-au^-i a. ::ML"i&1^d1ds 1 1-5 Functional Toxicology: A New Approach to Detect 1985 (5) that a body of data was presented which addressed the effect of environmental Biologically Active Xenobiotics estrogens on puberty in young children. John A. McLachlan The association between environmental fac- tors and precocious puberty remains un- National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 USA. established. However, concern was ex- pressed about the ubiquitous nature ofthese materials, their discordant potency, and xenobiotic estrogens, were summarized and their potential impact for public and en- evaluated. At that meeting, the possibilities vironmental health. A meeting held in 1992, for foreign chemicals to exert specific biolog- organized by the World Wildlife Fund, ical effects were clearly established. In fact, as explored the premise that these compounds shown in Figure 1, the theoretical basis for may have a deleterious effect on sexual environmental chemicals to exert hormone- development in a variety of wildlife species like effects is relatively straightforward. In (6). While these reports remain largely anec- the simplest model, chemicals can mimic a dotal at the present time, they again raised hormone by binding to its receptor and elic- the possibility that these molecules may iting a spectrum of biological effects. Con- have biological effects. versely, a foreign chemical that does not elic- For environmental estrogens, one may it these effects could bind a hormone recep- consider the work done with DES in experi- tor as an inactive compound and thus block mental systems, where there are human the response to the natural hormone. In studies for comparison, as a prototype. In either case, the result would be an alteration both experimental animals and humans, in the function of the hormone system. In prenatal exposure to the potent estrogen actuality, the processes involved are much DES is associated with, among other changes, more complex. vaginal cancer, paraovarian cysts, structural The preceding papers by Davis et al. and Many chemicals with different struc- abnormalities of the uterus and oviducts, Colborn et al. describe chemicals in the envi- tures were shown to exhibit weak estrogen- and reproductive dysfunction in the female ronment that may affect the immune, ner- like functions (Fig. 2). In addition to these offspring (4). vous, and endocrine system in ways that are diverse structures reported to be estrogenic, As with female offspring, male mice potentially deleterious to human health or other chemicals have been discovered. A exposed prenatally to DES express repro- the health ofa wide range ofanimals. Many study from our laboratory demonstrated the ductive tract lesions as adults which indude of the chemicals described exhibit biological estrogenic potential of some hydroxylated increased infertility, testicular abnormalities activities associated with the female sex hor- forms of polychlorinated biphenyls (PCBs) inconsistent with normal spermatogenesis, mone estrogen. The issue of biologically (4. More recently, the estrogenic substances epididymal cysts, and undescended testes. active environmental chemicals was raised at p-nonyl-phenol (3) and bisphenol-A (4) Similar findings have been reported for a symposium at NIEHS in 1979, "Estrogens have been shown to be released from plastic humans similarly exposed to DES in utero in the Environment" (1), in which concern under a variety ofconditions. (4 for the widespread distribution of advertent However, at the time of the 1979 meet- Exposure to estrogen during sexual hormones in the environment like diethyl- ing there were few, if any, documented development is also associated with femi- stilbestrol (DES), previously used in the cat- reports ofhuman health effects from xenoe- nization of the male reproductive system in tle industry, and inadvertent hormones in strogens. It wasn't until the next NIEHS mice (8) as well as in numerous other mam- the environment, such as DDT and other meeting, "Estrogens in the Environment" in malian, reptilian, and avian species; this, as yet, has not been reported in humans. I' I A Certainly, the lesions associated with devel- opmental exposure to a potent estrogen pro- vide indicators for potential effects associat- ed with much weaker environmental estro- gens. Although the possibilities for human HORMONAL health effects of hormonally active foreign chemicals remain theoretical, additional RESPONSE recent reports add to the growing sense that some diseases or dysfunctions associated with estrogens may have an environmental

*' * A : u component. For example, a recent report (S) suggests an association between organo- chlorines and breast cancer, and, in a com- B (HORMONE) mentary on the analysis that reported a sig- nificant decrease in human sperm count over the last 50 years (1J), the author specu- lates that this apparent drop in sperm may _>i NO EFFECT Address corrpespondence to J.A. McLachlan, NIEHS, PO Box 12233, Research Triangle Park, NC 27709 USA. Figure 1. Exogenous chemicals may act at hormone action sites. Received 14 July 1993; accepted 20 July 1993.

386 Environmental Health Perspectives - - e - - -- e*MP-a

OH OH ed with them; excitotoxins that interact with the NMDA, or excitatory amino acid receptor, would have another set of expect- HOJP HQ HO2X ed outcomes. Although this strategy would Diethystilbestrol Coumestrol in no way provide a complete toxicological Estradiol-17P profile of a chemical or replace full animal testing or epidemiology and public health strategies, it would generate essential, useful information for an important set of toxico- OH logical problems in a relatively short time and at relatively low cost. For example, HO 0 )o HO 000 using this strategy one could determine Equol Zearalenone 3,9-Dihydroxybenz[alanthracene what chemicals in the environment are DES-like and what their relative potency would be, at least, in an in vitro gene-activa- Ci Cl CI CH3 tion assay. Then one could develop further a plan to target either additional tests or HGl ci IC c ~~~~~OH public health approaches for these chemicals based on their functional toxicology. H3C O G5H11 CCl3 REFERENCS o,p-DDT Kepone Tetrahydrocannabinol (A9-THC) Figure 2 Chemicals reported to be estrogenic: steroidal 'natural" ovarian estrogens (estradiol); synthetic 1. McLachlan, JA, ed. Estrogens in the environ- potent estrogen (diethylstilbestrol); plant or phytoestrogens (coumestrol); estrogenic metabolites of plant ment. New YorklElsevier, 1980. substances (equol); mycotoxin estrogens (zearalenone); polycyclic aromatic hydrocarbon 'prohormone' 2. Korach KS, Sarver P, Chae K, McLachlan JA, (dimethylbenzanthracene); chlorinated hydrocarbon "prohormone" (o,p-DDT); fully chlorinated 'bioaccu- McKinney JD. Estrogen receptor binding activity mulative estrogen" (kepone); 'indirect" estrogen (tetrahydrocannabinol). See McLachlan et al. (13) for of polychlorinated hydroxybiphenyls: conforma- further details. tionally restricted structural probes. Mol Pharmacol 33:120-126(1988). be due to developmental exposure to estro- and gene activation. Thus, if a panel of 3. Soto AM, Justica H, Wray JW, Sonnensohein C. genic xenobiotics (11). receptor-containing cells were set up, one p-Nonyl-phenol: An estrogenic xenobiotic released from "modified" polystyrene. Environ These issues of biologically active xeno- could screen chemicals of unknown biology Health Perspect 92:167-173(1991). biotics have raised the need for a "new toxi- or toxicology and determine their function, 4. Krishnan AV, Stathis P, Permuth SF, Tokes L, cology" using available methodologies. A at least in regard to receptor-mediated gene Feldman D. Bisphenol-A an estrogenic substance great number of inter- and intracellular sig- activation. A related functional in vitro is released from polycarbonate flasks during auto- nals for cell differentiation, proliferation, screen for estrogens has been suggested by daving. Endocrinology 132:2279- 2286(1993). and function involve the interaction be- Soto et al. (6). 5. McLachlan JA, ed. Estrogens in the environment: influences on development. New York Elsevier, tween small molecules and their receptors. Screening for functionality could be 1985. A class of receptors that recognize most made fairly broad by having the panels 6. Colbom T, Clements C, eds. Chemically induced steroid hormones, thyroid hormones, indude the estrogen receptor, progesterone alterations in sexal and functional development: retinoids, and some vitamins are nuclear receptor, androgen receptor, glucocorticoid the wildlife/human connection. Princeton, NJ: transcription factors involved in gene regu- receptor, retinoid receptor, thyroid hor- Princeton Scientific Publishing, 1992. lation This dass and related families of mone receptor, peroxisome proliferator 7. Mori T, Nagasawa H, eds. Toxicity ofhormones (12). in perinatal life. Boca Raton, FL:CRC Press, genes also recognize some foreign chemicals, receptor, dioxin receptor, excitatory amino 1988. such as dioxin. Thus, some xenobiotics may acid receptors, and so on. At the end ofsuch 8. Newbold RR, Bullock BC, McLachlan JA. exert their effects, in part, through their an analysis, one could determine if a chemi- Miullerian remnants of male mice exposed prena- interaction with a nudear receptor and acti- cal of unknown biology and toxicology tally to diethylstilbestrol. Teratog Carcinog vation of the genes it regulates. In the case exhibited a known, measurable biological Mutagen 7:377-389(1987). of chemicals which interact with the estro- function. For a number of chemicals, one 9. Wolff MS, Toniolo P, Lee E, Rivera M, Dubin N. Blood levels of organochlorine residues and gen receptor, it is clear that their activity is could assign, in addition to melting point, the risk of breast cancer. J NatI Cancer Inst defined more by their function (estrogenici- molecular weight, and solubility, a func- 85(8):648-652(1993). ty) than by their structure. Thus, an under- tional description. This information could 10. Caisen E, Giweman A, Keiding N, Skakkebaek standing of receptor-mediated action of have public health significance because by NE. Evidence for decreasing quality of semen chemicals may help to define a function of understanding the functional basis of some during past 50 years. Br Med J 305:609-613 potential toxicological importance. toxicological responses, careful biological (1992). 11. Sharpe R, Skakkebaek NE. Are oestrogens To this end, we can suggest a research generalizations could be attempted. That is, involved in falling sperm counts and disorders of strategy referred to as "functional toxicolo- a chemical that is DES-like will have more the male reproductive tract? Lancet 341:1392- gy," in which chemicals are defined more by predictable, understandable toxicities associ- 1395, 1993. their function than by their chemistry. As ated with it, depending upon its dose, dura- 12. Parker, MG, ed. Nuclear Hormone Receptors. part of this strategy, cells, induding human tion, and time ofaction based on decades of London.Academic Press, 1991. cells, should be transfected with molecular with the 13. McLachlan JA, Newbold, RR, Korach KS, experience prototype compound Hogan M. Risk assessment considerations for constructs containing a specific receptor and and its paradigmic effects. Likewise, an reproductive and developmental toxicity of the reporter gene for that receptor. One unknown chemical functioning like retinoic oestrogenic xenobiotics. In: Human risk assess- then can assay in a relatively quick and acid, would have a different, yet, to some ment: the roles ofanimal selection and exmapola- straightforward way not only the binding of extent, predictable, set of toxicities associat- tion (Roloff MV, Wilson AW, eds). Lon- a chemical ligand to the receptor, but the ed with it. Chemicals that were dioxinlike don:Taylor and Francis, 1987;187-193. receptor occupancy of its response element would have another set oftoxicities associat-

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