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Glyphosate induces benign monoclonal gammopathy and promotes multiple myeloma progression in mice

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Background: Glyphosate is the most widely used herbicide in the USA and worldwide. There has been considerable debate about its carcinogenicity. Epidemiological studies suggest that multiple myeloma (MM) and non-Hodgkin lymphoma (NHL) have a positive and statistically significant association with glyphosate exposure. As a B cell genome mutator, activation-induced cytidine deaminase (AID) is a key pathogenic player in both MM and B cell NHL. Methods: Vk*MYC is a mouse line with sporadic MYC activation in germinal center B cells and considered as the best available MM animal model. We treated Vk*MYC mice and wild-type mice with drinking water containing 1000 mg/L of glyphosate and examined animals after 72 weeks. Results: Vk*MYC mice under glyphosate exposure developed progressive hematological abnormalities and plasma cell neoplasms such as splenomegaly, anemia, and high serum IgG. Moreover, glyphosate caused multiple organ dysfunction, including lytic bone lesions and renal damage in Vk*MYC mice. Glyphosate-treated wild-type mice developed benign monoclonal gammopathy with increased serum IgG, anemia, and plasma cell presence in the spleen and bone marrow. Finally, glyphosate upregulated AID in the spleen and bone marrow of both wild-type and Vk*MYC mice. Conclusions: These data support glyphosate as an environmental risk factor for MM and potentially NHL and implicate a mechanism underlying the B cell-specificity of glyphosate-induced carcinogenesis observed epidemiologically.
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R E S E A R C H Open Access
Glyphosate induces benign monoclonal
gammopathy and promotes multiple
myeloma progression in mice
Lei Wang
1,2
, Qipan Deng
1
, Hui Hu
1,3
, Ming Liu
1,4
, Zhaojian Gong
1,5
, Shanshan Zhang
1,6
, Zijun Y. Xu-Monette
7
,
Zhongxin Lu
3
, Ken H. Young
7
, Xiaodong Ma
1,8,9*
and Yong Li
1*
Abstract
Background: Glyphosate is the most widely used herbicide in the USA and worldwide. There has been considerable
debate about its carcinogenicity. Epidemiological studies suggest that multiple myeloma (MM) and non-Hodgkin
lymphoma (NHL) have a positive and statistically significant association with glyphosate exposure. As a B cell genome
mutator, activation-induced cytidine deaminase (AID) is a key pathogenic player in both MM and B cell NHL.
Methods: Vk*MYC is a mouse line with sporadic MYC activation in germinal center B cells and considered as the best
available MM animal model. We treated Vk*MYC mice and wild-type mice with drinking water containing 1000 mg/L of
glyphosate and examined animals after 72 weeks.
Results: Vk*MYC mice under glyphosate exposure developed progressive hematological abnormalities and plasma cell
neoplasms such as splenomegaly, anemia, and high serum IgG. Moreover, glyphosate caused multiple organ dysfunction,
including lytic bone lesions and renal damage in Vk*MYC mice. Glyphosate-treated wild-type mice developed benign
monoclonal gammopathy with increased serum IgG, anemia, and plasma cell presence in the spleen and bone marrow.
Finally, glyphosate upregulated AID in the spleen and bone marrow of both wild-type and Vk*MYC mice.
Conclusions: These data support glyphosate as an environmental risk factor for MM and potentially NHL and implicate a
mechanism underlying the B cell-specificity of glyphosate-induced carcinogenesis observed epidemiologically.
Keywords: Glyphosate, Multiple myeloma, Vk*MYC mice, Activation-induced cytidine deaminase
Introduction
Glyphosate is the most popular and profitable agrochem-
ical, being registered to use in over 160 countries and ac-
counting for around 25% of the global herbicide market. It
acts via inhibition of 5-enolpyruvylshikimate-3-phosphate
synthase (EPSPS) in the shikimate pathway, which is critical
to the growth of most plants but absent in animals. Since
the discovery of this herbicidal activity in 1974, glyphosate
usage has increased enormously, particularly with the re-
cent introduction of genetically modified crops carrying a
glyphosate-resistant version of EPSPS. Glyphosate is also
heavily used in crop pre-harvest desiccation. Glyphosate
has been detected in more than 50% of surface waters in
the USA, with a median concentration of ~ 0.02 μg/L and a
maximum concentration of 427 μg/L [1]. Around agricul-
tural basins, the median levels of glyphosate range from
0.08 to 4.7 μg/L, with the highest detected concentration of
430 μg/L [2]. Beyond surface water, glyphosate is found in
soil, air, and groundwater, as well as in food [3]. In a recent
report, urinary excretion levels of glyphosate among older
residents of Rancho Bernardo, CA, where glyphosate use is
significantly lower than in the US Midwest region, in-
creased from 0.024 to 0.314 μg/L from 1993 to 2016 [4].
Multiple epidemiological studies have investigated the
association of glyphosate exposure and cancer risk using
either cohort or case-control designs [5]. These studies
found no significant association between glyphosate
exposure and overall cancer risk but suggested that gly-
phosate exposure is positively associated with multiple
© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
* Correspondence: sciencema@hotmail.com;liy2@ccf.org
Lei Wang, Qipan Deng, and Hui Hu contributed equally to this project.
1
Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic,
Cleveland, OH, USA
Full list of author information is available at the end of the article
Wang et al. Journal of Hematology & Oncology (2019) 12:70
https://doi.org/10.1186/s13045-019-0767-9
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myeloma (MM) and non-Hodgkin lymphoma (NHL), as
concluded by a working group of the International
Agency for Research on Cancer (IARC), the cancer
agency of the World Health Organization (WHO) [5]. In
contrast, other national and international agencies like
the US Environmental Protection Agency (EPA), Euro-
pean Food Safety Authority, European Chemicals Au-
thority, and the Joint Food and Agriculture Organization
of the United Nations and WHO have maintained that
glyphosate is unlikely to pose a carcinogenic risk [6].
Three case-control studies performed in Iowa [7], France
[8], and Canada [9] suggest that glyphosate exposure
increases MM risk. The most recent update (2018) from
the Agricultural Health Study, however, found no associ-
ation between glyphosate exposure and either MM or
NHL [10]. Such inconsistencies likely reflect unidentified
confounders, recall bias, and the complex nature of hu-
man exposure that impact epidemiologic relationships,
underscoring the importance of investigations using ani-
mal models to test the effects of exposures in a controlled
environment. However, neither mouse nor rat studies
have been reported that specifically examine the impact of
glyphosate in the pathogenesis of MM, which is one of the
two cancer types relevant to humans reported to be asso-
ciated with glyphosate exposure thus far.
A hallmark of MM is that virtually all MM cases are
preceded by monoclonal gammopathy of undetermined
significance (MGUS) [11].Bergsagel and colleagues gen-
erated a mouse model of MM (Vk*MYC) under the
C57bl/6 genetic background with sporadic c-Myc activa-
tion in germinal center B cells, resulting in the develop-
ment of benign monoclonal gammopathy, a mouse
equivalent to MGUS, which then progresses to MM.
This is the best available MM animal model because it
recapitulates many biological and clinical features of hu-
man MM, including increased serum immunoglobulin G
(IgG), bone lesions, and kidney damage [12]. In this
work, we used Vk*MYC mice to test our hypothesis that
glyphosate has a pathogenic role in MM.
Materials and methods
Mouse model and treatments
All chronic and acute animal experiments were per-
formed in accordance with NIH guidelines and under
protocols approved by the Cleveland Clinic Institutional
Animal Care and Use Committee. Wild-type (WT)
C57Bl/6 mice were purchased from the Jackson Labora-
tory (Bar Harbor, ME). Vk*MYC mice in the C57Bl/6
genetic background were obtained from Dr. Leif Bergsa-
gel (Mayo Clinic, Scottsdale, AZ) [12]. Vk*MYC and
WT mice were intercrossed to obtain WT and Vk*MYC
littermates. Sex-matched WT and Vk*MYC mice (8
weeks old) were assigned to treatment or control groups
based on body weight. For chronic study of glyphosate
effects, treatment groups were provided 1.0 g/L glypho-
sate (Sigma-Aldrich, St. Louis, MO) in their drinking
water for 72 weeks. Regular drinking water was provided
for the control groups (Fig. 1a). Every 6 weeks, blood
was collected from the tail vein of mice, and the serum
IgG level was measured. We did not monitor the serum
concentration of glyphosate for mice due to sample
availability. However, the gross amount of drinking water
consumed by each group of studied mice was monitored
and no difference was observed between these groups.
Animal regulations prevented us from maintaining mice
till they died of cancer (natural death). Instead, mice had
to be euthanized whenever they reached humane end-
points (i.e., adverse health deterioration and serious
complications). Treated Vk*MYC mice began to reach
humane endpoints starting at week 60 with 4 surviving
until week 66 and 3 surviving to week 71. At week 72,
the remaining 3 surviving Vk*MYC mice reached hu-
mane endpoints. These 3 treated Vk*MYC mice were
used for M-spike detection and pathologic analyses,
along with mice from other groups. Other Vk*MYC
mice that were sacrificed before week 72 were analyzed
for total serum IgG levels, complete blood cell count,
and total serum creatinine. For comparison, mice from
other groups were euthanized at week 72 and their tis-
sues and blood analyzed. For acute treatment, 8-week-
old mice (n= 5 per group) were given 0, 1.0, 5.0, 10.0, or
30.0 g/L of glyphosate for 7 days before sacrifice. The
same variables were analyzed in the acute study.
Blood and post-mortem assays
Whole-blood complete blood count (CBC), IgG enzyme-
linked immunosorbent assay (ELISA), serum protein elec-
trophoresis, flow cytometry, and histological examinations
of relevant tissues were performed as described previously
[13]. Serum creatinine was measured by ELISA using a
creatinine assay kit (#ab65340, Abcam, Cambridge, MA)
according to the manufacturers protocol.
Western blotting analyses
Mouse tissues were processed for Western blotting as we
have described elsewhere [13]. The antibodies were from
Cell Signaling Technology (Danvers, MA, USA): AID
(L7E7) (#4975) and β-actin (#3700). Blotting was run with
3 technical replicates. Horseradish peroxidase-conjugated
anti-rabbitoranti-mouseIgGwasusedasthesecondary
antibody.
Statistics
Statistical analysis was carried out using GraphPad
InStat 3 software (GraphPad Software, Inc., San Diego,
CA, USA). The statistical significance between the
groups was determined by one-way or two-way analysis
of variance (ANOVA) with the appropriate post hoc
Wang et al. Journal of Hematology & Oncology (2019) 12:70 Page 2 of 11
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Fig. 1 Glyphosate reduced survival and induced splenomegaly in Vk*MYC mice. aSchematic diagram of the chronic glyphosate exposure
regimen in 4 groups of mice. bThe percentage of mice surviving under glyphosate exposure. The line (blue) to indicate untreated WT mice
aligned directly with that for WT treated mice and so was not visible. cMouse spleen weight at sacrifice. dThe total number of splenocytes per
spleen from mice at sacrifice. eRepresentative images of spleens from 4 groups (2 per group). fThe spleens from control and glyphosate-treated
mice were fixed, embedded in paraffin, sectioned, stained with H&E, and examined by light microscopy. Representative H&E-stained spleen
sections from glyphosate-treated Vk*MYC mice showing altered architecture with a reduction of lymphoid white pulp (WP) and an expansion of
hematogenous red pulp (RP). Scale bar = 500 μm (top), 200 μm (middle), or 100 μm (bottom). Data in cand dwere analyzed by one-way ANOVA
(spleen from one treated Vk*MYC animal was not included due to an incidental damage). The horizontal lines indicate the mean value. n=10
mice per group. *P0.05; **P0.01; ***P0.001
Wang et al. Journal of Hematology & Oncology (2019) 12:70 Page 3 of 11
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testing using Tukeys test. Statistical significance was ac-
cepted at P0.05. All data are shown as mean ± SEM
unless otherwise indicated.
Results
Chronic glyphosate exposure reduces survival and
induces splenomegaly in Vk*MYC mice
Eight-week-old Vk*MYC mice and their WT littermates
were provided 1.0 g/L glyphosate in drinking water for 72
weeks, and animals were monitored at regular intervals be-
fore sacrifice (Fig. 1a). Glyphosate significantly affected the
health of Vk*MYC mice, all of which had to be euthanized
by week 72 (Fig. 1b). Surviving mice in other groups were
sacrificed at week 72 (at age 80 weeks) for necropsy. Inspec-
tion of organs revealed a marked increase in spleen weight
and size in Vk*MYC mice treated with glyphosate com-
pared to the other 3 groups (Fig. 1c, e). Glyphosate signifi-
cantly augmented the splenocyte number in Vk*MYC mice
(Fig. 1d). Histopathologic analysis revealed distinct red and
white pulp in the spleens of untreated WT and Vk*MYC
mice, suggesting normal splenic organization. These histo-
logical characteristics were not preserved in the spleens
from WT mice treated with glyphosate, with predominant
red pulp involvement and poorly organized white pulp. The
spleens from Vk*MYC mice challenged with glyphosate
showed hematogenous red pulp without lymphoid white
pulp involvement, with more vacuoles and lymphocyte ne-
crosis. Additionally, marked histological disorganization
such as severe splenorrhagia was observed in some areas,
which blurred the boundariesbetweenredpulpandwhite
pulp (Fig. 1f). These findings indicate that glyphosate
induces splenomegaly in both WT and Vk*MYC mice.
Hematological abnormalities occur in Vk*MYC mice with
chronic glyphosate exposure
As illustrated in Fig. 2a, untreated Vk*MYC mice exhib-
ited higher IgG levels than untreated WT mice. Upon gly-
phosate exposure, WT mice showed moderate yet steady
increasing in IgG levels, suggesting that glyphosate
induces benign monoclonal gammopathy, a mouse
equivalent to human MGUS. Vk*MYC mice receiving gly-
phosate had greater IgG elevation, and by week 30, IgG
levels jumped to 11.78 g/L, more than 5-fold the 2.07 g/L
observed in untreated Vk*MYC mice. From week 36 to
week 72, the mean IgG level was significantly higher in
treated WTand Vk*MYC mice compared to the untreated
control groups, and Vk*MYC mice, treated or untreated,
had higher IgG levels than their WT counterparts (Add-
itional file 1: Figure S1). Overt MM diagnosis was deter-
mined by serum protein electrophoresis (SPEP) analysis to
detect the M-spike, which is a significant IgG monoclonal
peak. SPEP results showed that Vk*MYC mice treated
with glyphosate had a clear M-spike, whereas weaker M-
spike was observed in glyphosate-treated WT mice. No
clear M-spike was present in the untreated WT mice or
Vk*MYC mice (Fig. 2b). This is the direct in vivo evidence
that glyphosate exposure leads to M-spike, a cardinal
hematological abnormality consistent with MM.
Hematological abnormalities were present in glyphosate-
treated mice as compared to untreated control mice
(Fig. 2ci). The hemoglobin concentration was significantly
lower in glyphosate-treated Vk*MYC mice than in un-
treated Vk*MYC mice or glyphosate-treated WT mice. Gly-
phosate treatment slightly decreased the red blood and
white blood cell counts and increased mean red cell volume
in Vk*MYC mice compared with WT mice. The platelet
counts and hematocrit were also reduced in glyphosate-
treated Vk*MYC mice. Serum creatinine level is a marker
for kidney function, with higher levels indicating kidney
dysfunction. In glyphosate-treated Vk*MYC mice, the mean
serum creatinine concentration was 0.99 mg/dL, about 2-
fold of that in untreated Vk*MYC mice (0.48 mg/dL) and
treated WT mice (0.53 mg/dL). These data support the no-
tion that glyphosate induces multiple hematological abnor-
malities characteristic of MM in mice.
Vk*MYC mice chronically exposed to glyphosate develop
progressive plasma cell neoplasms
Plasma cells exhibit CD138
hi
B220
(high CD138 expres-
sion without B220 expression). Flow cytometric analyses
of cells harvested from the spleens and bone marrow
showed expansion of plasma cells in mice under glypho-
sate exposure. A marked increase in the numbers of
CD138
hi
B220
cells was detected in both WT and
Vk*MYC mice treated with glyphosate (Fig. 3a).
Glyphosate-treated Vk*MYC mice averaged 2.3%
CD138
hi
B220
plasma cells in the spleen, which was
significantly higher than the 0.98% in untreated
Vk*MYC mice and the 0.76% in treated WT mice
(Fig. 3b). Remarkably, the bone marrow of glyphosate-
treated WT and Vk*MYC mice harbored approximately
8.6% and 14.7% CD138
hi
B220
plasma cells, respect-
ively, significantly higher than their untreated counter-
parts (Fig. 3c).
To assess plasma cell localization and compartmentalization
in the spleen and bone marrow, we stained tissue sections
using antibodies against CD138
+
(plasma cells) and Ki67
+
(a
marker for proliferation). The number of plasma cells was
greater in both spleen and bone marrow of treated Vk*MYC
mice compared to treated WT mice (Fig. 3d, e). In
the spleens of Vk*MYC mice, most plasma cells
stained weakly for Ki67, indicating that these cells
were not plasmacytoma cells, which are generally pro-
liferative. These data demonstrate that glyphosate
treatment expands the plasma cell population in the
spleen and bone marrow in both WT and Vk*MYC
mice.
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Chronic glyphosate exposure triggers multiple organ
dysfunction
To determine whether target organ damage occurred in
glyphosate-treated mice, the femoral shaft, spleen, liver,
lung, and kidney were sectioned and stained with
hematoxylin and eosin (H&E). Severe destructive osteo-
lytic bone lesions in the femoral shaft were readily
detectable in glyphosate-treated Vk*MYC mice. Treated
WT mice showed smaller bone lesions. No lesions were
observed in the control groups (Fig. 4a). Plasma cells
with a perinuclear clear zone and eccentric round
nucleus were observed in glyphosate-treated WT and
Vk*MYC mice (Fig. 4b, c).
Next, we analyzed the histopathologic changes in the
liver, lung, and kidney. In glyphosate-treated mice,
hepatic fibrosis and collagen deposition were observed
in Vk*MYC mice, whereas WT mice showed less severe
liver damage; the 2 control groups had normal hepatic
tissue architectures (Fig. 4d). The lungs in treated
Vk*MYC mice were severely damaged, with large distal
air spaces filled by lymphocytes, neutrophils, cell debris,
and hyperplastic pneumocytes; those from untreated
WT mice had normal alveolar spaces and alveolar septa
lined with normal pneumocytes. The lungs from treated
WT mice and untreated Vk*MYC mice showed an inter-
mediate phenotype (Fig. 4e). Renal tubular obstruction
Fig. 2. Hematological abnormalities found in Vk*MYC mice treated with glyphosate. aTotal serum IgG in mice during 72 weeks of glyphosate
treatment. Mouse blood samples were collected and assayed for IgG every 6 weeks. bImmunoglobins from mice as determined by SPEP at week
72. Arrows indicate IgG clonal peaks (M-spike; γ-globulin peak). SPEP was performed for all mice in each group, and representative results of 2
mice per group are shown. chComplete blood cell counts in mice. Hemoglobin concentration (Hb, c), red blood cell count (d), white blood cell
count (e), mean red cell volume (MCV, f), platelet cell count (g), and hematocrit (HCT, h) are shown. iTotal serum creatinine in mice at week 72.
The horizontal lines indicated the mean value. Data were analyzed by two-way ANOVA (b) or one-way ANOVA (a,d,e). n= 10 mice per group
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Fig. 3 (See legend on next page.)
Wang et al. Journal of Hematology & Oncology (2019) 12:70 Page 6 of 11
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by large casts, indicative of necrotic tubular cells, were
detected in glyphosate-treated WT and Vk*MYC mice,
but not in the untreated groups; there were more and
larger casts in treated Vk*MYC kidneys than in WT
kidneys (Fig. 4f). Taken together, these data indicate that
glyphosate treatment damages multiple organs in both
WT and Vk*MYC mice with more severe damage occur-
ring in Vk*MYC mice.
Chronic glyphosate exposure induces AID upregulation
To investigate the underlying mechanisms of glyphosate-
mediated MGUS induction and MM progression, we
determined the expression of activation-induced cytidine
deaminase (AICDA, also known as AID) in mice treated
with 1.0 g/L glyphosate for 72 weeks. We found that AID
was upregulated in both the bone marrow and the spleen
of WT and Vk*MYC mice (Fig. 5a). For untreated animals,
AID expression was moderately higher in the bone
marrow of Vk*MYC mice. In our previous study [13], we
found that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a
contaminant in Agent Orange, induced MGUS in WT
mice and promoted MM progression in Vk*MYC mice.
We examined the expression of AID in WT and Vk*MYC
mice treated with TCDD chronically [13]. TCDD in-
creased AID expression in both bone marrow and spleen
of both WT and Vk*MYC mice (Fig. 5b).
Acute glyphosate exposure induces AID upregulation
To determine the acute effect of glyphosate, we treated 8-
week-old WT and Vk*MYC mice with increasing doses of
glyphosate (1, 5, 10, and 30 g/L) in drinking water for 7
days. This acute treatment neither increased spleen weight
nor affected body weight significantly. Only at the highest
dose (30 g/L, Additional file 1: Figure S2ac) did WT and
Vk*MYC mice have a detectable M-spike and significantly
higher serum IgG (Additional file 1:FigureS2d).The
serum creatinine level was not significantly affected (Add-
itional file 1: Figure S2e). The plasma cell populations in
the bone marrow, spleen, and lymph node of WT and
Vk*MYC mice were moderately increased in the treated
groups (Additional file 1: Figure S3). Next, we analyzed
the expression of AID in the spleen, bone marrow, and
lymph nodes and found that AID was upregulated in a
glyphosate dose-dependent manner in the spleen and
bone marrow of WT and Vk*MYC mice treated with 10
and 30 g/L of glyphosate (Fig. 5c). AID was highly
expressed in the spleen of untreated Vk*MYC mice but
was highest with 30 g/L glyphosate treatment. AID ex-
pression in lymph nodes was only higher in Vk*MYC mice
treated with 30g/L glyphosate. Lower doses (1 and 5 g/L)
did not upregulate AID expression in any organs of WT
or Vk*MYC mice (data not shown). For untreated ani-
mals, AID expression in the spleen, bone marrow, and
lymph nodes was higher in Vk*MYC mice than that in
WT mice, in agreement with previous results showing
that MYC transcriptionally upregulates AID expression
[14]. It is notable that the basal AID level in these acute
treatment groups differed from that in the chronic glypho-
sate study, likely due to the difference in ages at measure-
ment (9 weeks versus 80 weeks).
Given the role of AID in MM pathogenesis in the con-
text of its capacity to induce mutations and chromosome
translocations [12,15,16], these results from mice with
chronic and acute glyphosate treatment support an AID-
mediated mutational mechanism in the etiology of
MGUS and MM under glyphosate exposure.
Discussion
We have reviewed 9 studies testing glyphosate as a
single agent for carcinogenicity in either mice (2 studies)
or rats (7 studies) via chronic dietary or drinking water
administration (Additional file 2: Table S1). Both mouse
studies showed a positive trend toward increased inci-
dence of some rare cancers (kidney tumor [1719]or
hemangiosarcoma [20]) in male, but not female, CD-1
mice exposed to the highest doses of glyphosate. Of the
7 rat studies, 4 (including 1 in which animals received
drinking water ad lib containing 2700 mg/L glyphosate
for 24 months [21]) found no significant increase in can-
cer incidence in any groups of treated animals [20]. Two
other rat studies reported increased pancreas adenoma
incidence in males treated with intermediate glyphosate
doses; however, animals receiving the highest doses
developed these tumors at a lower incidence than those
receiving the intermediate doses [2225] (Additional file
2: Table S1). The last rat study is quite controversial, sci-
entifically and otherwise. Seralini et al. (2012) reported
that female Sprague-Dawley rats receiving 400 mg/L
(See figure on previous page.)
Fig. 3 Glyphosate-treated Vk*MYC mice developed progressive plasma cell neoplasms. aRepresentative flow cytometry plots detecting cell
surface markers CD138 (Y-axis) and B220 (X-axis) in splenocytes (upper panel) and bone marrow cells (lower panel). The numbers on the axes
denoted the log
10
values of fluorescence. The numbers in the inserts show the percentage of CD138
high
B220
-
cells in the entire cell population.
b,cBar graphs of the percentages of CD138
+
B220
-
and B220
+
cells from the spleen (b) and bone marrow (c). Data were analyzed by one-way
ANOVA. dConfocal microscopy images identifying Ki67
+
(green) and CD138
+
(red) expression with nuclear DAPI staining of cells in the spleen of
a representative WT (upper panel) and Vk*MYC mouse (lower panel), both treated with glyphosate. Scale bar = 10 μm. eConfocal microscopy
images identifying Ki67
+
(green) and CD138
+
(red) expression with nuclear DAPI staining of cells in the bone marrow of WT (upper panel) and
Vk*MYC mice (lower panel) treated with glyphosate. Scale bar = 10 μm. n= 10 mice per group. *P0.05; **P0.01; ***P0.001
Wang et al. Journal of Hematology & Oncology (2019) 12:70 Page 7 of 11
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glyphosate in drinking water for 24 months had an
increased mammary tumor incidence (100%) compared to
the no-glyphosate control (50%), yet the incidence was 90%
for the 2250 mg/L group [26]. Many challenged the patho-
logical and statistical analysis of this study [27,28].
The study was retracted [29], but some alleged the
retraction was influenced by the agrochemical giant Mon-
santo (acquired by Bayer AG) [30], a major manufacturer
of both glyphosate and glyphosate-resistant genetically
modified crop seeds. The authors (2014) then republished
this study without further review [31]. Largely based on
the results from these rodent studies and multiple
Fig. 4 Glyphosate led to multiple organ dysfunction. aHistological evaluation of bone morphology from 4 groups of mice. Bone lytic lesions
(indicated by arrows) were detected in the femoral shaft of Vk*MYC mice treated with glyphosate. Scale bar = 500 μm (top) or 100 μm (bottom).
bInfiltrating plasma cells in the bone marrow of glyphosate-treated mice. Scale bar = 20 μm. Arrows pointed to plasma cells. cInfiltrating plasma
cells in the spleen of glyphosate-treated mice. Scale bar = 20 μm. Arrows point to plasma cells. dCollagen deposition in the liver was observed in
glyphosate-treated Vk*MYC mice. n= 10 mice per group. Scale bar = 500 μm (top) or 200 μm (bottom). eDestruction of lung morphology was
observed in glyphosate-treated Vk*MYC mice. n= 10 mice per group. Scale bar = 500μm. fProtein deposition (indicated by arrows) in the kidney
was observed in glyphosate-treated Vk*MYC mice. n= 10 mice per group. Scale bar = 500 μm. All panels show 1 representative image each from
4 groups of mice unless otherwise indicated
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epidemiological studies, the IARC concluded that there is
sufficient evidence in experimental animals for the car-
cinogenicity of glyphosate[5], whereas the EPA, Euro-
pean Food Safety Authority, European Chemicals Agency,
and the Joint Food and Agriculture Organization of
United Nations and WHO Meeting on Pesticide Residues
(JMPR) concluded otherwise [6]. Specifically, JMPR stated
that administration of glyphosate [] at doses as high as
2000 mg/kg body weight by the oral route, the route most
relevant to human dietary exposure, was not associated
with genotoxic effects in an overwhelming majority of
studies conducted in mammals[20].
Our literature review, however, identifies a major draw-
back in these studiesthese strains of mice and rats gener-
ally do not develop MM, which is one of the only two
cancers that are linked to glyphosate exposure in epidemio-
logical studies. The availability of the Vk*MYC mouse
model, widely regarded as the best animal model for MM,
has allowed us to make the first direct determination of
whether glyphosate contributes to MM pathogenesis [12].
In this study, we demonstrate that glyphosate induces be-
nign monoclonal gammopathy (mouse equivalent to MGUS
in human) in WT mice and promotes MM progression in
Vk*MYC mice.In Vk*MYC mice, glyphosate causes
hematological abnormalities like anemia and multiple organ
dysfunction like lytic bone lesions and renal damage, which
are hallmarks of human MM. We examined the lymph
nodes located in armpits, groin, and neck of treated mice
and found no tangible lymphomas by week 72. Yet, we can-
not exclude the possibility that glyphosate may accelerate
lymphomagenesis in WT mice if longer glyphosate exposure
is applied.
Beyond epidemiology and animal models, the mechan-
ism of action is the third pillar required to define a com-
pound as a carcinogen. Numerous studies have revealed
that glyphosate may induce DNA damage, oxidative
stress, inflammation, and immunosuppression, as well as
modulate cell proliferation and death and disrupt sex hor-
mone pathways [5]. However, these mechanistic studies
have failed to explain why glyphosate exposure is only
positively associated with MM and NHL. Our results
demonstrate that glyphosate treatment, either at a chronic
low dose or acute high doses, upregulates the expression
of AID in the bone marrow and spleen of both WT and
Vk*MYC mice. AID is a B cell-specific genome mutator
[15] and a key pathogenic player in both MM [12]andB
Fig. 5 Glyphosate-induced AID upregulation. aWestern blotting analysis of mice treated with 1.0 g/L of glyphosate for 72 weeks. bWestern
blotting analysis of mice treated with TCDD. cWestern blotting analysis of mice treated with glyphosate for 7 days. One representative mouse
per treatment group is shown
Wang et al. Journal of Hematology & Oncology (2019) 12:70 Page 9 of 11
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
cell lymphoma [16], with the latter accounting for ~ 90%
of NHL cases. Specific to MM, the early genetic events are
dominated by translocations involving the IgH locus,
which are probably generated via abnormal somatic
hypermutation and class switch recombination mediated
by AID. We also noted that TCDD, a contaminant the
herbicide Agent Orange, also upregulates AID expression
(Fig. 5). Our data disclose, for the first time, that glypho-
sate elicits a B cell-specific mutational mechanism of ac-
tion in promoting carcinogenesis, as well as offering
experimental evidence to support the epidemiologic find-
ing regarding its tissue specificity in carcinogenesis (i.e.,
only increasing the risk for MM and NHL).
The acceptable daily intake (ADI)of glyphosate cur-
rently allowed in the USA, defined as the chronic refer-
ence dose as determined by EPA, is 1.75 mg/kg body
weight/day [32]; an average adult male or female in the
USA who weighs 88.8 or 76.4 kg [33] and drinks 2 L (8
glasses) water daily containing 77.7 (for male) or 66.9
(for female) mg/L glyphosate would reach the ADI. In a
previous study, rats subjected to 2700 mg/L glyphosate
for 24 months did not have a significantly higher cancer
incidence (Additional file 2: Table S1). Therefore, we
chose a dose of 1,000 mg/Lglyphosate in drinking water
(~ 15-fold the ADI) in this study, which caused signifi-
cant adverse effects and accelerated MM progression in
Vk*MYC mice, i.e., animals predisposed to MM. We are
cognizant that an individual would unlikely consume
such an excessive dose of glyphosate; however, our
results are of regulatory importance and suggest that the
ADI for glyphosate should be reassessed, particularly for
certain populations, such as MGUS patients.
Conclusions
Our data provide in vivo evidence to support that glypho-
sate induces MGUS and promotes disease progression to
MM. We uncover a B cell-specific mutational mechanism
for glyphosate exposure that increases MM and NHL risk,
providing a molecular basis for human epidemiological
findings. Given the increasing use of glyphosate in the
USA and worldwide, the present study supports epidemio-
logical reports and informs the EPA and other agencies
during the regulatory development of current and emer-
ging glyphosate-based herbicidal products.
Additional files
Additional file 1: Figures S1 and S2. Supplementary figures.
(PDF 1473 kb)
Additional file 2: Table S1. Supplementary table. (PDF 37 kb)
Abbreviations
ADI: Acceptable daily intake; AID: Activation-induced cytidine deaminase;
ANOVA: Analysis of variance; CBC: Complete blood count; ELISA: Enzyme-
linked immunosorbent assay; EPA: Environmental Protection Agency;
EPSPS: 5-Enolpyruvylshikimate-3-phosphate synthase; H&E: Hematoxylin and
eosin; IgG: Immunoglobin G; JMPR: The Joint Food and Agriculture
Organization of United Nations and WHO Meeting on Pesticide Residues;
MGUS: Monoclonal gammopathy of undetermined significance;
MM: multiple myeloma; NHL: Non-Hodgkin lymphoma; SPEP: Serum protein
electrophoresis; TCDD: 2,3,7,8-Tetrachlorodibenzo-p-dioxin; WHO: World
Health Organization; WT: Wild-type
Acknowledgements
The authors are grateful to Dr. Cassandra Talerico for editing the manuscript
and providing critical comments.
Authorscontributions
LW, QD, HH, and YL designed the research. All authors performed
experiments and/or contributed to data analyses. LW and YL wrote the
manuscript, and all authors provided critical review and revisions and
approved the final version of the manuscript.
Funding
YL is supported in part by NIH R01 grants (CA138688 and CA177810); LW is
supported by National Natural Science Foundation of China (NO. 31500326)
and Natural Science Foundation of Guangdong Province of China (NO.
2017A030313194).
Availability of data and materials
All data and materials supporting the conclusion of this study have been
included within the article and the supplemental data.
Ethics approval and consent to participate
Animal experiments are approved by the Cleveland Clinic Institutional
Animal Care and Use Committees. There is no human subject participation.
Consent for publication
This study does not include any individual persons data in any form.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic,
Cleveland, OH, USA.
2
School of Life Sciences, Institute of Modern
Aquaculture Science and Engineering, Guangdong Provincial Key Laboratory
for Healthy and Safe Aquaculture, South China Normal University,
Guangzhou 510631, China.
3
Department of Medical Laboratory, Central
Hospital of Wuhan, Wuhan, China.
4
State Key Laboratory of Respiratory
Diseases, Guangzhou Institute of Respiratory Diseases, The First Affiliated
Hospital of Guangzhou Medical University, Guangzhou Medical University,
Guangzhou, China.
5
Department of Stomatology, the Second Xiangya
Hospital, Central South University, Changsha, China.
6
Department of
Stomatology, Xiangya Hospital, Central South University, Changsha, China.
7
Department of Hematopathology, The University of Texas MD Anderson
Cancer Center, Houston, TX, USA.
8
Institute for Brain Research and
Rehabilitation, South China Normal University, Guangzhou 510631, China.
9
The Research Center of Basic Integrative Medicine, Guangzhou University of
Chinese Medicine, Guangzhou 510006, China.
Received: 6 May 2019 Accepted: 30 June 2019
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In rural areas of Paraguay are widely used herbicides, among which several are made of glyphosate, these are systemic broad spectrum and act by inhibiting the activity of enzymes that synthesize aromatic aminoacids in plants. Studies have shown that they don’t have genotoxic activity, however, other research indicates that they can alter cellular processes in animals, which can present an environmental and health risk factor. The main objective of this work is to evaluate the mutagenic activity of three glyphosate-based herbicides (commercial formulation) in Drosophila melanogaster through SMART assay. For this, third stage larvae of D. melanogaster obtained from standard crossing and high bioactivation cross were chronically treated orally for 72 ± 5 hours with different concentrations (0.01, 0.1, 1 and 10 mg.mL-1) of three glyphosate-based herbicides, Cyclophosphamide 2.61 and 0.78 mg.mL-1 were used as a mutagenic agent and as a control distilled water. The results obtained were not statistically significant, however, the herbicide GBH2 at the concentration of 10 mg.mL-1 showed significant differences compared to control in mutations number of small single spot type and total number of mutations observed.
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The present study was aimed to investigate the genotoxic and apoptotic effects of glyphosate (GLP) in Roundup formulation along with mitigation of two potent antioxidants i.e. vitamin C and E in caprine granulosa cells in vitro. The entire work was done in a dose and time dependent manner where different concentrations of GLP (0.1 mg/ml, 2.0 mg/ml and 4.0 mg/ml) in Roundup and antioxidants (0.5 mM and 1.0 mM) were employed to culture of granulosa cells for exposure durations of 24, 48 and 72 hours. Analysis of GLP‐induced geno‐toxicity was accomplished by using Single Cell Gel Electrophoresis (Comet assay) assay. Results have shown increased incidences of DNA fragmentation, evidenced by presence of different types of comets (Type 1‐ Type 4) in Roundup‐GLP‐ exposed groups in contrast to the control group (Type 0 comet). However, mitigation by both vitamin C and E was significant (p<0.05) in combating the GLP‐induced genotoxicity in granulosa cells in a concentration‐ and time‐dependent manner. The results of our study provide a clear indication of the ameliorative actions of vitamin C and E against Roundup‐GLP‐induced genotoxicity that instigate apoptosis in ovarian granulosa cells of caprine.
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Glyphosate is a universal herbicide with genital toxicity, but the effect of glyphosate on oocytes has not been reported. This study aimed to evaluate the effect of glyphosate (0, 10, 20, 50 and 100 mM) on bovine oocyte in vitro maturation. We showed that 50 mM glyphosate adversely affects the development of bovine oocytes. Exposure of oocytes to 50 mM glyphosate caused an abnormal reduction in oxidative (redox) levels compared with that in the control group, with a significantly higher reactive oxide species level (P < 0.05) and significantly lower glutathione (GSH) expression (P < 0.05). Additionally, the mRNA levels of antioxidant genes (SOD1, SOD2, SIRT2, SIRT3) and the mitochondrial membrane potential (MMP) were significantly reduced (P < 0.05). Furthermore, treatment with 50 mM glyphosate-induced apoptosis, and the mRNA levels of the apoptotic genes Caspase-3 and Caspase-4 were significantly higher than those in the control group (P < 0.05); however, the mRNA level of BAX was significantly higher than that in the control group (P < 0.01). Additionally, the mRNA levels of the anti-apoptotic genes Survivin and BCL-XL were significantly lower than those in the control group (P < 0.05), and oocyte quality was adversely affected. Together, our results confirmed that glyphosate impairs the quality of oocytes by promoting abnormal oocyte redox levels and apoptosis.
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A total of 243 samples of divers foodstuffs were analysed for glyphosate and AMPA using an LC/MS/MS method with a relatively low LOQ in the range of 0.0005 to 0.0025 mg kg⁻¹. Main contributors for dietary glyphosate and AMPA intake were cereals and pulses. The results suggest that pasta is a very important foodstuff for dietary glyphosate residue intake in Switzerland. Interestingly all samples of wine, fruit juice and nearly all samples of honey tested positive for glyphosate although at very low levels. A dietary risk assessment was conducted. Food products for analysis were not selected purely at random, rather products were selected for which high levels of glyphosate residues were suspected. However, even in samples where high residue levels were expected, no exceedances of MRLs were found. Consequently, human exposure did not exceed neither ADI nor ARfD. Therefore, glyphosate residues found in the sampled foodstuffs from the Swiss market were of no concern for human health.
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Although epidemiologic studies have suggested a possible association between occupational exposures to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the risk of development of multiple myeloma, definitive evidence in support of this association is lacking. In the present study, we employed the Vk*Myc mouse model of multiple myeloma to assess the impact of TCDD exposure on multiple myeloma pathogenesis. TCDD induced splenomegaly and multiple peripheral blood abnormalities, including anemia and high serum IgG levels. In addition, TCDD triggered bone lytic lesions, as well as renal tubular casts, a phenomenon associated with human myeloma kidney disease. Even in wild-type C57BL/6 mice, TCDD increased serum IgG levels, induced anemia, and increased plasma cell presence in the spleen and bone marrow, hallmarks of benign monoclonal gammopathy. Lastly, TCDD induced AKT activation and the DNA damage response, key pathogenic events in myeloma pathogenesis, in animal spleen and/or bone marrow. These data indicate that TCDD accelerates monoclonal gammopathy development and promotes progression to multiple myeloma in genetically-predisposed mice. This work offers the first direct experimental evidence establishing TCDD as an environmental risk factor for monoclonal gammopathy of undetermined significance and multiple myeloma.
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Since its initial sales in the 1970s, the herbicide glyphosate attained widespread use in modern agriculture, becoming the most commercially successful and widely used herbicide of all time as of 2016. Despite a primary mechanism that targets a pathway absent from animal cells and regulatory studies showing safety margins orders of magnitude better than many other, more directly toxic herbicides, the safety status of glyphosate, has recently been brought into question by a slow accumulation of studies evincing more insidious health risks, especially when considered in combination with the surfactants it is usually applied with. Current, official views of respected international regulatory and health bodies remain divided on glyphosate's status as a human carcinogen, but the 2015 IARC decision to reclassify the compound as Category 2a (probably carcinogenic to humans) marked a sea change in the scientific community's consensus view. The goal of this review is to consider the state of science regarding glyphosate's potential as a human carcinogen and genotoxin, with particular focus on studies suggesting mechanisms which would go largely undetected in traditional toxicology studies, such as microbiome disruption and endocrine mimicry at very low concentrations.
Article
Background: Glyphosate is the most commonly used herbicide worldwide, with both residential and agricultural uses. In 2015, the International Agency for Research on Cancer classified glyphosate as "probably carcinogenic to humans," noting strong mechanistic evidence and positive associations for non-Hodgkin lymphoma (NHL) in some epidemiologic studies. A previous evaluation in the Agricultural Health Study (AHS) with follow-up through 2001 found no statistically significant associations with glyphosate use and cancer at any site. Methods: The AHS is a prospective cohort of licensed pesticide applicators from North Carolina and Iowa. Here, we updated the previous evaluation of glyphosate with cancer incidence from registry linkages through 2012 (North Carolina)/2013 (Iowa). Lifetime days and intensity-weighted lifetime days of glyphosate use were based on self-reported information from enrollment (1993-1997) and follow-up questionnaires (1999-2005). We estimated incidence rate ratios (RRs) and 95% confidence intervals (CIs) using Poisson regression, controlling for potential confounders, including use of other pesticides. All statistical tests were two-sided. Results: Among 54 251 applicators, 44 932 (82.8%) used glyphosate, including 5779 incident cancer cases (79.3% of all cases). In unlagged analyses, glyphosate was not statistically significantly associated with cancer at any site. However, among applicators in the highest exposure quartile, there was an increased risk of acute myeloid leukemia (AML) compared with never users (RR = 2.44, 95% CI = 0.94 to 6.32, Ptrend = .11), though this association was not statistically significant. Results for AML were similar with a five-year (RRQuartile 4 = 2.32, 95% CI = 0.98 to 5.51, Ptrend = .07) and 20-year exposure lag (RRTertile 3 = 2.04, 95% CI = 1.05 to 3.97, Ptrend = .04). Conclusions: In this large, prospective cohort study, no association was apparent between glyphosate and any solid tumors or lymphoid malignancies overall, including NHL and its subtypes. There was some evidence of increased risk of AML among the highest exposed group that requires confirmation.
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The herbicide Roundup is sprayed onto genetically modified crops and applied as a desiccant to most small non–genetically modified grains. Use of this herbicide has increased since 1994 when genetically modified crops were introduced in the United States. Glyphosate, the primary ingredient in the herbicide, is found in these crops at harvest.¹ Environmental exposure through dietary intake of these crops has potential adverse health effects and can be assessed by measuring urinary excretion.²- 4 We measured excretion levels of glyphosate and its metabolite aminomethylphosphonic acid (AMPA) in participants from the Rancho Bernardo Study (RBS) of Healthy Aging.
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Anthropometry or human body measurements provide important indicators of nutritional status in children and adults (1). In children, measurements reflect general health status, dietary adequacy, and growth and development over time. In adults, body measurements are used to evaluate health and dietary status, disease risk, and body composition. This report presents anthropometric reference data for 2011-2014 for U.S. children and adults. All material appearing in this report is in the public domain and may be reproduced or copied without permission; citation as to source, however, is appreciated.
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A number of widely debated research articles claiming possible technology-related health concerns have influenced the public opinion on genetically modified food safety. We performed a statistical reanalysis and review of experimental data presented in some of these studies and found that quite often in contradiction with the authors’ conclusions the data actually provides weak evidence of harm that cannot be differentiated from chance. In our opinion the problem of statistically unaccounted multiple comparisons has led to some of the most cited anti-genetically modified organism health claims in history. We hope this analysis puts the original results of these studies into proper context.
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The health effects of a Roundup-tolerant NK603 genetically modified (GM) maize (from 11% in the diet), cultivated with or without Roundup application and Roundup alone (from 0.1 ppb of the full pesticide containing glyphosate and adjuvants) in drinking water, were evaluated for 2 years in rats. This study constitutes a follow-up investigation of a 90-day feeding study conducted by Monsanto in order to obtain commercial release of this GMO, employing the same rat strain and analyzing biochemical parameters on the same number of animals per group as our investigation. Our research represents the first chronic study on these substances, in which all observations including tumors are reported chronologically. Thus, it was not designed as a carcinogenicity study. We report the major findings with 34 organs observed and 56 parameters analyzed at 11 time points for most organs. Biochemical analyses confirmed very significant chronic kidney deficiencies, for all treatments and both sexes; 76% of the altered parameters were kidney-related. In treated males, liver congestions and necrosis were 2.5 to 5.5 times higher. Marked and severe nephropathies were also generally 1.3 to 2.3 times greater. In females, all treatment groups showed a two- to threefold increase in mortality, and deaths were earlier. This difference was also evident in three male groups fed with GM maize. All results were hormone- and sex-dependent, and the pathological profiles were comparable. Females developed large mammary tumors more frequently and before controls; the pituitary was the second most disabled organ; the sex hormonal balance was modified by consumption of GM maize and Roundup treatments. Males presented up to four times more large palpable tumors starting 600 days earlier than in the control group, in which only one tumor was noted. These results may be explained by not only the non-linear endocrine-disrupting effects of Roundup but also by the overexpression of the EPSPS transgene or other mutational effects in the GM maize and their metabolic consequences. Our findings imply that long-term (2 year) feeding trials need to be conducted to thoroughly evaluate the safety of GM foods and pesticides in their full commercial formulations.