Effects of dioxin isomers on induction of AhRs and CYP1A1 in early developmental stage embryos of medaka (Oryzias latipes)
Introduction
Dioxins have more than 200 isomers and are widespread environmental contaminants. The mammalian aryl hydrocarbon receptor (AhR) binds to polyaromatic compounds (PACs), including dioxins, and regulates the expression of CYP1A1, a cytochrome P450 which mediates their toxic effects (Hahn et al., 1997, Dong et al., 2004, Fujii-Kuriyama and Mimura, 2005). To evaluate the human and ecological risks associated with exposure to PACs, the concept of toxic equivalency factors (TEF) has been proposed and widely accepted (Berg et al., 1998). This concept is based on the relative toxicities of PACs to the toxicity of 2378T4CDD, one of the most potent dioxin isomers with a World Health Organization (WHO) 2005 TEF of 1 (Berg et al., 2006). The toxicity of a test sample is expressed as 2378T4CDD toxic equivalents based on the concentration of each isomer present, and requires the use of an animal model to predict the toxicity in humans. For evaluating the toxicity of man-made chemicals to humans, rodents are generally used, however, these models are costly, time-consuming, and are severely restricted and regulated (Seung et al., 2008). Recently, zebrafish have been widely used as an alternative to rats and mice in environmental toxicology to monitor environmental risks. Medaka (Oryzias latipes) is a promising vertebrate model for environmental toxicology studies due to their highly adaptive nature to a wide range of temperatures and osmotic pressures, daily egg production, clear chorionic membranes, and well characterized developmental stages. In addition to these attributes, a draft genome of medaka was recently published, further extending the potential usefulness of this organism (Sasaki et al., 2006, Kasahara et al., 2007). However, before medaka can be routinely used for evaluating chemical toxicity, it is necessary to first clarify whether the mammalian TEF concept can be applied in this fish model.
In this study, four AhR genes were located in the medaka genome, while three AhRs (AhR1a, AhR1b, and AhR2) are known for zebrafish (Danio rerio) and only a single AhR gene has been identified in mammals. Several experimental studies of the function and induced expression of CYP1A1 and AhRs upon 2378T4CDD exposure in zebrafish, lake trout, medaka, and mammals have been performed (Andreasen et al., 2002, Dong et al., 2004, Haarmann et al., 2007, Walker et al., 1991). However, as few studies have examined exposure to other dioxin isomers, it is uncertain whether the induced expression patterns and toxic functions of CYP1A1, AhRs and AhR repressor (AhRR) by other isomers are similar to those induced by 2378T4CDD. The TEF-concept can be applied to environmental risk assessment of dioxins, however, the WHO 2005 TEF for dioxins and dioxin-like compounds must be used according to Japanese law in Japan, using GC/MS analysis to quantify dioxins in the atmosphere, water, and exhaust gas. Therefore, there is a need to clarify whether the TEF concept can be applied to medaka embryos and hepatoma (DIT) lines, as well as mammals, for the evaluation of environmental biotoxicities. In this study, we determined the toxicity of several dioxin isomers in medaka embryos, and monitored the induction of the four AhRs, AhRR, and CYP1A1_ORYLA gene expression using quantitative real-time PCR. We confirmed that the dioxin isomers, including 2378T4CDD, caused significant toxicity and developmental abnormalities to medaka embryos with high correlation to their WHO 2005 TEF.
Section snippets
Chemicals
The dioxin isomers used in this study were as follows (WHO 2005 TEF are indicated in parentheses): 1M1CDD (0), 27D2CDD (0), 123T3CDD (0), 124T3CDD (0), 2378T4CDD (1), 12378P5CDD (1), 123478H6CDD (0.1), 123789H6CDD (0.1), 1234678H7CDD (0.01), O8CDD (0.0003), PCB77 (0.0001), PCB126 (0.1), and PCB180 (0). All isomers (99% pure) were purchased from Wellington Laboratories (Ontario, Canada) in a nonane solution, evaporated, and then re-dissolved in DMSO at the concentration of 5 μg m−1 L−1. All other
The effects of dioxin isomers on the hatching rate and development of medaka embryos
Fish preferentially accumulate 2378T4CDD, 2378T4CDD-like PCDD, PCDF isomers, and PCB isomers with high chlorine content (i.e., penta-, hexa-, hepta-chlorinated biphenyls) (Evans et al., 1991). As 2378T4CDD-induced disorders in medaka develop after organogenesis but prior to hatching, the appearance and severity of gross lesions were examined daily by observing embryos in microtiter plates under a dissecting microscope. For dioxin isomers which were found to cause toxicity to medaka embryos, the
Discussion
In this study, the exposure of medaka embryos and cultured cells to dioxin isomers with different WHO 2005 TEF revealed that their toxicities and induction of CYP1A1_ORYLA expression are highly related to their WHO 2005 TEF in mammals. In addition, among the 4 AhRs and 1 AhRR identified in the medaka genome, it was shown that AhR2a possibly mediates the adverse effects of 2378T4CDD. We also revealed that the expression profiles of medaka AhRR was dependent on the WHO 2005 TEF of the dioxin
Acknowledgments
We would like to express our thanks to Dr. Makoto Hirayama at Hiroshima University for his technical advices. This work was supported by a Grant-in-Aid for Scientific Research (S) [No. 21221003] from the Ministry of Education, Culture, Sports, Sciences and Technology, Japan to HM.
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