Cytotoxicity of RH1 and related aziridinylbenzoquinones: involvement of activation by NAD(P)H:quinone oxidoreductase (NQO1) and oxidative stress Au  sra Nemeikait _ e-  C _ eniene, a Jonas  Sarlauskas, b  Zilvinas Anusevi  cius, b Henrikas Nivinskas, b and Narimantas  C _ enas b, * a Institute of Immunology, Mol _ etu z Pl. 29, Vilnius 2021, Lithuania b Institute of Biochemistry, Lithuanian Academy of Sciences, Sector of Xenobiotics Biochemistry, Mokslininku z St. 12, Vilnius 2600, Lithuania Received 19 March 2003, and in revised form 27 March 2003 This paper is dedicated to Professor Pranas Sadauskas on the occassion of his 75th birthday Abstract It is supposed that the main cytotoxicity mechanism of antitumour aziridinyl-substituted benzoquinones is their two-electron reduction to alkylating products by NAD(P)H:quinone oxidoreductase (NQO1, DT-diaphorase, EC 1.6.99.2). However, other possible cytotoxicity mechanisms, e.g., oxidative stress, are studied insufficiently. In the single-electron reduction of quinones in- cluding a novel compound RH1 (2,5-diaziridinyl- 3-(hydroxymethyl)-6-methyl-1,4-benzoquinone), by NADPH:cytochrome P-450 reductase (EC 1.6.2.4, P-450R), their reactivity increased with an increase in the redox potential of quinone/semiquinone couple (E 1 7 ), reaching a limiting value at E 1 7 P  0:1V. The reactivity of quinones towards NQO1 did not depend on their E 1 7 . The cyto- toxicity of aziridinyl-unsubstituted quinones in bovine leukemia virus-transformed lamb kidney fibroblasts (line FLK) mimics their reactivity in P-450R-catalyzed reactions, exhibiting a parabolic dependence on their E 1 7 . The toxicity of aziridinyl-benzoquinones, although being higher, also followed this trend and did not depend on their reactivity towards NQO1. The action of azi- ridinylbenzoquinones in FLK cells was accompanied by an increase in lipid peroxidation, their toxicity decreased by desferriox- amine and the antioxidant N ,N 0 -diphenyl-p-phenylene diamine, and potentiated by 1,3-bis-(2-chloroethyl)-1-nitrosourea. The inhibitorofNQO1,dicumarol,protectedagainstthetoxicityofaziridinyl-benzoquinonesexceptof2,5-bis-(2 0 -hydroxyethylamino)- 3,6-diaziridinyl-1,4-benzoquinone (BZQ), which was almost inactive as NQO1 substrate. The same events except the absence of pronounced effect of dicumarol were characteristic in the cytotoxicity of aziridinyl-unsubstituted quinones. These findings indicate thatinadditiontotheactivationbyNQO1,theoxidativestresspresumablyinitiatedbysingle-electrontransferringenzymesmaybe an important factor in the cytotoxicity of aziridinylbenzoquinones. The information obtained may contribute to the understanding of the molecular mechanisms of aziridinylquinone cytotoxicity and may be useful in the design of future bioreductive drugs. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Aziridinylbenzoquinones; Cytotoxicity; NAD(P)H:quinone oxidoreductase; Oxidative stress Mammalian NAD(P)H:quinone oxidoreductase (NQO1, 1 DT-diaphorase, EC 1.6.99.2) is a dimeric fla- voenzyme containing one molecule of FAD per 31kDa subunit, catalyzing two-electron reduction of quinones and aromatic nitrocompounds ([1,2], and references therein). This enzyme plays an important, however con- Archives of Biochemistry and Biophysics 416 (2003) 110–118 www.elsevier.com/locate/yabbi ABB * Corresponding author. Fax: +11-370-2-72-91-96. E-mail address: ncenas@bchi.lt (N.  C_ enas). 1 Abbreviations used: E 1 7 , redox potential of quinone/semiquinone couple at pH 7.0; E 2 7 , redox potential of semiquinone/hydroquinone couple at pH 7.0; E 0 7 , potential of two-electron reduction of quinones (standard potential) at pH 7.0; cL 50 , the concentration of com- poundfor50%cellsurvival;NQO1,NAD(P)H:quinoneoxidoreductase; P-450R, NADPH:cytochrome P-450 reductase; Q, quinone; k cat , catalytic constant; k cat =K m , bimolecular rate constant; RH1, 2,5- diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone; BZQ, 2,5- bis(2 0 -hydroxy-ethylamino)-3,6-diaziridinyl-1,4-benzoquinone; DZQ, 2,5-diaziridinyl-1,4-benzoquinone; MeDZQ, 2,5-dimethyl-3,6-diazirid- inyl-1,4-benzoquinone; EO9, 3-hydroxymethyl-5-aziridinyl-1-methyl- 2-[1H-indole-4,7-dione]-prop-b-en-a-ol, DPPD, N ,N 0 -diphenyl-p-phe- nylene diamine; BCNU, 1,3-bis-(2-chloroethyl)-1-nitrosourea. 0003-9861/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0003-9861(03)00281-9