Food and Chemical Toxicology 66 (2014) 76–88

Contents lists available at ScienceDirect

Food and Chemical Toxicology

journal homepage: www.elsevier .com/locate/ foodchemtox

Urinary excretion of arsenicals following daily intake of various seafoodsduring a two weeks intervention q

http://dx.doi.org/10.1016/j.fct.2014.01.0300278-6915/� 2014 Elsevier Ltd. All rights reserved.

Abbreviations: AB, arsenobetaine; AC, arsenocholine; As (III), arsenite; As (V),arsenate; As, arsenic; As3MT, arsenic methyl transferase; CR, creatinine; CRM,certified reference material; CRP, C-reactive protein; DMA, dimethylarsinate; HPLC,high performance liquid chromatography; iAs, inorganic arsenic; ICP-MS, induc-tively coupled plasma mass spectrometry; \LOQ, limit of quantification; MA,methylarsonate; tAs, total arsenic; TETRA, tetramethyl arsonium ion.

q This research was supported by the Norwegian Research Council (project nr.142468/140), Oslo and Akershus University College of Applied Sciences, NationalInstitute of Nutrition and Seafood Research and the Norwegian Institute of PublicHealth. The study was approved by the National Committee for Research Ethics andwas carried out in accordance with The Code of Ethics of the World MedicalAssociation. Written informed consent was obtained from each participant.⇑ Corresponding author. Address: Atlantis Medical University College, P.O.

Box 4290 Nydalen, N-0402 Oslo, Norway. Tel.: +47 930 90 250; fax: +47 23 00 7351.

E-mail addresses: molinmarianne@gmail.com (M. Molin), sulven@hioa.no (S.M.Ulven), Lisbeth.Dahl@nifes.no (L. Dahl), walter.goessler@uni-graz.at (W. Goessler),Daniel.Fliegel@tine.no (D. Fliegel), marianneholck@yahoo.no (M. Holck),jjsl@food.dtu.dk (J.J. Sloth), arne.oshaug@hioa.no (A. Oshaug), Jan.Alexander@fhi.no(J. Alexander), Helle.Margrete.Meltzer@fhi.no (H.M. Meltzer), Trond.Arild.Yders-bond@ssb.no (T.A. Ydersbond).

M. Molin a,b,c,⇑, S.M. Ulven a, L. Dahl d, W. Goessler e, D. Fliegel d, M. Holck a, J.J. Sloth d,f, A. Oshaug a,J. Alexander g, H.M. Meltzer g, T.A. Ydersbond h

a Department of Health, Nutrition and Management, Faculty of Health Sciences, Oslo and Akershus University College of Applied Sciences, P.O. Box 4, St. Olavs Plass,NO-0130 Oslo, Norwayb Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, P.O. Box 1110, Blindern, NO-0317 Oslo, Norwayc Atlantis Medical University College, P.O. Box 4290 Nydalen, N-0402 Oslo, Norwayd National Institute of Nutrition and Seafood Research, P.O. Box 2029 Nordnes, N-5817 Bergen, Norwaye Institute for Chemistry-Analytical Chemistry, Stremayrgasse 16, 8010 Graz, Austriaf National Food Institute, Technical University of Denmark, DK-2860 Søborg, Denmarkg Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, N-0403 Oslo, Norwayh Statistics Norway, P.O. Box 8131 Dep, N-0033 Oslo, Norway

a r t i c l e i n f o

Article history:Received 3 December 2013Accepted 16 January 2014Available online 24 January 2014

Keywords:ArsenicSeafood safetySpeciationDietary interventionHumanMethylarsonate

a b s t r a c t

The excretion pattern of arsenic (As) species after seafood intake varies widely depending on speciesingested and individual handling. We have previously reported the 72 h urinary excretion of arsenicalsfollowing a single dose of seafood. Here, we report the excretion patterns in the same 37 subjects follow-ing 15 days daily consumption of either 150 g cod, salmon, blue mussels or potato (control), followed by a72 h period with a low-As diet. In all seafood groups, total As (tAs) in plasma and urinary excretion of tAs,arsenobetaine (AB) and dimethylarsinate (DMA) increased significantly after the intervention. Confirm-ing the single dose study AB and DMA excreted were apparently endogenously formed from other arsen-icals ingested. Total tAs excretion was 1386, 763 and 303 lg in the cod, blue mussel and salmon groups,respectively; about twice the amounts after the single dose study indicating accumulation of arsenicals.In the cod group, rapid excretion after the single dose was associated with lower total As in blood and lessaccumulation after two weeks with seafood indicating lower accumulation. In the blue mussels grouponly, inorganic As (iAs) excretion increased significantly, whilst methylarsonate (MA) strongly increased,indicating a possible toxicological concern of repeated mussel consumption.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

While it is well documented that a diet rich in seafood promoteshealth (Alexander et al., 2006; Mozaffarian and Rimm, 2006), sea-food may also contain potentially harmful compounds, like arsenic(As) (Alexander et al., 2006; Sirot et al., 2012). In addition to thewell-known toxicant inorganic As (iAs), thio-arsenicals, whichhave been little studied so far, constituted 10% of urinary As ex-creted after a bolus dose of blue mussels (Molin et al., 2012b).Thio-arsenicals are considered potentially toxic (Naranmanduraet al., 2011; Ochi et al., 2008; Rehman and Naranmandura, 2012).High concentrations of methylarsonate (MA) and dimethylarsinate(DMA) may also be of concern (EFSA, 2009; Fowler et al., 2007). Formost of the population within the EU, As exposure from drinkingwater is insignificant, but the possible risks of As exposure froma seafood rich diet are little studied (Borak and Hosgood, 2007;Choi et al., 2010; EFSA, 2009).

http://crossmark.crossref.org/dialog/?doi=10.1016/j.fct.2014.01.030&domain=pdfhttp://dx.doi.org/10.1016/j.fct.2014.01.030mailto:molinmarianne@gmail.commailto:sulven@hioa.nomailto:Lisbeth.Dahl@nifes.nomailto:walter.goessler@uni-graz.atmailto:Daniel.Fliegel@tine.nomailto:marianneholck@yahoo.nomailto:jjsl@food.dtu.dkmailto:arne.oshaug@hioa.nomailto:Jan.Alexander@fhi.nomailto:Helle.Margrete.Meltzer@fhi.nomailto:Trond.Arild.Ydersbond@ssb.nomailto:Trond.Arild.Ydersbond@ssb.nohttp://dx.doi.org/10.1016/j.fct.2014.01.030http://www.sciencedirect.com/science/journal/02786915http://www.elsevier.com/locate/foodchemtox

M. Molin et al. / Food and Chemical Toxicology 66 (2014) 76–88 77

The water-soluble specie arsenobetaine (AB) is the main arsen-ical in most seafood, usually constituting up to 95% of tAs in marinefish and up to 30–40% in blue mussel (EFSA, 2009). The iAs contentis generally low in most seafood; fin-fish like cod and salmon usu-ally contain

-7 7

24h

Baseline Day 21 Day 22 Day 23

21.0 21.1 21.2 21.3

Strictly controlled diet 2/period 2

24h 24h 24h 24h

Test meal 2 (day 21)

14 days with seafood lunch

Start of semi-controlled diet period

24h

Day 0 Day 1 Day 2

0.0 0.1 0.2 0.3

24h 24h 24h 24h

Test meal 1 (day 0)

Run-in

Strictly controlled diet 1/period 1

Plas

ma

Urin

eSp

ot

Spot

Spot

Spot

Day

Randomization

Molin et al. (2012a)* Present publication

Run-in

During the run-ins the subjects followed restrictions (no seafood, rice- and rice products, mushrooms or dietary supplements).

* The results assessing the urinary arsenical excretion after a bolus dose of seafood is published in Molin et al. (2012a).

Fig. 1. Study design. Thirty-seven subjects were randomized (the subjects were randomized, but not stratified by gender, and therefore by chance all subjects in the salmongroup were females) to four intervention groups: cod (9), salmon (11), blue mussel (7 (originally 38 subjects were part of the study, but data is missing for one subject in theblue mussel group for period 2)) and control (10). During the seven days of run-in (day �7 to day 0) and throughout the intervention (through day 23) the volunteers keptrestrictions regarding As-containing food. At day 0.0 and day 21.0 an identical controlled test meal were given and the following 72 h all urine were collected; the first 24 h inthree batches (test meal-2 pm, 2–7 pm, 7 pm until morning urine next day), the two last days in 24 h-batches. During the 72 h urine-collection periods, all volunteers got anequal, strictly controlled diet, identical for both periods. The urinary As excretion after test meal 1 reflects the urinary As excretion after ingestion of a single meal of seafood,whereas the urinary As excretion after ingestion of test meal 2 reflects the As excretion after a 15 consecutive days with seafood (14 days with seafood/potato lunch followedby test meal 2). The arrows pointing upwards indicate the time the urine and plasma samples were collected. Regarding the urine samples, it is indicated whether the samplewas a morning spot sample (spot) or part of a complete sampling (24 h).

78 M. Molin et al. / Food and Chemical Toxicology 66 (2014) 76–88

until all urine from each day/period was pooled. The total volume was then mea-sured and 15 ml of the urine was subsequently distributed into aliquots and keptfrozen (�70 �C) until analysis.

2.6. Adjustment for creatinine

The morning spot urine samples on day 7 and 21 were adjusted for the urinarycreatinine (CR) concentrations. The mean CR concentration on day 7 and day 21 was1.51 (0.51) g/L and 1.65 (0.64) g/L, respectively (Table 1). For the 72 h after the lastseafood meal (test meal 2), all urine was collected, thus the total amount (lg) Asexcreted was calculated and therefore not adjusted for the creatinineconcentrations.

2.7. Analytical methods

The tAs in the food, plasma and urine was determined using inductively coupledplasma mass spectrometry (ICP-MS) as previously described (Julshamn et al., 2007;Molin et al., 2012a). The arsenicals DMA, MA and iAs were measured with HPLC–ICP-MS using an anion-exchange column, while AB was measured with HPLC–ICP-MS using a cation-exchange column as previously described (Molin et al.,2012a). In all cases external calibration was used for the quantification of tAs andindividual arsenicals. The accuracy of the determination of tAs in the food was eval-uated by means of analysis of the certified reference materials (CRMs) DORM-2Dogfish muscle (National Research Council of Canada) and BCR CRM 627 Tuna fishtissue (IRMM, Geel, Belgium); the latter reference material was also used in theevaluation of the speciation analysis of the food. NIES No 18 Human urine (NationalInstitute for Environmental Studies, Ibaraki, Japan) was used to evaluate tAs as wellas iAs, MA, DMA and AB in the urine samples. All obtained results agreed well withthe respective CRMs as previously described in Molin et al. (2012a).

For the urinary creatinine determination, the urine samples prior to analysiswere defrosted at room temperature and centrifuged in an Eppendorf (5810R) cen-trifuge (15 min, 2000g and 4 �C). An aliquot of 200 lL was transferred to the testtube and placed in the Maxmat carousel. All sample treatment was automatic; sam-ples were diluted 1:20 with distilled water and reagents added. All samples wereanalyzed in one batch on the same day. Determination of CR was performed by acolorimetric enzymatic principle using MAXMAT PL II multidisciplinary diagnosticplatform using the Creatinine PAP kit, (Maxmat S.A., Montpellier, France).

2.8. Statistical analyses

The IBM� SPSS� Statistics (version 19) (SPSS Inc., Chicago, IL; USA) and R 2.10.1(http://cran.r-project.org/) were used for the statistical analyses. For maximalrobustness, non-parametric tests were preferably used due to the small samplesize: Mann Whitney U test were used to identify significant between-group differ-ences, while Wilcoxon paired samples test were used to identify within-groupchanges. Coefficients of correlation were calculated by the Spearman’s rho test. Inaddition, ANOVA/ANCOVA models together with pairwise t-tests (with Holm’s cor-rection for multiple comparisons) were used when appropriate, in particular whenlinear model estimates were needed. Non-quantified values (i.e. values below LOQ),

were set at LOQ/2 as suggested by international guidelines (FAO/WHO, 1995; Kroeset al., 2002). The following values (LOQ/2) were used in the present study: iAs(0.15 lg/kg); DMA (0.25 lg/kg); and MA (0.15 lg/kg).

3. Results

3.1. As content of the diet

The average daily tAs intake was calculated by analysing twoidentical samples of each of the test meals and the duplicate por-tions of the strictly controlled diet. The daily tAs intake was 670,180, 620 and 3.7 lg and the iAs intake was 2.8, 3.3, 13 and4.4 lg in the semi-controlled diets of the cod, salmon, blue musseland control group, respectively (Table 2, column 2).

3.2. Urinary and plasma As concentrations

Between the test meal 1 on day 0 and the start of the 15 dayswith seafood on day 7, the subjects ingested a diet practically freefrom As (Fig. 1). Thus, the morning urine As concentrations on day7 reflects the concentration after a one week wash-out of As, whilethe morning urine As concentrations on day 21 (before the lastmeal of the 15 days with seafood) is the resulting balance obtainedfollowing 14 days of repeated intake and elimination (Table 1). Themean urinary tAs concentrations at day 7 was below 20 lg/g CR forall intervention groups, comparable to that observed in unexposed,healthy subjects from the UK, who refrained from seafood threedays prior to urine sampling (17.5 lg/g CR) (Brima et al., 2006).The tAs concentrations in morning spot urine increased signifi-cantly (p < 0.05) for all seafood groups during the intervention,with the cod group showing the highest tAs concentration, fol-lowed by the blue mussel, salmon and control group, respectively(486 lg/g CR, 184 lg/g CR, 57 lg/g CR and 8.4 lg/g CR) (Table 1).The same pattern was seen for plasma, mean plasma tAs concen-trations at day 7 were below 0.9 lg/L, but increased significantly(p < 0.05) after the intervention for all seafood groups, with the fol-lowing plasma tAs concentrations on day 21: 12.6 lg/L, 4.6 lg/L,2.9 lg/L and 0.7 lg/L for the cod, blue mussel, salmon and controlgroup, respectively.

The AB spot urine concentrations were below 17 lg/g CR for allintervention groups on day 7 and increased significantly (p < 0.05)in all the seafood-consuming groups during the intervention. The

http://cran.r-project.org/

Table 1As concentrations of morning spot urine and tAs in plasma before (day 7a) and after (day 21) the 14 days semi-controlled diet with seafood (mean (±SD) and median (min–max)).

Total n = 38 (37)b Cod n = 9 Salmon n = 11 Blue mussel n = 8 (7) Control (potato) n = 10

lg/L lg/L p-Valuec lg/L p-Value lg/L p-Value lg/L p-Value

tAs plasmaBefore 0.70 (0.4) 0.50 (0.0) .008⁄ 0.9 (0.6) .008⁄ 0.7(0.3) .012⁄ 0.6 (0.3) NS

0.50 (0.5–2.10) 0.50 (0.50–0.50) 0.5(0.5–2.1) 0.5(0.5–1.2) 0.5 (0.5–1.4)After 5.0 (5.8) 12.6 (7.0) 2.9 (1.6) 4.6 (1.8) 0.7 (0.5)

3.8 (0.5–30.5) 10.6 (7.8–30.5) 3.0 (0.5–4.8) 4.9 (2.5–7.9) 0.5 (0.5–2.1)

g/L g/L p-Value g/L p-Value g/L p-Value g/L p-Value

CreatinineBefore 1.51 (0.51) 1.43 (0.47) NS 1.76 (0.48) NS 1.35 (0.41) 0.012 1.42 (0.62) NSAfter 1.65 (0.64) 1.68 (0.75) 1.58 (0.69) 1.76 (0.66) 1.65 (0.64)

lg As/g CR lg As/g CR p-Value lg As/g CR p-Value lg As/g CR p-Value lg As/g CR p-Value

tAs urineBefore 13.7 (11.2) 11.6 (6.04) .008⁄ 9.58 (6.06) .003⁄ 14.5 (6.79) .012⁄ 19.3 (18.7) NS

10.4 (2.92–59.7) 11.7 (5.04–25.2) 7.83 (3.57–23.6) 11.8 (8.1–27.2) 12.8(2.92–59.7)After 173 (233) 486 (276) 57.0 (21.1) 184 (107) 8.35 (8.23)

67.1 (2.98–1020) 463(113–1020) 44.6 (37.7–107) 183 (65.6–348) 5.6 (2.98–30.3)

lg As/g CR lg As/g CR p-Value lg As/g CR p-Value lg As/g CR p-Value lg As/g CR p-Value

AB urineBefore 11.5 (10.8) 10.0 (4.59) .008⁄ 7.17 (6.87) .004⁄ 12.8 (6.11) .028⁄ 16.7 (17.8) NS

8.94 (1.21–49.2) 9.68 (4.61–18.3) 4.64 (1.46–22.4) 10.6 (6.82–23.0) 11.7 (1.2–49.2)After 150 (224) 468 (238) 48.1 (25.6) 74.1 (39.3) 6.07 (10.4)

48.8 (0.20–917) 482 (119–917) 41.7 (22.1–96.6) 70.8 (25.3–137) 1.52 (0.20–32.7)

lg As/g CR lg As/g CR p-Value lg As/g CR p-Value lg As/g CR p-Value lg As/g CR p-Value

iAs urineBefore 0.25 (0.17) 0.30 (0.29) NS 0.20 (0.09) NS 0.20 (0.10) .028⁄ 0.28 (0.12) NS

0.20 (0.08–0.99) 0.18 (0.11–0.99) 0.19 (0.08–0.39) 0.18 (0.11–0.40) 0.24 (0.12–0.45)After 0.38 (0.47) 0.30 (0.42) 0.19 (0.10) 1.08 (0.71) 0.25 (0.12)

0.20 (0.07–2.21) 0.16 (0.12–1.41) 0.17 (0.07–0.41) 0.95 (0.30–2.21) 0.20 (0.16–0.54)

lg As/g CR lg As/g CR p-Value lg As/g CR p-Value lg As/g CR p-Value lg As/g CR p-Value

DMA urineBefore 2.25 (1.60) 1.85 (1.01) .008⁄ 1.97 (0.58) .003⁄ 2.47 (1.19) .028⁄ 2.75 (2.74) NS

1.84 (0.69–10.3) 1.55 (0.69–3.97) 1.84 (0.98–3.23) 1.97 (1.3–4.77) 1.9 (0.94–10.3)After 18.1 (31.8) 7.31 (3.1) 9.34 (2.74) 74.5 (46.4) 1.87 (0.87)

8.35 (0.68–137) 6.73 (2.84–12.5) 10.0 (3.74–12.2) 68.4 (26.0–137) 1.86 (0.68–3.11)

lg As/g CR lg As/g CR p-Value lg As/g CR p-Value lg As/g CR p-Value lg As/g CR p-Value

MA urineBefore 0.34 (0.20) 0.40 (0.29) NS 0.33 (0.12) NS 0.29 (0.18) .028⁄ 0.35 (0.19) NS

0.31 (0.14–1.06) 0.30 (0.15–1.06) 0.35 (0.14–0.50) 0.22 (0.15–0.68) 0.29 (0.14–0.77)After 4.65 (12.5) 0.48 (0.22) 0.73 (1.25) 24.6 (21.9) 0.33 (0.17)

0.40 (0.11–58.3) 0.40 (0.27–0.91) 0.38 (0.11–4.49) 15.2 (5.8–58.3) 0.26 (0.16–0.66)

a The subjects followed restrictions regarding As-containing foods one week prior to day 7.b The data for day 21–23 is missing for one subject.c p-Value for significant within-group changes (Wilcoxon paired samples test). Significant between-group changes when compared with control group (Mann Whitney U

test) are marked with an asterisk (⁄).

M. Molin et al. / Food and Chemical Toxicology 66 (2014) 76–88 79

cod group had the highest mean AB concentration on day 21 fol-lowed by the blue mussel, salmon and the control group (468 lg/g CR, 74 lg/g CR, 48 lg/g CR and 6.1 lg/g CR, respectively).

The mean spot urinary DMA concentrations increased signifi-cantly (p > 0.05) from below 2.8 lg/g CR for all the seafood groups,with highest concentrations on day 21 for the blue mussel group(74.5 lg/g CR), approximately ten times higher than the salmonand cod groups (Table 1). While the other groups all had MA con-centrations below 0.8 lg/g CR on day 21, the mean MA concentra-tion increased more than 80-fold in the blue mussel group fromday 7–21, from 0.29 to 24.6 lg/g CR (p = 0.028) (Table 1).

The iAs concentration in spot urine increased significantly onlyfor the blue mussel group during the intervention (from 0.20 lg/gCR to 1.08 lg/g CR, p = 0.028) (Table 1).

3.3. Urinary excretion of tAs, AB, iAs, DMA and MA during the 72 hfollow-up period

The urinary excretion of tAs and the arsenicals AB, iAs, DMA andMA during the 72 h follow-up period after test meal 2, is shown in

Table 2. The cod group excreted the largest amount of tAs(1390 lg, Table 2); 2-fold more than the blue mussel group, 4.5-fold more than the salmon group and 25-fold more than the con-trol group (Table 2, column 6). The AB excretion was highest inthe cod group (1240 lg) (Table 2).

The iAs excretion was about 4-fold higher in the blue musselgroup (3.8 lg) than the in the cod, salmon and the control groups(1 lg) (Table 2). The DMA excretion was highest after blue musselingestion (180 lg), about 6-fold and 9-fold higher than in the sal-mon and cod groups, respectively (Table 2). Likewise, the MAexcretion in the blue mussel group (34 lg) was more than 20-foldhigher than that found in the other intervention groups. 75–90% ofthe total excreted MA was excreted during the first 24 h.

3.4. Comparison of the plasma tAs concentrations in periods 1 and 2

The plasma response after the single meal in period 1 and thelast of 15 meals in period 2 was qualitatively similar, with a peakin the tAs plasma concentration approximately 2 h after the meals(Fig. 2A). After the peak at 2 h, the plasma tAs at first rapidly

Table 2Urinary As excretion (72 h) after 15 consecutive days with a semi-controlled seafood diet (mean ± SD) and the relative urinary excretion of arsenicals after 15 consecutive dayswith seafood compared to that after a bolus dose with seafood.

1Group/species

2Dailyamountingesteda

3Urine day 21

4Urine day 22

5Urine day 23

6Urine day 21–23

7% of tAs excretedday 21–23

8% of tAs excretedday 0–3

9p-Valueb

lg lg (SD) lg (SD) lg (SD) lg (SD) % of tAs % of tAs

Cod group (n = 9)Total As 670 787 (202) 347 (118) 252 (119) 1390 (412) 100 100AB 490 719 (150) 296 (114) 223 (120) 1240 (363) 89.3 88.5 NS⁄

Inorganic As 2.8 0.4 (0.1) 0.3 (0.1) 0.4 (0.1) 1.1 (0.4) 0.08 0.23 .008DMA 1.3 11 (2.6) 5.0 (1.4) 3.5 (1.3) 19 (4.6) 1.37 1.48 NSMA

0 50

05

1015

2025

30 A

Hours after last As-rich meal

Plas

ma

tAs

(µg/

L)

Plas

ma

tAs

(µg/

L)

SalmonCodMusselsControl

010

2030

40

10 20 30 40

0 5010 20 30 40

0 5010 20 30 40

0 5010 20 30 40

B

Hours after last As-rich meal

05

1015 C

Hours after last As-rich meal

05

1015 D

Hours after last As-rich meal

Plas

ma

tAs

(µg/

L)

Plas

ma

tAs

(µg/

L)

Fig. 2. Total As (tAs) in plasma, lg/L, as a function of time following intake of a single seafood meal, or the last of 15 consecutive seafood meals in period 1 (dotted lines) and 2(bold lines), respectively. Values are group averages and individual values for each study group. (A) Group averages during periods 1 and 2. (B) Individual values for the codgroup. Note the scale, the plasma concentrations in period 2 peaking at around 40 lg/L in the individuals with the highest values, about twice the maximum of period 1. Theaverage differences were much smaller. (C) Individual values for the blue mussels group. Note the scale, the plasma concentrations in period 2 peaking at around 13 lg/L,about twice the maximum of period 1. (D) Individual values for the salmon group. Note the scale, the plasma concentrations in period 2 peaking at around 9–13 lg/L, aboutthree times the maximum of period 1.

M. Molin et al. / Food and Chemical Toxicology 66 (2014) 76–88 81

The urinary AB concentrations patterns were qualitatively quitesimilar in the two periods (Fig. 4B). Since AB was the dominantarsenical excreted, the relationship between the AB excretion ratesat the end of periods 2 and 1 was similar to that for tAs excretion,with significantly higher rates for the cod and salmon groups(Fig. 4B and C). Individual curves for AB excretion in period 2 rela-tive to AB ingested in the cod and blue mussels groups are shownin Fig. 4D. Note the wide excretion range, from less than the testmeal content (about 50% of total AB intake) to about four timesthe content of the last meal.

For DMA, no significant differences in relative excretion werefound in any of the intervention groups after 15 consecutive dayswith seafood compared with that after a single dose of seafood(Table 2, column 7–8). The relative iAs excretion was below 1%for all seafood groups, for the single dose as well as for the 15 con-secutive days with seafood (Table 2, column 7–8).

The DMA excretion pattern was similar for period 1 and 2 forthe salmon and cod groups (Fig. 5B). However, for the blue musselsgroup it was significantly higher throughout period 2. The differ-ence is significant on day 23 (DMAday23 = 3.7 + 3.8 � DMAday2, p(slope 6 1) < 0.001, ANCOVA) (Fig. 5A–C). Individual curves for per-iod 2 excretion of DMA + iAs + MA relative to potential precursors(tAs minus dietary AB, which is considered to be relatively meta-bolically inert) ingested in the cod and blue mussels groups areshown in Fig. 5D. Note the wide excretion range, from approxi-mately 10% excreted as iAs + MA + DMA in individuals in the codgroup to about 20–60% excreted as iAs + MA + DMA of the

non-AB ingested in some blue mussel eaters. Also, the individualvariation was larger in the blue mussel group when compared tothe cod group.

The blue mussel group excreted more of iAs + DMA + MArelative to intake for both periods, in comparison with the otherseafood groups (Fig. 5D). The group means were 27%, 11%, 44%,and 48% in the control, cod, salmon and blue mussels groups,respectively. Except for the blue mussel vs salmon groups(0.1 < p < 0.2), all other group means were significantly(p < 0.0001) different.

The MA excretion relative to that of tAs was below 1% after 15consecutive days with cod and salmon, but 4.5% of tAs in the bluemussel group (Table 2, column 7). The amount of MA excreted rel-ative to tAs was significantly lower after a 15 consecutive dayswith cod, salmon and control than following a single meal. In con-trast, the relative amount of MA excreted was significantly higherafter 15 consecutive days with blue mussel when compared to asingle dose of blue mussel (p = 0.018) (Table 2, column 7–9).

Compared to the other groups, the MA excretion was remark-ably high immediately following the 15 consecutive days with bluemussels (period 2). However, the MA concentration rapidly de-creased after intake of the last blue mussel meal, especially duringthe first 48 h (Fig. 6B). Of the non-AB ingested with test meal 2, 3–10% was apparently excreted as MA in the blue mussel group, incomparison with less than 1% in the cod group (Fig. 6C). The MAexcretion on day 23 was not significantly different from that onday 2 in any group.

0 100 200 300 400 500 600 700

050

010

0015

0020

00 A

Urinary tAs excretion period 1

Urin

ary

tAs

excr

etio

n pe

riod

2 (µ

g)

SalmonCodMusselsControl

0 10 20 30 40 50 60 70

010

020

030

040

050

060

0

B

Hours after last As-rich meal

Urin

e tA

s co

ncen

tratio

n (µ

g/L)

0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70

050

010

0015

00 C

Hours after last As-rich meal

Tota

l tAs

exc

rete

d (µ

g)

050

100

150

200

250

300

D

Hours after last As-rich meal

Urin

ary

tAs

excr

etio

n, %

of t

As

0 5 10 15

02

46

810

12

E

Ratio day0/day2 tAs excretion

Plas

ma

tAs

day

23, µ

g/L

0.4 0.5 0.6 0.7 0.8 0.9 1.0

0.0

0.5

1.0

1.5 F

Ratio tAs excretion/tAs intake period 1

Rat

io tA

s ex

cret

ion/

tAs

inta

ke d

ay 2

1 in

gest

ed in

last

As-

rich

mea

l

Fig. 3. Urinary total As (tAs) in periods 1 and 2. (A) Total tAs excretion during period 2 vs total tAs excretion during period 1. In all study groups, total As excretion in period 2was about twice that of period 1. Within each study group, period 1 and 2 excretion was not strongly correlated. (B) Average urine tAs concentration during periods 1 and 2.Morning spot values for the first day in each period are not shown, as morning spot and full collection values are not comparable. Final urine concentrations were 2–4 timeshigher in period 2 compared to period 1. (C) Cumulative tAs excretion for each group during periods 1 (dotted lines) and 2 (bold dotted lines). (D) Individual cumulativevalues for the fraction (percentage) of the period 1 (dotted lines) and period 2 (bold lines) test meals excreted in the cod group (black) and blue mussels group (blue). Note thewide excretion range in period 2, from less than the test meal content (about 5% of total As intake days 8–21) to three times the meal content, corresponding to about 20% oftotal As intake. (E) Plasma tAs day 23 (period 2) vs ratio of day 0/day 2 tAs excretion (period 1). Regression lines for cod group (black) and the other groups pooled (green)shown. In the cod group, there was a significant (P < 0.01) association between rapid As excretion after the first test meal, as measured by the day 0/day 2 excretion ratio, andlower plasma tAs at the end of the study. A similar relationship was not observed for the control, salmon and blue mussels groups, where the slope differed significantly fromthe cod group (P < 0.001) and was positive (P < 0.01). (F) Excretion on day 21/intake of tAs in the last meal (day 21) vs excretion during the whole period 1/intake of singlemeal. The regression line (not shown) is y = 0.35 + 0.88x, which indicates an increased base level of excretion on day 21, but otherwise a fairly close association with tAsexcretion during period 1 (r2 = 0.22, p < 0.007). The y = x line is shown for comparison between the periods: Period 1 excretion relative to intake on day 0 may be taken as anindicator of absorption, and if a subject excretes a higher fraction on day 21 of the amount ingested in the last meal, i.e. plots above the y = x line, this implies that the subjecteither has increased absorption, or that the subject is in negative balance (or both). The y = 1 line is shown for comparison excretion/intake on day 21. (For interpretation ofthe references to color in this figure legend, the reader is referred to the web version of this article.)

82 M. Molin et al. / Food and Chemical Toxicology 66 (2014) 76–88

3.6. Excretion patterns

The data do not allow any detailed examination of As kineticsfrom seafood, but provide some information on individual andgroup-wise differences in the pattern of arsenical excretion andaccumulation. If the ratio of day 0/day 2 tAs excretion (after thesingle dose of seafood) is used as an indicator of individual excre-tion rate, strict first order kinetics with a half-life between 16–24 hwould give a ratio between 4 and 8. The actual ratios for the vari-ous seafood groups are shown (x-dimension) in Fig. 3E where theyrange from 6–15, 3–13 and 5–9 in the cod, salmon and blue musselgroups, respectively (pooled median 6.3).

In the cod group, those with slow elimination of the first singlemeal tended to end up with higher plasma As levels at the end ofthe study following 15 days of seafood. Hence, there was a signifi-cant negative association between rapid initial tAs excretion, andboth plasma tAs on day 23 (Fig. 3E) and tAs excretion during period2 (Fig. 3F). This would indicate As accumulation in parts of period2. In the other groups, there were no such significant associations,and by ANCOVA, for the plasma model (r2 = 0.54) differences be-tween the cod and salmon/blue mussels groups were significant(p < 0.05). In Fig. 3E the plasma association is shown, with regres-sion lines for the cod group and combined blue mussel and salmongroups.

0 100 200 300 400 500 600

050

010

0015

00 A

Urinary AB excreted period 1 ( µg)

Urin

ary

AB e

xcre

ted

perio

d 2

(µg)

SalmonCodMusselsControl

0 10 20 30 40 50 60 70

010

020

030

040

050

0

B

Hours after last As−rich meal

Urin

e AB

con

cent

ratio

n (µ

g/L)

0 10 20 30 40 50 60 70

020

0 40

0 60

080

012

00 C

Hours after last As−rich meal

Tota

l AB

excr

eted

(µg)

0 10 20 30 40 50 60 70

050

100

200

300

D

Hours after last As−rich meal

Urin

ary

AB, %

of A

B in

gest

ed in

last

As−

rich

mea

l

Fig. 4. Urinary AB in periods 1 and 2. (A) Total AB excretion during period 2 vs total AB excretion during period 1. In all study groups, total AB excretion in period 2 was two tofive times that of period 1. Within each study group, period 1 and 2 excretion was not strongly correlated. (B) Average urine AB concentration during periods 1 and 2. Morningspot values for the first day in each period are not shown, as morning spot and concentrations based on complete urine collections are not comparable. In period 2, final urineconcentrations in the cod group were about the same as initial concentrations in the salmon and blue mussels groups. (C) Cumulative AB excretion for each group duringperiods 1 (dotted lines) and 2 (bold lines). Note that while AB excretion was almost unchanged from period 1 to period 2 in the blue mussels group (blue), slightly changed inthe salmon group (red), it increased about threefold in the cod group (black). (D) Individual cumulative values for the fraction of the period 2 test meal excreted in the codgroup (black) and blue mussels group (blue). Note the wide excretion range, from less than to about four times the content of the last meal. (For interpretation of thereferences to color in this figure legend, the reader is referred to the web version of this article.)

M. Molin et al. / Food and Chemical Toxicology 66 (2014) 76–88 83

3.7. Associations between uptake and elimination in the two testperiods

There was a significant association (r2 = 0.22, p < 0.01) betweenthe amount tAs excreted (during days 0–2) relative to that ingestedof the single dose (range 0.41–0.99, median 0.63), and the corre-sponding ratio for day 21 (range 0.32–1.86, median 0.9) (Fig. 3F).Most of the subjects in the cod group and half the subjects in thesalmon group excreted as much as or more than they ingested inthe last of 15 meals. With regard to plasma concentrations fortAs these were equal at zero and 20 h except for the cod groupshowing a lower value at 20 h (Fig. 2A). All but two excreted moreAs relative to the amount ingested on day 21 than they did duringthe whole period 1 after the single dose with seafood. There was anestimated 4 h overlap between the 24 h excretion periods of days20 and 21, leading to somewhat higher expected excretion onday 21, but this could not account for the high values observed.

4. Discussion

In this study, accumulation of As was found in urine and plasmaafter 15 days of repeated intake of seafood. In addition, consider-able differences in the pattern, metabolism and excretion of Asfrom different sources of seafood As was found. The potentiallymost important finding in relation to seafood safety was the strongincrease in MA excretion, particularly within the first 24 h afteringestion of blue mussels for 15 consecutive days. Ingestion of bluemussels for 15 consecutive days also resulted in a 4-fold increasein the absolute excretion of iAs; however the amount of iAs relative

to tAs excreted after a single meal and following 15 consecutivedays with seafood were similar. This was also the case for DMAin all seafood consuming groups.

4.1. Urinary and plasma tAs concentrations

A single dose or fifteen consecutive days with seafood wereboth reflected in increased urinary and plasma tAs concentrations,and tAs concentrations in plasma were to some extent dose-dependent.

Non-exposed subjects seemed to excrete some As, despite verylow ingestion of As for one week (below 20 lg/g CR) (Table 1). Thisis in line with the results in non-exposed populations with low sea-food intake (Brima et al., 2006; Caldwell et al., 2009; Fowler et al.,2007; Navas-Acien et al., 2009). The day 21 plasma and urinary tAsconcentrations for the control group were also low (0.7 and 8.4 lg/L, respectively), which would indicate good compliance of the sub-jects regarding the food restrictions during the study period.

After 15 consecutive days with seafood, mean urinary tAs con-centration was 173 lg/g CR (Table 1). This is comparable with thelevels reported in a healthy population with frequent seafood con-sumption (Sirot et al., 2009), where mean urinary tAs concentra-tions of 94.8 and 59.7 lg/g CR was found in females and males,respectively (Table 1).

Following wash-out, on day 7 the urinary AB concentration(11.5 lg/g CR) was about sevenfold higher than the urinary ABconcentrations reported in two populations with low seafoodintake; 1.54 lg/g CR (Caldwell et al., 2009) and 0.5 lg/g CR(Navas-Acien et al., 2009). The DMA urinary concentration

20 40 60 80 100

050

100

150

200 A

Urinary DMA excreted period 1 ( µg)

Urin

ary

DM

A ex

cret

ed p

erio

d 2

(µg)

SalmonCodMusselsControl

0 10 20 30 40 50 60 70

020

4060

80

B

Hours after last As-rich meal

Urin

e D

MA

conc

entra

tion

(µg/

L)

0 10 20 30 40 50 60 70

050

100

150

200 C

Hours after last As-rich meal

Tota

l DM

A ex

cret

ed (µ

g)

0 10 20 30 40 50 60 70

010

20

3040

5060

70 D

Hours after last As-rich meal

Urin

ary

iAs+

MA+

DM

A, %

of n

on-A

B As

inge

sted

in la

st A

s-ric

h m

eal

Fig. 5. Urinary DMA in periods 1 and 2. (A) Total DMA excretion during period 2 vs total DMA excretion during period 1. In all study groups, total DMA excretion in period 2was two to three times that of period 1. Within each study group, period 1 and 2 excretion was not strongly correlated. For example, two individuals in the blue mussels groupexcreting about 50 and 100 lg DMA, respectively, during period 1, both excreted about 200 lg during period 2. (B) Average urine DMA concentration during periods 1 and 2.Morning spot values for the first day in each period are not shown, as morning spot and full collection values are not comparable. In the blue mussels group, initial DMAconcentrations in period 2 was 3–4 times those of period 1, and final concentrations in period 2 were comparable to initial concentrations in period 1. (C) Cumulative DMAexcretion for the blue mussels group during periods 1 (dotted lines) and 2 (bold lines). Note that the average excretion rate in this group was virtually unchanged from day 22to day 23. D: Individual cumulative values for the fraction of the period 2 test meal excreted as iAs + MA + DMA in the cod group (black) and blue mussels group (blue). Notethe wide excretion range, from above 60% of the test meal content of non-AB As in some blue mussels eaters, with large individual variation in the group, down to approx. 10%in the cod group, with much smaller individual variation. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of thisarticle.)

84 M. Molin et al. / Food and Chemical Toxicology 66 (2014) 76–88

(2.25 lg/g CR) was, however, somewhat lower than the DMA uri-nary concentrations reported by the same studies, 3.69 lg/g CR(Caldwell et al., 2009) and 5.0 lg/g CR (Navas-Acien et al., 2009).

Contrary to a Taiwanese study, which found little change inconcentrations of urinary iAs, DMA and MA excreted before andafter refraining from seafood for three days (Hsueh et al., 2002),we found that the urinary DMA concentrations increased signifi-cantly for all seafood consuming groups. However, the iAs andMA concentrations in urine increased strongly only in the bluemussel group after 15 consecutive days with seafood (Table 1).The reason for this is unknown.

4.2. Absolute excretion of tAs, AB, iAs, DMA and MA

Similar to period 1 and like in other seafood studies, AB was themain arsenical excreted with DMA as the second most abundantarsenical (Buchet et al., 1996; Heinrich-Ramm et al., 2002; Laiet al., 2004; Le et al., 1994). In agreement with previous studies,which have suggested that urinary DMA excretion may in part beformed from arsenosugars and/or arsenolipids (Francesconi et al.,2002; Raml et al., 2005, 2009; Schmeisser et al., 2006), the highestDMA excretion was found in the blue mussel group (180 lg); theintervention group that probably consumed the highest amountof arsenosugars. Recently, also a large cross-sectional study(NHANES) identified DMA as one of the main arsenicals excretedafter seafood intake (Navas-Acien et al., 2011). Urinary DMA mayresults from both the methylation process of iAs (EFSA, 2009),

ingested DMA and from bioconversion of organoarsenicals presentin seafood like arsenosugars and/or arsenolipids.

While the excretory pattern of most arsenicals was quite similarin periods 1 and 2, the absolute excretion of MA after 15 consecu-tive days with blue mussel was 4.5-fold higher when comparedwith the MA excretion after one single dose of blue mussel.

4.3. Excretion pattern

The urinary excretion patterns were basically similar after 15consecutive days with seafood and after a single dose, except forthe delayed excretion of AB in the cod group and DMA in the bluemussel group (Figs. 4D and 5D).

Like Choi et al. (2010), we found a rapid decrease in the urinaryarsenicals the first 24 h following the last seafood meal, but weobserved a slower decrease during the second day. This might bea result of 15 consecutive days of seafood consumption for oursubjects resulting in a higher body burden of arsenicals than the6 consumption days of subjects of Choi et al. They returned tobaseline levels after five days, while our subjects had still elevatedurinary tAs at the end of day 23 (Choi et al., 2010).

4.4. Associations between uptake and elimination in the two testperiods

The large increase in plasma tAs concentration and amount oftAs excreted in urine from periods 1 to 2 indicate increased As

0 10 20 30 40 50 60 70

05

1015

20 A

Hours after last As−rich meal

0 10 20 30 40 50 60 70

Hours after last As−rich meal

Urin

ary

MA

conc

entra

tion

(µg/

L)SalmonCodMusselsControl

010

2030

4050 B

Tota

l MA

excr

eted

(µg)

02

46

810 C

Urin

ary

MA,

% o

f non

−AB

As

0 10 20 30 40 50 60 70

Hours after last As−rich meal

inge

sted

in la

st A

s−ric

h m

eal

Fig. 6. Urinary MA. (A) Urinary MA concentration lg/L during period 2 and period1. During the first 48 h of period 2, average MA concentration decreased sixfold inthe blue mussels group. (B) Cumulative MA excretion during period 2 (bold lines)and period 1 (dotted lines) for individuals in the blue mussels group (lg). 75–90% oftotal individual excretion occurred during the first 24 h. (C) Cumulative MAexcretion during period 2 for individuals in the blue mussels (blue lines) and codgroups (black lines), as percentage of non-AB As ingested in test meal 2. As AB isconsidered mostly metabolically inert, most MA originates in the non-AB fraction ofAs. As much as 5–10% of ingested amount of these species were excreted as MA by 5of 7 subjects in the blue mussels group. In comparison, corresponding excretion inthe cod group was

86 M. Molin et al. / Food and Chemical Toxicology 66 (2014) 76–88

the relative urinary MA excretion (4.46%, Table 2, column 7) afterthe 15 consecutive days with blue mussels was comparable withthat in subjects occupationally exposed to iAs in a steel foundry(4.9%) (Soleo et al., 2008).

Only the blue mussel group excreted significantly more MAafter 15 consecutive days with seafood than after the single dose.Earlier studies of populations chronically exposed to iAs fromdrinking water, indicate that the relative proportion of urinaryMA might increase with increasing dose of iAs exposure (Del Razoet al., 1997; Hopenhayn-Rich et al., 1996; Kurttio et al., 1998; Liet al., 2008; Styblo et al., 1999). The observed decrease in methyl-ation capacity associated with the increased exposure to As-con-taminated drinking water, has been proposed to be a result ofthe inhibition of the second methylation step (limited capacity ofthe As3MT enzyme) when exposed to excessive iAs (Tseng,2009). We do not know whether such a mechanism is relevant inthe case of MA excretion following repeated intake of blue musselssince the pathway of formation and whether iAs might occurs as anintermediate is unknown. Neither is it known whether As bound asarsenosugars is converted directly mainly to DMA or whether iAsand MA are involved as intermediary metabolites.

4.6. Relative urinary excretion of iAs, DMA, MA

The relative distribution of iAs, MA and DMA in urine in variouspopulations is considered to be fairly constant and in the range of10–30%, 10–20% and 60–80%, respectively (Hopenhayn-Rich et al.,1996; Vahter, 1999). In line with this and similar to the relativedistribution after three days with seafood restrictions (8%, 6% and86%) found by Choi et al. (2010), the mean relative distributionof iAs, MA and DMA after one week’s seafood restrictions (day 7)was 9%, 12% and 79% in our study (Table 1). After 15 consecutivedays with seafood in our study group, excluding AB and other min-or organic arsenicals the percentage was lower for iAs (1.6%) butcomparable for MA (20.1%) and DMA (78%) (Table 1).

4.7. Inter-individual variations

We found large individual variations in the percentage ingestednon-AB in the last seafood meal that was excreted as iAs + MA + D-MA in the blue mussel group (Fig. 5D). This is in line with previousstudies of humans ingesting a single dose of arsenosugars in theform of algae (Le et al., 1994; Ma and Le, 1998) or synthetic arseno-sugar (Raml et al., 2009) and also after 6 consecutive days with al-gae/fish (Choi et al., 2010). In addition, large inter-individualvariations in AB and MA excretion were found in the blue musselgroup and for AB in the cod group.

Both race, gender, age, nutritional status as well as genetic poly-morphisms in the regulation of the enzymes (i.e. arsenic methyltransferase (As3MT)) involved in As metabolism, seem to have animpact on the urinary excretion of methylated arsenicals (EFSA,2009; Hughes, 2011). The observed inter-individual differencesfound in the metabolism of arsenicals in the present study, in par-ticular in the blue mussel group, might in addition to these factorsbe explained by variation in intestinal flora.

4.8. Safety of arsenicals in seafood

Although no signs of As toxicity have been reported in humansafter consumption of large quantities of seafood (Edmonds andFrancesconi, 1993) or seaweed (Andrewes et al., 2004), the toxicaction of iAs is believed to be related to its metabolism, i.e. the for-mation of methylated trivalent As species (Borak and Hosgood,2007), hence high urinary excretion of DMA and MA could beassociated with formation of trivalent species and therefore be ofhuman health concern.

MA (III) and DMA (III) are rapidly oxidized to their pentavalentcounterparts (Styblo et al., 2002; Thomas et al., 2001), andthese intermediates can initiate toxic effects, like DNA-damage(Mass et al., 2001; Nesnow et al., 2002), or be indirectly genotoxic(Hirano et al., 2004; Vahter, 2002). However, due to their reactivityand low stability, these could not be determined in the presentstudy.

A repeated dose of MA has been shown to have effects on theGI-tract, kidney, thyroid and the reproductive system (ATSDR,2007), and a higher proportion of urinary MA has been associatedwith increased risks of As-related diseases (Chen et al., 2005;Hsueh et al., 1997; Maki-Paakkanen et al., 1998; Steinmauset al., 2006; Tseng et al., 2005; Wu et al., 2006; Yu et al., 2000).DMA (V) has in animal studies been reported to promote carcino-genic effects in the urinary bladder, kidney, liver and the thyroidgland (Wanibuchi et al., 2004; Yamamoto et al., 1995) and has inaddition effects on the foetal development (ATSDR, 2007). Re-cently, both MA and DMA were classified as ‘‘possibly carcino-genic’’ to humans by the International Agency for Research onCancer (IARC) (IARC, 2009). Taken together with the possibilitythat toxic trivalent intermediates might occur in the formationof DMA and MA, the high DMA and MA excretion might be of pos-sible concern and the implication of this finding should be furtherinvestigated.

4.9. Strengths’ and weaknesses of the study

The main strength of this study is the study design, which en-abled a direct comparison if the urinary arsenical excretion patternafter a single dose and after 15 consecutive days with the identicalseafood species. Using a control group, other possible sources of Aswere minimized, all groups following restrictions of food itemshigh in As starting from seven days before study start (day 0)and throughout the study period. With three different seafood spe-cies providing large, but realistic doses of As, we obtained differentexcretion patterns with individual differences therein. Also, usingeach subject as his or her own control in the statistical analysiscould reduce variance.

One weakness of the study is that our laboratories could notprovide analysis of the arsenosugars and arsenolipids content onthe food matrix so those species could not be quantified in theingested foods.

5. Conclusion

We found clear indications of As accumulation after repeatedseafood intake, and after 15 days on a seafood diet. Only with highAB intake (the cod diet), a relation between slow initial As excre-tion speed and higher plasma and urinary As concentrations twodays after last seafood meal, was found.

While iAs excretion was relatively low in all study groups, thevery high MA excretion after repeated intake of blue musselsshould be further investigated. Also, since chronic low-level iAsexposure and high urinary excretion of MA has been related tohigher risk for adverse As-related health effects, a possible riskassociated with excessive blue mussel intake cannot be ruled out.

Conflict of Interest

The authors declare that there are no conflicts of interest.

Transparency Document

The Transparency document associated with this article can befound, in the online version.

http://dx.doi.org/10.1016/j.fct.2014.01.030

M. Molin et al. / Food and Chemical Toxicology 66 (2014) 76–88 87

Acknowledgements

This research was supported by the Norwegian Research Coun-

cil (project nr. 142468/140). We gratefully thank all volunteers and

personnel assisting during the seafood study and the analyticalwork by Siri Bargård and other lab staff at National Institute ofNutrition and Seafood Research.

References

Alexander, J., Frøyland, L., Hemre, G.I., Jacobsen, B.K., Lund, E., Meltzer, H.M., Skåre,J.U., 2006. A comprehensive assessment of fish and other seafood in theNorwegian diet. Norwegian Scientific Committee for Food Safety (VKM) Oslo,Norway.

Andrewes, P., Demarini, D.M., Funasaka, K., Wallace, K., Lai, V.W.M., Sun, H., Cullen,W.R., Kitchin, K.T., 2004. Do arsenosugars pose a risk to human health? Thecomparative toxicities of a trivalent and pentavalent arsenosugar. Environ. Sci.Technol. 38, 4140–4148.

ATSDR (Agency for Toxic Substances and Disease Registry), 2007. ToxicologicalProfile for Arsenic. U.S. Department of Health and Human Services, PublicHealth Service, Atlanta, GA.

Borak, J., Hosgood, H.D., 2007. Seafood arsenic: implications for human riskassessment. Regul. Toxicol. Pharmacol. 47 (2), 204–212.

Brima, E.I., Haris, P.I., Jenkins, R.O., Polya, D.A., Gault, A.G., Harrington, C.F., 2006.Understanding arsenic metabolism through a comparative study of arseniclevels in the urine, hair and fingernails of healthy volunteers from threeunexposed ethnic groups in the United Kingdom. Toxicol. Appl. Pharmacol. 216,122–130.

Brown, R.M., Newton, D., Pickford, C.J., Sherlock, J.C., 1990. Human metabolism ofarsenobetaine ingested with fish. Hum. Exp. Toxicol. 9, 41–46.

Buchet, J.P., Pauwels, J., Lauwerys, R., 1994. Assessment of exposure to inorganicarsenic following ingestion of marine organisms by volunteers. Environ. Res. 66,44–51.

Buchet, J.P., Lison, D., Ruggeri, M., Foa, V., Elia, G., 1996. Assessment of exposure toinorganic arsenic, a human carcinogen, due to the consumption of seafood.Arch. Toxicol. 70, 773–778.

Caldwell, K.L., Jones, R.L., Verdon, C.P., Jarrett, J.M., Caudill, S.P., Osterloh, J.D., 2009.Levels of urinary total and speciated arsenic in the US population: nationalhealth and nutrition examination survey 2003–2004. J. Expo. Sci. Environ.Epidemiol. 19, 59–68.

Challenger, F., 1947. Biological methylation. Sci. Prog. 35, 396–416.Chen, C.J., Hsu, L.I., Wang, C.H., Shih, W.L., Hsu, Y.H., Tseng, M.P., Lin, Y.C., Chou, W.L.,

Chen, C.Y., Lee, C.Y., Wang, L.H., Cheng, Y.C., Chen, C.L., Chen, S.Y., Wang, Y.H.,Hsueh, Y.M., Chiou, H.Y., Wu, M.M., 2005. Biomarkers of exposure, effect, andsusceptibility of arsenic-induced health hazards in Taiwan. Toxicol. Appl.Pharmacol. 206, 198–206.

Choi, B.-S., Choi, S.-J., Kim, D.-W., Huang, M., Kim, N.-Y., Park, K.-S., Kim, C.-Y., Lee,H.-M., Yum, Y.-N., Han, E.-S., Kang, T.-S., Yu, I.-J., Park, J.-D., 2010. Effects ofrepeated seafood consumption on urinary excretion of arsenic species byvolunteers. Arch. Environ. Contam. Toxicol. 58, 222–229.

Cleland, B., Tsuchiya, A., Kalman, D.A., Dills, R., Burbacher, T.M., White, J.W.,Faustman, E.M., Marien, K., 2009. Arsenic exposure within the Koreancommunity (United States) based on dietary behavior and arsenic levels inhair, urine, air, and water. Environ. Health Perspect. 117 (4), 632–638.

Del Razo, L.M., Garcia-Vargas, G.G., Vargas, H., Albores, A., Gonsebatt, M.E., Montero,R., Ostrosky-Wegman, P., Kelsh, M., Cebrian, M.E., 1997. Altered profile ofurinary arsenic metabolites in adults with chronic arsenicism. A pilot study.Arch. Toxicol. 71, 211–217.

Edmonds, J.S., Francesconi, K.A., 1993. Arsenic in seafoods: human health aspectsand regulations. Mar. Pollut. Bull. 26 (12), 665–674.

EFSA, 2009. Scientific Opinion on Arsenic in Food. EFSA Panel on Contaminants inthe Food Chain (CONTAM).

FAO/WHO, 1995. GEMS/Food-EURO Second Workshop on Reliable Evaluation ofLow-Level Contamination of Food. Report on a Workshop in the Frame ofGEMS/Food-EURO. Kulmbach, Federal Republic of Germany, 26–27 May1995.

Fowler, B.A., Chou, C.S.J., Jones, R.L., Chen, C.-J., 2007. Arsenic. In: Nordberg, G.,Fowler, B., Nordberg, M., Friberg, L. (Eds.), Handbook of the Toxicology ofMetals, third ed. Elsevier, Burlington, USA, pp. 367–406.

Francesconi, K.A., Tanggaar, R., McKenzie, C.J., Goessler, W., 2002. Arsenicmetabolites in human urine after ingestion of an arsenosugar. Clin. Chem. 48,92–101.

Freeman, H.C., Uthe, J.F., Fleming, R.B., Odense, P.H., Ackman, R.G., Landry, G.,Musial, C., 1979. Clearance of arsenic ingested by man from arseniccontaminated fish. Bull. Environ. Contam. Toxicol. 22, 224–229.

Gao, J., Yu, J., Yang, L., 2011. Urinary arsenic metabolites of subjects exposed toelevated arsenic present in coal in Shaanxi Province, China. Int. J. Environ. Res.Publ. Health 8, 1991–2008.

Hayakawa, T., Kobayashi, Y., Cui, X., Hirano, S., 2005. A new metabolic pathway ofarsenite: arsenic-glutathione complexes are substrates for human arsenicmethyltransferase Cyt19. Arch. Toxicol. 79, 183–191.

Heinrich-Ramm, R., Mindt-Prufert, S., Szadkowski, D., 2002. Arsenic speciesexcretion after controlled seafood consumption. J. Chromatogr. B 778, 263–273.

Hirano, S., Kobayashi, Y., Cui, X., Kanno, S., Hayakawa, T., Shraim, A., 2004. Theaccumulation and toxicity of methylated arsenicals in endothelial cells:important roles of thiol compounds. Toxicol. Appl. Pharmacol. 198, 458–467.

Hopenhayn-Rich, C., Biggs, M.L., Kalman, D.A., Moore, L.E., Smith, A.H., 1996. Arsenicmethylation patterns before and after changing from high to lowerconcentrations of arsenic in drinking water. Environ. Health Perspect. 104,1200–1207.

Hsueh, Y.M., Chiou, H.Y., Huang, Y.L., Wu, W.L., Huang, C.C., Yang, M.H., Lue, L.C.,Chen, G.S., Chen, C.J., 1997. Serum beta-carotene level, arsenic methylationcapability, and incidence of skin cancer. Cancer Epidemiol. Biomarkers Prev. 6,589–596.

Hsueh, Y.M., Hsu, M.K., Chiou, H.Y., Yang, M.H., Huang, C.C., Chen, C.J., 2002. Urinaryarsenic speciation in subjects with or without restriction from seafood dietaryintake. Toxicol. Lett. 133, 83–91.

Hughes, M., Beck, B.D., Chen, Y., Lewis, A.S., Thomas, D.J., 2011. Arsenic exposureand toxicology: a historical perspective. Toxicol. Sci.

IARC, I.A.f.R.o.C., 2009. Arsenic, Metals, Fibres and Dusts. A Review of HumanCarcinogens. World Health Organization (WHO), Lyon, France.

Julshamn, K., Maage, A., Norli, H.S., Grobecker, K.H., Jorhem, L., Fecher, P., 2007.Determination of arsenic, cadmium, mercury, and lead by inductively coupledplasma/mass spectrometry in foods after pressure digestion: NMKLinterlaboratory study. J. AOAC Int. 90, 844–856.

Kroes, R., Müller, D., Lambe, C., Löwik, M.R.H., van Klaveren, J., Kleiner, J., Massey, R.,Mayer, S., Urieta, I., Verger, P., Visconti, A., 2002. Assessment of intake from thediet. Food Chem. Toxicol. 40, 327–385.

Kurttio, P., Komulainen, H., Hakala, E., Kahelin, H., Pekkanen, J., 1998. Urinaryexcretion of arsenic species after exposure to arsenic present in drinking water.Arch. Environ. Contam. Toxicol. 34, 297–305.

Lai, V.W., Sun, Y., Ting, E., Cullen, W.R., Reimer, K.J., 2004. Arsenic speciationin human urine: are we all the same? Toxicol. Appl. Pharmacol. 198, 297–306.

Le, X.C., Cullen, W.R., Reimer, K.J., 1994. Human urinary arsenic excretion after one-time ingestion of seaweed, crab, and shrimp. (see comment). Clin. Chem. 40,617–624.

Li, L., Ekstrom, E.C., Goessler, W., Lonnerdal, B., Nermell, B., Yunus, M., Rahman, A.,El, A.S., Persson, L., Vahter, M., 2008. Nutritional status has marginal influenceon the metabolism of inorganic arsenic in pregnant Bangladeshi women.Environ. Health Perspect. 116, 315–321.

Ma, M., Le, X.C., 1998. Effect of arsenosugar ingestion on urinary arsenic speciation.Clin. Chem. 44, 539–550.

Maki-Paakkanen, J., Kurttio, P., Paldy, A., Pekkanen, J., 1998. Association betweenthe clastogenic effect in peripheral lymphocytes and human exposure to arsenicthrough drinking water. Environ. Mol. Mutagen 32, 301–313.

Mass, M.J., Tennant, A., Roop, B.C., Cullen, W.R., Styblo, M., Thomas, D.J., Kligerman,A.D., 2001. Methylated trivalent arsenic species are genotoxic. Chem. Res.Toxicol. 14, 355–361.

Molin, M., Ulven, S., Dahl, L., Telle-Hansen, V., Holck, M., Skjegstad, G., Ledsaak, O.,Sloth, J., Goessler, W., Oshaug, A., Alexander, J., Fliegel, D., Ydersbond, T.,Meltzer, H., 2012a. Humans seem to produce arsenobetaine anddimethylarsinate after a bolus dose of seafood. Environ. Res., 112.

Molin, M., Ydersbond, T.A., Ulven, S.M., Holck, M., Dahl, L., Sloth, J.J., Fliegel, D.,Goessler, W., Alexander, J., Meltzer, H.M., 2012b. Major and minor arseniccompounds accounting for the total urinary excretion of arsenic followingintake of blue mussels (Mytilus edulis): a controlled human study. Food Chem.Toxicol. 50, 2462–2472.

Mozaffarian, D., Rimm, E.B., 2006. Fish intake, contaminants, and human health:evaluating the risks and the benefits. JAMA 296, 1885–1899.

Naranmandura, H., Carew, M., Xu, S., Lee, J., Leslie, E., Weinfeld, M., Le, X., 2011.Comparative toxicity of arsenic metabolites in human bladder cancer EJ-1 cells.Chem. Res. Toxicol. 24, 1586–1596.

Navas-Acien, A., Umans, J.G., Howard, B.V., Goessler, W., Francesconi, K.A.,Crainiceanu, C.M., Silbergeld, E.K., Guallar, E., 2009. Urine arsenicconcentrations and species excretion patterns in American Indiancommunities over a 10-year period: the Strong Heart Study. Environ. HealthPerspect. 117, 1428–1433.

Navas-Acien, A., Francesconi, K.A., Silbergeld, E.K., Guallar, E., 2011. Seafood intakeand urine concentrations of total arsenic, dimethylarsinate and arsenobetainein the US population. Environ. Res. 111 (1), 110–118.

Nesnow, S., Roop, B.C., Lambert, G., Kadiiska, M., Mason, R.P., Cullen, W.R., Mass,M.J., 2002. DNA damage induced by methylated trivalent arsenicals is mediatedby reactive oxygen species. Chem. Res. Toxicol. 15, 1627–1634.

NNR, 2004. Nordic Nutrition Recommendations: NNR 2004: Integrating Nutritionand Physical Activity. Nordic Council of Ministers, Copenhagen.

Ochi, T., Kita, K., Suzuki, T., Rumpler, A., Goessler, W., Francesconi, K.A., 2008.Cytotoxic, genotoxic and cell-cycle disruptive effects of thio-dimethylarsinatein cultured human cells and the role of glutathione. Toxicol. Appl. Pharmacol.228, 59–67.

Olsen, V., Morland, J., 2004. Arsenic poisoning. Tidsskr Nor Laegeforen 124, 2750–2753.

Raml, R., Goessler, W., Traar, P., Ochi, T., Francesconi, K.A., 2005. Novel thioarsenicmetabolites in human urine after ingestion of an arsenosugar, 20 ,30-dihydroxypropyl 5-deoxy-5-dimethylarsinoyl-beta-D-riboside. Chem. Res.Toxicol. 18, 1444–1450.

Raml, R., Raber, G., Rumpler, A., Bauernhofer, T., Goessler, W., Francesconi, K.A.,2009. Individual variability in the human metabolism of an arsenic-containingcarbohydrate, 20 ,30-dihydroxypropyl 5-deoxy-5-dimethylarsinoyl-beta-D-

http://refhub.elsevier.com/S0278-6915(14)00044-1/h0010http://refhub.elsevier.com/S0278-6915(14)00044-1/h0010http://refhub.elsevier.com/S0278-6915(14)00044-1/h0010http://refhub.elsevier.com/S0278-6915(14)00044-1/h0010http://refhub.elsevier.com/S0278-6915(14)00044-1/h0395http://refhub.elsevier.com/S0278-6915(14)00044-1/h0395http://refhub.elsevier.com/S0278-6915(14)00044-1/h0395http://refhub.elsevier.com/S0278-6915(14)00044-1/h0020http://refhub.elsevier.com/S0278-6915(14)00044-1/h0020http://refhub.elsevier.com/S0278-6915(14)00044-1/h0025http://refhub.elsevier.com/S0278-6915(14)00044-1/h0025http://refhub.elsevier.com/S0278-6915(14)00044-1/h0025http://refhub.elsevier.com/S0278-6915(14)00044-1/h0025http://refhub.elsevier.com/S0278-6915(14)00044-1/h0025http://refhub.elsevier.com/S0278-6915(14)00044-1/h0030http://refhub.elsevier.com/S0278-6915(14)00044-1/h0030http://refhub.elsevier.com/S0278-6915(14)00044-1/h0040http://refhub.elsevier.com/S0278-6915(14)00044-1/h0040http://refhub.elsevier.com/S0278-6915(14)00044-1/h0040http://refhub.elsevier.com/S0278-6915(14)00044-1/h0035http://refhub.elsevier.com/S0278-6915(14)00044-1/h0035http://refhub.elsevier.com/S0278-6915(14)00044-1/h0035http://refhub.elsevier.com/S0278-6915(14)00044-1/h0045http://refhub.elsevier.com/S0278-6915(14)00044-1/h0045http://refhub.elsevier.com/S0278-6915(14)00044-1/h0045http://refhub.elsevier.com/S0278-6915(14)00044-1/h0045http://refhub.elsevier.com/S0278-6915(14)00044-1/h0050http://refhub.elsevier.com/S0278-6915(14)00044-1/h0055http://refhub.elsevier.com/S0278-6915(14)00044-1/h0055http://refhub.elsevier.com/S0278-6915(14)00044-1/h0055http://refhub.elsevier.com/S0278-6915(14)00044-1/h0055http://refhub.elsevier.com/S0278-6915(14)00044-1/h0055http://refhub.elsevier.com/S0278-6915(14)00044-1/h0060http://refhub.elsevier.com/S0278-6915(14)00044-1/h0060http://refhub.elsevier.com/S0278-6915(14)00044-1/h0060http://refhub.elsevier.com/S0278-6915(14)00044-1/h0060http://refhub.elsevier.com/S0278-6915(14)00044-1/h0065http://refhub.elsevier.com/S0278-6915(14)00044-1/h0065http://refhub.elsevier.com/S0278-6915(14)00044-1/h0065http://refhub.elsevier.com/S0278-6915(14)00044-1/h0065http://refhub.elsevier.com/S0278-6915(14)00044-1/h0070http://refhub.elsevier.com/S0278-6915(14)00044-1/h0070http://refhub.elsevier.com/S0278-6915(14)00044-1/h0070http://refhub.elsevier.com/S0278-6915(14)00044-1/h0070http://refhub.elsevier.com/S0278-6915(14)00044-1/h0075http://refhub.elsevier.com/S0278-6915(14)00044-1/h0075http://refhub.elsevier.com/S0278-6915(14)00044-1/h0090http://refhub.elsevier.com/S0278-6915(14)00044-1/h0090http://refhub.elsevier.com/S0278-6915(14)00044-1/h0090http://refhub.elsevier.com/S0278-6915(14)00044-1/h0095http://refhub.elsevier.com/S0278-6915(14)00044-1/h0095http://refhub.elsevier.com/S0278-6915(14)00044-1/h0095http://refhub.elsevier.com/S0278-6915(14)00044-1/h0100http://refhub.elsevier.com/S0278-6915(14)00044-1/h0100http://refhub.elsevier.com/S0278-6915(14)00044-1/h0100http://refhub.elsevier.com/S0278-6915(14)00044-1/h0105http://refhub.elsevier.com/S0278-6915(14)00044-1/h0105http://refhub.elsevier.com/S0278-6915(14)00044-1/h0105http://refhub.elsevier.com/S0278-6915(14)00044-1/h0110http://refhub.elsevier.com/S0278-6915(14)00044-1/h0110http://refhub.elsevier.com/S0278-6915(14)00044-1/h0110http://refhub.elsevier.com/S0278-6915(14)00044-1/h0115http://refhub.elsevier.com/S0278-6915(14)00044-1/h0115http://refhub.elsevier.com/S0278-6915(14)00044-1/h0115http://refhub.elsevier.com/S0278-6915(14)00044-1/h0120http://refhub.elsevier.com/S0278-6915(14)00044-1/h0120http://refhub.elsevier.com/S0278-6915(14)00044-1/h0120http://refhub.elsevier.com/S0278-6915(14)00044-1/h0125http://refhub.elsevier.com/S0278-6915(14)00044-1/h0125http://refhub.elsevier.com/S0278-6915(14)00044-1/h0125http://refhub.elsevier.com/S0278-6915(14)00044-1/h0125http://refhub.elsevier.com/S0278-6915(14)00044-1/h0130http://refhub.elsevier.com/S0278-6915(14)00044-1/h0130http://refhub.elsevier.com/S0278-6915(14)00044-1/h0130http://refhub.elsevier.com/S0278-6915(14)00044-1/h0130http://refhub.elsevier.com/S0278-6915(14)00044-1/h0135http://refhub.elsevier.com/S0278-6915(14)00044-1/h0135http://refhub.elsevier.com/S0278-6915(14)00044-1/h0135http://refhub.elsevier.com/S0278-6915(14)00044-1/h0140http://refhub.elsevier.com/S0278-6915(14)00044-1/h0140http://refhub.elsevier.com/S0278-6915(14)00044-1/h0400http://refhub.elsevier.com/S0278-6915(14)00044-1/h0400http://refhub.elsevier.com/S0278-6915(14)00044-1/h0150http://refhub.elsevier.com/S0278-6915(14)00044-1/h0150http://refhub.elsevier.com/S0278-6915(14)00044-1/h0150http://refhub.elsevier.com/S0278-6915(14)00044-1/h0150http://refhub.elsevier.com/S0278-6915(14)00044-1/h0155http://refhub.elsevier.com/S0278-6915(14)00044-1/h0155http://refhub.elsevier.com/S0278-6915(14)00044-1/h0155http://refhub.elsevier.com/S0278-6915(14)00044-1/h0160http://refhub.elsevier.com/S0278-6915(14)00044-1/h0160http://refhub.elsevier.com/S0278-6915(14)00044-1/h0160http://refhub.elsevier.com/S0278-6915(14)00044-1/h0165http://refhub.elsevier.com/S0278-6915(14)00044-1/h0165http://refhub.elsevier.com/S0278-6915(14)00044-1/h0165http://refhub.elsevier.com/S0278-6915(14)00044-1/h0170http://refhub.elsevier.com/S0278-6915(14)00044-1/h0170http://refhub.elsevier.com/S0278-6915(14)00044-1/h0170http://refhub.elsevier.com/S0278-6915(14)00044-1/h0405http://refhub.elsevier.com/S0278-6915(14)00044-1/h0405http://refhub.elsevier.com/S0278-6915(14)00044-1/h0405http://refhub.elsevier.com/S0278-6915(14)00044-1/h0405http://refhub.elsevier.com/S0278-6915(14)00044-1/h0180http://refhub.elsevier.com/S0278-6915(14)00044-1/h0180http://refhub.elsevier.com/S0278-6915(14)00044-1/h0185http://refhub.elsevier.com/S0278-6915(14)00044-1/h0185http://refhub.elsevier.com/S0278-6915(14)00044-1/h0185http://refhub.elsevier.com/S0278-6915(14)00044-1/h0190http://refhub.elsevier.com/S0278-6915(14)00044-1/h0190http://refhub.elsevier.com/S0278-6915(14)00044-1/h0190http://refhub.elsevier.com/S0278-6915(14)00044-1/h0195http://refhub.elsevier.com/S0278-6915(14)00044-1/h0195http://refhub.elsevier.com/S0278-6915(14)00044-1/h0195http://refhub.elsevier.com/S0278-6915(14)00044-1/h0195http://refhub.elsevier.com/S0278-6915(14)00044-1/h0200http://refhub.elsevier.com/S0278-6915(14)00044-1/h0200http://refhub.elsevier.com/S0278-6915(14)00044-1/h0200http://refhub.elsevier.com/S0278-6915(14)00044-1/h0200http://refhub.elsevier.com/S0278-6915(14)00044-1/h0200http://refhub.elsevier.com/S0278-6915(14)00044-1/h0205http://refhub.elsevier.com/S0278-6915(14)00044-1/h0205http://refhub.elsevier.com/S0278-6915(14)00044-1/h0210http://refhub.elsevier.com/S0278-6915(14)00044-1/h0210http://refhub.elsevier.com/S0278-6915(14)00044-1/h0210http://refhub.elsevier.com/S0278-6915(14)00044-1/h0220http://refhub.elsevier.com/S0278-6915(14)00044-1/h0220http://refhub.elsevier.com/S0278-6915(14)00044-1/h0220http://refhub.elsevier.com/S0278-6915(14)00044-1/h0220http://refhub.elsevier.com/S0278-6915(14)00044-1/h0220http://refhub.elsevier.com/S0278-6915(14)00044-1/h0215http://refhub.elsevier.com/S0278-6915(14)00044-1/h0215http://refhub.elsevier.com/S0278-6915(14)00044-1/h0215http://refhub.elsevier.com/S0278-6915(14)00044-1/h0225http://refhub.elsevier.com/S0278-6915(14)00044-1/h0225http://refhub.elsevier.com/S0278-6915(14)00044-1/h0225http://refhub.elsevier.com/S0278-6915(14)00044-1/h0410http://refhub.elsevier.com/S0278-6915(14)00044-1/h0410http://refhub.elsevier.com/S0278-6915(14)00044-1/h0235http://refhub.elsevier.com/S0278-6915(14)00044-1/h0235http://refhub.elsevier.com/S0278-6915(14)00044-1/h0235http://refhub.elsevier.com/S0278-6915(14)00044-1/h0235http://refhub.elsevier.com/S0278-6915(14)00044-1/h0240http://refhub.elsevier.com/S0278-6915(14)00044-1/h0240http://refhub.elsevier.com/S0278-6915(14)00044-1/h0245http://refhub.elsevier.com/S0278-6915(14)00044-1/h0245http://refhub.elsevier.com/S0278-6915(14)00044-1/h0245http://refhub.elsevier.com/S0278-6915(14)00044-1/h0245http://refhub.elsevier.com/S0278-6915(14)00044-1/h0245http://refhub.elsevier.com/S0278-6915(14)00044-1/h0245http://refhub.elsevier.com/S0278-6915(14)00044-1/h0245http://refhub.elsevier.com/S0278-6915(14)00044-1/h0245http://refhub.elsevier.com/S0278-6915(14)00044-1/h0250http://refhub.elsevier.com/S0278-6915(14)00044-1/h0250http://refhub.elsevier.com/S0278-6915(14)00044-1/h0250http://refhub.elsevier.com/S0278-6915(14)00044-1/h0250http://refhub.elsevier.com/S0278-6915(14)00044-1/h0250http://refhub.elsevier.com/S0278-6915(14)00044-1/h0250http://refhub.elsevier.com/S0278-6915(14)00044-1/h0250

88 M. Molin et al. / Food and Chemical Toxicology 66 (2014) 76–88

riboside, a naturally occurring arsenical in seafood. Chem. Res. Toxicol. 22,1534–1540.

Rehman, K., Naranmandura, H., 2012. Arsenic metabolism and thioarsenicals.Metallomics 4, 881–892.

Rumpler, A., Edmonds, J.S., Katsu, M., Jensen, K.B., Goessler, W., Raber, G.,Gunnlaugsdottir, H., Francesconi, K.A., 2008. Arsenic-containing long-chainfatty acids in cod-liver oil: a result of biosynthetic infidelity? Angew. Chem. Int.Ed. Engl. 47, 2665–2667.

Schmeisser, E., Rumpler, A., Kollroser, M., Rechberger, G., Goessler, W., Francesconi,K.A., 2005. Arsenic fatty acids are human urinary metabolites of arsenolipidspresent in cod liver. Angew. Chem. Int. Ed. Engl. 45, 150–154.

Schmeisser, E., Goessler, W., Francesconi, K.A., 2006. Human metabolism ofarsenolipids present in cod liver. Anal. Bioanal. Chem. 385, 367–376.

Sele, V., Sloth, J.J., Lundebye, A.-K., Larsen, E.H., Berntssen, M.H.G., Amlund, H., 2012.Arsenolipids in marine oils and fats: a review of occurrence, chemistry andfuture research needs. Food Chem. 133, 618–630.

Sirot, V., Guerin, T., Volatier, J.L., Leblanc, J.C., 2009. Dietary exposure andbiomarkers of arsenic in consumers of fish and shellfish from France. Sci.Total Environ. 407, 1875–1885.

Sirot, V., Leblanc, J.C., Margaritis, I., 2012. A risk-benefit analysis approach toseafood intake to determine optimal consumption. Br. J. Nutr. 107, 1812–1822.

Sloth, J.J., Julshamn, K., 2008. Survey of total and inorganic arsenic content in bluemussels (Mytilus edulis L.) from Norwegian fiords: revelation of unusual highlevels of inorganic arsenic. J. Agric. Food Chem. 56, 1269–1273.

Soleo, L., Lovreglio, P., Iavicoli, S., Antelmi, A., Drago, I., Basso, A., Di Lorenzo, L.,Gilberti, M.E., De Palma, G., Apostoli, P., 2008. Significance of urinary arsenicspeciation in assessment of seafood ingestion as the main source of organic andinorganic arsenic in a population resident near a coastal area. Chemosphere 73,291–299.

Steinmaus, C., Bates, M.N., Yuan, Y., Kalman, D., Atallah, R., Rey, O.A., Biggs, M.L.,Hopenhayn, C., Moore, L.E., Hoang, B.K., Smith, A.H., 2006. Arsenic methylationand bladder cancer risk in case-control studies in Argentina and the UnitedStates. J. Occup. Environ. Med. 48, 478–488.

Styblo, M., Del Razo, L.M., LeCluyse, E.L., Hamilton, G.A., Wang, C., Cullen, W.R.,Thomas, D.J., 1999. Metabolism of arsenic in primary cultures of human and rathepatocytes. Chem. Res. Toxicol. 12, 560–565.

Styblo, M., Drobna, Z., Jaspers, I., Lin, S., Thomas, D.J., 2002. The role ofbiomethylation in toxicity and carcinogenicity of arsenic: a research update.Environ. Health Perspect. 110 (Suppl 5), 767–771.

Taleshi, M.S., Jensen, K.B., Raber, G., Edmonds, J.S., Gunnlaugsdottir, H., Francesconi,K.A., 2008. Arsenic-containing hydrocarbons: natural compounds in oil from thefish capelin, Mallotus villosus. Chem. Commun., 4706–4707.

Taleshi, M.S., Edmonds, J.S., Goessler, W., Ruiz-Chancho, M.J., Raber, G., Jensen, K.B.,Francesconi, K.A., 2010. Arsenic-containing lipids are natural constituents ofsashimi tuna. Environ. Sci. Technol. 44, 1478–1483.

Tam, G.K., Charbonneau, S.M., Bryce, F., Sandi, E., 1982. Excretion of a single oraldose of fish-arsenic in man. Bull. Environ. Contam. Toxicol. 28, 669–673.

Thomas, D.J., Styblo, M., Lin, S., 2001. The cellular metabolism and systemic toxicityof arsenic. Toxicol. Appl. Pharmacol. 176, 127–144.

Tseng, C.-H., 2009. A review on environmental factors regulating arsenicmethylation in humans. Toxicol. Appl. Pharmacol. 235, 338–350.

Tseng, C.-H., Huang, Y.-K., Huang, Y.-L., Chung, C.-J., Yang, M.-H., Chen, C.-J., Hsueh,Y.-M., 2005. Arsenic exposure, urinary arsenic speciation, and peripheralvascular disease in blackfoot disease-hyperendemic villages in Taiwan(Erratum appears in Toxicol. Appl. Pharmacol. 2006 Mar 1; 211(2), 175).Toxicol. Appl. Pharmacol. 206, 299–308.

Vahter, M., 1999. Methylation of inorganic arsenic in different mammalian speciesand population groups. Sci. Prog. 82 (Pt 1), 69–88.

Vahter, M., 2002. Mechanisms of arsenic biotransformation. Toxicology 181–182,211–217.

Vahter, M., Marafante, E., Dencker, L., 1983. Metabolism of arsenobetaine in mice,rats and rabbits. Sci. Total Environ. 30, 197–211.

Wanibuchi, H., Salim, E.I., Kinoshita, A., Shen, J., Wei, M., Morimura, K., Yoshida, K.,Kuroda, K., Endo, G., Fukushima, S., 2004. Understanding arsenic carcinogenicityby the use of animal models. Toxicol. Appl. Pharmacol. 198, 366–376.

WHO, 2001. Arsenic and Arsenic Compounds. World Health Organization, Geneva.Wu, M.M., Chiou, H.Y., Hsueh, Y.M., Hong, C.T., Su, C.L., Chang, S.F., Huang, W.L.,

Wang, H.T., Wang, Y.H., Hsieh, Y.C., Chen, C.J., 2006. Effect of plasmahomocysteine level and urinary monomethylarsonic acid on the risk ofarsenic-associated carotid atherosclerosis. Toxicol. Appl. Pharmacol. 216, 168–175.

Xi, S., Zheng, Q., Zhang, Q., Sun, G., 2011. Metabolic profile and assessment ofoccupational arsenic exposure in copper- and steel-smelting workers in China.Int. Arch. Occup. Environ. Health 84, 347–353.

Yamamoto, S., Konishi, Y., Matsuda, T., Murai, T., Shibata, M.A., Matsui-Yuasa, I.,Otani, S., Kuroda, K., Endo, G., Fukushima, S., 1995. Cancer induction by anorganic arsenic compound, dimethylarsinic acid (cacodylic acid), in F344/DuCrjrats after pretreatment with five carcinogens. Cancer Res. 55, 1271–1276.

Yamauchi, H., Yamamura, Y., 1984. Metabolism and excretion of orally ingestedtrimethylarsenic in man. Bull. Environ. Contam. Toxicol. 32, 682–687.

Yu, R.C., Hsu, K.H., Chen, C.J., Froines, J.R., 2000. Arsenic methylation capacity andskin cancer. Cancer Epidemiol. Biomarkers Prev. 9, 1259–1262.

http://refhub.elsevier.com/S0278-6915(14)00044-1/h0250http://refhub.elsevier.com/S0278-6915(14)00044-1/h0250http://refhub.elsevier.com/S0278-6915(14)00044-1/h0255http://refhub.elsevier.com/S0278-6915(14)00044-1/h0255http://refhub.elsevier.com/S0278-6915(14)00044-1/h0260http://refhub.elsevier.com/S0278-6915(14)00044-1/h0260http://refhub.elsevier.com/S0278-6915(14)00044-1/h0260http://refhub.elsevier.com/S0278-6915(14)00044-1/h0260http://refhub.elsevier.com/S0278-6915(14)00044-1/h0270http://refhub.elsevier.com/S0278-6915(14)00044-1/h0270http://refhub.elsevier.com/S0278-6915(14)00044-1/h0270http://refhub.elsevier.com/S0278-6915(14)00044-1/h0265http://refhub.elsevier.com/S0278-6915(14)00044-1/h0265http://refhub.elsevier.com/S0278-6915(14)00044-1/h0275http://refhub.elsevier.com/S0278-6915(14)00044-1/h0275http://refhub.elsevier.com/S0278-6915(14)00044-1/h0275http://refhub.elsevier.com/S0278-6915(14)00044-1/h0280http://refhub.elsevier.com/S0278-6915(14)00044-1/h0280http://refhub.elsevier.com/S0278-6915(14)00044-1/h0280http://refhub.elsevier.com/S0278-6915(14)00044-1/h0285http://refhub.elsevier.com/S0278-6915(14)00044-1/h0285http://refhub.elsevier.com/S0278-6915(14)00044-1/h0290http://refhub.elsevier.com/S0278-6915(14)00044-1/h0290http://refhub.elsevier.com/S0278-6915(14)00044-1/h0290http://refhub.elsevier.com/S0278-6915(14)00044-1/h0295http://refhub.elsevier.com/S0278-6915(14)00044-1/h0295http://refhub.elsevier.com/S0278-6915(14)00044-1/h0295http://refhub.elsevier.com/S0278-6915(14)00044-1/h0295http://refhub.elsevier.com/S0278-6915(14)00044-1/h0295http://refhub.elsevier.com/S0278-6915(14)00044-1/h0300http://refhub.elsevier.com/S0278-6915(14)00044-1/h0300http://refhub.elsevier.com/S0278-6915(14)00044-1/h0300http://refhub.elsevier.com/S0278-6915(14)00044-1/h0300http://refhub.elsevier.com/S0278-6915(14)00044-1/h0305http://refhub.elsevier.com/S0278-6915(14)00044-1/h0305http://refhub.elsevier.com/S0278-6915(14)00044-1/h0305http://refhub.elsevier.com/S0278-6915(14)00044-1/h0310http://refhub.elsevier.com/S0278-6915(14)00044-1/h0310http://refhub.elsevier.com/S0278-6915(14)00044-1/h0310http://refhub.elsevier.com/S0278-6915(14)00044-1/h0320http://refhub.elsevier.com/S0278-6915(14)00044-1/h0320http://refhub.elsevier.com/S0278-6915(14)00044-1/h0320http://refhub.elsevier.com/S0278-6915(14)00044-1/h0315http://refhub.elsevier.com/S0278-6915(14)00044-1/h0315http://refhub.elsevier.com/S0278-6915(14)00044-1/h0315http://refhub.elsevier.com/S0278-6915(14)00044-1/h0325http://refhub.elsevier.com/S0278-6915(14)00044-1/h0325http://refhub.elsevier.com/S0278-6915(14)00044-1/h0330http://refhub.elsevier.com/S0278-6915(14)00044-1/h0330http://refhub.elsevier.com/S0278-6915(14)00044-1/h0335http://refhub.elsevier.com/S0278-6915(14)00044-1/h0335http://refhub.elsevier.com/S0278-6915(14)00044-1/h0340http://refhub.elsevier.com/S0278-6915(14)00044-1/h0340http://refhub.elsevier.com/S0278-6915(14)00044-1/h0340http://refhub.elsevier.com/S0278-6915(14)00044-1/h0340http://refhub.elsevier.com/S0278-6915(14)00044-1/h0340http://refhub.elsevier.com/S0278-6915(14)00044-1/h0345http://refhub.elsevier.com/S0278-6915(14)00044-1/h0345http://refhub.elsevier.com/S0278-6915(14)00044-1/h0350http://refhub.elsevier.com/S0278-6915(14)00044-1/h0350http://refhub.elsevier.com/S0278-6915(14)00044-1/h0355http://refhub.elsevier.com/S0278-6915(14)00044-1/h0355http://refhub.elsevier.com/S0278-6915(14)00044-1/h0360http://refhub.elsevier.com/S0278-6915(14)00044-1/h0360http://refhub.elsevier.com/S0278-6915(14)00044-1/h0360http://refhub.elsevier.com/S0278-6915(14)00044-1/h0415http://refhub.elsevier.com/S0278-6915(14)00044-1/h0370http://refhub.elsevier.com/S0278-6915(14)00044-1/h0370http://refhub.elsevier.com/S0278-6915(14)00044-1/h0370http://refhub.elsevier.com/S0278-6915(14)00044-1/h0370http://refhub.elsevier.com/S0278-6915(14)00044-1/h0370http://refhub.elsevier.com/S0278-6915(14)00044-1/h0375http://refhub.elsevier.com/S0278-6915(14)00044-1/h0375http://refhub.elsevier.com/S0278-6915(14)00044-1/h0375http://refhub.elsevier.com/S0278-6915(14)00044-1/h0380http://refhub.elsevier.com/S0278-6915(14)00044-1/h0380http://refhub.elsevier.com/S0278-6915(14)00044-1/h0380http://refhub.elsevier.com/S0278-6915(14)00044-1/h0380http://refhub.elsevier.com/S0278-6915(14)00044-1/h0385http://refhub.elsevier.com/S0278-6915(14)00044-1/h0385http://refhub.elsevier.com/S0278-6915(14)00044-1/h0390http://refhub.elsevier.com/S0278-6915(14)00044-1/h0390

Urinary excretion of arsenicals following daily intake of various seafoods during a two weeks intervention1 Introduction2 Subjects and methods2.1 Subjects2.2 Study design2.3 Test meals, the 72h controlled diet and the 14days semi-controlled diet2.4 Blood samples2.5 Urine samples2.6 Adjustment for creatinine2.7 Analytical methods2.8 Statistical analyses

3 Results3.1 As content of the diet3.2 Urinary and plasma As concentrations3.3 Urinary excretion of tAs, AB, iAs, DMA and MA during the 72 h follow-up period3.4 Comparison of the plasma tAs concentrations in periods 1 and 23.5 Comparison of the cumulative excretion of arsenicals in periods 1 and 23.6 Excretion patterns3.7 Associations between uptake and elimination in the two test periods

4 Discussion4.1 Urinary and plasma tAs concentrations4.2 Absolute excretion of tAs, AB, iAs, DMA and MA4.3 Excretion pattern4.4 Associations between uptake and elimination in the two test periods4.5 Chronic As exposure from seafood4.6 Relative urinary excretion of iAs, DMA, MA4.7 Inter-individual variations4.8 Safety of arsenicals in seafood4.9 Strengths’ and weaknesses of the study

5 ConclusionConflict of InterestTransparency DocumentAcknowledgementsReferences