Monitoring of Endocrine Disrupters in Transgenic Plants Carrying Aryl

Chapter 4

Monitoring of Endocrine Disruptors in Transgenic Plants Carrying Aryl Hydrocarbon Receptor and Estrogen Receptor Genes

Downloaded by COLUMBIA UNIV on September 7, 2012 | http://pubs.acs.org Publication Date: November 23, 2004 | doi: 10.1021/bk-2005-0892.ch004

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Hideyuki Inui , Hideaki Sasaki , Susumu Kodama , Nam-Hai Chua , and Hideo Ohkawa 1

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Kobe University, Rokkodai-cho 1-1, Nada-ku Kobe, Hyogo 657-8501, Japan Rockefeller University, 1230 York Avenue, New York, NY 10021

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Transgenic plants carrying mammalian hormone receptor genes were examined for monitoring of environmental chemicals including dioxins and endocrine disruptors. The transgenic tobacco plants expressing mouse arylhydrocarbon receptor gene detected 1ppb of the dioxin-like compound 20-methylcholanthrene in the cultured plants. In addition, the transgenic Arabidopsis plants expressing human estrogen receptor gene detected 1ppt of 17β-estradiol and ppb levels of the other endocrine disruptors such as bisphenol A, 4-t-octylphenol, and others in potted plants. These transgenic plants appear to be useful for on site monitoring of dioxins and estrogenic contaminants in the environment.

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© 2005 American Chemical Society In New Discoveries in Agrochemicals; Clark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

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Introduction Contamination of the environment and agricultural products with endocrine disruptors(EDs) including dioxins, industrial chemicals and certain pesticide residues is a serious problem. These chemicals were found in the environment and agricultural products at nano-level concentrations, and are suspected of affecting ecosystems and human health. The Ministry of the Environment of Japan surveyed contamination of EDs in rivers, showing that bisphenol A, 176-estradiol and nonylphenol were detected in 50.3%, 40.4% and 31.0% of the water samples, respectively(Table 1).

Table I. Contamination of environmental waters with endocrine disruptors in Japan Endocrine disruptor

a

Water sample* Detection point(%)

Maximum - RBA(%) conc.(ppb)

Bisphenol A

50.3

0.56

Πβ-Estradiol

40.4

0.0072

Nonylphenol

31.0

5.9

b

0.01 100 0.05

: Ministry of the Environment of Japan(Oct. 2002)(2)

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: The relative binding affinity(RBA) of each chemical was calculated as the ratio of concentrations of 17B-estradiol and chemical required to reduce the specific radioligand binding by 50%. RBA value for 17B-estradiol was arbitrarily set at 100(2).

Contamination level of these compounds was ppb to ppt levels. Thus, it is important to develop novel technologies to monitor contamination on site, since most conventional technologies are high cost, labor intensive and take a long time to obtain results. Particularly, development of novel technologies based on biological functions appears to be needed, because of the low risk potential for secondary contamination with chemicals. Certain plant species are grounding and have deep roots. These plants absorb and accumulate nano-level concentrations of EDs from a wide area through their root systems. On the other hand, it was found that certain

In New Discoveries in Agrochemicals; Clark, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.


42 biological systems containing an aryl hydrocarbon receptor(AhR) and an estrogen receptor(ER) specifically bind dioxins and estrogenic compounds at low concentrations, respectively. Binding of the complex of dioxinbound AhR and XhR nuclear translocator(Arnt) onto the xenobiotic responsive element(XRE) in a 5'-upstream region induces expression of CYP1A1 gene in mammals. Introduction of this system into plants seems to be possible for monitoring dioxins. It was also found that an estrogenic compound bound to ER induced expression of a number of genes in mammals. It was reported that the transgenic Arabidopsis plants with the recombinant transcription factors consisting of ligand binding domain of ER, LexA DNA binding domain(LexA) and virus VP16 transactivation domain(VP16) expressed a reporter gene by treatment with 170estradiol(E )(3). This system appears to be useful for monitoring estrogenic compounds. We attempted to produce genetically engineered plants carrying AhR and ER genes for monitoring nano-level concentrations of dioxins and estrogenic compounds on site, respectively. Mouse AhR and Arnt cDNAs were expressed in transgenic tobacco plants and examined for monitoring dioxin-like compounds. Also, the transgenic Arabidopsis plants carrying the LexA-VP16-ER recombinant gene were evaluated for monitoring estrogenic compounds. These transgenic plants have displayed no abnormal agronomic or physiological properties. 2

Transgenic tobacco plants carrying a mouse AhR gene The plasmid pSKAVAtG was constructed for dioxin-inducible expression system of GUS(B-glucuronidase) reporter gene and transformed into tobacco plants by Agrobacterium tumefaciensÇFigme V){4). The recombinant AhR bound to VP16 instead of transactivation domain of native AhR was constitutively expressed in transgenic tobacco plants(AhRV-GUS plant). The transcription factor bound dioxin may make a complex with Arnt in the nucleus and its heterodimer binds XRE upstream of GUS reporter gene, resulting in expression of GUS gene. After selection of transformants with kanamycin resistance, transgenic tobacco plants were incubated on a medium containing the dioxin-like compound 20-methylcholanthrene(MC) for two weeks. The transgenic plants showed MC-dependent inducible expression of GUS gene, suggesting that transactivation of VP16 had efficiently promoted GUS gene transcription. Dose-dependent expression of GUS gene was observed in AhRV-GUS plants treated with MC. The minimum concentration of


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MC enough to detect GUS activity was 5nM(approx. lppb) in the cultured plants(Figure 2). Histochemical staining of transgenic tobacco plants showed MC-dependent expression of GUS gene(Figure 3). These results indicated that transgenic tobacco plants absorbed MC through their roots and expressed GUS gene through AhR expression. Thus, the transgenic tobacco plants appear to be useful for monitoring dioxins in the environment.

Figure 1 The expression plasmid for arylhydrocarbon receptor-dependent inducible expression system for GUS reporter gene m35S-P, Τ 35S-P and NPTII represent minimal cauliflower mosaic virus(CaMV) 35S promoter, terminator, CaMV 35S promoter and kanamycin resistance gene, respectively. y

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AhRV-GUS plant

Figure 2 Dose-dependent GUS activity in the transgenic tobacco plants AhRV-GUS plant incubate in the culture mediad with MC


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DMSO treatment

MC treatment

Figure 3 Histochemical staining of GUS activity in transgenic tobacco plants AhRVGUS treated with MC

Transgenic Arabidopsis plants carrying a recombinant ER gene We constructed the recombinant transcription factor based on a ligand binding domain of ER, LexA and VP16. Two types of the expression plasmids pER8-GFP and pX6-GFP were constructed to express GFP(green fluorescence protein) reporter gene for the recombinant transcription factor LexA-VP16-ER in plants(Figure 4). Arabidopsis thaliana was transformed by Agrobacterium. The transgenic Arabidopsis plants transformed with pER8-GFP(XVE; LexA-VP16-ER) and pX6-GFP(CLX; Crc/loxP DNA excision system) show transient inducible expression and constitutive expression of GFP reporter gene, respectively. Namely, the Arabidopsis plants absorbed EDs through their roots, and then EDs bind the LexAVP16-ER transcription factor to change structural conformation. Then, the ED-transcription factor complex binds LexA operator region upstream of GFP gene, and induce transcription and translation of GFP gene


45 transiently in X V E plants(3). In case of CLX plants, ED-transcription factor complex binds upstream of Cre recombinase gene, and then Cre recombinase expressed truncates loxP sites located at downstream region of Gio-90 promoter and terminator for Cre recombinase. After truncation, Gio-9o promoter binds upstream of GFP gene, and GFP gene expresses constitutively(5). This system also brings us marker-free transgenic plants.


pER8-GFP(XVE plant)

Constitutive expression

Figure 4 Expression plasmids for estrogen receptor-dependent inducible expression system for GFP reporter gene Ρ, NPTII, 8xLexA-46 and Τ represent promoter, kanamycin resistance gene, 8 copies of LexA binding domain and terminator, respectively.

Dose-dependent transcription of GFP gene was observed by RT-PCR when O.OOOlppb to lOppb of E were treated. Minimum concentration of E detected was O.lppb and lppb of E in X V E and CLX plants, respectively, when plants were incubated for 7 days on a E -containing medium(data not shown)(6). Dose-dependent fluorescence of GFP was also observed(Figure 5). Furthermore, 11 of 19 chemicals tested induced transcription of GFP gene. Of these, estrone, atrazine and 4-i-oetylphenol were detected at lppb, O.lppm and O.lppm, respectively. These results revealed that X V E plants showed higher sensitivity than CLX plants, because transcription and translation of Cre recombinase gene are not required to induce GFP gene expression in XVE plants. Six EDs did not activate GFP gene transcription, suggesting that affinity of these EDs towards ER or uptake efficiency of these EDs into plants are low. Re labeled E and nonylphenol were taken into apical part of plants within 48 hours(Figure 6). Moreover, XVE and CLX plants were found to detect O.OOlppb and O.lppb of E in the potted Arabidopsis plants. 2

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2

2

2


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1E

0.1 E

D

100E

10E


pj?4/•/•!• IB/ Figure 5 Dose-dependent expression of GFP gene in transgenic Arabidopsis plants treated with 17fi-estradiol Right pictures of each column were taken by fluorescence microscopy. D, 0JE~100E represent treatment of 0.1% dimethyl sulfoxide, 0.1ppb~100ppb of Πβ-estradiol, respectively.

2H 1 R

E

4H 2

1 R

24H 2 R

1 R

48H 2

R

2_

_ ! R

R

R

2

NP

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Figure 6 Absorption of C-labeled llfi-estradiol and nonylphenol in transgenic Arabidopsis plants E and NP represent treatment of Πβ-estradiol and nonylphenol, respectively. R represent autoradiogram of plants treated with C-labeled compounds. 2

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Concluding remarks The transgenic plants expressing AhR and ER detected lppb of M G under cultured conditions and lppt of E under potted conditions, respectively. Basic technology was established for monitoring dioxins and EDs in the transgenic plants with the receptor genes. However, increase of the sensitivity towards other EDs with low affinity towards receptors is necessary. In order to overcome this problem, (l)sensitive receptors may be useful instead of mouse AhR and human ER. It is known that fish ERs were more sensitive to alkylphenols such as nonylphenol and octylphenol than mammalian ones. (2)Increase of uptake efficiency of dioxins and EDs is another way to increase sensitivity. Zucchini plants are known to have efficient translocation of polychlorinated dibenzo-p-dioxins and dibenzofurans(7). Furthermore, (3)genes synthesizing flower colors as reporter genes may be useful for visible monitoring of contaminations on site.


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References 1. Ministry of the Environment of Japan, (2002) 2. Kuiper, G. G. J. M.; Lemmen, J. G.; Carlsson, B.; Corton, J. C.; Safe, S. H.; van der Saag, P. T.; van der Burg, B.; Gustafsson, J-A.; Endocrinology, 1998, 139(10), 4252-4263 3. Zuo, J.; Niu, Q-W; Chua, N-H.; The Plant Journal, 2000, 24(2), 265-273 4. Kodama, S.; Okada, K.; Akimoto, K.; Inui, H.; Ohkawa, H.; 2003, submitted 5. Zuo, J.; Niu, Q-W; Moller, S. G.; Chua, N-H.; Nature Biotechnology, 2001, 19, 157-161 6. Inui, H.; Sasaki, H.; Chua, N-H.; Ohkawa, H.; 2003, submitted 7. Hülster, Α.; Müller, J. F.; Marschner, H.; Environmental Science and Technology, 1994, 28, 1110-1115


Monitoring of Endocrine Disrupters in Transgenic Plants Carrying Aryl

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