TCDD induces splenomegaly in Vk*Myc mice Vk*Myc mice and their WT littermates were given intraperitoneal injections of 2.5 g/kg TCDD or corn oil for 24 weeks. evidence establishing TCDD as an environmental risk factor for monoclonal gammopathy of undetermined significance and multiple myeloma. oncogene is activated in 67% of cases of multiple myeloma but not in MGUS, suggesting that activation may be key in the transition to MM [13, 14]. Bergsagel and colleagues generated a conditional transgenic mouse model (Vk*Myc) with sporadic, activation- induced deaminase-dependent Myc activation in germinal center B cells. These mice develop benign monoclonal Cephalothin gammopathy, which progresses to multiple myeloma after 70 weeks . This model is regarded Cephalothin as the best available mouse model of multiple myeloma because it recapitulates many of the biological and clinical features of human disease, including progression from MGUS to MM, target organ damage, anemia, and the M-spike (elevated serum IgG) 27 It should be noted that the Vk*Myc mouse line is generated in a C57bl/6 genetic background and that 2-year-old wild-type (WT) C57bl/6 mice have a high incidence of benign monoclonal gammopathy, which rarely progress to multiple myeloma . Thus, aging WT C57bl/6 and Vk*Myc mice are genetically prediposed to monoclonal gammopathy and multiple myeloma and represent suitable models for studying MGUS and multiple myeloma. Given that no rodent models of multiple myeloma have been used in the past to study the impact of TCDD in myeloma pathogenesis, we hypothesized that exposure to TCDD in the Vk*Myc model would reveal whether TCDD is a promoter of multiple myeloma progression. Our data provide compelling evidence that TCDD accelerates benign monoclonal gammopathy in WT mice and promotes multiple myeloma progression in Vk*Myc mice, providing the first direct experimental animal evidence to support TCDD as an environmental causative factor for multiple myeloma. 2.?Materials and methods 2.1. Mice Vk*Myc mice in the C57Bl/6J genetic background were generated and obtained by Dr. Bergsagel and genotyping was performed using PCR as previously reported . Vk*Myc and wild-type (WT) C57BL/6J mice were crossed to Ptprc obtain littermates of WT and Vk*Myc mice. Sex-matched WT and Vk*Myc mice (8 weeks old) were assigned to a treatment or control group based on body weight. The dosing regimen employed is presented in Figure 1a. The treatment group was injected intraperitoneally with 2.5 g TCDD /kg body weight (Sigma-Aldrich, St. Louis, MO) diluted 1:10 in corn oil once a week per mouse for 24 weeks, for a total calculated accumulated TCDD dose of 60 g/kg body weight. Equal volumes of corn oil per body weigh were injected into WT and Vk*Myc mice as vehicle control groups. Treated mice were then housed without any further treatment for an additional 24 weeks (Figure 1a). Every 6 weeks, blood from 5 arbitrarily selected mice from each group was drawn and the IgG levels measured. At the end of the 48 weeks, mice were euthanized by isoflurane overdose followed by cervical dislocation. All animal experimentation was carried out in accordance with NIH guidelines and under protocols approved by the Cleveland Clinic Institutional Animal Cephalothin Care and Use Cephalothin Committee. Open in a separate window Figure 1. TCDD treatment promotes splenomegaly in Vk*Myc mice.(a) Schematic diagram of the experimental exposure regimen used in all 4 groups. (b) Mouse spleen weight at 48 weeks. (c) Representative images of spleens Cephalothin from 4 groups of mice (2 from each group). (d) The total number of splenocytes per spleen from mice at sacrifice. (e) Representative H&E-stained spleen sections from TCDD-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=200 m (top) or 25 m (bottom). Data in (b and d) were analyzed by one-way ANOVA. The horizontal lines were presented as the mean value. n=5 mice per group. *, P 0.05; **, P 0.01; ***, P 0.001. 2.2. Blood assays A complete blood count (CBC) was determined using the Advia 120 Hematology System (Siemens Healthineers, Erlangen, Germany). To measure total serum IgG, blood was collected into Eppendorf tubes, allowed to coagulate at room temperature for 5 minutes, and centrifuged for 10 minutes at 850 Total IgG in serum was then determined by ELISA using the Easy- TiterMouse IgG Assay Kit and Mouse IgG Isotype Control (Thermo Fisher Scientific, Rockford, IL) according to the manufacturers protocol. For serum protein electrophoresis, sera were diluted 1:2.