Chemosphere 119 (2015) 757–762
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Westernized diets lower arsenic gastrointestinal bioaccessibility but increase microbial arsenic speciation changes in the colon Pradeep Alava a,⇑, Gijs Du Laing a, Filip Tack a, Tine De Ryck b, Tom Van De Wiele b a b
Laboratory of Analytical Chemistry and Applied Ecochemistry, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000 Ghent, Belgium Laboratory of experimental cancer research, Department of Radiation Oncology and Experimental Cancer Research, Faculty of medicine and health sciences, Ghent, Belgium
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Effect of diet matrix on
bioaccessibility of different arsenic species in diet. Effect of diet matrix on biotransformation of different arsenic species in diet. The difference in formation of new toxic arsenic species basing on diet matrix. How difference in diet matrix might be important in case of oral arsenic exposure.
a r t i c l e
i n f o
Article history: Received 18 April 2014 Received in revised form 13 July 2014 Accepted 1 August 2014 Available online 3 September 2014 Handling Editor: X. Cao Keywords: Arsenic Speciation Asian diet Western diet Presystemic metabolism Simulator of human gastrointestinal microbial ecosystem
a b s t r a c t 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 (iAsV) and dimethyl arsinate (DMAV) 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 iAsV and DMAV (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. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Arsenic (As) is a naturally occurring element in food, soil, air, and water. The major sources of exposure are from food and water. ⇑ Corresponding author. http://dx.doi.org/10.1016/j.chemosphere.2014.08.010 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.
A variety of adverse health effects, e.g., skin and internal cancers, cardiovascular, and neurological effects, have been attributed to As exposure (Chen et al., 1992). Human health effects from chronic As exposure have also been reported mainly in populations with low socioeconomic status and high levels of malnutrition. Diet composition has a significant
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effect on contaminant bioaccessibility and thereby also oral bioavailability. We previously (Alava et al., 2012a) demonstrated that lipids have a large impact on arsenic bioaccessibility, but also that this depends on the As speciation. Some other studies stated that in protein rich rice (Narukawa and Chiba, 2010) AsIII is tightly bound to the thiol groups of the proteins and may influence its bioaccessibility. Previous studies demonstrated a relationship between diet composition and levels of MMAIII found in urine after ingesting inorganic As (Steinmaus et al., 2005). Additionally, some studies reported that people, who consumed diets relatively high in roots and gourds as opposed to meat or other kinds of vegetables, were less likely to develop As-related skin lesions and also showed different metabolic rate of consumed arsenic (Lammon and Hood, 2004). Differences in dietary matrix composition may affect release of As from the food source after ingestion. Food contains both organic and inorganic As, whereas drinking water primarily contains inorganic forms of As. In a scenario of dietary As exposure, rice has been demonstrated to be one of the major foodstuffs contributing to human As exposure (Williams et al., 2007) and also widely consumed (Raab et al., 2009). Interestingly, As biotransformations by human gut microbiota are even reported in case of As contaminated rice (Sun et al., 2012). Moreover, rice is an important carbohydrate sources in both Asian and Western style diets. Trenary et al. reported bioaccessibility of arsenic species from different types of rice in USA by using in vitro setup. These people extended their results to estimate the exposure level to American people by using probabilistic exposure model. All these above observations and reports lead to an interesting and important question; what is the effect of diet matrix on bioavailability of arsenic from rice? As extension to the above question the second part to focus is presystemic metabolism. Upon oral exposure of As contaminated food, gastrointestinal digestion may release a fraction of the matrix-bound As: this is termed the bioaccessible fraction, which is considered a conservative estimator of the oral bioavailable As fraction. The As fraction that is not absorbed across the small intestinal epithelium and the remaining matrix-bound As will reach the colon region. Here, microbial breakdown of the remaining food matrix may further contribute to As release, while the released As in general may get subjected to the diverse metabolic potency of the vast endogenous microbial community. Such conversion of As by colon microbiota is termed presystemic metabolism. Notably, some studies have revealed that microorganisms are important contributors to arsenic speciation changes. A wide range of microbial metalloid biotransformations have been revealed, including oxidation, reduction, methylation and thiolation (DiazBone and Van de Wiele, 2010). Recently, As biotransformations by human gut microbiota have been characterized using As contaminated soils and rice (Van De Wiele et al., 2010; Alava et al., 2012b). Summarizing the above paragraphs; as rice being one of the major source of arsenic through food and diet components like lipids and fiber affecting the bioaccessibility of ingested arsenic species we aimed to study whether and how bioaccessibility of As is affected by diet composition and further how these diets effect the biotransformations of ingested As. Arsenic through rice is major problem in western countries like America (Trenary et al., 2012) and Eastern countries like Bangladesh, India and Taiwan (Meharg and Rahman, 2003, Chowdhury et al., 2000, Pal et al., 2009), and there is significant difference between diet compositions of these two regions; Asian diet (low in fat, protein and high in carbohydrate) and Western diet (high fat and protein) (Suhana et al., 1999 and Park et al., 2006). We choose these two diet compositions to elucidate whether and how bioaccessibility of As is affected by diet composition, further how these diets effect the biotransformations of As.
2. Experimental 2.1. Arsenic standards and samples Standards used were: NaAsO2 solution (VWR, Belgium) for Arsenite (AsIII), Na2HAsO47H2O (Fluka, Switzerland) for Arsenate (AsV), (CH3)2AsO2Na3H2O (Fluka, Switzerland) for dimethyl arsinous acid (DMAV), and (CH3)AsNa2O36H2O (Chemservice, Belgium) for mono methyl arsonous acid (MMAV). Monomethylmonothioarsinic acid (MMMTA) was prepared in the lab. Stability, purity and procedure were already published (Alava et al., 2012). The structure of the product was checked by LC–ESI–MS and MS/MS. MMAIII and DMAIII were purchased from Argus Chemicals (Italy). 2.2. Certified reference material (CRM) and rice sample NIST 1568a Rice Flour (National Institute of Standards and Technology, NIST, USA) was used to check the recovery of total arsenic. The certified value of total As in NIST SRM 1568a is 290 ± 30 lg kg 1, while the As speciation in this reference material is not defined. Basmati rice from Indian origin was purchased from a local supermarket (Colruyt, Gent, Belgium). 2.3. Analysis of rice samples for As content All rice samples were microwave digested at 80 °C for 30 min using water as extraction solvent. This method has previously been proven to be successful for extracting total As from rice (Alava et al., 2012). NIST 1568a Rice Flour (National Institute of Standards and Technology, NIST, USA) was used to check the recovery of total arsenic. The certified value of total As in NIST SRM 1568a is 290 ± 0.03 lg kg 1. Digested samples were filtered using a 0.45 lm syringe-type PVDF membrane filter and the filtrate was diluted to 25 mL using double distilled deionized water. This filtrate was analyzed for total arsenic content using ICP–MS (Table 1). The same filtrate was used for speciation analysis using HPLC–ICP– MS (Alava et al., 2011). 2.4. Setup To better mimic in vivo gastrointestinal behavior of As, all regions – stomach, small intestine, and colon – were simulated. The IVG method was previously validated against in vivo data for As bioaccessibility (Rodriguez and Basta, 1999) in the upper digestive tract (gastric, small intestine), whereas the SHIME has been validated against in vivo data for the colon microbial community composition and metabolic activity toward drugs and phytoestrogens (Molly et al., 1994; Possemiers et al., 2006). To mimic the inter variability of microorganisms in different persons, fecal matter from ten different people representing both Asian and Western countries is collected, mixed well into a homogenous mixture and used as a fecal inoculate in SHIME reactor to develop the microbial source.
Table 1 Optimized instrumental settings for ICP-MS. Detection DRC mode (As) Instrument Plasma RF power Nebulizer flow rate Lens voltage and autolens Dwell times m/z Reaction gas Cell parameters Rpq
Perkin Elmer Elan DRC-e 1250 W 0.7–1.1 mL min 1 (optimized daily) Optimized for AsO daily 91 (AsO 500 ms) O2, 0.75 mL min 1 0.6
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Asian diet and western diet were prepared (Table 2) with rice as source of As. Diets were incubated in gastric solution (1L) at a liquid to solid ratio of 5. One hundred ml/h of gastric juice was added during 2 h to perform gastric digestion. By adjusting the pH and adding pancreatin and bile salts according to the IVG protocol, intestine digestion was performed for 3 h. Samples were collected each hour. Subsequently, 30 mL of gastrointestinal solution was incubated with 30 mL of distal colon suspension from the SHIME system. The vessels containing the colon digests were capped with butyl rubber stoppers and subsequently flushed with N2 for 30 min to obtain anaerobic conditions. They were incubated on a shaker at 150 rpm and 37 °C for 48 h. Samples were collected after 0, 5, 8, 24 and 48 h. All experiments were performed in triplicate and analyzed for As species using HPLC–ICP–MS.
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2.7. Absorption percentage of As species through Caco-2 cells To assess the absorption of bioaccessible As across the gut epithelium an in vitro Caco-2 cell line model was used. Caco-2 cells were cultivated until differentiation and brought in contact with 100 lg/L of each As species in both Asian diet and Western diet matrix separately. Caco-2 cell viability was measured with an MTT assay and epithelial transport was evaluated by measuring the As concentration in the apical and basolateral compartment of a Transwell insert experimental setup. A more detailed description (Blaser, 2007) and discussion of these epithelial transport experiments can be consulted in Alava et al. (under review). All experiments are done in triplicates. 2.8. Stability and storage of solutions
2.5. Sample treatment To preserve the As speciation all samples were immediately stored in freezer at 80 °C. Before performing the analysis, samples were thawed and upon complete thawing, samples were centrifuged at 10 400 relative centrifugal forces to separate solid-bound (pellet) As and As released to the liquid fraction (i.e. the bio-accessible fraction). Samples were filtered using a 0.45 lm syringe-type PVDF membrane filter. The filtrate was injected onto a HPLC column to perform As speciation analysis using HPLC–ICP–MS (Alava et al., 2011). The total As amount is also analyzed in filtrate using ICP-MS and it was considered the bio-accessible fraction. Solidbound As (As remained in pellet) was extracted using microwave digestion. Total As content was analyzed in both filtrate and pellet samples to calculate chromatographic recovery and mass balance. 2.6. Instrumentation A MarsX system (CEM, Matthews, NC, USA) was used for the microwave-assisted extraction. A HPLC–ICP–MS system consisting of a liquid chromatographic system (PerkinElmer Series 200, Sunnyvale, CA, USA) hyphenated to an inductively coupled plasma mass spectrometer (ICP-MS, Elan DRC-e, PerkinElmer, Sunnyvale, CA, USA) was used for speciation analysis. A Hamilton PRP-X100 anion exchange column (Grace, Belgium) and a Zorbax XDB C18 column (250 mm 4.6 mm I.D., 5 lm, 120 A, Agilent, United Kingdom) were used as stationary phases. By using solution from PerkinElmer (Masscall solution, Sunnyvale, CA, USA) the instrument was tuned to maintain the ratio of oxides (CeO/Ce) and doubly charged ions (140Ce2+/140Ce+) lower than 0.03 to minimize the potential interferences, as prescribed by PerkinElmer. Oxygen was used as reaction gas in both methods, resulting in detection of Arsenic as AsO (arsenic oxide) at 91 amu. This procedure is designed to overcome interferences caused by ArCl+ and CaCl+ upon Arsenic detection at 75 amu.
First, the stabilities of several As standards and stock solutions during storage at 4 °C was checked by measuring their concentrations after 1 day, 2 days, 1 week, 2 weeks and 4 weeks. Individual standards were prepared by diluting the stock solutions to 100 lg As/L. A mixed standard, containing all compounds, at a level of 100 lg As/L each, was also prepared. MMAIII and DMAIII stock solutions were found to be unstable when stored for more than 1 week. In these solutions, increasing intensities of respectively MMAV and DMAV peaks were measured. Therefore, they were stored in a freezer at 80 °C, as those standards were stable at this temperature. Stability was also evaluated under these conditions after 15 days and one month. After one month when they are analyzed, a higher variability that is 2% of RSD was observed compared to initial results. Except for MMAIII and DMAIII stock solutions, other stock solutions were stored in a fridge at 4 °C until their analysis within four weeks after preparation. Standards prepared in SHIME matrix instead of MQ water were freshly prepared from the stored stock solutions. Samples collected from the SHIME colon compartments or batch experiments were immediately stored at 80 °C and analyzed before 3 days from collection point of time. 2.9. Statistical analysis One-way analysis of variance (ANOVA) was applied to detect possible differences in bioaccessible As content and biotransformation of As content. A significance level of P < 0.05 was adopted for all comparisons. Statistical analysis was performed using Origin 8.0 (Originlab, North Hampton, MA). 3. Results 3.1. Analysis of rice samples for As content
Compound
Source
Asian diet (g/meal)
Western diet (g/meal)
Protein
Casein Gluten
5.5 19.2
22.0 10.9
Arsenic was extracted from all rice types using microwave digestion. Extraction efficiencies were calculated on a NIST 1568a certified reference rice matrix and were found to be almost 100% (Table 3). The digested solution was analyzed for total As content and As speciation. The total amount of arsenic was 395.1 ± 8.3 lg kg 1 in basmati rice (Table 3). DMAV represented the most abundant species followed by AsV and AsIII (Table 3). The distribution of organic versus inorganic As was around 70% versus 30% at starting point (Table 3).
Fat
Corn oil Butter
22.9 0.0
8.1 33.1
3.2. Quality control
Carbohydrates
Basmati rice (only source of As) Starch Sucrose Dietary fiber (pectin)
56.0
56.0
58.0 13.9 3.0
0.0 52.7 0.0
Table 2 Composition of different diets.
The amount of As remaining in the pellet was analyzed and the sum of As in pellet and bioaccessible As was used to calculate a mass balance. In both diets the As recovery ranged between 97.2% and 98.8%. Arsenic speciation is an important determinant
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in this study; so we calculated chromatographic recovery from the sum of all chromatographically detected species, so that to check whether we are missing some unknown peaks. The chromatographic recoveries for all colon digests were satisfactorily high: 97 ± 3% (mean ± SD). 3.3. Bioaccessibility of As in cooked rice The bioaccessibility of As from Asian and Western diets upon the gastric phase was 95% and 83%, respectively (Fig. 1). Upon 3 h of intestinal digestion, the bioaccessible As fraction significantly decreased from 95% to 83% for Asian diet and from 83% to 63% for Western diet. Subsequently, the bioaccessible As fraction was decreased to 59.7% for Asian diet and to 56.1% for Western diet by the end of the 48 h colon digestion (Fig. 1).
Fig. 1. Bioaccessibility of total As in rice at the stomach, small intestine and colon stage at different time intervals.
3.4. Arsenic speciation at the simulated colon stage The most important observation at the simulated colon stage was the occurrence of significant levels of MMAIII and MMMTAV upon digestion of both diets (Table 4). The concentration of MMAIII accounted for 18% and 21% of absolute total As (22.4 lg) in digested Asian diet and Western diet respectively (Fig. 2). Due to thiolation of pentavalent methylated arsenicals, MMMTAV production accounted for 15% and 18% of absolute total As in digested Asian and Western diet respectively (Fig. 2). 3.5. Absorption percentage of As species through Caco-2 cells Given the diversity of As speciation changes upon incubation with gut microbiota, we evaluated the potency for these As metabolites to be absorbed across the gut epithelium. Yet, given the diversity in toxicity, we first evaluated the inherent toxicity of the different As species towards Caco-2 cells by monitoring their viability with an MTT assay upon incubation. Exposure to 100 lg/L of each As species did not result in any viability loss, except for the trivalent methylated As species (50% loss of viability). This was also reflected by an decrease in transepithelial electrical resistance (TEER). Trivalent methylated species were more efficiently absorbed across a confluent Caco-2 cell layer, with DMAIII displaying 71% of transport from the apical to basal compartment. The point to be noted here is the toxic effect of trivalent species on Caco2-cell lines may also has an additional affect on those species transport through the cell line. On the other hand, pentavalent methylated species and DMAV in particular displayed the lowest absorption (28%), while thiolated methylarsenicals displayed a similar absorption (49%) as inorganic (iAs) species (46%). As iAs species are only available for absorption for around 46% at the intestine level, the remaining amount of As (2.2 lg in case of an Asian diet and 2.0 lg compared to a Western diet) will become available to colon microbes. 4. Discussion A first important observation in this study is that methylation of inorganic As appeared to be a diet dependent process with the Western diet displaying a delayed but more efficient methylation
(P < 0.05) than Asian diet. Although higher absolute amounts of bioaccessible As reached the colon upon incubation of Asian diet, the more efficient methylation during colon digestion of Western diets finally resulted in a higher production of trivalent and thiolated methylarsenicals (Table 4). Human gut microbiota (Van de Wiele et al., 2010) and mice caecum microbiota (Pinyayev et al., 2011) were previously reported to metabolize soil-derived As and form methylated and thiolated arsenicals. This difference in production trivalent and thiolated methylarsenicals between the diets illustrates that the contaminated matrix affects As speciation changes by human colon microbiota. Assessing the internal exposure to As species upon ingestion of a contaminated matrix may therefore require a better characterization of matrix composition. The finding of trivalent methylarsenicals and thiolated methylarsenicals at significant concentrations is of toxicological concern. The LD50 of MMAIII and DMAIII is reported to be lower than that of AsIII, suggesting that MMAIII and DMAIII, intermediates in inorganic As methylation, is more toxic (styblo et al., 2002) and cytotoxic (Naranmandura et al., 2007). Moreover, trivalent organoarsenic compounds display a higher transport across epithelial cells in comparison with inorganic arsenic and the pentavalent organic species (Calatayud et al., 2010; Calatayud et al., 2011). Dopp et al., 2004, also stated that DMAIII has higher membrane permeability then MMAIII. Another important observation was the formation of MMMTAV. MMMTAV toxicity towards all human cells remains to be resolved. Yet, other thiolated methylarsenicals like DMMTAV (dimethyl monothio arsenic acid) have displayed cytotoxic and genotoxic effects similar to trivalent arsenicals (Yoshida et al., 2001; Kuroda et al., 2004). The cytotoxicity of MMMTAV in human bladder cells is DMAIII, DMMTAV > iAsIII, iAsV > MMMTAV > MMAV, DMAV, and DMDTAV (Naranmandura et al., 2011). Eventhough MMMTAV is comparatively less toxic, our transport experiments showed that MMMTAV was efficiently absorbed across Caco-2 cells (50%), similar to both iAs species, indicating chances of their high exposure after their presystemic formation. These observations emphasize the need for considering presystemic metabolism of As in the colon as a relevant process when assessing As toxicokinetics. Following diet composition affect on As speciation changes next important observation is As bioaccessibility is largely affected by
Table 3 As speciation by HPLC–ICP–MS and total As results by ICP–MS in basmati rice, NIST 1568a (290 lg kg undetected.
NIST 1568a Basmati
1
reported value). Tabulated as average ± standard deviation, n = 3; ND:
AsIII (lg/kg)
AsV (lg/kg)
DMAV (lg/kg)
MMAV (lg/kg)
Total As (lg/kg)
Extraction efficiency (%)
53.3 ± 1.0 2.4 ± 0.2
46.1 ± 0.7 90.4 ± 2.1
172.3 ± 1.7 298.8 ± 6.2
13.2 ± 0.3 ND
286.8 ± 4.1 395.1 ± 8.3
98.9
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Table 4 The total As amount and amounts of different As species in supernatants by end of stomach, intestinal phase and 48 h incubation of colon extracts (average ± standard deviation), n = 3; ND: undetected. DMAV (lg)
MMAV (lg)
AsV (lg)
MMMTAV (lg)
MMAIII (lg)
DMAIII (lg)
Sum of species (lg)
Initial total As (lg)
Total bioaccessible As (%)
Asian diet (end of stomach phase) Asian diet (end of intestine phase) Asian diet (end of 48 h colon incubation) Western diet (end of stomach phase) Western diet (end of intestine phase) Western diet (end of 48 h colon incubation)
ND
15.9 ± 0.9
ND
5.3 ± 0.2
ND
ND
ND
21.2
22.4
94.6
ND
14.1 ± 0.8
ND
4.1 ± 0.3
ND
ND
ND
18.2
22.4
81.2
ND
2.1 ± 0.2
1.6 ± 0.2
2.2 ± 0.3
3.4 ± 0.4
4.1 ± 0.4
ND
13.4
22.4
59.7
ND
14.4 ± 0.9
ND
4.4 ± 0.3
ND
ND
ND
18.8
22.4
83.9
ND
11.4 ± 0.5
ND
2.8 ± 0.3
ND
ND
ND
14.2
22.4
63.4
1.8 ± 0.003
ND
3.9 ± 0.003
4.8 ± 0.003
ND
12.6
22.4
56.2
ND
2.1 ± 0.003
Asian diet 100 80 60 40
DMA
30
V
MMA
V
MMA
III
DMA
III
20 V
MMMTA
10
0 0
20
40
Bioaccessible As (%) and As in pellet
AsIII (lg)
Bioaccessible As (%) and As in pellet
Treatment
Western diet
40
20
40
60
Time (hr)
III
As
40
V
As
Pellet
30
20
10
0 60
Time (hr)
Bioaccessible As (%) and As in pellet
Bioaccessible As (%) and As in pellet
V
0
0
Asian diet
40
As
20
60
50
20
III
Pellet
Time (hr)
0
As
Western diet 100 80 60 40 40
DMA
30
MMA
V
MMA
III
DMA
20
V
III
MMMTAV
10 0 0
20
40
60
Time (hr)
Fig. 2. Arsenic species in the aquatic phase and total As in the solid residue (pellet) as a function of time while incubating Asian diet and Western diet with As-polluted rice in colon suspension, expressed as percentage of total As in suspension.
the composition of diet. The acidic conditions from the gastric phase resulted in higher As bioaccessibility than those from the intestine, thereby confirming similar findings on gastric vs. intestinal bioaccessibility in earlier studies (Juhasz et al., 2006 and Denys et al., 2012). However, Sun et al., 2012 reported higher bioaccessibility at small intestine level, this difference might be attributed to difference in whole diet matrix ingested and also to sample pretreatment. In this study samples are centrifuged at 21 000 rpm. Incubation with an Asian diet displayed higher As bioaccessibility values in both gastric as intestine conditions than incubation with a Western diet (Fig. 1). A detailed analysis of how lipids affect As bioaccessibility can be consulted in Alava et al. (2013). While it
must be acknowledged that bioaccessibility values are only estimators of in vivo bioavailability, our in vitro findings are quite similar to earlier in vitro and in vivo observations with cooked contaminated rice. Laparra et al. (2005) found 63% to 99% of the total As to be bioaccessible from cooked rice, while the in vivo bioavailability of As was found to be 89% in swine that were fed cooked rice (Juhasz et al., 2006). On a concluding note there is high amount of arsenic released from the food matrix at gastric level which may alter by composition of the food matrix. Thus difference in diet composition may also differ the amount of As reaching colon level and amount of As available for pre-systemic metabolism.
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Next, we investigated to what extent bioaccessibility is species dependent. iAsV was proportionally more easily released from the rice matrix during gastrointestinal digestion than DMAV. The acidic environment in the simulated stomach (pH 2.0) will result in a more efficient breakdown of the rice protein fraction (Goodman, 2010) and it has been found that inorganic As species are more likely to bind to the protein matrix of endosperm cells (Sanz et al., 2007; Narukawa et al., 2008 and Moore et al., 2010) more particularly by complexing to thiol containing amino acids (Lombi et al., 2009). The observed higher release of iAs from the rice matrix into the supernatant of the gastric digest is therefore quite plausible. In summary, we can conclude that the food matrix affected the efficiency with which the released As species got metabolized by gut microbiota from human origin. In addition, release of As from contaminated food matrices during gastrointestinal digestion depends on the food matrix composition. Finally, different As species are released to a different extent upon gastrointestinal incubation of a contaminated food matrix. These findings ask for a more thorough characterization of the ingested matrix, especially when different As species are present. Additionally, these findings imply that the internal exposure assessment to As from an ingested matrix should entail (i) a speciation analysis of arsenicals present in the food matrix and (ii) a thorough characterization of the ingested matrix as both the bioavailability as microbial metabolic potency towards As seems to rely on matrix composition. Acknowledgement This study was funded by Federal public service of health, food chain safety and environment Contract (BIOTRAS RF 6247). References Alava, P., Du laing, G., Tack, F., Van De Wiele, T., 2012. HPLC ICP MS method development to monitor arsenic speciation changes by human gut microbiota. Biomed. Chromatogr. 26 (4), 524–533. Alava, P., Du laing, G., Tack, F., Van De Wiele, T., 2012a. Towards an optimal sample preparation procedure: Arsenic speciation is preserved during extensive grinding and pressurized extraction from rice. Anal. Methods 4 (5), 1237–1243. Alava, P., Tack, F., Laing, G.D., de Wiele, T.V., 2012b. Arsenic undergoes significant speciation changes upon incubation of contaminated rice with human colon micro biota. J. Hazard. Mater. http://dx.doi.org/10.1016/j.jhazmat.2012.05.042. Alava, P., Du Laing, G., Tack, F., Van De Wiele, T., 2013. Arsenic bioaccessibility upon gastrointestinal digestion is highly determined by its speciation and lipid-bile salt interactions. J. Environ. Sci. Health A. Tox. Hazard. Subst. Environ. Eng. 48 (6), 656–665. http://dx.doi.org/10.1080/10934529.2013.732367. Blaser, D., 2007. Determination of Drug Absorption Parameters in Caco-2 Cell Monolayers with a Mathematical Model Encompassing Passive Diffusion, Carrier-Mediated Efflux, Non-Specific Binding and Phase II Metabolism. University of Basel (Thesis). Calatayud, M., Gimeno, J., Vélez, D., Devesa, V., Montoro, R., 2010. Characterization of the intestinal absorption of arsenate, monomethylarsonic acid, and dimethylarsinic acid using the Caco-2 cell line. Chem. Res. Toxicol. 23, 547–556. Calatayud, M., Gimeno, J., Vélez, D., Devesa, V., Montoro, R., 2011. In vitro study of intestinal transport of arsenite, monomethylarsonous acid, anddimethylarsinous acid by Caco-2 cell line. Toxilogical Lett. 204 (2–3), 547– 556. Chen, C.J., Chen, C.W., Wu, M.M., Kuo, T.L., 1992. Cancer potential in liver, lung, bladder and kidney due to ingested inorganic arsenic in drinking water. Br. J. Cancer 66, 888–892. Chowdhury, T.R., Basu, G.K., Mandal, B.K., et al., 2000a. Arsenic poisoning in the Ganges delta (vol 401, pg 545, 1999). Nature 404. 36–36. Chowdhury, U.K., Biswas, B.K., Chowdhury, T.R., et al., 2000b. Arsenic groundwater contamination and sufferings of people in West Bengal – India and Bangladesh. Trace Elem. Man Anim. 10, 645–650. Denys, S., Caboche, J., Tack, K., Rychen, G., Wragg, J., Cave, M., Jondreville, C., Feidt, C., 2012. In Vivo validation of the unified BARGE method to assess the bioaccessibility. Environ. Sci. Technol. 46 (11), 6252–6260. Diaz-Bone, R.A., Van de Wiele, T., 2010. Biotransformation of metal(loid)s by intestinal microorganisms. Pure Appl. Chem. 82, 409–427. Dopp, E., Hartmann, L.M., Florea, A.-M., von Recklinghausen, U., Pieper, R., Shokouhi, B., Rettenmeier, A.W., Hirner, A.V., Obe, G., 2004. Uptake of inorganic and organic derivatives of arsenic associated with induced cytotoxic and genotoxic effects in Chinese hamster ovary (CHO) cells. Toxicol. Appl. Pharmacol. 201, 156–165.
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Lombi, E., Scheckel, K.G., Pallon, J., Carey, A.M., Zhu, Y.G., Meharg, A.A., 2009. Speciation and distribution of arsenic and localization of nutrients in rice grains. New Phytol. 184, 193–201. Meharg, A.A., Rahman, M., 2003. Arsenic contamination of Bangladesh paddy field soils: implications for rice contribution to arsenic consumption. Environ. Sci. Technol. 37, 229–234. Molly, K., Van de Woestyne, M., De Smet, I., Verstraete, W., 1994. Validation of the simulator of the human intestinal microbial ecosystem (SHIME) reactor using microorganism- associated activities. Microbial Ecol. Health Disease 7, 191– 200. Moore, K.L., Schroder, M., Lombi, E., Zhao, F.J., McGrath, S.P., Hawkesford, M.J., Shewry, P.R., Grovenor, C.R.M., 2010. Nano SIMS analysis of arsenic and selenium in cereal grain. New Phytol. 185, 434–445. Naranmandura, H., Ibata, K., Suzuki, K.T., 2007. Toxicity of dimethylmonothioarsinic acid toward human epidermoid carcinoma A431 cells. Chem. Res. Toxicol. 20, 1120–1125. Naranmandura, H., Carew, M.W., Xu, S., Lee, J., Leslie, E.M., Weinfeld, M., Le, X.C., 2011. Comparative toxicity of arsenic metabolites in human bladder cancer EJ-1 cells. Chem. Res. Toxicol. 24, 1586–1596. Narukawa, T., Chiba, K., 2010. Heat-assisted aqueous extraction of rice flour for arsenic speciation analysis. J. Agric. Food Chem. 58, 8183–8188. Narukawa, T., Inagaki, K., Kuroiwa, T., Chiba, K., 2008. The extraction and speciation of arsenic in rice flour by HPLC–ICP–MS. Talanta 77, 427–432. Pal, A., Chowdhury, U.K., Mondal, D., et al., 2009. Arsenic burden from cooked rice in the populations of arsenic affected and nonaffected areas and Kolkata City in West-Bengal, India. Environ. Sci. Technol. 43, 3349–3355. Park, S., Park, C.H., Jang, J.S., 2006. Antecedent intake of traditional Asian-style diets exacerbates pancreatic b-cell function, growth and survival after Western-style diet feeding in weaning male rats. J. Nutr. Biochem. 17, 307–318. Pinyayev, T.S., Kohan, M.J., Herbin-Davis, K., Creed, J.T., Thomas, D.J., 2011. Preabsorptive metabolism of sodium arsenate by anaerobic microbiota of mouse cecum forms a variety of methylated and thiolated arsenicals. Chem. Res. Toxicol. 24, 475–477. Raab, A., Baskaran, C., Feldmann, J., Meharg, A.A., 2009. Cooking rice in a high water to rice ratio reduces inorganic arsenic content. J. Environ. Monit. 11, 41–44. Rodriguez, R.R., Basta, N.T., 1999. An in vitro gastrointestinal method to estimate bioavailable arsenic in contaminated soils and solid media. Environ. Sci. Technol. 33, 642–649. Sanz, E., Munoz-Olivas, R., Camara, C., Sengupta, M.K., Ahamed, S., 2007. Arsenic speciation in rice, straw, soil, hair and nails samples from the arsenic-affected areas of Middle and Lower Ganga plain. J. Environ. Sci. Health, Part A 42, 1695– 1705. Steinmaus, C., Carrigan, K., Kalman, D., Atallah, R., Yuan, Y., Smith, A.H., 2005. Dietary intake and arsenic methylation in a US population. Environ. 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Trenary, H., Creed, A.P., Andrea, R.Y., Madhavi, M., Carol, A.S., Jianping, X., Michael, J.K., Karen, Herbin-Davis, David, J.T., Joseph, A.C., Creed, J.T., 2012. An in vitro assessment of bioaccessibility of arsenicals in rice and the use of this estimate within a probabilistic exposure model. J. Exposure Sci. Environ. Epidemiol. 22 (4), 369–375. Van de Wiele, T., Gallawa, C.M., Kubachka, K.M., Creed, J.T., Basta, N., Dayton, E.A., Whitacre, S., Du Laing, G., Bradham, K., 2010. Arsenic metabolism by human gut microbiota upon in vitro digestion of contaminated soils. Environ. Health Perspect. 118, 1004–1009. Williams, P.N., Villada, A., Deacon, C., Raab, A., Figuerola, J., Green, A.J., Feldmann, J., Meharg, A.A., 2007. Greatly enhanced arsenic shoot assimilation in rice leads to elevated grain levels compared to wheat and barley. Environ. Sci. Technol. 41, 6854–6859. Yoshida, K., Kuroda, K., Inoue, Y., Chen, H., Date, Y., Wanibuchi, H., 2001. 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Chemosphere journal homepage: www.elsevier.com/locate/chemosphere
Westernized diets lower arsenic gastrointestinal bioaccessibility but increase microbial arsenic speciation changes in the colon Pradeep Alava a,⇑, Gijs Du Laing a, Filip Tack a, Tine De Ryck b, Tom Van De Wiele b a b
Laboratory of Analytical Chemistry and Applied Ecochemistry, Faculty of Bioscience Engineering, Ghent University, Coupure Links 653, 9000 Ghent, Belgium Laboratory of experimental cancer research, Department of Radiation Oncology and Experimental Cancer Research, Faculty of medicine and health sciences, Ghent, Belgium
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Effect of diet matrix on
bioaccessibility of different arsenic species in diet. Effect of diet matrix on biotransformation of different arsenic species in diet. The difference in formation of new toxic arsenic species basing on diet matrix. How difference in diet matrix might be important in case of oral arsenic exposure.
a r t i c l e
i n f o
Article history: Received 18 April 2014 Received in revised form 13 July 2014 Accepted 1 August 2014 Available online 3 September 2014 Handling Editor: X. Cao Keywords: Arsenic Speciation Asian diet Western diet Presystemic metabolism Simulator of human gastrointestinal microbial ecosystem
a b s t r a c t 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 (iAsV) and dimethyl arsinate (DMAV) 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 iAsV and DMAV (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. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Arsenic (As) is a naturally occurring element in food, soil, air, and water. The major sources of exposure are from food and water. ⇑ Corresponding author. http://dx.doi.org/10.1016/j.chemosphere.2014.08.010 0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.
A variety of adverse health effects, e.g., skin and internal cancers, cardiovascular, and neurological effects, have been attributed to As exposure (Chen et al., 1992). Human health effects from chronic As exposure have also been reported mainly in populations with low socioeconomic status and high levels of malnutrition. Diet composition has a significant
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effect on contaminant bioaccessibility and thereby also oral bioavailability. We previously (Alava et al., 2012a) demonstrated that lipids have a large impact on arsenic bioaccessibility, but also that this depends on the As speciation. Some other studies stated that in protein rich rice (Narukawa and Chiba, 2010) AsIII is tightly bound to the thiol groups of the proteins and may influence its bioaccessibility. Previous studies demonstrated a relationship between diet composition and levels of MMAIII found in urine after ingesting inorganic As (Steinmaus et al., 2005). Additionally, some studies reported that people, who consumed diets relatively high in roots and gourds as opposed to meat or other kinds of vegetables, were less likely to develop As-related skin lesions and also showed different metabolic rate of consumed arsenic (Lammon and Hood, 2004). Differences in dietary matrix composition may affect release of As from the food source after ingestion. Food contains both organic and inorganic As, whereas drinking water primarily contains inorganic forms of As. In a scenario of dietary As exposure, rice has been demonstrated to be one of the major foodstuffs contributing to human As exposure (Williams et al., 2007) and also widely consumed (Raab et al., 2009). Interestingly, As biotransformations by human gut microbiota are even reported in case of As contaminated rice (Sun et al., 2012). Moreover, rice is an important carbohydrate sources in both Asian and Western style diets. Trenary et al. reported bioaccessibility of arsenic species from different types of rice in USA by using in vitro setup. These people extended their results to estimate the exposure level to American people by using probabilistic exposure model. All these above observations and reports lead to an interesting and important question; what is the effect of diet matrix on bioavailability of arsenic from rice? As extension to the above question the second part to focus is presystemic metabolism. Upon oral exposure of As contaminated food, gastrointestinal digestion may release a fraction of the matrix-bound As: this is termed the bioaccessible fraction, which is considered a conservative estimator of the oral bioavailable As fraction. The As fraction that is not absorbed across the small intestinal epithelium and the remaining matrix-bound As will reach the colon region. Here, microbial breakdown of the remaining food matrix may further contribute to As release, while the released As in general may get subjected to the diverse metabolic potency of the vast endogenous microbial community. Such conversion of As by colon microbiota is termed presystemic metabolism. Notably, some studies have revealed that microorganisms are important contributors to arsenic speciation changes. A wide range of microbial metalloid biotransformations have been revealed, including oxidation, reduction, methylation and thiolation (DiazBone and Van de Wiele, 2010). Recently, As biotransformations by human gut microbiota have been characterized using As contaminated soils and rice (Van De Wiele et al., 2010; Alava et al., 2012b). Summarizing the above paragraphs; as rice being one of the major source of arsenic through food and diet components like lipids and fiber affecting the bioaccessibility of ingested arsenic species we aimed to study whether and how bioaccessibility of As is affected by diet composition and further how these diets effect the biotransformations of ingested As. Arsenic through rice is major problem in western countries like America (Trenary et al., 2012) and Eastern countries like Bangladesh, India and Taiwan (Meharg and Rahman, 2003, Chowdhury et al., 2000, Pal et al., 2009), and there is significant difference between diet compositions of these two regions; Asian diet (low in fat, protein and high in carbohydrate) and Western diet (high fat and protein) (Suhana et al., 1999 and Park et al., 2006). We choose these two diet compositions to elucidate whether and how bioaccessibility of As is affected by diet composition, further how these diets effect the biotransformations of As.
2. Experimental 2.1. Arsenic standards and samples Standards used were: NaAsO2 solution (VWR, Belgium) for Arsenite (AsIII), Na2HAsO47H2O (Fluka, Switzerland) for Arsenate (AsV), (CH3)2AsO2Na3H2O (Fluka, Switzerland) for dimethyl arsinous acid (DMAV), and (CH3)AsNa2O36H2O (Chemservice, Belgium) for mono methyl arsonous acid (MMAV). Monomethylmonothioarsinic acid (MMMTA) was prepared in the lab. Stability, purity and procedure were already published (Alava et al., 2012). The structure of the product was checked by LC–ESI–MS and MS/MS. MMAIII and DMAIII were purchased from Argus Chemicals (Italy). 2.2. Certified reference material (CRM) and rice sample NIST 1568a Rice Flour (National Institute of Standards and Technology, NIST, USA) was used to check the recovery of total arsenic. The certified value of total As in NIST SRM 1568a is 290 ± 30 lg kg 1, while the As speciation in this reference material is not defined. Basmati rice from Indian origin was purchased from a local supermarket (Colruyt, Gent, Belgium). 2.3. Analysis of rice samples for As content All rice samples were microwave digested at 80 °C for 30 min using water as extraction solvent. This method has previously been proven to be successful for extracting total As from rice (Alava et al., 2012). NIST 1568a Rice Flour (National Institute of Standards and Technology, NIST, USA) was used to check the recovery of total arsenic. The certified value of total As in NIST SRM 1568a is 290 ± 0.03 lg kg 1. Digested samples were filtered using a 0.45 lm syringe-type PVDF membrane filter and the filtrate was diluted to 25 mL using double distilled deionized water. This filtrate was analyzed for total arsenic content using ICP–MS (Table 1). The same filtrate was used for speciation analysis using HPLC–ICP– MS (Alava et al., 2011). 2.4. Setup To better mimic in vivo gastrointestinal behavior of As, all regions – stomach, small intestine, and colon – were simulated. The IVG method was previously validated against in vivo data for As bioaccessibility (Rodriguez and Basta, 1999) in the upper digestive tract (gastric, small intestine), whereas the SHIME has been validated against in vivo data for the colon microbial community composition and metabolic activity toward drugs and phytoestrogens (Molly et al., 1994; Possemiers et al., 2006). To mimic the inter variability of microorganisms in different persons, fecal matter from ten different people representing both Asian and Western countries is collected, mixed well into a homogenous mixture and used as a fecal inoculate in SHIME reactor to develop the microbial source.
Table 1 Optimized instrumental settings for ICP-MS. Detection DRC mode (As) Instrument Plasma RF power Nebulizer flow rate Lens voltage and autolens Dwell times m/z Reaction gas Cell parameters Rpq
Perkin Elmer Elan DRC-e 1250 W 0.7–1.1 mL min 1 (optimized daily) Optimized for AsO daily 91 (AsO 500 ms) O2, 0.75 mL min 1 0.6
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Asian diet and western diet were prepared (Table 2) with rice as source of As. Diets were incubated in gastric solution (1L) at a liquid to solid ratio of 5. One hundred ml/h of gastric juice was added during 2 h to perform gastric digestion. By adjusting the pH and adding pancreatin and bile salts according to the IVG protocol, intestine digestion was performed for 3 h. Samples were collected each hour. Subsequently, 30 mL of gastrointestinal solution was incubated with 30 mL of distal colon suspension from the SHIME system. The vessels containing the colon digests were capped with butyl rubber stoppers and subsequently flushed with N2 for 30 min to obtain anaerobic conditions. They were incubated on a shaker at 150 rpm and 37 °C for 48 h. Samples were collected after 0, 5, 8, 24 and 48 h. All experiments were performed in triplicate and analyzed for As species using HPLC–ICP–MS.
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2.7. Absorption percentage of As species through Caco-2 cells To assess the absorption of bioaccessible As across the gut epithelium an in vitro Caco-2 cell line model was used. Caco-2 cells were cultivated until differentiation and brought in contact with 100 lg/L of each As species in both Asian diet and Western diet matrix separately. Caco-2 cell viability was measured with an MTT assay and epithelial transport was evaluated by measuring the As concentration in the apical and basolateral compartment of a Transwell insert experimental setup. A more detailed description (Blaser, 2007) and discussion of these epithelial transport experiments can be consulted in Alava et al. (under review). All experiments are done in triplicates. 2.8. Stability and storage of solutions
2.5. Sample treatment To preserve the As speciation all samples were immediately stored in freezer at 80 °C. Before performing the analysis, samples were thawed and upon complete thawing, samples were centrifuged at 10 400 relative centrifugal forces to separate solid-bound (pellet) As and As released to the liquid fraction (i.e. the bio-accessible fraction). Samples were filtered using a 0.45 lm syringe-type PVDF membrane filter. The filtrate was injected onto a HPLC column to perform As speciation analysis using HPLC–ICP–MS (Alava et al., 2011). The total As amount is also analyzed in filtrate using ICP-MS and it was considered the bio-accessible fraction. Solidbound As (As remained in pellet) was extracted using microwave digestion. Total As content was analyzed in both filtrate and pellet samples to calculate chromatographic recovery and mass balance. 2.6. Instrumentation A MarsX system (CEM, Matthews, NC, USA) was used for the microwave-assisted extraction. A HPLC–ICP–MS system consisting of a liquid chromatographic system (PerkinElmer Series 200, Sunnyvale, CA, USA) hyphenated to an inductively coupled plasma mass spectrometer (ICP-MS, Elan DRC-e, PerkinElmer, Sunnyvale, CA, USA) was used for speciation analysis. A Hamilton PRP-X100 anion exchange column (Grace, Belgium) and a Zorbax XDB C18 column (250 mm 4.6 mm I.D., 5 lm, 120 A, Agilent, United Kingdom) were used as stationary phases. By using solution from PerkinElmer (Masscall solution, Sunnyvale, CA, USA) the instrument was tuned to maintain the ratio of oxides (CeO/Ce) and doubly charged ions (140Ce2+/140Ce+) lower than 0.03 to minimize the potential interferences, as prescribed by PerkinElmer. Oxygen was used as reaction gas in both methods, resulting in detection of Arsenic as AsO (arsenic oxide) at 91 amu. This procedure is designed to overcome interferences caused by ArCl+ and CaCl+ upon Arsenic detection at 75 amu.
First, the stabilities of several As standards and stock solutions during storage at 4 °C was checked by measuring their concentrations after 1 day, 2 days, 1 week, 2 weeks and 4 weeks. Individual standards were prepared by diluting the stock solutions to 100 lg As/L. A mixed standard, containing all compounds, at a level of 100 lg As/L each, was also prepared. MMAIII and DMAIII stock solutions were found to be unstable when stored for more than 1 week. In these solutions, increasing intensities of respectively MMAV and DMAV peaks were measured. Therefore, they were stored in a freezer at 80 °C, as those standards were stable at this temperature. Stability was also evaluated under these conditions after 15 days and one month. After one month when they are analyzed, a higher variability that is 2% of RSD was observed compared to initial results. Except for MMAIII and DMAIII stock solutions, other stock solutions were stored in a fridge at 4 °C until their analysis within four weeks after preparation. Standards prepared in SHIME matrix instead of MQ water were freshly prepared from the stored stock solutions. Samples collected from the SHIME colon compartments or batch experiments were immediately stored at 80 °C and analyzed before 3 days from collection point of time. 2.9. Statistical analysis One-way analysis of variance (ANOVA) was applied to detect possible differences in bioaccessible As content and biotransformation of As content. A significance level of P < 0.05 was adopted for all comparisons. Statistical analysis was performed using Origin 8.0 (Originlab, North Hampton, MA). 3. Results 3.1. Analysis of rice samples for As content
Compound
Source
Asian diet (g/meal)
Western diet (g/meal)
Protein
Casein Gluten
5.5 19.2
22.0 10.9
Arsenic was extracted from all rice types using microwave digestion. Extraction efficiencies were calculated on a NIST 1568a certified reference rice matrix and were found to be almost 100% (Table 3). The digested solution was analyzed for total As content and As speciation. The total amount of arsenic was 395.1 ± 8.3 lg kg 1 in basmati rice (Table 3). DMAV represented the most abundant species followed by AsV and AsIII (Table 3). The distribution of organic versus inorganic As was around 70% versus 30% at starting point (Table 3).
Fat
Corn oil Butter
22.9 0.0
8.1 33.1
3.2. Quality control
Carbohydrates
Basmati rice (only source of As) Starch Sucrose Dietary fiber (pectin)
56.0
56.0
58.0 13.9 3.0
0.0 52.7 0.0
Table 2 Composition of different diets.
The amount of As remaining in the pellet was analyzed and the sum of As in pellet and bioaccessible As was used to calculate a mass balance. In both diets the As recovery ranged between 97.2% and 98.8%. Arsenic speciation is an important determinant
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in this study; so we calculated chromatographic recovery from the sum of all chromatographically detected species, so that to check whether we are missing some unknown peaks. The chromatographic recoveries for all colon digests were satisfactorily high: 97 ± 3% (mean ± SD). 3.3. Bioaccessibility of As in cooked rice The bioaccessibility of As from Asian and Western diets upon the gastric phase was 95% and 83%, respectively (Fig. 1). Upon 3 h of intestinal digestion, the bioaccessible As fraction significantly decreased from 95% to 83% for Asian diet and from 83% to 63% for Western diet. Subsequently, the bioaccessible As fraction was decreased to 59.7% for Asian diet and to 56.1% for Western diet by the end of the 48 h colon digestion (Fig. 1).
Fig. 1. Bioaccessibility of total As in rice at the stomach, small intestine and colon stage at different time intervals.
3.4. Arsenic speciation at the simulated colon stage The most important observation at the simulated colon stage was the occurrence of significant levels of MMAIII and MMMTAV upon digestion of both diets (Table 4). The concentration of MMAIII accounted for 18% and 21% of absolute total As (22.4 lg) in digested Asian diet and Western diet respectively (Fig. 2). Due to thiolation of pentavalent methylated arsenicals, MMMTAV production accounted for 15% and 18% of absolute total As in digested Asian and Western diet respectively (Fig. 2). 3.5. Absorption percentage of As species through Caco-2 cells Given the diversity of As speciation changes upon incubation with gut microbiota, we evaluated the potency for these As metabolites to be absorbed across the gut epithelium. Yet, given the diversity in toxicity, we first evaluated the inherent toxicity of the different As species towards Caco-2 cells by monitoring their viability with an MTT assay upon incubation. Exposure to 100 lg/L of each As species did not result in any viability loss, except for the trivalent methylated As species (50% loss of viability). This was also reflected by an decrease in transepithelial electrical resistance (TEER). Trivalent methylated species were more efficiently absorbed across a confluent Caco-2 cell layer, with DMAIII displaying 71% of transport from the apical to basal compartment. The point to be noted here is the toxic effect of trivalent species on Caco2-cell lines may also has an additional affect on those species transport through the cell line. On the other hand, pentavalent methylated species and DMAV in particular displayed the lowest absorption (28%), while thiolated methylarsenicals displayed a similar absorption (49%) as inorganic (iAs) species (46%). As iAs species are only available for absorption for around 46% at the intestine level, the remaining amount of As (2.2 lg in case of an Asian diet and 2.0 lg compared to a Western diet) will become available to colon microbes. 4. Discussion A first important observation in this study is that methylation of inorganic As appeared to be a diet dependent process with the Western diet displaying a delayed but more efficient methylation
(P < 0.05) than Asian diet. Although higher absolute amounts of bioaccessible As reached the colon upon incubation of Asian diet, the more efficient methylation during colon digestion of Western diets finally resulted in a higher production of trivalent and thiolated methylarsenicals (Table 4). Human gut microbiota (Van de Wiele et al., 2010) and mice caecum microbiota (Pinyayev et al., 2011) were previously reported to metabolize soil-derived As and form methylated and thiolated arsenicals. This difference in production trivalent and thiolated methylarsenicals between the diets illustrates that the contaminated matrix affects As speciation changes by human colon microbiota. Assessing the internal exposure to As species upon ingestion of a contaminated matrix may therefore require a better characterization of matrix composition. The finding of trivalent methylarsenicals and thiolated methylarsenicals at significant concentrations is of toxicological concern. The LD50 of MMAIII and DMAIII is reported to be lower than that of AsIII, suggesting that MMAIII and DMAIII, intermediates in inorganic As methylation, is more toxic (styblo et al., 2002) and cytotoxic (Naranmandura et al., 2007). Moreover, trivalent organoarsenic compounds display a higher transport across epithelial cells in comparison with inorganic arsenic and the pentavalent organic species (Calatayud et al., 2010; Calatayud et al., 2011). Dopp et al., 2004, also stated that DMAIII has higher membrane permeability then MMAIII. Another important observation was the formation of MMMTAV. MMMTAV toxicity towards all human cells remains to be resolved. Yet, other thiolated methylarsenicals like DMMTAV (dimethyl monothio arsenic acid) have displayed cytotoxic and genotoxic effects similar to trivalent arsenicals (Yoshida et al., 2001; Kuroda et al., 2004). The cytotoxicity of MMMTAV in human bladder cells is DMAIII, DMMTAV > iAsIII, iAsV > MMMTAV > MMAV, DMAV, and DMDTAV (Naranmandura et al., 2011). Eventhough MMMTAV is comparatively less toxic, our transport experiments showed that MMMTAV was efficiently absorbed across Caco-2 cells (50%), similar to both iAs species, indicating chances of their high exposure after their presystemic formation. These observations emphasize the need for considering presystemic metabolism of As in the colon as a relevant process when assessing As toxicokinetics. Following diet composition affect on As speciation changes next important observation is As bioaccessibility is largely affected by
Table 3 As speciation by HPLC–ICP–MS and total As results by ICP–MS in basmati rice, NIST 1568a (290 lg kg undetected.
NIST 1568a Basmati
1
reported value). Tabulated as average ± standard deviation, n = 3; ND:
AsIII (lg/kg)
AsV (lg/kg)
DMAV (lg/kg)
MMAV (lg/kg)
Total As (lg/kg)
Extraction efficiency (%)
53.3 ± 1.0 2.4 ± 0.2
46.1 ± 0.7 90.4 ± 2.1
172.3 ± 1.7 298.8 ± 6.2
13.2 ± 0.3 ND
286.8 ± 4.1 395.1 ± 8.3
98.9
761
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Table 4 The total As amount and amounts of different As species in supernatants by end of stomach, intestinal phase and 48 h incubation of colon extracts (average ± standard deviation), n = 3; ND: undetected. DMAV (lg)
MMAV (lg)
AsV (lg)
MMMTAV (lg)
MMAIII (lg)
DMAIII (lg)
Sum of species (lg)
Initial total As (lg)
Total bioaccessible As (%)
Asian diet (end of stomach phase) Asian diet (end of intestine phase) Asian diet (end of 48 h colon incubation) Western diet (end of stomach phase) Western diet (end of intestine phase) Western diet (end of 48 h colon incubation)
ND
15.9 ± 0.9
ND
5.3 ± 0.2
ND
ND
ND
21.2
22.4
94.6
ND
14.1 ± 0.8
ND
4.1 ± 0.3
ND
ND
ND
18.2
22.4
81.2
ND
2.1 ± 0.2
1.6 ± 0.2
2.2 ± 0.3
3.4 ± 0.4
4.1 ± 0.4
ND
13.4
22.4
59.7
ND
14.4 ± 0.9
ND
4.4 ± 0.3
ND
ND
ND
18.8
22.4
83.9
ND
11.4 ± 0.5
ND
2.8 ± 0.3
ND
ND
ND
14.2
22.4
63.4
1.8 ± 0.003
ND
3.9 ± 0.003
4.8 ± 0.003
ND
12.6
22.4
56.2
ND
2.1 ± 0.003
Asian diet 100 80 60 40
DMA
30
V
MMA
V
MMA
III
DMA
III
20 V
MMMTA
10
0 0
20
40
Bioaccessible As (%) and As in pellet
AsIII (lg)
Bioaccessible As (%) and As in pellet
Treatment
Western diet
40
20
40
60
Time (hr)
III
As
40
V
As
Pellet
30
20
10
0 60
Time (hr)
Bioaccessible As (%) and As in pellet
Bioaccessible As (%) and As in pellet
V
0
0
Asian diet
40
As
20
60
50
20
III
Pellet
Time (hr)
0
As
Western diet 100 80 60 40 40
DMA
30
MMA
V
MMA
III
DMA
20
V
III
MMMTAV
10 0 0
20
40
60
Time (hr)
Fig. 2. Arsenic species in the aquatic phase and total As in the solid residue (pellet) as a function of time while incubating Asian diet and Western diet with As-polluted rice in colon suspension, expressed as percentage of total As in suspension.
the composition of diet. The acidic conditions from the gastric phase resulted in higher As bioaccessibility than those from the intestine, thereby confirming similar findings on gastric vs. intestinal bioaccessibility in earlier studies (Juhasz et al., 2006 and Denys et al., 2012). However, Sun et al., 2012 reported higher bioaccessibility at small intestine level, this difference might be attributed to difference in whole diet matrix ingested and also to sample pretreatment. In this study samples are centrifuged at 21 000 rpm. Incubation with an Asian diet displayed higher As bioaccessibility values in both gastric as intestine conditions than incubation with a Western diet (Fig. 1). A detailed analysis of how lipids affect As bioaccessibility can be consulted in Alava et al. (2013). While it
must be acknowledged that bioaccessibility values are only estimators of in vivo bioavailability, our in vitro findings are quite similar to earlier in vitro and in vivo observations with cooked contaminated rice. Laparra et al. (2005) found 63% to 99% of the total As to be bioaccessible from cooked rice, while the in vivo bioavailability of As was found to be 89% in swine that were fed cooked rice (Juhasz et al., 2006). On a concluding note there is high amount of arsenic released from the food matrix at gastric level which may alter by composition of the food matrix. Thus difference in diet composition may also differ the amount of As reaching colon level and amount of As available for pre-systemic metabolism.
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Next, we investigated to what extent bioaccessibility is species dependent. iAsV was proportionally more easily released from the rice matrix during gastrointestinal digestion than DMAV. The acidic environment in the simulated stomach (pH 2.0) will result in a more efficient breakdown of the rice protein fraction (Goodman, 2010) and it has been found that inorganic As species are more likely to bind to the protein matrix of endosperm cells (Sanz et al., 2007; Narukawa et al., 2008 and Moore et al., 2010) more particularly by complexing to thiol containing amino acids (Lombi et al., 2009). The observed higher release of iAs from the rice matrix into the supernatant of the gastric digest is therefore quite plausible. In summary, we can conclude that the food matrix affected the efficiency with which the released As species got metabolized by gut microbiota from human origin. In addition, release of As from contaminated food matrices during gastrointestinal digestion depends on the food matrix composition. Finally, different As species are released to a different extent upon gastrointestinal incubation of a contaminated food matrix. These findings ask for a more thorough characterization of the ingested matrix, especially when different As species are present. Additionally, these findings imply that the internal exposure assessment to As from an ingested matrix should entail (i) a speciation analysis of arsenicals present in the food matrix and (ii) a thorough characterization of the ingested matrix as both the bioavailability as microbial metabolic potency towards As seems to rely on matrix composition. Acknowledgement This study was funded by Federal public service of health, food chain safety and environment Contract (BIOTRAS RF 6247). References Alava, P., Du laing, G., Tack, F., Van De Wiele, T., 2012. HPLC ICP MS method development to monitor arsenic speciation changes by human gut microbiota. Biomed. Chromatogr. 26 (4), 524–533. Alava, P., Du laing, G., Tack, F., Van De Wiele, T., 2012a. Towards an optimal sample preparation procedure: Arsenic speciation is preserved during extensive grinding and pressurized extraction from rice. Anal. Methods 4 (5), 1237–1243. Alava, P., Tack, F., Laing, G.D., de Wiele, T.V., 2012b. Arsenic undergoes significant speciation changes upon incubation of contaminated rice with human colon micro biota. J. Hazard. Mater. http://dx.doi.org/10.1016/j.jhazmat.2012.05.042. Alava, P., Du Laing, G., Tack, F., Van De Wiele, T., 2013. Arsenic bioaccessibility upon gastrointestinal digestion is highly determined by its speciation and lipid-bile salt interactions. J. Environ. Sci. Health A. Tox. Hazard. Subst. Environ. Eng. 48 (6), 656–665. http://dx.doi.org/10.1080/10934529.2013.732367. Blaser, D., 2007. Determination of Drug Absorption Parameters in Caco-2 Cell Monolayers with a Mathematical Model Encompassing Passive Diffusion, Carrier-Mediated Efflux, Non-Specific Binding and Phase II Metabolism. University of Basel (Thesis). Calatayud, M., Gimeno, J., Vélez, D., Devesa, V., Montoro, R., 2010. Characterization of the intestinal absorption of arsenate, monomethylarsonic acid, and dimethylarsinic acid using the Caco-2 cell line. Chem. Res. Toxicol. 23, 547–556. Calatayud, M., Gimeno, J., Vélez, D., Devesa, V., Montoro, R., 2011. In vitro study of intestinal transport of arsenite, monomethylarsonous acid, anddimethylarsinous acid by Caco-2 cell line. Toxilogical Lett. 204 (2–3), 547– 556. Chen, C.J., Chen, C.W., Wu, M.M., Kuo, T.L., 1992. Cancer potential in liver, lung, bladder and kidney due to ingested inorganic arsenic in drinking water. Br. J. Cancer 66, 888–892. Chowdhury, T.R., Basu, G.K., Mandal, B.K., et al., 2000a. Arsenic poisoning in the Ganges delta (vol 401, pg 545, 1999). Nature 404. 36–36. Chowdhury, U.K., Biswas, B.K., Chowdhury, T.R., et al., 2000b. Arsenic groundwater contamination and sufferings of people in West Bengal – India and Bangladesh. Trace Elem. Man Anim. 10, 645–650. Denys, S., Caboche, J., Tack, K., Rychen, G., Wragg, J., Cave, M., Jondreville, C., Feidt, C., 2012. In Vivo validation of the unified BARGE method to assess the bioaccessibility. Environ. Sci. Technol. 46 (11), 6252–6260. Diaz-Bone, R.A., Van de Wiele, T., 2010. Biotransformation of metal(loid)s by intestinal microorganisms. Pure Appl. Chem. 82, 409–427. Dopp, E., Hartmann, L.M., Florea, A.-M., von Recklinghausen, U., Pieper, R., Shokouhi, B., Rettenmeier, A.W., Hirner, A.V., Obe, G., 2004. Uptake of inorganic and organic derivatives of arsenic associated with induced cytotoxic and genotoxic effects in Chinese hamster ovary (CHO) cells. Toxicol. Appl. Pharmacol. 201, 156–165.
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