Glyphosate induces benign monoclonal gammopathy and promotes multiple myeloma progression in mice
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.
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.
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.
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.
KeywordsGlyphosate Multiple myeloma Vk*MYC mice Activation-induced cytidine deaminase
Acceptable daily intake
Activation-induced cytidine deaminase
Analysis of variance
Complete blood count
Enzyme-linked immunosorbent assay
Environmental Protection Agency
Hematoxylin and eosin
The Joint Food and Agriculture Organization of United Nations and WHO Meeting on Pesticide Residues
Monoclonal gammopathy of undetermined significance
Serum protein electrophoresis
World Health Organization
Glyphosate is the most popular and profitable agrochemical, being registered to use in over 160 countries and accounting 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 recent 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 . Around agricultural basins, the median levels of glyphosate range from 0.08 to 4.7 μg/L, with the highest detected concentration of 430 μg/L . Beyond surface water, glyphosate is found in soil, air, and groundwater, as well as in food . 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, increased from 0.024 to 0.314 μg/L from 1993 to 2016 .
Multiple epidemiological studies have investigated the association of glyphosate exposure and cancer risk using either cohort or case-control designs . These studies found no significant association between glyphosate exposure and overall cancer risk but suggested that glyphosate exposure is positively associated with multiple 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) . In contrast, other national and international agencies like the US Environmental Protection Agency (EPA), European Food Safety Authority, European Chemicals Authority, and the Joint Food and Agriculture Organization of the United Nations and WHO have maintained that glyphosate is unlikely to pose a carcinogenic risk . Three case-control studies performed in Iowa , France , and Canada  suggest that glyphosate exposure increases MM risk. The most recent update (2018) from the Agricultural Health Study, however, found no association between glyphosate exposure and either MM or NHL . Such inconsistencies likely reflect unidentified confounders, recall bias, and the complex nature of human exposure that impact epidemiologic relationships, underscoring the importance of investigations using animal 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 associated with glyphosate exposure thus far.
A hallmark of MM is that virtually all MM cases are preceded by monoclonal gammopathy of undetermined significance (MGUS) . Bergsagel and colleagues generated a mouse model of MM (Vk*MYC) under the C57bl/6 genetic background with sporadic c-Myc activation in germinal center B cells, resulting in the development 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 human MM, including increased serum immunoglobulin G (IgG), bone lesions, and kidney damage . 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
Blood and post-mortem assays
Whole-blood complete blood count (CBC), IgG enzyme-linked immunosorbent assay (ELISA), serum protein electrophoresis, flow cytometry, and histological examinations of relevant tissues were performed as described previously . Serum creatinine was measured by ELISA using a creatinine assay kit (#ab65340, Abcam, Cambridge, MA) according to the manufacturer’s protocol.
Western blotting analyses
Mouse tissues were processed for Western blotting as we have described elsewhere . 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-rabbit or anti-mouse IgG was used as the secondary antibody.
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 testing using Tukey’s test. Statistical significance was accepted at P ≤ 0.05. All data are shown as mean ± SEM unless otherwise indicated.
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 before 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. Inspection of organs revealed a marked increase in spleen weight and size in Vk*MYC mice treated with glyphosate compared to the other 3 groups (Fig. 1c, e). Glyphosate significantly 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 histological 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 necrosis. Additionally, marked histological disorganization such as severe splenorrhagia was observed in some areas, which blurred the boundaries between red pulp and white 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
Hematological abnormalities were present in glyphosate-treated mice as compared to untreated control mice (Fig. 2c–i). The hemoglobin concentration was significantly lower in glyphosate-treated Vk*MYC mice than in untreated Vk*MYC mice or glyphosate-treated WT mice. Glyphosate 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 notion that glyphosate induces multiple hematological abnormalities characteristic of MM in mice.
Vk*MYC mice chronically exposed to glyphosate develop progressive plasma cell neoplasms
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 proliferative. These data demonstrate that glyphosate treatment expands the plasma cell population in the spleen and bone marrow in both WT and Vk*MYC mice.
Chronic glyphosate exposure triggers multiple organ dysfunction
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 intermediate phenotype (Fig. 4e). Renal tubular obstruction 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 occurring in Vk*MYC mice.
Chronic glyphosate exposure induces AID upregulation
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 S2a–c) did WT and Vk*MYC mice have a detectable M-spike and significantly higher serum IgG (Additional file 1: Figure S2d). The serum creatinine level was not significantly affected (Additional 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 expression in lymph nodes was only higher in Vk*MYC mice treated with 30 g/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 animals, 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 . It is notable that the basal AID level in these acute treatment groups differed from that in the chronic glyphosate study, likely due to the difference in ages at measurement (9 weeks versus 80 weeks).
Given the role of AID in MM pathogenesis in the context 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.
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 incidence of some rare cancers (kidney tumor [17, 18, 19] or hemangiosarcoma ) 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 ) found no significant increase in cancer incidence in any groups of treated animals . 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 [22, 23, 24, 25] (Additional file 2: Table S1). The last rat study is quite controversial, scientifically and otherwise. Seralini et al. (2012) reported that female Sprague-Dawley rats receiving 400 mg/L 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 . Many challenged the pathological and statistical analysis of this study [27, 28]. The study was retracted , but some alleged the retraction was influenced by the agrochemical giant Monsanto (acquired by Bayer AG) , a major manufacturer of both glyphosate and glyphosate-resistant genetically modified crop seeds. The authors (2014) then republished this study without further review . Largely based on the results from these rodent studies and multiple epidemiological studies, the IARC concluded that “there is sufficient evidence in experimental animals for the carcinogenicity of glyphosate” , whereas the EPA, European 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 . 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” .
Our literature review, however, identifies a major drawback in these studies—these strains of mice and rats generally do not develop MM, which is one of the only two cancers that are linked to glyphosate exposure in epidemiological 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 . In this study, we demonstrate that glyphosate induces benign 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 cannot exclude the possibility that glyphosate may accelerate lymphomagenesis in WT mice if longer glyphosate exposure is applied.
Beyond epidemiology and animal models, the mechanism of action is the third pillar required to define a compound 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 hormone pathways . 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  and a key pathogenic player in both MM  and B cell lymphoma , 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 glyphosate elicits a B cell-specific mutational mechanism of action in promoting carcinogenesis, as well as offering experimental evidence to support the epidemiologic finding regarding its tissue specificity in carcinogenesis (i.e., only increasing the risk for MM and NHL).
The “acceptable daily intake (ADI)” of glyphosate currently allowed in the USA, defined as the chronic reference dose as determined by EPA, is 1.75 mg/kg body weight/day ; an average adult male or female in the USA who weighs 88.8 or 76.4 kg  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/L glyphosate in drinking water (~ 15-fold the ADI) in this study, which caused significant 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.
Our data provide in vivo evidence to support that glyphosate 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 epidemiological reports and informs the EPA and other agencies during the regulatory development of current and emerging glyphosate-based herbicidal products.
The authors are grateful to Dr. Cassandra Talerico for editing the manuscript and providing critical comments.
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.
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).
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 person’s data in any form.
The authors declare that they have no competing interests.
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