human exposure to endocrine disrupters: standardisation of a marker of estrogenic exposure in...

APMIS 109: 185-97, 2001 Copyrinht 8 A P M I S 2001 @lease use this original reference for citation) Printed in Denmark. All rights reserved

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APUN ISSN 0903-4641

Human exposure to endocrine disrupters: Standardisation of a marker of estrogenic exposure in adipose tissue

ANA RIVAS, MARIANA E FERNANDEZ, ISABEL CERRILLO, JESUS IBARLUZEA, M. FATIMA OLEA-SERRANO, VICENTE PEDRAZA and NICOLAS OLEA

Laboratory of Medical Investigations, Radiology Dept, S Cecilio Univ Hosp; (M.F.0-S) Nutrition and Food Science Dept, Univ 18071-Granada; (J.1) Delegaci6n de Sanidad de Guipuzcoa,

Health Dpt. Basque Goverment, Spain

Rivas A, Fernandez MF, Cerrillo I, Ibarluzea J, Olea-Serrano F, Pedraza V & Olea N. Human exposure to endocrine disrupters: Standardisation of a marker of estrogenic exposure in adipose tissue. APMIS 2001;109: 185-97.

In many epidemiological studies based on the direct measurement of exposure to organochlorines, the chemicals of concern are determined directly from adipose tissue samples. Although the measurement of all possible organochlorines, their metabolites, isomers and congeners may be desirable, it is expensive and time-consuming and many chemicals with hormonal activity may not yet have been identified. Testing systems are therefore required to screen for estrogenicity and to identify appropriate biomarkers of human exposure. To address this issue, we developed and standardised a method to assess the total estrogenic xenobiotic burden in human adipose tissue. The method extracts and separates the more lipophilic xenoestrogens from ovarian estrogens, with a subsequent bioassay determination of the cumulative effect of the xenoestrogens. It was applied to 400 women, using 200 mg of adipose tissue: 65% of samples showed measurable estrogenicity in the fraction where most non-polar xenoestrogens eluted, and 76% of fractions where ovarian estrogens eluted were positive for estrogenicity. Residues of 16 organochlorine pesticides were determined. No correlation was found between pesticide content and estrogenicity of the samples. The high percentage of positive samples suggests that the method is sensitive enough to be used as a biomarker of human exposure to estrogenic xenobiotics and can be applied in epidemiological studies.

Key words: organochlorines; estrogenicity; xenoestrogens; endocrine disrupters.

N. Olea. Laboratory of Medical Investigations, Radiology Dept., S. Cecilio Univ. Hosp., 1807 1-Gran- ada, Spain. e-mail: [email protected].

Confirmation that humans are exposed to chemicals that interfere with the hormonal system (endocrine disrupters) has prompted many epidemiological studies but an association between chemical residues and disease remains elusive. Exposure to these chemicals occurs mainly through diet, water, environment, in- door air, dust, soil, and also in an occupational setting. For example, organochlorine pesticides and polychlorinated biphenyls (PCB) enter the

Received December 4, 2000. Accepted January 15, 2001.

human organism via food and water but they also may reach humans by inhalation and con- tact, especially among those professionally exposed in industrial and agricultural settings. Many of these organochlorine derivatives accumulate in adipose tissues because of their solubility in lipids and their inefficient met- abolism.

Since 1970, the use of some organochlorines has been banned, leading to a reduction in their accumulation in the environment and a de- crease, albeit slower, in the human body burden (1). Other organochlorine pesticide, including

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endosulfan, are still used and frequently found in the environment (2) and in human tissues and fluids (3). Historic exposure to bioaccumulative xenoestrogens is reconstructed through study of the body burden, mostly blood and adipose tissue content (4) and of medical records and with the use of questionnaires. These methods permit investigation of the association between exposure to persistent organochlorines and long latency diseases such as breast cancer. Over the past 16 years, many studies have reported a wide range of pesticide content in the adipose tissue of breast cancer patients, not only dichlo- rodiphenyltrichloroethane (DDT) and its metabolites (5-14), but also hexachlorobenzene (7- 8, 10, 13-14) mirex, chlordane, and P-hexachlorocyclohexane (14). Experimental data confirmed the estrogenicity of many of these fat- soluble chemicals, which are now classified as xenoestrogens (14). However, there has been no demonstration to date of a clear connection between exposure to these chemicals and hormone-dependent diseases, or of the role of these xenobiotics in the modulation of cancer growth (1 5-1 6).

It was stated (17) that the design of retrospec- tive breast cancer studies based on the analysis of human samples should consider the hypothesis to be tested, the chemicals to be measured, and the biological activity to be analysed. However, these considerations are frequently disregarded. For example: i) many studies have included chemicals that are not hormonally active, taking no account of the role of estrogens in the pathogenesis of the disease, ii) other studies have only measured a single residue because of the time, resources and the large sample sizes required to determine more chemicals, and iii) most of the studies have disregarded cumulative effects and interactions between chemicals. There is a need to develop markers of estrogenic exposure that go further than the quantification of isolated xenoestrogens. It has been proposed (18) that rather than measuring the levels of identified xenoestrogens, it might be more meaningful to assess the risk of endocrine disruption by measuring the biological activity resulting from the xenobiotics.

We adopted this approach and developed a system to estimate the total estrogenic xenobiotic burden in serum samples (19) using the E- Screen, an in vitro bioassay of proven utility for

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the identification of xenoestrogens (20). This bioassay compares the cell yield between cultures of human breast cancer MCF-7 cells treated with estradiol and those treated with different concentrations of xenobiotics suspected of being estrogenic (18, 21-22). We then developed a methodology to extract xenoestrogens from human fat samples, separating sex steroids from bioaccumulated xenoestrogens and testing them in the E-Screen bioassay (23-24).

The purpose of the present study was to stan- dardise and apply a method to extract xenoestrogens from the adipose tissue of 400 patients and test them for estrogenicity using the E- Screen bioassay. In addition, residues of sixteen organochlorine pesticides and their metabolites were quantified in the fat of the same patients. Some of the chemicals tested are already identified as estrogenic mimics but others are de- fined here as estrogenic agonist for the first time.

MATERIAL AND METHODS

Pa tien ts Four hundred women with different diseases who

were undergoing surgical treatments were included in the study; the mean age was 53 years. Fat samples were taken from mammary or abdominal fatty tissue. Adipose tissue collected during surgical treatment at Granada University Hospitals and Almeria Hospital was placed into a glass vial on ice, coded, and frozen to -70” C, always within 30 min of being excised, and stored at the same temperature at the Laboratory of Medical Investigations until they were processed.

Reagents AU solvents used were of high purity grade for

HPLC: Methanol, 2-isopropanol, hexane, ethanol, chloroform, hydrochloric acid (Panreac, Barcelona, Spain). Patterns: Aldrin, dieldrin, endrin, lindane, methoxychlor, endosulfan I and 11, mirex, p-p’-DDT and o-p’-DDT (Supelco, Bellefonte, PA); o,p’-DDD, PCB 77, 126 and 169 and p-p’dichlorobenzophenone (Dr Ehrenstorfer Lab., Ausburg, Germany); p,p’- DDE (Chem Service, West Chester, PA); endosulfan metabolites, endosulfan diol, sulfate, lactone and ether (Hoechst Schering AgrEvo, Frankfurt, Ger- many); biochanin A, a-zearalenol, p-zearalenol, di- ethylestilbestrol, 17~-estradiol,17a-estradiol, estrone, 17a-ethynylestradiol, progesterone and testosterone (Sigma, St. Louis, MI); bisphenol-A (Aldrich Chemi- cal Company, Milwaukee, WI).

Bisphenol derivatives with substituents at the central carbon and phenol ring, 1,l bis (4-hydroxyphenyl) ethane (MMl), 1,l bis (4-hydroxyphenyl) propane

A MARKER OF ESTROGENIC EXPOSURE

(MM2), 2,2 bis (4-hydroxyphenyl) butane (MM3), 3,3 bis (4-hydroxyphenyl) pentane (MM4), 4,4 bis (4- hydroxyphenyl) heptane (MM5), 2,2 bis (Chydroxy- 3-methylphenyl) propane (MM6), 2,2 bis (Chydroxy- phenyl) perfluoropropane (MM7), bis (Chydroxy- phenyl) ketone (MM8) and 2,2-bis(4-hydroxyphenyl) propanol (MM9) were synthesized by Dr. M. Metzler of Karlsruhe University (Karlsruhe, Germany). Chlorinated bisphenols, mono-, di-, tri and tetrachlo- ro-bisphenol-A were a gift from Dr. J.L. Vilchez (Analytical Chemistry Dpt. University of Granada, Spain). p-Nonylphenol was purchased from Fluka Chemika (Buchs, Switzerland), bromine-p-nonylphenol acetate (BrNPAc), bromine-p-nonylphenol monoethoxylated acetate (BrNPlEOAc) and bromine-p-nonylphenol diethoxylated acetate (BrNP2EOAc) were a gift from Dr. E Ventura (Ag- bar, Barcelona, Spain). The purity of all of these chemicals was at least 97%. The chemicals were dissolved in ethanol to a final concentration of 1 mM and stored at -20°C.

Apparatus The HPLC procedure was done with a Waters

Model 50 1 Millipore apparatus (Marlborough, MA) equipped with two pumps and a U6K injector of 500 pL load capacity. Ultravioletlvisible detector was a Waters Model 490 Millipore set at 280 nm and Mil- lennium Chromatography Manager software was used. A Lichrocart column (25 cmX4.6 mm) was used (Merck, Darmstadt, Germany) packed with Lichrospher Si-60, of 5 pm particle size. Gas chromatography was done with a Varian-3350 machine (Walnut Creek, CA) with electron capture detector (63Ni) and using Millennium Chromatography Man- ager software. A methyl silicone column (30 mX0.25 mm) was used. Gas chromatography-Mass spectrometry was done with a Saturn 2000 Varian Instru- ment (Walnut Creek, CA) with automatic injector 8200, SPI/1078; a DB5-MS capillary column (30 mx0.25 mm) was used.

Extraction of xenoestrogens from adipose tissue and separation of sex steroids

Sample extraction. Samples were processed in batches of six. Two quality control samples were run every two batches. Bioaccumulative compounds were extracted from adipose tissue by a previously described method (25), with slight modifications. An aliquot of 200 mg adipose tissue was dissolved in hexane and eluted in a glass column filled with Alumine Merck 90 (76230 mesh) no. 1097 that had been dried at 600°C for 4 h and rehydrated with the addition of 5% water. The eluate obtained was concentrated at reduced pressure and then under a stream of nitrogen to a volume of 1 mL and then injected twice (500 pL) into the preparative HPLC. Spiked fat samples were run in parallel in order to assess the recovery of pesticides from adipose tissue. Aliquots were spiked with

a known amount of standard solutions of pesticides, homogenised and allowed to equilibrate for 2 hours at room temperature. The recovery and reproducibility of the process was established by running ten fat samples.

High performance liquid chromatography (HPLC). A preparative liquid chromatography method was developed to allow the separation of the more lipophilic xenoestrogens from natural estrogens without destroying them. Extracts were eluted by a gradient with two mobile phases: n-hexane (phase A) and n- hexane: methanol: 2-isopropanol (40:45: 15)(v/v) (phase B) at a flow rate of 1.0 mL/min. The gradient programme was based on a previously described method (26) with modifications. Working conditions were: gradient t = O min, 100% phase A; t=17 min, 60% phase A; t=25 min, 100% phase B; t=32 min, 100% phase A. Three pooled fractions, named a , x and p, were separated by HPLC. The a fraction is collected in the first 11 minutes, the x fraction is collected between minutes 11 and 12, and the I3 fraction is collected between minutes 12 and 25. The method was employed under similar working conditions except for an injection volume of 20 pL of standard solutions, in order to establish the retention times of the chemicals eluted in the a, x and I3 fractions.

Gas chromatography (GC) . a-fractions of HPLC chromatography were dried, dissolved in n-hexane, spiked with an internal standard (p,p'-dichlorobenzo- fenone) and then injected into a gas chromatograph with an electron capture detector (ECD). In the gas chromatography, no organochlorine chemicals were detected in the x and I3 fractions. Standard solutions of organochlorine pesticides were previously analysed by gas chromatography in order to determine the retention times and calibration curves of these chemicals. The working conditions were: ECD at 300°C; injector at 250°C; Program: Initial T 130°C (1 min); 20"C/min to 150°C; 10" C/min to 200°C; 20"C/min to 260°C (20 min). The carrier gas was Nitrogen at a flow of 30 mL/min and the auxiliary gas Nitrogen at a flow of 40 mL/min. The injection volume was 1 pL. The reproducibility of the process was established by running ten fat samples 10 times. The presence of organochlorine in fraction a was confirmed by gas chromatography and mass spectrometry ( G C I M S ) , following the working conditions previously described (27-28). The working conditions were: Oven T 80°C (2.5min), 50"C/min until 140"C, 5"C/min to 260°C during 3 min; Injector T initial temperature 90°C (0.1 min), 200"C/min to 280°C; gas He; injector flow: lpL/sec. Injection volume 1 pL.

Lipid determination. Total lipid content was quantified gravimetrically: 100 mg of adipose tissue were homogenised in 2.5 mL of chloroform:methanol:hydrochloric acid (20:lO:O.l). After repeating the process, 5 mL of HC1 0.1N were added and centrifuged at 3000 rpm for 10 min. The organic phase was collected; the non-organic phase was extracted again

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and added to the first extraction product. After dry- ing under a nitrogen stream, the tubes were weighed and the total lipid expressed in g of lipid per g of adipose tissue.

Bioassay for measuring estrogenicity MCF-7 cell line. Cloned MCF-7 cancer cells were

grown for routine maintenance in Dulbecco’s modi- fication of Eagle’s medium supplemented with 5% fe- tal bovine serum (BioWittaker, Walkersville, MD) in an atmosphere of 5% C02195% air under saturating humidity at 37°C. The cells were subcultivated at weekly intervals using a mixture of 0.05% trypsin and 0.01% EDTA.

Charcoal-dextran treatment of serum. Plasma-de- rived human serum was prepared from outdated plasma by adding calcium chloride to a h a 1 concentration of 30 mM to facilitate clot formation. Sex steroids were removed from serum by charcoal-dextran stripping. Briefly, a suspension of 5% charcoal (Norit A, Sigma Chemical Co, St. Louis, MO) with 0.5% dextran T-70 (Pharmacia-LKB, Uppsala, Sweden) was prepared. Aliquots of the charcoal-dextran suspension of a volume similar to the serum aliquot to be processed were centrifuged at 1000 g for 10 min. Supernatants were aspirated and serum aliquots were mixed with the charcoal pellets. This charcoal-serum mixture was maintained in suspension by rolling at 6 cycleslmin at 37°C for 1 h. The suspension was centrifuged at 1000 g for 20 min, and the supernatant was then filtered through a 0.22 pm filter (Millipore). Charcoal dextran-treated human serum (CDHuS) was stored at -20°C until needed. In addition manufactured CDHuS from Irvine Scientific (Santa h a , CA) was also employed.

Cell proliferation experiments. MCF-7 cells were used in the test of estrogenicity according to a tech- nique slightly modified (22) from that originally described (20). Briefly, cells were trypsinized and plated in 24-well plates (Limbro, McLean, VA) at initial concentrations of 20.000 cells per well in 5% FBS in DME. Cells were allowed to attach for 24 h; then the seeding medium was replaced with 10% CDHuS- supplemented phenol red-free DME. Different concentrations of the test compound were added, and the assay was stopped after 144 h by removing medium from wells, fixing the cells and staining them with sulforhodamine-B (SRB). The cells were treated with cold 10% trichloroacetic acid and incubated at 4°C for 30 min, washed five times with tap water and left to dry. Trichloroacetic-fixed cells were stained for 10 min with 0.4% (wthol) SRB dissolved in 1% acetic acid. Wells were rinsed with 1% acetic acid and air dried. Bound dye was dissolved with 10 mM Tris base (pH 10.7) in a shaker for 20 min. Finally, aliquots were transferred to a 96-well plate and read in a Titer- tek Multiscan apparatus (Flow, Irvine, CA) at 492 nm. The linearity of the SRB assay with cell number was verified prior to the cell growth experiments. The 1000/0 proliferative effect (PE) was calculated as the

ratio between the highest cell yield obtained with 50 pM of estradiol and the proliferation of hormone- free control cells.

Quantitative evaluation of estrogenicity of chemicals. Ethanol solutions of pure chemicals were prepared at 1 mM and tested in quadruplicate in three different assays in a range of 0.1 mM to 1 pM. The proliferative effect (PE) of any chemical indicates the lowest concentration needed for maximal cell yield. The relative proliferative effect (RPE) is calculated as 100 X (PE-1) of the test compound/(PE-1) of estradi- 01. A value quantitatively similar to estradiol (RPE= 100) indicates that the compound tested is a full agonist, a value of 0 indicates that the compound lacks estrogenicity at the doses tested, and a value of significantly lower than estradiol suggest that the xenobiotic is a partial agonist. Relative proliferative potency (RPP) is the ratio between the estradiol and xenobiotic doses needed to produce maximal yield X 100. All the compounds designated as full or partial agonists (RPE> 15) significantly increased cell yields compared to the controls without hormones (peO.05).

Quantitative evaluation of estrogenicity of fa t extracts: Duplicated dry pooled a and 13 fractions obtained by preparative HPLC chromatography were resuspended in 5 mL of CDHuS supplemented phenol red-free medium, vigorously shaken and left at rest for 30 min, then filtered throughout a 0.22 pm filter and tested in the assay for estrogenicity at dilutions from 1:l to 1:lO. Each sample was analysed in triplicate with a negative (vehicle) and a positive (estradiol 10 pM) control in each plate. The PE of a and 13 fractions was referred to the maximal proliferative effect obtained with estradiol and transformed into estradiol equivalent units (Eeq) by reading from a dose-response curve prepared using estradiol (Con- centration range 0.1 pM to 10 nM).

Statistical analysis Results from cell proliferation experiments were ex-

pressed as means? SD. Proliferation yield experi-

TABLE 1. Recovery of organochlorine pesticides from spiked fa t samples

Organochlorine Recovery YO Low High pesticides

M SDf?) M SD f?) p,p’-DDE 90.10 3.72 98.23 2.40

Endosulfan I 85.04 2.53 90.23 2.17 Lindane 83.45 1.50 95.18 2.64 Dieldrin 98.80 3.41 102.12 3.23 M, mean; SD, standard deviation. Samples were spiked with a mixture of five organochlorine pesticides to give Low (10 nglg of lipid) or High (500 nglg p,p’-DDT; 100 nglg p,p’-DDT and 50 ng/g of endosulfan I, lindane and dieldrin) fhal concentrations.

p,p’-DDT 92.51 2.65 97.3s 3.55

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TABLE 2. Retention times of selected xenoestrogens and natural estrogens in the DreDarative HPLC

Chemicals Retention time (min)

PCB 126 BrNPAc BrNP 1 EOAc PCB 169 PCB 77 Tetra Cl-BPA BrNP2EOAc Tri Cl-BPA Di Cl-BPA Mono C1-BPA p-Nonylphenol Progesterone Estrone 4,4 bis (4-hydroxyphenyl) heptane

(MM5) Testosterone 2,2 bis (4-hydroxy-3-methylphenyl)

17a-estradiol 3,3 bis (4-hydroxyphenyl)

2,2 bis (4-hydroxyphenyl)

17P-estradiol 1,l bis (4-hydroxyphenyl)

2,2 bis (Chydroxyphenyl)

DES 17a-ethynylestradiol 1,l bis (4-hydroxyphenyl)

ethane (MM1) Bisphenol-A (BPA) a-zearalenol bis (4-hydroxyphenyl) ketone (MM8) 2,2 bis(4-hydroxyphenyl)

S-zearalenol

propane (MM6)

pentane (MM4)

butane (MM3)

propane (MM2)

peduoropropane (MM7)

propanol (MM9)

2.36 2.48 2.53 2.80 2.91 4.31 4.33 9.63 9.93

10.85 11.34 12.36 13.71 15.21

15.30 15.38

15.75 16.00

16.26

16.16 16.35

16.41

16.43 16.51 16.63

16.65 17.03 17.65 20.46

21.60 Biochamin A 25.78

ments conducted in quadruplicate or triplicate wells were repeated at least 3 times. The mean cell numbers from each experiment were normalised to the steroid- free control cultures to correct for differences in the initial seeding density. The differences between the different chemical treatment groups were assessed by analysis of variance and the a posteriori Shaffe’s test. The correlations between organochlorine and its derivatives, and between these compounds and the total xenoestrogen burden expressed as estradiol equivalent (Eeq)/g of lipid were tested by calculating the Spearman’s correlation coefficient. A p value of <0.05 was regarded as significant.

RESULTS

Extraction and separation of non-polar xenoestrogens from sex-steroids by HPLC

The methodology developed allowed the quantitative extraction of xenoestrogens from human adipose tissues. The recovery percen- tages of samples spiked with a mixture of organochlorines were always higher than 83% (Table 1). Extracted xenoestrogens and natural sex- steroids were separated by HPLC into three different fractions: the first 11 mL (0-11 min) of elution corresponded to the a fraction, the next 2 mL (1 1-12 min) to the x fraction and the last 13 mL (12-25 min) collected to the 13 fraction. This normal-phase column separated xenoestrogens according to their polarity; with the most lipophilic compounds retained for the shortest time. Organochlorine pesticides, chlorinated bisphenols and brominated p-nonylphenol eluted notably earlier than the steroidal estrogens (Table 2). These data indicate that the non-polar xenoestrogens can be successfully separated from sex steroids, phytoestrogens, mycoestrogens and bisphenols. The a, x and p fractions were analysed by GC/ECD and also tested in the bioassay for estrogenicity. The x fraction is a consequence of the gradient used in the elution process; it has the meaning of a safety fraction with no chemicals recorded in UV or GC-ECD, which separates the a fraction containing the

TABLE 3. Retention times and detection (LOD) limits and quantification (QL) of organochlorine pesti-

cides in gas chromatography Organochlorine Retention LOD QL pesticides time (min) (nglmL) (nglmL) Lindane 7.98 0.5 1 Endosulfan-eter 8.86 0.1 0.5 Aldrin 9.77 1 3 Endosulfan-lactone 10.19 2 5 Endosulfan-diol 10.48 0.5 1 Endosulfan I 11.04 0.5 1 p,p’-DDE 1 1.29 5 10 o,p’-DDD 11.41 5 10 Dieldrin 11.53 1 5 Endrin 11.80 3 25 Endosulfan I1 11.87 2 10 o,p’-DDT 12.13 0.5 1 Endosulfan-sulfate 12.6 1 0.5 5 p,p’-DDT 12.71 5 10 Methoxychlor 13.81 1 5 Mirex 15.62 10 5

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TABLE 4. Reproducibility of gas chromatography Organochlorine M SD C.1 pesticides (nglg) (+I (%I Lindane 2.3 1 0.16 7.26 Endosulfan-ether 0.48 0.05 10.95 Aldrin 20.62 2.19 10.62 Endosulfan-diol 3.26 0.22 7.00

p,p’-DDT 27.91 1.01 3.63 Fat samples were processed ten times by gas chromatography. M, mean; SD standard deviation; C . l coefficient of variation.

p,p’-DDE 520.73 23.11 4.43

more lipophilic substances from the p fraction where natural estrogens and more polar xenoestrogens eluted. The mean percentage of lipid content in the fat samples gathered in the present study was higher than 80%.

Identification and quantification of organochlorine pesticides

The GC demonstrated that organochlorine pesticides were eluted in the a fraction. The retention times of these chemicals are shown in Table 3, together with the detection (LOD) and quantification limits (QL). The calibration linearity of the 16 pesticides in pure and processed standards was greater than 0.98. The reproducibility for the GC/ECD results in an adipose tissue sample tested 10 times is shown in Table 4. The mean values of the organochlorine pesticides measured in the extracted adipose tissue

samples are shown in Table 5. p,p’-DDE was the commonest residue found in our series of patients and 98% of the assayed samples showed measurable amounts of this pesticide. It was fol- lowed in frequency by endosulfans and some of their metabolites (Range 52-11%) and by hexachlorocyclohexane lindane (42%). The order of organochlorine pesticides by concentration level was: p,p’-DDE >o,p’-DDD > endosulfan-sulfate > mirex > endrin > lindane > among the pesticides that showed levels of >40 ng/g of lipid concentration. The p,p’-DDT and some of the cyclodiene derivatives occupied the next highest positions, whereas endosulfans and their metabolites and dieldrin were found at lower concentrations. GC/MS always confirmed the presence of the organochlorine pesticides in the a-fraction (data not shown). No organochlorine pesticides were found in the x or D fractions.

Estrogenic effect of chemicals Culture bioassays for estrogenicity commonly

use MCF-7 breast cancer cells, an estrogen-sensitive and -dependent cell line. We used an E- Screen MCF-7 based bioassay to test the chemicals suspected of being hormone-mimics. The addition of estradiol to CDHuS-supplemented medium increased the number of MCF-7 cells in the culture. The maximum PE was obtained at concentrations of 50 pM estradiol and higher (Figure 1). The cell yields were 6-fold greater

TABLE 5. QuantiJication of organochlorine pesticides in extracted adipose tissue Organochlorine M Median S D (k) Frequency

Lindane 41.29 15.82 92.62 42.26

Dieldrin 14.92 12.78 12.62 22.40 Endrin 44.50 49.30 16.92 12.70 Mirex 48.52 57.51 17.92 6.92 p,p’-DDE 504.87 393.57 571.32 97.92 o,p’-DDD 215.13 180.38 170.63 20.78 o,p’-DDT 15.54 15.14 1 1.62 18.70 p,p’-DDT 39.10 37.34 31.09 23.09 Methoxychlor 31.71 13.54 53.21 4.15 Endosulfan-ether 3.49 0.94 10.27 51.96

Endosulfan-diol 13.84 5.15 42.13 28.86 Endosulfan I 13.21 2.54 54.99 20.32

Endosulfan-sulfate 80.00 15.34 187.74 10.85

pesticide (ng/g lipid) (ng/g lipid) (%)

Aldrin 35.52 20.03 53.46 30.48

Endosulfan-lactone 7.09 2.02 23.59 14.78

Endosulfan I1 21.80 20.33 11.37 10.85

M, mean; SD, standard deviation.

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Fig. 1. Dose-response curves of endosulfans, chlorinated bisphenols and brominated alkylphenols. The cells were harvested after 6 days of exposure. The data points represent the mean of the ratios between the proliferation of treatedcontrol groups for a set of three experiments; the error bars represent SD. Dotted line: negative control, cell yield in the absence of chemicals. A: estradiol (unfilled circle), endosulfan I (unfilled square), endosulfan I1 (filled square), endosulfan diol (unfilled triangle), endosulfan ether (filled triangle), endosulfan sulfate (unfilled diamond) and endosulfan lactone (filled diamond). B: estradiol (unfilled circle), bisphenol-A (unfilled square), mono-chlorine bisphenol-A (filled square), di-chlorine bisphenol-A (unfilled triangle), tri- chlorine bisphenol-A (filled triangle), and tetra-chlorine bisphenol-A (unfilled diamond). C: estradiol (unfilled circle), p-nonylphenol (unfilled square), bromine p-nonylphenol acetate, (filled square) bromine p-nonylphenol monoethoxylated acetate (unfilled triangle), bromine p-nonylphenol diethoxylated acetate (Wed triangle).

TABLE 6. Estrogenic effect of endosulfans, chlorinated bisphenols and brominated p-nonylphenols as measured by the E-Screen assay

Chemicals Concentration RPE % RPP %

Endosulfan I 10 pmoYL 69 0.0005 Endosulfan I1 10 pmol/L 74 0.0005 Endosulfan-diol 10 pmoUL 61 0.0005 Endosulfan-ether 100 VmoYL 48 0.00005

Estradiol 50 pmol/L 100 100

Endosulfan-lactone 100 pmoYL - - Endosulfan-sulfate 100 pmoVL - -

Bisphenol-A 1 pmoYL 85 0.005 Mono Cl-BPA 10 pmoYL 71 0.0005 Di Cl-BPA 10 pmoYL 71 0.0005 Tri Cl-BPA 10 pmoYL 63 0.0005 Tetra Cl-BPA 10 pmoVL 30 0.0005 Nonylphenol 1 pmoYL 75 0.005 BrNPAc 5 pmoVL 65 0.001 BrNF'lEOAc 5 pmoYL 48 0.001 BrNP2EOAc 5 pmoVL 36 0.001

RPE was calculated as 100 X (PE-1) of the test compound/(PE-1) of estradiol, where PE is the lowest concentration needed for maximal cell yield (PE); RPP is the ratio between estradiol and xenobiotic doses needed to produce maximal yield X 100. All of the compounds that were designated as full or partial agonists (RPE>15) significantly increased cell yields compared with the controls without hormones (pCO.05). - no effect observed.

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TABLE 7. Estimated values of total xenobiotic estrogenic burden for the a and,&-fractions of 400 adipose tissue samples

HPLC Fraction Frequency M (pM) Median (pM) SD(+) a-Fraction 3051400

EeqlmL 24.4 3.7 53.3 Eeqlg lipid 750.5 105.2 330.4

EeqlmL 29.8 5.7 46.6 Eeqlg lipid 903.2 170.6 320.5

The Eeqs of the a and p-fractions were interpolated from the estradiol dose-response curve (Figure 3). The means and medians are expressed as the estradiol equivalent (Eeq) estimated for 1 mL of culture medium in the E-Screen assay and are expressed in Eeqlg of lipids after adjustment for the sample weight, lipid content and dilution factor of the dried extracts. M, mean; SD, standard deviation.

P-Fraction 3331400

than in control cultures after 6 days (mean?SD, 6.01 5 1.03 in 10 experiments). In the absence of estradiol (controls), the cells proliferated mini- mally. The PEs of endosulfans, brominated alkylphenols and chlorinated bisphenols are shown in Figure 1 and Table 6. PE was significantly greater than 1 for all the compounds tested except for endosulfan lactone and sulfate, which were negative in the bioassay. In comparison with the RPE of estradiol, all the positive compounds showed a full to partial agonistic response that produced cell yields ranging from 85% of estradiol-induced yield for bisphenol-A to 30% for tetrachlorine bisphenol-A derivative.

Testing the estrogenic activity of the adipose tissue extract

Fractions a , x and 13 were tested in the E- Screen bioassay in order to determine the total estrogenic xenobiotic burden corresponding to each sample. The first step towards this end was to define the dose-proliferative response curve for estradiol and MCF-7 cells, as a standard (Figure 2). At concentrations below 1 pM estradiol, equivalent to 1 fmol in 1 ml of culture medium, the mean of cell numbers did not differ significantly from that of the steroid-free control. Thus, 1 fmol estradiouwell was determined as the lowest detectable estrogen amount in this assay. The proliferative effects obtained with 1 : 1 to 1:lO dilutions of a , x and D fractions were converted to estradiol equivalents (Eeq) by interpolation on the estradiol dose-response curve. No activity was found when testing the x fraction, therefore no data are presented. Figure 3 shows the proliferative effect corresponding to the dilutions of a and 13 fractions of a fat sample chosen from among those giving a positive re-

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sponse. Table 7 lists the mean Eeq/g lipid values for a and D-fractions in all the 400 samples studied; 78% of samples showed measurable estrogenic activity in the a-fraction, with a mean value of 750 pM (range, 2.7 pM to 1.8 nM). In addition, 83 YO of D-fractions were positive in

Fig. 2. Dose-response curve of estradiol on MCF-7 cells in culture. Cells were harvested after 6 days of exposure to variable amounts of estradiol. Ordinate: RPE calculated as 100 X (Proliferative effect - 1) of each estradiol concentratiod(Pro1iferative effect - 1) of plateau estradiol concentrations (0.1 nM estradiol and higher). Data points represent the mean of sets of four experiments repeated every twenty tests; the error bars represent the SD. Logit transformation of data fit a straight line, with a correlation coefficient r=0.956.


Fig. 3. Bioactivity of an adipose tissue extract on MCF-7 cells in culture. Data points represent the mean of proliferation treatedkontrol ratio of serial dilutions of a (unfilled circle) and 13 (filled circle) fractions. Dotted line: negative control, cell yield in the absence of chemicals. The proliferative effect of a and 13 fractions was referred to the proliferative effect of estradiol (Dose response curve of estradiol) and transformed into Eeq units; the results were 124 pM/ mL and 20 pM/mL, respectively.

the E-Screen assay, with a mean value of 903 pM (range, 3.4 pM to 5.6 nM). Absence of toxic activity in the extracts was demonstrated when partial agonist effect was shown in the presence of 100 pM of estradiol at maximal dilution of the extracts (data not shown).

Quality control of the method To study the precision of the procedure, 24

aliquots of 200 mg were extracted from two fat samples previously assigned as having a low (8 pM/mL) and high (120 pM/mL) level of estrogenicity, respectively. The aliquots underwent preparative HPLC and were assayed in the E- Screen. The calculation of Eeq/mL was made independently for each sample. At each step throughout the process four samples were pooled out (Pool), eluted, and re-included in the process. A good reproducibility (mean? SD, 120.0+38.1 pM; post-extraction [pool]

mean+SD, 111.1k27.3 pM; post HPLC Ipool] meankSD, 136.3225.2 pM) for the whole process was obtained among samples with high estrogenicity, whereas there were some variations among those with lower estrogenicity (mean-+SD, 8.92 1.5 pM; post-extraction [pool] meankSD, 8.120.9 pM; post HPLC [pool] meankSD, 6.321.3 pM).

DISCUSSION

Humans are exposed to endocrine disrupters of very varied origin through diet and in other ways, especially through different types of exposure at the workplace. For example, individuals employed in intensive greenhouse agricul- ture in Southern Europe have been shown to be subject to high exposure levels (3, 29). Once in- side the body, endocrine disrupters can act cumulatively or in combination with endoge- nous hormones. Some of these chemicals, such as organochlorine chemicals, accumulate and persist in adipose tissue due to their lipid solubility and resistance to metabolisation, reaching levels 200-1000-fold those found in serum (14). Therefore, fat deposits can maintain elevated levels of organochlorine and PCBs in the circu- lation for years, (4) even when there has been no exposure during this period. Serum levels of organochlorines are frequently used as biomarkers of exposure, on the assumption that they represent fat reservoir content and are re- liable indicators of total body burden (30). However, a perfect relationship between serum levels and adipose tissue content is not always evident. Several authors have recommended the direct measurement of adipose tissue content (4, 17, 14).

The procedures described in the present paper constitute the standardisation of a marker of human exposure to biocumulative xenoestrogens that permits the quantitation of estrogenicity. We measured the combined estrogenic effect of chemicals extracted from adipose tissue by determining the cumulative effect of the xenoestrogens with the E-screen bioassay for estrogenicity. The process ended with assessment of the proliferative effect, expressed in estradiol equivalents per gram of lipid con tent (Eeqlg), and assignation of the total estrogenic burden to each patient. We demonstrated a high pro-

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portion of individuals with estrogenic activity attributable to estrogens accumulated in adipose tissue. The mean value of Eeq/g of lipid for the a-fraction was established at 750 pM/g for 304 samples (76% of the series), equivalent to 24 fmoVmL of estradiol in the culture medium, corresponding to a segment of the standard curve that provides more precise discrimi- nation. After taking into account the sensitivity of the method (1 fmoVmL) and the dilution factor, the percentage of samples considered positive was adjusted to 65%.

We do not know which chemicals are respon- sible for estrogenicity of the samples, but organochlorine pesticides, PCBs and halogenated bisphenols and alkylphenols emerged as candi- dates because they were collected in the most lipophilic, non-polar a-fraction of the preparative HPLC. The organochlorine concentrations measured in the a-fraction are comparable to those previously reported in adipose tissue from different sites. For example, The mean value of 505 ng of p,p’-DDE Ig of lipid that we found is similar to a recently reported finding of 596-693 pg/Kg for breast adipose tissue (14) but much lower than values reported in the USA in 1987 (8). Results for residues of the parent compound of p,p’-DDE show some discrepancies. The p,p’- DDT value in our series was two-fold that reported in other series (14). These differences were even more marked for residues of other organochlorines such as mirex, which we found at five times the concentrations described by other authors (14), although only in 7% of our series. We included DDT isomers and metabolites, cyclodiene derivatives and hexachlorocyclohexane pesticides in the present study because of the interest in running epidemiological studies on these chemicals. For example, these organochlorines have been studied in relation to breast cancer, either because they fit the xenoestrogen hypothesis that links hormonally active chemicals to the risk of hormonal disease (31), or because they may act as direct carcinogens. We also included in the study a group of endosulfans, isomers and metabolites, because some of them are weak estrogens and may therefore be related to hormonal imbalance, although our findings cannot be compared because of the absence of published data on endosulfans.

The results for DDT isomers and those for their metabolites correlated with each other. We

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found a significant correlation between o,p’- DDT and p,p’-DDT (p=0.116; p<0.016), per- haps because commercial grade DDT, mainly the p,p’-isomer, typically has 15% of the o,p’-isomer. The concentrations of o,p’-isomers in humans are frequently low and may be undetectable in specimens of the size normally collected (17). However, these good correlations have led to pro- posals for the p,p’-measurement to be used as a surrogate biomarker suitable for epidemiological purposes. p,p’-DDE was also significantly correlated with p,p’-DDT (p=O. 190; p<O.OOOl) but not with p,p’-DDT or DDD. p,p’-DDE is the most prominent environmental residue of DDT, a metabolite that constitutes approximately 90% of the total residues in humans.

Endosulfans I and I1 and their metabolites were also related to each other. We found a significant correlation between endosulfan I and I1 (p=0.114, p<0.018) and both of them correlated with endosulfan ether, lactone, and diol but not with endosulfan sulfate. The endosulfan sulfate concentrations were only related to the levels of endosulfan diol. Finally, a clear correlation was found between the cyclodiene compounds. These correlations were significant for the pairs dieldrin-aldrin (p=O. 182; p<O.OOOl) and endrin-aldrin (p=0.185, p<O.OOl). This suggests a relationship between parent compounds and derivatives.

Interestingly, given the purpose of our study, no correlation was found between the content of a single pesticide in the a-fraction and the estrogenicity determined in the bioassay, No single organochlorine could predict the estrogenicity of a sample. There may be several rea- sons for this lack of concordance: i) the estrogenic effects depicted in the E-Screen bioassay are a consequence of the combined effect of several organochlorines; ii) the proliferative effect is due to other chemicals not measured, either other organochlorine pesticides or other lipophilic compounds. The present work sheds some new light on this issue. Whereas the estrogenicity of endosulfan I and I1 was already known (21), we also identified endosulfan diol and ether as estrogenic in the E-Screen bioassay (range 10-100 pM). All of these four chemicals were positively identified among the organochlorine pesticides that bioaccumulated in the fat samples of the present series. They eluted in the a-fraction of the preparative HPLC and


may therefore account for the positivity of the samples in the E-Screen.

Bisphenol-A, fluorine-containing bisphenol- A (bisphenol-AF) and other bisphenol derivatives are estrogenic for MCF-7 cells, promoting cell proliferation and increasing the synthesis and secretion of cell-type specific proteins (32). We have also demonstrated, to our knowledge for the first time, the estrogenic activity of four chlorinated bisphenols. All of them induced the proliferation of MCF-7 cells in culture at concentrations of 1 pM and higher. These chlorinated compounds eluted in the a-fraction together with the organochlorine pesticides. Inter- estingly, other halogenated bisphenols such as prefluorinated bisphenol (MM7) and non-halogenated bisphenol A-related compounds eluted at longer times and were collected in the HPLC D-fraction.

Since 1991 , p-nonylphenol has been identified as an estrogen agonist released from modified polystyrene (32) but no information has been presented on the estrogenicity of their halogenated derivatives. Three brominated derivatives of p-nonylphenol were included in the present study and all were estrogenic in the E- Screen bioassay. These brominated alkylphenols were also collected in the HPLC a-fraction, re- flecting their fat solubility. The presence in adipose tissue of these brominated alkylphenols and the chlorinated bisphenols has yet to be demonstrated. However, if they do bioaccumul- ate in adipose tissue, this may also account for the estrogenicity of the a-fractions.

Finally, the D-fraction, where additives and monomers of plastics eluted, such as bisphenol A, phytoestrogens, mycoestrogens and sex-hormones, was estrogenic in 83% of the samples assayed. The mean value of Eeq/g of lipid for the D-fraction was established at 903 pM/g of lipid for 333 patients. After taking into account the quantification limit, 76% of the samples were finally considered to be positive. When analysing the estrogenicity of 8-fractions, it must be borne in mind that natural estrogens are produced in adipose tissue and subsequently accumulate (33-34). The role of natural estrogens and xenoestrogens in the estrogenicity of the D-fraction is an intriguing issue that war- rants further investigation. Our laboratory is currently examining the estrogenic contribution of the phyto- and myco-estrogens, synthetic

hormones and bisphenols that eluted in the present HPLC D-fraction.

Concerns about the role of environmental and dietary estrogens as contributors to the increasing incidence of hormone-related diseases have prompted epidemiologists and clinicians to design patient-based studies in which to test their hypotheses. However, the study of etiologic fac- tors in diseases is not a simple task when the period of exposure is far removed from the clin- ical presentation of the disease. This can be observed in studies that have attempted to link breast cancer risk to organochlorines content in adipose tissue. A positive association was found for some PCBs (1 1-14) but was not clearly established for a mixture of PCBs, DDE, HCB, j3- HCH, mirex and chlordane (14). It is possible that a relationship between long-latency diseases and organochlorine exposure cannot be demonstrated from the levels of a single xenoestrogen or of only a few of them. Most studies have estimated the circulating levels or adipose content of one or a small number of chemicals, and have ig- nored the impact of other chemicals and also the cumulative effects of chemical mixtures in the environment, which cannot be assessed by the testing of isolated chemicals. The method we proposed may help to establish a relationship between the content of xenoestrogens in adipose tissue and the risk of hormonal diseases.

We thank Dr. E Ventura and Dr. J.L. Vilchez for providing the brominated and chlorinated bisphen- 01s. We thank Richard Davies for editorial assistance. Ana Rivas is on a fellowship from the Spanish Minis- try of Education. This research was supported by grants from the Spanish Ministry of Health (FIS, 00/ 543), the Andalusian Regional Government, Depart- ment of Health (Consejeria de Salud, JA, 96/99) and the EU Commission, SMT Programme 1995 (C5- 148/07), PRISTINE (PL970916) and QLK4-1999- 01422.

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DISCUSSION

Patrick Thonneau (Toulouse, France) Have you any information about reproductive history and reproductive outcomes among these 400 women? Have you studied the time to conceive, and are there any differences in outcomes between the women positive and negative for oestrogens?

Nicolas Olea (Granada, Spain) We performed a case control study of women with breast cancer including more than 700 women using a 150 question questionnaire and we analysed reproductive history and exposure assessment. We also assessed exposure by chemical analysis to give a total xenoestrogenic profile. There were 280 breast cancer patients and the rest were paired case controls. The results of our analysis are not yet available because the epidemiologist will need to assess the entire data before divulg- ing parts of the study.

Finn Bro-Rasmussen (Copenhagen, Denmark) Your presentation has stressed the necessity for considering the complexity of chemical mixtures present in the environment. You detected the presence of Aldrin, Dieldrin, Endrin, Mirex etc., but the use of these chemicals has been phased out in most of Europe. Do you think that the presence of these substances indicates recent use, or are they persisting in the environment as a result of earlier use?

Nicolas Olea We analysed samples from

women aged 35-65 years old and children who may have been exposed to the "- drins" previously, because these chemicals have been re- stricted or prohibited for the last 15 years. We do know that these chemicals have been used in cotton fields in Southern Spain and have per- sisted in the environment. The women and children in our study were highly contaminated suggesting that the persistent contaminants have entered the food chain, and also may pass from mother to child during pregnancy or lac- tation. There may be residual contamination of the water supply. We think that one of the most important sources for the children is from their mothers.

Finn Bro-Rasmussen It is surprising that you found Aldrin in human milk and tissues. Dr Brock indicated that Aldrin is metabolised to Dieldrin and therefore should not be detected.

Jose Russo (Philadelphia, USA) In your fat studies, did you always take adipose tissue from the same part of the body for extraction?

Nicolas Olea We obtained fat tissue from breast from the cases, and from the abdominal wall from abdominal surgery in the controls because our analysis was part of the breast cancer case control study. In order to correct for the quality of fat from the different locations we expressed our results as both pesticide content and total xeno-oestrogen burden per gram of lipid

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content in fat samples rather than by weight of tissue. We could not use fat from benign breast disease as controls as our epidemiologist indicated that this would introduce bias.

Ana Soto (Boston, USA) We have found good correlation between the oestrogen activity of water detected by the E-SCREEN assay, and the expected activity calculated from the concentrations of the oestrogenic contaminants in the water. Have you tested whether this correlation is also apparent in your fat samples com- paring chemical with biological assays?

Nicolas Olea We have not yet calculated the expected oestrogenicity from the amount of organochlorine residue content in fat samples. However, we are sure that the oestrogenic activity is not due to a single chemical but is a result of a combination of substances. We have correlated the oestrogenic activity with the total concentration of pesticides present. We measure 16 different pesticides at present and we are adding more to the list. It is possible that we are miss- ing the important compound, which has the greatest oestrogenic activity.

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human exposure to endocrine disrupters: standardisation of a marker of estrogenic exposure in...

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