Kirk, Charlotte
(2024)
The bioactive properties of selenium enriched Allium sativum (garlic).
PhD thesis, University of Nottingham.
Abstract
Selenium (Se) is an essential micronutrient for human health, low Se status has been found to predispose people to multiple health conditions. Se is obtained through dietary sources, however intake of Se is decreasing within the UK population, due to multiple factors including low Se soils and shifts in dietary patterns. Crops are generally low in Se, biofortification methods aim to increase the Se content. However, there is a narrow therapeutic window of Se supplementation and the toxicity of Se is poorly understood therefore there is concern that enriching food crops with Se without further understanding of toxicity could have adverse effects. The aim of this work was to enrich garlic, a widely consumed food crop with Se, and assess the bioactivity of the plant tissues and further the understanding of Se toxicity mechanisms.
Results found that Se foliar application of 25μM or 50μM of sodium selenate had no significant effect on the majority of growth measures (p<0.05). Although significant differences in clove weight were seen with increasing Se concentration of the cultivars Lyubasha, Bulbils and Solent (p<0.05) as well as increased pseudostem diameter in the cultivar Solent at 25μM Se application (p=0.034), however none of these differences impacted significantly on overall bulb weight. Foliar application was found to not significantly impact on Se accumulation of any of the cultivars as compared to control (p>0.05). Significant decreases in antioxidant capacity of clove tissues was also seen with increasing concentration of Se treatments in all cultivars (p>0.05) using the ABTS assay.
In a second series of experiments hydroponic systems were used to enrich the cultivars with Se and this approach resulted in a significant accumulation of Se within tissues of up to 230mg Se/kg DW as compared to 0.16mg Se/kg DW in control tissues (p<0.05). The highest levels of Se accumulated in the roots > shoot> clove with levels differing between cultivars. Chemical speciation analysis as determined using HPLC-ICP-MS analysis showed that Se application promoted the accumulation of organic and inorganic forms namely Selenomethionine, Selenocystine, Selenate and Selenomethylselenocysteine. Interestingly, Se accumulation did not significantly influence the cytotoxicity of the majority of cultivar extracts (p>0.05). This finding indicating that other factors in addition to Se levels and speciation are responsible for cytotoxicity. However, significant differences in IC50 values between control and enriched clove tissues of the cultivars Mersley and Solent (p<0.001) were found. Se speciation results suggest that difference in cytotoxicity is not solely due to presence of Se species.
Further, analysis of synthetic Se species was conducted to better define the cytotoxic effects of Se species found in Se enriched crops. Using a human hepatoma HepG2 model, SeCys2 and Selenite were found to be the most cytotoxic Se species, however, the cytotoxicity of selenite was found to be enhanced by the presence of 500μM of cellular antioxidant glutathione (p<0.05). We also confirm that the cytotoxicity of selenite + GSH is due to the presence of a short-lived reaction intermediate (<10 minutes) previously proposed to be the superoxide anion. However, despite increasing ROS production in mammalian cells caused by this reaction mix over time (p<0.05), incubation with 500μM of radical scavengers (Ascorbic acid, N-acetyl cysteine, Uric acid, Sodium hydrosulphide, Cytochrome C, Trolox and Catalase) failed to prevent toxicity (p>0.05). Therefore, we postulate that H2Se, a central metabolite in Se metabolism and generated in reaction between selenite + GSH, plays an important role in mediating cell death when exposed to selenite + GSH.
To further explore this possibility, we synthesised a novel slow release H2Se donor, TDN1042 (SeGYY). HepG2 exposure to SeGYY resulted in a concentration dependent decrease in cell viability as well as LDH leakage (p=0.032), DNA damage, ROS production (p<0.05), caspase activation (p<0.001) and PARP cleavage (p<0.001). However, its toxicity was not as dramatic as Selenite likely due to the slow release of H2Se in cell culture media. Therefore, we postulate that H2Se could be a fourth gaseous mediator alongside CO, NO and H2S, however, we acknowledge additional research is needed to confirm this.
In conclusion, further work is needed to assess the bioactivity and cytotoxicity of Se enriched crops intended for human consumption, further work is also needed to understand the role of H2Se in Se toxicity.
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