transformation of natural and synthetic estrogens by maize seedlings

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Article

Transformation of Natural and Synthetic Estrogens by Maize SeedlingsMarcella L. Card, Jerald L. Schnoor, and Yu-Ping Chin

Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es3040335 • Publication Date (Web): 14 Mar 2013

Downloaded from http://pubs.acs.org on March 18, 2013

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Transformation of Natural and Synthetic Estrogens 1

by Maize Seedlings 2

Marcella L. Card*, Jerald L. Schnoor, and Yu-Ping Chin 3

Environmental Science Graduate Program, The Ohio State University, 125 South Oval Mall, 4

Columbus, OH 43210; Dept of Civil and Environmental Engineering, College of Engineering, 5

University of Iowa, 4119 Seamans Center, Iowa City, IA 52242; School of Earth Sciences, The 6

Ohio State University, 125 South Oval Mall, Columbus, OH 43210 7

* Corresponding author e-mail: [email protected]; phone: +61 426 194 558; 8

Current address: National Research Centre for Environmental Toxicology, 39 Kessels Road, 9

Coopers Plains QLD 4108 Australia 10

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Abstract 12

In agricultural fields, crop plants may transform or degrade hormonally-active compounds in 13

manure used as fertilizer and thereby affect the overall endocrine disrupting activity of 14

agricultural runoff. This study examined the transformation of two natural steroid estrogens 15

(17β-estradiol [17β-E2] and estrone [E1]) and two synthetic estrogen mimics (zeranol [α-ZAL] 16

and zearalanone [ZAN]) by maize seedlings. Growing whole maize seedlings in hydroponic 17

solutions of target estrogens resulted in both oxidative (i.e., 17β-E2 to E1 and α-ZAL to ZAN) 18

and reductive transformations (i.e., E1 to 17β-E2 and ZAN to α-ZAL). Although all four 19

estrogens accumulated in maize roots both as parents and products, the shoots contained only 20

17β-E2 and α-ZAL regardless of whether they were the parent or the product. Crude plant 21

enzyme extracts led to substantial reductive transformations, but created only trace amounts of 22

oxidation products. In contrast, only oxidative transformations occurred in solutions exposed to 23

plant-associated microbes. Thus, the combined effects of plant enzymes and plant-associated 24

microbes account for the reversible transformations observed with whole plants. These effects 25

are expected to generally decrease the overall estrogenicity of runoff from manure-fertilized 26

fields. 27

Introduction 28

Livestock manure contains a myriad of hormones and pharmaceuticals, which may enter the 29

environment via runoff from manure fertilized crop fields. These contaminants are frequently 30

detected in surface waters impacted by agriculture (1) and may have deleterious effects on 31

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exposed wildlife and humans. Hormonally active contaminants can impact reproduction, 32

growth, and development, or increase cancer risks at nanomolar concentrations (2-5). 33

The hormones in livestock manure may include both endogenous hormones and synthetic 34

mimics. Endogenous hormones, which are produced and excreted by all mammals, include 35

estrogens such as 17β-estradiol (17β-E2) and estrone (E1). The synthetic mimics of those 36

estrogens are α-zearalanol (commonly known as zeranol or α-ZAL) and zearalanone (ZAN) (Fig. 37

1). Both 17β-E2 and α-ZAL are approved for use in the United States as hormonal growth 38

promoters (6), which are administered to livestock to increase the muscle mass per unit of feed. 39

Soil microbes and crop or buffer plants may transform or degrade estrogens, which can affect 40

the overall estrogenicity of the runoff. Reversible transformation between 17β-E2 and E1 by 41

algae, aquatic macrophytes, and terrestrial plants has previously been reported (7- 9). However, 42

crops are the first plants with which hormones in manure fertilizer interact, and are exposed to 43

the hormones before dilution by overland runoff. To develop a more complete understanding of 44

the environmental fate of manure borne estrogens, this study evaluated the temporal 45

transformation of 17β-E2, E1, α-ZAL, and ZAN by whole maize seedlings, enzyme extracts of 46

maize roots and shoots, and plant-associated microbes. Finally, this investigation combined all 47

of the uptake and transformation data into mass balances to trace the temporal distribution of 48

parent and product estrogens. 49

Experimental Methods and Materials 50

Chemicals. 17β-E2 (98%), E1 (99%), zearalanone (98%), and N,O-51

Bis(trimethylsilyl)trifluoro-acetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) were 52

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purchased from Sigma-Aldrich, Inc. (St. Louis, MO). α-ZAL was extracted from Ralgro 53

Magnum (Schering-Plough Animal Health Corp., Union, NJ) (10). Solvents, inorganic nutrients 54

for hydroponic solutions, and other chemicals were purchased from Fisher Scientific (Pittsburgh, 55

PA). 56

Hydroponic uptake. Uptake of 17β-E2, E1, α-ZAL, and ZAN by maize seedlings (Zea 57

mays, Golden Cross Bantam [Hybrid]; Ferry-Morse Seed Co, Fulton, KY) from hydroponic 58

solutions was evaluated as described previously (10 and refer to supporting information [SI]). 59

Briefly, 9-mL glass vials were filled with 2 µM solutions of 17β-E2, E1, α-ZAL, or ZAN in ½-60

strength Hoagland nutrient solution (pH 6.8) (11) and maize roots were submerged in the 61

solution (refer to the abstract art). Seedlings were grown in Hoagland solution without spiked 62

estrogens as blank controls while glassware controls were estrogen solutions without seedlings. 63

At the beginning of each experiment, seeds, solutions, and glassware were sterilized as described 64

previously (10). At given time intervals, four replicates of each treatment including controls 65

were destructively sampled. The aqueous phase, root, and shoot tissues were collected, 66

processed, and analyzed for estrogen content as described in the SI. 67

Transformation in tissue enzyme extracts. Enzymatic transformation in plants was tested 68

using crude enzyme extracts from plants previously unexposed to 17β-E2, E1, α-ZAL, or ZAN. 69

Briefly, crude enzyme extracts of maize seedling root, seed, and shoot tissues were prepared in 70

50 mM potassium phosphate buffer (pH 7.0; approximately 0.2 g fresh weight [fw] root tissue or 71

0.5 g fw leaf or seed tissue per 1 mL). Solutions of 2 µM 17β-E2, E1, α-ZAL, or ZAN were 72

prepared in 50 mM potassium phosphate buffer (pH 7.0) and equilibrated to 25 °C in a water 73

bath. After the addition of crude enzyme extract to an estrogen solution, the reaction solution 74

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was incubated and finally quenched by the addition of glacial acetic acid. Estrogen 75

concentrations in the resulting solutions were quantified using reverse-phase high-pressure liquid 76

chromatography (RP-HPLC). Additional details are available in the SI. 77

Transformation in microbe-inoculated solutions. The transformation of estrogens by 78

plant-associated microbes was tested in solutions that had been exposed to corn seedlings. Corn 79

seedlings were grown in ½ strength Hoagland solution, and after 18 d the aqueous phases were 80

collected, combined, and diluted in fresh ½ strength Hoagland solution at a 1:1 ratio. From this 81

stock, 2 µM solutions of 17β-E2, E1, α-ZAL, or ZAN were made. Solutions were transferred 82

into vials and, at given times, collected and analyzed by RP-HPLC and gas chromatography 83

coupled with mass spectrometry (GC-MS) as described for maize-exposed samples. 84

RP-HPLC analysis of estrogen concentrations. Estrogen concentrations were quantified 85

using a Waters RP-HPLC (1515 isocratic pump and 717plus autosampler; Milford, MA) with a 86

Waters Sunfire C18 column and UV-Vis detection (Waters 2487 dual λ absorbance detector). 87

Injection volumes were 150 µL and the mobile phase was 50:50 v/v acetonitrile:water with 1 mL 88

min-1

flow rate. Detection wavelengths are listed in Table SI-1, and under these conditions the 89

limit of quantification was 1 nM. 90

Transformation product identification by GC-MS. Transformation products were 91

qualitatively identified using GC-MS (12, 13). Briefly, replicates of maize-exposed and 92

microbe-inoculated treatment and control were prepared as described above and analytes from 93

the filtered solutions were concentrated onto Oasis HLB Plus solid phase extraction packs 94

(Waters Corp.). Solid phase extraction pack eluents were dissolved in hexane and the solutions 95

derivatized with trimethylsilane (TMS; added as 50 µL BSTFA [1% TMCS]). Derivatized 96

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solutions were then analyzed by GC-MS (HP 6890 series GC system and 5973 mass selective 97

detector; Agilent Technologies) with a 15 m Restek RTX-5MS column (Restek Corporation, 98

Bellefonte, PA). The MS was set to scan mode (m/z 50 to 650) or select ion mode (SIM) for the 99

parent ion of each estrogen and m/z 73 (TMS mass). Further details are available in the SI. 100

Results and Discussion 101

Transformation of maize-exposed estrogens. Transformation products were identified in 102

all hydroponic solutions following exposure to maize seedlings. The GC chromatograms and 103

mass spectra of plant-exposed and glassware control samples demonstrate that exposure to maize 104

resulted in both oxidation (i.e., 17β-E2 to E1 and α-ZAL to ZAN) and reduction transformations 105

(i.e., E1 to 17β-E2 and ZAN to α-ZAL) (Fig. SI-1, Fig. SI-2). No estrogens were detected in the 106

solutions or tissues from blank controls. 107

The rate at which maize seedlings removed parent estrogens from solution and formed 108

products varied considerably (Fig. 2a,b; Table SI-2). The concentrations at which transformation 109

products were produced also varied among parent estrogens, but transformation product 110

concentrations increased rapidly in all solutions for the first 5 d of exposure to maize seedlings 111

(Fig. 2). After 5 d of exposure, the concentrations of 17β-E2, E1, and α-ZAL as products 112

decreased to levels that were nearly undetectable. In α-ZAL solutions, however, the ZAN 113

transformation product continued to accumulate and did not decrease until after 11 days. The 114

accumulation of ZAN in α-ZAL solutions can be attributed to slow uptake (10). 115

Both the alcohols (17β-E2 and α-ZAL) and the ketones (E1 and ZAN) were measured in root 116

tissue extracts, but the alcohols were present in much higher concentrations (Fig. 3; Table SI-3). 117

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In contrast, the only estrogens detected in the shoot tissues were the reduced forms, regardless of 118

whether they were the parent or product. Thus, either the oxidized forms are prevented from 119

being translocated into the shoots, or only the reduction reactions occur in the shoots and/or 120

roots. 121

The reversible transformation between 17β-E2 and E1 has previously been observed in algae 122

(7), aquatic plants (8), and terrestrial plants (9) but this paper is the first to report the reversible 123

transformation between α-ZAL and ZAN by plants. The reversible transformation between α-124

ZAL and ZAN is clearly demonstrated in the maize-exposed ZAN solutions. In those samples, 125

the major transformation product (α-ZAL) concentrations peaked after 5 d of exposure, and then 126

rapidly decreased. A spike in ZAN concentrations occurred concurrently to the decrease in α-127

ZAL concentrations, which is attributed to the re-oxidation of α-ZAL to ZAN (Figure 2) while 128

the remaining α-ZAL is lost to further transformation products. 129

The oxidation of 17β-E2 and reduction of E1 in mammals have both been attributed to one 130

class of oxidoreductases, 17β-hydroxysteroid dehydrogenases (17β-HSD; e.g., International 131

Union of Biochemistry and Molecular Biology enzyme class 1.1.1) (14). These enzymes have 132

also been found in plants, and it has been reported that a maize 17β-HSD can reduce the 17β-133

hydroxyl moiety on testosterone, although no activity towards 17β-E2 was detected (15). 134

The estrogenic activities of α-ZAL and ZAN are significantly lower than that of 17β-E2 135

because α-ZAL and ZAN conformations must change slightly in order to bind to estrogen 136

receptors, which decreases their apparent affinity (16). Similarly, the oxidoreductase(s) that 137

transform 17β-E2 and E1 also likely interact with α-ZAL and ZAN in the correct conformations. 138

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Therefore α-ZAL and ZAN showed slower transformation relative to 17β-E2 and E1 due to α-139

ZAL and ZAN having a lower affinity to the oxidoreductase. 140

Mass balances. The initial aqueous concentration of estrogens used in this study (2 µM) 141

was orders of magnitude greater than might be expected in manure-fertilized fields, but the 142

estrogen concentrations in crop fields are likely patchy depending on localized conditions (e.g., 143

manure application and rainfall infiltration rates). Despite this high initial concentration, 144

however, the estrogens were rapidly transformed, translocated, and in some cases degraded to 145

nondetectable levels. We combined the aqueous, root, and shoot concentrations of parent and 146

product estrogens for each of 17β-E2, E1, α-ZAL, and ZAN to generate mass balances and 147

assess the distribution of estrogens at specific time points within the treatment period (Fig. 4, 148

Fig. SI-3; Table SI-4). The areas of the pie charts in Fig. 4 and Fig. SI-3 correspond to the total 149

mass of measured estrogens remaining in the systems at the time (smaller areas denote less mass 150

over time). Decreasing mass (smaller pie charts) of measured estrogens is attributed to other 151

processes such as transformation to products that were not identified or measured, formation of 152

conjugation products which were not measured, irreversible sorption to plant tissues, and 153

mineralization of estrogens. 154

Concentrations of the product in E1 solutions were significantly lower than product 155

concentrations in 17β-E2 solutions between 1 and 6 d of exposure (Student’s t test, df=6, 2.75 < t 156

< 11.2 for these time points, p < 0.05), after which period the product concentrations approached 157

the limit of quantification. Despite these differences, the total mass of measured estrogens 158

remaining in 17β-E2 and E1 solutions were statistically equal at each sampling time (Student’s t 159

test, df=6, t < 1.45 for all time points, p > 0.1; Fig. 4). Total α-ZAL and ZAN mass balances also 160

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changed at similar rates (Fig. SI-3), but decreased much more slowly than 17β-E2 and E1. 161

Further, the distribution of α-ZAL and ZAN parent and product was very similar throughout the 162

experiment, with the largest difference being accumulation of α-ZAL in the root tissues in α-163

ZAL samples. 164

Despite this rapid and complete uptake, estrogens and other hormones are frequently detected 165

in agriculturally impacted surface water. We attribute this to the relatively small volume of soil 166

inhabited by the seedlings and the large patches of soil between rows of maize where surface 167

runoff can move unimpeded. Considering the observed rapid uptake rates and transformations, 168

planted buffer strips should decrease the estrogen concentrations in runoff (22). In densely-169

planted buffer strips, the roots inhabit a larger volume of soil, slowing water movement and 170

allowing for longer interactions between the roots and chemicals contained in the runoff. 171

Transformation products in tissue enzyme extracts. The transformation of estrogens was 172

assessed in enzyme extracts from maize roots, seeds, or leaves (Fig. 5; Table SI-5). As 173

compared to the oxidized estrogens (E1 or ZAN), concentrations of the reduced forms of each 174

pair (17β-E2 or α-ZAL) decreased rapidly in all three enzyme extracts, in many cases reaching 175

undetectable levels 30 min. The oxidation products (E1 or ZAN) were detected at small 176

concentrations in solutions of all three extracts, but not in the solutions of leaf extracts until 30 177

min into the exposure (Fig. 5c). 178

The oxidized forms were removed much more slowly, with 78% or more remaining in 179

solution after 30 min of incubation with the enzyme extracts. Reduction products appeared in 180

the solutions exposed to seed or leaf enzymes, with the products accumulating in the leaf enzyme 181

solutions (Fig. 5b,c). The concentrations of 17β-E2 or α-ZAL as products in E1 or ZAN 182

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solutions exposed to leaf enzymes reach concentrations between one and three orders of 183

magnitude greater than the product concentrations in any other samples. In contrast, E1 or ZAN 184

were not detected in initial 17β-E2 or α-ZAL solutions incubated with leaf enzyme extracts. 185

Considering these results, it is postulated that only the reduced forms were detected in the 186

shoots of whole plants because E1 or ZAN translocated into the leaves may be enzymatically 187

reduced into 17β-E2 or α-ZAL, whereas 17β-E2 or α-ZAL translocated into the leaves will not 188

be oxidized. However, these data do not explain why products of both oxidation and reduction 189

were present in the maize-exposed hydroponic solutions. It had been hypothesized that some 190

tissues could perform both oxidative and reductive transformations, but oxidative 191

transformations occurred only to a small degree in the solutions exposed to any of the enzyme 192

extracts. Microbes in maize-exposed solutions may perform the observed oxidations, or the 193

transformations observed in the crude enzyme extracts may not be representative of those that 194

occur in whole plants (17). 195

Transformation products formed in microbe-inoculated solutions. The observed 196

degradation and transformation of maize-exposed 17β-E2, E1, α-ZAL, and ZAN may also be 197

attributed to metabolism by plant-associated microbes. All equipment and experimental 198

components were carefully sterilized, and seeds were surface-sterilized, so microbes present in 199

maize-exposed solutions were likely endophytic or otherwise closely associated with the plants. 200

Concentrations of 17β-E2 and α-ZAL decreased more quickly than E1 and ZAN in microbe-201

inoculated solutions, but all four target estrogens decreased to undetectable concentrations within 202

15 d of exposure (Fig. 2c, Table SI-6). This is in contrast to degradation by plant tissue enzyme 203

extracts, where the majority of the oxidized forms remained in solution. The target estrogens all 204

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demonstrate similar sorption to roots (10), but the reduced forms are removed by microbial and 205

plant enzymatic processes more quickly than the oxidized forms. Indeed, 17β-E2 and α-ZAL 206

were removed slightly more quickly than for E1 and ZAN in solutions exposed to whole maize 207

seedlings. 208

Although mammals can reduce E1 to 17β-E2, microbial processes can perform this 209

transformation only under strongly reducing (methanogenic and nitrate-, sulfate-, and iron-210

reducing) conditions (18, 19). Based on pathways known to occur in aerobic or slightly 211

anaerobic conditions, microbial oxidation of 17β-E2 to E1 and α-ZAL to ZAN can be predicted 212

a priori while microbial reduction of E1 to 17β-E2 and ZAN to α-ZAL cannot (20). Aerobic 213

conditions were maintained throughout the sampling period (Fig. SI-4, Table SI-7), and the 214

products of oxidation processes (i.e., E1 in 17β-E2 solutions and ZAN in α-ZAL solutions) were 215

detected as expected in microbe-inoculated samples, using RP-HPLC and GC-MS (Fig. 2c, Fig. 216

SI-1c, Fig. SI-2c). These transformations may be mediated by microbial oxidoreductases (e.g., 217

21). 218

Trace amounts of 17β-E2 or α-ZAL (at the limits of detection) were also detected in 219

microbe-inoculated E1 or ZAN solutions using GC-MS (Fig. 2c, Fig. SI-1c, Fig. SI-2c), but were 220

not detectable in the RP-HPLC chromatograms and therefore not quantifiable. The source of 221

these compounds remains a mystery as microbial production of alcohols from ketones was not 222

expected for aerobic solutions and no 17β-E2 or α-ZAL impurities were detected in stock 223

solutions of E1 or ZAN. Because the microbial inoculations were made from plant-exposed 224

solutions, exudates and root cells shed into solution may play a role in the observed reductions. 225

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This study derived no other hypotheses to explain with any greater plausibility the mechanism by 226

which E1 or ZAN may be reduced in aerobic microbial cultures. 227

In all cases, though, the parent estrogen and transformation product concentrations quickly 228

decreased to undetectable concentrations in microbe-inoculated solutions. Thus the microbes, 229

like the maize seedlings, were ultimately able to degrade or mineralize the estrogens into 230

products that were not measured in the present study. Further, the pattern of product formation is 231

quite similar between maize-exposed and microbe-inoculated 17β-E2 and α-ZAL solutions. 232

However, the rates of removal by microbe-inoculated and maize-exposed solutions cannot be 233

directly compared because the concentrations and compositions of microbial communities may 234

have differed between treatments in the presence and absence of maize seedlings. 235

The microbes did not reductively transform estrogens, and in this way maize metabolism 236

more closely resembles that of other eukaryotic organisms (e.g., mammals). Solutions incubated 237

with tissue enzyme extracts primarily underwent reductive transformations whereas the microbes 238

primarily performed oxidative transformations; thus the two processes cumulatively accounted 239

for the transformations observed with whole plants. 240

The extent to which these results may be extrapolated to crops and buffers is yet to be 241

established. The plants were grown without soil, limiting the rhizosphere to a layer of epiphytes. 242

Further, the nutrient-rich hydroponic solution, selected to ensure plant survival through the 243

period of the study, may support microbial communities that are not commonly found in soils. 244

Effects on overall estrogenicity. The plant- and microbe-mediated transformations of 245

hormones as observed in this study may affect the estrogenicity of runoff. For instance, 17β-E2 246

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is a significantly more potent estrogen than E1. In the environment where uptake of estrogens by 247

plants is incomplete, plant transformation of E1 may contribute to the 17β-E2 load and therefore 248

overall estrogenicity in runoff. Microbial oxidation of 17β-E2, on the other hand, will decrease 249

the estrogenicity of runoff. Similarly, ZAN is a significantly less potent estrogen than α-ZAL 250

(23). However, the observed reduction of ZAN to α-ZAL does not necessarily indicate a 251

significant increase in estrogenic activity. Mammalian metabolism reduces ZAN to a racemic 252

mixture of α-ZAL and β-ZAL, where β-ZAL is only slightly more estrogenic than ZAN 253

(approximately 19% the estrogenicity of α-ZAL) (24). The present study did not separate α-ZAL 254

and β-ZAL, the transformation product observed in maize-exposed ZAN solutions is likely a 255

racemic mixture with lower overall estrogenicity than that of α-ZAL alone. As a product, the 256

racemic mixture of α- and β-ZAL is only four times as estrogenic as ZAN, as compared to α-257

ZAL, which is more than seven times as estrogenically active as ZAN (24). 258

These results demonstrate that plants and associated microbes are able to efficiently take up 259

and transform 17β-E2, E1, α-ZAL, and ZAN. These effects are expected to generally decrease 260

the overall estrogenicity of runoff from manure-fertilized fields, and indicate that plants may be 261

used to effectively inhibit the movement of hormones from agricultural fields into surface 262

waters. 263

The removal of both oxidized (E1 and ZAN) and reduced (17β-E2 and α-ZAL) estrogens 264

from solution and reversible oxidation and reduction of estrogens in plant-exposed systems is a 265

result of both plant and microbial activity. The interactions between formation of products by 266

one organism and degradation of that product by another (e.g., reduction of E1 to 17β-E2 by 267

plant enzymes followed by degradation of 17β-E2 by plant and microbial enzymes) contribute to 268

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rapid transformation of the estrogens. Although separating the effects of plants and plant-269

associated microbes, especially because endophytic microbes can significantly contribute to 270

plant-mediated degradation of contaminants (25), this interaction warrants further study. 271

Supporting information available 272

Mass spectra of transformation products, aqueous and tissue estrogen concentrations, mass 273

balances for maize-exposed estrogen solutions, estrogen degradation data for enzyme extracts 274

and plant-associated microbes, and hydroponic solution water chemistry data. This information 275

is available free of charge on the internet at http://pubs.acs.org. 276

Acknowledgements 277

This work was funded by grants from the National Science Foundation (CBET-0965863 and 278

CBET- 0966683) and a National Science Foundation Graduate Research Fellowship awarded to 279

M. L. Card. This is also a contribution from the W. M. Keck Phytotechnologies Laboratory at 280

the University of Iowa. 281

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TOC/Abstract Art

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Figure 1. Estrogens and estrogen mimics used in this study.

OH

OH

OH

O

O

OOH

OH

OH

O

OOH

OH

O

17ββββ-E2

E1 α-ZAL ZAN

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Figure 2. Aqueous concentrations of parent and product estrogens. Shaded symbols are maize-

exposed and microbe-inoculated samples and open symbols are glassware controls of 17β-E2 (

, ), E1 ( , ), α-ZAL ( , ) and ZAN ( , ). Error bars show standard deviation (SD),

n=4.

a. Maize-exposed

b. Transformation product

in maize-exposed solutions

(note different vertical axes)

c. Microbe-inoculated

Aq

ueo

us

con

cen

tra

tio

n (

µM

)

17β-E2 solutions

E1 solutions

α-ZAL solutions

ZAN solutions

Time (d)

0

0.5

1

1.5

2

0 5 10 15 20

0

0.1

0.2

0.3

0 5 10 15 20

0

0.5

1

1.5

2

0 5 10 15 20

0

0.5

1

1.5

2

0 5 10 15 20-0.03

0

0.03

0.06

0.09

0 5 10 15 200

0.5

1

1.5

2

0 5 10 15 20

-0.5

0

0.5

1

1.5

2

0 5 10 15 20

-0.2

0

0.2

0.4

0.6

0 5 10 15 200

0.5

1

1.5

2

0 5 10 15 20

0

0.5

1

1.5

2

0 5 10 15 20

0

0.1

0.2

0.3

0.4

0 5 10 15 20

0

0.5

1

1.5

2

0 5 10 15 20

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Figure 3. Concentrations of 17β-E2 ( ), E1 ( ), α-ZAL ( ), and ZAN ( ) in root and shoot

tissues of exposed maize seedlings. Error bars indicate SD, n=4.

a. Root tissue b. Shoot tissue

Co

nce

ntr

ati

on

in

tis

sue

(µm

ol

g-1

wet

wei

gh

t)

17β-E2 solution

E1 solution

α-ZAL solution

ZAN solution

Time (d)

0

0.1

0.2

0 5 10 15 20

0

0.01

0.02

0.03

0 5 10 15 20

0

0.05

0.1

0.15

0 5 10 15 20

0

0.0005

0.001

0 5 10 15 20

0

0.002

0.004

0 5 10 15 20

0

0.0005

0.001

0 5 10 15 20

0

0.002

0.004

0.006

0 5 10 15 20

0

0.00005

0.0001

0 5 10 15 20

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Figure 4. Distribution of 17β-E2 and E1 over time of exposure to maize seedlings. Total pie

area shows relative mass of estrogens remaining in the system.

Days 17ββββ-E2 solutions E1 solutions

0

1

4

8

12

17β-E2 aqueous E1 aqueous 17β-E2 root E1 root 17β-E2 shoot

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Figure 5. Parent and product estrogen concentrations during of exposure to tissue enzyme

extracts. 17β-E2, E1, α-ZAL, ZAN. Markers are omitted where products were below

the limit of detection. Error bars indicate SD.

a. Root enzymes b. Seed enzymes c. Shoot enzymes

Aq

ueo

us

con

cen

tra

tio

n (

µM

)

17β-E2 solutions

E1 solutions

α-ZAL solutions

ZAN solutions

Time (min)

0

1

2

0 10 20 30

0

1

2

0 10 20 30

0

1

2

0 10 20 30

0

1

2

0 10 20 30

0

1

2

0 10 20 30

0

1

2

0 10 20 30

0

1

2

0 10 20 30

0

1

2

0 10 20 30

0

1

2

0 10 20 30

0

1

2

0 10 20 30

0

1

2

0 10 20 30

0

1

2

0 10 20 30

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transformation of natural and synthetic estrogens by maize seedlings

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