Sumontha Nookabkaew

Determination of Arsenic Species in Rice from Thailand and Other Asian Countries Using Simple Extraction and HPLC-ICP-MS Analysis

Human exposure to arsenic (As) via rice consumption is of increasing concern. In the present study, the extraction and HPLC-ICP-MS analysis for As speciation in rice were investigated. A simple extraction with water and digestion with α-amylase followed by the analysis using ion-paring mode HPLC separation was developed. The method showed good extraction efficiencies (generally >80%) and column efficiencies (>90%) for rice samples. The optimization of mobile phase showed well separated peaks of As species. The limits of quantification (LOQ) of As(III), As(V), MMA, and DMA that were calculated based on sample mass were 1.6, 2.0, 2.0, and 1.6 μg kg(-1), respectively. A total of 185 rice samples (various types of rice) collected from different four regions in Thailand and some other Asian countries were analyzed. The total As and inorganic As in rice samples were in the ranges of 22.51-375.39 and 13.89-232.62 μg kg(-1), respectively. The estimated weekly intake of inorganic As from rice by Thai people accounted for 13.91-29.22% of the provisional tolerable weekly intake (PTWI).

Evaluation of trace elements in selected foods and dietary intake by young children in Thailand

Elemental concentrations in rice, animal products, eggs, vegetables, fruits, infant formulas and drinking water were determined in 667 food samples randomly collected from local markets, big supermarkets and grocery stores in Bangkok, Thailand, during the period October 2005–August 2008. Samples were digested with nitric acid and analysed by inductively coupled plasma–mass spectrometry. Arsenic and cadmium levels in most foods were below the maximum levels as set by international organisations. Filtered and bottled drinking water, rice, vegetables and banana contained low concentrations of arsenic, cadmium and lead. Non-polished rice had higher magnesium, calcium, manganese, iron and selenium concentrations than polished rice. Banana was a major source for manganese and selenium. Pig kidney and liver contained high levels of arsenic and cadmium. Manganese, cadmium, lead and aluminium concentrations in soybean milk could also be of concern. With respect to food safety for children, the amounts of arsenic and cadmium ingested with poultry, pig liver or rice corresponded to high weekly or monthly intake.

Immunomodulatory Effects of Cadmium and Gynostemma pentaphyllum Herbal Tea on Rat Splenocyte Proliferation

Gynostemma pentaphyllum Makino (GP) is a herbal tea widely grown in Southeast Asia. However, this herbal tea can be contaminated with some heavy metals, especially cadmium (Cd), from agricultural areas, which may affect human health. The objective of this study is to evaluate the immunomodulatory effects of Cd contaminated in GP herbal tea and inorganic Cd on rat splenocytes. Rats were divided into groups and treated with drinking water (control), high CdCl 2 in drinking water (HCd; 0.05 mg/L), GP herbal tea containing 0.05 mg/L Cd (GP-HCd) for 4 months, low CdCl 2 in drinking water (LCd; 0.006 mg/L), and GP herbal tea containing 0.006 mg/L Cd (GP-LCd) for 6 months. After the treatments, Cd accumulation in organs and blood was detected by using a graphite furnace atomic absorption spectrophotometer. In spleen, HCd-treated rats had 4-fold higher Cd accumulations than GP-HCd-treated rats. Cd accumulation in liver and kidney in the HCd group also increased significantly. There were no significant changes in total leucocyte and lymphocyte counts; however, these parameters tended to decrease slightly in LCd, GP-LCd, and GP-HCd groups. The HCd group (ex vivo) significantly produced suppressive effects on T cell mitogen-induced splenocyte proliferation, with 1 mug/mL Con A and PHA-P. In addition, 0.5 mug/mL PWM-induced B cell proliferation, through T cell functions, was also significantly inhibited by HCd as compared to the control group, while GP-HCd had no effects. However, both GP-LCd- and LCd-treated rats had a slight increase in Con A-stimulated splenocyte proliferation. This study indicated that high Cd contamination in drinking water alone had suppressive effects on T cell functions, but these effects could not be found with the same Cd level contamination in GP herbal tea.

Control of zinc homeostasis in Agrobacterium tumefaciens via zur and the zinc uptake genes znuABC and zinT.

The Agrobacterium tumefaciens zinc uptake regulator (Zur) was shown to negatively regulate the zinc uptake genes znuABC, encoding a zinc transport system belonging to the ATP-binding cassette (ABC) transporter family, and zinT, which encodes a periplasmic zinc-binding protein. The expression of znuABC and zinT was inducible when cells were grown in medium containing a metal chelator (EDTA), and this induction was shown to be specific for zinc depletion. The expression of znuABC was reduced in response to increased zinc in a dose-dependent manner, and zinT had a less pronounced but similar pattern of zinc-regulated expression. The inactivation of zur led to constitutively high expression of znuABC and zinT. In addition, a zur mutant had an increased total zinc content compared to the WT NTL4 strain, whereas the inactivation of zinT caused a reduction in the total zinc content. The zinT gene is shown to play a dominant role and to be more important than znuA and znuB for A. tumefaciens survival under zinc deprivation. ZinT can function even when ZnuABC is inactivated. However, mutations in zur, znuA, znuB or zinT did not affect the virulence of A. tumefaciens.

Concentrations of Trace Elements in Organic Fertilizers and Animal Manures and Feeds and Cadmium Contamination in Herbal Tea (Gynostemma pentaphyllum Makino)

Thailand is predominantly an agriculture-based country. Organic farming is enlisted as an important national agenda to promote food safety and international export. The present study aimed to determine the concentrations of trace elements in commercial organic fertilizers (fermented and nonfermented) composed of pig and cattle manures available in Thailand. Pig and cattle manures as well as animal feeds were also collected from either animal farms or markets. The results were compared to the literature data from other countries. Fermented fertilizer composed of pig manure contained higher concentrations of nitrogen (N) and phosphorus (P) than fertilizer composed of cattle manure. High concentrations of copper (Cu) and zinc (Zn) were also found in fertilizers and manures. Some organic fertilizers had high concentrations of arsenic (As), cadmium (Cd), and lead (Pb). The range of As concentration in these fertilizers was 0.50-24.4 mg/kg, whereas the ranges of Cd and Pb were 0.10-11.4 and 1.13-126 mg/kg, respectively. Moreover, pig manure contained As and Cd (15.7 and 4.59 mg/kg, respectively), higher than their levels in cattle manure (1.95 and 0.16 mg/kg, respectively). The use of pig manure as soil supplement also resulted in high Cd contamination in herbal tea (Gynostemma pentaphyllum Makino; GP). The Cd concentration in GP plants positively correlated with the Cd concentration in the soil. Therefore, the application of some organic fertilizers or animal manures to agricultural soil could increase some potentially toxic elements in soil, which may be absorbed by plants and, thus, increase the risk of contamination in agricultural products.

Regulation of the Cobalt/Nickel Efflux Operon dmeRF in Agrobacterium tumefaciens and a Link between the Iron-Sensing Regulator RirA and Cobalt/Nickel Resistance

ABSTRACT The Agrobacterium tumefaciens C58 genome harbors an operon containing the dmeR (Atu0890) and dmeF (Atu0891) genes, which encode a transcriptional regulatory protein belonging to the RcnR/CsoR family and a metal efflux protein belonging to the cation diffusion facilitator (CDF) family, respectively. The dmeRF operon is specifically induced by cobalt and nickel, with cobalt being the more potent inducer. Promoter-lacZ transcriptional fusion, an electrophoretic mobility shift assay, and DNase I footprinting assays revealed that DmeR represses dmeRF transcription through direct binding to the promoter region upstream of dmeR. A strain lacking dmeF showed increased accumulation of intracellular cobalt and nickel and exhibited hypersensitivity to these metals; however, this strain displayed full virulence, comparable to that of the wild-type strain, when infecting a Nicotiana benthamiana plant model under the tested conditions. Cobalt, but not nickel, increased the expression of many iron-responsive genes and reduced the induction of the SoxR-regulated gene sodBII. Furthermore, control of iron homeostasis via RirA is important for the ability of A. tumefaciens to cope with cobalt and nickel toxicity. IMPORTANCE The molecular mechanism of the regulation of dmeRF transcription by DmeR was demonstrated. This work provides evidence of a direct interaction of apo-DmeR with the corresponding DNA operator site in vitro. The recognition site for apo-DmeR consists of 10-bp AT-rich inverted repeats separated by six C bases (5′-ATATAGTATACCCCCCTATAGTATAT-3′). Cobalt and nickel cause DmeR to dissociate from the dmeRF promoter, which leads to expression of the metal efflux gene dmeF. This work also revealed a connection between iron homeostasis and cobalt/nickel resistance in A. tumefaciens.

Roles of Agrobacterium tumefaciens C58 ZntA and ZntB and the transcriptional regulator ZntR in controlling Cd2+/Zn2+/Co2+ resistance and the peroxide stress response.

The putative zinc exporters ZntA (a P1B-type ATPase) and ZntB (2-TM-GxN family) in Agrobacterium tumefaciens were characterized. The expression of the zntA gene is inducible by CdCl2, ZnCl2 and CoCl2, of which CdCl2 is the most potent inducer, whereas zntB is constitutively expressed. The metal-induced expression of zntA is controlled by the MerR-like regulator ZntR. The zntA and zntR mutants were highly sensitive to CdCl2 and ZnCl2, and CoCl2 sensitivity was demonstrated to a lesser extent. By contrast, the zntB mutant showed similar levels of metal resistance to the WT strain. Even in the zntA mutant background, zntB did not play an apparent role in metal resistance under the conditions tested. The inactivation of zntA increased the accumulation of intracellular cadmium and zinc, and conferred hyper-resistance to H2O2. Thus, the metal transporter ZntA and its regulator ZntR are important for controlling zinc homeostasis and cadmium and cobalt detoxification. The loss of either the zntA or zntR gene did not affect the virulence of A. tumefaciens in Nicotiana benthamiana.

Agrobacterium tumefaciens Zur Regulates the High-Affinity Zinc Uptake System TroCBA and the Putative Metal Chaperone YciC, along with ZinT and ZnuABC, for Survival under Zinc-Limiting Conditions

ABSTRACT Agrobacterium tumefaciens has a cluster of genes (Atu3178, Atu3179, and Atu3180) encoding an ABC-type transporter, here named troA, troB, and troC, respectively, which is shown here to be a zinc-specific uptake system. Reverse transcription (RT)-PCR analysis confirmed that troA, troB, and troC are cotranscribed, with troC as the first gene of the operon. The yciC (Atu3181) gene is transcribed in the opposite orientation to that of the troCBA operon and belongs to a metal-binding GTPase family. Expression of troCBA and yciC was inducible under zinc-limiting conditions and was controlled by the zinc uptake regulator, Zur. Compared to the wild type, the mutant strain lacking troC was hypersensitive to a metal chelator, EDTA, and the phenotype could be rescued by the addition of zinc, while the strain with a single yciC mutation showed no phenotype. However, yciC was important for survival under zinc limitation when either troC or zinT was inactivated. The periplasmic zinc-binding protein, ZinT, could not function when TroC was inactivated, suggesting that ZinT may interact with TroCBA in zinc uptake. Unlike many other bacteria, the ABC-type transporter ZnuABC was not the major zinc uptake system in A. tumefaciens. However, the important role of A. tumefaciens ZnuABC was revealed when TroCBA was impaired. The strain containing double mutations in the znuA and troC genes exhibited a growth defect in minimal medium. A. tumefaciens requires cooperation of zinc uptake systems and zinc chaperones, including TroCBA, ZnuABC, ZinT, and YciC, for survival under a wide range of zinc-limiting conditions. IMPORTANCE Both host and pathogen battle over access to essential metals, including zinc. In low-zinc environments, physiological responses that make it possible to acquire enough zinc are important for bacterial survival and could determine the outcome of host-pathogen interactions. A. tumefaciens was found to operate a novel pathway for zinc uptake in which ZinT functions in concert with the high-affinity zinc importer TroCBA.

Arsenic Speciation and Accumulation in Selected Organs after Oral Administration of Rice Extracts in Wistar Rats

Despite its nutritional values, rice also contains arsenic. There has been increasing concern about health implications associated with exposure to arsenic through rice consumption. The present study evaluated arsenic accumulation and its speciation in selected organs of Wistar rats after 28 day repeated oral administrations of polished or unpolished rice and their control arsenic compounds (sodium arsenite or dimethylarsinic acid; DMA). Only the treatment of sodium arsenite (2 μg/kg body weight), significantly increased total arsenic concentrations in blood when compared to the distilled water control group. In all groups, total arsenic concentrations were highest in kidney (1.54-1.90 mg/kg) followed by liver (0.85-1.52 mg/kg), and the predominant arsenic form in these organs was DMA. However, there was no significant difference in arsenic accumulation in the measured organs among the control and rice-treated groups. Therefore, the repeated 28 day administration of arsenic-contaminated rice did not cause significant arsenic accumulation in the animal organs.