ENVIRONMENT
Environmental Hormone Disrupters Focus Of Major Research Initiatives • Synthetic hormone disrupters in environment adversely affect wildlife, but human effects are not yet established Bette Hileman, C&EN Washington
T
he hypothesis that chemicals in the environment disrupt hormone systems in the human fetus and thereby cause male birth defects, lower sperm counts, and testicular cancer has received a great deal of media attention recently. The attention was prompted in part by a spate of new scientific articles on the subject, and by the publication of the book "Our Stolen Future," by Theo Colborn, Dianne Dumanoski, and John Peterson Myers (C&EN, April 1, page 34). The intense public concern over the issue and serious interest in the scientific and regulatory community have given rise to major government and industry research initiatives. In the popular press, the hypothesis is often characterized as either a confirmed scary reality or chimeric science. In fact, the truth probably lies somewhere between these two extremes, say most experts in the area. The hypothesis is supported, although not proved, by hundreds of peer-reviewed scientific studies, and therefore seems plausible to many specialists. The National Institute for Occupational Safety & Health, for example, reports that individual exposures to more than 1,000 chemicals found in the workplace cause reproductive effects in lab animals. A report on an Environmental Protection Agency-sponsored workshop last year on endocrine disrupters cites almost 200 studies. But the crucial link between chemicals in the environment and adverse effects on the general population has not 28
MAY 13,1996 C&EN
been well established. The critical issue is whether there are sufficiently high levels of synthetic endocrine-disrupting chemicals in the ambient environment to exert effects on the general population. And if synthetic endocrine disrupters are harmful, what is the role of the natural endocrine disrupters found in many plants? Despite the uncertainties, some scientists, such as chemist Nicholas A. Ashford, professor of technology and policy at Massachusetts Institute of Technology, believe that the potential danger from synthetic endocrine disrupters is so great that they are calling for immediate preventative action. They say that the time has come to place an international ban on many substances that are known to be persistent, notably dioxins, polychlorinated biphenyls (PCBs), which are still in use, and those pesticides, such as DDT, that have clearly been shown to harm the endocrine system. Other specialists are more cautious. "When you scrutinize the data," says John P. Giesy, a toxicologist at Michigan State University who has worked on wildlife contaminants such as dioxins and PCBs for many years, "it doesn't seem like there is very strong evidence that there are widespread effects in human populations." However, he doesn't rule out that possibility. "These compounds cause effects at very low dose and there is reason to be concerned, because in wildlife we have some effects that can be attributed to contaminants." Consequently, "we should probably screen chemicals more than we do," he says. A third position is espoused by the American Crop Protection Association (ACPA), which contends that, based on current evidence, no new tests are needed to detect endocrine disruption in screens of pesticide safety. It and many other groups say no additional regulatory action is needed unless studies establish clear links between
human exposure to hormone disrupters and adverse effects. A number of studies are being planned by academic, government, and industry scientists that will seek to prove or disprove such links. However, carrying out research in this area is not an easy task, according to Paul M. D. Foster, head of endocrine disrupter research at the Chemical Industry Institute of Toxicology. CUT, a nonprofit research institute located in Research Triangle Park, N.C., is supported by nearly 40 companies. Foster points out some of the complications involved. In vitro tests can give useful data, he says, but they cannot provide conclusive information about whether a substance will act as an endocrine disrupter in living animals. He poses three important questions: • What are the metabolites? • Do they cross the placental barrier in sufficient amounts to be active? • Do they have a structure that can interfere with hormones in the fetus? Dose-response studies with endocrine disrupters are also complicated, Foster says. In typical cancer bioassays, rather high doses are used, and from the effects seen at high doses, the re-
Foster: endocrine system is dynamic
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[email protected] mal offspring of diethylstilbestrol (DES), the one synthetic estrogen that was given to mothers during pregnancy; and epidemiological research on sperm counts, testicular cancer, and male birth defects. What makes the hypothesis plausible to many scientists is that some of the physiological changes now being seen in human populations can be induced in lab animals with exceedingly low levels of various endocrine disrupters—disrupters that are found in all humans and in the environment. However, major unGuillette examines alligator from Lake Apopka, certainties surround the searcher extrapolates to predict effects human data. First, it is not clear whether at low doses. "But when you get high or to what degree sperm counts are fallsuperphysiological concentrations of ing, and if they are declining in some hormones [or endocrine disrupters], places, whether this is caused by chemithe system starts to self-regulate/' he cal exposure. The incidence of testicular explains. "It shuts itself down." As cancer is rising almost everywhere, but it hormone levels get higher and higher, is not known if this can be attributed to the response lessens because the cell exposure to synthetic chemicals. Male starts to become desensitized to the ef- birth defects in the reproductive tract are fects of the hormone. A researcher increasing markedly in some places, but could find something wrong after giv- again the link to chemical exposure is ing a massive dose of an endocrine dis- not clear. The relative importance for hurupter, but that effect wouldn't predict mans of plant estrogens versus synthetic the subtler effects at lower doses, Fos- hormone disrupters also is not known. ter says. And finally, only a few synthetic Another problem is that the endo- chemicals have been tested for endocrine crine system is dynamic, Foster says. disruption, so the universe of substances "It's supposed to go up and down. that could be disrupting hormones is That's the normal state. So it's not like known only in part. Some of those other toxic endpoints where you look chemicals that appear in in vivo or in for a zero response. What you are look- vitro tests to disrupt hormones are dioxing for is some kind of modulation of ins; planar PCBs; DDT; DDE, the prodthe normal, which is supposed to be in uct of the metabolic breakdown of DDT; a dynamic phase." Kepone; the fungicide vinclozolin; the Given these uncertainties and diffi- plastic monomer bisphenol-A; and nonculties, it is important to look at how ylphenol, which is used in plastics and is the endocrine disruption hypothesis a breakdown product of nonylphenol arose. Scientists need to look at what is ethoxylates in some detergents. known to support it with reasonable Scientists have published thousands certainty and what the important un- of studies showing that chemicals in fairanswered questions are. ly heavily contaminated areas cause endocrine disruption in wildlife. For examOrigin of hypothesis ple, Louis J. Guillette Jr., a reproductive The hypothesis emerged mainly from endocrinologist at the University of Florthree major types of studies: research on ida, Gainesville, found that 60% of male the effects of endocrine-disrupting alligators in central Florida's Lake Apopchemicals on wildlife and lab animals; ka, which was the site of a pesticide (distudies of the effects on human and ani- cofol) spill in 1980, have shorter than
normal penises and sex hormone levels close to those of female alligators. The dicofol was contaminated with DDT at a level of 15%, which has metabolized into DDE, a known antiandrogen hormone disrupter. Recently, Charles J. Henny, leader of the Northwest Research Station of the Interior Department's National Biological Service, Corvallis, Ore., completed a major study of river otters. He examined nine- or 10-month-old male otters living in the lower Columbia River and compared them with otters of the same age inhabiting much less polluted waters outside the Columbia River Basin. He found that the weights of the otters' testicles and baculums (a bony structure in the penis) correlated inversely with the degree of contamination in the animals' livers, which reflects the contamination in the part of the river where they lived. In Columbia River Basin otters, most of the baculums are about half the normal size, and the testicles are about one-seventh normal weight, whereas otters outside the Columbia River Basin have reproductive organs of normal size. Because the contaminants in the Columbia River—PCBs, dioxins, furans, insecticides, and some metals—are all intercorrelated, it is currently impossible to tell which ones are causing the problem, Henny explains. The nearly extinct Florida panthers that inhabit the Everglades—only 30 to 50 animals remain—have similar problems, according to Charles F. Facemire, recently retired from his position as an environmental toxicologist at the U.S. Fish & Wildlife Service in Atlanta, who has been studying these panthers for many years. A few years ago, nearly all scientists at the service believed the problems—a variety of endocrine and reproductive system defects, including abnormal sperm, low sperm counts, undescended testicles, and thyroid dysfunction—were most likely the result of inbreeding. Now, however, Facemire and some others believe they result from the panthers' diet of fish-eating raccoons, which results in DDE levels as high as 57 ppm in panther fat. "The genetic diversity in the Horida panther ranges from slightly below to slightly above average when other species of large cat are considered," Face mire says. Undescended testicles have never been reported in any other species of nondomestic cat regardless of the deMAY 13,1996 C&EN
29
ENVIRONMENT
The two baculums (the bony part of the penis) on the left are from nine- or 10-month-old river otters living in rural coastal range streams outside the Columbia River Basin. The six on the right are from nine- or 10-month-old river otters taken from the lower Columbia River. The weight of the baculums is inversely related to the degree of contamination in the otters' livers, which reflects the contamination in the part of the river where the otters lived. The five baculums on the right are from relatively urban areas.
gree of inbreeding, he adds. For example, Texas and California cougars, which feed mainly on deer, do not have a problem with undescended testicles. However, managers at the Fish & Wildlife Service have decided that the Florida panthers' problems do result from inbreeding and have released eight Texas cougars—which have a somewhat different genetic makeup— in the Everglades to mate with the panthers, enlarge the gene pool, and, perhaps, lower the number of defects.
Laboratory studies In recent years, scientists have conducted many studies that show that 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD, the most toxic dioxin), DDE, vinclozolin, and some planar PCBs disrupt hormones in lab animals. When very small amounts of such chemicals are given to pregnant rodents, the male offspring have reduced sperm counts, reduced testicular weights, and tend to have genital defects. For example, L. Earl Gray Jr., a reproductive toxicologist at EPA's Health Effects Research Laboratory, Research Triangle Park, N.C., has conducted a number of tests in which he administered one meal containing a minute quantity of 2,3,7,8-TCDD to the pregnant rat on gestational day 15—the most sensitive time for development of the fetus' reproductive tract. The doses 30
MAY 13,1996 C&EN
in his most recent tests were exceedingly low: 50, 200, and 800 ng per kg of adult body weight. In the male offspring, the 50-ng dose caused subtle effects. The 200-ng and 800-ng doses caused reduced sperm counts. Linda S. Birnbaum, head of the environmental toxicology division at the same EPA lab, examined how much of a single meal of dioxin given to the pregnant rat reaches the rat fetus. When radiolabeled 2,3,7,8-TCDD is given to the rodent mother at a level of 1,000 ng per kg of adult body weight on gestational day eight, the resulting levels in the fetus are 20 to 40 ng per kg (20 to 40 parts per trillion). These levels cause reduced sperm counts in the male offspring and malformations in the female offspring. Birnbaum's experiments show that dioxin levels that are not a great deal higher than background levels found in the human population can induce developmental problems in rodents, Gray says. Gray also has studied the effects of vinclozolin, which is widely used to kill fungus on fruit. When doses of 100 and 200 mg per kg of body weight—which are far below the maximum tolerated dose—are given to the pregnant female rat in the feed, the male offspring have reduced sperm counts, undescended testicles, and hypospadias (a condition in which the urethra opens on the underside rather than the end of the penis).
Vinclozolin binds to the rats' androgen receptors, blocking the normal action of the male hormone testosterone. Gray points out that the effects seen in rodents with antiandrogenic chemicals would probably occur in humans if they received a dose analogous to what the rat was given. "We've shown that the active metabolites of vinclozolin bind to the human androgen receptor with the same affinity they show for the rat androgen receptor, and we know a lot about what androgens do in human fetal development," he says. Fortunately, vinclozolin residues on fruit would probably not provide enough of the fungicide to affect the human fetus, Gray says. But if a pregnant woman was working as a pesticide applicator, her level of vinclozolin could be comparable to the levels used in the test rats.
Human data One reason that the lab experiments on animals may have relevance for humans, especially those performed at very low levels, is that everyone on the planet who has ever been tested has been found to have accumulated body burdens of synthetic chemicals in their fat. Since 1971, the Centers for Disease Control & Prevention (CDC) has conducted four nationwide surveys of human chemical contamination, each of which sampled blood from about 20,000 people. According to James L. Pirkle, who works in the laboratory division of CDC's National Center for Environmental Health, Atlanta, these surveys show that the average U.S. resident has hundreds of chemicals accumulated in fat. Among these are an average of at least 17 dioxin- and furan-related compounds and at least half-a-dozen PCB isomers. PCBs and some of the persistent pesticides usually are found at low parts-perbillion and high parts-per-trillion levels, and 2,3,7,8-TCDD often is found in the low parts-per-trillion range. One proven human endocrine disrupter is the synthetic estrogen DES, which was prescribed for pregnant women from 1948 to 1972 to prevent miscarriage. It caused clear-cell carcinoma in the vagina and reproductive abnormalities in female offspring and a much higher than normal rate of genital defects in the males. These defects included undescended testicles and stunted testicles. As many as 35% of
and the percentage of motile sperm decreased 0.6% per year. D. Stewart Irvine, a gynecologist at the Medical Research Council's Reproductive Biology Unit in Edinburgh, Scotland, found in a study of 3,729 men that the median sperm count declined from 128 million per mL for semen donors born in the 1940s to 75 million per mL for donors born in the late 1960s. However, in Finland, sperm counts seem to have remained steady from 1958 to 1992, reports Jyrki Suominen of the department of medical biology at the University of Turku. In fact, in 1992 the mean sperm count of 114 million per mL was nearly double that reported by Skakkebaek for men worldwide. Fisch: difficult to study general population And Harry Fisch, director the male offspring of women who took of the Male Reproductive Center at CoDES have such defects. Some studies lumbia Presbyterian Medical Center in indicate that the male offspring have New York City, studied the sperm charbelow-normal sperm counts, but others acteristics of 1,283 men who banked seconclude that DES did not affect sperm men before having a vasectomy in New York City, Los Angeles, and Roseville, counts. The evidence for and against a de- Minn. In a study published May 1 in cline in human sperm counts has re- Fertility & Sterility [65,1009 (1996)], Fisch ceived the most attention recently. The reports that over the past 25 years, while first study that attracted widespread the men's sperm count increased slightscientific interest was a meta-analysis ly, volume and motility did not change. of 61 sperm count studies from 20 coun- The average sperm count in New York, tries done by Niels E. Skakkebaek, chief however, was about twice that in Los of the department of growth and repro- Angeles. duction at the University Hospital in Specialists have very different reacCopenhagen, and his colleagues. The tions to the sperm count studies comanalysis, which combined data from the pleted thus far. Fisch claims that the men 61 studies into a single study, included included in his study are more represen14,947 men of proven fertility and tative of the general population than the showed a decline in average sperm con- men in the European studies because the centration from 113 million per mL in donations of all men who wanted to 1938 to 66 million per mL in 1990. bank sperm before a vasectomy were inSkakkebaek's analysis prompted sev- cluded in his analysis. In most of the Eueral laboratories to evaluate their data on ropean studies, only selected sperm dosemen quality. A French team led by nors were used and the criteria for selecJacques Auger analyzed data on sperm tion could have changed over the years, from 1,351 healthy fathers who volun- he says. He concludes from his work teered in Paris for sperm donation. The that there has not been a worldwide demode of recruitment of the men and the cline in sperm counts, and that the studmethod of semen analysis remained the ies showing a decline in European men same over the study period. Auger may not represent the general populafound that the sperm concentration de- tion. The bottom line, he notes, "is it is clined from 89 million per mL in 1973 to very difficult to study the general popu60 million per mL in 1993. During the lation" because one has to use some sesame period, the percentage of normal lection criteria. Stephen H. Safe, professor of veterispermatozoa decreased 0.5% per year
nary physiology and pharmacology at Texas A&M University, believes that the declines reported in at least three locations in Europe are real. However, the U.S. data show sperm count is not declining worldwide, he says. "It looks to me now that there is not a worldwide sperm count decrease, but there are regional problems," he notes. He contends that because most of the Western countries where the studies were performed have had "pretty similar chemical exposure," the most likely cause of sperm count declines is not chemicals. "It's not proof that chemicals aren't causing it, but it means you'd better start looking at other things," he says. Other experts say that if endocrine disrupters are causing a decline in sperm counts, decreases would not be seen everywhere because the degree of background contamination and other factors varies from place to place. So they expect to see nonuniform changes. The incidence of male birth defects in the reproductive tract seems to have increased sharply in several countries over the past few decades. Data from Sweden, Norway, and Denmark are probably the most convincing because they are based on a national register that includes the whole population. In Denmark, the incidence of hypospadias increased from 7.5 per 10,000 births in 1970 to 14 per 10,000 births in 1989. In a few weeks, results of a nationwide survey done by CDC will be published that show a sharp increase in hypospadias in the U.S. Because so many endocrine disrupting chemicals cause hypospadias in lab animals, some scientists, such as Richard M. Sharpe of the U.K.'s Medical Research Council's Reproductive Biology Unit in Edinburgh, say that the increases could be caused by chemical exposures in the womb. Several studies also have indicated that the incidence of undescended testicles is rising in some places. And data from many countries around the world collected by the World Health Organization's International Agency for Research on Cancer, based in Lyons, France, and other groups show that testicular cancer incidence is rising almost everywhere where data are available. It is a disease mostly of young men in their 20s and 30s and is nearly always detected. In many countries, it is now the most common malignancy Continued on page 34 MAY 13,1996 C&EN
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