Enantioselective induction of estrogen-responsive gene expression by permethrin enantiomers in embryo-larval zebrafish

Abstract

Enantioselectivity in the separation, toxicology, biodegradation and estrogenic activity of chiral pesticides has become a groundbreaking topic recently. In this study, real-time, quantitative polymerase chain reaction was adapted to investigate the induction of estrogen-responsive gene expression in embryo-larval zebrafish after 7 d of exposure to permethrin (PM) enantiomers. The PM enantiomers were completely separated by a chiral HPLC column. The in vivo study found that a 7 d exposure to 250 ng L⁻¹ PM racemate and its enantiomers was sufficient to stimulate vtg1, esrα and cyp19b expression, while 1000 ng L⁻¹ exposure significantly induced gene expression in a pattern similar to that of the control (50 ng L⁻¹ E2), except for vtg2. Significant differences were detected between the enantiomers in the induction of estrogen-responsive gene expression. At the exposure level of 1000 ng L⁻¹, the vtg1, esrα and cyp19b responses to the (−)-trans enantiomer were about 3.2-, 1.8- and 1.5-fold higher, respectively, than those in the group treated with (+)-trans enantiomer (p < 0.05). In the two cis-enantiomer treatment groups, (+)-cis increased the mRNA level of the cyp19b gene about 1.5-fold higher than the (−)-cis-enantiomer did. Of the four enantiomers, the (−)-trans enantiomer showed the greatest estrogenic activity. The results strongly indicate the occurrence of significant enantioselectivity in estrogenic activity of PM enantiomers exposed to embryo-larval zebrafish. These findings add to a growing body of evidence concerning enantioselectivity in the toxicity, endocrine-disrupting activity, and environmental biodegradation of chiral pesticides.

Introduction

Over the past decades, increasing concern has arisen regarding environmental compounds with the capacity to alter normal endocrine function of wildlife and human (McKinlay et al., 2008). Such compounds are known as endocrine-disrupting chemicals (EDCs), and it has become evident that many currently used chemicals, including insecticides, have such hormonal activity. These compounds may interfere with developmental and reproductive functions in aquatic species, including altering sex ratios, delaying sexual differentiation, damaging gonad development, and changing plasma steroid levels. Furthermore, evidence is now emerging that EDCs may influence reproductive and neuroendocrine systems, senescence, and even carcinogenesis in humans and wildlife (Rhind, 2005, Patisaul et al., 2006).

Manmade pesticides are among the most common sources of EDCs worldwide. Approximately 25% of currently used pesticides are chiral, and this ratio is still increasing (Liu et al., 2005a). In most cases, enantiomers are known to interact selectively with biological systems, and may behave as totally different compounds (Müller and Kohler, 2004). Recent studies have shown that enantiomers of chiral pesticides behave enantioselectively in their effects on aquatic toxicity and environmental fate (Lewis et al., 1999, Liu et al., 2005b, Liu et al., 2005c).

Permethrin (PM), one of the most common synthetic pyrethroids, is widely used for the control of agricultural and indoor pests. PM has two asymmetric positions and contains two pairs, including four ((+)-cis, (−)-cis, (+)-trans, and (−)-trans)) enantiomers. It is valuable to point out that the use of pyrethroids is expected to increase further because of restrictions on the use of organophosphorus pesticides (Qin and Gan, 2006). In fact, the Chinese government gave SPs the first priority to be used for preventing the occurrence of infectious disease after the great 2008 Wenchuan earthquake. Although PM is highly hydrophobic and immobile in soil, it is easily transported offsite into aquatic systems via runoff or soil erosion. As a result, its use will influence aquatic biological systems, even in very low concentrations. In fact, a previous study reported PM concentrations was around 1–5.5 ng g⁻¹ in dry weight basis collected at the lower Missouri River of USA (Echols et al., 2008). The endocrine-disrupting activity of PM has been suggested in some in vitro and in vivo experimental systems (Go et al., 1999, Perry et al., 2007). Moreover, our recent study strongly suggested the occurrence of significant enantioselectivity in estrogenic activity of PM enantiomers in adult male zebrafish (Danio rerio) (Jin et al., 2008a). However, whether or how PM executes its potential hormonal activity in other life stages still remain unknown.

Zebrafish have been used as a predominant test model for hormonal activity assessment of EDCs. Toxicity investigations have been applied to different developmental stages in zebrafish-embryo, larvae, juvenile and adult (Brion et al., 2004). The use of fish in their early development stages in exposure tests has proven to be a convenient and effective approach to examining aquatic toxicity (Kishida et al., 2001, Andersen et al., 2003). Thus, embryo-larval zebrafish are an ideal model to analyze estrogenic activity in aquatic environment.

The aim of this study was to evaluate enantioselectivity in the endocrine-disrupting potential of PM, using embryo-larval zebrafish as the standard laboratory model. In this study, we adopted a real-time polymerase chain reaction (PCR) to determine the estrogenic effects of PM enantiomers by detecting the expression levels of estrogen-responsive genes in embryo-larval zebrafish exposed to PM enantiomer solutions for a period of 7 d immediately post-fertilization.