ROMANIAN JOURNAL OF PHYSICS

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mushrooms were collected according with Codex Methods of Sampling [9]. For each species were sampled several individuals from the same location, but also.
DETERMINATION OF SEVERAL ELEMENTS IN EDIBLE MUSHROOMS USING ICP-MS ANDREEA ANTONIA GEORGESCU1,6, ANDREI FLORIN DANET2, CRISTIANA RADULESCU3, CLAUDIA STIHI3, IOANA DANIELA DULAMA4, DANIELA ELENA CHELARESCU5 1

Valahia University of Targoviste, Faculty of Environmental Engineering and Food Science, 130004 Targoviste, Romania E-mail: [email protected] 2 University of Bucharest, Faculty of Chemistry, 050657 Bucharest, Romania 3 Valahia University of Targoviste, Faculty of Sciences and Arts, 130024 Targoviste, Romania 4 Valahia University of Targoviste, Multidisciplinary Research Institute for Sciences and Technologies, 130004 Targoviste, Romania 5 Horia Hulubei Institute of Physics and Nuclear Engineering, 077125 Magurele, Romania 6 University of Bucharest, Doctoral School in Chemistry, 050657, Bucharest, Romania Corresponding author: Cristiana Radulescu E-mail: [email protected] Received July 23, 2015 The determination of elements content in the fruiting bodies of mushrooms is essential in dietary intake studies, because this aliment is used in the diet of many countries. In this study the contents of several elements, including Mn, Fe, Cu, and Zn, in cap and stipe of ten edible mushroom species, were determinate by using ICPMS technique. These edible mushrooms species were collected from four sites of Dambovita County, Romania. After this study it was observed that mushrooms accumulate elements in different levels, in caps and stipes, depending on its species. It is well known that some elements cause a metal stress in the cells, resulting in the formation of ROS, survival of the microorganism in this stress is attributed to the induced biosynthesis of enzymes responsible for antioxidant defense. Key words: edible mushrooms, metal content, ICP-MS, ROS, peroxidase.

1. INTRODUCTION

Reactive oxygen species (ROS) are continuously produced at low level during norMAC metabolic processes [1]. All aerobic organisms generate reactive oxygen species, especially through aerobic respiration [1, 10]. But in biological systems, increasing the synthesis of ROS is one of the initial responses to different stress factors [2, 5, 7]. Reactive oxygen species are formed during normal cellular metabolism, but when present in high concentration they become toxic. ROS induce damage to the biomolecules through peroxidation of Rom. Journ. Phys., Vol. 61, Nos. 5–6, P. 1087–1097, Bucharest, 2016

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membrane lipids, alteration of protein functions, DNA mutation, damage to chlorophyll and disruption of some of metabolic pathways (electron transport chain and ATP production) [14]. Superoxide, hydrogen peroxide and hydroxyl radicals, which are mutagens produced by radiation, are also by-products of normal metabolism. Therefore the tolerance of mushrooms to stress conditions depends on their ability to make balance between the production of toxic oxygen derivatives and capacity of antioxidative defense systems. Mushrooms have complex ROS scavenging mechanisms at the molecular and cellular levels. These mechanisms with inhibition or slow the oxidation of biomolecules and oxidative chain reactions [4, 12] decrease the cellular oxidative damage and increase resistance to heavy metals. The mushroom antioxidant defense systems include antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POX), catalase (CAT). SOD catalyzes the dismutation of superoxide anion to hydrogen peroxide and molecular oxygen. Hydrogen peroxide is degraded by CAT and POX, enzymes that act synergistically to protect cells. CAT is an enzyme able to catalyze the breakdown of hydrogen peroxide (H2O2) to water and oxygen. The search for new products with antioxidant properties is a very active research field. Mushrooms have been used as food and food-flavouring material in soups and sauces for centuries, due to their unique and subtle flavour. Recently, they have become attractive as functional foods and as a source of physiologically beneficial medicines, while being devoid of undesirable side-effects [5]. Mushrooms were also found to be medically active in several therapies, such as antitumor, antibacterial, antiviral, hematological and immunomodulating treatments. In particular, mushrooms, useful against cancers of the stomach, esophagus, lungs, etc., are known in China, Japan, Korea, Russia, United States and Canada. The antioxidative and free radical scavenging properties of the phenolic content of mushroom methanolic extracts have been reported, suggesting possible protective roles of these compounds, due to their ability to capture metals, inhibit lipoxygenase and scavenge free radicals [16]. Transition metals that may undergo redox cycling, such as Cu, Fe, Zn and Mn, may act as potent catalysts in some of the reactions generating ROS [17]. These elements are essential for the human metabolism but in low concentrations, because they are enzyme activators. They become toxic in the situation of increasing their concentrations too much. In the same time mushrooms are natural indicators of pollution. Mushrooms easily accumulate metals, pesticides, radioactive substances. A chemical analysis of fungi can show the state of pollution of a habitat. Thus, research on the

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remediation environment deals with the development of biotechnology based on reducing the metal content in soil with mushroom crop. Composition and content of metals in enzymes of fungi differ by species, ontogenetic stage of development. They are correlated with different parts (cap, stipe) of the fruiting body, microclimate conditions, and nutritional substrate on which it grows. In this study the biological material is part of the families: Russulaceae, Lepiotaceae, Tricholomataceae, Pleurotaceae, Cantharellaceae, Boletaceae, all belonging to Basidiomycota order. Harvested mushrooms were edible species, such as Cantharellus cibarius, Russula alutacea, Russula atropurpurea, Russula cyanoxantha, Russula nigrescens, Macrolepiota procera, Macrolepiota excoriata, Boletus edulis, Armillaria mellea, and Pleurotus ostreatus. Mushroom species were collected from different sites of Dambovita County (i.e. Adanca, Ungureni, Manesti, and Picior de Munte). For analyses were chosen only cap and stipe from each selected mushroom species. This research aims to investigate the effects of four elements, including Mn, Fe, Cu, and Zn, on some biomarkers of oxidative stress in order to study relationship between metal toxicity and oxidative stress. The results have indicated if the defense systems against free radicals fungus were induced by different concentrations of metals. In this investigation the content of elements (i.e. Mn, Fe, Cu, and Zn) were determined by inductively coupled plasma-mass spectrometer (ICP-MS). POX and CAT activity of fungi was determined in order to find correlations between the oxidoreductase activity and concentration of heavy metals. It was found that POX activity was low in metal hyperaccumulators species; in contrast, CAT activity was slightly influenced.

2. MATERIALS AND METHODS

2.1. DESCRIPTION OF SITES

Sampling points were chosen such that: to rural areas without industrial pollution point sources (factories, plants), is not close to roads and roads with car traffic, do not fall under the possible pollution of agricultural products (pesticides, fertilizers) (Fig. 1).

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Fig. 1 – Sampling points in Dambovita County.

2.2. SAMPLING PROCEDURE

Ten mushroom species were collected. Details concerning the families and the edibility about the analyzed mushroom species are presented in Table 1. The mushrooms were collected according with Codex Methods of Sampling [9]. For each species were sampled several individuals from the same location, but also from different locations (n = 5). Samples were collected in order to contain all the fruiting body: cap, stipe, blades, and cuticle. They were taken for analysis samples stipe and cap for each species. Freshly collected fruiting bodies of mushrooms were carefully cleaned of vegetal wastes and soil using deionized water and were cut with a plastic knife in small pieces. After that, the samples were dried at 60°C between 24 and 30 hours (Binder drying system) until the total elimination of water from tissues, then grinded until to fine powder and finally weighed. Thus, the obtained powders were weighed and then analyzed by ICP-MS spectrometry.

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Table 1 Species of mushrooms used for analysis Family

Genus

Russulaceae

Russula

Lepiotaceae

Macrolepiota

Tricholomataceae Pleurotaceae Cantharellaceae Boletaceae

Armillaria Pleurotus Cantharellus Boletus

Mushrooms species Russula nigrescens Russula cyanoxantha Russula alutacea Russula atropurpurea Macrolepiota procera Macrolepiota excoriata Armillaria mellea Pleurotus ostreatus Cantharellus cibarius Boletus edulis

Edibility Edible Edible Edible Edible Edible Edible Edible Edible Edible Edible

2.3. ICP-MS TECHNIQUE

Sample preparation About 200 mg powdered samples (stipe and cap for each mushroom species) were digested with 5 mL hydrogen peroxide (31% H2O2 Sigma Aldrich) and 3 mL nitric acid (65% HNO3 Sigma Aldrich) in Teflon vessel using TOPwave microwave-assisted pressure digestion system (Analytik Jena – Table 2). Table 2 The parameters of digestion process Steps 1 2 3 4 5

Temperature (°C) 170 200 50 50 50

Pressure (bar) 50 50 0 0 0

Ramp 5 1 1 1 1

Time (minutes) 10 15 10 10 1

Power (W) 90 90 0 0 0

Then, after digestion the vessels have cooled at room temperature (about 30 minutes). The solutions were filtered and brought to 50 mL graduated flasks with deionized double-distilled water (produced by Thermo Scientific UV/UF system). Sample analysis The Cu, Zn, Fe, and Mn contents in the samples of mushrooms were determined using a Thermo Scientific iCAP Qc ICP-MS system. The system of

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sample introduction consist of low flow nebulizers, cyclonic spray chambers and wide bore injectors that ensure the highest plasma robustness. Water cooled Peltier is supplied as standard to ensure the highest sample introduction stability. All quantitative measurements in triplicates were performed in the standard mode (STD) using the instruments software Qtegra and the relative standard deviation (RSD) values was less than 10%. This software allows an automated interpretation of the interest spectrum. Several well-known isobaric interferences were automatic corrected. The oxide levels were kept below 2%. The instrumental analysis and data acquisition parameters are described in detail in Table 3. All samples contents were reported as mg/kg dried weight material. Table 3 Instrumental analyses and data acquisition parameters Instrumental Parameters Plasma Power Nebulizer Ar flow Plasma Ar flow Sample uptake rate

Sensitivity (kcps/ppb)

1548.6 W

7

1 L/min

59

10.75 L/min

115

0.4 mL/min

238

Data acquisition parameters quantitative mode

Li

50

Co

100

In

220

U

300

Detection Limits (ppt) 9

Be

< 0.5

standard

115

In

< 0.1

Point per peak

1

209

Bi

< 0.1

Integration time

3000 ms

Replicates

3

Measuring mode

Oxides (%) CeO/Ce

< 2.00

2.4. DETERMINATION OF PEROXIDASE ACTIVITY

The chosen method is a colorimetric method and is based on property POX to oxidize in the presence of hydrogen peroxide or other peroxide compounds such as aromatics. To determine the POX activity using fresh biological material (5 g sample for each species of mushrooms) and POX activity was determined by reading absorbance at 420 nm wavelength, using SPEKOL device. The unit of POX activity was defined as the oxidation of one micromole H2O2 per minute at 25°C (pH = 7.0).

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2.5. DETERMINATION OF CATALASE ACTIVITY

To determine the activity of CAT, the enzyme extract obtained from vegetable raw material (5 g) is placed to act on a certain amount of hydrogen peroxide at room temperature for few minutes. Then, in an acid medium, unconverted hydrogen peroxide was titrated with potassium permanganate. The unit of CAT activity was defined as the amount of enzyme, which decomposes one micromole H2O2 per minute at 25°C, according to the method.

3. RESULTS AND DISCUSSION

Minerals enter into the composition of chemical compounds with structural role, but have great physiological importance, as activators or inhibitors of the enzymatic systems or components of enzymes, coenzymes, and so on [8]. Mineral content is characteristic for a species or a plant organ and varies quite large, depending on climatic factors, soil and crop technology. Measurements performed on selected species of mushrooms shows that they are rich in Cu, Zn, Fe, Mn, metals that have a role in activation of enzymes and enzyme systems. Metals are important for living organisms, but, if they exceed a certain level after bioaccumulation, are toxic [8]. The mean of heavy metals concentrations in species edible mushrooms parts are presented in Table 4. From Table 4 and Figure 2 (a) it can see that the copper concentration exceeded the maximum admitted concentration (MAC) according with the MAC values (e.g. Ord. 975/1998 from Romanian legislation [20] and JECFA 2005) for fresh or congealed vegetables/raw vegetable leaves (5 mg/kg) in all analyzed samples. Maximum permissible contents for copper are in Pleurotus ostreatus species, both in cap and stipe. Zinc is a good nutrient for vegetable, which help the chlorophyll formation in plant leaves. The zinc concentration was exceeded the MAC value according with Ord. 975/1998 and JECFA 2005, in most of the samples (except Macrolepiota excoriata, Macrolepiota procera, Russula alutacea and Cantharellus cibarius stipe species). The comparison of results from present study to the published literature revealed that for mushroom species studied, the contents of cooper and zinc are higher [6, 18, 19]. A highest level of zinc was found in Russula atropurpurea (105.54 mg/kg in cap and 100.81 mg/kg in stipe) and Russula nigrescens stipe species 94.81 mg/kg for Zn (Table 3 and Figure 2 b), but these species having lower contents of Fe and Mn (Figure 3). From Figure 2 it can see that the majority of mushroom species analyzed have accumulated higher contents of copper in cap than stipe. However, zinc was accumulated at similar contents in both parts of the mushrooms, except Russula nigrescens species.

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Table 4 Mean of heavy metals concentrations in species edible mushrooms parts Component parts Russula nigrescens Cap Stipe Russula cyanoxantha Cap Stipe Russula alutacea Cap Stipe Russula atropurpurea Cap Stipe Macrolepiota excoriate Cap Stipe Macrolepiota procera Cap Stipe Armillaria mellea Cap Stipe Pleurotus ostreatus Cap Stipe Boletus edulis Cap Stipe Cantharellus cibarius Cap Stipe MAC for fresh or congealed vegetables / raw vegetable leaves (Ord. 975/1998 and JECFA 2005 RSD [%] Mushrooms species

(a)

Mean of heavy metals concentrations [mg/kg d.w.] Mn Fe Cu Zn 34.21 107.03 13.10 25.41 57.41 141.30 6.72 94.81 32.21 107.98 23.54 42.91 7.81 24.58 13.80 27.42 26.76 118.17 14.19 15.11 19.58 130.88 13.28 17.84 36.35 192.32 8.58 105.55 18.81 48.96 17.28 100.81 45.68 164.86 48.37 16.76 102.51 603.93 35.55 12.96 14.04 49.77 36.69 16.62 20.47 715.14 12.27 10.27 31.87 454.89 10.06 14.34 49.59 489.67 8.94 11.32 4.73 31.61 5.62 34.92 1.97 22.69 5.04 20.19 12.65 38.89 21.82 26.62 130.72 178.8 17.02 24.71 6.98 36.73 7.585 6.91 15.43 172.96 21.29 26.04 –



5.0

15.0

0.43–2.91

0.55–5.92

0.45–3.65

0.44–4.62

(b)

Fig. 2 – Metal concentrations in selected edible mushroom species: (a) Cooper concentration; (b) Zinc concentration.

In macromycete species, Mn and Fe accumulates differentially higher contents being found in the body of fructification stipe Russula species, except species Russula nigrescens, as can be seen in Figure 3 (a) and (b). High

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concentrations of Mn and Fe are stipe species Macrolepiota excoriata (102.51 mg/kg for Mn; 603.94 mg/kg for Fe), Boletus edulis (130.73 mg/kg for Mn) and Macrolepiota procera (715.14 mg/kg for Fe). In Romanian legislation is not specified the MAC for manganese and iron in vegetables.

(a)

(b)

Fig. 3 – Metal concentrations in edible mushroom species: (a) Manganese concentration; (b) Iron concentration

CAT is important antioxidant enzyme which converts H2O2 to water in the peroxisomes [13]. In this organelle, H2O2 is produced from β-oxidation of fatty acids and photorespiration [13]. Higher activity of CAT decreases H2O2 level in cell and increases stability of membranes and CO2 fixation, because several enzymes of Calvin cycle within chloroplasts are extremely sensitive to H2O2. High level of H2O2 directly inhibits CO2 fixation [11]. This enzyme by catalyzing H2O2 to H2O and O2 via two-electron transfer [15] prevent the generation of OH• and protect proteins, nucleic acids and lipids against ROS.

Fig. 4 – Catalase and peroxidase activities in edible mushrooms species studied.

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From Figure 4 is seen as catalase activity is increased in species of mushrooms that have accumulated copper levels over the maximum admitted level (Macrolepiota excoriata, Macrolepiota procera, Russula cyanoxantha, Russula atropurpurea, Chantarellus cibarius). Because heavy metals cause a metal stress in the cells resulting in the formation of ROS, survival of the microorganism in these stresses is attributed to the induced biosynthesis of enzymes responsible for antioxidant defense. Peroxidase activity is lower in mushroom species Macrolepiota excoriata and Armilaria mellea, probably due to high contents of cooper, zinc and iron that inhibit its activity. Catalase activity was not influenced by the high levels of these metals.

4. CONCLUSIONS

From this study it can see that the copper and zinc concentrations exceeded the maximum admitted concentrations (MAC) according with MAC values from Romanian legislation (i.e. Ord. 975/1998) and from JECFA 2005, for fresh or congealed vegetables/raw vegetable leaves (5 mg/kg, respectively 15 mg/kg) in all analyzed samples. Maximum permissible levels for cooper are in Pleurotus ostreatus species, both in cap and stipe, while zinc has allowed concentrations in species Macrolepiota excoriata, Macrolepiota procera and Armilaria mellea. High concentrations of Mn and Fe are stipe species Macrolepiota excoriata, Boletus edulis and Macrolepiota procera. The main cause in the variation of different activity of the detoxification enzymes (POX and CAT) can be that they exist in different parts of the cell and having different threshold tolerance to metals. The obtained results showed that these mushrooms have a high tolerance to different heavy metals stress and can survive under high levels of these metals. Excessive accumulation of some metals cause oxidative stress in the cell, results in the formation of ROS. In conditions of oxidative stress, antioxidant enzyme activity increases in all species of studied mushrooms.

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