Cytotoxicity Of Quinones Research Articles

Kinetics of Flavoenzyme-Catalyzed Reduction of Tirapazamine Derivatives: Implications for Their Prooxidant Cytotoxicity.

Derivatives of tirapazamine and other heteroaromatic N-oxides (ArN→O) exhibit promising antibacterial, antiprotozoal, and tumoricidal activities. Their action is typically attributed to bioreductive activation and free radical generation. In this work, we aimed to clarify the mechanism(s) of aerobic mammalian cell cytotoxicity of ArN→O performing the parallel studies of their reactions with NADPH:cytochrome P-450 reductase (P-450R), adrenodoxin reductase/adrenodoxin (ADR/ADX), and NAD(P)H:quinone oxidoreductase (NQO1); we found that in P-450R and ADR/ADX-catalyzed single-electron reduction, the reactivity of ArN→O (n = 9) increased with their single-electron reduction midpoint potential (E17), and correlated with the reactivity of quinones. NQO1 reduced ArN→O at low rates with concomitant superoxide production. The cytotoxicity of ArN→O in murine hepatoma MH22a and human colon adenocarcinoma HCT-116 cells increased with their E17, being systematically higher than that of quinones. The cytotoxicity of both groups of compounds was prooxidant. Inhibitor of NQO1, dicoumarol, and inhibitors of cytochromes P-450 α-naphthoflavone, isoniazid and miconazole statistically significantly (p < 0.02) decreased the toxicity of ArN→O, and potentiated the cytotoxicity of quinones. One may conclude that in spite of similar enzymatic redox cycling rates, the cytotoxicity of ArN→O is higher than that of quinones. This is partly attributed to ArN→O activation by NQO1 and cytochromes P-450. A possible additional factor in the aerobic cytotoxicity of ArN→O is their reductive activation in oxygen-poor cell compartments, leading to the formation of DNA-damaging species similar to those forming under hypoxia.

A focused library synthesis and cytotoxicity of quinones derived from the natural product bolinaquinone.

Bolinaquinone is a natural product that is a structurally complex, cytotoxic sesquiterpene quinone. A scaffold simplification and focused library approach using a microwave-assisted Suzuki coupling gave 32 bolinaquinone analogues with good-to-excellent cytotoxicity profiles. Mono-arylbenzoquinones, Library A, were preferentially toxic towards BE2-C (neuroblastoma) cells with growth inhibition (GI50) values of 4–12 µM; only the 3,4-dimethoxyphenyl 23 and 3-biphenyl 28 variants were broad-spectrum active—HT29 (colon carcinoma), U87 and SJ-G2 (glioblastoma), MCF-7 (breast carcinoma), A2780 (ovarian carcinoma), H460 (lung carcinoma), A431 (skin carcinoma), Du145 (prostate carcinoma), BE2-C (neuroblastoma), MIA (pancreatic carcinoma) and SMA (spontaneous murine astrocytoma). Library B with a second aryl moiety exhibited broad-spectrum cytotoxicity with MCF-7 cells’ GI50 values of 5.6 ± 0.7 and 5.1 ± 0.5 µM for 2,5-dimethoxy-3-(naphthalene-1-yl)-6-(naphthalene-3-yl) 33 and 2,5-dimethoxy-3-(biaryl-2-yl)-6-(naphthalene-3-yl) 36, respectively. Similar potencies were also noted with 2,5-dimethoxy-3,6-diphenyl 30 against A2780 (GI50 = 5.9 ± 0.0 µM) and with 2,5-dimethoxy-3-(biaryl-3-yl)-6-(naphthalene-3-yl) 37 against HT29 (GI50 = 5.4 ± 0.4 µM), while the 3,4-dimethoxy mono-aryl analogue 23 exhibited good levels of activity against A2780 (GI50 = 3.8 ± 0.75 µM), the neuroblastoma cell line BE2-C (GI50 = 3 ± 0.35 µM) and SMA (GI50 = 3.9 ± 0.54 µM). Introduction of the amino-substituted Library C gave 2-(naphthalen-1-yl)-5-(naphthalen-3-yl)-3,6-bis(propylamino) 43, with excellent activity against HT29 (0.08 ± 0.0 µM), MCF-7 (0.17 ± 0.1 µM), A2780 (0.14 ± 0.1 µM), A431 (0.11 ± 0.0 µM), Du145 (0.16 ± 0.1 µM), BE2-C (0.08 ± 0.0 µM) and MIA (0.1 ± 0.0 µM).

Neuroglobin Modification by Reactive Quinone Species

The physiological functions of neuroglobin (Ngb), the heme protein of the globin family expressed in the nervous tissue, have not yet been clarified. Besides O2 storage and homeostasis, Ngb is thought to play a role in neuroprotection as a scavenger of toxic reactive species generated in vivo under conditions of oxidative stress. Herein, the interaction of Ngb with the quinones generated by oxidation of catecholamines (dopamine, norepinephrine) and catechol estrogens (2-hydroxyestradiol and 4-hydroxyestradiol), which have been implicated in neurodegenerative pathologies like Parkinson's and Alzheimer's diseases, has been investigated. The cytotoxicity of quinones has been ascribed to the derivatization of amino acid residues (mainly cysteine) in proteins through the formation of covalent bonds with the aromatic rings. Combined studies of tandem mass spectrometry and protein unfolding indicate the presence of quinone-promoted modifications in all of the Ngb derivatives analyzed (i.e., obtained employing either catecholamines or catechol estrogens as the source of the reactive species). Among protein residues, the highest reactivity of cysteines (Cys46, Cys55, and Cys120 in human Ngb) toward quinone species has been confirmed, and the dependence of the extent of protein modification on the method employed for catechol oxidation has been observed. When the oxidation reaction proceeds by one-electron steps, the involvement of semiquinone reactivity has been observed. The whole analysis of the data of Ngb modification suggests that the catecholamine-oxidation products can extensively modify proteins (likely by catecholamine oligomers, the compounds initially formed during the transformation of catecholamine to melanin). The modification mediated by catechol estrogens is less pronounced but strongly affects the interactions with the solvent as well as the protein stability.

Synthetic cannabinoid quinones: Preparation, in vitro antiproliferative effects and in vivo prostate antitumor activity

Chromenopyrazolediones have been designed and synthesized as anticancer agents using the multi-biological target concept that involves quinone cytotoxicity and cannabinoid antitumor properties. In cell cytotoxicity assays, these chromenopyrazolediones have antiproliferative activity against human prostate cancer and hepatocellular carcinoma. It has been shown that the most potent, derivative 4 (PM49), inhibits prostate LNCaP cell viability (IC50 = 15 μM) through a mechanism involving oxidative stress, PPARγ receptor and partially CB1 receptor. It acts on prostate cell growth by causing G0/G1 phase arrest and triggering apoptosis as assessed by flow cytometry measurements. In the in vivo treatment, compound 4 at 2 mg/kg, blocks the growth of LNCaP tumors and reduces the growth of PC-3 tumors generated in mice. These studies suggest that 4 is a good potential anticancer agent against hormone-sensitive prostate cancer.

Contribution of Reductase Activity to Quinone Toxicity in Three Kinds of Hepatic Cells

Two mechanisms have been proposed to explain quinone cytotoxicity: oxidative stress via the redox cycle, and the arylation of intracellular nucleophiles. The redox cycle is catalyzed by intracellular reductases, and therefore the toxicity of redox cycling quinone is considered to be closely associated with the reductase activity. This study examined the relationship between quinone toxicity and the intracellular reductase activity using 3 kinds of hepatic cells; rat primary hepatocytes, HepG2 and H4IIE. The intracellular reductase activity was; primary hepatocyte >>HepG2>H4IIE. The three kinds of cells showed almost the same vulnerability to an arylating quinone, 1,4-naphthoquinone (NQ). However, the susceptibility to a redox cycling quinone, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) was; primary hepatocyte>HepG2>H4IIE. In addition, the cytotoxicity elicited by DMNQ was significantly attenuated in HepG2 cells and almost completely suppressed in primary hepatocytes by diphenyleneiodonium chloride, a reductase inhibitor. These data suggest that cells with a high reductase activity are susceptible to redox cycling quinones. This study provides essential evidence to assess the toxicity of quinone-based drugs during their developmental processes.

Aminopyrimidoisoquinolinequinone (APIQ) redox cycling is potentiated by ascorbate and induces oxidative stress leading to necrotic-like cancer cell death

Several phenylaminopyrimidoisoquinolinequinones (APIQs) were tested for their cytotoxicity against different cancer cell lines (K562, T24, HepG2) in the presence or absence of ascorbate. Ascorbate enhanced the toxic effects of quinones with first half-wave potential E(I) (1/2) values in the range of -480 to -660mV. Phenylaminoquinones that were unsubstituted at position 6 exhibited greater cytotoxic activity than did their 6-methyl-substituted analogues. Two groups of compounds were further selected, namely 8-10 and 20-22, to study the cellular mechanisms involved in quinone cytotoxicity. Indeed, these compounds have the same range of redox potentials but differed considerably in their capacity to induce cell death. In the presence of ascorbate, the cell demise induced by compounds 8-10 was not caspase-3 dependent, as shown by the lack of activation of caspase-3 and the absence of cleaved PARP fragments. In addition, an index of ER stress (eIF2α phosphorylation) was activated by these compounds. Quinones 8-10 decreased the cellular capacity to reduce MTT dye and caused marked ATP depletion. Taken together, our results show that ascorbate enhances quinone redox-cycling and leads to ROS formation that inhibits cell proliferation and provokes caspase-independent cell death. Interestingly, we also observed that quinone 8 had a rather selective effect given that freshly isolated peripheral blood leukocytes from human healthy donors were more resistant than human leukemia K562 cells.

Role of glutathione in augmenting the anticancer activity of pyrroloquinoline quinone (PQQ)

Pyrroloquinoline quinone (PQQ), a bacterial redox co-factor and antioxidant, is highly reactive with nucleophilic compounds present in biological fluids. PQQ induced apoptosis in human promonocytic leukemia U937 cells and this was accompanied by depletion of the major cellular antioxidant glutathione and increase in intracellular reactive oxygen species (ROS). Treatment with glutathione (GSH) or N-acetyl-L-cysteine (NAC) did not spare PQQ toxicity but resulted in a 2–5-fold increase in PQQ-induced apoptosis in U937 cells. Cellular GSH levels increased following treatment by NAC alone but were severely depleted by co-treatment with NAC and PQQ. This was accompanied by an increase in intracellular ROS. Alternatively, depletion of glutathione also resulted in increased PQQ cytotoxicity. However, the cells underwent necrosis as evidenced by dual labeling with annexin V and propidium iodide. PQQ-induced cytotoxicity is thus critically regulated by the cellular redox status. An increase in GSH can augment apoptosis and its depletion can switch the mode of cell death to necrosis in the presence of PQQ. Our data suggest that modulation of intracellular GSH can be used as an effective strategy to potentiate cytotoxicity of quinones like PQQ.

Differential effects of redox-cycling and arylating quinones on trans-plasma membrane electron transport.

Cytotoxicity of quinones has been attributed to free radical generation and to arylation of cellular nucleophiles. For redox-cycling quinones, cell injury is associated with mitochondrial permeability transition, whereas arylating quinones directly depolarise the mitochondrial membrane and deplete ATP. Like mitochondrial electron transport, plasma membrane electron transport (PMET), plays a multifaceted role in cellular redox homeostasis but the effects of quinones on PMET are unknown. Here we investigate the effects of redox-cycling 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), arylating 1,4-benzoquinone (BQ) and mixed mechanism 2-methyl-1,4-naphthoquinone (MNQ) on PMET, viability and growth of P815 mouse mastocytoma cells.BQ and MNQ rapidly and extensively inhibited PMET as determined by WST-1 /mPMS reduction (IC50 3.5-5 microM at 30 min) whereas the effects of DMNQ were less pronounced. In contrast, MTT reduction (cytosolic NADH dehydrogenase activity over 30 min) was weakly inhibited by BQ (IC50 20 microM) but not by MNQ or DMNQ and cell viability was unaffected. Inhibition of WST-1/mPMS reduction by BQ and MNQ but not DMNQ was fully reversed by NAC. Treatment with DMNQ, MNQ and to a lesser extent BQ inhibited cell proliferation as determined by MTT reduction at 48 h. The effects of BQ and MNQ were reversed by NAC through covalent bonding to BQ and MNQ, but not DMNQ. These results show that arylating quinones are more potent inhibitors of PMET than pure redox-cycling quinones, but that redox-cycling quinones are more cytotoxic.

Synthesis of 1-/2-substituted-[1,2,3]triazolo[4,5- g]phthalazine-4,9-diones and evaluation of their cytotoxicity and topoisomerase II inhibition

Studies on antitumor heterocyclic quinones containing nitrogens revealed that the number and position of nitrogens on the heterocyclic ring have significance on cytotoxicity of quinones. In our continuous effort to find more cytotoxic quinone compounds, we designed triazolophthalazine analogues in order to introduce more nitrogens on the heterocyclic quinones. 1-/2-Substituted-[1,2,3]triazolo[4,5- g]phthalazine-4,9-diones were synthesized by 1,3-dipolar addition of phthalazine-5,8-dione and 4-methoxybenzyl azide by modification of previously reported method. The cytotoxicity of the synthesized compounds was evaluated by a SRB (sulforhodamine B) assay against nine types of human cancer cell lines and inhibition against topoisomerase II (Topo II) of them was assessed by a decatenation assay. Most of the synthesized compounds showed considerably higher cytotoxicity than that of doxorubicin. Also, topoisomerase II inhibitory activity of the tested compounds was higher than that of etoposide and IC 50 values of the compounds were 19.4–64.5 μM.

Modelling and Measuring Redox Cycling and Cytotoxicity of Quinones

The roles of alkylation and redox cycling in quinone toxicity were investigated. In general the more cytotoxic quinones produced the highest responses in an assay monitoring redox activity. No evidence of alkylation of high molecular weight protein thiols was detected. We conclude quinone toxicity is dominated by redox cycling.

Redox Cycling and Thiol Reactivity in Quinone Cytotoxicity

not Available.

NRH:quinone oxidoreductase 2 (NQO2) catalyzes metabolic activation of quinones and anti-tumor drugs

NRH:quinone oxidoreductase 2 (NQO2) is a cytosolic flavoprotein that utilizes NRH as electron donor. The present studies investigate the role of NQO2 in metabolic detoxification/activation of quinones and quinone based anti-tumor drugs. Chinese hamster ovary (CHO) cells stably overexpressing cDNA derived mouse NQO2 and mouse keratinocytes from DMBA-induced skin tumors in wild-type and NQO2-null mice were generated. The CHO cells overexpressing NQO2 and mouse keratinocytes expressing or deficient in NQO2 were treated with varying concentrations of mitomycin C (MMC), CB1954, MMC analog BMY25067, EO9, menadione and BP-3,6-quinone, in the absence and presence of NRH. The cytotoxicity of the drugs was evaluated by colony formation. The CHO cells overexpressing higher levels of mouse NQO2 showed significantly increased cytotoxicity to menadione, BP-3,6-quinone and to the anti-tumor drugs MMC and CB1954 when compared to CHO cells expressing endogenous NQO2. The cytotoxicity increased in presence of NRH. Similar results were also observed with BMY25067 and EO9 treatments, but to a lesser extent. The results on keratinocytes deficient in NQO2 supported the data from CHO cells. The inclusion of NRH had no effect on cytotoxicity of quinones and drugs in keratinocytes deficient in NQO2. Mouse NQO2 protein was expressed in bacteria, purified and used to study the role of NQO2 in MMC-induced DNA cross-linking. Bacterially expressed and purified NQO2 efficiently catalyzed MMC activation that led to DNA cross-linking. These results concluded that NQO2 plays a significant role in the metabolic activation of both quinones and anti-tumor drugs leading to cytotoxicity and cell death.

Mechanism of arylating quinone toxicity involving Michael adduct formation and induction of endoplasmic reticulum stress

Quinones permeate our biotic environment, contributing to both homeostasis and cytotoxicity. All quinones generate reactive oxygen species through redox cycling, while partially substituted quinones also undergo arylation (Michael adduct formation) yielding covalent bonds with nucleophiles such as cysteinyl thiols. In contrast to reactive oxygen species, the role of arylation in quinone cytotoxicity is not well understood. We found that the arylating quinones, including unsubstituted 1,4-benzoquinone (1,4-BzQ) and partially substituted vitamin E congener gamma-tocopherol quinone (gamma-TQ), were cytotoxic, with gamma-TQ > 1,4-BzQ, whereas the fully substituted nonarylating vitamin E congener alpha-tocopherol quinone was not. In vitro, both arylating quinones formed Michael adducts with the thiol nucleophile N-acetylcysteine (NAC) at rates where 1,4-BzQ > gamma-TQ. In cultured cells, concurrent addition of NAC eliminated 1,4-BzQ caused toxicity, but preincubation was required for the same NAC detoxification effect on gamma-TQ. These data clearly established the role of arylation in quinone toxicity and revealed that arylating quinone structure affects cytotoxicity by governing detoxification through the rate of adduct formation. Furthermore, arylating quinones induced endoplasmic reticulum (ER) stress by activating the pancreatic ER kinase (PERK) signaling pathway including elF2alpha, ATF4, and C/EBP homologous protein (CHOP). Detoxification by NAC greatly attenuates CHOP induction in arylating quinone-treated cells, suggesting that ER stress is a cellular mechanism for arylating quinone cytotoxicity.

Enhancement of DMNQ-induced hepatocyte toxicity by cytochrome P450 inhibition

Two mechanisms have been proposed to explain quinone cytotoxicity: oxidative stress via the redox cycle and the arylation of intracellular nucleophiles. As the redox cycle is catalyzed by NADPH cytochrome P450 reductase, cytochrome P450 systems are expected to be related to the cytotoxicity induced by redox-cycling quinones. Thus, we investigated the relationship between cytochrome P450 systems and quinone toxicity for rat primary hepatocytes using an arylator, 1,4-benzoquinone (BQ), and a redox cycler, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). The hepatocyte toxicity of both BQ and DMNQ increased in a time- and dose-dependent manner. Pretreatment with cytochrome P450 inhibitors, such as SKF-525A (SKF), ketoconazole and 2-methy-1,2-di-3-pyridyl-1-propanone, enhanced the hepatocyte toxicity induced by DMNQ but did not affect BQ-induced hepatocyte toxicity. The production of superoxide anion and the levels of glutathione disulfide and thiobarbituric-acid-reactive substances were increased by treatment with DMNQ, and SKF pretreatment further enhanced their increases. In addition, NADPH oxidation in microsomes was increased by treatment with DMNQ and further augmented by pretreatment with SKF, and a NADPH cytochrome P450 reductase inhibitor, diphenyleneiodonium chloride completely suppressed NADPH oxidations increased by treatment with either DMNQ- or DMNQ + SKF. Pretreatment with antioxidants, such as alpha-tocopherol, reduced glutathione, N-acetyl cysteine or an iron ion chelator deferoxamine, totally suppressed DMNQ- and DMNQ + SKF-induced hepatocyte toxicity. These results indicate that the hepatocyte toxicity of redox-cycling quinones is enhanced under cytochrome P450 inhibition, and that this enhancement is caused by the potentiation of oxidative stress.

A physiological threshold for protection against menadione toxicity by human NAD(P)H:quinone oxidoreductase (NQO1) in Chinese hamster ovary (CHO) cells

NAD(P)H:quinone oxidoreductase 1 (NQO1) has often been suggested to be involved in cancer prevention by means of detoxification of electrophilic quinones. In the present study, a series of Chinese hamster ovary (CHO) cell lines expressing various elevated levels of human NQO1 were generated by stable transfection. The level of NQO1 over-expression ranged from 14 to 29 times the NQO1 activity in the wild-type CHO cells. This panel of cell lines, allowed investigation of the protective role of NQO1 in quinone cytotoxicity. It could be demonstrated that menadione toxicity was significantly reduced in all NQO1-transfected CHO clones compared to the wild-type cells, but the clones did not show differences in their level of protection against menadione. This observation pointed at a critical threshold concentration of NQO1 above which a further increase does not provide further protection against quinone cytotoxicity. Additional studies in which the NQO1 activity was inhibited by dicoumarol showed that only dicoumarol concentrations of about five times the ec 50 for NQO1 inhibition were able to reduce NQO1 levels below the apparent threshold, making the cells more sensitive. The level of this threshold was estimated to be in the range of base line NQO1 activities observed in several tissues and species. Thus, the results of the present study indicate that beneficial effects of NQO1 induction by, for example, cruciferous vegetables might be absent or present depending on the NQO1 activity threshold for optimal protection and the basal level of NQO1 expression in the tissue and species of interest.

Role of NAD(P)H:quinone oxidoreductase 1 (DT diaphorase) in protection against quinone toxicity

NQO1−/− mice, along with Chinese hamster ovary (CHO) cells, were used to determine the in vivo role of NAD(P)H:quinone oxidoreductase 1 (NQO1) in cellular protection against quinone cytotoxicity, membrane damage, DNA damage, and carcinogenicity. CHO cells permanently expressing various levels of cDNA-derived P450 reductase and NQO1 were produced. Treatment of CHO cells overexpressing P450 reductase with menadione, benzo[ a]pyrene-3,6-quinone (BPQ), and benzoquinone led to increased cytotoxicity as compared with CHO cells expressing endogenous P450 reductase. In a similar experiment, overexpression of NQO1 significantly protected CHO cells against the cytotoxicity of these quinones. Knockout ( NQO1−/−) mice deficient in NQO1 protein and activity had been generated previously in our laboratory and were used in the present studies. Wild-type ( NQO1+/+) and knockout ( NQO1−/−) mice were given i.p. injections of menadione and BPQ, followed by analysis of membrane damage and DNA damage. Both menadione and BPQ induced lipid peroxidation in hepatic and non-hepatic tissues, indicating increased membrane damage. Exposure to BPQ also resulted in increased hepatic DNA adducts in NQO1−/− mice as compared with NQO1+/+ mice. The skin application of BPQ alone and BPQ + 12- O-tetradecanoylphorbol-13-acetate (TPA) failed to induce papillomas, or other lesions, for up to 50 weeks in either NQO1+/+ or NQO1−/− mice. The various results from CHO cells and NQO1−/− mice indicated that NQO1 protects against quinone-induced cytotoxicity, as well as DNA and membrane damage. The absence of BPQ-induced skin carcinogenicity in NQO1−/− mice may be related to the strain (C57BL/6) of mice used in the present study and/or due to poor BPQ absorption into the skin and/or due to detoxification of BPQ by cytosolic NRH:quinone oxidoreductase 2 (NQO2).

Redox cycling of o-naphthoquinones in yrypanosomatids : Superoxide and hydrogen peroxide production

β-Lapachone and structurally related lipophilic o-naphthoquinones, namely, CG 8-935, CG 9-442, CG 10-248 and mansonones A, C, E, and F, were investigated for redox cycling, production of reactive oxygen species, and cytotoxicity in the trypanosomatids Crithidia fasciculata and Leptomonas seymouri. Structural analysis of the assayed quinones indicated that a tricyclic structure, including a naphthalene ring, a 1,2 b or l,8 bc pyran ring, and two ortho-carbonyl groups were required for quinone activities. The contribution of oxygen radical production to quinone cytotoxicity was supported by: (a) spectroscopic observation of quinone redox cycling; (b) production of the semiquinone radical; (c) H 2O 2 and O 2 − production; (d) the effect of β-lapachone on thiol pools in C. fasciculata; (e) the effect of quinones on cell respiration; (f) Superoxide dismutase inactivation after incubation of C. fasciculata with CG 8-935; and (g) the effect of quinones on cell growth.

Mutagenicity and cytotoxicity of naphthoquinones for Ames Salmonella tester strains.

The molecular mechanisms involved in quinone cytotoxicity, especially mutagenicity, are still largely unknown. In order to better understand the molecular aspects of the mechanisms of quinone mutagenicity and cytotoxicity, we examined them by using a series of 13 simple structural naphthoquinone (NQ) derivatives for 9 Ames Salmonella mutagenicity tester strains in the presence of absence of liver homogenate S9 mix from rats induced with phenobarbital and 5,6-benzoflavone. Most NQs used in this study showed mutagenicity with and/or without S9 mix. The most potent mutagenic NQ was 2,3-dichloro-1,4-NQ, with mutagenicity of 18 induced revertants/nmol/plate for strain TA104 without S9 mix. Among the strains used, TA104, which is sensitive to oxidative mutagens, was the most sensitive to the NQs, and the second most sensitive strain was TA2637, which detects bulky DNA adducts. The relationship of mutagenic potency to the one-electron reduction potential with TA104 suggested that the higher redox potential NQs were more mutagenic than the lower redox potential NQs. Significant reduction of the mutagenicity of 1,4-naphthoquinone without S9 mix was observed in the presence of catalase. Enhancement of the mutagenic potential of the NQs by the pKM101 plasmid implicated in error-prone repair was also observed. The most cytotoxic NQ was 2,3-dichloro-5,8-dihydroxy-1,4-NQ, and the least cytotoxic NQ was beta-NQ-4-sulfonic acid potassium salt, a 700-fold range in potency. The cytotoxic effect of the NQs was largely dependent on the structures of their substituents.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective inhibition of adenylate cyclase in bovine cortex by quinones: a novel cellular substrate for quinone cytotoxicity.

Quinones are widely distributed substances of often potential toxicological significance. On the other hand, cyclic AMP is known to promote a cell-survival response and to retard apoptosis [Berridge, Tan and Hilton (1993) Exp. Hematol. 21, 269-276]. Therefore the effects of quinones on adenylate cyclase were tested. Adenylate cyclase is rapidly inhibited by quinones, with IC50 values of 40-45 microM for p-benzoquinone (BQ) or 200 microM for dichlorophenol-indophenol (DCIP), with 2-substituted quinones being inactive. Membrane solubilization decreases the IC50 values for BQ and DCIP to 18 microM and 40 microM respectively. The inhibition is not affected by GTP, GDP or analogues, or by cholera and pertussis toxins; therefore it is not mediated by a G-protein or the activation of a defined receptor. Further, the inhibition stoichiometrically competes with forskolin activation of adenylate cyclase, equimolar concentrations of quinone and forskolin restoring the enzyme activity to its basal value. Reduction of BQ with sodium dithionite stoichiometrically prevents the inhibition of adenylate cyclase; in turn, oxidation of hydroquinone with ferricyanide fully restores it, indicating that the oxidized state of the quinone is required for inhibition. In addition, BQ is cytotoxic in vivo on HepG2, a human hepatocellular carcinoma cell line, but the effect can be prevented with forskolin. In plasma membranes, BQ tightly binds only one major and two minor proteins; these BQ-binding proteins were purified by means of labelling with [14C]BQ followed by PAGE under native conditions. Together these observations indicate that the action of quinone can be traced to targeting a limited number of proteins at the plasma membrane in a highly selective way and to affecting key enzymes such as adenylate cyclase.

In vitro cytotoxicities of 1,4-naphthoquinone and hydroxylated 1,4-naphthoquinones to replicating cells.

Using the human hepatoma cell line, HepG2, and the BALB/c mouse fibroblast cell line, 3T3, as the bioindicators in the neutral red cytotoxicity assay, the effect of hydroxyl substitution on the toxicity of 1,4-naphthoquinone was studied. The sequence of potency for the quinones was 5,8-dihydroxy-1,4-naphthoquinone > 5-hydroxy-1,4-naphthoquinone > 1,4-naphthoquinone >> 2-hydroxyl-1,4- naphthoquinone. Pretreatment of the cells with dicoumarol, an inhibitor of DT-diaphorase, enhanced the cytotoxicity of 1,4-naphthoquinone but not of the hydroxylated naphthoquinones. Pretreatment of the BALB/c cells with buthionine sulfoximine, an inhibitor of glutathione synthesis, enhanced the sensitivity of the cells to all the hydroxylated naphthoquinones but not to 1,4-naphthoquinone. A similar pretreatment of the HepG2 cells with buthionine sulfoximine enhanced the toxicity of the 2-hydroxy- and 5,8-dihydroxy-1,4-naphthoquinones but not of 5-hydroxy-1,4-naphthoquinone or of 1,4-naphthoquinone. Some differences were noted in the responses to the hydroxylated 1,4-naphthoquinones between buthionine sulfoximine-treated replicating cells and buthionine sulfoximine-treated isolated rat hepatocytes, a nonreplicating cell in culture. The use of a replicating cell system in studying the mechanisms of the cytotoxicity of quinones may be an important adjunct to studies using the isolated rat hepatocytes, which is the standard model system.