Cytotoxicity of catechol towards human glioblastoma cells via superoxide and reactive quinones generation

Neurochemestry and Cellular Laboratory, Health Sciences Institute, Universidade Federal da Bahia. This work was supported by grants from CNPq/CTPETRO and UFBA/CADCT/PRODOC. abstract It is known that the exposure to benzene in the petroleum industry causes lympho-haematopoietic cancer among workers. However, there is little data concerning the toxicity of benzene to the central nervous system. Benzene easily penetrates the brain where it is metabolized to catechol. Since catechol autoxidizes in physiological phosphate buffer, we hypothesized that it could be toxic towards glial cells due to the generation of reactive oxygen species and quinones. In this work we studied the cytotoxic properties of catechol towards human glioblastoma cells. We found that catechol was toxic towards these cells after 72 hours and this toxicity was related to the formation of quinones. Catechol at 230μM killed 50% of cells. The catechol-induced cytotoxicity was prevented by the addition of 100U superoxide dismutase, which also inhibited the formation of quinones. These data suggest that catechol induces cytotoxicity via the extracellular generation of superoxide and quinones. Catechol


resumo
Sabe-se que a exposição de trabalhadores ao benzeno na indústria petrolífera é uma causa de câncer do sistema linfo-hematopoiético. Pouco se sabe, contudo, a respeito da toxicidade do benzeno no sistema nervoso central.O benzeno penetra facilmente no cérebro, onde é metabolizado a catecol.Como o catecol se auto-oxida em tampão fosfato no pH fisiológico, supôs-se que esse composto poderia ser tóxico para células gliais por gerar espécies reativas do oxigênio e quinonas.Nesse trabalho estudou-se a citotoxicidade do catecol para células de glioblastoma humano.O catecol foi tóxico após 72 horas e essa toxicidade relacionou-se com a formação de quinonas.O catecol a 230µM matou metade das células em cultura.A toxicidade do catecol e a produção de quinonas foram inibidas por 100U de superóxido dismutase.Esses dados sugerem que a toxicidade induzida pelo catecol deve-se à produção extracelular de superóxido e quinonas reativas.L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine and also other drugs bearing a catechol group like apomorphine is well-documented in in vitro cell studies (7,17,18) .However, there are few data concerning the toxicity of catechol toward glial cells.Although the human glioma cell line GL15 is poorly differentiated, these cells preserve many characteristics of astrocytes (20) and proliferate rapidly in culture.Glioma cells have been largely used for the study of astrocyte properties (4) .In this study we evaluate the toxicity of catechol toward GL-15 cells and its relation with the production of superoxide by the measurement of mitochondrial dehydrogenases activity and quinones formation in cell cultures with and without superoxide dismutase (SOD) and catalase.

Measurement of catechol autoxidation in a cell-free system
Neutral aqueous solutions of catechol undergo spontaneous oxidative decomposition.Autoxidation is a multi-step reaction resulting in ROS and quinone derivatives formation.In the present study, the autoxidation rates were measured at 320nm in 1mM HCl, 50mM phosphate buffer (pH 7.4).The catechol oxidation (final concentration of 1mM) was carried out with or without 17U SOD or 100IU catalase in a volume of 3ml.Catechol oxidation was also measured by means of a Clark-type oxygen electrode fitted to an oxygen monitoring system (YSI model 53 oxygen monitor).

Introduction
Occupational hygienists have investigated the exposure to benzene (13) in the petroleum industry because it causes lympho-haematopoietic cancer among workers.The wastewater generated by the petroleum industry contains an appreciable amount of benzene, toluene, ethylbenzene and xylene (BTEX) that requires specific treatment to avoid environmental contamination (15) or to remove these compounds from the ground or surface water resources (6) .Certain tasks, such as the loading and unloading of petroleum products in the marine, railcars, trucks and so forth, produce a high risk of exposure to benzene (26) .Moreover, benzene is also present in tobacco smoke.The metabolism of benzene by human liver microsomes forms phenol, hydroquinone and catechol (1,2-dihydroxybenzene) (23) .Benzene, at concentrations up to 10mM, was neither genotoxic nor cytotoxic toward peripheral blood mononuclear cells in vitro.However, its metabolites benzenetriol and catechol are genotoxic at micromolar concentrations (9) .
Catechol easily autoxidizes to form potentially toxic quinones and reactive oxygen species (ROS) that induce stress proteins in Acinetobacter calcoaceticus (2) .Trace elements in tobacco smoke may act as catalysts for the formation of ROS and various free radicals by redox cycling of catechol, thus greatly enhancing the potential for tissue damage (24) .Catechol at micromolar concentrations induces a time-dependent release of iron from ferritin in vitro and causes lipid peroxidation in rat brain homogenates (1) .Furthermore, catechol and cigarette smoke condensates are reported to induce oxidative regulation of protein kinase C and influence the invasive capacity and metastatic spread of lung carcinoma cells (14) .Tobacco smoking is also a co-factor lowering the blood serum immunoglobulin concentration in subjects with occupational exposure to organic solvents containing BTEX (21) .Generally speaking, chronic smoking and exposure to BTEX are relevant problems to human health.In addition, the use of tobacco regularly associated with inhalation of solvents among street children has been reported as a social problem in Brazil (10) .
There is an increasing interest to study the toxicity of catechols toward cells from the central nervous system (CNS) since it has been speculated that the toxicity of these compounds is associated with neurodegenerative diseases.Among these cells astrocytes are the most numerous cellular elements in the CNS, where they play a critical role in physiological and pathological events.The dose-dependent toxicity of endogenous catechols like 2001 (22) .Experiments were initiated after cells had become confl uent.Cell cultures were divided into seven groups.The control group was treated with 2 × 10 -5 M HCl, which was used as the solvent of catechol.The other six groups were treated with concentrations of catechol ranging between 100 and 1000µM for 72 hours.In each group, cells were seeded into six Petri dishes (35mm in diameter) to obtain a fi nal density of 3 × 10 5 cells/dish.In order to study the role of the production of superoxide on the toxicity of catechol, cells were treated with this compound in the presence of 100U SOD for 72 hours.Catalase at 500IU was used to assess the role of hydrogen peroxide in the catechol-induced toxicity.The catechol oxidation in the medium was monitored spectrophotometrically at 405nm, based on quinones and melanin-like pigment formation (18) .The cell viability was quantifi ed by the conversion of yellow MTT to purple MTT formazan by living cell mitochondrial dehydrogenases (16) .

Calculation
The rates of oxygen consumption and formation of quinones were obtained from a linear regression plot.Linear regression was carried out by using the least square method.This calculation was also used to determine the concentration of catechol that killed 50% of cells (IC 50 ), since the plot was linear for catechol concentrations ranging between 100 and 500µM (y = -0.6085x+ 63.99; r 2 = 0.9927).Data obtained for the catechol oxidation in a cell-free system were compared by the Student's t test.The signifi cance of results obtained from toxicity studies was analyzed by ANOVA, with the Student-Newman-Keuls post hoc test.

Catechol autoxidation in a cell-free system
The oxygen consumption during autoxidation of 1mM catechol was estimated quantitatively.Catechol, at 1mM, oxidizes consuming oxygen in a rate of 1.2 ± 0.3µM.min - .From Figure 1 it can be seen that the addition of 17U SOD significantly inhibited oxygen consumption by 83.7%.The addition of 100IU catalase did not inhibit catechol autoxidation.The rate of quinones formation was also decreased by 37% in the presence of 17U SOD (Figure 2) but no signifi cant difference was found when 100U catalase was added to the system.Altogether, these data suggest that superoxide is produced during the oxidation of catechol.On the other hand, the production of hydrogen peroxide was not evidenced by the use of catalase.

Catechol toxicity towards GL-15 cells
In order to determine the nature of cytotoxicity induced by catechol, GL-15 cells were treated for 72 hours with several concentrations of this compound.As shown in Figure 3, catechol treatment resulted in a marked loss of cell viability in a dose-dependent manner.The IC 50 value for catechol-induced cytotoxicity towards GL-15 cells after 72 hours was 230µM.Autoxidation of catechol also formed quinones in the culture medium in a dosedependent manner (Figure 3).The cytotoxic effects induced by catechol appeared to correlate with the formation of quinones in cell cultures.
The addition of 100U SOD to cells treated with 300µM catechol for 72 hours inhibited the formation of quinones by 63.5% (Figure 4).However, the addition of the same amount of heat-inactivated SOD (iSOD) or 500IU catalase did not inhibit catechol oxidation.Moreover, the addition of 100U SOD combined with 500IU catalase did not increase the inhibition induced by SOD alone.The presence of SOD at 100U also signifi cantly prevented catechol-induced cytotoxicity towards GL-15 cells (Figure 5).Cell survival increased from 49.5% ± 4.2% in cells treated with 200µM catechol (n = 5) to 71.9% ± 3.9% in the group incubated in the presence of 100U SOD (n = 5).Cell survival also increased from 47.4% ± 6.5% in the group treated with 300µM catechol (n = 5) to 61.6% ± 6.9% in that incubated in the presence of SOD (n = 4).The addition of iSOD or 500IU catalase did not protect the cells.Once again, SOD combined with catalase did not increase the protective effect of SOD.

Discussion
The present results indicate that the reaction between catechol and oxygen is inhibited by SOD in a cell-free system.One of two phenolic hydroxyl groups of the catechol molecule is ionized in alkaline solutions and, therefore, electron transfer from this ionized group to a dissolved oxygen occurs easily (27) .Consequently, this results in superoxide formation during the catechol autoxidation.On the other hand, the formed superoxide reacts with catechol accelerating the formation of quinones, since this reaction was also inhibited by SOD.Since catalase did not inhibit the oxygen consumption or the quinone formation, this means that superoxide and not peroxide was the main ROS formed in the autoxidation.The oxidative property of catechol seems important because endogenous catechols like catecholamines and catechol estrogens (5) are produced in mammal brains and other tissues.Thus, catechol oxidation can be used for structure-activity relationship studies.
Reactive species produced during the autoxidation of catechols may also be toxic to cells.In this study catechol was toxic towards human GL-15 cells after 72 hours and this was related to the formation of quinones.The IC 50 value for catechol-induced cytotoxicity was 230µM.Some drugs that contain catechol moieties, like apomorphine, also have the same oxidative properties (8) .We have found that apomorphine-induced toxicity towards glial C6 cells depends on time and dose with an IC 50 value of 200µM after 48 hours (7) .Catecholamines are also toxic at high concentrations (> 50-100µM) (25) .The IC 50 values for L-DOPA-and dopamine-induced cytotoxicities towards SH-SY5Y neuroblastoma cells were 194 and 184µM respectively, after 24-hour drug exposure (17) .Thus, it seems that molecules bearing a catechol group are toxic to cells from the CNS in vitro.However, catechols at low concentrations (1µM), like norepinephrine and catechol, provide protection to dopaminergic neurons in mesencephalic cell cultures (25) .
In this study it has been shown that SOD protected cells against catechol-induced cytotoxicity and also inhibited quinones formation.Superoxide is the most common intracellular free radical leading to the formation of other reactive' cell-damaging species, when present in excessive amounts.It is well accepted that SOD represents the first line of defense against oxygen toxicity.SOD also protected SH-SY5Y neuroblastoma cells against the neurotoxic properties of L-DOPA and dopamine-related compounds (17) .In the present study, catalase did not prevent catecholinduced cytotoxicity towards GL-15 cells.Similarly, catalase had no effect on semiquinone radicals formation in dopamine oxidation, although this enzyme showed a small preventive effect against dopamine-induced toxicity towards SH-SY5Y cells (17) .It has been determined both by animal and clinical studies that SOD protects brain against injury by ischemic stroke and trauma (12) .
To increase the efficacy of L-DOPA in the treatment of Parkinson's disease, this agent is sometimes associated with inhibitors of catechol-O-methyltransferases (COMT, EC 2.1.1.6)and amino acid decarboxylase (AADC, EC 4.1.1.28).These inhibitors also present catechol groups in their structures.This association between L-DOPA with COMT or AADC inhibitors increased protein carbonyls, lipid peroxidation and oxidative DNA damage levels in the cerebral cortex of normal cynomologus monkeys (19) .Consistent with these results, microdialysis experiments carried out in rats showed that the association between L-DOPA, carbidopa (an AADC inhibitor) and tolcapone (a central and peripheral COMT inhibitor) increased striatal hydroxyl radical production.Thus, it is thought that cell death induced by catechols involves oxidative damage.
In conclusion, these results revealed a possible mechanism of catechol-induced cytotoxicity in glioblastoma GL-15 cells.Catechol, which is a metabolite of benzene, probably exerts cytotoxicity mainly due to the generation of superoxide and quinones during its autoxidation.

Figure 3 -Figure 4 -Figure 5 -
Figure 3 -Concentration-response curves for catechol-induced cytotoxicity and quinones formation in GL-15 cell cultures after 72h.Cell viability () was determined by the MTT test and expressed as a percentage of control treated with 2 × 10 -5 M HCl.Each point represents the percentage of cell viability after treatment with catechol in several concentrations.The formation of quinones during the autoxidation of catechol was also measured by spectrophotometry at 405nm ().Data are represented by the mean ± SD, n = 5