Pradeep Alava

Arsenic in cooked rice: Effect of chemical, enzymatic and microbial processes on bioaccessibility and speciation in the human gastrointestinal tract

Rice, used as staple food for half of the world population, can easily accumulate arsenic (As) into its grain, which often leads to As contamination. The health risk induced by presence of As in food depends on its release from the food matrix, i.e., its bioaccessibility. Using an in vitro gastrointestinal simulator, we incubated two types of cooked rice (total As: 0.389 and 0.314 mg/kg). Arsenic bioaccessibility and speciation changes were determined upon gastrointestinal digestion. Washing with deionized water and cooking did not result in changes of As speciation in the rice although the arsenic content dropped by 7.1-20.6%. Arsenic bioaccessibility of the cooked rice in the small intestine ranged between 38 and 57%. Bioaccessibility slightly increased during digestion in the simulated small intestine and decreased with time in the simulated colon. Significant speciation changes were noted in the simulated colon, with trivalent monomethylarsonate (MMA(III)) becoming an important species.

Arsenic Thiolation and the Role of Sulfate-Reducing Bacteria from the Human Intestinal Tract

Background: Arsenic (As) toxicity is primarily based on its chemical speciation. Although inorganic and methylated As species are well characterized in terms of metabolism and formation in the human body, the origin of thiolated methylarsenicals is still unclear. Objectives: We sought to determine whether sulfate-reducing bacteria (SRB) from the human gut are actively involved in the thiolation of monomethylarsonic acid (MMAV). Methods: We incubated human fecal and colon microbiota in a batch incubator and in a dynamic gut simulator with a dose of 0.5 mg MMAV in the absence or presence of sodium molybdate, an SRB inhibitor. We monitored the conversion of MMAV into monomethyl monothioarsonate (MMMTAV) and other As species by high-performance liquid chromatography coupled with inductively coupled plasma mass spectrometry analysis. We monitored the sulfate-reducing activity of the SRB by measuring hydrogen sulfide (H2S) production. We used molecular analysis to determine the dominant species of SRB responsible for As thiolation. Results: In the absence of sodium molybdate, the SRB activity—primarily derived from Desulfovibrio desulfuricans (piger)—was specifically and proportionally correlated (p < 0.01) to MMAV conversion into MMMTAV. Inactivating the SRB with molybdate did not result in MMAV thiolation; however, we observed that the microbiota from a dynamic gut simulator were capable of demethylating 4% of the incubated MMAV into arsenous acid (iAsIII), the trivalent and more toxic form of arsenic acid (iAsV). Conclusion: We found that SRB of human gastrointestinal origin, through their ability to produce H2S, were necessary and sufficient to induce As thiolation. The toxicological consequences of this microbial As speciation change are not yet clear. However, given the efficient epithelial absorption of thiolated methylarsenicals, we conclude that the gut microbiome—and SRB activity in particular—should be incorporated into toxicokinetic analysis carried out after As exposure. Citation: DC.Rubin SS, Alava P, Zekker I, Du Laing G, Van de Wiele T. 2014. Arsenic thiolation and the role of sulfate-reducing bacteria from the human intestinal tract. Environ Health Perspect 122:817–822; http://dx.doi.org/10.1289/ehp.1307759

HPLC-ICP-MS method development to monitor arsenic speciation changes by human gut microbiota.

Inorganic arsenic (iAs) has been classified as a type 1 carcinogen and has also been linked to several noncancerous health effects. Prior to 1995, the As(V) methylation pathway was generally considered to be a detoxification pathway, but cellular and animal studies involving MMA(III) (mono metyl arsonous acid) and DMA(III) (dimethyl arsinous acid) have indicated that their toxicities meet or exceed that of iAs, suggesting an activation process. In addition, thiolated arsenic metabolites were observed in urine after oral exposure of inorganic arsenic in some studies, for which the toxicological profile was not yet fully characterized in human cells. Studies have revealed that microorganisms from the gut environment are important contributors to arsenic speciation changes. This presystemic metabolism necessitates the development of protocols that enable the detection of not only inorganic arsenic species, but also pentavalent and trivalent methylated, thiolated arsenicals in a gastrointestinal environment. We aim to study the biotransformation of arsenic (As) using a Simulator of the Human Intestinal Microbial Ecosystem (SHIME). To be able to analyze the arsenicals resulting from biotransformation reactions occurring in this system, a method using liquid chromatography hyphenated to an inductively coupled plasma mass spectrometer (HPLC-ICP-MS) was developed. A Hamilton PRP-X100 anion exchange column was used. The method allowed separation, identification and quantification of As(III) (arsenite), As(V) (arsenate), DMA(V) (dimethylarsinicacid), MMA(V) (monomethylarsonicacid) and MMMTA (monomethylmonothioarsenate). Attempts to optimize the same method for also separating MMA(III) and DMA(III) did not succeed. These compounds could be successfully separated using a method based on the use of a Zorbax C₁₈ column. The properties of the column, buffer strength, pH and polar nature of mobile phase were monitored and changed to optimize the developed methods. Linearity, sensitivity, precision, accuracy and resolution of both methods were checked. The combination of the two methods allowed successful quantification of arsenic species in suspensions sampled in vitro from the SHIME reactor or in vivo from the human colon and feces.

Arsenic undergoes significant speciation changes upon incubation of contaminated rice with human colon micro biota

Cellular and animal studies involving MMA(III) (monomethyl arsonous acid) and DMA(III) (dimethyl arsinous acid) have indicated that their toxicities meet or exceed that of iAs. Thiolated arsenic metabolites were observed in urine after oral exposure of inorganic arsenic in some studies. For these species, the toxicological profile was not yet fully characterized in human cells. Some studies revealed that trivalent organoarsenic species are well absorbed in the intestine compared to iAs. However, other studies also indicated that a significant amount of rice-bound As reaches the colon, which may be attributed to the fibre-rich nature of the rice. Studies have revealed that microorganisms from the gut environment are important contributors to arsenic speciation changes. We aimed to study how the gut microbial metabolism affects As in different rice matrices. This was done in vitro using colon suspension from the Simulator of the Human Intestinal Microbial Ecosystem (SHIME system). Significant amounts of MMA(III), DMA(III) and MMMTA(V) were formed due to microbial metabolic processes like methylation and thiolation. These results suggested that presystemic metabolism by human gut micro biota should not be neglected in risk assessment studies. In this context, also toxicity and absorption of thiolated species by mammalian cells should be further investigated.

Westernized diets lower arsenic gastrointestinal bioaccessibility but increase microbial arsenic speciation changes in the colon

Arsenic (As) is an important contaminant present in food and water. Several studies have indicated that the occurrence of As based skin lesions is significantly different when root and gourd rich diets are consumed compared to meat rich diets. Additionally, urinary As speciation from orally exposed individuals appears to depend on the composition of the diet. These observations imply that diet composition can affect both the bioavailable As fraction as the As speciation in the body. In this study, we used the in vitro gastrointestinal method (IVG) to evaluate how an Asian type diet (fiber rich) and a Western type diet (fat and protein rich), differ in their capability to release inorganic As (iAs(V)) and dimethyl arsinate (DMA(V)) from a rice matrix following gastrointestinal digestion. Moreover, we used a validated dynamic gut simulator to investigate whether diet background affects As metabolism by gut microbiota in a colon environment. An Asian diet background resulted in a larger As bioaccessibility (81.2%) than a Western diet background (63.4%). On the other hand, incubation of As contaminated rice with human colon microbiota in the presence of a Western type diet resulted in a larger amount of hazardous As species - monomethyl arsonite and monomethylmonothio arsonate - to be formed after 48 h. The permeability of these As species (60.5% and 50.5% resp.) across a Caco-2 cell line was significantly higher compared to iAs(V) and DMA(V) (46.5% and 28% resp.). We conclude that dietary background is a crucial parameter to incorporate when predicting bioavailability with bioaccessibility measurements and when assessing health risks from As following oral exposure.

Arsenic bioaccessibility upon gastrointestinal digestion is highly determined by its speciation and lipid-bile salt interactions

The release of arsenic (As) from a contaminated food matrix during gastrointestinal digestion, i.e., its bioaccessibility, is an important estimator of its bioavailability, and therefore also its health risk. In addition, As toxicity is primarily determined by its speciation and it is not clear how different As species behave during digestion in the upper digestive tract. Here, we evaluated to what extent digestive parameters like gastric pH and bile concentration, but also food matrix constituents affect the bioaccessibility of 8 As species (AsIII, AsV, MMAV, DMAV, MMAIII, DMAIII, MMMTAV, DMMTAV). Bioaccessibility of all As standards ranged between 85% and 90% under pH 1.8. Bioaccessibility of methylated and thiolated arsenicals was decreased from 85% to 50% with increasing gastric pH to 4, yet an increasing bile salts concentration up to 30 g/L lowered the bioaccessibility of inorganic species from 83% to 70% due to interaction with Fe present in bile salts. With respect to food matrices, we noted that the fiber content did not affect As bioaccessibility, yet the presence of fat resulted in an increased bioaccessibility of both inorganic and organic arsenicals in the presence of bile salts. With respect to inorganic arsenic, the intestinal presence of trivalent Fe appeared to be the predominant factor for bioaccessibility of iAs. These data demonstrate that species dependent bioaccessibility must be considered upon ingestion and gastrointestinal digestion.

Bioaccessibility of selenium from cooked rice as determined in a simulator of the human intestinal tract (SHIME)

BACKGROUND As an essential but also potentially toxic element, both overexposure and underexposure to selenium (Se) can significantly affect public health. Rice is a common source of Se, especially in Asia. Not all Se may be released from the rice and become available for absorption into the bloodstream upon digestion in the gastrointestinal tract. Therefore, the bioaccessibility of Se in cooked white (polished) rice was assessed in vitro using the static gastrointestinal simulator SHIME (Simulator of the Human Intestinal Microbial Ecosystem). RESULTS The common cooking procedure in China prior to consumption [i.e. boiling at low rice:water ratios (1:3) until all of the water is absorbed into the rice] did not change total Se levels in the rice. Gastrointestinal digestion of the cooked rice matrix revealed a Se bioaccessibility of 67-76% of total Se. Subsequent microbial activity in the colon reduced the accessibility of Se in the cooked rice to 51-62%. CONCLUSION Not all Se present in cooked white rice should be considered as being bioavailable in the small intestine. A minor part is transferred with the remaining food matrix to the colon, where it is available for the microbial metabolism.