A rapid, physiologic protocol for testing transcriptional effects of thyroid-disrupting agents in premetamorphic Xenopus tadpoles

Abstract

Increasing numbers of substances present in the environment are postulated to have endocrine-disrupting effects on vertebrate populations. However, data on disruption of thyroid signaling are fragmentary, particularly at the molecular level. Thyroid hormone (TH; triiodothyronine, T3) acts principally by modulating transcription from target genes; thus, thyroid signaling is particularly amenable to analysis with a transcriptional assay. Also, T3 orchestrates amphibian metamorphosis, thereby providing an exceptional model for identifying thyroid-disrupting chemicals. We combined these two advantages to develop a method for following and quantifying the transcriptional action of T3 in Xenopus laevis tadpoles. This technology provides a means of assessing thyroid activity at the molecular level in a physiologically relevant situation. Moreover, translucent tadpoles are amenable to "on-line" imaging with fluorescent reporter constructs that facilitate in vivo measurement of transcriptional activity. We adapted transgenesis with TH-responsive elements coupled to either luciferase or green fluorescent protein to follow T3-dependent transcription in vivo. To reduce time of exposure and to synchronize responses, we optimized a physiologic pretreatment protocol that induced competence to respond to T3 and thus to assess T3 effects and T3 disruption within 48 hr. This pretreatment protocol was based on a short (24 hr), weak (10(-12) M) pulse of T3 that induced TH receptors, facilitating and synchronizing the transcriptional responses. This protocol was successfully applied to somatic and germinal transgenesis with both reporter systems. Finally, we show that the transcriptional assay allows detection of the thyroid-disrupting activity of environmentally relevant concentrations (10(-8) M) of acetochlor, a persistent herbicide.

Figure 1

A significant transcriptional response induced by a 24-hr pretreatment, or priming, pulse of 10⁻¹² M T₃ within 48 hr in tadpoles later exposed to 10⁻⁸ M T₃. NS, not significant. (A) A linear schema indicating the timing of the pretreatment/rinse/exposure protocol. Tadpoles were pretreated with or without T₃ 24 hr, rinsed in water, and fed during 24 hr before injection with 200 ng TH/bZIP-luc construct in the caudal muscle. (B) Measured TH/bZIP-luc transcription in pretreated tadpoles exposed or not exposed to T₃ (10⁻⁸ M) for 48 hr. Values shown are mean ± SE (n = 12/group). In each case, the experiment was repeated three times, providing similar results. **p < 0.01.

Figure 2

Induction of TR-β expression by a weak T₃ pulse. To evaluate the effect of the pretreatment protocol on TR-β expression, tadpoles were exposed for 6 hr to 10⁻¹² M T₃. Total RNA was extracted from caudal muscles and used for RT-PCR analysis of TR-β expression. Rpl8 was used as the internal control. (A) Typical scan obtained after 22 cycles of PCR amplification. (B) Same results quantified by Phosphoimager scanning (Molecular Dynamics, Sunnyvale, CA, USA). Values shown are mean ± SE of five independent experiments expressed as multiples of induction, where 1 is equal to expression in the absence of T₃ (untreated tadpole; Rpl8) as the control level. For each sample, densitometry readings were normalized against the value for Rpl8. *p < 0.05.

Figure 3

Action of TH agonists assessed using the somatic gene transfer method and pretreatment. Tadpoles were pretreated 24 hr with 10⁻¹² M T₃ and then rinsed and fed during 24 hr before injection of 200 ng TH/bZIP-luc construct in the caudal skeletal muscle. TH/bZIP-luc transcription was measured in injected tadpoles exposed to 5 × 10⁻⁸ M T₃ or 5 × 10⁻⁸ M TRIAC for 48 hr. Values shown are mean ± SE (n = 12/group). Each experiment was repeated three times, providing similar results. *p < 0.05. ** < 0.01.

Figure 4

Dose dependency of TH effects on transcriptional responses. NS, not significant. (A) Transcriptional response in tadpoles pretreated for 24 hr with 10⁻¹² M T₃ and then rinsed and fed during 24 hr before injection of 200 ng TH/bZIP-luc construct in the caudal skeletal muscle. The TH/bZIP-luc transcription was measured in injected tadpoles exposed or not exposed to 5 × 10⁻⁸ M, 5 × 10⁻⁹ M, or 5 × 10⁻¹⁰ M T₃ for 48 hr. (B) Transcriptional response in tadpoles injected with 500 ng TH/bZIP-luc construct in the brain, pretreated 24 hr with 10⁻¹² M T₃, and then rinsed and fed during 24 hr before exposure to 10⁻⁷ M, 10⁻⁸ M, or 10⁻¹⁰ M T₃. TH/bZIP-luc transcription was measured after 48 hr. Values shown are mean ± SE (n = 12/group). Each experiment was repeated three times, providing similar results. *p < 0.05. **p < 0.01. ^#p < 0.001.

Figure 5

(A–F) Transcription responses of the TRE-containing transgene to endogenous TH induced by natural metamorphosis in germinally transgenic F₀ embryos at NF stages 51 (A–C) and 62 (D–F). (A, D) Brain. (B–F) Limb buds. TH/bZIP-eGFP is expressed first in the brain (A, yellow arrowhead) and then in other tissues, and persists throughout larval development (A, D). No fluorescence above background is present in limb buds at NF stage 51 (C, yellow arrow). The signal increases throughout larval development until metamorphosis is reached (NF stage 62), when it increases strongly (F). Bars = 1.6 mm (A); 0.5 mm (B, C); 1.8 mm (D); 1.7 mm (E, F). (G–L) The pretreatment protocol significantly reduced time for response to T₃ and to TH analogues in TH/bZIP-eGFP transgenic F₀ tadpoles (G, H, head; I–L, hindlimb). Tadpoles were pretreated for 24 hr with 10⁻¹² M T₃ at NF stages 51–52, and then rinsed and fed during 24 hr before being exposed to 10⁻⁸ M T₃ (J) or to 5 × 10⁻⁸ M TRIAC (L). Fluorescence in the CNS (H) and in hindlimb buds (J, L) was observed after 2 days of treatment. Yellow arrows indicate limb buds; arrowhead indicates the brain area. White arrows indicate crystallin-RFP expression in the eye. Bars = 2 mm (G, H); 0.8 mm (I–L).

Figure 6

The TH enhancing activity of the pesticide acetochlor revealed in the brains of germinally transgenic premetamorphic tadpoles. Abbreviations: a, anterior; NS, not significant; p, posterior. (A) NF stage 50–52 germinally transgenic tadpoles bearing a TH/bZIP-eGFP transgene were pretreated for 24 hr with 10⁻¹² M T₃ and then rinsed and fed for 24 hr.Fluorescence was measured in tadpole brains after 48 hr exposure to 10⁻¹⁰ M T₃ or to 10⁻¹⁰ M T₃ plus 10⁻⁸ M acetochlor. Values shown are mean ± SE of three experiments expressed as multiples of induction, where 1 = control expression in the absence of T₃. Data were normalized and analyzed by Student’s t-test. (B) Representative photographs of strongly fluorescent tadpoles brain from each group (n = 15 tadpoles per group). Bars = 0.4 mm. *p < 0.05. ^#p < 0.001.