Cadmium-binding protein components of flaxseed: Influence of cultivar and location

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Abstract

The distribution of cadmium-(Cd)-binding components from five flaxseed cultivars grown at three locations in southern Manitoba was investigated to examine genotypic and environmental effects. Three protein fractions with different electrostatic properties, eluting at 0.10, 0.25 and 0.50 M NaCl by ion-exchange chromatography DEAE-Sephacel, represented 12%, 66% and 7% of the bound (extracted) protein, respectively, while 15% of the protein remained unbound. Cadmium and other divalent metal (zinc, copper and calcium) contents of protein fractions were strongly influenced by location. Cultivar differences in protein and cadmium contents of the protein fractions were highly significant. Cadmium and zinc accumulated similarly in the 0.10 and 0.25 M protein fractions at 51% and 40–43%, respectively. Transfer of copper occurred prominently in the 0.50 M fraction while most of the calcium (55%) remained unbound. The distribution of cadmium, zinc, copper and calcium in fractions of flaxseed proteins was strongly influenced by cultivar and location, indicating differences in their accumulation, migration and transfer.

Introduction

Cadmium (Cd) absorbed from the soil and translocated in plants can enter the food supply, thereby presenting a food safety concern. High intake of Cd (over the provisional tolerable weekly intake of 400–500 μg per individual) by humans results in excessive Cd loads that may lead to impairment of kidney function and other chronic toxicities (FAO/WHO, 1995). The toxicity of cadmium is still not completely understood and different processes are apparently involved (Romero-Puertas, Palma, Gomez, Del Rio, & Sandalio, 2002). These mechanisms include lipid peroxidation, inhibition of chlorophyll biosynthesis, oxidative stress induced by oxygen free radical production or decreased antioxidants. Cadmium produces a concentration-dependent imbalance in the antioxidant status of pea plants, resulting specifically in depressed superoxide dismutase and catalase activities that, in turn, increase lipid peroxidation rate. Cadmium treatment also impedes the cellular antioxidant defence system by reducing copper zinc superoxide dismutase (CuZn-SOD) activity by 95%, probably due to modifications of CuZn-SOD biosynthesis at translational or transcriptional level (Romero-Puertas et al., 2002). The FAO/WHO (1995) provisional guideline level of 0.1 ppm for Cd in flaxseed is currently under debate.

On the positive side, a recent report (Waalkes & Diwan, 1999) indicates that cadmium can effectively inhibit tumor formation in the liver and lung in mice when administered at non-toxic doses and even when given well after tumor formation. Cadmium dose-dependently reduces tumor growth and metastasis of human lung carcinoma xenografts, independent of apoptosis. Therefore, cadmium-induced tumor suppression could be accomplished with non-toxic doses, which would be a positive attribute for any cancer chemotherapy. The cytotoxic impact of cadmium is often a function of the level of cellular metallothionein, a cadmium-inducible metal-binding protein encoded by the MT gene which sequesters cadmium and thereby mitigates its toxicity (Masters et al., 1994, Waalkes and Goering, 1990). Studies cited by Waalkes and Diwan (1999) have shown that metallothionein is poorly expressed in liver and lung tumors from mice, in human hepatocellular carcinomas and pulmonary small cell carcinoma, possibly predicting a common sensitivity to cadmium. The cytotoxic effect of cadmium is often a function of the level of cellular metallothionein; low methallothionein levels generally correspond to high cadmium-induced cytotoxicity (Waalkes & Diwan, 1999). Therefore, the hypersensitivity of liver tumors, and possibly lung tumors, may be due to poor expression of metallothionein in the tumor cells.

In flax, a high concentration of Cd, exceeding the dietary critical value or maximum level of 0.3 ppm (Marquard, Bohm, & Freidt, 1990), is known to accumulate in the seeds, even at low soil Cd level. Further, cadmium accumulation in flaxseed is genotype- and environment-dependent (Becher et al., 1997, Grant et al., 2000, Li et al., 1997). However, the underlying mechanism of the genotypic difference, as well as the distribution of Cd in flaxseed is still unclear. Our previous study (Li-Chan, Sultanbawa, Losso, Oomah, & Mazza, 2002) elucidated the presence of Cd-binding components in protein extracts of dehulled and defatted flaxseed cultivar NorMan with phytochelatin-like components present in two fractions eluting at high salt concentrations of 0.45 and 0.50 M NaCl. A recent investigation (Lei, Li-Chan, Oomah, & Mazza, 2003) of the distribution of Cd in flaxseed protein of cultivar NorMan showed that about 80% and 73% of the extracted proteins and cadmium, respectively, from flaxseed bound to the ion-exchange column. The Cd-binding protein was distributed mainly in the 0.10 and 0.25 M fractions with 66% and 25% of the Cd, respectively. Distribution of zinc was similar to that of cadmium with 44%, 36% and 20% of eluted zinc in the 0.10, 0.25 M NaCl and unbound fractions, respectively. The copper contents were approximately equally distributed (∼41%) in the 0.10 and 0.25 M fractions. Calcium was distributed in every protein fraction with the highest and lowest contents, 51% and 11%, in the unbound and 0.10 M NaCl fraction, respectively.

The present study is a continuation of our earlier investigations (Lei et al., 2003, Li-Chan et al., 2002) aimed at determining the genotypic difference and distribution of Cd in flaxseed. Since cadmium can affect the activity of metalloenzymes, CuZn-SOD, in particular (Romero-Puertas et al., 2002), the contents of copper, zinc and calcium in flaxseed protein fractions were also examined. Information on the genotypic and environmental effects of cadmium accumulation and distribution in flaxseed is paramount in the long-term objective of reducing the risk of Cd toxicity and ensuring the continued safety with increased flaxseed utilization in functional foods and the nutraceutical industry.

Section snippets

Source of materials and sample preparations

Tris Ultrapure [Tris-(hydroxymethyl)aminomethane] was from ICN Biomedicals, Inc., Costa Mesa, CA. Sodium chloride and hydrochloric acid (ACS certified) were from Fisher Scientific, Nepean, ON, Canada. 2-Mercaptoethanol electrophoresis reagent was from Sigma–Aldrich Canada Ltd., Oakville, ON, Canada. Bicinchoninic acid (BCA) protein assay reagents A and B were from Pierce Chemical Company, Rockford, IL. The deionized distilled water (∼18 MΩ) used for all experiments was produced by a Barnstead

Results and discussion

Average protein content of flaxseed cultivars grown at three locations in southern Manitoba was 19% (N × 6.25) (Table 1). Processing, particularly lipid removal (defatting), more than doubled the protein content. The amount of protein loaded on the column ranged from 472 to 609 mg and differed significantly among cultivars, AC McDuff and Flanders, in particular. The highest amount of bound protein was eluted by 0.25 M NaCl, followed by 0.10 and 0.50 M NaCl. The protein content of the major fraction

Acknowledgements

This research was funded by grants from the Flax Council of Canada, the Natural Sciences and Engineering Research Council of Canada and Agriculture and Agri-Food Canada.

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