Mutagenic and Genotoxic Potentialof Direct
Electric Current in Escherichia coliand
Salmonella thyphimurium Strains
Marina das Neves Gomes,1Janine Simas Cardoso,2Alvaro Costa Leit~ao,2 and Carla Holandino Quaresma3*
1N!ucleo de Ci^encias Biom!edicas Aplicadas, Instituto Federal de EducaSc~ao, Ci^encia e
Tecnologia do Rio de Janeiro, Brazil
2Laborat!orio de Radiobiologia Molecular, Programa de Biologia Molecular, Instituto de
Biof!lsica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil
3Laborat!orio Multidisciplinar de Ci^encias Farmac^euticas, Departamento de
Medicamentos, Faculdade de Farm!acia, Centro de Ci^encias da Sa!ude, Universidade Federal do Rio de Janeiro, Brazil
Direct electric current has several therapeutic uses such as antibacterial and antiprotozoal action, tissues scarring and regeneration, as well as tumor treatment. This method has shown promising results in vivo and in vitro, with significant efficacy and almost no side effects. Considering lack of studies regarding direct electric current mutagenic and/or genotoxic effects, the present work evaluated both aspects by using five different bacterial experimental assays: survival of repair-deficient mutants, Salmonella-histidine reversion mutagenesis (Ames test), forward mutations to rifampicin resistance, phage reactivation, and lysogenic induction. In these experimental conditions, cells were submitted to an approach that allows evaluation of anodic, cathodic, and electro-ionic effects generated by 2 mA of direct electric current, with doses ranging from 0.36 to 3.60 Coulombs. Our results showed these doses did not induce mutagenic or genotoxic effects. Bioelectromagnetics. 37:234–243, 2016. © 2016 Wiley Periodicals, Inc.
Key words: electrochemical treatment; survival of repair-deficient mutants; phage reactivation; lysogenic induction; Ames test
INTRODUCTION
Direct current (DC) has been largely used in several biological systems with different purposes. The literature has registered destructive and regenera-tive DC aspects, and some of them are dependent on specific applied and intrinsic biological character-istics. Among destructive ones, its antibacterial [Tiehm et al., 2009; Mahapatra et al., 2011; Wei et al., 2011] and antiprotozoal actions [Hejazi et al., 2004; Gomes et al., 2012] are well known. Considering DC regenerative properties, skin lesion healing [Huckfeldt et al., 2007; Talebi et al., 2007; Jennings et al., 2008; Zhao et al., 2012; Zuzzia et al., 2013; Beheregaray et al., 2014], bone fracture recovery [Wang et al., 1998; Ciombor and Aaron, 2005], as well as nerve repair and regeneration [MendonSca et al., 2003; Ackermann et al., 2011] were described.
Another important biological DC property is its anticancer potential, commonly known as electro-chemical treatment (EChT) [Nordenstr€om, 1989, 1994; Liu et al., 1994; Nilsson et al., 2000; Ciria et al., 2004, 2013]. Advantages of EChT include its
local action (i.e., damage can be focused on the tumor), with minor effects to surrounding tissues, slight and self-limited adverse effects, as well as low cost [Ciria et al., 2013; Cury et al., 2015]. Besides, EChT uses inert electrodes, which can be distributed in different positions and distances, as well as linked
Grant sponsors: FundaSc~ao de Amparo #a Pesquisa no Estado do Rio de Janeiro (FAPERJ). Conselho Nacional de Desenvolvi-mento Cient!ıfico e Tecnol!ogico (CNPq). FundaSc~ao Jos!e Bonif!acio (FUJB).
Conflicts of interest: None.
*Correspondence to: Carla Holandino, Faculdade de Farm!acia, Departamento de Medicamentos, Universidade Federal do Rio de Janeiro, Rua Professor Rodolfo Rocco s/n, CCS, Bloco B sub-solo, salas 11, 34. Cidade Universit!aria, 21941-902 Rio de Janeiro, Brazil. E-mail: cholandino@gmail.com; cholandino@pharma.ufrj.br Received for review 22 March 2015; Accepted 4 March 2016 DOI: 10.1002/bem.21970
Published online 28 March 2016 in Wiley Online Library (wileyonlinelibrary.com).
to specific polarities [Miklav$ci$c et al., 1993, 1994; Li et al., 1997; Jim!enez et al., 2011]. Most DC effects on EChT have been attributed to electrolytic products resulting from anodic or cathodic reactions [Veiga et al., 2000; Cury et al., 2015]. Anodic reactions, generated by anodic flow induced dryness and local acidity. In contrast, pH alkalization can be detected in cathodic microenvironment from cathodic flow, leading to swollen tissue, lysis, and several cellular alterations [Veiga et al., 2000, 2005; Holandino et al., 2001].
Veiga et al. [2000] developed an experimental system in which cellular suspensions were separately exposed to three different flows: anodic flow (AF); cathodic flow (CF); and electro-ionic flow (EIF), which is the part of the system that received the electro-ionic flow through filter paper bridges. This experimental situation is generated by EIF, suggesting that generation of electrolytic products might be essential for occurrence of the most important cellular modifications [Veiga et al., 2000; Holandino et al., 2001; De Campos et al., 2010].
Many works have shown positive EChT effects in several tumor models, such as in cellular cultures [Holandino et al., 2000, 2001; Veiga et al., 2000, 2005; De Campos et al., 2010], animals [David et al., 1985; Morris et al., 1992; Miklav$ci$c et al., 1993, 1994; Griffin et al., 1994; Chou et al., 1997; Li et al., 1997; Robertson et al., 1998; Cabrales et al., 2000; Nilsson et al., 2000; Ciria et al., 2004; Schaefer et al., 2008; Cury et al., 2015] and humans [Wojcicki et al., 1999; Fosh et al., 2002, 2003; Li et al., 2006; Jarque et al., 2007; Vogl et al., 2007; Oji and Ani, 2010]. EChT has proved to be a safe, effective, easy-to-use and low-cost therapy, when compared to conventional therapeutics and can even be used in non-responsive patients [David et al., 1985; Marino et al., 1986; Miklav$ci$c et al., 1993; Nordenstr€om, 1994; Robertson et al., 1998; Wemyss-Holden et al., 2000; Tell!o et al., 2004; Jarque et al., 2007; Vogl et al., 2007].
However, mutagenic and genotoxic potential involved in DC use has yet to be elucidated. So far, there is no record of possible genetic alterations on in vivo and in vitro assays—an important factor for interpretation of carcinogenicity studies. In the present work, mutagenic and genotoxic potential of 2 mA of DC treatment was evaluated using an in vitro experimental model, developed by Veiga et al. [2005], in which AF, CF, and EIF effects were extensively studied in different cellular models. Those very promising studies can be complemented by these new results, which will be an important step for regulatory agencies to recognize EChT as an alterna-tive and complementary antineoplastic therapy.
MATERIALS AND METHODS
Bacterial Strains and Bacteriophages
Microorganism strains used in this work were Escherichia coli, obtained from Paul Howard-Flanders—(Yale University, New Haven, CT), and Salmonella typhimurium, acquired from Bruce N. Ames—(University of California, Berkeley, CA). Escherichia coli strains were WP2s(l), RJF013 (lysogenic induction test), AB1157 (wild type), AB1886 (uvrA6), and AB2463 (recA13). Salmonella typhimurium strains were TA97, TA98, TA100, and TA102 [Maron and Ames, 1983]. Bacteriophage l was used in reactivation studies.
Bacteria and Bacteriophage Media
E. coli strains were grown overnight in fresh liquid Lysogeny-Broth (LB) medium (Becton, Dick-inson and Company, Franklin Lakes, NJ) at 37 8C in a shaking incubator (Innova/USA, New Brunswick Scientific, Edison, NJ). An aliquot of 250 ml was then inoculated in fresh liquid LB medium (1:40 v/v) until bacteria reached exponential growth phase. S. typhi-murium strains were grown overnight in Oxoid nutrient broth No. 2 (OXOID, Basingstoke, UK) at 37 8C in the same shaking incubator to a density of 1 ! 109–2 ! 109 colony forming units (CFU)/ml [Maron and Ames, 1983]. Bacteriophages l were produced in E. coli AB1157, quantified, and stored in a 10"2M solution of magnesium sulfate (MgSO
4 -7 H2O; Quimibr!as Industrias Quimicas SA, Rio de Janeiro, Brazil).
DC Stimulation
DC experimental system (Fig. 1) consisted of a 24-well culture plate equipped with symmetri-cally arranged platinum-rhodium (90–10%) electro-des, which were connected to a DC source (FA-305, Instrutherm, S~ao Paulo, Brazil) and a professional digital multimeter—this last one, to monitor DC intensity (Icel Manaus-6500; Manaus, Brazil). Cellular suspensions (2 ml) were diluted in
phosphate buffered saline (PBS–pH 7.4,
308 mOsm). Some PBS features are important for preventing cellular damage, such as tonicity, pH range similar to physiological environment, and presence of chloride ions, which is indispensable for DC electrical conductivity and generation of oxidant species [Veiga et al., 2005]. Cellular suspensions were distributed in each specific well, identified by AF, EIF, and CF. These three wells were connected in series by filter paper bridges (5.3 ! 0.8 cm) and fitted with electrodes in their extremities, which were immersed in cell
suspension [De Campos et al., 2010; Gomes et al., 2012]. Each small-plate-like electrode measuring 1.0 (width) ! 1.5 (height) and 0.1 (depth) was immersed in cellular suspensions, located 7.3 cm from each other (Fig. 1). Using this approach, it is possible to treat cells with three different flows (AF, CF, and EIF), and consequently evaluate their effects. In EIF, no pH alterations occur; however, electrons can flow through filter paper bridges, as described previously [Veiga et al., 2000, 2005]. Control cells were under same experimental con-ditions, except for DC treatment. Cell suspensions were treated with different DC charges, using a range from 0.36 to 3.60 Coulombs (C), considering E.coli and S. typhimurium sensibilities.
Total electric charge applied was generated by DC source, which corresponded to dose of direct electric current used (“C”). This dose was calculated for each experimental situation, according to the formula I ¼ C/t, where “I” is amount of direct electric current applied in amperes (in our experiments, fixed in 2 mA), “C” is charge in Coulombs; and “t” is time of DC-exposure, in seconds. DC measurements were monitored by a multimeter connected to DC source by cables (Fig. 1).
All treatments were performed at room tempera-ture and the highest value achieved was 25.4 8C, after 30 min of stimulation. Alterations in pH were
monitored after each period of treatment, and cellular suspension variation was similar to other systems [Veiga et al., 2000, 2005; Holandino et al., 2001; Gomes et al., 2012]. This standardized experimental model guaranteed reproducibility of results.
Viability Assays
To assess bacterial viability, each strain was stimulated with DC, considering its intrinsic cellular sensibility, and electrochemical doses currently used in therapeutic procedures [Holandino et al., 1998; Cabrales et al., 2000]. For this, E. coli AB1157, AB1886, AB2463, and WP2s(l) were treated with 0, 0.36, 0.72, 1.08, and 1.44 C; and S. typhimurium TA97, TA98, TA100, and TA102 were stimulated by 0, 1.44, 2.16, 2.88, and 3.60 C. After DC treatment, cells were plated and N/N0 relation calculated as survival fractions, in which N is number of colony-forming units (CFUs) after DC treatment and N0 is CFU number of non-treated samples.
Genotoxicity Studies
Lysogenic induction assays. WP2s(l) and RJF013 strains of E. coli B/r were used in lysogenic induction assays using a similar protocol to the Inductest (lysogenic induction assay) [Moreau et al., 1976]. E. coli strain WP2s(l) contains l prophage and a mutation in uvrA gene (excision repair). This mutation was used because it confers high sensitivity to agents capable of causing DNA lesions [Blanco et al., 1998]. The quantification of infectious centers was measured in the indicator bacterium (strain RJF013) after DC stimulation. The results were presented as mean values obtained from three independent duplicate experiments. A culture of WP2s(l) strain was treated with 0.36, 0.72, and 1.08 C. After DC application, RJF013 suspension was diluted and plated on solid LB medium (Becton, Dickinson), to verify bacterial cytotoxicity. It was mixed with 0.3 ml of the indicator bacteria (RJF013) in the stationary growth phase and plated in solid LB medium containing ampicillin 20 mg/ml (Sigma–Aldrich) to detect lysogenic induc-tion. After overnight incubation at 37 8C, number of plaques induced in E. coli WP2s(l) was calculated and expressed as the number of infective centers. Positive controls consisted of cultures exposed to a single dose of UV-C at 254 nm (2 J/m2).
Bacteriophage reactivation assays. Bacteriophage l (107phages/ml) solution was stimulated with 0.36, 0.72, and 1.08 C. Afterwards, it was diluted in 0.1 M Mg2SO4 (Quimibr!as Industrias Quimicas, Rio de Janeiro, Brazil), and added to each indicator strain
Fig. 1. Schematic representation of 24-well plate used for DC treatment of bacterial strains. Cell suspensions are distributed over each individual well and connected to a positive pole (AF) and negative pole (CF). Anintermediary well, namelyelectroionic flow (EIF), is connected to other wells by filter paper bridges moistened with phosphate saline buffer. Platinum electrodes are inserted into AF and CF wells, allowing the system to be connected to a DC source and an amperimeter to monitor DC intensity. This distribution permits separate exposure of cells to cathodic and anodic reactions, and to FEI. Control cells are exposed to same conditions, except DCuse.
(E. coli AB1157, AB1886, and AB2463), for 20 min at 37 8C, which were bacteriophage adsorption con-ditions. Next, this solution was plated on solid LB medium containing agar (Becton, Dickinson and Company, Franklin Lakes, NJ). Finally, as positive control, bacteriophage reactivation was induced by UV-C irradiation, at 254 nm, using 50 J/m2 (AB1157 and AB2463) and 10 J/m2(AB1886) doses.
Mutagenicity Studies
Forward mutation to rifampicin resistance RifS
!RifR. Both spontaneous and induced mutagen-esis experiments were evaluated using a rifampicin (Rif) resistance system. E. coli AB1157 suspension was stimulated with 0.36, 0.72, and 1.08 C to evaluate induced mutagenesis. Number of Rif-resistant mutants (RifS!RifR) was quantified and compared to those resulting from spontaneous mutagenesis. After DC-stimulation, aliquots were removed, incubated in an inclined tube containing LB liquid medium (Becton, Dickinson and Company, Franklin Lakes, NJ), and
grown overnight at 37 8C, under shaking. Cells in exponential growth phase were diluted and plated on solid LB medium, whereas undiluted aliquots were plated on solid LB containing 100 mg/ml Rif (Sigma-Aldrich, St. Louis, Missouri). After incubation at 30 8C for 24–48 h, colonies were scored and muta-tion frequency was determined [Maron and Ames, 1983]. Mutagenesis was expressed as frequency of RifR mutants per 108 cells. As a mutagenesis positive control, AB1157 strain was irradiated with UV-C at 254 nm (40 J/m2).
Salmonella-histidine reversion mutagenesis assays (Ames test). The Ames test [Maron and Ames, 1983] was performed using the histidine S. typhimurium auxotroph mutant strains TA97, TA98, TA100, and TA102. Cell suspensions were treated with 1.44 C, in order to induce 10% of viable cells (DL10). After treatment, they were incubated in reaction medium for 10, 20, and 30 min to verify DC mutagenic effects. These results were presented as mean values obtained from three independent duplicate experiments (n ¼ 3).
Fig. 2. Survival fraction (N/N0) of E. coli strains, in which N is number of colony-forming units (CFUs) after DC treatment and N0is CFUnumberofnon-treated samples. E. colistrains AB1157 (A), AB2463 (B), AB1886 (C), and WP2sl (D) were treated by Anodic Flow, AF (O); Electro-Ionic Flow, EIF (&); and Cathodic Flow, CF (D). Results are expressed as means $ SD of three independent duplicate experiments (n ¼ 3). Statistical analyses were performed by standard analyses of vari-ance (ANOVA).%Thereisa statisticalsignificant difference (P < 0.05).
Mutagenesis rate was calculated using the number of histidine revertants, and expressed as the fold increase number over spontaneous mutants. For a mutagenesis positive control, all strains were irradiated with UV-C (254 nm) at the specific following doses: 2 J/m2 (TA97, TA98, and TA100) and 40 J/m2(TA 102). Statistical Analysis
Results are expressed as mean $ standard devia-tion (SD) and were calculated for each specific experiment. Significant differences in all experiments were assessed by standard analysis of variance (ANOVA), followed by Dunnett’s test using Action version 2.4 (Estatcamp, S~ao Carlos, Brazil).
RESULTS
E. coli viability assays showed that all strains analyzed (AB1157, AB2463, AB1886, and WP2sl)
presented a 90% death rate when treated by amounts of electrical charge above 1.08 C (Fig. 2A–D). In contrast, S. typhimurium strains (TA97, TA98, TA100, and TA102) were treated with 1.44 C in order to reach DL10 (Fig. 3A–D). Because of this, the subsequently mutagenic and genotoxic assays were done with these specific doses, 1.08 for E. coli and 1.44 C for S. typhimurium, respectively.
Lysogenic induction analyses (Fig. 4) revealed no statistical significant difference (P > 0.05) among DC groups (AF, EIF, CF), considering number of infective centers quantified after 0.36, 0.72, and 1.08 C charges applied. As a positive control, WP2s(l) was irradiated with an UV-C dose (2 J/m2) originating 3.6 ! 107 infective centers. These results were expressed as mean values obtained from three independent duplicate experiments (n ¼ 3), and clearly indicate that DC is unable to promote lyso-genic induction.
Fig. 3. Survival fraction (N/N0) of Salmonella thyphimurium strains, in which N is the number of colony-forming units (CFUs) after DC treatment and N0is the CFU number of non-treated samples. S. thyphimurium strains TA97 (A), TA98 (B), TA100 (C), and TA102 (D) were treated by Anodic Flow, AF (O); Electro-Ionic Flow, EIF (&); and Cathodic Flow, CF (D). Results are expressed as means þ SD of three independent duplicate experiments (n ¼ 3). Statistical analysis were performed by standard analysis of variance (ANOVA).%Statistically significant
In bacteriophage reactivation assay, a bacterio-phage l suspension containing 107phages/ml was stimulated with 0.36, 0.72, and 1.08 C, and then AB1157, AB2463, and AB1886 were added as indicator bacteria. No statistical significant difference (P > 0.05) was detected in the amount of plaque-forming units considering all E. coli strains tested. Figure 5 shows AB2463 reactivation assay as a representative result of these experiments.
The rifampicin (Rif) resistance system indicated no significant number of mutants when E. coli
AB1157 suspension was stimulated with 0.36, 0.72, and 1.08 C. This result was expressed as a fold increase over E. coli spontaneous mutation and indicated no mutation in the rpoB gene. In contrast, using an UV-C dose (40 J/m2), a 42-fold increase in number of E. coli AB1157 spontaneous revertants was observed (Fig. 6), suggesting that our experimen-tal conditions were correct. These results are pre-sented as mean values and were obtained from four independent duplicate experiments (n ¼ 4).
The Ames test performed using the histidine S. typhimurium auxotroph mutant strains TA97, TA98, TA100, and TA102 confirmed absence of DC mutagenicity potential, considering all experimental situations (AF, EIF, CF). In this assay, results were expressed as fold increase number over spontaneous S. typhimuriummutants (Table 1). Values lower than 2.0 characterized absence of mutagenic process, as we also registered in our results. In positive control, S. typhimurium strains were irradiated with 2 J/m2 of UV-C (TA97, TA98, TA100) and 40 J/m2 (TA102) and showed increases of 6.8, 28.1, 34.9, and 32.8, respectively. These results are expressed as mean values and were obtained from three independent duplicate experiments (n ¼ 3).
DISCUSSION
To evaluate mutagenic and genotoxic effects of DC, an experimental system previously validated by our group [Holandino et al., 2000; Veiga et al., 2005; De Campos et al., 2010; Gomes et al., 2012] was
Fig. 4. Number of infective centers induced in E. coli WP2s(l) after electrical stimulation. (O) Anodic Flow, AF; (&) Electro-Ionic Flow, EIF; and (D) Cathodic Flow, CF. Results are expressed as mean $ SD of three independent duplicate experiments (n ¼ 3). Statistical analyses, performed by stan-dard analyses of variance (ANOVA), showed no significant statistical differences (P > 0.05) in the number of infective cen-ters, indicating absence of DC-genotoxic potential.
Fig. 5. Bacteriophage reactivation assay. One solution contain-ing 107l phages/ml was stimulated with 0.36, 0.72, and 1.08 C, diluted in Mg2SO4and added to E. coli 2463. (O) An-odic Flow, AF; (&) Electro-Ionic Flow, EIF; and (D) CathAn-odic Flow, CF. Results are expressed as mean $ SD of five indepen-dent duplicate experiments (n ¼ 5). Statistical analyses, per-formed by standard analyses of variance (ANOVA), showed no significant statistical differences (P > 0.05) among treat-ments.
Fig. 6. A fold increase over E. coli (AB 1157) spontaneous mutation in Rifampicin Resistance System after treatments with 0.36, 0.72, and 1.08 C of (&) Anodic, (&) Electro-Ionic Flow, EIF; and ( ) Cathodic Flow, CF. Results are expressed as mean $ SD of four independent duplicate experiments (n ¼ 4). Statistical analyses, performed by standard analyses of variance (ANOVA), showed no significant statistical differen-ces (P > 0.05) among treatments. As a mutagenesis positive control, AB1157 strain was irradiated with UV-C (40 J/m2) gen-erating a 42-fold increase number of spontaneous revertants.
used. This system allowed us to analyze, in an independent way, effects of AF, CF, and EIF. In our former works, we used the term “flow” to express movement of electrons and ions generated by DC. Acid and alkaline pH values were detected in AF and CF, respectively [Veiga et al., 2000]. It is well known that electrolysis products are formed in the vicinity of electrodes during electric treatment, and that the nature of these chemical species depends on polarity on which electrodes were connected [Miklav$ci$c et al., 1993; Veiga et al., 2000; Gravante et al., 2011; Cury et al., 2015].
Additionally, other important antitumor mecha-nisms are triggered by electrochemical therapy, such as reactive oxygen species (ROS) generation [Miklav$ci$c et al., 1993], which can be involved with necrosis and histopathological findings detected in DC-treated cells [Veiga et al., 2000; Cabrales et al., 2001; Holandino et al., 2001]. Cabrales et al. [2001] showed significant alterations in mouse organs (liver and spleen), in which necrosis and other histopathological changes were detected, with con-comitant tumor volume reduction, after 5 C of DC. Besides, another interesting aspect in these experi-ments was spleen weight increase related to immune system activation [Cabrales et al., 2001].
Different groups have registered some mecha-nisms by which electrochemical therapy works, such as electrolysis products, immune system activation, necrotic and apoptosis events [Liu et al., 1994; Cabrales et al., 2001; Ciria et al., 2004; Veiga et al., 2005; Gomes et al., 2012; Cury et al., 2015]. However, DC can generate ROS, which constitutes a threat for cellular homeostasis, because their pro-inflammatory character, which can cause adverse effects, such as local pain and tissue swelling [Jarque et al., 2007]. Cabrales et al. [2001] suggested that ROS generation should occur after higher DC-doses, indicating that reactive oxygen species may has an essential role in the electrochemical treatment.
In fact, ROS production is directly related to the amount of electric charge applied, and considering intrinsic cellular sensitivity, it is important to evaluate ROS generation in each specific system.
In order to verify the role of ROS in cell death induction, A549 human lung cells were treated with AF (from 0.24 to 3.60 C/cm3), in the presence of a ROS scavenger—reduced glutathione (GSH). These results indicated no significant changes when a 1.20 C dose was used, even at the highest GSH concentration. However, using higher AF doses, cellular death was prevented by increasing concentrations of GSH. These findings suggested that ROS generation should occur after higher DC-doses; confirming that ROS may
TABLE 1. Fold Increase Number Over Spontaneous Mutants of TA97, TA98, TA100, and TA102 Salmonella typhimurium Strains After Direct Electric Current, at Time Zero (Immediately After Electrical Stimulation) and at 10, 20, and 30 min After Respective DC Treatment TA97 TA98 TA100 TA102 Amount of electrical charge (C)
Time after DC (min)
AF EIF CF AF EIF CF AF EIF CF AF EIF CF 1.44 0 1.6 $ 0.4 1.3 $ 0.5 1.3 $ 0.6 1.7 $ 0.2 1.5 $ 0.5 1.4 $ 0.2 1.7 $ 0.4 1.4 $ 0.5 1.5 $ 0.3 1.0 $ 0.5 1.0 $ 0.6 0.8 $ 0.7 1.44 10 0.9 $ 0.3 1.0 $ 0.2 1.2 $ 0.3 1.2 $ 0.4 0.6 $ 0.2 1.3 $ 0.6 1.6 $ 0.6 1.2 $ 0.3 1.3 $ 0.4 1.3 $ 0.2 0.9 $ 0.2 1.0 $ 0.5 1.44 20 1.2 $ 0.2 1.2 $ 0.6 0.8 $ 0.1 1.3 $ 0.5 0.9 $ 0.3 1.0 $ 0.3 1.3 $ 0.2 1.0 $ 0.2 1.3 $ 0.7 1.0 $ 0.3 0.7 $ 0.2 1.2 $ 0.7 1.44 30 1.5 $ 0.5 0.9 $ 0.2 1.2 $ 0.5 1.0 $ 0.5 0.7 $ 0.3 1.2 $ 0.6 1.6 $ 0.5 1.2 $ 0.2 1.5 $ 0.8 0.9 $ 0.4 0.8 $ 0.1 0.9 $ 0.4 There was no significant statistical difference (P > 0.05) among S. thyphimurium strains. The results are expressed as the mean $ SD of three independent duplicate experiments (n ¼ 3).
have an essential role in electrochemical treatment, in agreement with Cabrales et al. [2001]. Additionally, Wartenberg et al. [2008] also detected a strict correla-tion between apoptosis and ROS when oral mucosa cancer cells were submitted to electrical field treat-ment (4 V/m, 24 h). Therefore, mutagenic and/or genotoxic effects need to be investigated using differ-ent DC charges.
In AF microenvironment, oxidative reactions lead to pH acidification, as well as ROS and oxidized chlorine species production. In addition, hypochlorous acid and chlorine gas, both involved with these electrolytic reactions, can also yield chloramines species, which are known as apoptosis inductors [Wagner et al., 2002; Veiga et al., 2005]. On the other hand, cathodic flow produces hydroxyls species with alkalization of medium accompanied by intense necrotic events [Veiga et al., 2000; Cury et al., 2015].
In our experimental approach, these pH changes are easily evaluated and were involved with DC antitumoral properties [Holandino et al., 2001; De Campos et al., 2010]. However, additional mechanisms of cell death may be triggered by electro-ionic flow (EIF), a third experimental condition that can be evaluated by our system (Fig. 1). In fact, vincristine-resistant derivative K562-Lucena 1 cells presented identical aspects to untreated cells, after EIF treatment, except for the impaired growth profile [Holandino et al., 2001]. These experimental results indicated that cellular alterations occurred after EIF, even in the absence of electrolytic products. Furthermore, these findings lead to the hypothesis that DC triggers cellular proliferation inhibition and/or additional tumoral death mechanisms, which can explain some successful clini-cal DC reports [Xin, 1994; Xin et al., 1997].
The literature cited different cellular models to evaluate mutagenic aspects, including E. coli strains and their mutants. The E. coli AB1886 and AB2463 also differ from the wild type, AB157. The first one presents an uvrA6 mutation, which inactivates the UvrA protein—component of UvrABC nuclease exci-sion. This enzyme is important for pyrimidine dimers repair and for other adducts in DNA. The strain AB2463, on the other hand, has the recA13 mutation, which causes lack of active RecA protein and problems in recombination, post replication repair, and SOS repair induction [MacEntee, 1977; Sancar and Rupp, 1983]. We thus verified that applied DC produced neither excision-repairable nor genetic recombination repairable DNA lesions, since viability of AB1886 and AB2463 mutants did not show any difference from the wild type AB1157 viability (Fig. 6A–C).
Genotoxic tests, performed in reactivation assays, evidenced that when bacteriophages are
dam-aged, they cannot be self-repaired and need an enzymatic system in the host. The wild-type bacte-rium is able to repair phages, but mutant bacteria are only able to repair them through mechanisms present in their host. In the present work, no statistical significant difference (P > 0.05) was detected in the amount of plaques, indicating that DC doses applied do not generate lesions in viral DNA. Furthermore, this damage is not liable to be repaired by either AB1886 or AB2463 strains.
In the present work, DC mutagenicity aspects were evaluated by forward mutation to rifampicin resistance and by Ames test assays. The site of rifampicin action is the b subunit of the RNA polymerase; in rifampicin-resistant mutants, the b subunit is altered due to mutations in the rpoB gene [Jin and Gross, 1988]. Our results indicated absence of mutation in the rpoB gene, since there was no increase in number of spontaneous mutants (untreated system). Ames mutagenicity assay, using the TA97, TA98, TA100, and TA102 Salmonella typhimurium strains confirmed that DC was not mutagenic. Recently, our group has been deepening mutagenic and genotoxic properties using similar DC doses, in animal models (C57Bl/6 mice). These results showed that 2.4 C of DC did not induce chromosome segrega-tion in animal peripheral blood when micronucleus assay was performed. These recent results also confirm, in an animal model, absence of mutagenic and genotoxic effects of DC, corroborating results obtained with microorganism strains.
This report is innovative since it is the first research that investigates DC genotoxic and muta-genic potential effects.
CONCLUSIONS
These results add new important information about DC use. Besides having few side effects and good clinical responses, electrotherapy did not induce mutagenic and genotoxic effects on microorganisms strains. These aspects reinforce the importance of this therapy for different pathologies, including neoplasia. REFERENCES
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