It is not possible under the experimental conditions outlined to state that '0.985 _+ 0.01 nmoles Oi- was generated with 0.1 /~g/ml KO2'. This figure does not provide an accurate estimate of the dose received by CHO cells during their 1-h period with potassium superoxide. Ann Hanham Mutagenesis Section, Environmental Health Centre, Tunney's Pasture, Ottawa, Ont. KIA OL2 (Canada)