C Pharmacology & Toxicology 2000, 86, 24–29. Copyright C Printed in Denmark . All rights reserved ISSN 0901-9928 Endocrine-Disrupting Agents on Healthy Human Tissues Guglielmo Paganetto 1 , Franca Campi 2 , Katia Varani 2 , Adriano Piffanelli 2 , Gloria Giovannini 2 and Pier Andrea Borea 2 1 Polyolefins ‘‘G. Natta’’ Research Center, Montell, 44100 Ferrara, and 2 Department of Clinical and Experimental Medicine, University of Ferrara, 44100 Ferrara, Italy (Received April 21, 1999; Accepted August 5, 1999) Abstract: A vast number of substances have been suggested as possibly contributing to perturbation of the endocrine system. Several have been tested with different approaches ranging from yeast expression system of human oestrogenic receptors to human breast cancer cells assays. Surprisingly, no inhibition-binding experiments to steroid receptors on healthy human tissue have been performed so far. Our study provides inhibition binding experiments to oestrogens, progesterone, testosterone and retinoic acid receptors in prostate and uterine human tissue of organochlorine pesticides, phthalate esters, oestrogenic constituents derived from plants and phenol derivates. Affinities of significant extent of phthalates on oestrogenic, progestinic and androgenic receptors have not been detected. As for retinoic acid receptors, mono(2-ethylexyl)phthalate provokes a notable reduction of the binding of the tritiated retinoic acid, phtalic acid ethyl- n-butyl ester and 4-octylphenol show an affinity comparable to that of isoflavonoid genistein, whereas 4-nonylphenol reduces the binding of retinoic acid in prostate. The possibility of wide-spread adverse human and wild-life impacts from environmental chemicals causing interference with normal endocrine function has received increasing attention from the scientific community, the media, and other parties concerned over the last several years. Several studies deal with this matter. The incidence of female breast cancer has increased substantially since 1950. A frequently cited article in 1993 suggested a connection between breast cancer and man-made oestrogen-like chemicals (Davis et al. 1993). The effects most often cited as possibly due to indus- trial ‘‘endocrine disruptors’’ are declining sperm counts, dramatic increases in prostate and testicular cancer, and in- crease in the incidence of cryptorchidism, and abnormalities in the reproductive organ (McLeod & Wang 1979). Most frequently cited are sex differentiation and reproductive problems in fish-eating birds, feminization of trout and re- productive abnormalities and failures among alligators in Florida’s Lake Apopka (Rolland et al. 1995). In the last few years the basic hypothesis has been intro- duced that a wide variety of man-made chemicals in com- monly used throughout the world, and particularly the ones that persist and accumulate in the environment and human and wild-life tissues, may be interfering with the normal functioning of human and wild-life endocrine systems thus causing the above mentioned effects. Recently new studies have given rise to an international debate which has brought the terms of the problems into focus. Arnold et al. (1996) have found 150 to 1600 times Author for correspondence: Pier Andrea Borea, Department of Clinical and Experimental Medicine, University of Ferrara, Via Fossato di Mortara, 17–19, 44100 Ferrara, Italy (fax π39 0532 291205, e-mail bpa/dns.unife.it). increase in synergistic interactions between binary mixtures of the weak oestrogenic pesticides endosulfan, dieldrin, tox- aphene, and chlordane in competitive oestrogen receptor binding assays and in an oestrogen-response assay in yeast. On the basis of these data, it has been suggested that the oestrogenic potency of some environmental chemicals, when tested singly, may be underestimated. Not only have synergistic effects not been detected in previous studies (Ko- hono et al. 1994; Simons 1996) but they have not been con- firmed (Ramamoorthy et al. 1997) and have been later re- tracted (McLahlan 1997). Most of these authors have per- formed competitive binding experiments on assays based on several tissues such as liver of rainbow trout (Jobling et al. 1995), human breast cancer cells (Jobling et al. 1995; Soto et al. 1995; Harris et al. 1997) and yeast (Arnold et al. 1996; Simons 1996; Ramamoorthy et al. 1997) but, surprisingly, no binding studies on human healthy tissues have been per- formed. The first aim of the present study was to evaluate the affinity of single molecules of environmental and indus- trial interest by direct binding assays on oestrogenic and progestinic receptors from female uterus and on androgen receptors from human prostate gland. Moreover, as retinoic acid receptors are involved in a signalling call-talk with oes- trogen receptor through oestrogen-responsive elements (Keller et al. 1995), we decided to extend the study to evalu- ate also the affinity of tested compounds to retinoic acid receptors in prostatic and uterine human tissue. Materials and Methods Chemicals. [2, 4, 6, 7, 3 H] 17b-oestradiol (96 Ci/mmol), [ 3 H]16a- ethyl-21 hydroxy-19 norpreg-4 ene 3, 20-dione (ORG-2058, 52 Ci/ mmol), [17a methyl- 3 H]- 17a methyltrienolone (R1881 85 Ci/ mmol), [20-methyl- 3 H]. All trans retinoic acid (74 Ci/mmol) were