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)

Gunderson, Mark P.; Veldhoen, Nik; Skirrow, Rachel C.; Macnab, Magnus K.; Ding, Wei; Aggelen, Graham van; Helbing, Caren C.

doi:10.1016/j.aquatox.2010.12.019

Cited by 30 publications

(15 citation statements)

References 79 publications

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“…AZ has been reported to cause endocrine disruption in mammals, birds, reptiles, fish and amphibians by affecting normal reproductive function and development in these organisms (Holloway et al, 2008;Gunderson et al, 2011;Kloas et al, 2009;de la Casa-Resino, 2012;Rohr and McCoy, 2010).…”

Section: Introductionmentioning

confidence: 99%

Gene expression profiles in testis of developing male Xenopus laevis damaged by chronic exposure of atrazine

Sai¹,

Dong

Li³

et al. 2016

Chemosphere

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As a widely used herbicide, atrazine (AZ) has been extensively studied for its adverse effects on the reproductive system, especially feminization in male animals. However, the relationship of gene expression changes and associated toxicological endpoints remains unclear. In this study, developing Xenopus laevis tadpoles were exposed to concentration of AZ at 0.1, 1, 10 or 100 μg/L continuously. Compared with froglets in the control group, there were no significant differences in body length, body weight, liver weight and hepatosomatic index (HSI) of males in groups treated with AZ for 90 d. At 100 μg/L AZ treatment caused a significant reduction of gonad weight and gonadosomatic index (GSI) of males (p < 0.01). In addition, AZ at all dose levels caused testicular degeneration, especially in froglets from the groups with 0.1 and 100 μg/L which exhibited U-shaped dose-response trend. We further investigated the gene expression changes associated with the testicular degeneration induced by AZ. We found that the expression of 1165 genes was significantly altered with 616 upregulated and 549 downregulated compared to the expression profile of the control animals. KEGG analysis showed that genes which were significantly affected by AZ are mainly involved in arginine and proline metabolism, cell cycle, riboflavin metabolism, spliceosome, base excision repair and progesterone-mediated oocyte maturation pathway. Our results show that AZ may affect reproductive and immune systems by interference with the related gene expression changes during the male X. laevis development. The findings may help to clarify the feminization mechanisms of AZ in male X. laevis.

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Section: Introductionmentioning

confidence: 99%

Gene expression profiles in testis of developing male Xenopus laevis damaged by chronic exposure of atrazine

Sai¹,

Dong

Li³

et al. 2016

Chemosphere

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“…Thus, rpl8 is used as the reference gene for analyzing gene expression in X. laevis research. So far, however, the expression stability of rpl8 has not been validated in Rana, although rpl8 has been used as the reference gene for analyzing gene expression in some species of the Rana genus including R. nigromaculata (Gunderson et al, 2011;Hogan et al, 2007). For example, Zhang et al (2013) used rpl8 to normalize mRNA expression of sex-related genes to investigate toxic effects of microcystin-LR on R. nigromaculata using qRT-PCR, without validation of the suitability of rpl8 as the reference gene.…”

Section: Discussionmentioning

confidence: 99%

“…Quantitative analysis of gene expression using quantitative reverse transcription polymerase chain reaction (qRT-PCR) typically requires the use of a constitutively expressed reference gene, which is adequately expressed in certain tissues with minimal variability in expression among samples under the experimental conditions used, as an internal control to normalize for differences in starting the cDNA template among samples (Arukwe, 2006;Huggett et al, 2005;Morse et al, 2005). Ribosomal protein L8 (rpl8) is a frequently used reference gene for normalizing gene expression in amphibians using qRT-PCR in previous studies (Gunderson et al, 2011;Hogan et al, 2007). However, nucleotide sequences of rpl8 were characterized in only three species of the Rana genus, i.e., R. catesbeiana, Rana clamitans, and Rana sylvatica, in NCBI (www.ncbi.nlm.nih.gov).…”

Section: Introductionmentioning

confidence: 99%

Molecular characterization and mRNA expression of ribosomal protein L8 in Rana nigromaculata during development and under exposure to hormones

Lou

Cao

et al. 2014

Journal of Environmental Sciences

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Like Xenopus laevis, some species of the Rana genus are also used to study endocrine disrupting chemicals (EDCs). Although ribosomal protein L8 (rpl8) is the most-used reference gene for analyzing gene expression by quantitative reverse transcription polymerase chain reaction in Rana, its suitability as the reference gene has never been validated in any species of the Rana genus. We characterized rpl8 cDNA in Rana nigromaculata, a promising native species in East Asia for assaying endocrine disrupting effects. We found that the rpl8 cDNA consisted of 919 bp and encoded 257 amino acids, exhibiting high identities of amino acid sequence with known rpl8 in other Rana species. Then, we examined the stability of mRNA expression during development. Compared with elongation factor 1 alpha 1, another common housekeeping gene, neither stage-specific nor tissue-specific expression of the rpl8 gene was found in all tissues examined (brain, liver, intestine, tail, testis and ovary) during R. nigromaculata development. Finally, we investigated rpl8 expression under exposure to hormones. No change in rpl8 mRNA expression was found under exposure to thyroid hormone (T4) and estrogen (estradiol), whereas expression of the corresponding biomarker genes was induced.Our results show that rpl8 is an appropriate reference gene for analyzing gene expression by quantitative reverse transcription polymerase chain reaction for assaying EDCs using R. nigromaculata, and might also provide support for using rpl8 as a reference gene in other Rana species due to the high conservation of rpl8 among the Rana genus.

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“…Total cDNA was prepared from 1 μg total RNA using the High-Capacity cDNA Reverse Transcription Kit (Life Technologies) as per the manufacturer’s recommended protocol and diluted 20-fold prior to qPCR analysis. Gene-specific amplification was achieved using cyp19a1, nr5a1, and ribosomal protein L8 ( rpl8 ) qPCR primers designed and validated for R. catesbeiana (Table 1) (Gunderson et al, 2011). Reactions were assembled and run on a MX3005P Real-Time PCR System (Agilent Technologies Canada Inc, Mississauga, ON, Canada) as described previously (Gunderson et al, 2011).…”

Section: Methodsmentioning

confidence: 99%

Estrogenic environmental contaminants alter the mRNA abundance profiles of genes involved in gonadal differentiation of the American bullfrog

Wolff

Veldhoen

Helbing

et al. 2015

Science of The Total Environment

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Wildlife and human populations are exposed to anthropogenic mixtures of chemicals in the environment that may adversely influence normal reproductive function and development. We determined the effects of exposure to estrogenic chemicals and wastewater effluent (WWE) on developing gonads of the American bullfrog, Rana (Lithobates) catesbeiana, a species whose widespread distribution make it an ideal model for environmental monitoring for endocrine effects of chemical contaminants. Premetamorphic bullfrog tadpoles were exposed to treatment vehicle, 17β-estradiol (E2; 10−9 M) or 4-tert-octylphenol (OP; 10−9 M, 10−8 M, and 10−7 M). Additionally, gonadal differentiation was evaluated in bullfrog tadpoles from a WWE-containing site versus those from a reference location receiving no WWE. In both studies, phenotypic sex, steroidogenic factor-1 (nr5a1), and aromatase (cyp19a1) mRNA levels using quantitative real-time PCR were determined. Exposure to E2 or OP did not alter sex ratios. In controls, both nr5a1 and cyp19a1 transcript levels exhibited sexual dimorphism, with males demonstrating higher levels of nr5a1 and females greater abundance of cyp19a1. However, E2 exposure increased cyp19a1 mRNA abundance in testes and decreased levels in ovaries, eliminating the sexual dimorphism observed in controls. E2-exposed males exhibited increased nr5a1 transcript levels in the testes compared to controls, while females demonstrated no E2 effect. OP treatment had no effect on female cyp19a1 mRNA abundance, but exposure to 10−7 M OP increased testicular transcript levels. Treatment with 10−9 and 10−8 M OP, but not 10−7 M, resulted in decreased abundance of nr5a1 transcript in both ovaries and testes. Animals from the field had sexually dimorphic gonadal levels of cyp19a1, but both sexes from the WWE site exhibited elevated cyp19a1 transcript abundance compared to the reference location. Individual chemical compounds and anthropogenic wastewater effluent dispersed within the environment influence the levels of gonadal mRNA encoding key proteins involved in gonadal differentiation.

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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)

Cited by 30 publications

References 79 publications

Gene expression profiles in testis of developing male Xenopus laevis damaged by chronic exposure of atrazine

Gene expression profiles in testis of developing male Xenopus laevis damaged by chronic exposure of atrazine

Molecular characterization and mRNA expression of ribosomal protein L8 in Rana nigromaculata during development and under exposure to hormones

Estrogenic environmental contaminants alter the mRNA abundance profiles of genes involved in gonadal differentiation of the American bullfrog

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