Evaluation of the teratogenic effects of three traditional Chinese medicines, Si Jun Zi Tang, Liu Jun Zi Tang and Shenling Baizhu San, during zebrafish pronephros development

Abstract

The aim of this study was to evaluate the teratogenic effects of three common Chinese medical prescriptions, Si Jun Zi Tang (SJZT), Liu Jun Zi Tang (LJZT) and Shenling Baizhu San (SLBS), during zebrafish pronephros development. We used the transgenic zebrafish line Tg(wt1b:EGFP) to assess the teratogenic effects using 12 different protocols, which comprised combinations of 4 doses (0, 25, 250, 1,250 ng/mL) and 3 exposure methods [methods I, 12-36 hours post fertilization (hpf), II, 24-48 hpf, and III, 24-36 hpf]. As a result, few defects in the kidneys were observed in the embryos exposed to 25 ng/mL of each medical prescription. The percentage of kidney malformation phenotypes increased as the exposure concentrations increased (25 ng/mL, 0-10%; 250 ng/mL, 0-60%; 1,250 ng/mL, 80-100%). Immunohistochemistry for α6F, which is a basolateral and renal tubular differentiation marker, revealed no obvious defective phenotypes in either SJZT- or LJZT-treated embryos, indicating that these Chinese medical prescriptions had minimal adverse effects on the pronephric duct. However, SLBS-treated embryos displayed a defective phenotype in the pronephric duct. According to these findings, we suggest (1) that the Chinese medical prescriptions induced kidney malformation phenotypes that are dose dependent and (2) that the embryonic zebrafish kidney was more sensitive to SLBS than SJZT and LJZT.

Keywords: Chinese medical prescriptions; kidney; nephrotoxicity; zebrafish.

Fig. 1.

(A) Exposure methods of CMP treatments used in this study. Three CMP exposure methods were designed based on exposure onset and duration: method I, 12–36 hpf; method II, 24–48 hpf; and method III, 24–36 hpf. (B, C, D) Survival rates of zebrafish embryos after exposure to water containing 25, 250; and 1,250 ng/mL of CMP (SJZT, LJZT, SLBS) using exposure methods I, II; and III. (E) The Tukey-Kramer HSD (honestly significant difference) test reported the marginal mean survival rates and corresponding 95% confidence intervals for the three dose level groups, which were adjusted for method and CMP effects. Two group means are significantly different if their intervals are disjoint; and are not significantly different if their intervals overlap.

Fig. 2.

Visible and defective phenotypes of Tg(wt1b:EGFP) embryos after exposure to SJZT (250 ng/mL). (A) Lateral and (B) dorsal view of Tg(wt1b:EGFP) embryos. (C) No defect. (D) Separated glomerulus. (E) Curved pronephric tube and duct and swollen glomerulus. Figures C, D and E were obtained from a dorsal view and at the developmental stage of 48 hpf. gl, glomerulus; pd, pronephric duct; pt, pronephric tube. An arrow, star and triangle indicate the defective sites in gl, pt; and pd, respectively. Scale bar: 80 μm (A, B), 40 μm (C–E).

Fig. 3.

CMP-induced kidney malformation phenotypes are dose dependent. Morphological changes of Tg(wt1b:GFP) embryos after exposure to 25, 250, and 1,250 ng/mL of CMP (SJZT, LJZT, SLBS). Phenotype percentages of zebrafish embryos after CMP treatments using exposure methods I, II, and III (A, B, C). All photos were taken from a dorsal view at the developmental stage of 48 hpf. (D) The Tukey-Kramer HSD (honestly significant difference) test reported the marginal mean malformation rates and corresponding 95% confidence intervals for combinations of the method and CMP groups, which were adjusted for dose effect. Two group means are significantly different if their intervals are disjoint; and are not significantly different if their intervals overlap. An arrow, star and triangle indicate the defective sites in gl, pt; and pd, respectively. Scale bar: 40 μm.

Fig. 4.

Immunohistochemical staining of the basolateral marker Na⁺/K⁺-ATPase alpha1 subunit (α6F) was performed on transverse sections of the gl, pt and pd from a 48 hpf zebrafish larvae without (A) or with SJZT (B), LJZT (C) and SLBS (D) treatment (250 ng/mL). (A’–D’) Sections were stained with a monoclonal antibody against α6F and counterstained with hematoxylin. gl, glomerulus, pd, pronephric duct; pt, pronephric tube. An arrow indicate the defective sites in pd. Scale bar: 40 μm (A–D); 2 μm (A’–D’).