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
As illustrated in Fig. 2a, untreated Vk*MYC mice exhibited higher IgG levels than untreated WT mice. Upon glyphosate 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 glyphosate 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 WT and Vk*MYC mice compared to the untreated control groups, and Vk*MYC mice, treated or untreated, had higher IgG levels than their WT counterparts (Additional file 1: Figure S1). Overt MM diagnosis was determined 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. 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
Plasma cells exhibit CD138hi B220– (high CD138 expression without B220 expression). Flow cytometric analyses of cells harvested from the spleens and bone marrow showed expansion of plasma cells in mice under glyphosate exposure. A marked increase in the numbers of CD138hi B220– cells was detected in both WT and Vk*MYC mice treated with glyphosate (Fig. 3a). Glyphosate-treated Vk*MYC mice averaged 2.3% CD138hi 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% CD138hi B220– plasma cells, respectively, significantly higher than their untreated counterparts (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 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
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 osteolytic 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 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
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 increased 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 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 [14]. 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.