Tamoxifen Increased Parasite Burden and Induced a Series of Histopathological and Immunohistochemical Changes During Chronic Toxoplasmosis in Experimentally Infected Mice

Abstract

The global distribution of breast cancer and the opportunistic nature of the parasite have resulted in many patients with breast cancer becoming infected with toxoplasmosis. However, very limited information is available about the potential effects of tamoxifen on chronic toxoplasmosis and its contribution to the reactivation of the latent infection. The present study investigated the potential effects of tamoxifen on chronic toxoplasmosis in animal models (Swiss albino mice). Following induction of chronic toxoplasmosis and treatment with the drug for 14 and 28 days, the anti-parasitic effects of tamoxifen were evaluated by parasitological assessment and counting of Toxoplasma cysts. In addition, the effects of the drug on the parasite load were evaluated and quantitated using TaqMan real-time quantitative PCR followed by investigation of the major histopathological changes and immunohistochemical findings. Interestingly, tamoxifen increased the parasite burden on animals treated with the drug during 14 and 28 days as compared with the control group. The quantification of the DNA concentrations of Toxoplasma P29 gene after the treatment with the drug revealed a higher parasite load in both treated groups vs. control groups. Furthermore, treatment with tamoxifen induced a series of histopathological and immunohistochemical changes in the kidney, liver, brain, and uterus, revealing the exacerbating effect of tamoxifen against chronic toxoplasmosis. These changes were represented by the presence of multiple T. gondii tissue cysts in the lumen of proximal convoluted tubules associated with complete necrosis in their lining epithelium of the kidney section. Meanwhile, liver tissue revealed multiple T. gondii tissue cysts in hepatic parenchyma which altered the structure of hepatocytes. Moreover, clusters of intracellular tachyzoites were observed in the lining epithelium of endometrium associated with severe endometrial necrosis and appeared as diffuse nuclear pyknosis combined with sever mononuclear cellular infiltration. Brain tissues experienced the presence of hemorrhages in pia mater and multiple T. gondii tissue cysts in brain tissue. The severity of the lesions was maximized by increasing the duration of treatment. Collectively, the study concluded novel findings in relation to the potential role of tamoxifen during chronic toxoplasmosis. These findings are very important for combating the disease, particularly in immunocompromised patients which could be life-threatening.

Keywords: chronic toxoplasmosis; histopathology; immunohistochemistry; real-time PCR; tamoxifen.

Figure 1

Timeline of the experimental protocol explaining the day (D) number, treatments, and sampling.

Figure 2

Average brain parasite load (ABPL) of tamoxifen-treated animals as compared with control at different time points.

Figure 3

Photomicrograph of kidney sections from experimental mice groups stained with Hx&E stain: (A,B) represent tissue sections from control group showing normal histological architectures compromising in normal glomerular structure in the cortical area (A, arrows), surrounded with normal convoluted tubules. Normal tubular structures in renal medulla area (B, arrows); (C) renal tissue section from control positive group infected by Toxoplasma gondii showing; the presence of multiple T. gondii tissue cysts in the lumen of proximal convoluted tubules (arrow) associated with complete necrosis in their lining epithelium. (D) Kidney sections from the treated group for 14 days showing: the presence of marked infested multiple T. gondii tissue cysts in the lumen of proximal convoluted tubules (red arrowheads), severe necrosis in the lining epithelium of proximal convoluted tubules (arrows) associated with moderate extravasation of blood among renal tubules (arrowheads). (E) Renal tissue section from the treated group for 28 days showing: severe infested multiple T. gondii tissue cysts of ME49 Avirulent strain in the lumen of proximal convoluted tubules (arrows) associated with complete necrosis in their lining epithelium. The bar size of each picture was indicated under each picture.

Figure 4

Photomicrograph of liver sections from experimental mice groups stained with H&E stain: (A,B) a mice liver from the control group demonstrating normal hepatic architectures. (A) Central vein (CV), sinusoids (S), and Hepatocytes (H). (B) Intact portal area; Portal Vein (P.V), Hepatic artery (arrow), and Bile duct (star). (C) Hepatic tissue section from the control positive group infected by Toxoplasma gondii showing; the presence of bradyzoites appeared as pseudocyst of T. gondii among degenerated hepatocytes (arrow). (D,E) Liver sections from the group treated for 14 days showing: (D) The presence of a cluster of tachyzoites appeared as pseudocyst of T. gondii among some degenerated hepatocytes (arrow), severe dilatation in hepatic sinusoids (S). (E) Focal aggregations of inflammatory cells in hepatic tissue (arrow). (F) Hepatic tissue section from treated group for 28 days showing: severe infested multiple T. gondii tissue cysts in hepatic parenchyma (arrows) which alters hepatic structure. The bar size of each picture was indicated under each picture.

Figure 5

Photomicrograph of Uterus sections from experimental mice groups stained with Hx&E stain: (A,B) a mice uterus from the control group demonstrating normal uterine architectures. (A) Normal endometrial epithelium (arrows). (B) Normal endometrial glands (arrow). (C) Uterine tissue section from control positive group infected by Toxoplasma gondii showing; severe endometrial necrosis appeared as diffuse nuclear pyknosis in most of the endometrial epithelium (arrows), severe mononuclear cellular infiltration (red arrowheads). (D) Uterus sections from the treated group for 14 days showed: tachyzoites infestation in connective tissue stroma of the endometrial lamina propria submucosa (arrow). (E) Uterine tissue section from the treated group for 28 days showing: exhibited the presence of a cluster of tachyzoites intracellularly in the lining epithelium of endometrium (arrow). The bar size of each picture was indicated under each picture.

Figure 6

Photomicrograph of brain sections from experimental mice groups stained with Hx&E stain: (A) a mice brain from the control group demonstrating normal brain architectures. (B) Brain tissue section from control positive group infected by Toxoplasma gondii showing; the presence of T. gondii cysts (arrowhead). (C) Brain sections from the group treated for 14 days showing: degenerated T. gondii tissue cysts in the white matter of the infected cerebrum (arrow). (D,E) Brain tissue section from the treated group for 28 days showing: (D) hemorrhages in pia mater (arrow), (E) exhibited the presence of multiple T. gondii tissue cysts in brain tissue (arrowhead). The bar size of each picture was indicated under each picture.

Figure 7

Photomicrograph of immunohistochemical staining for anti-Toxoplasma gondii anti IgG in kidney sections from experimental mice groups showed positive golden brown immunoreactive T. gondii tissue cysts fill most of the infected renal tubules using IHC technique, DAB stain; (A) control positive group infected with ME49 Avirulent strain of T. gondii showing mild tissue IHC reaction. (B) Treated group for 14 days showed moderate IHC tissue reaction. (C magnified in D): from the treated group for 28 days; strong positive results by IHC represented by the presence of multiple golden-brown positive immunoreactivity T. gondii tissue cysts nearly occludes the infected renal tubules. The bar size of each picture was indicated under each picture.

Figure 8

Histomorphometry graph showing quantitative measurements of parasitic cyst in (A) kidney, (B) liver, (C) uterus, and (D) brain tissue among the experimental groups. Data are expressed as means ± standard deviations. Significant differences vs. the control group are marked by different asterisks through one-way ANOVA with Tukey’s post-hoc test: ^**p ≤ 0.01, ^***p ≤ 0.001.