Effects of Sanionia uncinata extracts in protecting against and inducing DNA cleavage by reactive oxygen species

Abstract

When mosses are exposed to increased quantities of ultraviolet (UV) radiation, they produce more secondary metabolites. Antarctica moss Sanionia uncinata (Hedw.) Loeske has presented high carotenoid contents in response to an increase in UVB radiation. This moss has been recommended as a potential source of antioxidants. In the present work, the protective and enhancing effects of aqueous (AE) and hydroalcoholic (HE) extracts of S. uncinata on the cleavage of supercoiled DNA were evaluated through topological modifications, quantified by densitometry after agarose gel electrophoresis. Total phenolic contents reached 5.89 mg/g. Our data demonstrated that the extract does not induce DNA cleavage. Furthermore, both extracts showed antioxidant activity that protected the DNA against cleavage induced by (i) O₂^•−, 89% (AE) and 94% (HE) (P < 0.05), and (ii) .OH, 17% (AE) and 18% (HE). However, the extracts intensified cleavage induced by Fenton-like reactions: (i) Cu²⁺/H₂O₂, 94% (AE) and 100% (HE) (P < 0.05), and (ii) SnCl₂, 62% (AE) and 56% (HE). DNA damages seem to follow different ways: (i) in the presence of Fenton-like reactions could be via reactive oxygen species generation and (ii) with HE/Cu²⁺ could have also been triggered by other mechanisms.

Keywords:

• Sanionia uncinata
• Reactive oxygen species
• DNA cleavage
• Antioxidants
• Antarctica

Introduction

Research on Antarctic mosses has provided an interesting perspective on the influence of the increase in ultraviolet (UV) B levels (280–315 nm) caused in recent decades by ozone depletion.¹ When they are exposed to greater solar UV radiation, mosses produce more of certain secondary metabolites, mainly flavonoids² and carotenoids,³ which can be understood as antioxidants that protect them against UV radiation.⁴

Antarctica moss Sanionia uncinata (Hedw.) Loeske has presented high contents of the carotenoids violaxanthin, and nexanthin in the presence of higher levels of natural UVB radiation.⁵ Moreover, no significant increase in cyclobutane pyrimidine dimers has been detected in the S. uncinata genome exposed to natural UVB radiation in Antarctica. This tolerance suggests its acclimatization well to environmental stresses via DNA repair or an increase in the synthesis of UV chromophores.⁶

Correlations between phenolic content and responses to in vitro antioxidant activity have been found using a hydroalcoholic extract of the moss collected from King George Island, Antarctica. Based on this, it has been suggested that S. uncinata could be an important natural source of antioxidants for applications in medicine and cosmetics.⁷

Antioxidants protect plant cells from damage induced by free radicals and reactive oxygen species (ROS). ROS accumulation causes damage to the DNA, proteins, lipids, and various molecules in the cells and can lead to cell death.⁸ DNA oxidative damage is particularly implied in mutagenesis and carcinogenesis processes.⁹ In fact, UV radiation can induce the damage of DNA chains¹⁰ via a reduction in molecular oxygen and singlet oxygen-mediated photo-oxidation, leading to the formation of ROS, which are directly responsible for DNA damage.¹¹

In the present study, using agarose gel electrophoresis, we investigated DNA topological modifications to evaluate whether aqueous (AE) and hydroalcoholic (HE) extracts from S. uncinata acted as protectors against ROS or as inductors of cleavage in DNA plasmid. As biological systems involve higher complexity of interactions, the present investigation focused in vitro assays to aim primary screening on effects of the extracts following basic mechanisms of ROS generations.

Materials and methods

Chemicals, bacterial strains, and plasmid DNA

Stannous chloride anhydrous (SnCl₂, CAS 7772-099-78), l-histidine (CAS 7048-02-4), pyrocatechol (CAS 120-80-9), ethylenediaminetetraacetic acid (EDTA, CAS 60-00-4), and sodium azide (NaN₃, CAS 26628-22-8) were purchased from Sigma Co (St Louis, MO, USA). Analytical-grade dimethyl sulfoxide (DMSO) and 30% H₂O₂ were purchased from Merck (Darmstadt, Germany). Pyrogallol (99%, CP95 Pharm grade) was obtained from Heco Trading (Hamburg, Germany). All other reagents were analytical grade. Highly purified E-pure water with a resistivity of 18.1 MΩ-cm (Barnstead/Thermolyne, Dubuque, IA, USA) was used throughout the preparations and assays. Plasmid pUC18 (2686 bp, double strand) was isolated from Escherichia coli DH5alpha strain using the standard alkaline lysis procedure.¹²

Sampling site at King George Island

Samples of S. uncinata were collected from King George Island, in the South Shetland Islands, Antarctic Peninsula, in February 2004. Throughout the year, the monthly mean air temperature at the island ranges from +2 to −10°C. On King George Island, there are ice-free areas in the summer (December to late March), when soil and air temperatures typically remain above freezing. During this period, mosses growing on rocky terrains are exposed to direct UV radiation. From Autumn to Spring, the mosses are covered by a snow/ice layer that varies from a few centimeters to ∼2 m in depth. At sub-Antarctic sites such as King George Island, UV radiation levels reaching the surface are ∼4 times higher in the summer (in terms of maximum daily UV index) than in the winter or autumn, leading to a high gradient in seasonal UV irradiation. Samples were collected in the vicinity of the Brazilian Comandante Ferraz Antarctic Station (62°05′S, 58°24′W) using aseptic procedures, stored in plastic bags and kept frozen until being processed in laboratory conditions.

Preparation and characterization of the extracts

The S. uncinata specimen was identified by Dr Denise Pinheiro da Costa of the Botanical Garden Research Institute of Rio de Janeiro (Rio de Janeiro, Brazil). Moss material (9 g) was washed four-fold with 200 ml water for 15 minutes under magnetic agitation for the removal of visible debris and sediments. The vegetal material was extracted by boiling for 15 minutes consecutively with 300 ml water and 300 ml 70% v/v ethanol. The extracts were filtered through Whatman no. 1 filter paper, centrifuged at 3900 × g for 20 minutes at 4°C, and then the supernatant was sterilized by membrane filtration (nylon, 0.22 µm pore size). The ethanol was then evaporated under 300 mTorr pressures in an SC-110 SpeedVac^® (Savant Instruments, Holbrooks, NY, USA). The extracts were freeze-dried and further stored in a desiccator in the dark at −20°C until use. The yields from wet moss were 1.88 and 0.73% (w/w), respectively, for AE and HE. The total phenolic content (mg pyrocatechol equivalent per g of dried extract) was determined in triplicate by the Folin–Ciocalteu method as described previously,¹³ using a standard curve (with five pyrocatechol concentration levels) at 760 nm in a Shimadzu UV-160A spectrophotometer.

DNA treatment

Plasmid DNA (pUC18, 394 µg/ml) was incubated in a tris-acetate-EDTA buffer (TAE), pH 7.8, with increasing concentrations of AE and HE (20, 500, and 900 µg/ml) for 60 minutes at 37°C under aerobic conditions to evaluate its genotoxic potential. SnCl₂ at 200 µg/ml was used as a positive control of DNA cleavage using the same incubation conditions as previously described.¹⁴ Reaction mixtures (20 µl) containing S. uncinata extracts (fixed at 900 µg/ml) and plasmid DNA in TAE were incubated at 37°C under aerobic conditions as described below.

The protective activity of the extracts was evaluated, including ROS inducers that had been diluted in TAE. The experimental design was adapted from previous studies of DNA cleavage caused by ROS.^15–18 Thus, pyrogallol (250 µM), H₂O₂ (2 mM), Fe²⁺/H₂O₂ (200 mM, 50 mM, Fenton reaction), and Cu²⁺/H₂O₂ (1.25 mM, 0.125 mM, Fenton-like reaction) were included individually in the reaction medium (20 µl) and this was incubated for 60 minutes (except for H₂O₂, 30 minutes). DMSO 5% was added as an inhibition control since this compound is a scavenger of O₂^•− and OH.⁹

The extracts were also incubated in the presence of SnCl₂ (200 µg/ml) for 60 minutes. Well-known antioxidants or ROS scavengers were included following previously reported procedures²⁰ butylated hydroxytoluene (BHT, 20 mM), sodium benzoate (benz, 2 M), l-histidine (50 mM), DMSO (5%), and NaN₃ (100 mM).

All experiments were performed in triplicate.

DNA electrophoresis

Agarose gel (1.5%) electrophoresis was performed using standard procedures as previously described¹² and as used elsewhere^{14,15,21–23} in order to separate different structural conformations found after DNA incubation: form I (supercoiled DNA); form II (relaxed circular) resulting from a single-strand break; and form III (linear) resulting from double-strand breaks. As such, aliquots (15 µl) from the incubation mixture were mixed with a loading buffer (0.25% xylene cyanol FF; 0.25% bromophenol blue; 30% glycerol in water), added to a horizontal gel electrophoresis chamber in TAE buffer, pH 8.0, and then electrophoresed at 60 V for 90 minutes. The final volume was 20 µl. After electrophoresis, the distinct DNA bands were stained with 0.5 µg/ml ethidium bromide, visualized under UV light using a UV transilluminator (UVP, Cambridge, UK), and then digitally photographed (Kodak Digital ID, EDAS 120, Rochester, NY, USA). The stained bands were scanned by densitometry using the ImageJ 1.34a software (National Institute of Health; Bethesda, MD, USA) and expressed as an intensity ratio ranging between form II and total (I + II + III). Differences among the densitometry data were statistically evaluated using z test of proportions.

Results and discussion

Phenolic content estimation

The phenolic content of AE and HE was 1.04 ± 0.27 and 5.89 ± 2.05 mg/g, respectively. The statistically higher content (P < 0.05) in the HE was probably due to the intense presence of derivatives with few or no combined glycoside groups. Natural phenolic compounds can contain glycosides in their chemical structures, and glycosilation usually leads to high solubility in water and low solubility in low-polarity and non-polar solvents.²⁴ The results found in HE were two times lower as those values reported elsewhere⁷ because the data in the literature are based on freeze-dried moss material.

Effects on DNA topology

Fig. 1 shows the structural conformation of pUC18 DNA after incubation with AE and HE at three concentrations. Lane 1 shows the results from incubation without the extracts, representing the negative control. SnCl₂ was used as a positive control (lane 5) since it is a genotoxic agent that induces DNA damages via Fenton-like reactions.^14,21–23 No induction of DNA cleavage by either extract was detected (lane 2–4), meaning that molecular genotoxicity cannot be attributed to them.

Figure 1. Agarose gel electrophoresis of plasmid DNA (pUC18, 394 µg/ml) incubated for 1 hour at 37^oC with aqueous (A) or hydroalcoholic (B) extracts of S. uncinata (lanes 2–4). Lane 1 represents the negative control. Stannous chloride at 200 µg/ml was used as a positive control (lane 5). The direction of electrophoresis is top to bottom. Supercoiled (I) and relaxed circular (II) plasmid DNA forms are indicated. The gels are representative of experiments with similar results.

Protective effects against induction of ROS generation

In view of the above results, the possible protective effects of the extracts at 900 µg/ml against the induction of ROS were investigated using four experimental designs performed independently. The electrophoresis analysis of the DNA topology after incubation is illustrated in Fig. 2, in which lanes 1 and 2 are the negative and DNA damage controls, respectively. The relative signal intensities of form II obtained by densitometry analysis are presented in Table 1.

Figure 2. Agarose gel electrophoresis of plasmid DNA (pUC18, 394 μg/ml) incubated for 1 hour at 37^oC with aqueous and hydroalcoholic extracts of S. uncinata at 900 µg/ml in the presence and absence of ROS generation systems: (A) Pyrogalol 250 µM for O₂^•−, (B) Fe²⁺/H₂O₂ (200 µM, 50 mM) for ^•OH, via Fenton reaction, (C) Cu²⁺/H₂O₂ (200 µM, 50 mM) for ^•OH and O₂^−•, via Fenton-like reaction, (D) SnCl₂ (200 µg/ml) for ^•OH and O₂^−•, via in situ Fenton-like reaction. ROS inhibitors: DMSO 5%, except for SnCl₂ in which EDTA 25 mM was used. The direction of electrophoresis is top to bottom. Supercoiled (I), relaxed circular (II), and linear (III) forms are indicated. The gels are representative of experiments with similar results.

Fig. 2A shows the agarose gel electrophoresis of plasmid DNA after incubation with pyrogallol, an inducer of topological changes in DNA from form I to II via O₂^•− generation.^15,25 In Table 1, the action of both extracts in inhibiting pyrogallol-induced conformational changes (lane 2) (P < 0.05) can be observed, since AE and HE inhibited 89 and 94% (lanes 3 and 4, respectively) of DNA cleavage in form II (lane 2). This inhibition effect was statistically identical to the negative control.

Table 1. Intensity (%, mean relative to total band density and standard deviation) of form II DNA (plasmid pUC18) treated with the aqueous (AE) and hydroalcoholic (HE) extracts of S. uncinata in the absence (–) or presence (+) of ROS generation systems and without (wo) or with (w) ROS inhibitors

Lane^a	Test		ROS generation systems (Figure)^b
			Pyrogallol	Fe^2+/H2O2	Cu^2+/H2O2	SnCl2
			(Fig. 2A)	(2B)	(2C)	(2D)
1	—	– (wo)	0 ± 0	0.6 ± 0.1	1.7 ± 0.5	5.0 ± 0.9
2	—	+ (wo)	44.8 ± 2.3^d*	86.1 ± 4.1^c,d	43.5 ± 5.7^d*	55.7 ± 2.0^d*
3	AE	+ (wo)	5.0 ± 2.9^e*	71.1 ± 4.8^d	84.6 ± 7.2^d,e*	90.2 ± 0.7^d
4	HE	+ (wo)	2.7 ± 1.2^e*	70.4 ± 4.1^d	97.8 ± 3.1^d,e*	87.1 ± 6.0^d
5	—	– (w)	2.0 ± 1.8^e*	1.3 ± 0.3^e	2.6 ± 3.7^e*	7.0 ± 3.1
6	—	+ (w)	2.3 ± 1.7^e*	4.1 ± 0.3^e	25.6 ± 4.5	22.7 ± 5.1
7	AE	+ (w)	1.8 ± 1.2^e*	0.6 ± 0.4^e,f	21.1 ± 14.3^f*	36.6 ± 16.2^f*
8	HE	+ (w)	0.6 ± 0.8^e*	0.3 ± 0.4^e,g	74.7 ± 9.8^d,g*	28.3 ± 3.0^g*

^aLane showed in the gels in Fig. 2.

^bDetails of ROS generation systems, incubation conditions, and ROS inhibitors are described in Fig. 2 and in the text.

^cForm III was observed only under these conditions and at a relative intensity of 13.9 ± 4.1. Statistically significant differences (z test for proportions, P < 0.01 or *P < 0.05) relative to

^dnegative control (lane 1),

^elane 2,

^flane 3,

^glane 4.

All experiments were performed in triplicate.

Hydrogen peroxide is known to be the second ROS formed by the reduction of O₂. The induction of single- and double-strand breaks of plasmid DNA by 50–400 µM H₂O₂ has been demonstrated.^16,26 However, in our experiments 50 µM to 2 mM H₂O₂ did not induce pUC18 DNA cleavage under the same conditions in the absence or presence of either extract (data not shown) after 30 minutes of incubation. Since OH generation by H₂O₂ in the presence of appropriate inducers has been considered an appropriate model for ROS generation,¹⁹ these results suggest that neither extract contained compounds that induce .OH generation.

The effects of AE and HE were investigated in the situations where DNA cleavage was induced by the Fenton reaction (Fig. 2B). In this case, the reduction of H₂O₂ was observed, particularly by Fe²⁺, producing ^•OH.¹⁷ Extensive DNA cleavage in forms II and III induced by the Fenton reaction can be observed in lane 2 (P < 0.01). The inhibition control was effective (lanes 5–8), showing that ROS were responsible for DNA cleavage in the assay. AE and HE protected the plasmid DNA (lanes 3 and 4) against cleavage induced by the Fenton reaction, with the intensity of the form II band decreasing by up to 17% for AE and by up to 18% for HE (Table 1). Form III generation was also inhibited. No statistically significant differences between the effects of the extracts (P > 0.05) were found, regardless of the presence of an inhibitor (comparing lanes 3 and 4, and 7 and 8).

Interaction with Fenton-like reactions

Fenton-like reactions have a significant role in the induction of oxidative DNA damage²⁷ following the generation of ROS via the reduction of H₂O₂ by metal ions, except in ferrous salts.^28,29 As such, the effect of both extracts on DNA cleavage induced by Fenton-like reactions was also investigated (Fig. 2C) using the Cu²⁺/H₂O₂ system. In contrast with the protective effect of both extracts against Fenton reaction-induced cleavage, both AE and HE induced a statistically significant (P < 0.01) increase in DNA cleavage induced by a Fenton-like reaction (lanes 3 and 4). The maximum increase observed was 94% for AE and 100% for HE (P < 0.05) (Table 1). DMSO, which was used as an inhibition control, decreased damage by Fenton-like reactions, even in the presence of AE (lanes 6 and 7). However, this inhibition was lower in the presence of HE/Fenton-like reactions (comparing lanes 6 and 7 with 8).

Antioxidant activity of natural phenolic compounds it is well known. However, prooxidant effects of these compounds have been also observed with the participation of transition metal ions and dissolved oxygen. The resulting rise to oxidative damage has also been detected.³⁰ The antioxidant and prooxidant activities of these substances are generally concentration dependents.^31–33 Some natural phenolic compounds associated with Cu²⁺ promote extensive in vitro DNA cleavage under aerobic conditions.^15,25 Even so, no topological modification was observed when plasmid DNA was treated for 1 hour with this ion in the presence of AE and HE (data not shown), excluding any possibility of a reaction between Cu²⁺ and the extracts causing DNA cleavage, such as observed above in the Fenton-like reactions.

The distinct effect of HE in the presence of Fenton-like reactions can be associated on the higher amounts of phenolic ingredients. Furthermore, the antioxidant and the copper-initiated prooxidant activities of phenolic compounds depend on the number and position of –OH substituents in its backbone structure, apart from the presence of glycosides, and the overall degree of conjugation are important in determining activity.^32,33

Fenton-like reaction with hydrogen peroxide formed in situ

Fig. 2D shows the conformational forms observed for plasmid DNA treated with both extracts in the presence of SnCl_2. In this system, H₂O₂ is generated in situ after the reduction of molecular oxygen (in aerobic conditions) by the metal ion.²¹ Moreover, Sn²⁺ also presents direct mutagenic effects via the oxidation of DNA resulting in 8-oxoguanine.²²

The SnCl₂-induced DNA cleavage (lanes 3 and 4, in Table 1) may have occurred via Fenton-like reactions (P < 0.01), since this effect was also previously observed in the Cu²⁺/H₂O₂ system assay (Fig. 2C). Both extracts increased the damage to the plasmid DNA: AE by 62% and HE by 56%. EDTA, a metal chelating compound that inhibits DNA damage by SnCl₂, was included as a control (lanes 5–8). EDTA also inhibited the DNA cleavage induced by SnCl₂ in the presence of each extract. Thus, we suggest that the increased activity of the extracts was associated with the presence of the non-complexed metal ion.

The effects of ROS scavengers (DMSO for O^•−₂ and ^•OH; NaN₃ and histidine for singlet oxygen¹⁹) and antioxidants (BHT and sodium benzoate³⁴) on the induction of DNA cleavage by the AE and HE were also investigated. The electrophoresis analysis is illustrated in Fig. 3 and the densitometry results are shown in Table 2. DNA cleavage fell sharply and significantly (P < 0.01) in the presence of ROS scavengers and antioxidants. These results indicate that the increased SnCl₂-induced DNA cleavage induced by the extracts was caused via ROS generation, but without generation of specific ROS.

Figure 3. Agarose gel electrophoresis of plasmid DNA (pUC18, 394 µg/ml) incubated for 1 hour at 37^oC with aqueous (A) and hydroalcoholic (B) extracts of S. uncinata at 900 µg/ml in the presence of SnCl₂ (200 µg/ml) with ROS scavengers or antioxidants: l-histidine 50 mM (his), DMSO 5%, NaN₃ 100 mM, BHT 20 mM, and sodium benzoate 2 M (benz). The direction of electrophoresis is from top to bottom. Supercoiled (I) and relaxed circular (II) forms are indicated. The gels are representative of experiments with similar results.

Table 2. Intensity (%, mean relative to total band density and standard deviation) of form II DNA (plasmid pUC18) treated with the aqueous (AE) and hydroalcoholic (HE) extracts of S. uncinata in the absence (wo) or presence (w) of SnCl₂ and without (–) or with (+) ROS scavengers and antioxidants

Lane^a	SnCl2	ROS scavengers or antioxidants^b		Extract
				AE	HE
1	wo^c	—	wo	0.0 ± 0.0	0.0 ± 0.0
2	w	—	wo	54.1 ± 4.2^d*,f	76.7 ± 9.1^d
3	w	—	w	99.2 ± 1.1^d	99.2 ± 1.2^d,e
4	w	Histidine	w	11.7 ± 3.3^f	34.2 ± 0.5^f
5	w	DMSO	w	0.3 ± 0.0^f	0.2 ± 0.2^e,f
6	w	NaN3	w	8.0 ± 7.1^f	0.0 ± 0.0^e,f
7	w	BHT	w	1.2 ± 1.7^f	0.0 ± 0.0^e,f
8	w	Benz	w	15.0 ± 9.4^f	8.0 ± 3.3^e,f

^aLane showed in the gels in Fig. 3. No form III was observed.

^bDetails of ROS scavengers or antioxidants, incubation conditions, and ROS inductor are described in Fig. 3 and text.

^cAbsence (wo) or presence (w) of SnCl₂ or extracts. Statistically significant differences (z test for proportions, P < 0.01 or *P < 0.05) relative to

^dnegative control (lane 1),

^elane 2, and

^flane 3. All experiments were performed in triplicate.

Conclusion

The aqueous and hydroalcoholic extracts of the moss S. uncinata induced neither plasmid DNA cleavage nor ROS generation in the presence of H₂O₂. Furthermore, our results also showed that the potential antioxidant activity of extracts of S. uncinata, which was previously shown elsewhere using conventional chemical antioxidant assays,⁷ effectively protected the plasmid DNA against cleavage induced by O₂^•− (by pyrogallol) and .OH (interanging with Fenton reaction); and that this effect is likely due to the presence of antioxidant phenolic compounds. On the other hand, both extracts intensified the DNA cleavage induced by Fenton-like reactions. EDTA, ROS scavengers, and antioxidants inhibited the enhancing effect of both extracts on Fenton-like reactions, which take place via in situ ROS generation. Nevertheless, DMSO did not inhibit the HE-induced plasmid DNA damage in the Cu²⁺/H₂O₂ system, suggesting that the HE increased the induction of DNA cleavage by Fenton-like reactions, and that this could have been via mechanisms other than ROS generation. For instance, the main sequences of events in this type of DNA damage starts with Cu²⁺ reducing to Cu⁺, which rapidly binds to DNA with high affinity.³⁵ The combination of this complex with H₂O₂ primarily produces DNA modifications,^36–37 such as 8-oxoguanine, and can be an important source of mutagenesis, carcinogenesis, and the aging process.^38–40

It has been suggested that the moss S. uncinata has potentially promising qualities for photoprotection and medical and cosmetic applications. Our results showed that the AE and HE of this moss had protective effects against plasmid DNA cleavage by ROS. However, potential risks in the direct use of these extracts were also identified, in view of the various possible combinations involving metal ions in formulations.

Acknowledgments

The authors wish to thank Dr Denise Pinheiro da Costa from the Botanical Garden Research Institute of Rio de Janeiro (Brazil) for the identification of the moss. This work was supported by CNPq (Brasília, Brazil) and Faperj (Rio de Janeiro, Brazil). We also thank PROANTAR (CNPq556971/2009–4) and INCT-Criosfera for allowing sampling in Antarctica.