Effect of low dose exposure to the herbicide atrazine and its metabolite on cytochrome P450 aromatase and steroidogenic factor-1 mRNA levels in the brain of premetamorphic bullfrog tadpoles (Rana catesbeiana)

Abstract

The transcriptional regulator steroidogenic factor 1 (SF-1) and the enzyme cytochrome P450 aromatase (CYP19) play a central role in modulation of a broad range of tissue-specific developmental processes associated with hormone homeostasis that includes differentiation of the central nervous system. SF-1 and CYP19 expression may be targeted by a variety of endocrine disruptive agents prevalent within the environment. In the present study, we cloned and characterized partial sequences for bullfrog (Rana catesbeiana) SF-1 and CYP19 and examined the effects of a 48h exposure to 1 and 100μg/l of the herbicide atrazine (ATZ) and its major metabolite desethylatrazine (DEA), as well as 5ng/l of the estrogenic chemical, 17α-ethynylestradiol (EE(2)), and 673ng/l of the thyroid hormone, 3,5,3'-triiodothyronine (T(3)), on SF-1 and CYP19 mRNA abundance in the brains of premetamorphic bullfrog tadpoles. Quantitative RT-PCR analysis showed an increase in CYP19 mRNA following a 48h exposure to EE(2) but not T(3) while no significant changes in SF-1 transcript levels occurred. We observed a strong positive correlation between CYP19 and SF-1 transcript abundance in the ATZ-exposed animals which was not evident with DEA- or hormone-exposed tadpoles. Our results are intriguing in light of reported behavioral changes in ATZ-exposed frogs and suggest that further research is warranted to examine the relationship and role of CYP19 and SF-1 in amphibian brain development.

Figure 1

Putative protein sequence alignment of CYP19 from R. catesbeiana brain with other amphibian species. Clustal alignment were performed using ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/) with amino acid positions identical to R. catesbeiana shown by a dash. Gap alignment positions are denoted by an asterisk. Amino acid positions relative to the start of the known putative sequence and sequence similarity scores generated by MatGAT 2.02 (http://bitincka.com/ledion/matgat/) compared to R. catesbeiana CYP19 are shown on the right. Tissue-specific mRNA forms observed in some species are denoted by (B) brain and (G) gonad. The asterisk indicates notable differences between the bullfrog sequence and the fish brain and gonadal isoforms.

Figure 2

Putative protein sequence alignment of SF-1 from R. catesbeiana brain with other amphibian species. Clustal alignment were performed using ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/) with amino acid positions identical to R. catesbeiana shown by a dash. Gap alignment positions are denoted by an asterisk. Amino acid positions relative to the start of the known putative sequence and sequence similarity scores generated by MatGAT 2.02 (http://bitincka.com/ledion/matgat/) compared to R. catesbeiana SF-1 are shown on the right.

Figure 3

Relative fold difference in CYP19 and SF-1 mRNA abundance in whole brain tissue of premetamorphic Rana catesbeiana tadpoles exposed for 48 h to 673 ng/L T₃, 50 ng/L EE₂, or isopropyl alcohol (IPA) vehicle alone. The results from two separate experiments are shown. The medians are shown as solid black lines within the box, and the box indicates the first and third quartiles. The whiskers indicate minimum and maximum values. Outlier (cases between 1.5 and 3.0 box lengths from the upper or lower edge of the box) and extreme values (cases >3.0 box lengths from the upper or lower edge of the box) are indicated by an open circle and asterisk, respectively. Significance is indicated by an ‘a’ (p= 0.007) relative to the IPA control.

Figure 4

Relative fold difference in CYP19 and SF-1 mRNA abundance in whole brain tissue of premetamorphic Rana catesbeiana tadpoles exposed for 48 h to isopropyl alcohol (IPA) vehicle alone or 1 μg/L and 100 μg/L of ATZ or DEA. A bevel indicates increasing concentrations of ATZ or DEA. The medians are shown as solid black lines within the box, and the box indicates the first and third quartiles. The whiskers indicate minimum and maximum values. Outlier (cases between 1.5 and 3.0 box lengths from the upper or lower edge of the box) and extreme values (cases >3.0 box lengths from the upper or lower edge of the box) are indicated by an open circle and asterisk, respectively.