Introduction
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 [
1]. 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 [
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, increased 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 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) [
5]. 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 [
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 association between glyphosate exposure and either MM or NHL [
10]. 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) [
11]
. 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 [
12]. In this work, we used Vk*MYC mice to test our hypothesis that glyphosate has a pathogenic role in MM.
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 incidence of some rare cancers (kidney tumor [
17‐
19] 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 cancer 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 [
22‐
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 [
26]. Many challenged the pathological and statistical analysis of this study [
27,
28]. The study was retracted [
29], but some alleged the retraction was influenced by the agrochemical giant Monsanto (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 epidemiological studies, the IARC concluded that “there is sufficient evidence in experimental animals for the carcinogenicity of glyphosate” [
5], 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 [
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 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 [
12]. 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 [
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] and B 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 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 [
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/
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.
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