Doktorarbeit

Thioarsenates in Rice Plants and Grains: Implications for Food Safety

Andrea Colina Blanco (10/2019-04/2024)

Betreuer: Britta Planer-Friedrich

Rice is typically grown under flooded conditions which mobilizes naturally occurring arsenic (As) from paddy soils. Besides the well-known oxyAs species, inorganic As (iAs: arsenite and arsenate), monomethylarsenate (MMAV ), and dimethylarsenate (DMAV ), inorganic and methylated thioarsenates have recently been found in paddy soil pore waters. Moreover, the uptake of thioarsenates by rice plants has been confirmed. Amongst methylated thioarsenates, dimethylmonothioarsenate (DMMTA) is particularly relevant because of its high toxicity in mammalian cells. Despite mounting evidence of the ubiquitous presence of thioarsenates in soil pore waters little is known about their interaction with plants and accumulation in grains.

This thesis aimed to investigate the relevance of thioarsenates, particularly DMMTA, for food safety and rice plants. The occurrence of thioarsenates in rice grains and products was examined. Additionally, the toxic effects of DMMTA on plants were assessed and the path of DMMTA to the grains, specifically (trans)formation, accumulation, transport, and translocation, was studied in rice plants.

The first two studies investigated the occurrence of thioarsenates in rice grains and products. Since routine acid-based extractions co-determined DMMTA as DMAV , a method to detect thioarsenates was developed. The method consists of a two-step enzymatic extraction followed by chromatographic separation with 2.5-100 mmol L -1 NaOH as eluent. The extraction efficiency of the method was confirmed by using a rice-certified reference material. Contents of DMAV , iAs, and sum of As species were all in agreement with the certified values. Formation of DMMTA during the enzymatic extraction was ruled out by the complete recovery of a DMAV spike added before extraction. Analysis of commercial samples showed that thioarsenates, namely DMMTA, dimethyldithioarsenate (DMDTA), and monothioarsenate (MTA) accumulated in the grains. Notably, puffed rice cakes had particularly higher contents of thioarsenates in comparison to the rice samples. Screening of a larger dataset of commercial puffed rice cakes (n = 80) revealed that DMMTA and DMDTA were widely present in the samples, accounting for up to 38 and 46% of total As, respectively. Additionally, MTA, dithioarsenate (DTA), monomethylmonothioarsenate (MMMTA), and monomethyldithioarsenate (MMDTA) were also widely present as minor species. A comparison between the As speciation of rice grains and their respective puffed rice cake revealed that the high content of thioarsenates detected in the puffed rice is a consequence of the high temperatures employed during the puffing treatment. Reduced sulfur originating from the thermal degradation of sulfur-containing compounds in rice was speculated to cause thiolation.

The following two studies focused on the behavior of DMMTA in plants. Hydroponic experiments with Arabidopsis thaliana demonstrated DMMTA caused a strong root growth inhibition, more than arsenite and by far more than DMAV . Unlike arsenite, DMMTA exposure did not lead to the accumulation of reactive oxygen species but caused deformation of root epidermal cells revealing different toxicity mechanisms between the As species. Furthermore, like DMAV , the phytochelatin pathway did not contribute to DMMTA detoxification, hence speciation in roots and shoots revealed efficient translocation of DMMTA within plants. Shoot growth and development were also severely affected by the translocated DMMTA. Alterations such as curling of the leaves due to dehydration, decrease in chlorophyll a and b and carotenoids, and accumulation of anthocyanins were observed. Hydroponic experiments with rice plants during grain filling revealed that both DMMTA- and DMAV -exposed plants accumulated similar shares of DMMTA in the leaves, grains, and husks and DMDTA in the leaves and grains, unveiling in planta (de)thiolation processes. Stem-girdling experiments indicated preferential transport of DMMTA in the phloem. Phloem transport was further confirmed by the detection of DMMTA in grains after flag leaf-feeding experiments. For the first time, arsenite and MMAV in planta thiolation was observed in rice seedlings. In planta thiolation of DMAV was also detected in A. thaliana and the kinetics of DMAV thiolation showed this process is not purely abiotic. Significantly lower DMAV thiolation was observed in glutathione (GSH) deficient mutants compared to wild-type A. thaliana plants, thus suggesting GSH concentration as an important parameter influencing in planta thiolation of As species.

The fifth study merged our findings and other evidence available on thioarsenates occurrence in rice and thioarsenates detection challenges and put it in context with cytotoxicity data and regulatory limits for As in rice worldwide. The study highlighted that overlooking thioarsenates poses a potential food safety threat, particularly when it comes to the misidentification of unregulated DMAV as highly cytotoxic DMMTA.

Overall, our findings revealed the importance of studies on the interaction of thioarsenates, specially DMMTA, with plants with respect to food safety. DMMTA was shown to be highly mobile and toxic for plants. Even in the absence of thioarsenates in soil pore water, in planta thiolation of DMAV can lead to DMMTA and DMDTA accumulation in grains. The occurrence of thioarsenates in commercial rice grains and puffed rice cakes indicates that further monitoring and risk assessment characterization of ingesting thioarsenate-containing rice is urgently needed.