Acta 1315, 176- 184. Received 19 March 1996. The intracellular reducing environment modulates cytoregulation and cytotoxicity by reactive oxygen species.
Biochemical Society Transactions
16 Meister, A. and Anderson, M. E. (1983) Annu. Rev. Biochem. 52, 71 1-760 17 Sics, €I., Akerboom, 7‘. and Ishikawa, ‘r. (1989) in Glutathione Centennial (Taniguchi, N., Higashi, T., Sakamoto, Y. and Meister, A., eds.), pp. 357-367, Academic Press, San Diego 18 Lou, M. F., McKellar, R. and Chyan, 0. (1986) Exp. Eye Kes. 42, 607-616 19 Pirie, A., van Heyningen, R. and h a g , J. W. (1953) Hiochem. J. 54, 682-688 20 Institoris, E., Eid, H., Bodrogi, I . and Bok, M. (1995) Anticancer Res. 15, 1371-1374 21 Lovell, M. A., Ehmann, W. D., Butler, S. M. and Markesbery, W. R. (1995) Neurology 45, 1594- 1601 22 IIarding, J. J. (1973) J. Chromatogr. 77, 191-199 23 Rathbun, W. B., Bovis, M. G. and Ilolleschau, A. M. (1986) Ophthalmic Kes. 18, 282-287 24 Dovrat, A. and Gershon, I). (1981) Exp. Eye Kes. 33, 65 1-66 I 25 Bours, J., Fink, H. and Hockwin, 0. (1988) Curr. Eye Res. 7,449-455
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Received 19 March 1996
The intracellular reducing environment modulates cytoregulation and cytotoxicity by reactive oxygen species D. T.-Y. Chiu,* H. P. Monteirot and A. Stern*§ *Chang Gung College of Medicine and Technology, Department of Medical Technology, Tao-Yuan, Taiwan, tFPS/Hemocentro de SZo Paulo, SZo Paulo, Brazil, and $ N e w York University Medical Center, Department of Pharmacology, N e w York, N Y I00 16, U.S.A.
Introduction The involvement of reactive oxygen species (ROS) in cellular regulatory and cytotoxic pro-
cesses implies that t h e cellular redox enviroment plays a major role in their action. T h e outcome depends o n t h e concentration of ROS a n d t h e availability of reducing equivalents, both intraa n d extra-cellularly. T h e potential toxicity of ROS has been well studied since the inception of superoxide dismutase as a probe of one-electron reduction reactions of oxygen. Although m u c h is understood about t h e involvement of ROS in cytotoxic processes, several important issues require further elucidation, particularly those related to t h e role of t h e reducing environment. Recent evidence indicates that, besides their
Abbreviations used: KOS, reactive oxygen species; GOPD, glucose-6-phosphate dehydrogenase; Pl’P, protein tyrosine phosphatase; SNP, sodium nitroprusside; SNAP, S-nitroso-N-acetylpenicillamine; MAP kinase, mitogen-activated protein kinase. $ T o whom correspondence should be addressed.
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capacity to cause cytotoxicity, ROS can promote cell growth [l], stimulate protein tyrosine phosphorylation associated with growth-factor-mediated signalling [Z], activate transcriptional regulators [ 3 ] a n d enhance t h e expression of proto-oncogenes [4]. In this paper we present two approaches for elucidating t h e role of the reducing environment in cytoregulation a n d cytotoxicity: a n investigation of t h e involvement of ROS and other oxidants in growth-factor-mediated signalling a n d in glucose-bphosphate dehydrogenase (G6PD) deficiency.
ROS and growth-factor-mediated signalling Protein tyrosine phosphorylation, which is primarily triggered by association of a growth factor with its specific receptor at t h e cell surface, is essential to signalling. Cellular levels of tyrosine phosphorylation are tightly regulated by a family of enzymes called protein tyrosine phosphatases ( P T P s ) [5,6]. All PTPs have a n essential cysteine residue in their catalytic domain, and total
Keeping the Cell Reduced: Enzymes Influencing Redox Reactions
ROS and growth-factor-mediated signalling Protein tyrosine phosphorylation, which is primarily triggered by association of a growth factor with its specific receptor at the cell surface, is essential to signalling. Cellular levels of tyrosine phosphorylation are tightly regulated by a family of enzymes called protein tyrosine phosphatases (PTPs) [5,6]. All PTPs have an essential cysteine residue in their catalytic domain, and total activity of these enzymes requires the cysteine residue in its reduced form [7]. Stress responses (e.g. oxidative stress and heat shock) can also induce protein tyrosine phosphorylation of proteins in growth-factor-mediated signalling pathways [8]. T h e importance of the reducing environment in the regulation of growth-factor-mediated signalling was initially apparent in studies with the thiol oxidant diamide, which inhibited cellular PTP activities [9]. This inhibition was prevented in cells pre-exposed to the reducing agent P-mercaptoethanol. Further studies with the ROS-generating system ascorbic acid/iron [ 101 confirmed the observations made with the di-
amide system. Hydrogen peroxide can also inhibit cellular PTP activities, but such activities are spontaneously recoverable. When cells were treated with the glutathione synthase inhibitor i,-buthionine sulfoximine, no additional effect was observed on hydrogen peroxide-induced inhibition of PTP activities. However, recovery of the activities was retarded, suggesting that the availability of glutathione is required in the reduction of the target site of hydrogen peroxide to restore the activity of the enzymes [ 113. NO, because of its redox behaviour towards thiols, was recently shown to inhibit P T P activities in murine fibroblasts [12]. We have also demonstrated that NO can induce protein tyrosine phosphorylation as detected by immunoblotting using anti-phosphotyrosine antibodies after SDS/PAGE [12]. A set of proteins (125, 56 and 43 kDa) was phosphorylated on tyrosine when murine fibroblasts were exposed to the NO donors sodium nitroprusside (SNP) and Snitroso-N-acetylpenicillamine (SNAP) (Figure 1). Of the three proteins, the 43 kDa protein has been tentatively identified (D. T.-Y. Chiu, H. P. Monteiro and A. Stern, unpublished work) as a
Figure I Effects of SNP and SNAP on tyrosine phosphorylation levels in murine fibroblasts in culture Cells were starved for 48 h and treated with 0 I rnM SNP or SNAP Tyrosine phosphorylation levels in each protein band were determined by laser-densitometric analysis of the autoradiographed irnmunoblots The data represent means fS E M for three independent experiments (Adapted from ref [ I21 with permission ) m, SNP, P , SNAP T
c
126 kDa
56 kDa
43 kDa
I996
885
Biochemical Society Transactions
Table I Plasma vitamin levels in G6PD-deficient individuals and matching controls Each value is expressed as mean _+ S.E.M. (adapted from ref. [24]).
886
Level in controls Number of G6PD-deficient individuals P -value Number of matching controls
45.59 f2.50 32.09 & 2. I4
0.560+ 0.088
0.690 0.020 0.575 f0.034
< 0.05 60
