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

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: Activation-induced cytidine deaminase; Glyphosate; Multiple myeloma; Vk*MYC mice.

Fig. 1

Glyphosate reduced survival and induced splenomegaly in Vk*MYC mice. a Schematic diagram of the chronic glyphosate exposure regimen in 4 groups of mice. b The 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. c Mouse spleen weight at sacrifice. d The total number of splenocytes per spleen from mice at sacrifice. e Representative images of spleens from 4 groups (2 per group). f The 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 c and d were 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. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001

Fig. 2.

Hematological abnormalities found in Vk*MYC mice treated with glyphosate. a Total serum IgG in mice during 72 weeks of glyphosate treatment. Mouse blood samples were collected and assayed for IgG every 6 weeks. b Immunoglobins 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. c–h Complete 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. i Total 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

Fig. 3

Glyphosate-treated Vk*MYC mice developed progressive plasma cell neoplasms. a Representative 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 log₁₀ values of fluorescence. The numbers in the inserts show the percentage of CD138^highB220^- cells in the entire cell population. b, c Bar 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. d Confocal 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. e Confocal 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. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001

Fig. 4

Glyphosate led to multiple organ dysfunction. a Histological 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). b Infiltrating plasma cells in the bone marrow of glyphosate-treated mice. Scale bar = 20 μm. Arrows pointed to plasma cells. c Infiltrating plasma cells in the spleen of glyphosate-treated mice. Scale bar = 20 μm. Arrows point to plasma cells. d Collagen 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). e Destruction of lung morphology was observed in glyphosate-treated Vk*MYC mice. n = 10 mice per group. Scale bar = 500 μm. f Protein 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

Fig. 5

Glyphosate-induced AID upregulation. a Western blotting analysis of mice treated with 1.0 g/L of glyphosate for 72 weeks. b Western blotting analysis of mice treated with TCDD. c Western blotting analysis of mice treated with glyphosate for 7 days. One representative mouse per treatment group is shown