Developing a Marker of Exposure to gull embryos exposed in ovo to DDT and other pesticides (4). Similarly, the decreased Xenoestrogen Mixtures in Human Serum reproductive success of alligators and tur- tles in Lake Apopka, Florida, was linked to Ana M. Soto, Mariana F. Fernandez, Maria F. Luizzi, a spill of kelthane, a pesticide formulation Anita S. Oles Karasko, and Carlos Sonnenschein containing DDE (5). Other evidence that certain chemicals acted as endocrine dis- Tufts University School of Medicine, Department of Anatomy ruptors was revealed when various occupa- and Cellular Biology, Boston, Massachusetts tional exposures in humans were reported. In 1949, aviation crop dusters handling It has been hypothesized that environmental estrogens may play a role in the increasing DDT were found to have reduced sperm incidence of breast cancer, testicular cancer, and other problems of the reproductive system. counts (6). While a single causal agent can While a single causal agent can be identified in cases in which humans have had occupational be identified in cases in which humans exposures, wildlife showing signs of reproductive damage have usually been exposed to a have had occupational exposures, wildlife combination of endocrine disruptors that may act cumulatively. The development of appropriate showing signs of reproductive damage are biomarkers of cumulative exposure, and their measurement at developmental points where usually exposed to a combination of exposure is critical, are required to test the environmental estrogen hypothesis. Measuring levels endocrine disruptors. Additionally, some of of each of the xenoestrogens in blood is a better approximation of real exposure at the target these chemicals may traverse the human organ level than inferring cumulative exposure by estimating from mass balance of dietary levels. placenta and have the potential to However, the cumulative estrogenicity of mixtures cannot be directly concluded from individual adversely affect the developing fetus (7). xenoestrogen plasma levels. Two approaches may be used to assess total load: a) the development Although some of these effects may be of methods to study mixtures of these xenoestrogens, to quantify their cumulative effects, and to obvious upon birth, others may not mani- begin to understand their interactions (i.e., additivity, synergy, antagonism, or independent maturity has action), so that plasma concentrations may be translated into units of activity such as "estradiol fest themselves until sexual equivalents"; and b) the development of methods to separate xenoestrogens from ovarian been reached. For example, boys exposed estrogens in blood and to directly measure the estrogenic activity of the xenoestrogen extract in utero to PCBs appeared normal during using a bioassay. The cumulative activity may be used as a marker of exposure to xenoestrogens. childhood, but at puberty their penises This article reports the development of a method to extract and separate xenoestrogens from were significantly smaller than those of ovarian estrogens using human serum as a source, followed by using a bioassay for determination nonexposed controls (7). of the cumulative xenoestrogen load as "estradiol equivalents." Environ Health Perspect It has been hypothesized that environ- 1 05(Suppl 3):647-654 (1997) mental estrogens may play a role in the declining quantity and quality of human Key words: xenoestrogens, estradiol equivalents, environmental exposure, p,p'-DDE, DDD, semen during the last 50 years, as well as in BBP, BPA, PCBs the increased incidence of testicular cancer and cryptorchidism in males and breast cancer incidence in both females and Introduction males in the industrialized world (8-10). Regarding breast cancer, the main risk fac- Xenoestrogens include several lipophilic, biphenyl (PCB) congeners. More recently, tor for its development is exposure to estro- persistent compounds to which humans nonchlorinated compounds used as antiox- gens throughout an entire lifetime (11). and wildlife have been exposed. Among idants and plasticizers were found to be Moreover, increased plasma levels of "bio- these synthetic chemicals are a number of estrogenic (1-3). The detrimental effects available" ovarian estrogens (not bound to chlorinated organics, such as the insecti- ofenvironmental exposure to xenoestrogens sex hormone-binding globulin [SHBG]) in cides kepone, dieldrin, DDT and its are evident when observing the abnormal postmenopausal women correlate positively metabolites, and some polychlorinated development of the reproductive system in with breast cancer (12). Among the estrogenic xenobiotics, PCBs and DDT were considered suitable This paper was presented in part at the Workshop on Hormones, Hormone Metabolism, Environment, and markers of exposure for breast cancer Breast Cancer held 28-29 September 1995 in New Orleans, Louisiana. Manuscript received at EHP 6 June 1996; manuscript accepted 8 August 1996. because they were released massively into This work was partially supported by grants from the W. Alton Jones Foundation, U.S. Environmental the environment beginning approximately Protection Agency (CR 820301), National Institutes of Health (CA-13410), National Science Foundation (DCB- 50 years ago and they are persistent; their 9105594), the Silent Spring Institute, and the Massachusetts Department of Public Health (DPH 79005-214 Hi 1). The assistance of The Center for Reproductive Research at Tufts University (P30 HD 28897) is gratefully presence in serum may represent cumula- acknowledged. We are also grateful to C. Michaelson for her skillful technical assistance. tive exposure during a lifetime. Three Address correspondence to Dr. A.M. Soto, Tufts University School of Medicine, Department of Anatomy recent studies showed a correlation between and Cellular Biology, Center for Reproductive Research, 136 Harrison Avenue, Boston, MA. Telephone: (617) the occurrence ofbreast cancer and levels of 636-6954. Fax: (617) 636-6536. E-mail: [email protected] Abbreviations used: BBP, benzylbutylphthalate; BPA, bisphenol-A; CD, charcoal-dextran; CDHuS, CD human xenoestrogens. Wolff et al. (13) found that serum in phenol red-free DMEM; DDD, dichlorodiphenyldichloroethane; DDE, dichlorodiphenyltrichloroethyl- serum DDE levels correlated with breast ene; DDT, dichlorodiphenyltrichloroethane; DES, diethylstilbestrol; DMEM, Dulbecco's modified Eagle's cancer incidence in a study of 58 breast medium; DMSO, dimethyl sulfoxide; E2, 17,Bestradiol; EEqs, estradiol equivalents; HCB, hexachlorobiphenyl; HPLC, high-performance liquid chromatography; Pa, pascals; PCBs, polychlorinated biphenyls; PgR, proges- cancer patients and 171 controls that were terone receptor; RPE, relative proliferative effect; SHBG, sex hormone-binding globulin; tD, doubling time. well matched for risk factors and age. Environmental Health Perspectives * Vol 105, Supplement 3 * April 1997 647 SOTO ET AL. Another study documented that estrogen- determine the effect of xenoestrogen mix- immediately before use. A 5% charcoal- receptor positive breast cancer correlated tures by means of the E-SCREEN bioassay are 0.5% dextran T70 (Pharmacia-LKB, with higher concentrations of DDE in their presented. This bioassay measures the pro- Uppsala, Sweden) suspension was pre- tissues (14). Krieger et al. (15) studied 150 liferative effect of estrogens on their target pared. CD suspension aliquots of a volume women with breast cancer and 150 controls; cells. The proliferative effect of xenoestro- similar to that of serum aliquots to be each set consisted of 50 African-American, gens is expressed as "estradiol equivalents." processed were centrifuged at 100xg for 10 50 Caucasian, and 50 Asian-American min. Supernatants were aspirated and women. When the data from all ethnic Materials and Methods serum aliquots were mixed with the char- groups were pooled, no significant correla- Chemicals coal pellets. This charcoal-serum mixture tion was observed between plasma levels of was maintained in suspension by rolling at DDE and breast cancer (15). However, Estradiol-17P (E2) was obtained from 4 cycles/min at 370C for 1 hr. This suspen- when the cases and matching controls were Calbiochem (Richmond, CA). DDT sion was centrifuged at 2000xgfor 20 min. evaluated separately, according to their eth- (technical grade), o,p'-DDT, p,p'-DDT, The supernatant was then filtered through nic group, high serum DDE levels were cor- o,p'-DDD, p,p'-DDD, p,p'-DDE, a PCB a Nalgene filter with a pore size of 0.45 related with breast cancer incidence in congener (2,2'3,3'6,6'-hexachlorobiphenyl pm. More than 99% of serum sex steroids Caucasian and African-American women [2,2',3,3',6,6'-HCB]), o-phenylphenol, were removed by this treatment, as mea- but there was no significant correlation in m-phenylphenol, p-phenylphenol, and ben- sured by removal of 3H-estradiol (17); Asian-American women. Evidence of a link zylbutylphthalate were from Ultra Scientific estradiol concentrations after CD treat- between exposure to PCBs and breast cancer (North Kingstown, RI). Bisphenol-A was ment were less than 0.01 pg/ml as mea- incidence is equivocal (16). However, these purchased from Aldrich Chemical Co. sured by radioimmunoassay. CD-treated studies correlated exposure to total PCBs, (Milwaukee, WI). Each of these chemicals sera were stored at -20°C until needed. rather than to the levels ofspecific congeners. was dissolved in ethanol or dimethyl sulf- Samples kept in the freezer for 1 year Epidemiological studies should be oxide