Methylated Phenylarsenical Metabolites Discovered in Chicken Liver

Article abstract of DOI:10.1002/anie.201700736

We report the discovery of three toxicologically relevant methylated phenylarsenical metabolites in the liver of chickens fed 3-nitro-4-hydroxyphenylarsonic acid (ROX), a feed additive in poultry production that is still in use in several countries. Methyl-3-nitro-4-hydroxyphenylarsonic acid (methyl-ROX), methyl-3-amino-4-hydroxyphenylarsonic acid (methyl-3-AHPAA), and methyl-3-acetamido-4-hydroxyphenylarsonic acid (or methyl-N-acetyl-ROX, methyl-N-AHPAA) were identified in such chicken livers, and the concentration of methyl-ROX was as high as 90 μg kg^?1, even after a five-day clearance period. The formation of these newly discovered methylated metabolites from reactions involving trivalent phenylarsonous acid substrates, S-adenosylmethionine, and the arsenic (+3 oxidation state) methyltransferase enzyme As3MT suggests that these compounds are formed by addition of a methyl group to a trivalent phenylarsenical substrate in an enzymatic process. The IC₅₀ values of the trivalent phenylarsenical compounds were 300–30 000 times lower than those of the pentavalent phenylarsenicals.

Full text of DOI:10.1002/anie.201700736

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International Edition: DOI: 10.1002/anie.201700736
German Edition: DOI: 10.1002/ange.201700736
Metabolism
Methylated Phenylarsenical Metabolites Discovered in Chicken Liver
Hanyong Peng, Bin Hu,* Qingqing Liu, Jinhua Li, Xing-Fang Li, Hongquan Zhang, and
X. Chris Le*
Abstract: We report the discovery of three toxicologically
relevant methylated phenylarsenical metabolites in the liver of
chickens fed 3-nitro-4-hydroxyphenylarsonic acid (ROX),
a feed additive in poultry production that is still in use in
several countries. Methyl-3-nitro-4-hydroxyphenylarsonic acid
(methyl-ROX), methyl-3-amino-4-hydroxyphenylarsonic acid
(methyl-3-AHPAA), and methyl-3-acetamido-4-hydroxyphe-
nylarsonic acid (or methyl-N-acetyl-ROX, methyl-N-AHPAA)
were identified in such chicken livers, and the concentration of
methyl-ROX was as high as 90 mgkgÀ1, even after a five-day
clearance period. The formation of these newly discovered
methylated metabolites from reactions involving trivalent
phenylarsonous acid substrates, S-adenosylmethionine, and
the arsenic (+ 3 oxidation state) methyltransferase enzyme
As3MT suggests that these compounds are formed by addition
of a methyl group to a trivalent phenylarsenical substrate in an
enzymatic process. The IC50 values of the trivalent phenyl-
arsenical compounds were 300–30000 times lower than those
of the pentavalent phenylarsenicals.
ROX may be metabolized and potentially produce new
arsenic species of toxicological significance.
To gain an understanding of the possible metabolism of
ROX, we have conducted a controlled feeding study that
involved 1600 chickens of two common commercial strains.
The chickens were given either a standard control feed or
a ROX-supplemented feed. Chicken liver samples were
collected for characterization of arsenic species. We previ-
ously identified eight arsenic species in chicken liver, breast
meat, and waste.[4] However, several arsenic-containing
species were not identified, and their chemical nature
remained unknown. We herein report the discovery of three
methylated phenylarsenical metabolites of ROX in chicken
livers and show the toxicological implications of these new
arsenic metabolites because of the involvement of possible
enzymatic methylation processes in the formation of these
metabolites. Using chromatographic separation coupled with
both elemental and molecular mass spectrometry tech-
niques,[5] we identified a group of new arsenic metabolites,
namely methylated 3-nitro-4-hydroxyphenylarsonic acid
A
rsenic consistently ranks first on the priority list of
(methyl-ROX),
3-amino-4-hydroxyphenylarsonic
acid
environmental contaminants because of the occurance, per-
sistence, and toxicity of various arsenic compounds. Chronic
exposure to high concentrations of arsenic puts more than
100 million people around the world at risk of developing
cancer and other adverse health effects.[1] The general
population is exposed to arsenic mainly through ingestion of
water and food. The practice of feeding 3-nitro-4-hydroxy-
phenylarsonic acid (roxarsone, ROX; see the Supporting
Information, Figure S1 for its structure) to poultry and swine
lasted for more than 60 years[2] before the European Union
and the United States stopped its use. Many other countries
continue to use phenylarsenicals in the poultry industry.[3]
Ingestion of such poultry meat and meat products results in
exposure to residual arsenic. However, it remains unclear how
(methyl-3-AHPAA), and 3-acetamido-4-hydroxyphenylar-
sonic acid (methyl-N-AHPAA; see Figure S1), in liver
samples of chickens that had been fed ROX. We further
demonstrated the involvement of an arsenic methyltransfer-
ase (As3MT) in the methylation of the trivalent substrates
ROXIII and 3-AHPAAIII to the corresponding methylated
products of these phenylarsenicals. These intermediate tri-
valent arsenicals and methyl-ROXIII are much more cytotoxic
than their pentavalent counterparts, with IC50 values in T24
cells that are lower by factors of 300–30000. Therefore, the
detection of these methylation metabolites and the implica-
tions of the trivalent intermediates are toxicologically sig-
nificant in relation to human exposure to arsenic.
We first used HPLC and inductively coupled plasma mass
spectrometry (ICP-MS) to identify arsenic species in the
extracts of liver samples from chickens fed either a control
diet or a ROX-supplemented diet. Representative chromato-
grams in Figure 1 show that ROX (Figure 1a) is present in the
ROX-fed chicken (Figure 1c) and not in the control chicken
(Figure 1b). Because of the selective ion monitoring of m/z 75
(As+), the HPLC-ICP-MS analyses revealed the presence of
eleven arsenic species in the ROX-fed chicken (Figure 1c)
and traces of five arsenic species in the control chicken
(Figure 1b). We then separately added a known amount of
arsenobetaine (AsB), arsenite (AsIII), dimethylarsinic acid
(DMA), monomethylarsonic acid (MMA), arsenate (AsV),
3-AHPAA, N-AHPAA, and ROX to aliquots of the chicken
liver sample. Repeated HPLC-ICP-MS analyses of these
spiked samples (Figure 1d–k) showed that the chromato-
graphic retention times of these arsenic species match with
[*] Dr. H. Peng, Prof. B. Hu
Key Laboratory of Analytical Chemistry for Biology and Medicine
(Ministry of Education)
Department of Chemistry, Wuhan University
Wuhan, 430072 (China)
E-mail: binhu@whu.edu.cn
Dr. H. Peng, Dr. Q. Liu, J. Li, Prof. X.-F. Li, Prof. H. Zhang,
Prof. X. C. Le
Division of Analytical and Environmental Toxicology
Department of Laboratory Medicine and Pathology
Faculty of Medicine and Dentistry, University of Alberta
Edmonton, Alberta, T6G 2G3 (Canada)
E-mail: xc.le@ualberta.ca
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.201700736.
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Figure 1. Chromatograms from HPLC-ICP-MS analyses of ROX and
chicken liver samples from a ROX-fed chicken and a control chicken.
a) ROX standard. b) A liver sample from a control chicken fed the
basal diet. c) A liver sample from a chicken fed the ROX-containing
diet. d–k) Analysis of the same chicken liver sample after replicate
aliquots were separately spiked with AsB (d, peak 1), AsIII (e, peak 2),
DMA (f, peak 3), MMA (g, peak 4), AsV (h, peak 6), 3-AHPAA (i,
peak 7), N-AHPAA (j, peak 9), and ROX (k, peak 11). Peaks 5, 8, and
10 did not correspond to any available arsenic standards.
eight of the eleven arsenic species detected in the chicken
liver sample. These results, which are in agreement with
previous reports,[4a,6] indicate the presence of 3-AHPAA,
N-AHPAA, and ROX in ROX-fed chicken liver, in addition
to the five arsenic species (AsB, AsIII, DMA, MMA, and AsV)
that are commonly present as background in both control and
ROX-fed chicken liver samples. However, the retention times
of three arsenic species (peaks 5, 8, and 10) did not match
those of any available arsenic standards. These new arsenic
species have thus not been reported previously. Therefore, we
subsequently focused on characterizing these new arsenic
metabolites arising from ROX.
Figure 2. Identification of peak 10 in liver extracts using ESI-TOF-MS
analysis. A) High-resolution TOF-MS analysis shows the accurate mass
of peak 10 at m/z 259.9548, with a mass error of 0.8 ppm compared to
the theoretical mass (259.9546) of methyl-ROX (C7H7NAsO5À). B) The
product ion spectrum of m/z 259.9548 shows the specific fragment
peaks.
detection of peak 10 (Figure S3) in the analysis of the chicken
liver extract using HPLC-ESI-TOF-MS. The compound of
interest [MÀ1]À has an accurate mass of m/z 259.9548, which
matches that of methyl-ROX. The measured value (259.9548)
is in excellent agreement with the theoretical value (259.9546)
for methyl-ROX, deviating by a very small mass error
(Dm/m) of only 0.8 ppm. The spectrum (Figure 2B) generated
from the precursor ion at m/z 259.9548 also showed expected
characteristic fragment ions at m/z 90.9180 (AsOÀ), 106.9109
To identify these new metabolites of unknown nature
without standards, we developed a strategy that comple-
mented HPLC-ICP-MS with a series of electrospray ioniza-
tion mass spectrometry (ESI-MS) measurements (Figure S2
and Section S6). This approach made use of the characteristic
fragment ion information of the known arsenic compounds
(Table S1) to build a precursor ion scan method in the HPLC-
ESI-MS analysis of the sample. Parallel HPLC-ICP-MS
analysis of the same sample (Figure S3A) provided the
retention time of the arsenic-containing peak. This approach
allowed us to tentatively identify peak 10 in the HPLC-ICP-
MS and HPLC-ESI-MS chromatograms as methyl-ROX. The
arsenic-containing precursor ion at m/z 260 from the detec-
tion of peak 10 showed characteristic arsenic-containing
À
À
À
(AsO2 ), 120.9249 (CH2AsO2 ), 138.0200 (C6H4NO3 ), and
À
212.9490 (C7H6AsO3 ), supporting the identification of
methyl-ROX. The measured and theoretical values of these
fragment ions are in good agreement (Table S2).
We then synthesized methyl-ROX (procedures shown in
Section S7 and characterization shown in Figure S4 and
Table S3) and used it to further confirm the detection of
this compound in chicken liver. We combined HPLC sepa-
ration with simultaneous detection by ICP-MS and ESI-MS
(Figure 3A). Using this technique, we analyzed an extract of
chicken liver as well as the same extract spiked with the
synthesized methyl-ROX species. The element-specific ICP-
fragment ions at m/z 91 (AsOÀ), 107 (AsO2 ), 121
À
À
(CH2AsO2 ), and 123 (AsO3À; Figure S3C).
We further complemented the molecular and fragment
ion information with accurate mass measurements by high-
resolution time-of-flight mass spectrometry (TOF-MS). Fig-
ure 2A shows a representative mass spectrum from the
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peak 10 detected with ICP-MS (Figure 3B), further confirm-
ing the identity of peak 10 as methyl-ROX, a methylation
metabolite of ROX.
Using the same strategy (Figure S2) and the complemen-
tary techniques, we also identified the other two new arsenic
compounds as methyl-3-AHPAA (Figures S5–S8 and
Tables S5 and S6) and methyl-N-AHPAA (Figures S9 and
S10 and Table S7). Thus the three identified arsenic metab-
olites are a group of methylated analogues of ROX,
3-AHPAA, and N-AHPAA.
Having discovered the three new methylation metabolites
of phenylarsenicals, we further investigated how these
phenylarsenicals are methylated. Methylation of inorganic
arsenic is known to involve arsenic (+ 3 oxidation state)
methyltransferase (As3MT), which catalyzes the addition of
a methyl group from S-adenosylmethionine (SAM, as the
methyl donor) to trivalent arsenicals.[7] As3MT has been
shown to be responsible for the methylation of inorganic
arsenic to methylarsenicals in experimental rats, mice, and
algae.[8] There is no report on the methylation of phenyl-
arsenicals.
To test whether a similar pathway as for the methylation
of inorganic arsenic can take place for the methylation of
phenylarsenicals, we first incubated the trivalent phenyl-
arsenicals as substrates with the As3MT enzyme and the
methyl donor SAM, and monitored the formation of the
methylated phenylarsenicals. Figure 4 shows experiments for
testing the methylation of trivalent ROXIII to methyl-ROX
(Figure 4A) and representative chromatograms from analy-
ses of the treated reaction mixture at the beginning of the
reaction and after 6 h (Figure 4B). Note that while the
reaction used trivalent ROXIII as the substrate (Figure 4A),
the chromatographic analysis involved pretreatment of the
sample aliquots with 0.1% hydrogen peroxide. Thus the
trivalent ROXIII was oxidized to the pentavalent ROX prior
to analysis, and the peak of ROX in the chromatograms
(Figure 4B) represents the trivalent ROXIII in the reaction
mixture. HPLC analysis with both ICP-MS detection (Fig-
ure S11A) and ESI-MS detection (Figure S11B) showed the
formation of methyl-ROX and its increasing amount over the
6 h period of reaction. The amount of methyl-ROX accounts
for approximately 36% of the total arsenic in the reaction
mixture (Figure 4C). Our positive control using MMAIII as
a known substrate of As3MT[9] provided the expected results.
Approximately 50% of MMAIII was methylated to dimethy-
larsinic acid (DMAV) in 3 h (Figure S12), which is consistent
with previous results.[10]
Figure 3. Identification of arsenic species by combining HPLC separa-
tion with simultaneous detection by both ICP-MS and ESI-MS.
A) Features of ICP-MS and ESI-MS to provide complementary detec-
tion for HPLC. B) HPLC-ICP-MS analyses of a chicken liver sample and
the same sample spiked with synthesized methyl-ROX. C) HPLC-ESI-
MS analyses of a chicken liver sample and the same sample spiked
with synthesized methyl-ROX. Two ion transitions (260/107 and 260/
138) of methyl-ROX were monitored in the MRM mode.
Similarly, the incubation of the trivalent 3-AHPAAIII
substrate with the As3MT enzyme and the SAM methyl
donor resulted in the formation of methyl-3-AHPAA (Fig-
ure S13). Both HPLC-ICP-MS and HPLC-ESI-MS measure-
ments showed increasing amounts of methyl-3-AHPAA with
increasing enzymatic reaction times from 0 to 6 h. More than
50% of the substrate 3-AHPAAIII had been converted into
methyl-3-AHPAA after 6 h of the enzymatic reaction.
We further tested whether the pentavalent phenylarsen-
icals ROX and 3-AHPAA could serve as substrates for the
As3MT-catalyzed methylation reaction. No methyl-ROX or
methyl-3-AHPAA was detectable. Only when we added high
MS detection of 75As revealed the presence of eleven arsenic-
containing compounds (Figure 3B). Analysis of the extract
supplemented with the synthesized methyl-ROX shows an
increase in peak 10, supporting that peak 10 is methyl-ROX.
The simultaneous detection by ESI-MS (Figure 3C) revealed
a consistent increase of both characteristic ion transitions
(260/138 and 260/107; Table S4) in multiple reaction monitor-
ing (MRM) mode. This peak has the same retention time as
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ically significant. Previous studies of other trivalent arsenicals
have consistently shown that their toxicity is higher than that
of the pentavalent arsenicals.[12] Our toxicological tests with
T24 human bladder carcinoma cells showed that the 24 h IC50
values for the trivalent ROXIII, methyl-ROXIII, and
3-AHPAAIII species were 0.2 mm, 0.4 mm, and 22 mm, respec-
tively, whereas the IC50 values for the respective pentavalent
ROX, methyl-ROX, and 3-AHPAA compounds were
5700 mm, 4700 mm, and 680 mm (Table S8). Thus the trivalent
ROXIII, methyl-ROXIII, and 3-AHPAAIII compounds are
approximately 300–30000 times more cytotoxic than the
pentavalent arsenic compounds ROX, methyl-ROX, and
3-AHPAA. This finding agrees with previous studies, which
consistently reported higher toxicities for trivalent than for
pentavalent methylarsenicals.[12]
Chicken is the most consumed meat among all meat types
in North America on a per capita basis,[13] averaging 80 g per
day. Although the European Union and the United States
have discontinued the use of ROX in the poultry industry,
many other countries continue to use ROX. Our analyses of
liver samples from eight chickens fed ROX-supplemented
food for 28 days showed 73 Æ 24 mgkgÀ1 methyl-ROX, 387 Æ
92 mgkgÀ1 methyl-3-AHPAA, and 32 Æ 4 mgkgÀ1 methyl-N-
AHPAA (Table S9). Compared to the eight previously
characterized arsenic species, the three methylated phenyl-
arsenicals accounted for 42% of the total arsenic in these
chicken liver samples. The standard practice in poultry
industry requires a five-day clearance period after feeding
ROX to chickens. We have determined the concentrations of
arsenic species in liver samples collected from chickens (n =
8) five days after cessation of feeding ROX. The concen-
trations of the three methylated phenylarsenical metabolites
were 92 Æ 23 mgkgÀ1, 29 Æ 8 mgkgÀ1, and 11 Æ 5 mgkgÀ1, for
methyl-ROX, methyl-3-AHPAA, and methyl-N-AHPAA,
respectively. These residual arsenic species in chicken liver
are relevant to human exposure if chicken livers are
consumed. In addition, the differences in toxicity among the
ROX metabolites make the assessment of human exposure to
phenylarsenicals important.
Figure 4. Methylation of the ROXIII substrate into methyl-ROX. A) For-
mation of methyl-ROX from ROXIII. B) Chromatograms from HPLC-
ICP-MS analyses of a reaction mixture that contained the ROXIII
substrate, the SAM methyl donor, and the As3MT enzyme. C) Percent-
age of methyl-ROX and ROX in the reaction mixture over the 6 h
reaction period.
Acknowledgements
We thank Alberta Health, Alberta Innovates, the Canada
Research Chairs Program, the Canadian Institutes of Health
Research, the Natural Sciences and Engineering Research
Council of Canada, and the National Natural Science
Foundation of China (21575107 and 21375097) for financial
support. H.P., Q.L., and J.L. acknowledge support from the
China Scholarship Council. We thank Dr. Martin J. Zuidhof
for assistance in the chicken feeding experiments, Dr. David
Thomas and Dr. Miroslav Styblo for providing the As3MT
enzyme, and Dr. Rongfu Huang, Xiufen Lu, and Zonglin
Yang for technical assistance.
concentrations of reducing agents, such as 10 mm glutathione
(GSH) and 1 mm tris(2-carboxyethyl)phosphine (TCEP),
could we observe traces of methyl-ROX (Figure S14A) and
methyl-3-AHPAA (Figure S14B). These results suggest that
reduction of the pentavalent phenylarsenicals to the trivalent
phenylarsenical intermediates is required, which in turn serve
as the substrates for their enzymatic methylation. The finding
that pentavalent arsenicals are reduced to the trivalent
arsenicals followed by enzymatic methylation is consistent
with the classic pathway for the biomethylation of inorganic
arsenicals.[10]
The implication that trivalent phenylarsenical intermedi-
ates are formed during the methylation process is toxicolog-
4
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[6] a) S. D. Conklin, N. Shockey, K. Kubachka, K. D. Howard, M. C.
Conflict of interest
Carson, J. Agric. Food Chem. 2012, 60, 9394 – 9404.
[7] a) W. R. Cullen, K. J. Reimer, Chem. Rev. 1989, 89, 713 – 764;
b) M. Vahter, Toxicology 2002, 181, 211 – 217; c) S. Lin, Q. Shi,
F. B. Nix, M. Styblo, M. A. Beck, K. M. Herbin-Davin, L. L.
Hall, J. B. Simeonsson, D. J. Thomas, J. Biol. Chem. 2002, 277,
10795 – 10803; d) D. J. Thomas, J. X. Li, S. B. Waters, W. B. Xing,
B. M. Adair, Z. Drobna, V. Devesa, M. Styblo, Exp. Biol. Med.
2007, 232, 3 – 13; e) A. Ajees, B. P. Rosen, Geomicrobiol. J. 2015,
32, 570 – 576.
The authors declare no conflict of interest.
Keywords: arsenic · cytotoxicity · metabolism · methylation ·
roxarsone
[8] a) J. Qin, C. R. Lehr, C. G. Yuan, X. C. Le, T. R. McDermott,
B. P. Rosen, Proc. Natl. Acad. Sci. USA 2009, 106, 5213 – 5217;
b) S. Suzuki, L. L. Arnold, K. L. Pennington, B. W. Chen, H.
Naranmandura, X. C. Le, S. M. Cohen, Toxicol. Appl. Pharma-
col. 2010, 244, 99 – 105; c) Z. Drobna, H. Naranmandura, K. M.
Kubachka, B. C. Edwards, K. Herbin-Davis, M. Styblo, X. C. Le,
J. T. Creed, N. Maeda, M. F. Hughes, D. J. Thomas, Chem. Res.
Toxicol. 2009, 22, 1713 – 1720; d) H. Naranmandura, K. Rehman,
X. C. Le, D. J. Thomas, Anal. Bioanal. Chem. 2013, 405, 1885 –
1891; e) J. M. Currier, C. Douillet, Z. Drobna, M. Styblo, J.
Environ. Sci. 2016, 49, 104 – 112.
[1] a) D. K. Nordstrom, Science 2002, 296, 2143 – 2145; b) A. H.
Smith, E. O. Lingas, M. Rahman, Bull. World Health Org. 2000,
78, 1093 – 1193; c) M. F. Naujokas, B. Anderson, H. Ahsan, H. V.
Aposhian, J. H. Graziano, C. Thompson, W. A. Suk, Environ.
Health Perspect. 2013, 121, 295 – 302.
[2] a) H. D. Chapman, Z. B. Johnson, Poult. Sci. 2002, 81, 356 – 364;
b) F. T. Jones, Poult. Sci. 2007, 86, 2 – 14; c) J. F. Stolz, E. Perera,
B. Kilonzo, B. Kail, B. Crable, E. Fisher, M. Ranganathan, L.
Wormer, P. Basu, Environ. Sci. Technol. 2007, 41, 818 – 823;
d) K. E. Nachman, P. A. Baron, G. Raber, K. A. Francesconi, A.
Navas-Acien, D. C. Love, Environ. Health Perspect. 2013, 121,
818 – 824; e) D. J. Fisher, L. T. Yonkos, K. W. Staver, Environ.
Sci. Technol. 2015, 49, 1999 – 2012.
[9] a) M. Styblo, L. M. Del Razo, E. L. LeCluyse, G. A. Hamilton,
C. Q. Wang, W. R. Cullen, D. J. Thomas, Chem. Res. Toxicol.
1999, 12, 560 – 565.
[10] L. Ding, R. J. Saunders, Z. Drobna, F. S. Walton, P. Xun, D. J.
Thomas, M. Styblo, Toxicol. Appl. Pharmacol. 2012, 264, 121 –
130.
[3] a) S. Mafla, R. Moraga, C. G. Leon, V. G. Guzman-Fierro, J.
Yanez, C. T. Smith, M. A. Mondaca, V. L. Campos, World J.
Microbiol. Biotechnol. 2015, 31, 1267 – 1277; b) T. Gul Kazi,
A. Q. Shah, H. I. Afridi, N. A. Shah, M. B. Arain, Ecotoxicol.
Environ. Saf. 2013, 87, 120 – 123; c) K. E. Nachman, G. Raber,
K. A. Francesconi, A. Navas-Acien, D. C. Love, Sci. Total
Environ. 2012, 417, 183 – 188; d) L. X. Yao, L. X. Huang, Z. H.
He, C. M. Zhou, W. S. Lu, C. H. Bai, Sci. Total Environ. 2016,
566, 1152 – 1158.
[4] a) H. Peng, B. Hu, Q. Liu, Z. Yang, X. Lu, R. Huang, X.-F. Li,
M. J. Zuidhof, X. C. Le, J. Chromatogr. A 2014, 1370, 40 – 49;
b) Q. Liu, H. Peng, X. Lu, M. J. Zuidhof, X.-F. Li, X. C. Le,
Environ. Health Perspect. 2016, 124, 1174 – 1181; c) Z. Yang, H.
Peng, X. Lu, R. Huang, B. Hu, G. Kachanoski, M. J. Zuidhof,
X. C. Le, Environ. Sci. Technol. 2016, 50, 6737 – 6743.
[5] a) S. A. Viczek, K. B. Jensen, K. A. Francesconi, Angew. Chem.
Int. Ed. 2016, 55, 5259 – 5262; Angew. Chem. 2016, 128, 5345 –
5348; b) H. R. Hansen, R. Pickford, J. Thomas-Oates, M.
Jaspars, J. Feldmann, Angew. Chem. Int. Ed. 2004, 43, 337 –
340; Angew. Chem. 2004, 116, 341 – 344; c) A. Rumpler, J. S.
Edmonds, M. Katsu, K. B. Jensen, W. Goessler, G. Raber, H.
Gunnlaugsdottir, K. A. Francesconi, Angew. Chem. Int. Ed.
2008, 47, 2665 – 2667; Angew. Chem. 2008, 120, 2705 – 2707.
[11] a) F. Challenger, Chem. Rev. 1945, 36, 315 – 361; b) W. R. Cullen,
B. C. McBride, J. Reglinski, J. Inorg. Biochem. 1984, 21, 45 – 60;
c) W. R. Cullen, Chem. Res. Toxicol. 2014, 27, 457 – 461.
[12] a) M. J. Mass, A. Tennant, B. C. Roop, W. R. Cullen, M. Styblo,
D. J. Thomas, A. D. Kligerman, Chem. Res. Toxicol. 2001, 14,
355 – 361; b) J. S. Petrick, F. Ayala-Fierro, W. R. Cullen, D. E.
Carter, H. V. Aposhian, Toxicol. Appl. Pharmacol. 2000, 163,
203 – 207; c) T. Schwerdtle, I. Walter, I. Mackiw, A. Hartwig,
Carcinogenesis 2003, 24, 967 – 974; d) B. Moe, H. Peng, X. Lu, B.
Chen, L. W. L. Chen, S. Gabos, X.-F. Li, X. C. Le, J. Environ. Sci.
2016, 49, 113 – 124.
[13] U. S. Department of Agriculture, Economic Research Service,
2014. Food availability (per capita) data system; http://www.ers.
usda.gov/data-products/food-availability-per-capita-data-
system/ [accessed January 20, 2017].
Manuscript received: January 21, 2017
Revised: March 16, 2017
Final Article published: && &&, &&&&
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Toxic chicken: Three toxicologically rele-
vant methylated phenylarsenical metabo-
lites were detected in the liver of chickens
fed 3-nitro-4-hydroxyphenylarsonic acid
(roxarsone), a feed additive in poultry
production that is still in use in several
countries. These compounds are likely
formed by methylation of trivalent phe-
nylarsonous acid substrates in the pres-
ence of an arsenic methyltransferase.
H. Peng, B. Hu,* Q. Liu, J. Li, X.-F. Li,
H. Zhang, X. C. Le*
&&&& — &&&&
Methylated Phenylarsenical Metabolites
Discovered in Chicken Liver
6
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Angew. Chem. Int. Ed. 2017, 56, 1 – 6
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