Changes of thyroid hormone levels and related gene expression in Chinese rare minnow (Gobiocypris rarus) during 3-amino-1,2,4-triazole exposure and recovery

Abstract

Thyroid hormones (THs) play an important role in the development and metabolism of fish through their influences on genetic transcription and are targets for endocrine disruptive agents in the aquatic environment. Amitrole is a pesticide potentially interfering with thyroid hormone regulation. In this study, the rare minnow (Gobiocypris rarus) was exposed to different levels of 3-amino-1,2,4-triazole (amitrole) and allowed to recover in clean water. Plasma TH levels and the expression of TH-related genes, including transthyretin (ttr), deiodinases (d1 and d2), and the thyroid hormone receptor (trα) from the livers and brains were evaluated. After exposure, the plasma TH levels did not change. Histopathological observations showed that livers were degenerated at 10,000 ng/l and these damages could be recovered by the withdrawal of amitrole. However, the ttr, d1, and d2 mRNA levels in the livers of males were significantly up-regulated in all exposure groups (p < 0.05). The ttr and d2 mRNA levels were significantly up-regulated at 10,000 ng/l and 10, 100, and 1000 ng/l in the livers of females, respectively (p < 0.05). In the brains of males, a twofold increase of d2 mRNA levels at ≥100 ng/l and a fivefold decrease of trα mRNA levels at ≥10 ng/l were observed (p < 0.05), whereas no significant differences were observed in the expression of d2 and trα in the brains of females. After a recovery period, the ttr, d1, and d2 mRNA levels in the livers of males returned to control levels, but the trα mRNA levels were irreversibly decreased at all treatments (p < 0.05). In addition, the d2 mRNA levels in the livers of females were significantly induced at ≥100 ng/l. Moreover, the d2 mRNA levels in the brains of males and females were up-regulated at 10,000 ng/l. These results indicated that amitrole exposure could result in alternations of ttr, d1, d2, and trα gene expression in different tissues of the rare minnow. The expression of these TH-related genes in males was more sensitive to amitrole than those of females. Recovery in clean water was associated with the selective regulation of TH-related gene transcription in the rare minnow. Therefore, these TH-related genes can serve as biomarkers to screen the effects of thyroid disruption chemicals in rare minnow.

Introduction

The thyroid hormones (THs) triiodothyronine (T₃) and thyroxine (T₄) have a wide range of biological effects in physiological processes of vertebrates (Power et al., 2001, Yen, 2001). T₃ is derived from outer ring deiodination of T₄ mediated by deiodinase enzyme (D1 and D2) resulting in an increased binding to TH receptors (Marchand et al., 2001, Orozco and Valverde, 2005) and exert its full biological activity (Orozco and Valverde, 2005). Transthyretin (TTR) is a specific TH transport protein in fish, and is a major carrier to transfer the hormones into the target cells (Morgado et al., 2007b). There is growing evidence that thyroid hormone synthesis, metabolism, and distribution may be targets for the interference by endocrine disruption chemicals (Cocco, 2002, Arukwe and Jenssen, 2005).

Many environment pollutants including aromatic hydrocarbons, halogenated aromatic hydrocarbons (dioxin, PCBs), organochlorine, organophosphorus, carbamate pesticides, chlorinated paraffins, cyanide compounds, methyl bromide, phenol, ammonia, metals (aluminum, arsenic, cadmium, lead and mercury), environmental steroids, and a variety of pharmaceutical agents are ubiquitous in the aquatic environment (Lans et al., 1993, Brouwer et al., 1998, Brouwer et al., 1999, Arukwe and Jenssen, 2005, Beck et al., 2006). Some ecotoxicological studies have indicated they could cause disturbances of thyroid hormone homeostasis, hypothyroidism and thyroid gland abnormalities in fish and humans (Langer et al., 2005, Langer et al., 2007, Zoeller, 2005).

The use of pesticides on agricultural land has resulted in contamination of adjacent surface waters (Guo et al., 2008, Singh and Singh, 2008). One of these pesticides, 3-amino-1,2,4-triazole (amitrole) has been shown to interfere normal function of thyroid system, which has a potential risk to aquatic organisms because of its relatively high water solubility (Zen et al., 2000). Measurable concentrations of amitrole in ground water were 1.1 μg/l (Bobeldijk et al., 2001). Previous studies confirmed that the primary target for the toxicity of amitrole is on the thyroid peroxidase with subsequent inhibitory actions on thyroid hormone synthesis (Strum and Karnovsky, 1971), but the impacts of amitrole contamination in aquatic ecosystems and, in particular, the effects on fish and potential thyroid axis disrupting capabilities remain to be elucidated.

Rare minnow (Gobiocypris rarus) is considered to be an appropriate species for the assessment of endocrine disrupting chemicals due to its small size, ease of culture, short life cycle and prolific egg production with high fertilization and hatching rates (Zha et al., 2007, Zha et al., 2008). Rare minnow is an ideal model fish in the laboratory. We have previously published the effects of environmental pollutants on expression of the vitellogenin and estrogen receptor genes in Chinese rare minnow (Zhang et al., 2008).

The changes of TH-related genes of fish have not been throughout investigated so far. The objectives of this study were as follows: to clone partial cDNA sequences of ttr, d1, d2, and trα in the rare minnow and quantify transcription rates using real-time polymerase chain reactions (PCR); to determine the regulation of mRNA transcription levels of these genes from liver and brain in adult rare minnow exposed to amitrole; to examine plasma T₃ and T₄ levels and to discuss the possible molecular mechanisms underlying toxic response.