Vitamin Para-Aminobenzoic Acid (PABA) Controls Generation of Nitric Oxide (NO) In Vitro and its Biological Functions in the Bacterial Cells

In 1984, S.V. Vasilieva was the first who experimentally substantiated the hypothesis about a selective inhibition of PABA-inducible SOS DNA repair response in E. coli bacterial cells, -regulation of the error-prone” DNA mutagenesis, W-reactivation and W-mutagenesis, prophage induction [10], sfiA-dependent filament formation and other functions [11-13]. The results obtained in a study In Vitro of the molecular mechanisms of these phenomena using methods of differential spectrophotometry and thermal denaturation of DNA, optical activity (circular dichroism) and fluorescence became the basis for the hypothesis about a direct chemical interaction of PABA and DNA. Experiments In Vitro proved the formation of electrostatic bonds between the components [14].


Introduction
In the aerobic environment, all structures of a cell -DNA, proteins and lipids of membranes -are constantly influenced by reactive species of oxygen (ROS) and nitrogen (RNS). In mammals and humans their generation induces the systems of molecular and genetic protection from stress, just as regulators of aging processes and development of major pathologies do.
Para-aminobenzoic acid (PABA), being a B vitamin, is a cofactor and precursor in the synthesis of folic acid, purines and thymine in most species of bacteria, algae and higher plants [1].
In 1984, S.V. Vasilieva was the first who experimentally substantiated the hypothesis about a selective inhibition of PABA-inducible SOS DNA repair response in E. coli bacterial cells, -regulation of the error-prone" DNA mutagenesis, W-reactivation and W-mutagenesis, prophage induction [10], sfiA-dependent filament formation and other functions [11][12][13]. The results obtained in a study In Vitro of the molecular mechanisms of these phenomena using methods of differential spectrophotometry and thermal denaturation of DNA, optical activity (circular dichroism) and fluorescence became the basis for the hypothesis about a direct chemical interaction of PABA and DNA. Experiments In Vitro proved the formation of electrostatic bonds between the components [14].
In 2004 the PABA/NO complex was synthesized and proposed for use as a targeted catalyst of glutathione-S transferase P1 [15]. This enzyme selectively super-expresses in cancer cells, leads to changes in their redox balance in the cell and launches many potentially lethal cellular responses [16]. PABA functions as a protector of cells against ROS radicals, directly interacting with OH hydroxyl radicals and inhibiting oxidation of deoxyribose in Fenton reaction [7,8]. It stimulates biosynthesis of endogenous interferon in animals and humans; the antiviral drug "AKTIPOL" was developed on the basis of PABA and introduced into practice for treatment of herpes and adenovirus eye infections [17].
On the whole, vitamins are widely used in complex therapy for In the present study we focus on the genetic and physiological evidence in favor of interference of NO-donors and PABA in bacterial cells with DNA repair gene expression and biofilm formation, depending on the rate of NOdonating In Vitro and intracellular ROS/RNS accumulation in the cells. The crystalline dinitrosyl iron complexes (NO-29 and NO-33) with thiourea as the ligands and 3 crystalline tetranitrosyl iron complexes with thiosulfate (TNIC thio ) -and with sulfur-containing aliphatic ligands -cysteamine and penicillamine were studied first as the NO-donors in pure solutions and in the combination with PABA.
In E. coli cells with the combined action of PABA (0.01-5 mM) with nitric oxide donors we observed an inhibition of NO-signaling potency in the SOS (sfiA gene)-and the SoxRS (soxS gene) DNA repair pathway up to 3.5 fold, depending on the dose of PABA. PABA tested at 0.5 mM afforded 24% protection against the level of biofilm formation induced by TNIC thio .
Using the antioxidant-capacity assay, we observed a many-fold decrease in the ROS/RNS level production in the samples of E. coli cells with PABA and NO-donor-TNIC thio .
Vitamin Para-Aminobenzoic Acid (PABA) Controls Generation of Nitric Oxide (NO) In Vitro and Its Biological Functions in the Bacterial Cells dependent on the sfiA expression. sfiA gene expression was monitored as described by Quillardet et al. [23]. E. coli TN530 wt [soxS::lacZ] strain was used for the soxS gene expression according to [25]. The quantitative level of β-gal activity in the cells was determined according to Miller [27]. Briefly, an overnight E. coli culture was diluted 1:50 into Luria-Bertani broth (LB) medium and grown for 3,5 h to OD600=0.3-0.4, which corresponded to the early log-phase of growth. Cells were treated with NO donors and/ or PABA for 30 min at 37°C and further incubated in the presence of the chromogen o-nitrophenyl-β-D-galactopyranoside (ONPG). The β-gal activity was measured by PD-303UV digital spectrophotometer (APEL Co. Ltd., Japan), at 420 nm. To calculate the β-gal activity (E), an equation E=1000•OD420/t, where OD420 is the optical density at 420 nm and t is the time of incubation with the chromogen, was used. As the positive controls, 26.3 μM 4-nitroquinoline oxide, 4NQO, was used for E. coli PQ37 and 0.5 mM menadione for E. coli TN530.

Plankton cell culture and biofilm processing
The basic protocol of the method described in [28]. The plankton cell growth was determined by OD 600 value. 0.1% (wt/v) crystal violet was used for 10 min to stain the attached cells. Unattached dye was rinsed away by washing two times with distilled water and the stained biomass was dissolved with 1:4 (v/v) mixtures of acetone and ethanol. After 15 min, the OD 570 was measured to quantify the biofilm biomass. Antibiotic ciprofloxacin (CF) was used as a positive control.

Antioxidant-capacity assay and quantitative study of ROS/ RNS production in E. coli cells
The sum of reactive oxygen (ROS) and nitrogen RNS species [being formed in the cells treated with iron-sulfur nitrosative agents (NOdonors) in aerobic conditions] was measured using antioxidant capacity assay, that employed the reactive oxygen species (ROS)-sensitive probe 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H 2 DCFDA). Because of the dominance of oxidative processes caused by RNS, the term nitroxidative stress is proposed, emphasizing the oxidative (as opposed to nitrosative or nitrative) stress that dominates RNS actions under biological conditions, in the cells [29]. The antioxidant-capacity assay was used to examine the intracellular ROS in E. coli TN530.
E. coli TN530 cells were grown to logarithmic growth phase in LB medium in accordance with Wu and Outten [28], washed twice in phosphate saline buffer pH 7.4 (PBS) and resuspended therein. PABA solution aliquots in PBS were added to the cell suspension with 1•10 6 titer. The concentration of NO donor TNIC thio in all variants -0.05 mM. 198 ml of the TNIC thio +/-PABA treated cell suspension were transferred to a 2 ml Eppendorf with radical probe, 5-(and-6)chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H 2 DCFDA) (Molecular Probes, Eugene, OR, USA) at the conc. of 100 mM and incubated at 37°C. ROS production was measured by fluorescence intensity (conv) on QUBIT PORTABLE FLUOROMETER (INVITROGEN, Turner BioSystems, USA) at 488 nm (excitation) and 525-530 nm (emission) every 15 min during 1 h.
CM-H 2 DCFDA (inactive for ROS) is converted to DCFH (dichlorofluorescein diacetate, active for ROS), by being taken into the cell and acted upon by an intracellular enzyme esterase. The H 2 O 2 • or O 2 •oxidizes intracellular DCFH (non-fluorescent) to DCF (fluorescent) [30].

Statistical Analysis
Results are presented as the mean values of at least three experiments inhibition of oxygen free radicals ROS; however, much less is known about the role of vitamins in regulation of reactive nitrogen species (RNS) and their generation in various biological systems.
The aim of our work is to study the mechanisms of regulation by para-aminobenzoic acid of DNA-repair systems of oxidative and nitrosative stress as well as biofilm antibiotic resistance induced by nitric oxide donors in E. coli cells.  [18][19][20][21].

NO donors
NO donors were dissolved immediately before each experiment in distilled water or in phosphate-buffer saline (PBS), pH=7.4.
Para-aminobenzoic acid (PABA) was recrystallized from the hot aqueous solution and dried in the air. Water/PBS solutions with pH=7.4 were studied.

Quantification of NO (+/-PABA) releasing
NO generated by the NO donors (TNIC thio and PenA) was quantitated using the sensor electrode amiNO-700 of the INNO NITRIC OXIDE MEASURING SYSTEM (Innovative Instruments, United States) [22]. The concentration of NO was detected for maximum 4000 s at 2 s intervals. The electrochemical sensor was calibrated using the standard 100 μM NaNO 2 aqueous solution. All experiments were performed under aerobic or anaerobic conditions at 23°C; pH of solutions was measured with an HI 8314 membrane pHmeter (HANNA Instruments, Germany).
In P. aeruginosa, the non-lethal dose of NO donor (TNIC thio 0.02 mM) inhibited the level of biofilm formation with and without of PABA application.
In the sample of (TNIC thio 0.02 mM+PABA 0.5 mM) the level of the biofilm formation inhibition corresponded to 45% as compared to the control and it was higher than that in the sample with CF ( Figure  3). PABA inhibited the levels of bacterial biofilm formation in E. coli MC4100 induced by NO donors CysA, PenA and TNIC thio (Figure 4). CysA was the most effective inhibitor in the biofilm formation process.
The kinetic curves of NO donating by NO-donors (nM) and PABA influence were shown in Figures 5 and 6. We studied the process in the aerobic and anaerobic conditions in the experiments with PenA, exclusively. On the initial stage of the donor incubation (up to 1350 s) the level of NO donating by TNIC thio decreased in the sample with PABA, but then it was higher. On Figure 6 we observed the same dependence (samples 1 and 3). We observed the highest level of NOdonating in anaerobic conditions ( Figure 6, sample 2). In common, the process of NO-donating depended on PABA concentration. The growth in PABA dose promoted the reduction of NO-generation in aerobic conditions. And the maximum level of NO-donating inhibition was observed in the sample of [PenA+PABA (1:10)] in aerobic conditions.
Using the antioxidant-capacity methodology Nakajima et al. [30] tested an influence of PABA on the ROS\RNS production in E. coli cells treated with TNIC thio as NO-donor, (DCFDA fluorescence, in relative units) (Figure 7). The NO donor was tested in the constant conc. of 0.05 mM, while the doses of PABA were variable from 0.05 mM to 5 mM. We observed the significant inhibition of the ROS\RNS production in all samples with TNIC thio in the combination with PABA. The change in NO:PABA=1:0 to NO:PABA=1:100 the level of ROS\RNS was decreased five-fold.

Discussion
Our work is the first to study the function of para-aminobenzoic acid vitamin in the regulation of protective reactions of bacterial cells to cyto-and genotoxic stress caused by mono-and binuclear iron-sulphurnitrosyl complexes -crystalline NO donors. All studied donors easily penetrate through the bacterial cell membrane [31][32][33] and are thought to form intracellularly, on the basis of donated nitric oxide and cellular iron, main NO transport and functional structures -dinitrosyl iron complexes -S-nitrosothiols and dinitrosyl iron complexes (DNICs), which are more stable and ensure NO transport in vivo [34][35][36].
Such complexes were first observed and identified in all biological systems by their specific EPR signals [37,38]. The mechanisms of DNIC appearance in the bacterial cells are not quite clear. These NO compounds  In enteric bacteria, nitric oxide is produced mostly as a byproduct of anaerobic metabolism; NO resembles superoxide -a natural agent of the respiratory process.
In 1999, Vasilieva et al. were the first to substantiate and experimentally confirm a SOS-inducing activity of NO-donors [31]. The authors used the method of quantitative assessment of the sfiA gene expression of the SOS regulon in E. coli strain PQ37 by S-nitrosothiols (GSNO, SNAP) and iron-sulphur-nitrosyl complexes (DNICs). It was subsequently proved that all NO-donors have a strong SOS-inducing activity and the level of sfiA gene expression is dose-dependent [22,[39][40][41].
EPR-spectroscopic study of the cells treated with NO-donors showed characteristic for intracellular protein DNICs signal with the anisotropic factor g=2.03. The appearance of these signals correlated with the increase in the expression of the sfiA gene of the SOS response regulating the repair of DNA damage during oxidative stress. The stress was triggered by peroxynitrite which is formed as a result of interaction between NO molecules donated by the donor and superoxide anion. A direct dependence was found between the intensity of the SOSinduction and the content of intracellular iron since iron chelator-o-     phenanthroline-inhibited NO-induced expression of the sfiA gene in the cells; also the decrease in the intensity of signal g=2.03 was observed in the EPR spectra of these cells as a result of a lower level of intracellular protein DNICs.
These data have become direct evidence in favor of interconnection between formation in the cells of two signals: 1) EPR signal g=2.03 of the DNIC-protein complex and 2) the signal initiating induction of the SOS-box multi enzyme complex to launch the DNA-SOS repair system. To date, not all details of the molecular structures comprising the DNA-SOS-repair system of E. coli have been established. However, the presence in the DNA structure of double-strand breaks that cannot be repaired by other known DNA repair systems is undeniable [42].
Resistance of E. coli cells to superoxide anion and nitric oxide is selectively controlled by multi-functional SoxRS-regulon. A two-step mechanism of induction of SoxRS-regulon has been studied in detail and is associated with interaction of the iron-sulfur cluster of the sensory SoxR[2Fe-2S] protein with superoxide anion O 2 •or other redox agent (including DNICs), as well as with the changes in the redox state of the cluster of this protein and activation of the soxS structural gene with the induction of at least 15 repair enzymes of antioxidant protection, including against most antibiotics that generate ROS radicals [43][44][45].
According to Lancaster Jr. [29] the peak in oxidative reactions In Vitro with 1:1 fluxes of NO• and O 2 •does not occur under biological conditions, in the cells and 1) the quantitatively dominant (92-99.6%) process in vivo is oxidation, compared to nitrosation and nitration; 2) only five of the possible RNS reactions with thiol are quantitatively important biologically. Because of the dominance of oxidative processes caused by Lancaster Jr. [29] proposed the term nitroxidative stress, emphasizing the oxidative (as opposed to nitrosative or nitrative) stress that dominates RNS actions in the cells. The conducted study has shown that a combined action of PABA (0.01-5 mM) and nitric oxide donors inhibits induction of the signal activity of SOS (sfiA) and SoxRS (soxS) genes of E. coli regulons up to 3.5 times maximum, depending on the dose of PABA (Figures 1 and 2).
Antioxidative activity of PABA towards inhibiting the levels of formation of biofilms with antibiotic resistance -as a response to oxidative stress -was first found in our experiments on a combined action of PABA and TNIC thio donors in P. aeruginosa cells (Figure 3) and TNIC thio , PenA and CysA in E. coli 4100 (Figure 4). In all cases, the application of PABA resulted in the increase of the effectiveness of NO donors as inhibitors of biofilm formation.
We were the first to reveal on E. coli cells that the nitric oxide donor PenA modified cytotoxic effects of UVC (254 nm) radiation under aerobic and anaerobic conditions. The effect of NO on the cytotoxic action of UV radiation depended on the genotype of the cells -the activity of DNA excision repair system Uvr ABC and expressed either as a 4-or 10-fold protective effect (in aerobic environment) or as a 5-fold sensitization of cells under hypoxic conditions to the ROS/RNS radicals [19].
The obtained results on the nitric oxide donors are consistent with the conclusions made by Hu et al. [7] that PABA effectively absorbs ROS radicals (OH• radical, singlet oxygen [ 1 O 2 -] and HOCL) and protects DNA from UVC (254 nm) radiation. It is the radical damages to calf thymus DNA that is potentiated by the Fenton reaction system containing iron. We suppose that in our experiments with the ironcontaining NO-donating agents we obtained exactly the same situation that has been described by Hu et al. [7].
Our study provides convincing evidence for the dependence of ROS/ RNS radical products in E. coli cells TN530 when they are individually or in combination with PABA treated with TNIC thio donor ( Figure 7). The total level of reactive oxygen species in the cells (in relative units) decreased with the increase of the PABA dose (at the constant TNIC thio concentration, 0.05 mm).
On the base of the results obtained we conclude that a combined application of PABA and iron-sulphur-nitrosyl complexes -nitric oxide donors, in which NO can be coordinated or bound with different ligands, aimed at regulating protective reactions of cells against stress, enables PABA to selectively interact with ROS/RNS radicals that are formed during hydrolysis of the donors and their metabolism. At the same time, PABA effectively absorbed NO generated by NO-donating agents in the experiments In Vitro.

Conclusion
For a long time para-aminobenzoic acid (PABA) considered a vitamin and a precursor of folic acid for certain bacteria, fungi and plants. On the basis of our results (the present paper and the previous priority publications) we conclude that in the bacterial cell PABA functions a potent inhibitor of DNA repair pathways induced by nitric oxide and a powerful antioxidant; an inhibitor of the bacterial biofilm formation and a regulator of nitric oxide generation In Vitro. Our data are of significance since the natural compounds with immense therapeutic effects and the medicine agents of the new class on the basis of the stable dinitrosyl-iron complexes (NO-donors) for the treatment of infections caused by E. coli and other invasive pathogens are of great concern.