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Carotenoids-antioxidants of D. radiodurans stimulate regeneration in mice.

Introduction

It is known that the active formation of reactive oxygen species (ROS) occurs during the early stages of regeneration. ROS have both bactericidal and regulatory function [1]. However, in wounds in case of chronic inflammation there is no decrease in ROS which is one of the main causes of poor wound healing in patients with diabetes, atherosclerosis, as well as in the elderly ones. The development of topical medications with antioxidant activity, which would allow to eliminate inflammation and to create conditions for normal regeneration of chronic wounds, is extremely relevant today due to the high prevalence of diabetes mellitus (DM). The formation of nonhealing wounds is one of the most dangerous complications of DM. In some cases carotenoids promote healing of wounds [2,3].

A promising source of carotenoid antioxidants is Deinococcus radiodurans. Natural strains of these bacteria unlike colorless (unpigmented) mutants are 30 times more resistant to ionizing radiation than E. coli and 1,000 times more resistant than the human cells [4]. Carotenoid complex of D. radiodurans is based on deinoxantin and astaxanthin [5].

Genetic apparatus is one of the main cellular targets of ROS. Therefore, one should assess the ability of antioxidants to inactivate ROS as well as DNA-protective activity when performing the screening.

The aim of this study was to compare the antioxidant and antimutagenic activity of the carotenoid fraction of D. radiodurans in express tests involving bacteria and the ability to stimulate the healing of superficial wounds in mice with streptozotocin-induced diabetes.

Methods

Carotenoids

The protocol of preparation and chemical composition of the carotenoid fraction of D.radiodurans is described in [5].

Bioluminescence test

Antioxidant and DNA-protective activity of the carotenoid preparation was investigated by recombinant biosensor strains of E. coli MG1655 (pSoxS-lux), E. coli MG1655 (pKatG-lux), E. coli MG1655 (pRecA-lux). Biosensors with promoters PkatG and PsoxS detect the presence of oxidants that are able to form hydroperoxides and superoxide anion radicals in the cell. Biosensor with plasmid pRecA detects the presence of factors that cause DNA damage [6]. Testing protocol and the formula to calculate the protective activity are specified in Ref. [7].

We used paraquat (1,1'-dimethyl-4,4bipyridylium dichloride), dioxidine (1,4-dioxide 2,3-quinoxalinedimethanol) and hydrogen peroxide as an oxidative stress inducers.

Determination of the frequency of spontaneous and induced mutagenesis

To study an antimutagenic activity, we determined the frequency of mutants resistance to rifampicin (100 mg/l) in the presence of potential protectors and without them. Dioxidine was used as an inducer of mutagenesis. An overnight culture of E. coli was grown at 37[degrees]C in the presence of an inducer for 18-20 h (with the same volume of sterile saline in the control culture). On Day 2, the strain culture was diluted with fresh LB medium (Luria-Bertani) up to 1-2 McFarland units (3-6 x [10.sup.8] CFU). An optical density of the solution was measured using a densitometer DEN-1 B. Then we made serial dilutions of culture 1:10 in saline (taking the original culture as 1). Diluted culture was applied on plates with LB agar with and without antibiotic by a surface seeding. A count of colonies was performed after 48 h.

Experiments to study the process of regeneration in mice

Type I DM was induced in 10-11 week old male mice (CD-1 outbred stock) by intraperitoneal administration of streptozotocin [8]. The disease was diagnosed in mice with glucose concentration of 14 mmol/l and polyuremia. After 3-4 weeks, we made full-thickness skin wound by removing the skin flap of 7-12 mm in diameter with an area of 1.5% relative to the surface area of the mouse body. Carotenoid fraction of D. radiodurans dissolved in olive oil was applied to the wound surface once a day. In case of oral administration, we used a probe. The control animals received olive oil that has passed all the processing steps used to obtain a preparation of carotenoids in similar forms and doses. We used three methods of drug administration--on-skin (0.5 mg/day), oral (250 mg/kg/day) and their combination. Measurement of the wound surface area (planimetric study) in the process of wound healing was performed using digital macro photography on Days 1,3, 7, 14, and 21 after wounding.

To assess the dynamics of wound healing on the background of DM, we have calculated an overall percentage of reduction in wound area between consecutive measurements using the ImageJ program (National Institutes of Health (NIH) http:/rsb.info.nih.gov/ij/). The extent of spontaneous and metal-catalyzed oxidation of proteins in the serum was determined by the method of Levine [9] in the modification of Dubinina [10]. We used a similar dose of lycopene as a positive control. Totally 96 mice were used. The statistical significance was determined by Student t-test for independent samples with p-value < 0.05.

Experiments on animals were performed in compliance with the principles of the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes.

Results and Discussion

Antioxidant and DNA-protective activity of carotenoids of D. radiodurans using bioluminescent assay

Antioxidant and DNA-protective concentrations for carotenoid fraction of D. radiodurans were registered in the range from 0.001 to 10 mg/ml. The data on the maximum protective activities in this range are shown in Table 1. The results obtained with a-tocopherol in the similar series of tests are also specified.

As can be seen from the presented data, the mixture of carotenoids of D. radiodurans, in contrast to [alpha]-tocopherol, is able to reduce the level of generation of superoxide anion radicals by the action of hydrogen peroxide and dioxidine and considerably enhance the work of cellular mechanisms that provide decomposition of hydrogen peroxide when it is administered exogenously. At the same time, the mixture of carotenoids of D. radiodurans shows a lower level of DNA activity when compared to [alpha]-tocopherol.

Further experiments showed that carotenoids of D. radiodurans considerably reduce the frequency of mutations induced by dioxidine: concentration of 15.5 mg/l is able to reduce the frequency of mutations by 85.9%; concentration of 1.55 mg/l - by 53.2% if compared to the control group. In Figure 1, the results of a quantitative comparison of the number of mutants in a culture treated with various concentrations of carotenoids, and in the control group, are demonstrated.

Thus, there is a direct connection between the antioxidant and antimutagenic effects of carotenoid fraction of D. radiodurans. This gives reason to assume that the antimutagenic activity of the preparation may be partially or entirely based on the ability of its components to neutralize ROS.

In experiments on mice, it was found that on-skin administration of extract of bacterial carotenoid improves the rate of regeneration of dermal wounds more effectively compared to the control compound--lycopene (see Figure 2). Maximum effect was obtained with combined administration (healing rate was improved by 21% (p < 0.05) in comparison to the control group, Figure 3).

On-skin administration of carotenoids of D. radiodurans caused statistically significant reduction in wound area during both formation and maturation of the granulation tissue and scarring (Figure 4). Histological studies demonstrate the ability of carotenoids of D. radiodurans to reduce neutrophil infiltration of the wound. This process accelerates the first (exudative) regeneration phase and increases the rate of wound healing in diabetic mice.

The results of biochemical tests suggest that this is an antioxidant effect that causes the observed acceleration of the regeneration process. Thus, during the analysis of spontaneous and metal-catalyzed degradation of serum proteins of mice with type 1 DM, we have found that the intensity of oxidative modification of proteins was decreased by more than 30% (p < 0.05) during the combined administration of carotenoids of D. radiodurans (Table 2).

Conclusion

Thus, the experimental results show that the carotenoids extract of D. radiodurans has high antioxidant activity that promotes healing of chronic wounds in experimental animals.

Furthermore, we demonstrated the possibility to predict antimutagenic activity based on express test using bacterial biosensors, responding to DNA damage and an increase in the level of ROS in a cell. The use of this technique for express screening in the study of newly synthesized or extracted compounds and pharmaceutical preparations seems to be very promising.

Acknowledgment

The work was supported by the Ministry of Education and Science of the Russian Federation, the task No. 6.1202.2014/K "Study of microbial resistance to antimicrobial agents caused by the use of mutagen drugs". Analytical work was carried out on the equipment of Centers for collective Use of Southern Federal University "High Technology", grant RFMEFI5941 4X0002.

References

[1.] Menke NB, Ward KR, Witten TM, Bonchev DG, Diegelmann RF (2007) Impaired wound healing. Clinics in Dermatology 25(1): 19-25.

[2.] Chan KC, Pen PJ, Yin MC (2012) Anticoagulatory and antiinflammatory effects of astaxanthin in diabetic rats. Journal of Food Science 77(2): 76-80.

[3.] Rao AV, Rao LG (2007) Carotenoids and human health. Pharmacological Research 55(1): 207-216.

[4.] Slade D, Radman M (2011) Oxidative stress resistance in Deinococcus radiodurans. Microbiology and Molecular Biology Review 75(1): 133-191.

[5.] Lysenko VS, Chistyakov VA, Zimakov DV, Soier VG, Sazykina MA, et al. (2011) Separation and mass spectrometry identification of carotenoid complex from radioresistant bacteria Deinococcus radiodurans. Journal of Analytical Chemistry 66(13): 1281-1284.

[6.] Zavilgelsky GB, Kotova VY, Manukhov IV (2007) Action of 1,1-dimethylhydrazine on bacterial cells is determined by hydrogen peroxide. Mutation Research/Genetic Toxicology and Environmental Mutagenesis 634(1): 172-176.

[7.] Chistyakov VA, Prazdnova EV, Gutnikova LV, Sazykina MA, Sazykin IS (2012) Superoxide scavenging activity of plastoquinone derivative 10-(6'-plastoquinonyl) decyltriphenylphosphonium (SkQ1). Biochemistry (Moscow) 77(7): 776-778.

[8.] Kunjathoor VV, Wilson DL, LeBoeuf RC (1996) Increased atherosclerosis in streptozotocin-induced diabetic mice. The Journal of Clinical Investigation 97: 1767-1773.

[9.] Levine RL, Garland D, Oliver CN, Amici A, Climent I, et al. (1990) Determination of carbonyl content in oxidatively modified proteins. Methods in Enzymology 186: 464-478.

[10.] Dubinina EE, Burmistrov SO, Hodov DA, Porotov GE (1995) Oxidative modification of proteins of the human serum. Methods for its determination. Issues of Medical Chemistry 41(1): 24-26.

Published: 19th Dec 2014

Evgeniya Valer'evna Prazdnova*, Svetlana Vladimirovna Dem'yanenko, Vladimir Anatol'evich Chistyakov, Vladimir Sergeevich Lysenko, Mikhail Mikhailovich Batyushin

Academy of Biology and Biotechnology of Southern Federal University, Stachki Avenue 194/1, Rostov-on-Don, 344090, Russia.

* Corresponding author

Table 1: Protective activity (P) of D. radiodurans carotenoids
and tocopherol in the biosensors.

Antioxidants     Biosensors     Inductors of oxidative
                                        stress

                                   Hydrogen peroxide

                                P (%)   Concentration
                                        ([micro]g/ml)

A mixture of   E. coli MG1655    23      [10.sup.-3]
carotenoids     (pSoxS-lux)
of D.

radiodurans    E. coli MG1655    100     [10.sup.-3]
                (pKatG-lux)

               E. coli AB1157    54           1
                (pRecA-lux)

Tocopherol     E. coli AB1157   49.3     [10.sup.-5]
               (pRecA-lux) *

Antioxidants     Biosensors     Inductors of oxidative
                                        stress

                                        Paraquat

                                P (%)   Concentration
                                        ([micro]g/ml)

A mixture of   E. coli MG1655     0          --
carotenoids     (pSoxS-lux)
of D.

radiodurans    E. coli MG1655    37           1
                (pKatG-lux)

               E. coli AB1157    28      [10.sup.-1]
                (pRecA-lux)

Tocopherol     E. coli AB1157     0          --
               (pRecA-lux) *

Antioxidants     Biosensors     Inductors of oxidative
                                        stress

                                        Dioxidine

                                P (%)   Concentration
                                        ([micro]g/ml)

A mixture of   E. coli MG1655   64.9          1
carotenoids     (pSoxS-lux)
of D.

radiodurans    E. coli MG1655    --          --
                (pKatG-lux)

               E. coli AB1157    36      [10.sup.-1]
                (pRecA-lux)

Tocopherol     E. coli AB1157   70.4     [10.sup.-5]
               (pRecA-lux) *

Antioxidants     Biosensors        Types of
                                   activity

A mixture of   E. coli MG1655     Removal of
carotenoids     (pSoxS-lux)       superoxide
of D.                           anion radical

radiodurans    E. coli MG1655     Removal of
                (pKatG-lux)        hydrogen
                                   peroxide

               E. coli AB1157   DNA protection
                (pRecA-lux)

Tocopherol     E. coli AB1157   DNA protection
               (pRecA-lux) *

* No statistically significant effects were found for tocopherol
in the experiments on the remaining strains.

Table 2: Oxidative modification of proteins (arbitrary units-ml)
of the serum of CD I mice during the administration of
carotenoids of D. radiodurans or lycopene (M [+ or -] m, n = 2-
6) per os.

Groups                     Spontaneous           Metal-catalyzed
                            oxidative               oxidative
                         modification of         modification of
                             proteins               proteins

Control, olive oil     5.89 [+ or -] 0.33     31.46 [+ or -] 0.80
per os and per os +
on-skin, 21 days

Carotenoids of D.      4.76 [+ or -] 0.64     29.92 [+ or -] 2.37
radiodurans per os
(125
[micro]g/kg/day) and
on-skin (0.05
[micro]g) + per os
(125
[micro]g/kg/day), 21
days

Carotenoids of D.      4.03 [+ or -] 0.31 *   27.82 [+ or -] 1.42 *
radiodurans per os
(250
[micro]g/kg/day) and
on-skin (0.5
[micro]g) + per os
(250
[micro]g/kg/day), 21
days

Lycopene per os (125   5.06 [+ or -] 0.87     30.13 [+ or -] 2.08
[micro]g/kg/day), 21
days

Lycopene per os (250   4.90 [+ or -] 0.97     30.42 [+ or -] 2.18
[micro]g/kg/day), 21
days

* Differences from the control are statistically significant,
t-test, p < 0.05.

Figure 1: Quantity of [Rif.sup.r] mutants per 1 ml of culture
([10.sup.8] cells) in the presence of dioxidine and carotenoids of
D. radiodurans.

1--control group; 2--in the presence of 15.5 mg/l of carotenoids
of D. radiodurans; 3--in the presence of dioxidine 2.25 x [10.sup.-5]
M; 4--in the presence of dioxidine and carotenoids of D. radiodurans
1.55 mg/l; 5--in the presence of dioxidine and carotenoids of
D. radiodurans 15.5 mg/l.

     cfu/ml

1    4.73
2    3.13
3   63.73
4   30.67
5    9.13

Note: Table made from bar graph.

Figure 2: The dynamics of wound area reduction, an average value
per week during the administration of the protective compound
on-skin in the dose of 0.5 mg/day.

1--control group; 2--carotenoids of D. radiodurans; 3--lycopene. *
p < 0.05 (t-criterion).

    Reducing the wound
    area, week average, %

1      54.8
2      59.6 *
3      57.9

Note: Table made from bar graph.

Figure 3: The dynamics of wound area reduction, an average value
per week during combined administration of the protective compound
(on-skin and oral administration).

1--control group; 2--carotenoids of D. radiodurans. *
p < 0.05 (t-criterion).

    Reducing the wound
    area, week average, %

1     52.8
2     76.1 *

Note: Table made from bar graph.
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Article Details
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Title Annotation:Research Article
Author:Prazdnova, Evgeniya Valer'evna; Dem'yanenko, Svetlana Vladimirovna; Chistyakov, Vladimir Anatol'evic
Publication:Biology and Medicine
Article Type:Report
Date:Jul 1, 2014
Words:2338
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