human exposure to endocrine-disrupting chemicals: assessing the total estrogenic xenobiotic burden

trends in analyticalchemistry, vol. 16, no. 10, 1997 613

Human exposure to endocrine-disrupting chemicals: assessing the total estrogenic xenobiotic burden

Ana Rivas, Nicolh Olea* Labora tory of Medical lnves tiga tions, Department of Radiology, University of Granada, 18071 Granada, Spain

F6tima Olea-Serrano Department of Nutrition and Food Science, School of Pharmacy, University of Granada, 18071 Granada, Spain

A relationship has been hypothesized between adverse effects on human and wildlife reproductive health and a number of chemical substances capable of altering hormonal homeostasis. A testing system to screen for endocrine activities and the development of appropriate biomarkers of cumulative exposure are required. This article reports the work of our group in the following areas: (i) the identification of chemical agents with estrogenic hormonal activity, (ii) existing evidence on forms and sources of human exposure, and (iii) developing a methodology to assess the total estrogenic burden, defined as the estrogenic activity in a bioassay of human samples from which ovarian estrogens have been previously eliminated.

0 1997 Elsevier Science B.V.

1. Introduction

The existence of chemical substances capable of altering hormonal homeostasis (endocrine-disrupting chemicals, EDCs) has been known for some time. Almost 50 years ago, Singer reported a clinical suspicion of an association between exposure to certain pesticides and the failure of sperm production in workers exposed to chemical compounds in commercial formulations [ 11. However, despite clinical reports of associations between

*Corresponding author.

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exposure and disease and the identification of hormonal xenobiotics, causal relationships have not always been established. Nevertheless, clinical reports have generated sufficient ‘reasonable suspicion’ to make the following types of study manda- tory:

Epidemiological analyses of health effects on exposed populations Basic research to elucidate the mechanisms of activity of these chemical substances Construction of models that to a more or less simplified degree reproduce the phenomena of exposure, impregnation, and metabolism and demonstrate consequent biological effects. The body of information from experimental

models is sufficiently complex to generate high levels of concern, uncertainty and scepticism and is interpreted in very distinct ways by the different parties involved. Industry, government, regulators and health researchers are confronted by a complex puzzle with missing pieces and try respectively, according to the best criteria available and in the light of their own convictions, to produce more and better chemical products that the user can have confidence in, to regulate exposure, to prevent health risks, and to give reliable and useful scientific information.

Environmental studies showing an association between exposure to chemical contaminants and harmful effects on development and hormonal homeostasis in animal species are much more numerous than those studying effects on humans [ 2,3]. It has often been assumed that what occurs in the animal world is an advance warning of a phenomenon subtly occurring in the human species but that is yet to manifest itself in its full form [ 41. Knowledge gained in studying the animal world suggests that the absence of clear effects in humans may be due to a number of different circumstances, including the long human life cycle and the multi- ple causes of associated diseases, which also often have long latency periods.

In spite of the difficulties in identifying causes of diseases that are linked to development, hormonal

0 1997 Elsevier Science B.V. All rights reserved.

614 trends in analytical cbemisfry, vol. 76, no. IO, 1997

homeostasis and the balance between cell production and cell death, progress in our understanding of these morbid processes is essential, especially given the large numbers and severity of these diseases. For this reason, a great effort is being made in many fields of biology and medicine to identify the causes and pathogenesis of diseases related to exposure to chemical substances with hormonal activity. These etiological studies aim both to identify causal agents -chemical compounds capable of interfering with the hormonal system in very different ways (agonist, antagonist, additive, syner- gistic) - and also to describe pathogenic mechanisms.

We present the work of our group in the following areas: ( 1) the identification of chemical agents with estrogenic hormonal activity, (ii) gathering evidence on forms and sources of human exposure, and (iii) developing a methodology to assess the total estrogenic burden, defined as the estrogenic activity in a bioassay of human samples (serum, fat) from which ovarian estrogens have been previously eliminated.

2. Catalogue of endocrine-disrupting chemicals

Numerous studies have defined the estrogenicity of certain pesticides [ 5,6]. DDT and its metabolites, kepone, methoxychlor, toxaphene, dieldrin and endosulfan are some of the compounds that present estrogenic activity in different models and systems. Residues of these organochlorine pesticides have been found in human fat, breast milk, urine and blood [ 7 1.

Polychlorinated bisphenols (PCBs) are another group of xenoestrogens, which are widely used as electrical isolators and because of their stability have been used as plastifiers, in paints, lubricants, isolating coverings, etc. There is clear evidence that these products can alter endocrine function in fish, birds and mammals, including humans [ 8 1. The presence of some PCB congeners has been described in almost all kinds of organism and in almost every part of the world [ 7 1.

Recently, the possibility has been studied that some man-made chemicals present in surface waters and aquatic sediments may adversely affect reproduction in fish [ 9 1. Alkylphenol polyethoxy- lates and their biodegradation products belong to a group of non-ionic surfactants with proven estro- genie activity [ 10,111. High concentrations of

these products have been found in the sediment around sewer effluents [ 121 and have been shown to bioaccumulate in several species [ 13 1.

Another class of EDCs that mimic estrogens is a group of diphenylalkanes known as bisphenols. Many reports have described the estrogenicity of some of these products [ 14,15 1, which are widely used in the plastics industry. Bisphenol A is used as prime material in the manufacture of polycarbon- ates, epoxy resins, phenolic resins, polyesters and polyacrylates, which gives some idea of the exten- sive production of bisphenols and of the enormous quantities released into the environment and to which humans and wildlife are exposed.

Alteration of endocrine or reproductive function is not always due to man-made products. Phytoes- trogens, for example, are naturally occurring estrogens found in plants and have a long history of co- evolution with humans and animals [ 16 1.

One of the fullest and most recent accounts of chemical compounds related to estrogenic activity, published in 1995, is already out of date [ 6 1. New substances that can mimic sexual hormones have since been discovered; new compounds with estro- genie hormonal activity are swelling the ranks of the xenoestrogens, such as the phlalates [ 61, or the diphenylalkanes of similar structure to bisphenol A [ 17 1. Furthermore, complementary hormonal activities have now been assigned to some of the chemical products gathered in 1995. Many products classified as environmental estrogens can act by binding to more than one type of steroid receptor; e.g., o,p-DDT and chlordecone can bind to either estrogen or progesterone receptors with similar affinities [ 18 1. Nonylphenol and the metabolites of methoxychlor, 2,2-bis-(p-hydroxyphenyl)- 1 ,l, 1 -trichloroethane, have the ability to inhibit binding to estrogen, progesterone and androgen receptors [ 18 1. This is also true for endosulfan, which has recently been demonstrated to be a good competitor for the progesterone receptor [ 19 1, and for DDE, which appears to be an excel- lent competitor in the displacement of dihydrotes- tosterone for its binding to the androgen receptor 1201.

3. Identification of endocrine-disrupting chemicals

There is an urgent need to establish validated in vivo and in vitro assays to test the activity of endocrine-disrupting chemicals [ 21-23 1. Predictive

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tests are complex and it is difficult to select the appropriate dosage for these studies as the chemicals may not follow the classic dose-response model [ 22 1. Moreover, the different molecular structures of estrogenic xenobiotics, to take one example, make it impossible to predict which chemical may act as an endocrine disrupter, so that all substances have to be tested.

The E-SCREEN test stands out among the different in vitro tests so far proposed; otherwise known as the MCF7 cell proliferation assay, the E- SCREEN test is very sensitive, easy to perform and can screen many compounds over a wide range of concentrations. E-SCREEN was used to identify the longest series of estrogenic xenobiotics to date [ 61 and it is striking that no false positives or false negatives were observed among the estrogens and non-estrogens tested.

The induction of cell proliferation is regarded as the hallmark of estrogenic action, but an estrogenicity marker could also be sought in the quantity of proteins synthesized by estrogenic stimulus. Tests using MCF7 breast cancer cells offer a wide range of end-points, from estimation of cellular proliferation to that of progesterone receptor (PgR) induction, and including the synthesis and secretion of cell-type specific proteins such as pS2. The PgR assay in MCF7 cells is a good model because the synthesis and cell availability of the progesterone receptor are estrogen-dependent. Study of the pS2 protein synthesized in estrogen-dependent cells after exposure to estradiol has demonstrated that the accumulation of protein in the culture medium reflects not only the growth of the cell population but also the over-expression of pS2 [ 6,15, 17,241.

To summarize, we need a combination of in vivo and E-SCREEN-type in vitro assays to identify the hormonal activity of any chemical. A single test able to characterize any hormonal activity is not feasible, and we must establish a sequence of new tests able to reveal the hormonal character of chemical compounds. It would be economical and useful to extend the end-points of routine toxicology tests in order to gather information related to endocrine activity, which is inadequately assessed in existing protocols.

Tests that identify EDCs could also be useful for assessing risk, offering to epidemiological studies estimations of human exposure when the substance

has already been used, and informing regulatory authorities of the potential hazards if the chemical product is new.

4. Human exposure to endocrine- disrupting chemicals

The greatest problem in determining exposure to the xenobiotics in which we are interested is that this exposure is environmental. This is true for estrogenically active chemical compounds in pesticides, electrical insulation materials, detergent additives or plastic goods. Despite restrictions imposed on the manufacture and employment of some of these, their use is currently very wide- spread.

The main source of exposure to estrogenic xenobiotics is food and to a lesser degree water. Estro- genie activity has, for example, been detected in foods preserved in cans, and bisphenol A was identified to be responsible for this [ 141.

Another possible contribution to human exposure to EDCs is via dental composites and sealants, which contain bisphenol A and its derivatives. When saliva samples were analyzed after standard treatments, those with a large number of these compounds showed estrogenicity in the biological assay [ 151.

Once possible sources of exposure to EDCs are analyzed it is necessary to establish how the levels found in the environment can reflect impregnation of individuals and what the observable effects are. The life stage when the exposure occurs plays a very important role and the relative impact of exposure at different stages of development must be established. Mother-child exposure seems to be of greater importance than might at first have been thought. The accumulation of hormonal xenobiot- its in fat tissue during the life of the mother may be a major source of exposure for the child, both during gestation and via breast-feeding, which would provide an explanation for the levels of some xenobiotics detected in the fat tissue of young children [ 25 1. Humans, especially children, may be inad- vertently exposed to these xenobiotics, which may act cumulatively and in combination with endoge- nous substances.

Human exposure to EDCs may come from many sources and is not limited to populations at high

616 trends in analytical chemistry, vol. 16, no. 10, 1997

risk, and we set ourselves the problem of how to measure the levels of these products to which individuals have been exposed during their life.

5. Total estrogenic xenobiotic burden (TEXB)

Diseases with a long latency period and with many factors implicated, such as cancer in sex hormone-dependent organs (breast, testicle or pros- tate), could be related to human exposure to EDCs. The data are not conclusive, and while some studies have related breast cancer to exposure to DDT and its derivatives [ 26 1, the evidence for this relation is very controversial [ 271.

One handicap in the design of experimental models is the isolated analysis of the chemicals. Humans are not exposed to just a single product but rather to a complex mixture of substances. Bio- markers of exposure to xenoestrogens must be established to relate total estrogenic burden to the risk of suffering a particular disease. Many of the xenoestrogens that have been identified are lipophilic, so that they accumulate and persist in human tissue. Diseases with a long latency may be related to environmental exposure to particular xenoestrogens.

Measurement of the activity of these products in fat samples could provide one indicator of the exo- genous estrogenic burden.

The assessment of the total estrogenic burden - defined as the cumulative estrogenic effect of xenobiotics - and the identification of the contaminants responsible could be correlated with the risk of suffering a tumoral disease and with the previous estimation of exposure.

Based on this hypothesis we are running a hos- pital-based epidemiological study of paired cases and controls. This continuing study includes more than 200 patients surgically treated for breast cancer and a matching group of women with no sign of estrogen-dependent disease. We analyzed samples of fat tissue and serum from the study population and identified and quantified the estrogenic substances in them. The low concentrations of these products that we found led us to carefully address the analytical determination and quality assurance to obtain accurate measurements of biological exposure.

The analytical measurements of xenoestrogens in cases and controls are being considered along-

side other risk factors, which are to be gathered in a questionnaire for a parallel analysis.

6. Extraction of xenoestrogens from fat tissue

We extracted xenoestrogens from fat tissue by the method described by Okond’ Ahoka et al. with modifications [ 28 1. A solid-liquid procedure used Pyrex glass columns; the column was filled with Alumine Merck 90 No. 1097; 0.1 g of breast fat tissue (from non-tumoral surgery in controls) was dissolved in hexane. It was then eluted in column with the same solvent. The eluate obtained was concentrated at reduced pressure, under a stream of nitrogen, to a volume of 0.5 ml for later purifi- cation by preparative liquid chromatography.

7. Semi-preparative high performance liquid chromatography (HPLC)

A preparative liquid chromatography method was developed to allow the separation of xenoestrogens from natural estrogens without destroying the xenoestrogens. Xenoestrogens were eluted by a gradient with two mobile phases: n-hexane (phase A) and n-hexane-methanol-2_propanol(40:45: 15 v/v) (phase B) at a flow rate of 1 .O ml/min. The gradient program follows the technique described by Medina et al. [ 29 ] modified for adaptation to the preparative HPLC procedure. Three fractions (a, x and p) are separated by HPLC: fraction a is the eluate collected in the first 11 min and contains xenobiotics from pesticides (DDT and metabolites, endosulfan, dieldrin, chlordecone and methoxychlor); fraction x is collected between 11 and 13 min; fraction p is collected between 13 and 25 min and contains additives and monomers from plastics, such as bisphenols, and natural hormones. Fig. 1 depicts the chromatographic profile by preparative HPLC of a study sample of fat tissue.

8. Gas-liquid chromatography (GLC)

The three fractions are dried, dissolved in hexane, marked with an internal pattern (2,3,4-PCB ) and then injected into a gas chromatographer to determine in which fraction the pesticides elute and to assess the performance of the extraction. In gas chromatography with electron capture detec-


Fig. 1. Chromatographic profile by preparative HPLC of a fat extracted from a breast cancer fat tissue sample.

Fractions a, x and p are defined by elution time. In fraction a lipophilic organochlorine pesticides eluted as

demonstrated by gas chromatography.

tion (ECD ) the x and p fractions are silent. Table 1 lists the percentage recovery for each product.

Table 1 Recovery of pesticides from a spiked fat tissue sample

Product Amount addeda Recovery (%)

Lindane Aldrin Dieldrin Endosulfan Mirex Kepone Endrin

50 ng/4 ml 90.5 f 3.1 50 ng/4 ml 82.6zt2.7 50 ng/4 ml 97.1 ct 2.3 50 ng/4 ml 95.1 f 4.5 50 ng/4 ml 87.4k4.1 50 ng/4 ml 94.2 f 3.5 50 ng/4 ml 98.8 zt 2.4

“Corresponds to 0.1 g of fat tissue in 4 ml of hexane.

A search of the literature showed that the best technique for determining organochlorine pesticides in different types of samples is gas chromatography with ECD [ 30,3 11. Our group is using gas chromatography to assess contamination levels presumably existing in the environment and in humans in order to calculate the estrogenic burden of individuals and correlate it with the risk of developing breast cancer.

9. Quantitative evaluation of estrogenic activity

The a and p fractions obtained by preparative liquid chromatography (the fractions expected to

618 trends in analytical chemistry, vol. 76, no. 10, 1997

g 120 t

$ 80- $

10_‘0 Estradiol [M]

Fig. 2. Dose-response curve to estradiol of human breast cancer MCF7 cells. The a fraction collected after preparative HPLC of 0.1 g extracted fat sample shown in Fig. 1 was tested in the E-SCREEN. The proliferative effects of l:l, 15 and 1:lO dilutions were read in the dose-response curve. An estimation of 0.16 nMestradiol equivalents was assigned to this sample.

contain xenoestrogens), were dried and then tested in the E-SCREEN to determine the total estrogenic load. To this end we calculated the dose-response curve to estradiol of MCF7 cells (Fig. 2). The two fractions were resuspended in 5 ml of the charcoal dextran-treated culture medium and a series of dilutions were made (l:l, 1:5, 1:lO). The dilutions were tested by E-SCREEN. Cell numbers were converted to estradiol equivalents, calculated from the dose-response curve for estradiol.

10. Summary

Many studies support the idea of a relationship between adverse effects on human and wildlife reproductive health and a number of environmental contaminants that have demonstrated estrogenic activity in laboratory studies. There is a growing demand by the public and by regulatory agencies for an evaluation of the role of xenoestrogens in human health.

A testing system must be created because many chemicals already available or about to come on the market have not been routinely screened for endocrine activities. We must clarify basic mechanisms of activity and cross-species differences in pharma- cokinetics and pharmacodynamics and establish the relative importance of uterine/fetal compared with adult exposure.

Wildlife and humans are continuously exposed to xenoestrogens from different sources, such as diet or environment, and we must know the effects of mixtures of these compounds on health. Data on the presence of environmental and dietary estrogens suggest that these compounds may interact. Soto et al. [ 321 demonstrated that the effect of a mixture of xenoestrogens is higher than expected assuming simple additivity.

A new toxicology approach is required that uses existing methodologies. McLahan has proposed a research strategy in functional toxicology by which chemical products are defined more by their function than by their chemical structure [ 33 1. The E- SCREEN test is concerned with function, and can rapidly analyze chemical products of unknown biology and toxicology to determine their biological function; it can measure the estrogenic activity of mixtures of xenoestrogens, expressed as ‘estradiol equivalents’. The use of this parameter in extracts of biological samples in our ongoing epidemiological study gives us an indication of real impregnation and biological function. The subse- quent gas chromatography analysis tells us which contaminants are responsible for the effect observed in the biological assay. When we incor- porate the risk factor analysis included in our study we shall be able to correlate total estrogenic burden with the development of breast cancer.

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Professor Nicola’s Olea and Dr. Ana Rivas are at the Labora tory of Medical lnvestiga tions, Department of Radiology, University of Granada, 18071 Granada, Spain. Professor Fa’tima Olea-Serrano is at the Department of Nutrition and Food Science, School of Pharmacy of the University of Granada, 180 17 Granada, Spain. The research group that they represent has been involved for the last 10 years in the study of endocrine-disrupting chemicals from a multivariate approach: identification, human exposure, tissue burden and health effects.

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human exposure to endocrine-disrupting chemicals: assessing the total estrogenic xenobiotic burden

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