Great differences in antioxidant properties exist between 56 apple cultivars and vegetation seasons

The contents of ascorbate, thiols, and phenolic compounds and antioxidative enzyme activity were measured in the apple peel of 56 genotypes after harvest in two vegetation seasons, 2003 and 2004. The main reason of great interest in these bioactive compounds is their well-established physiological role in all living systems. The biggest differences between tested genotypes were noted for ascorbate peroxidase and glutathione reductase (GR) activity, followed by total ascorbate, phenolics, and glutathione concentration; the least difference was observed in the case of catalase. A large cultivar variation was noted in the anthocyanins and flavonols contents. Distinguishing the cultivars with the lowest, highest, relatively stable or those in which antioxidant content greatly differed depending on growing seasons was attempted. The GR activity is proposed as an environmental stress marker of apple fruit.     

Apple peel as a contributor to whole fruit quantity of potentially healthful bioactive compounds. Cultivar and year implication

On the basis of the fresh weight of apple fruit and its peel and the concentration of bioactive compounds, the total quantity of L-cysteine, glutathione, ascorbate, flavonols, and anthocyanins as well as phenolics was evaluated in a wide range of cultivars and two growing seasons. Apple peel as a contributor to the entire apple quantity of the examined components considerably differed in relation to the investigated compounds and was also highly cultivar dependent. A great amount of flavonols was found in apple peel (approximately 40%), followed by ascorbate (approximately 30%) and total phenolics (approximately 20%), while the lowest contribution was assessed for thiols (approximately 11% and 14% for L-cysteine and total quantity of glutathione, respectively), based on average values for both years. Seasonal variations in the quantity of antioxidants was more pronounced in apple peel, whereas the contribution of apple peel to the whole fruit was predominantly affected by the genotype. A very high positive correlation existed between apple peel and the whole fruit quantity of antioxidants.     

Active oxygen detoxifying enzymes and phenylalanine ammonia-lyase in the ethylene-induced chilling tolerance in citrus fruit

The effects of applying ethylene (2 microL x L(-)(1)) during cold storage of Fortune mandarins on the development of chilling-induced peel damage and on changes in the activities of the enzymes of the antioxidant system, superoxide dismutase, catalase (CAT), ascorbate peroxidase, guaiacol peroxidase, and glutathione reductase, and on phenylalanine ammonia-lyase (PAL) have been investigated. Chilling damage was reduced by applying ethylene during fruit storage at 1.5 degrees C. PAL activity increased in response to cold stress and was higher in fruit held under ethylene than under air during the whole storage period, whereas CAT was temporarily higher in ethylene-treated fruit. In contrast, the activities of the other enzymes were not increased by ethylene. The global results suggest that the ethylene-induced chilling tolerance in Fortune mandarins might be due to increased PAL and CAT activities.     

Changes in oxygen-scavenging systems and membrane lipid peroxidation during maturation and ripening in blackberry

Maturation and ripening of blackberry (Rubus sp.) fruit was accompanied by decreased activities of oxygen-scavenging enzymes [superoxide dismutase (EC 1.15.1.1), glutathione-peroxidase (EC 1.11.1.9), catalase (EC 1.11.1.6)] and enzymes in the ascorbate-glutathione cycle [ascorbate peroxidase (EC 1.11.1.11), monodehydroascorbate reductase (EC 1.6.5.4), dehydroascorbate reductase (EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2)]. Nonenzyme components in the ascorbate-glutathione cycle such as ascorbate (AsA), dehydroascorbate (DHAsA), glutathione (GSH), and oxidized glutathione (GSSG) and the ratios of AsA/DHAsA, GSH/GSSG were also decreased. These decreases in antioxidant capacity were correlated with increases in the ratios of saturated to unsaturated fatty acid of polar lipids and free sterols to phospholipids, thus contributing to decreased fluidity, enhanced lipid peroxidation, and membrane deterioration, which may be associated with ripening and senescence in blackberry fruit.     

Apple and pear peel and pulp and their influence on plasma lipids and antioxidant potentials in rats fed cholesterol-containing diets

The aim of this study was to assess the bioactive compounds of apple and pear peel and pulp in vitro and their influence on plasma lipids and antioxidant potentials in vivo. The antioxidant potentials measured by 1,1-diphenyl-2-picrylhydrazyl (DPPH), beta-carotene bleaching (beta-carotene), and nitric oxide inhibition radical scavenging (NO) tests in apple peel and pulp were significantly higher than in pear peel and pulp, respectively. The ethanol extract of apple peels showed the strongest inhibition of lipid peroxidation as a function of its concentration and was comparable to the antioxidant activity of butylated hydroxyanisole. The pear pulp extract had the weakest antioxidant ability, whereas other extracts such as apple pulp and pear peel were nearly equal. The antioxidant activities comprised contributions from polyphenols, phenolic acids, and flavonoids and correlated well with polyphenols and flavonoids. The correlation coefficients between polyphenols and antioxidant activities by DPPH, beta-carotene, and NO were as follows: 0.9207, 0.9350, and 0.9453. Contrarily, the correlation coefficient between the content of dietary fiber and the antioxidant activities test was low. The content of all studied indices in apple and pear peel was significantly higher than in peeled fruits (p < 0.05). Diets supplemented with fruit peels exercised a significantly higher positive influence on plasma lipid levels and on plasma antioxidant capacity of rats than diets with fruit pulps.     

Effect of reddening-ripening on the antioxidant activity of polyphenol extracts from cv. 'Annurca' apple fruits

Apple is among the most consumed fruits worldwide, and several studies suggest that apple polyphenols could play a role in the prevention of degenerative diseases. 'Annurca' apple fruit undergoes, after harvest, a typical reddening treatment to turn the apples' skin red, and it is noted for its high firmness. This paper reports the effect of reddening-ripening treatment on polyphenol concentration and antioxidant activity of both peel and flesh extracts. The in vitro antioxidant properties have been compared with the protective effect against the cytotoxic effects of reactive oxygen species using Caco-2 cells as model system. Pretreatment of cells with different polyphenolic apple extracts provides a remarkable protection against oxidative damage. This effect seems to be associated with the antioxidant activity of 'Annurca' apple polyphenolic compounds. The flesh has antioxidant properties comparable to those possessed by the peel. Neither the reddening nor the fruit conservation causes changes in the antioxidant properties possessed by this apple variety. The data indicate that polyphenolic compounds in 'Annurca' apples are relatively stable in the peel and also in the flesh; therefore, the health benefits of polyphenols should be maintained during long-term storage. Finally, a diet rich in apple antioxidants could exert a beneficial effect in the prevention of intestinal pathologies related to the production of reactive oxygen species.     

Antioxidant systems and their relationship with the response of pepper fruits to storage at 20 degrees C

Fresh peppers (Capsicum annuum L., variety California) in their green and red ripe stages were stored at 20 degrees C for 7 and 19 days to determine the effects of storage on whole fruit antioxidant capacity (TAA) and ascorbate (ASC) content, as well as on some antioxidant enzyme activities, such as catalase (CAT), superoxide dismutase (SOD), and those of the ASC-glutathione cycle. At least one Mn-SOD, two Fe-SODs, and three CuZn-SODs were detected in the fruit extract after native polyacrylamide gel electrophoresis. All of the SOD isozymes and glutathione reductase had higher activity levels in the red control fruits than in the green fruits, whereas the activities of monodehydroascorbate and dehydroascorbate reductase were higher in green fruits. Ascorbate peroxidase (APX) was found to be similar in both fruits. SODs, CAT, and APX seem to be involved in pepper fruit ripening and senescence during storage at 20 degrees C, perhaps influencing the active oxygen species levels in the fruit. TAA, as well as the ASC content, was higher in red peppers than in green, and storage increased the ASC in both green and red fruits.     

Combined salicyclic acid and ultrasound treatments for reducing the chilling injury on peach fruit

The effects of salicylic acid (SA; 1 mmol L(-1)) and ultrasound treatment (40 kHz, 10 min) either separately or combined on the chilling injury (CI) in cold-stored peach fruit ( Prunus persica Batsch cv. Baifeng) were investigated. The results showed that SA treatment alone alleviated CI during storage. Ultrasound alone had no influence, but when it was combined with SA, it resulted in greater inhibition of CI than SA alone. The activities of antioxidant enzymes, such as catalase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase, were induced by a combination of SA with ultrasound. In addition, the combined treatment also increased the endogenous SA concentrations in peaches. These results suggested that the induced tolerance against CI by the combination of ultrasound and SA treatment in cold-stored peach fruit was related to the induction of antioxidant enzymes and the increase in the SA concentration.     

Pre-extraction preparation (fresh, frozen, freeze-dried, or acetone powdered) and long-term storage of fruit and vegetable tissues: effects on antioxidant enzyme activity

Activities of the antioxidant enzymes ascorbate peroxidase, catalase, dehydroascorbate reductase, glutathione reductase, guaiacol peroxidase, monodehydroascorbate reductase, and superoxide dismutase were assayed in honeydew (Cucumis melo L.) fruit and spinach (Spinacia oleracea L.) leaves either as fresh, frozen to -80 degrees C, frozen in liquid nitrogen, freeze-dried, or acetone powder, representing the various ways tissues are treated prior to enzyme extraction. Treated tissues were analyzed following treatment or stored for up to 8 weeks at -80 degrees C. Enzyme activities in fruit frozen with or without liquid nitrogen and leaves frozen with or without liquid nitrogen or freeze-dried were equal to those of fresh tissue. Enzyme activities in freeze-dried or acetone-powdered fruit and leaves and in acetone-powdered tissues were significantly higher or lower than those in fresh tissue. Enzyme activities in both tissues frozen with or without liquid nitrogen and stored for 8 weeks at -80 degrees C changed little; those in freeze-dried and acetone-powdered tissues, however, significantly increased/decreased over the same period. Fresh tissue should be used in antioxidant enzyme assays, but if storage is necessary, tissues should be placed directly into a -80 degrees C freezer.     

Major phytochemicals in apple cultivars: contribution to peroxyl radical trapping efficiency

Forty-one samples of apples (peel plus pulp), obtained from eight cultivars, were examined for concentration of some important phytochemicals and for antioxidant activity expressed as peroxyl radical trapping efficiency. Five major polyphenolic groups plus ascorbate were identified and quantified by HPLC in the apple varieties. Oligomeric and polymeric proanthocyanidins were found to be about two-thirds of total polyphenols. The antioxidant efficiency of the apple extracts and of representative pure compounds for each group of phytochemicals was measured in a micellar system mimicking lipid peroxidation in human plasma. Although the amount of polyphenols measured by HPLC is similar to that measured by standard methods, the antioxidant efficiency calculated on the basis of the contribution of the pure compounds was lower than the antioxidant efficiency of the apple extracts. The higher efficiency of apples appears to be strictly related to the overwhelming presence of oligomeric proanthocyanidins.     

Boron stress,oxidative damage and antioxidant protection in potato cultivars (Solanum tuberosum L.)

Six potato cultivars grown in Turkey in boron-prone areas and differing in their tolerance towards high boron were studied to reveal whether boron causes oxidative stress. To assess stress level, chlorophyll fluorescence and growth parameters were measured. Oxidative damage was assessed as malondialdehyde level, and antioxidant protection was evaluated as ascorbate (AA), dehydroascorbate, reduced glutathione (GSH) and oxidized glutathione amounts and superoxide dismutase, catalase, ascorbate peroxidase (APX) and glutathione reductase (GR) activities. High boron stress affected photosynthesis negatively in a threshold-dependent manner and inhibited growth. No pronounced changes in oxidation of lipids occurred in any cultivar. Activation of APX suggested the involvement of an ascorbic acid–reduced glutathione cycle in the protection against oxidative stress caused by high boron. Efficient work of this antioxidant system was probably hindered by boron complexation with NAD(P)⁺/NAD(P)H and resulted in the inhibition of GR and a decrease in AA and GSH. Hence, oxidative stress associated with high boron is a secondary component of boron toxicity which arises from metabolic changes caused by boron interference with major metabolites. Potato cultivars tolerate excess boron stress well and show damage only in very high boron concentrations. The potato cvs best suited for high boron soils/breeding purposes are cvs Van Gogh and Agria.

Abbreviations: AA: ascorbic acid; APX: ascorbate peroxidase; CAT: catalase; DHA: dehydroascorbic acid; DHAR: dehydroascorbate reductase; DTNB: 5; 5′-dithiobis-2-nitrobenzoic acid; DTT: dithiotreitol; F_v/F_m: photosynthetic efficiency at the dark-adapted state; GR: glutathione reductase; GSH: reduced glutathione; GSSG: oxidized glutathione; MDA: malondialdehyde; ROS: reactive oxygen species; SOD: superoxide dismutase; TCA: trichloroacetic acid     

Antioxidant capacity, vitamin C, phenolics, and anthocyanins after fresh storage of small fruits.

Fresh strawberries (Fragaria x ananassa Duch.), raspberries (Rubus idaeus Michx.), highbush blueberries (Vaccinium corymbosum L.), and lowbush blueberries (Vaccinium angustifolium Aiton) were stored at 0, 10, 20, and 30 degrees C for up to 8 days to determine the effects of storage temperature on whole fruit antioxidant capacity (as measured by the oxygen radical absorbing capacity assay, Cao et al., Clin. Chem. 1995, 41, 1738-1744) and total phenolic, anthocyanin, and ascorbate content. The four fruit varied markedly in their total antioxidant capacity, and antioxidant capacity was strongly correlated with the content of total phenolics (0.83) and anthocyanins (0.90). The antioxidant capacity of the two blueberry species was about 3-fold higher than either strawberries or raspberries. However, there was an increase in the antioxidant capacity of strawberries and raspberries during storage at temperatures >0 degrees C, which was accompanied by increases in anthocyanins in strawberries and increases in anthocyanins and total phenolics in raspberries. Ascorbate content differed more than 5-fold among the four fruit species; on average, strawberries and raspberries had almost 4-times more ascorbate than highbush and lowbush blueberries. There were no ascorbate losses in strawberries or highbush blueberries during 8 days of storage at the various temperatures, but there were losses in the other two fruit species. Ascorbate made only a small contribution (0.4-9.4%) to the total antioxidant capacity of the fruit. The increase observed in antioxidant capacity through postharvest phenolic synthesis and metabolism suggested that commercially feasible technologies may be developed to enhance the health functionality of small fruit crops.     

Utility of Metabolomics toward Assessing the Metabolic Basis of Quality Traits in Apple Fruit with an Emphasis on Antioxidants

A gas chromatography-mass spectrometry approach was employed to evaluate the use of metabolite patterns to differentiate fruit from six commercially grown apple cultivars harvested in 2008. Principal component analysis (PCA) of apple fruit peel and flesh data indicated that individual cultivar replicates clustered together and were separated from all other cultivar samples. An independent metabolomics investigation with fruit harvested in 2003 confirmed the separate clustering of fruit from different cultivars. Further evidence for cultivar separation was obtained using a hierarchical clustering analysis. An evaluation of PCA component loadings revealed specific metabolite classes that contributed the most to each principal component, whereas a correlation analysis demonstrated that specific metabolites correlate directly with quality traits such as antioxidant activity, total phenolics, and total anthocyanins, which are important parameters in the selection of breeding germplasm. These data sets lay the foundation for elucidating the metabolic basis of commercially important fruit quality traits.     

The impact of selenium application on enzymatic and non-enzymatic antioxidant systems in Zea mays roots treated with combined osmotic and heat stress

Selenium (Se), regarded as an antioxidant, has been found beneficial for plants growing under stressed conditions. To investigate whether the Se application helps to improve stress tolerance, sodium selenite (Na₂SeO₃ · 5H₂O, 5–15 μM) was hydroponically applied to Zea mays variety OSSK-713-roots under heat and/or PEG-induced osmotic stress (25% PEG-6000) for 8 h. The individual/combined stress caused accumulation of reactive oxygen species (ROS). While only superoxide dismutase (SOD) increased with heat stress alone, the activities of SOD, catalase (CAT) and ascorbate peroxidase (APX) increased under PEG exposure. The combination of these stresses resulted in an induction of both SOD and CAT activities. Lipid peroxidation (TBARS) levels were also high in all the stress treatments, especially under the combination treatment. Addition of Se not only improved the activities of SOD, APX and glutathione reductase (GR) in stress-treated roots, but it also changed the activities of monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR). The findings reveal that Se has a positive effect on heat and/or osmotic stress mitigation mainly by regulating the ascorbate-glutathione cycle, especially in PEG-treated plants. Under the combined stress treatment, addition of 5 µM of exogenous Se was most effective.     

Maturity and storage influence on the apple (Malus domestica) allergen Mal d 3, a nonspecific lipid transfer protein

Consumption of apples can provoke severe allergic reactions, in susceptible individuals, due to the presence of the allergen Mal d 3, a nonspecific lipid transfer protein, found largely in the fruit skin. Levels of Mal d 3 were determined in peel as a function of apple cultivar, position of the fruit growing on the tree, apple maturity, and postharvest storage by ELISA. As the apples mature, Mal d 3 levels increased, although the rate was dependent on cultivar and tree position. During storage, levels of Mal d 3 decreased in all cultivars (cvs. Cox, Jonagored, and Gala), the rate of overall decrease being greatest under controlled atmosphere conditions. There was no correlation between Mal d 3 levels and total apple peel protein, indicating specific alterations in Mal d 3 expression. Thus pre- and postharvest treatments (i.e., storage) can modify the allergen load in apple peel, the highest levels being found in overly mature and freshly harvested fruits.     

Modulation of oxidative damage due to salt stress using salicylic acid in Hordeum vulgare

Oxidative status of salicylic acid (SA) treated barley (Hordeum vulgare) plants grown under saline conditions was examined in a two-year field study during 2012–2013 and 2013–2014 growing seasons. Salinity caused a marked oxidative stress which was manifested as increased concentration of hydrogen peroxide (H₂O₂) and reduced grain yield of barley. Barley plants induced antioxidant system to tolerate salt stress, so that activities of four antioxidant enzymes: peroxidase, catalase, superoxide dismutase and glutathione reductase were enhanced by 22%, 28%, 57% and 44% in the first and by 33%, 20%, 45% and 68% in the second year, respectively. Foliar application of SA in concentration over 0.5 mM enhanced the activities of all four antioxidant enzymes and reduced H₂O₂ content and so enhanced yield. However, higher concentrations of SA reduced grain yield in salt stressed plants in both years, which could be attributed to the negative interaction of antioxidant enzymes with higher concentrations of SA as a non-enzymatic antioxidant. Furthermore, SA is in benzoic acid group and at very higher concentrations can act as an herbicide. It was concluded that SA modulated the oxidative stress through enhanced activities of antioxidant enzymes and reduced the salt-induced adverse effect thereby improving grain yield.     

Apple peels as a value-added food ingredient

There is some evidence that chronic diseases, such as cancer and cardiovascular disease, may occur as a result of oxidative stress. Apple peels have high concentrations of phenolic compounds and may assist in the prevention of chronic diseases. Millions of pounds of waste apple peels are generated in the production of applesauce and canned apples in New York State each year. We proposed that a valuable food ingredient could be made using the peels of these apples if they could be dried and ground to a powder without large losses of phytochemicals. Rome Beauty apple peels were treated with citric acid dips, ascorbic acid dips, and blanches before being oven-dried at 60 degrees C. Only blanching treatments greatly preserved the phenolic compounds, and peels blanched for 10 s had the highest total phenolic content. Rome Beauty apple peels were then blanched for 10 s and dried under various conditions (oven-dried at 40, 60, or 80 degrees C, air-dried, or freeze-dried). The air-dried and freeze-dried apple peels had the highest total phenolic, flavonoid, and anthocyanin contents. On a fresh weight basis, the total phenolic and flavonoid contents of these samples were similar to those of the fresh apple peels. Freeze-dried peels had a lower water activity than air-dried peels on a fresh weight basis. The optimal processing conditions for the ingredient were blanching for 10s and freeze-drying. The process was scaled up, and the apple peel powder ingredient was characterized. The total phenolic content was 3342 +/- 12 mg gallic acid equivalents/100 g dried peels, the flavonoid content was 2299 +/- 52 mg catechin equivalents/100 g dried peels, and the anthocyanin content was 169.7 +/- 1.6 mg cyanidin 3-glucoside equivalents/100 g dried peels. These phytochemical contents were a significantly higher than those of the fresh apple peels if calculated on a fresh weight basis (p < 0.05). The apple peel powder had a total antioxidant activity of 1251 +/- 56 micromol vitamin C equivalents/g, similar to fresh Rome Beauty peels on a fresh weight basis (p > 0.05). One gram of powder had an antioxidant activity equivalent to 220 mg of vitamin C. The freeze-dried apple peels also had a strong antiproliferative effect on HepG(2) liver cancer cells with a median effective dose (EC(50)) of 1.88 +/- 0.01 mg/mL. This was lower than the EC(50) exhibited by the fresh apple peels (p < 0.05). Apple peel powder may be used in a various food products to add phytochemicals and promote good health.     

Orchard studies utilizing fluidized bed material

Abstract

Fluidized bed material (FBM, a coal/limestone combustion byproduct) was used as a Ca source and lime substitute in established apple (Malus domestica Borkh) and peach (Prunus persia L.) orchards. FBM or limestone had little effect on apple tree Ca status over three growing seasons. Peach leaf Ca concentrations were significantly greater from FBM applied at three times the soil lime requirement (on a weight basis) than limestone applied at the lime requiremnt or a non‐amended control. Peach peel and flesh Ca, however, were not significantly altered. Tissue trace element concentrations (Mn, Fe, Cu, Zn, B ,Al, Sr, Pb) were not affected by treatments. FBM applied at twice the lime requirement, on a weight basis, maintained soil pH at levels equivalent to agricultural limestone applied at the lime requirement during the three growing seasons in both studies. FBM was found to be a satisfactory substitute for agricultural limestone under orchard conditions and when applied at mutiples up to three times the soil lime requirement.     

Comparative analysis of polyphenolic profiles and antioxidant and antimicrobial activities of tunisian pome fruit pulp and peel aqueous acetone extracts

Pome trees, apple, pear, and quince, are classified into the subfamily Pomoideae, belonging to the Rosaceae family. Their autumnal fruits are consumed worldwide in different forms, that is, fresh or transformed into jams, jelly, juices, etc. Their well-established beneficial properties to human health were found mainly related to their phenolic content. Pulp and peel aqueous acetone extracts obtained from Tunisian fruits at commercial maturity were comparatively evaluated for their phenolic profiles and antioxidant and antimicrobial potentials. The phenolic compounds present in the extracts were identified and quantified using RP-HPLC-DAD and ESI-MS techniques. Significant differences in the chromatographic profiles among these fruits, as well as between pulp and peel extracts of each fruit, were observed. Quince, followed by 'Red Delicious', peel extracts showed the highest phenolic content (160.33 and 110.90 mg/100 g of fresh weight). The stronger inhibitory effect on DPPH radicals corresponded to those obtained from peel materials. A comparative analysis of the antimicrobial potential against a range of microorganism strains was also carried out. Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus cereus were the most sensitive to the active extracts. Among the examined phenolic extracts, 'Red Delicious' and quince peels showed the highest effects for inhibiting bacteria growth. Minimum inhibitory and bactericide concentrations ranged from 10(2) to 10(4) microg of polyphenol/mL. Red skin apple and quince peels could be of great interest as important antioxidant and antimicrobial polyphenol sources.     

Responses of Components of Antioxidant System in Moongbean Genotypes to Cadmium Stress

Four genotypes (Pusa 9531, Pusa 9072, Pusa Vishal, PS‐16) of moongbean [Vigna radiata (L.) Wilczek] grown in earthen pots were treated with cadmium at 0, 25, 50, and 100 mg kg^?1 soil. Cadmium tolerance (CdT), the ability of a plant to maintain growth at high levels of cadmium (Cd), was calculated as the ratio of dry‐matter production in the untreated and the Cd‐treated soils. The moongbean genotypes showed a differential response to Cd concentrations; Pusa 9531 was identified as Cd tolerant, whereas PS 16 was Cd susceptible. To find out the physiological basis of these differences, we investigated the possible role of antioxidant (enzymatic and nonenzymatic) defense systems. Activities of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6), ascorbate peroxidase (EC 1.11.1.11), and glutathione reductase (EC 1.6.4.2) and the amounts of ascorbate and glutathione were monitored in the Cd‐tolerant and Cd‐sensitive moongbean genotypes. The results revealed the presence of a strong antioxidant defense system in the Cd‐tolerant genotype (Pusa 9531) for providing adequate protection against oxidative stress caused by Cd.