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

Abstract

Nitric oxide (NO) serves either a universal signaling molecule or extremely toxic agent, depending on the dose. Up to date there have been a very limited number of natural compounds serving as effective regulators of NO signaling and toxic potencies. NO acts in concert with H2S to coordinate cell responses; however, how exactly this interaction is achieved is not known. Both agents have an effect on the accumulation of both reactive chemical species, ROS and RNS and can give rise to other reactive species. Para-aminobenzoic acid (PABA) is an essential metabolite for certain organisms. Once considered a vitamin, PABA, functions as an effective inhibitor of inducible SOS DNA repair processes in E. coli. 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 (TNICthio) - 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 TNICthio. 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-TNICthio.

Full text

Research Article Open Access
Vasilieva et al., Adv Tech Biol Med 2016, 4:4
DOI: 10.4172/2379-1764.1000195
Research Article OMICS Iinternational
Advanced Techniques in
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ISSN: 2379-1764
Volume 4 • Issue 4 • 1000195
Adv Tech Biol Med, an open access journal
ISSN: 2379-1764
Keywords: Nitric oxide (NO); p-aminobenzoic acid (PABA); E. coli;
Oxygen reactive species
Introduction
In the aerobic environment, all structures of a cell - DNA, proteins
and lipids of membranes - are constantly inuenced 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].
It was established, that vitamins of B group, incl. РАВА and beta-
carotene (provitamin A) and D-alpha-tocopherol (vitamin E), possess
the UV-absorbing property and are widely used to protect from solar
radiation, photocarcinogenic exposures and inammatory agents [1-9].
Molecular and genetic mechanisms of these processes in mammalian
cells are poorly understood.
In 1984, S.V. Vasilieva was the rst 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], sA-dependent lament formation and
other functions [11-13]. e results obtained in a study In Vitro of
the molecular mechanisms of these phenomena using methods of
dierential spectrophotometry and thermal denaturation of DNA,
optical activity (circular dichroism) and uorescence 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]. is
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
*Corresponding author: Svetlana V Vasilieva, N.M. Emanuel Institute of Biochemical
Physics, Russian Academy of Sciences, 4 Kosygin Street, Moscow, 119334, Russia,
Tel: 74959397293; Fax: 74991374101; E-mail: svasilieva@chph.ras.ru
Received November 23, 2016; Accepted November 28, 2016; Published
November 30, 2016
Citation: Vasilieva SV, Petrishcheva MS, Gusarova EI, Osipov AN (2016) Vitamin
Para-Aminobenzoic Acid (PABA) Controls Generation of Nitric Oxide (NO) In Vitro
and Its Biological Functions in the Bacterial Cells. Adv Tech Biol Med 4: 195. doi:
10.4172/2379-1764.1000195
Copyright: © 2016 Vasilieva SV, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the
original author and source are credited.
Abstract
Nitric oxide (NO) serves either a universal signaling molecule or extremely toxic agent, depending on the dose.
Up to date there have been a very limited number of natural compounds serving as effective regulators of NO
signaling and toxic potencies. NO acts in concert with H2S to coordinate cell responses; however, how exactly this
interaction is achieved is not known. Both agents have an effect on the accumulation of both reactive chemical
species, ROS and RNS and can give rise to other reactive species. Para-aminobenzoic acid (PABA) is an essential
metabolite for certain organisms. Once considered a vitamin, PABA, functions as an effective inhibitor of inducible
SOS DNA repair processes in E. coli.
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 biolm formation, depending on the rate of NO-
donating 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 (TNICthio)
- and with sulfur-containing aliphatic ligands – cysteamine and penicillamine were studied rst 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 (sA 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 biolm formation
induced by TNICthio.
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-TNICthio.
Vitamin Para-Aminobenzoic Acid (PABA) Controls Generation of Nitric
Oxide (NO)
In Vitro
and Its Biological Functions in the Bacterial Cells
Svetlana V Vasilieva1*, Maria S Petrishcheva1, Elizaveta I Gusarova1 and Andreyan N Osipov2,3
1N.M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, 4 Kosygin Street, Moscow, 119334, Russia
2N.N. Semenov Institute of Chemical Physics, Russian Academy of Sciences, 4 Kosygin Street, Moscow, 119991, Russia
3A.N. Burnasyan Federal Medical Biophysical Center of Federal Medical Biological, Moscow, 23 Marshal Novikov Street, 123098, Russia

Citation: Vasilieva SV, Petrishcheva MS, Gusarova EI, Osipov AN (2016) Vitamin Para-Aminobenzoic Acid (PABA) Controls Generation of Nitric
Oxide (NO) In Vitro and Its Biological Functions in the Bacterial Cells. Adv Tech Biol Med 4: 195. doi: 10.4172/2379-1764.1000195
Page 2 of 6
Volume 4 • Issue 4 • 1000195
Adv Tech Biol Med, an open access journal
ISSN: 2379-1764
dependent on the sA expression. sA 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]. e quantitative level
of β-gal activity in the cells was determined according to Miller [27]. Briey,
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). e 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 biolm processing
e basic protocol of the method described in [28]. e plankton
cell growth was determined by OD600 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.
Aer 15 min, the OD570 was measured to quantify the biolm biomass.
Antibiotic ciprooxacin (CF) was used as a positive control.
Antioxidant-capacity assay and quantitative study of ROS/
RNS production in E. coli cells
e sum of reactive oxygen (ROS) and nitrogen RNS species [being
formed in the cells treated with iron-sulfur nitrosative agents (NO-
donors) in aerobic conditions] was measured using antioxidant capacity
assay, that employed the reactive oxygen species (ROS)-sensitive probe
5-(and-6)-chloromethyl-2',7'-dichlorodihydrouorescein diacetate,
acetyl ester (CM-H2DCFDA). 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]. e 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 buer pH 7.4 (PBS) and resuspended therein.
PABA solution aliquots in PBS were added to the cell suspension with
1•106 titer. e concentration of NO donor TNICthio in all variants
- 0.05 mM. 198 ml of the TNICthio +/- PABA treated cell suspension
were transferred to a 2 ml Eppendorf with radical probe, 5-(and-6)-
chloromethyl-2', 7'-dichlorodihydrouorescein diacetate, acetyl ester
(CM-H2DCFDA) (Molecular Probes, Eugene, OR, USA) at the conc.
of 100 mM and incubated at 37°C. ROS production was measured by
uorescence 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-H2DCFDA (inactive for ROS) is converted to DCFH
(dichlorouorescein diacetate, active for ROS), by being taken into
the cell and acted upon by an intracellular enzyme esterase. e
H2O2
• or O2
•- oxidizes intracellular DCFH (non-uorescent) to DCF
(uorescent) [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.
e 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 biolm antibiotic resistance induced by
nitric oxide donors in E. coli cells.
Materials and Methods
NO donors
Water-soluble nitrosyl iron-sulfur complexes – crystalline NO
donors with various ligands, were rst synthesized at the Institute of
Problems of Chemical Physics RAS: tetranitrosyl iron complex with
thiosulfate Na2[Fe2(S2O3)2(NO)4]2-⋅4H2O (TNICthio), nitrosyl iron
complexes with aliphatic ligands of natural origin cysteine – cisaconite:
(CysA) - [Fe2(S(CH2)2NH3)2(NO)4]SO4 2.5 H2O and penicillamine –
penaconite (PenA) [Fe2(S(C(CH3)2CH(NH3)COOH))2(NO)4]SO4 5H2O
and two cationic dinitrosyl iron complexes (DNICs) with thiourea -
Fe(SС(NH2)2)2(NO)2]2SO4∙H2O (NO-29) and [Fe(SС(NH2)2)2(NO)2]
Cl∙H2O (NO-33). Structure and physico-chemical characteristics of the
donors were studied in [18-21].
NO donors were dissolved immediately before each experiment in
distilled water or in phosphate-buer 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.
Quantication of NO (+/-PABA) releasing
NO generated by the NO donors (TNICthio and PenA) was
quantitated using the sensor electrode amiNO-700 of the INNO
NITRIC OXIDE MEASURING SYSTEM (Innovative Instruments,
United States) [22]. e concentration of NO was detected for
maximum 4000 s at 2 s intervals. e electrochemical sensor was
calibrated using the standard 100 μM NaNO2 aqueous solution. All
experiments were performed under aerobic or anaerobic conditions at
23°C; pH of solutions was measured with an HI 8314 membrane pH-
meter (HANNA Instruments, Germany).
Bacterial strains
e bacterial strains of Escherichia coli were studied: E. coli PQ37
sA::Mud (APlac) cts lac ΔU169 mal+ uvrA galE galY PhoG rda F– thr
leu his pyrD thi trp::Muc+ srl300::Tn10 was kindly provided by Hofnung
(Pasteur Institute, Paris) [23];
E.coli MC4100 F- [araD139]B/r Δ(argF-lac)169 & lambda-e14-
hD5301
Δ(fruK-yeiR)725 (fruA25) relA1 rpsL150(strR) rbsR22 Δ(mB-
mE)632(::IS1) deoC1 was a gi of Ding, USA [24];
E.coli TN530 F2 Δ(lacZYA-argF)U169 rpsL λ(soxS'-lacZ) soxRS+
was a gi of Nunoshiba, Japan [25].
P. aeruginosa PAO1 (the clinical isolate, lab. collection) was used in
the biolm experiments [26].
β-galactosidase (β-gal) assay
e level of the sA-gene expression (the SOS-regulon) was studied
in E. coli PQ37 with the [sA::lacZ] operon fusion and a deletion in the
chromosomal lac operon, so that β-galactosidase activity was strictly

Citation: Vasilieva SV, Petrishcheva MS, Gusarova EI, Osipov AN (2016) Vitamin Para-Aminobenzoic Acid (PABA) Controls Generation of Nitric
Oxide (NO) In Vitro and Its Biological Functions in the Bacterial Cells. Adv Tech Biol Med 4: 195. doi: 10.4172/2379-1764.1000195
Page 3 of 6
Volume 4 • Issue 4 • 1000195
Adv Tech Biol Med, an open access journal
ISSN: 2379-1764
and SEM, condence intervals are shown for P value=0.05. Statistical
analysis of the experimental results was performed using the Microso
Excel and OriginPro 7.0 soware packages.
Results
In Figure 1 we summarized the experimental data of DNA repair
gene expression obtained in E. coli and PABA (0-5.0 mM) inuence in
the samples with 0.1 mM NO-33 and 0.05 mM NO-29. Pure aqueous
PABA solutions did not inuence the gene expression. PABA tested at 5
mM decreased the expression of the soxS and the sA gene expression
by 2.5-3.5 folds. e low PABA doses were much less eective.
PABA aorded 36% protection against PenA damages in the sample
with PABA: PenA (1:10) in E. coli PQ37; but in the experiment with
TNICthio:PABA (1:100) PABA inuence was less eective in E. coli
TN530 (Figures 2a and 2b).
In P. aeruginosa, the non-lethal dose of NO donor (TNICthio 0.02
mM) inhibited the level of biolm formation with and without of PABA
application.
In the sample of (TNICthio 0.02 mM+PABA 0.5 mM) the level of
the biolm 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 biolm formation in E. coli
MC4100 induced by NO donors CysA, PenA and TNICthio (Figure 4).
CysA was the most eective inhibitor in the biolm formation process.
e kinetic curves of NO donating by NO- donors (nM) and PABA
inuence 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 TNICthio 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 NO-
donating in anaerobic conditions (Figure 6, sample 2). In common, the
process of NO-donating depended on PABA concentration. e 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 inuence of PABA on the ROS\RNS production in E. coli cells
treated with TNICthio as NO-donor, (DCFDA uorescence, in relative
units) (Figure 7). e 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 signicant inhibition of the ROS\RNS production in
all samples with TNICthio in the combination with PABA. e change
in NO:PABA=1:0 to NO:PABA=1:100 the level of ROS\RNS was
decreased ve-fold.
Discussion
Our work is the rst 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-sulphur-
nitrosyl complexes - crystalline NO donors. All studied donors easily
penetrate through the bacterial cell membrane [31-33] 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 [34-36].
Such complexes were rst observed and identied in all biological
systems by their specic EPR signals [37,38]. e mechanisms of DNIC
appearance in the bacterial cells are not quite clear. ese NO compounds
Figure 1: Expression of the soxS gene (SoxRS regulon) in E. coli TN530 (1,2)
and the sA gene (SOS regulon) in E. coli PQ37 (3,4), induced by 0.1 mM NO-
33 and 0.05 mM NO-29 and PABA inuence.
Figure 2: Expression of the soxS gene (SoxRS regulon) in E. coli TN530 (a) and the sA gene (SOS regulon) in E. coli PQ37 (b), induced by NO donors and PABA
inuence (a): 1-control; 2-PABA 0.01-5 mM; 3-TNICthio 0.01 mM; 4-TNICthio+PABA (1:10); 5-TNICthio+PABA (1:100); 6-TNICthio+PABA (1:500); 7-menadione 0.5 mM
(positive control); (b): 1-control; 2-PABA 0.5 mM; 3-PenA 0.05 mM; 4-PenA+PABA (1:1); 5-PenA+PABA (1:10); 6-4NQO (positive control).

Citation: Vasilieva SV, Petrishcheva MS, Gusarova EI, Osipov AN (2016) Vitamin Para-Aminobenzoic Acid (PABA) Controls Generation of Nitric
Oxide (NO) In Vitro and Its Biological Functions in the Bacterial Cells. Adv Tech Biol Med 4: 195. doi: 10.4172/2379-1764.1000195
Page 4 of 6
Volume 4 • Issue 4 • 1000195
Adv Tech Biol Med, an open access journal
ISSN: 2379-1764
trigger the signal pathways of transduction associated with physiological
and pathological responses of cells to dierent types of stress. Despite a
characteristic for all DNICs EPR signal with anisotropic factor g=2.03,
the reactivity and functions of DNICs are dierent, depending on
physiological conditions and the genotype of cells.
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 a l. were the rst to substantiate and experimentally
conrm a SOS-inducing activity of NO-donors [31]. e authors used
the method of quantitative assessment of the sA 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
sA gene expression is dose-dependent [22,39-41].
EPR-spectroscopic study of the cells treated with NO-donors
showed characteristic for intracellular protein DNICs signal with the
anisotropic factor g=2.03. e appearance of these signals correlated
with the increase in the expression of the sA gene of the SOS response
regulating the repair of DNA damage during oxidative stress. e 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 SOS-
induction and the content of intracellular iron since iron chelator-o-
Figure 3: Biofilm formation in P. aeruginosa: 1-control; 2-CF 0.05 μM; 3-TNICthio
0.02 mM; 4-TNICthio 0.02 mM+PABA 0.5 mM.
Figure 4: Biofilm formation in E. coli MC4100: 1-control; 2-PABA 0.01 mM-
PABA 0.05 mM; 3-TNICthio 0.01 mM; 4-TNICthio+PABA (1:1); 5-CysA 0.01 mM;
6-CysA+PABA (1:1); 7-PenA 0.05 mM; 8-PenA+PABA (1:1); 9-CF 0.07 μg/ml.
Figure 5: Time dependence of NO (nM) generated by TNICthio (1•10-5М) in
water solution at 25°C: 1-TNICthio; 2-TNICthio+PABA (1:1).
Figure 6: Time dependence of NO (nM) generated by PenA (1•10-5М) in buffer
pH=7.4 under aerobic (+O2) and anaerobic (–O2) conditions at 25°C: 1-PenA
(+O2), without PABA; 2-PenA (–O2), without PABA; 3-PenA (+O2), with PABA
(1:1); 4-PenA (–O2), with PABA (1:1); 5-PenA (+O2), with PABA (1:10).
Figure 7: The level of ROS/RNS production in E. coli TN530 cells treated with
0.05 mM TNICthio and PABA inuence: 1-control; 2-menadione 0.5 mM; 3-PABA
5 mM; 4-TNICthio+PABA 5 mM; 5-TNICthio+PABA 0.5 mM; 6-NICthio+PABA 0.05
mM; 7-TNICthio 0.05 mM; 8-CF 0.05 mM. The levels of all samples are indicated
by deducing «0» for PBS uorescence.

Citation: Vasilieva SV, Petrishcheva MS, Gusarova EI, Osipov AN (2016) Vitamin Para-Aminobenzoic Acid (PABA) Controls Generation of Nitric
Oxide (NO) In Vitro and Its Biological Functions in the Bacterial Cells. Adv Tech Biol Med 4: 195. doi: 10.4172/2379-1764.1000195
Page 5 of 6
Volume 4 • Issue 4 • 1000195
Adv Tech Biol Med, an open access journal
ISSN: 2379-1764
phenanthroline-inhibited NO-induced expression of the sA 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.
ese 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 O2•– 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 [43-45].
According to Lancaster Jr. [29] the peak in oxidative reactions In
Vitro with 1:1 uxes of NO• and O2•– 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 ve 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.
us, when protecting from stress and invasive pathogens in
aerobic environment, nitrogen oxide in composition with DNICs and
superoxide anion O2•– are getting involved in the formation of the body
of reactive compounds and their precursors, ROS and RNS (О2
–•, NO
–•, ООNO–, OH–, NO2•–). NO can function in 3 Redox states of NO+,
NO•, NO– and in each of them nitric oxide has the unique chemical
activity in relation to its main biological targets – SH-groups and [Fe-S]
clusters.
Our In Vitro studies have shown that PABA signicantly inhibits
generation of nitric oxide by NO donors - TNICthio and PenA in the
buer (pH 7.4), with a predominant drop in the indices of NO-donation
in the aerobic environment (Figures 5 and 6) and with dose dependence
on the concentration of PABA. A 5-fold inhibition of NO-generation
was observed in a variant with the PenA+PABA ratio (1:10).
e 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 (sA) 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 biolms with antibiotic resistance - as a response to
oxidative stress - was rst found in our experiments on a combined
action of PABA and TNICthio donors in P. aeruginosa cells (Figure 3)
and TNICthio, PenA and CysA in E. coli 4100 (Figure 4). In all cases, the
application of PABA resulted in the increase of the eectiveness of NO
donors as inhibitors of biolm formation.
We were the rst to reveal on E. coli cells that the nitric oxide donor
PenA modied cytotoxic eects of UVC (254 nm) radiation under
aerobic and anaerobic conditions. e eect 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 eect (in aerobic environment) or as a
5-fold sensitization of cells under hypoxic conditions to the ROS/RNS
radicals [19].
e obtained results on the nitric oxide donors are consistent with
the conclusions made by Hu et al. [7] that PABA eectively absorbs
ROS radicals (OH• radical, singlet oxygen [1O2
–] 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 iron-
containing 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 TNICthio donor (Figure 7).
e total level of reactive oxygen species in the cells (in relative units)
decreased with the increase of the PABA dose (at the constant TNICthio
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 dierent
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 eectively 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 biolm
formation and a regulator of nitric oxide generation In Vitro. Our
data are of signicance since the natural compounds with immense
therapeutic eects 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.
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Citation: Vasilieva SV, Petrishcheva MS, Gusarova EI, Osipov AN (2016) Vitamin Para-Aminobenzoic Acid (PABA) Controls Generation of Nitric
Oxide (NO) In Vitro and Its Biological Functions in the Bacterial Cells. Adv Tech Biol Med 4: 195. doi: 10.4172/2379-1764.1000195
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Adv Tech Biol Med, an open access journal
ISSN: 2379-1764
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Citation: Vasilieva SV, Petrishcheva MS, Gusarova EI, Osipov AN (2016)
Vitamin Para-Aminobenzoic Acid (PABA) Controls Generation of Nitric Oxide
(NO) In Vitro and Its Biological Functions in the Bacterial Cells. Adv Tech Biol
Med 4: 195. doi: 10.4172/2379-1764.1000195