Cytotoxicity of anticancer aziridinyl-substituted benzoquinones in primary mice splenocytes

The anticancer activity of aziridinyl-quinones is mainly attributed to their NAD(P)H:quinone oxidoreductase 1 (NQO1)-catalyzed two-electron reduction into DNAalkylating products. However, little is known about their cytotoxicity in primary cells, which may be important in understanding their side effects. We found that the cytotoxicity of aziridinyl-unsubstituted quinones (n = 12) in mice splenocytes with a low amount of NQO1, 4 nmol × mg–1 × min–1, was caused mainly by the oxidative stress. Aziridinyl-benzoquinones (n = 6) including a novel anticancer agent RH1 were more cytotoxic than aziridinylunsubstituted ones with the similar redox properties, and their cytotoxicity was not decreased by an inhibitor of NQO1, dicumarol. The possible reasons for their enhanced cytotoxicity are discussed.

were characterized by a melting point and 1 H-NMR, UV, and IR spectroscopy.
Cell culture growth and cytotoxicity studies.In the studies of primary mice splenocytes, 4-to 8-week old male and female BALB/c mice (24±2.0g) were kept under standard conditions, and were given food and water ad libitum.The mice were sacrificed by decapitation, their spleen was removed according to an established procedure (Stack et al., 1999).These experiments were approved by the Lithuanian Veterinary and Food Service (License No. 0215, 2011).For each experiment, spleens of 3-5 mice were used as a source of splenocytes.Erythrocytes were lysed by 5 min exposure in lysis solution (0.155 M NH 4 Cl, 10 mM KCl, 0.1 mM EDTA).After washing the cells twice with RPMI 1640 medium, they were resuspended at the concentration of 10 6 cells /ml in RPMI 1640 medium with 5% fetal bovine serum and antibiotics, and were used for the further experiments.Cell viability was determined after 24 h of incubation of splenocytes with the examined compounds in 96-well cell culture plates (200 µl suspension per well), according to a Trypan blue exclusion test.The stock solutions of examined compounds were made in DMSO.The total concentration of DMSO in all the experiments was equal to 0.2%, and did not affect the viability of the cells, 98-96%, after their 24 h incubation.
Enzyme activity determination.Splenocytes were washed with PBS twice, and sonicated on ice in four cycles of 20 s.The homogenate was centrifuged at 14 000 × g for 45 min and the resulting supernatant with added 1.0 mM PMSF was used for the further analysis.Protein amount was determined according to the method of Bradford.All the spectrometric measurements were performed using a Hitachi-557 spectrophotometer at 25°C in 0.1 M K-phosphate (pH 7.0) containing 1mM EDTA.The activity of catalase was determined following the decomposition of 10 mM H 2 O 2 (Schallreuter et al., 1991).The activity of superoxide dismutase was determined from the inhibition of reduction of nitroblue tetrazolium by xanthine oxidase/xanthine system (Oberley & Buettner, 1979).One unit of superoxide dismutase activity corresponds to an amount of protein needed to inhibit the reduction of nitroblue tetrazolium by 50%.The activity of NAD(P)H:oxidase was determined according to the rate of oxidation of 100 µM NAD(P)H (∆ε 340 = 6.2 mM -1 cm -1 ).The activity of NAD(P)H:cytochrome c reductase was determined according to the rate of reduction of 50 µM cytochrome c (∆ε 550 = 20 mM -1 cm -1 ) in the presence of 100 µM NAD(P)H.The activity of glutathione reductase was determined according to the rate of oxidation of 100 µM NADPH in the presence of 1.0 mM GSSG.The activity of NAD(P)H:quinone oxidoreductase 1 (NQO1) was determined following the rate of reduction of 50 µM cytochrome c in the presence of 10 µM menadione (2-methyl-1,4-naphthoquinone) and 100 µM NADPH, as the difference between the reduction rate in the absence of dicumarol, and in its presence (20 µM) (Lind et al., 1990).In this assay, Tween 20 (0.01%) and bovine serum albumin (0.25 mg × ml -1 ) were used as the activators of NQO1.
Statistical analysis.Statistical data analysis was performed using Statistica (version 4.3, Statsoft Inc., 1993).Values are shown as standard error of mean.Where appropriate, the data were analyzed using a Student's t-test.
Next, we examined the 24-h cytotoxicity of model aziridinyl-unsubstituted quinones (n = 12) in splenocytes.The data of Table 1 show that their concentrations for 50% cell survival (cL 50 ) decreased with an increase in their single-electron reduction potential (E 1 7 ), and ranged from 4.0±0.4µM (5-hydroxy-1,4-naphthoquinone, E 1 7 = -0.09V) to 1000±100 µM (2-hydroxy-1,4-naphthoquinone, E 1 7 = -0.41V).The dependence of log cL 50 on E 1 7 of quinones is parabolic (Fig. 2), being in line with the previously observed parabolic or linear log cL 50 vs.E 1 7 relationships with Δlog cL 50 /Δ E 1 7 ~ -10 V -1 in their aerobic cytotoxicity in several mammalian cell lines (Nemeikaitė-Čėnienė et al., 2003, and references therein).It points to the dominating role of their oxidative stress-type cytotoxicity.It reflects the ease of the singleelectron reduction of quinones by P-450R or by similar single-electron transferring flavoenzymes, which initiate their redox cycling.The reaching of the limiting value of cL 50 of quinones at high redox potentials may be explained by the instability of high potential partly-substituted quinones, i.e., their rapid trapping by reduced glutathione (GSH) and other cellular nucleophiles, and/ or their impaired redox cycling ability (Wardman et al., 1995).Importantly, aziridinyl-substituted quinones RH1, MeDZQ, trimethyl-2-aziridinyl-1,4-benzoquinone, and, especially, BZQ (Fig. 1) exhibited higher cytotoxicity than it may be expected from their E 1 7 value (Fig. 2).It points to the additional mechanisms of their action.
Further, we found that the cytotoxicity of RH1 at close to cL 50 concentration was partly protected by the antioxidant N,N´-diphenyl-p-phenylene diamine (DPPD), the iron ion chelator desferrioxamine, and potentiated by the prooxidant BCNU, the latter inactivating the antioxidant enzyme glutathione reductase, and depleting GSH (Öllinger & Brunmark, 1991) (Fig. 3).Importantly, the cytotoxicity of RH1 was not protected by an inhibitor of NQO1, dicumarol (Fig. 3).Analogous protection/potentiation effects by DPPD, desferrioxamine, and BCNU were observed in the cytotoxicity of tetramethyl-1,4-benzoquinone (duroquinone) and BZQ (data not shown).Duroquinone is a nonalkylating analogue of RH1 with similar reactivity towards the single-electron transferring P-450R and two-electron transferring NQO1 (Nemeikaitė-Čėnienė et al., 2003).On the other hand, BZQ undergoes redox cycling and directly alkylates DNA, but is not a substrate for NQO1 (DiFrancesco et al., 2004).Analogously, dicumarol also did not protect against the cytotoxicity of duroquinone and BZQ (data not shown).

DISCUSSION
According to our best knowledge, the previous primary cell cytotoxicity studies of aziridinyl-benzoquinones were scarce, and did not disclose their specific mode(s) of action.They involved the short-time testing of AZQ in rat hepatocytes (O'Brien, 1991), a study of trenimon derivatives in primary human skin fibroblasts (Huang et al., 2009), and the related genotoxic studies of AZQ in lymphocytes (Klingerman et al., 1988), as well as the cytotoxicity of AZQ and DZQ in mice epidermal cells (Li et al., 1999).In contrast, our data provide a more quantitative insight into the cytotoxicity of aziridinyl-substituted quinones in primary cells with a low amount of NQO1.In our opinion, the primary splenocytes used in this study, are a more advantageous model as compared to primary hepatocytes, because of the possibility to perform longer 24-h cytotoxicity tests.
Previously, we found that the cytotoxicity of aziridinyl-benzoquinones in transformed cells with high activity of NQO1, 250 nmol × mg -1 × min -1 , was 10-100 times higher than that of aziridinyl-unsubstituted quinones possessing the same E 1 7 values (Nemeikaitė-Čėnienė et al., 2003).The current data show that in spite of the low activity of NQO1 in splenocytes, 4.0 nmol × mg -1 × min -1 , the cytotoxicity of aziridinyl-benzoquinones is still enhanced (Fig. 2).The much enhanced cytotoxicity of BZQ (Fig. 2) may be explained by its direct alkylation of DNA without the involvement of enzymatic activation (Butler et al., 1990).In contrast, MeDZQ and RH1 do not alkylate DNA directly (Lee et al., 1992;DiFrancesco et al., 2004), but they still possess increased cytotoxicity, which, in the case of RH1, is not related to the action of NQO1, because it is not inhibited by dicumarol (Fig. 3).The protective effects of DPPD and desferrioxamine against the action of RH1 (Fig. 3) indicate that its cytotoxicity is partly attributed to the oxidative stress.However, in our opinion, the higher cytotoxicity of RH1, MeDZQ and aziridinyl-benzoquinones with the higher values of E 1 7 (Table 1, Fig. 2) may not be attributed to their enhanced redox cycling, because both aziridinyl-substituted and -unsubstituted quinones follow the same log (rate constant) vs. E 1 7 relationship in their P-450R-catalyzed single-electron reduction (Nemeikaitė-Čėnienė et al., 2003).On the other hand, it may be caused by other currently insufficiently addressed NQO1-independent cytotoxicity modes (Tudor et al., 2005), e.g., a net two-electron reduction of aziridinyl-benzoquinones by single-electron transferring enzymes into their DNA-alkylating hydroquinones under partly anaerobic conditions (Anusevičius et al., 2013), which may take place in relatively anaerobic cell compartments, or, possibly, the action of dihydronicotinamide riboside:qui- ).The dependences of the concentrations of aziridinyl-unsubstituted (A) and -substituted (B) quinones causing the 50% survival of the cells (cL 50 ) are presented as the second order approximations, the numbers of quinones are taken from Table 1.none oxidoreductase 2 (NQO2) (Yan et al., 2008).These problems warrant a more thorough examination.However, the enhanced cytotoxicity of RH1 in splenocytes (Fig. 2) is in line with its significant suppressive effects on the cells of the immune system, which were observed during its Phase-I clinical trials (Danson et al., 2011).It shows that this novel compound may be not devoid of significant side effects even in the cells with low content of NQO1, which may limit its further clinical application.

Figure 2 .
Figure 2. The dependence of the cytotoxicity of aziridinyl-unsubstituted and -substituted quinones in primary splenocytes on their single-electron reduction potential (E 1 7).The dependences of the concentrations of aziridinyl-unsubstituted (A) and -substituted (B) quinones causing the 50% survival of the cells (cL 50 ) are presented as the second order approximations, the numbers of quinones are taken from Table1.