Manuscript received 27 April 1995; manuscript accepted 15 March 1996.
Introduction
In 1992, a research group at the Department of Growth and Reproduction, the National University Hospital (Rigshospitalet), and the Panum Institute, Copenhagen, Denmark, published in the British Medical Journal a metaanalysis of the data from the international literature that revealed a significant decrease in sperm concentration and semen volume in otherwise normal men over the period 1938 to 1990. During the same time period, the incidence of testicular cancer had markedly increased in many countries. These and other observations provided a clue that this apparent decline in male reproductive health might be caused by some common environmental factors. It was recognized that similar abnormalities of the male reproductive system were caused by administration of estrogens during pregnancy in humans and experimental animals; therefore, a hypothesis was put forward that environmental chemicals having estrogenic effects were contributing agents. In particular, it was suggested that fetal exposure to an excess of estrogenic compounds was a key risk factor.
After an intense public debate in the Danish news media on the possible role of environmental chemicals, such as pesticides, detergents, plasticizers, and other industrial chemicals, the Danish Environmental Protection Agency (DEPA) of the Ministry of Environment and Energy in September 1994 decided to support the preparation of a review summarizing the current knowledge on male reproductive disorders and environmental chemicals with estrogenic effects. In addition, the review was to identify gaps in knowledge and address research needs and requirements in order for researchers to perform adequate risk assessments. The DEPA asked Professor Niels E. Skakkebæk, Department of Growth and Reproduction at the National University Hospital and John Chr. Larsen, Division Head, Institute of Toxicology at the Danish National Food Agency to prepare the report. The project received additional financial support from the Ministry of Health through its National Research Centre for Environmental Medicine.
Dr. Jorma Toppari, Departments of Pediatrics and Physiology, University of Turku, Finland, prepared a draft of the report. This draft was then discussed by a group of Danish and international experts invited to a one-week workshop that was held at Rigshospitalet in Copenhagen, January 23-27, 1995. The participants who are the authors of the present publication actively contributed to the endeavor both during the workshop and afterward. The final manuscript was edited by Jorma Toppari together with Niels E. Skakkebæk and John Chr. Larsen. The present review is a revised version of the official report (1), which was printed by the Danish Ministry of Environment and Energy mainly for circulation in Denmark.
The review addresses the possible effects of environmental chemicals known to possess estrogenic activity on male reproductive health. The term xenoestrogen is often used for such compounds, whereas the term synthetic estrogens refers to medical drugs mainly used for contraception and treatment of various diseases.
A number of other environmental chemicals have been implicated as environmental hormones or endocrine disruptors. Although not shown (and in many instances not adequately tested) to possess direct estrogenic activity, some of these compounds may in some cases also affect male reproduction. The mechanisms of action are not known in detail but they may involve, for example, antiandrogenic activity; modulatory effects on enzymes controlling sex hormone metabolism; or direct influence on the hormone-producing organs such as the thyroid gland, pituitary gland, and adrenal glands. These compounds may also affect estrogen levels through indirect feedback mechanisms.
The authors are well aware that the decline in semen quality and the increase in the incidence of testicular cancer may be caused by many other environmental, life-style, or genetic factors. For example, some chemicals that are now known as occupational toxicants were shown to affect the semen quality of the workers through a toxic action on the gonads, without any apparent estrogenic effects. Such toxic effects are not the object of the present report, but should be kept in mind in any consideration or scientific investigation of the adverse effects of environmental chemicals on male reproductive health.
Secular Trends in the Incidence of Male Reproductive Disorders
Trends in Semen Quality
Several reports in the literature have suggested a possible decline in human semen quality during the last 50 to 60 years (2-4). However, most of these reports were based on data from men attending infertility clinics or from very selected groups of fertile men and, therefore, the decline in sperm counts was presumed just to reflect changes in the policy of infertility treatment or a bias in selection of patients rather than a true biological phenomenon. A systematic metaanalysis of 61 studies that included 14,947 normal men revealed a significant decrease in sperm concentration (113 million/ml vs 66 million/ml; Figure 1) and semen volume (3.40 ml vs 2.75 ml) over the period of 1938 to 1990 (5). This report stimulated extensive discussion and some criticisms on the basis of possible technical errors and known limitations of metaanalysis (6,7). Carlsen and co-workers responded to these criticisms (8). Although the data for 1970 to 1990 were compatible with a decrease as well as with no change or an increase in semen quality, the cautious general conclusion was that a real decline occurred during the 50-year period (9). The findings of Carlsen et al. (5) were also compared (6) to those of MacLeod and Wang (10) from the United States. This comparison is not relevant, however, because the metaanalysis was based on semen analyses of normal men, whereas the American study examined men who were clients of an infertility clinic.
Figure 1. Linear regression of mean sperm density reported in 61 publications (represented by circles the area of which is proportional to the logarithm of the number of subjects in the study), each weighted according to number of subjects, 1938-1990. The figure is based on the data reported by Carlsen et al. (5). A corresponding figure in that paper was incomplete.
The metaanalysis of Carlsen et al. (5) prompted several laboratories to evaluate their data on the quality of semen obtained during recent years. In a French study of 1351 healthy men volunteering for sperm donation, a 2.1% decrease in sperm concentration per year from 89 million/ml in 1973 to 60 million/ml in 1992 (
p<0.001) was found (11). Furthermore, the percentages of motile and normal spermatozoa also decreased significantly (Figure 2), whereas semen volume remained unchanged (3.8 ml). It is notable that the year of birth of the study subjects contributed significantly to the results. Multiple regression analysis (which allows for separate effects of age and calendar year at birth) showed yearly decreases of 2.6% in sperm concentration, 0.3% in motility percentage, and 0.7% in the percentage of normal spermatozoa according to the year of birth of the men (all changes,
p<0.001) (11). Similar results were obtained in a Scottish study (12) of 577 semen donors where a correlation was found between the median sperm count and the year of birth; the median sperm concentration decreased from 98 million/ml among donors born before 1959 to 78 million/ml among donors born after 1970 (
p=0.002). The total number of sperm in the ejaculate fell from 301 million to 214 million (
p=0.0005)(12). The association between declining semen quality and a more recent year of birth lends support to the concept that adverse prenatal factors may influence the sperm production capacity in adult life. Deterioration of sperm counts as well as motility among semen donor candidates during the past two decades was also observed in a smaller Belgian study (13). Ginsburg and Hardiman (14) reported a decrease in sperm concentrations (105 million/ml in 1978-1983 vs 76 million/ml in 1984-1989) of the partners of women treated for infertility and living in the Thames water supply area of London, whereas no decrease was found among those who lived in other water supply areas of London. However, the mean percentage of abnormal spermatozoa increased in all areas during the study period (18-19% vs 30-32%) (14). The data in all the studies cited above originated in individual laboratories that used consistently the same methods for semen analysis throughout the period.
The decreasing trend in semen quality may not be global. In contrast to the Paris area, no change in sperm concentration was found in the Toulouse area in France during 1977 to 1992 (15). The mean sperm count of samples from 302 healthy fertile donors was 83 million/ml (15). Furthermore, the sperm concentration in semen of Finnish men has remained unchanged between 1958 and 1992 (111 million/ml vs 124 million/ml) and is higher than elsewhere in Europe (16). It is of interest that the incidence of testicular cancer, and perhaps also hypospadias, in Finland is much lower than that in other Nordic countries (below), suggesting that these phenomena may be related in some way. The reason remains unknown, but further examination may provide important clues to the etiology of decreasing sperm quality worldwide. Urban areas (e.g., Paris) appear to have a declining trend in sperm counts, whereas rural areas (e.g., Toulouse or Finland) seem to have stable sperm concentrations in semen.
Incidence of Testicular Cancer
Testicular cancer is now the most common malignancy of young men in many countries; and although it is still rare compared to the malignant diseases most prevalent in old age, the lifetime risk of developing testicular cancer now approaches 1% in a country such as Denmark. The incidence of testicular cancer has increased for several decades (17). On the basis of data from cancer registries, increases in incidence are evident in England and Wales (18,19), Scotland (20), the Nordic and Baltic countries (21,22), Australia (23), New Zealand (24,25), and the United States (26). The observed increase has been approximately 2 to 4% per annum in men under 50 years of age (Figures 3, 4) and occurred in the same age group in which testicular cancer incidence peaks, i.e., young adults (Figure 5). Table 1 displays the changes that have occurred during the last 25 years. There are marked racial and geographic differences. For example, Denmark has a 4-fold higher incidence of testicular cancer than does nearby Finland, and Caucasians are 3-fold more susceptible to this disease than are African Americans in the United States. Nevertheless, it is obvious that there is a worldwide trend toward an increased incidence of testicular cancer as illustrated in Figures 3-5. The incidences of both seminomas and nonseminomas have increased (17). Mortality due to testicular cancer increased from the beginning of this century until the early 1970s when, because of the development of good medical treatment, mortality began to decline (17). However, we still do not know the etiology of testicular cancer and cannot therefore develop any preventive measures.
Figure 3. Secular, racial, and geographic trends in the incidence of testicular cancer, 1953-1987. Compilation of data from IARC (318-323).
Figure 4. Trends in age-standardized (world standard population) incidence rates of testicular cancer. From Adami et al. (22); reprinted with permission from Wiley-Liss, Inc.
Figure 5. Age-specific incidence of testicular cancer 1985-1989 in the Nordic countries, Poland, and former East Germany. From Adami et al. (22); reprinted with permission from Wiley-Liss, Inc.
Incidence of Cryptorchidism
Birth data from several reports have indicated a substantial increase in the incidence of cryptorchidism (maldescent of the testis). However, estimates of the prevalence of cryptorchidism obtained from different studies are difficult to compare. It is often not clear how a cryptorchid testis was defined, and inclusion of different proportions of boys with retractile testes could account for the reported differences. The sources of data used in these reports also differ considerably. The prevalence rates have varied between 0.03 and 13.4% on the basis of data from birth to 1 year of age from hospital or central registers (including different proportions of preterm babies) (27-48); 0.16 to 13.3% in surveys from school, army, etc. (36,38,49-57); and 2 to 4.7% in cohort studies based on discharge diagnosis (41,58,59). A few studies include ethnic data on non-Caucasians: birth data from India (33), Formosa (Taiwan) (38), and Korea (44) indicated prevalences of 1.6, 1.4, and 0.7% of cryptorchidism, respectively. A school survey from Nigeria (56) indicated a prevalence of 0.5%. The incidence of cryptorchidism among African Americans was reported to be only one-third that among whites (34), although another study (48) did not find a significant difference. Racial and ethnic data are pooled in most studies. Unfortunately, very few studies exist examining temporal changes in the incidence of cryptorchidism, confined to the same population and geographic areas and using an identical definition of the condition.
Discharge data from the Hospital Inpatient Enquiry from England and Wales showed that the proportion of boys undergoing orchidopexy (operation to bring the testis into the scrotum) before 15 years of age increased from 1.4% for a 1952 birth cohort to 2.9% for a 1977 birth cohort (58). However, it is not known whether this is a reflection of a true increase in the prevalence of cryptorchidism or whether a considerable number of boys with retractile testes were undergoing orchidopexy. In Scotland, the annual number of discharges of boys 0 to 14 years of age with the diagnosis of cryptorchidism also showed a substantial increase during 1961 to 1985 (41). In Denmark, the prevalence of cryptorchidism at birth in male infants weighing > 2500 g varied between 1 and 1.8% in three different data sets obtained in the late 1950s (60). School surveys suggested higher prevalence rates up to 7% during 1940 to 1966, but these figures apparently included retractile testes (55). Cohort analysis of data from the Danish National Register of Hospital In- and Outpatients, from the period 1982 to 1985, indicated an incidence of cryptorchidism of approximately 2% (59).
In a study from the late 1950s that examined more than 3500 male infants delivered in a hospital in London and followed up to 1 year of age, Scorer (31) found that the incidences of cryptorchidism at 3 months of age in boys with birth weights <2500 g and >2500 g were 1.74 and 0.91%, respectively. Scorer used very accurate definitions of the positions of testes, and therefore, this well-conducted study has served as a reference for later research. In a large study from the 1980s, comprising 7441 male infants from Oxford (47), the very same examination technique and definitions of cryptorchidism were used; the rates of cryptorchidism at the age of 3 months in boys with birth weights <2500 g and >2500 g were 5.2 and 1.61%, respectively, indicating a significant increase compared to Scorer's figures. In another study from the late 1980s composed of 6935 male infants from New York (48) (using identical study techniques and case definition), prevalence rates of cryptorchidism at the age of 3 months in boys with birth weights <2500 g and >2500 g were 1.94 and 0.91%, respectively. However, the study population was racially and ethnically heterogeneous. From these three large studies, one can conclude that there has been a significant increase in the incidence of cryptorchidism in England, but the incidence in the racially and ethnically mixed population of New York is similar to that reported in the 1950s in England.
The epidemiological data indicative of an increasing incidence of cryptorchidism are not unequivocal. This important issue necessitates large regional prospective studies in which standard criteria are adopted for an accurate description of cryptorchidism.
Incidence of Hypospadias
Birth data from several reports have indicated a substantial increase in prevalence of hypospadias (Figures 6, 7). Figures of birth prevalence of hypospadias in the world literature vary considerably--from 0.37 to 41 per 10,000 infants (61,62)--and are difficult to compare. There are several factors that may contribute to the reported differences: different levels of ascertainment, different inclusion of minor forms of hypospadias or differences in ethnical origin of the population. As reported for cryptorchidism, very few longitudinal studies confined to the same population and geographic area exist. The increasing incidence of hypospadias has been reported primarily in England and Wales (39), Hungary (63,64), Sweden (65-67), Norway (67,68) and Denmark (66,67). No increasing trend was noticed in Finland, Spain, New Zealand, Australia, or Czechoslovakia (67).
Figure 6. Prevalence of hypospadias at birth (rate per 10,000 births) in England and Wales, and Hungary. Based on data from WHO (67).
Figure 7. Prevalence of hypospadias at birth (rate per 10,000 births) in the Scandinavian countries. Based on data from WHO (67).
England and Wales. The data from England and Wales are based on the national register that includes the whole population. Analysis of the data indicated a steady increase in the prevalence of hypospadias from 7.3 per 10,000 births in 1964 to approximately 16 per 10,000 births in the early 1980s, when the number of cases stabilized (Figure 6). In 1990 the prevalence of hypospadias showed a decrease to 11.7 per 10,000.
Hungary. The Hungarian data are based on the national register of the whole population. As shown in Figure 6, there was a rapid increase in the prevalence of hypospadias from 5.5 per 10,000 births in 1964 to 23.9 per 10,000 in 1978. Since then, the prevalence of hypospadias, although fluctuating, has remained at approximately the same level.
Scandinavian Countries. The data on the incidence of hypospadias in Scandinavia (Figure 7) are all based on the national registers that include the whole populations. The Danish data from 1970 to 1981 indicated a significant increase for this period (from approximately 7.5 to 12 per 10,000 births) (66). A further increase was noticed during the period 1982 to 1988 (67). However, this increase may be difficult to interpret as a new registration system was introduced. Nevertheless, this increase was similar to that reported for the years 1970 to 1981.
The data from Sweden also indicated a marked increase in the early 1970s: prevalence of hypospadias at birth was 40% higher between 1974 and 1982 than for the period 1965 to 1968 (66,69). However, the data obtained in the earlier period could be more complete, because they included both hospital records and registry data.
Also in Norway the prevalence of hypospadias at birth increased from 7 to 8 per 10,000 births between 1967 and 1971 to 13 per 10,000 in 1973 (68). In 1988, the prevalence was 20.7 per 10,000 births (67).
Ethnic Differences. The incidence of hypospadias in the United States is higher in Caucasians than in African Americans (ratio of 1.3-3.9:1) (70-73). In British Columbia, Canada, Native Americans were reported to have a lower incidence of hypospadias than the general Caucasian population, with a ratio of 1:6.7 (74,75).
Geographic Variation. Considerable variation exists in the prevalence of hypospadias among different countries. Interestingly, some populations with a low incidence of testicular cancer (e.g., Finland) (22) have a very low prevalence of hypospadias (Figure 7). Furthermore, there seems to be considerable geographic variation in the prevalence of hypospadias within different countries (66,76).
Incidence of Male Breast Cancer
As xenoestrogens were implicated as possible factors involved in the pathogenesis of breast cancer in women (77), the trends in the incidence of this disease in males should be monitored. Male breast cancer is a rare disease; only a few studies exist on geographical and temporal trends. Ewertz et al. (78) studied the incidence of male breast cancer in four Nordic countries and found a weak increase with calendar time in Denmark (1943-1982) but no change in Finland, Norway, and Sweden over the period 1953 (1958 for Sweden) to 1982. Ewertz et al. noted a remarkable geographical trend, with Denmark having an incidence about twice that of Finland.
Summary
Semen quality has deteriorated in many countries during the last 50 years. The incidence of testicular cancer has been increasing almost invariably worldwide. Increases in the incidences of cryptorchidism and hypospadias have been observed in countries in which longitudinal studies have been performed. However, there are clear regional differences. The prevalence of male breast cancer has been rising and is higher in Denmark than in Finland.
Changes in Male Reproduction in Wildlife. Estrogenic Effects on Developing Animals
Changes in male reproduction in wildlife involve such issues as feminization, demasculinization, reduced fertility, reduced hatchability, reduced viability of offspring, impaired hormone secretion or activity, and altered sexual behavior (79). Since it is not possible to review all of the data in detail, the reader is referred to a recent workshop publication titled "Chemically-induced Alterations in Sexual and Functional Development: The Wildlife/Human Connection" (80). Many of the reproductive disorders listed above have been associated with xenoestrogenic effects on the fetus. It has usually not been possible to ascribe lowered reproductive success and signs of feminization and demasculinization in wildlife to a single agent; in these cases, chemical analyses of specimens have revealed the presence of multiple compounds, some of which are known to have hormonelike activity. There is experimental evidence that xenoestrogens act cumulatively, i.e., 10 compounds administered simultaneously, each one at 1/10 of their effective dose, resulted in a potent estrogenic response (81). Cumulative exposure to nongonadal estrogens is easily documented in male birds, amphibians, and fish by measuring plasma vitellogenin levels.
Gastropod Species
Pseudohermaphroditism or imposex-- females developing male characteristics--in marine gastropod species has been reported worldwide: the northeastern United States (82), the United Kingdom (83), Alaska (84), and Southeast Asia (85). This phenomenon is caused by tributyltin (TBT) compounds leached from marine antifouling paints used on ships, boats, and mariculture pen nets (83). Very low concentrations (1 ng/liter) of TBT-derived tin are effective in induction of imposex (83), and marine areas averaging 6 to 8 ng/liter of TBT suffer reproductive failure and local extinction due to female sterility (86). The use of TBT was restricted in the 1980s, and recovery of some species has been reported after that (87). However, imposex still remains a problem for gastropod species in several marine areas. The mechanism of action of TBT is unknown. The effect mimics that of an antiestrogen, and therefore presents an example of the drastic hormonelike effect of a xenobiotic threatening the existence of several species, even though higher species seem to be far less sensitive to TBT.
Reptiles
Lake Apopka, Florida, was extensively polluted with dicofol (86), 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) (and its metabolites 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane [DDD], 1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene [DDE] and chloro-DDT), and sulfuric acid spilled accidentally from a chemical company in 1980. Further contamination of the lake by agricultural sewage dumping has made this lake one of Florida's most polluted wetlands. A number of these pollutants are known to have estrogenic or endocrine-disrupting effects (89). The chemical spill was followed in the subsequent 3 years by a significant decline in the number of juvenile alligators, whereas alligator populations elsewhere were increasing or stable at the same time (90). The population decline was associated with reproductive disorders that were hypothesized to be caused by endocrine-disrupting xenobiotics (91). There are extensive data supporting the hypothesis (92,93): female alligators from Lake Apopka had plasma estradiol concentrations 2 times that of normal females from the control lake, Lake Woodruff. The females exhibited abnormal ovarian morphology. Likewise, in males abnormal germ cells were observed in the testes. Furthermore, Lake Apopka male alligators had abnormally small phalli. Basal and luteinizing hormone (LH)-stimulated plasma testosterone concentrations of male juvenile alligators in Lake Apopka were significantly lower than those of Lake Woodruff males, equaling those of females. The plasma estrogen concentrations of male alligators from these two lakes did not differ. However, testes from Lake Apopka alligators produced significantly more estradiol in vitro than testes from control alligators; testosterone production was similar (93). The discordance in the in vitro and in vivo findings suggests additional significant differences in steroid metabolism and liver function between the alligators from these two lakes, emphasizing the complexity of environmental influences. Reptiles have temperature-sensitive sex determination that can be altered by estrogen treatment (94). This has been demonstrated both in alligators (95) and turtles (96). The data from Lake Apopka suggested that the gonads of alligators had been permanently modified, altering steroidogenesis and inhibiting normal sexual maturation (91,97,98).
A recent study (99) has demonstrated that a number of PCB metabolites are capable of acting as synthetic estrogens. As with crocodilians, many turtles exhibit environmental sex determination so that the temperature at which the egg is incubated determines the sex of the offspring (97). Turtle eggs incubated at 26šC produce 100% males. However, if eggs incubated at a male-producing temperature are "painted" with either one of two PCB metabolites (2´,4´,6´-trichloro-4-biphenylol or 2´,3´,4´,5´-tetrachloro-4-biphenylol) sex reversal occurs as if the eggs were treated with the natural estrogen, 17ß-estradiol (99). Interestingly, if eggs are treated with a dose of both compounds (100 µg of each)--a dose that produced a small percentage of sex-reversed turtles (20 and 0%, respectively)--they act synergistically, producing sex reversal in 80% of the eggs treated. Thus, animals that should have developed as males are modified so that their internal and external morphology is that of a normal female. It is unknown if this sex reversal produces fertile adult females.
Fish
Modifications in reproductive functioning of male fish living downstream from kraft pulp mills have been well documented. For example, white sucker (Catostomis commersoni) collected from sites receiving primary effluent exhibited delayed maturation, smaller gonads, and an absence of secondary sex characteristics (100). Furthermore, these males had significantly reduced serum testosterone concentrations and dysfunctional hypothalamic control of the pituitary-gonadal axis. A similar reduction in gonad size was reported for both male and female perch (Perca fluviatilis) inhabiting sites receiving primary effluent from a kraft mill on the coast of the Gulf of Bothnia (101). The effects observed in these perch were graded along the pollution gradient (2-8 km) from the mill. Masculinization and behavioral changes of female mosquito fish (Gambusia affinis) were observed in Florida rivers downstream from paper and pulp mills (102). Laboratory studies corroborated the hypothesis that kraft mill effluents containing large amounts of the plant steroids sitosterol and stigmastanol caused the changes (103). However, the etiologic agents in paper-mill effluents have not been identified unequivocally. Other effects of paper-mill effluents on fish have been reviewed by Owens (104) and Leatherland (105).
The effect of PCB exposure on testicular function in fish also has been examined. In the Atlantic cod (Gadus morhua), testicular steroidogenesis is disrupted by dietary exposure to PCBs (106). Additionally, Freeman et al. (107) observed that cod exposed to PCBs in vivo exhibited increased metabolism of steroid hormones in vitro by kidney and liver tissues. Further, dietary exposure of juvenile males to PCBs precluded the rise in plasma testosterone concentrations associated with sexual maturity. These data suggest that PCBs modify both testicular androgen synthesis and steroid utilization/degradation in peripheral tissues. Exposure to crude oil also induced a decrease in plasma testosterone concentrations in the winter flounder, Pseudopleuronectus americanus (108).
Organochlorines have also been implicated in a number of developmental and reproductive abnormalities in fish living in the Laurentian Great Lakes of North America. Male coho salmon (Oncorhynchus kisutch) living in Lake Erie exhibit a number of abnormalities, including decreased fertility, lower plasma concentrations of gonadotropins and steroids (testosterone, 11-ketotestosterone), poor expression of secondary sex characteristics, and high precocious sexual maturation (109,110). It is unknown at this time if the above deficiencies are all related or represent different effects. It is hypothesized that some may be due to modifications of the developing embryo (organizational effects), whereas others may be due to disruptive activational events in adults.
The observation of an increased prevalence of hermaphroditism in fish in sewage treatment water (STW) lagoons in England and Wales initiated a series of studies examining environmental estrogens using a bioassay involving vitellogenin synthesis in STW-exposed rainbow trout (Oncorhynchus mykiss) (111,112). Vitellogenin is produced in the liver under estrogen control by female fish for the growth of ova (113). Males produce it only after exogenous estrogen treatment (114). Vitellogenin production by male fish can therefore be used as a biomarker of environmental estrogenic activity. STW exposure of caged rainbow trout induced increases of 500- to 100,000-fold in plasma vitellogenin concentration, and males were shown to achieve vitellogenin levels almost as high as females, indicating the contamination of water by estrogenic compounds (112). In vitro studies with trout hepatocytes established a dose-response relationship with estrogen exposure and vitellogenesis (111). Using this culture system, it was demonstrated that degradation products of several alkylphenol-polyethoxylates, a major group of surfactants present in sewage, are estrogenic to fish (111). Their effect on trout hepatocytes could be blocked with an antiestrogen, Tamoxifen, demonstrating that the compounds act through the estrogen receptor. Although the estrogenic activity of individual xenobiotics was low, their effects may be additive in nature.
Birds
Feminization of gulls and terns in several locations along the Pacific coast of the United States has been associated with DDT and DDE pollution (115,116). Fry and Toone (115) demonstrated the feminizing capacity of some DDT compounds by injecting gull eggs. Feminization leads to a skewed male/female ratio, which is known to increase female-female pairing. Supernormal clutches, i.e., five to six gull eggs per nest instead of the normal three, are often laid after this type of pairing, and the fertility of the eggs is poor (117). There is some controversy as to whether feminization of males or differential mortality of males resulted in a skewed sex ratio (118). Fox (86) considered both the feminization of males and their increased mortality, when compared to females, as possible reasons for the female-female pairing and supranormal clutches in the gulls.
Mammals
Although the mechanism is unknown at this time, elevated PCBs and DDE concentrations are associated with a decrease in plasma testosterone concentrations in Dall's porpoises, Phocoenoides dalli (119). Testicular steroidogenesis in vitro has been studied in the gray seal (Halichoerus grypus) in association with exposure to methyl mercury (MeHg), cadmium (Cd), arsenic (As), selenium (Se), and a PCB mixture (Aroclor 1254). All contaminants except As and Se, stimulated testosterone synthesis in vitro from seal testicular tissue (120). The mechanism by which this stimulation occurs is unknown. Gonadal steroidogenesis is not the only target for PCBs, as a number of PCB metabolites have been shown to decrease thyroid function in vivo in the common seal, Phoca vitulina (121).
The majority of the remaining (approximately 35 individuals) endangered Florida panthers (Felis concolor coryi) exhibit a number of developmental abnormalities and reproductive defects (122). Specifically, males (n=12) showed low ejaculate volume, low sperm concentrations (3-15
106 sperm/ml semen), poor sperm motility, and a very high proportion (92.9%) of sperm with morphological abnormalities (123). Cryptorchidism (both uni- and bilateral) has increased exponentially in male cubs since 1975 so that today >90% of the male population exhibits this phenomenon (eight of nine cubs born since 1985). Male sterility may be a problem as well. Female panthers (n=3) have high body burdens of various contaminants including p,p´-DDE (5.45-57.65 mg/g lipid fresh weight), PCBs (7.32-27.06 mg/g lipid fresh weight), oxychlordane (<0.0098-2.00 mg/g lipid fresh weight) and trans-nonachlor (<0.0098-4.82 mg/g lipid fresh weight) (122). Panthers also have elevated tissue levels of mercury, methoxychlor, and other lipophilic organochlorine compounds. These contaminants are derived primarily from their major food item, the raccoon (124). The reproductive abnormalities described above were suggested to be due to the contamination of mothers by endocrine-disrupting environmental xenobiotics rather than to problems associated with inbreeding (122).
Summary
Reproductive disorders in gastropod species, reptiles, fish, birds, and mammals have been associated with environmental factors. Several of the disorders, such as sex reversal in reptiles and vitellogenin production by male fish, may result from estrogenic action of chemicals in the environment. Fewer data are available concerning the mammals. However, some endangered species such as Florida panthers that are exposed to estrogenic and/or other endocrine-disrupting contaminants show reproductive disorders comparable to those found in the human.
Sexual Differentiation and the Physiological Role of Estrogens
Sexual Differentiation
Sexual differentiation occurs during the first trimester of human pregnancy (125). An indifferent gonad develops into a testis under the influence of the SRY gene on the Y chromosome. In addition to SRY, there are several downstream effectors and autosomal genes (e.g., SOX9 and SF-1) that are required for normal differentiation of the testis (126,127). Sertoli cells in the newly differentiated testis produce Müllerian inhibiting substance (MIS), which induces regression of the Müllerian ducts that would otherwise develop into the oviducts, uterus, and upper part of the vagina. Sertoli cells also regulate development and early function of the Leydig cells that secrete testosterone to promote differentiation of the embryonic Wolffian ducts into the male accessory sex organs, epididymides, seminal vesicles, and vasa deferentia. Masculinization of the external genitalia and the rest of the body, except the brain, is also controlled by androgens and occurs after conversion of testosterone from the testis into 5
-dihydrotestosterone in the target tissue. Female reproductive organs develop in the absence of SRY and thereby in the absence of the testis (128). The female developmental pattern seems to be a genetic default pathway, and it is largely independent of hormonal regulation. Disturbances in sexual differentiation occur when factors in the male developmental cascade go wrong or when the genetic female is exposed to an elevated plasma-androgen concentration. In the first instance, a genetic male will develop a female phenotype and in the latter case a female will be virilized.
Disorders of Genital Development and Testicular Malignancy
The association between disorders of genital development and sexual differentiation and gonadal malignancy has been observed since the beginning of this century and is now well established (129-131). The most frequent abnormality leading to neoplasia is gonadal dysgenesis with the presence of Y chromosomal material; other disorders include true hermaphroditism and androgen-insensitivity syndrome. Although the general prevalence of disorders of sex differentiation is low, the high incidence of germ cell tumors makes the intersex gonad a good model for the study of factors involved in the pathogenesis of germ-cell neoplasia. Malignant growth frequently appears in the intersex gonad in early childhood, thus suggesting that the carcinogenic process begins in utero. The intersex syndrome comprises a variety of genetic disorders, as different from each other as, for example, XY/XO mosaicism and a mutation in the androgen receptor gene. The phenomenon of heterogeneous genetic defects leading to a common result, malignancy of germ cells, indicates that any disruption of early gonadal development may render the germ cells susceptible to neoplastic transformation by yet unknown mechanisms. There are some hypotheses concerning possible mechanisms of neoplastic transformation --e.g., arrested differentiation of immature germ cells (132)--or an imbalance in fetal hormonal environment (133). Androgen-insensitivity syndrome provides a clue that the lack of the appropriate inductive hormonal environment may arrest fetal gonadal differentiation and lead to neoplasia later in life. It is possible that high levels of testicular androgens have a protective function during gonadal development; for example, a relative excess of maternal testosterone during early pregnancy was shown in black women compared to a matched white group, providing a possible explanation for the lower incidence of testicular cancer in black men (134). There is some experimental evidence that androgens and estrogens may have opposite effects on certain pathways in the developing gonad; e.g., free estrogen may decrease expression of MIS (135,136), whereas androgens seem to have a stimulatory effect (137).
The Physiological Role of Estrogens in Sexual Differentiation
Estrogens act through a nuclear receptor that is a ligand-activated transcription factor. In addition, steroid hormones may effect the cell membrane. Estrogens are essential in the development of female secondary sexual characteristics and in the female reproductive cycle, fertility, and maintenance of pregnancy. In the developing male, the physiological role(s) of estrogens is unclear, though by analogy to the situation in adulthood, they probably play a role in regulating the differentiation and function of Leydig cells (138). The role of estrogen action in embryonic sexual differentiation is controversial. In rats and rabbits (139,140), estrogen synthesis is activated in male and female embryos at the time of blastocyst implantation in the uterus. Estrogen receptor mRNA can be detected in blastocysts and two-cell stage embryos (141). Immunohistochemical studies by Greco et al. (142) demonstrated estrogen receptor expression in both male and female mouse gonads on fetal day 13 and 15. The gonads lose their estrogen receptor expression at later ages. These studies suggested a role for estrogens in development of the gonads (143). A putative molecular target of estrogens could be the MIS gene that contains a DNA sequence similar to the estrogen response element in the upstream regulatory region (144). In contrast, the classical organ ablation studies by Jost (145) demonstrated that gonadectomy of the embryo always resulted in a female phenotype. However, maternal and placental estrogens might still have contributed to this developmental pattern.
Defects in the Estrogen Receptor Gene
Recent reports on estrogen receptor gene-deleted mice (146) and a male patient with a defective estrogen receptor (147) have begun to clarify the possible importance of endogenous estrogens in sexual development. Lubahn et al. (146) disrupted the estrogen receptor gene by targeted deletion that resulted in complete estrogen resistance. Both male and female mice survived to adulthood without apparent morphological anomalies. However, females were infertile with hypoplastic uteri and hyperemic ovaries that contained no corpora lutea. Fertility of the males was also decreased. Only 3 of 15 males that paired with normal females produced any offspring, and even those that were initially fertile lost their ability to sire subsequent litters. Testicular weights were low and sperm counts (in the testis and epididymis) were only 10% of control. Weights of seminal vesicles and coagulating gland were normal. These findings suggest that estrogens are necessary but not indispensable for fetal sexual development; i.e., development is overtly normal, but the sexual organs do not reach their normal size and function.
There is only one reported patient case of estrogen resistance--a 28-year-old white male (147). This man had incomplete epiphyseal closure and therefore continued linear growth into adulthood despite otherwise normal pubertal development. He was normally masculinized and had normal male genitalia with bilateral descended testes (20 and 25 ml) and a normal-sized prostate gland. His sexual functions were normal including morning erections and nocturnal emissions. However, his semen quality was subnormal: sperm concentration was 25 million/ml and viability 18% (normal values: >20 million; >50%, respectively). His serum testosterone concentration was normal, whereas estradiol, estrone, follicle-stimulating hormone (FSH), and LH concentrations were elevated. The elevated gonadotropin concentrations suggest that estrogens play a role in the regulation of gonadotropin secretion in males, and thereby may have several indirect effects. The patient case and the receptor gene-deleted mice demonstrate that a normal male phenotype develops in the absence of estrogen receptor-mediated influences, but semen quality and probably fertility may be compromised as a result.
Overexpression of the Estrogen Receptor
In transgenic mice that overexpress the estrogen receptor, normal differentiation of sexual organs was observed (148). However, females in several transgenic lines had fertility problems and their gestational length was significantly prolonged, resulting in loss of litters due to difficulties in parturition. No major fertility problems were reported in the male transgenics. The only abnormalities described in the males (as well as in the females) were hernias of the abdominal wall musculature. This is of interest, since estrogen administration has been reported to induce inguinal hernias in male mice (149), and dogs (150,151), and there is some indication that inguinal hernias are a risk factor for testicular cancer (152). It is not known yet whether transgenic male mice that overexpress the estrogen receptor will develop testicular tumors later in life.
Nonmammalian Vertebrates
Amphibians and birds differ from mammals in their sexual differentiation (153). That is, the female phenotype in birds develops under estrogen control, whereas the male phenotype appears in the absence of estrogen (154). Exogenous estrogen administration causes sex reversal in male birds and frogs. Some reptiles (crocodilians and some turtles) have temperature-dependent sex determination; for example, in turtles, female hatchlings are produced by incubation of the eggs at a higher temperature than males, but an excess of estrogen causes feminine differentiation also at low temperatures typical of males (155,156). Estrogen-induced sex reversal can be used as a biomarker of the estrogenicity of an environmental pollutant, as demonstrated recently for PCBs (99). Estrogen receptors are expressed both in female and male chick embryos in the Müllerian ducts (154). Interestingly, the left Müllerian duct that develops into an oviduct in females (regresses in male) has higher estrogen binding capacity than the right, which regresses in both sexes (157). Treatment of chick embryos with diethylstilbestrol (DES) on day 5 prevented regression of the Müllerian ducts in both sexes (158). Similar findings in the mouse are reviewed in the section "Effects of Synthetic Estrogens on the Testis in Animal Models."
Estrogens and Sperm Production Capacity
Sperm production is dependent on permissive actions of FSH and testosterone (and therefore LH). Sperm production capacity depends on the number of Sertoli cells in the seminiferous tubules (which is directly related to the length of the tubules) (159), since each Sertoli cell supports a finite number of germ cells (160). Sertoli cells proliferate quickly in rats from embryonic day 19 to day 15 after birth, then slow down and cease multiplication approximately on postnatal day 20 (161,162). Multiplication is largely dependent on FSH stimulation (162). In humans, regulation of Sertoli-cell proliferation may be very similar. Men with hypogonadotropic hypogonadism do not develop normal-sized testes after gonadotropin treatment, which may be a consequence of inadequate Sertoli cell multiplication in early childhood. This hypothesis is supported by findings in the monkey (163): Sertoli cells proliferate in the neonatal and infantile period but not during or after puberty. Estrogens suppress gonadotropin production in animals at all ages preceding puberty (164). It is hypothesized that this is the case also in humans. Decreased gonadotropin stimulation during the critical developmental phase may result in inadequate Sertoli cell proliferation and small testes (165). Specific FSH gene deletion experiments also demonstrated that FSH regulates the size of the testis (TR Kumaar, personal communication). At present it is not known whether the number of Sertoli cells in human testes has decreased and whether this might be a reason for decreased sperm counts.
Summary
Normal masculine differentiation occurs under the influence of the SRY gene and several other autosomal genes, and androgens are required for this process. Disorders of gonadal development are frequently associated with testicular germ cell neoplasia. Estrogens act through a specific nuclear receptor. Normal masculine differentiation occurs even in the absence of a functioning estrogen receptor, but the patient with the receptor defect had poor semen quality. Estrogen receptor-deficient male mice were subfertile and few were able to sire one litter. Estrogens are involved in the feedback regulation of gonadotropin secretion, and the suppression of FSH secretion during the period of Sertoli cell proliferation (perinatal period) may result in small testes and a low sperm production capacity in adult life.
Occurrence of Abnormalities in the Reproductive System of the Sons of Women Exposed to Diethylstilbestrol during Pregnancy
Exposure
Diethylstilbestrol (DES) was prescribed to more than five million pregnant women from the late 1940s to the early 1970s to prevent abortions and pregnancy complications (166). Dieckmann and co-workers performed a double-blind placebo-controlled study on the therapeutic value of DES during pregnancy in the early 1950s (167). DES was given to 840 pregnant women and placebo to 806 controls. Compliance was verified by a dye indicator in the urine during the whole study. The women entered the study between weeks 7 and 20 of pregnancy (the majority during weeks 10-12) and received increasing doses of DES until pregnancy week 35 (5-150 mg/day). This study clearly indicated that the medication was not efficacious in the indications for which it was used (167). Instead, in the reanalysis of the material of Dieckmann et al. (167), DES was associated with significant increases in abortions, neonatal deaths, and premature births (168). When Herbst and co-workers (169,170) reported the high incidence of a very rare cancer, clear cell adenocarcinoma of the vagina, in pubertal girls exposed to DES in utero, the U.S. Food and Drug Administration (FDA) banned the use of DES during pregnancy. Medical authorities in Europe that had allowed DES use for pregnant women soon followed FDA regulations. In Europe, approximately 200,000 French, more than 150,000 Dutch, 63,000 Czechoslovakian, and 7000 British women were exposed to DES, whereas in the United States 4.8 million women were prescribed DES during pregnancy. In addition, DES was used as an anabolic agent in livestock, and the general population that used dairy products and meat may have been exposed to the hormone via this route to an unknown, and probably variable, extent. Some of the DES-exposed daughters and sons have been followed since the 1970s and a significant number of abnormalities in the structure and function of reproductive organs have been described (171).
Structural Anomalies
Structural anomalies of the reproductive organs that are significantly more frequent in DES-exposed male subjects than in controls include meatal stenosis (12.9 vs 1.8%); hypospadias (4.4 vs 0%); epididymal cysts (20.8 vs 4.9%); testicular abnormalities, including hypoplastic testis, cryptorchidism, and capsular induration (11.4 vs 2.9%); and microphallus (4 cases vs 0 cases) (172-174). The data of Bibbo and Gill and their co-workers (173,174) are based on the follow-up studies of the offspring of mothers who took part in the double-blind study of the effects of DES on pregnancy in 1953 (167), and therefore the studies can be considered prospective. There were 308 men exposed to DES and 307 men exposed to placebo included in the study; 31.5% of men exposed to DES had an abnormality of their reproductive tract, whereas only 7.8% of controls had an anomaly (174). In the recent follow-up study of these males, it was found that the men who were exposed to DES before week 11 of gestation had twice as high a frequency of genital anomalies than did those who were exposed only later (175). This finding indicates the importance of the timing of the exposure (time of organogenesis). In a small study comprising 17 DES-exposed men, 12 nonexposed volunteers, and 11 fertile control men, genital anomalies (varicocele, epididymal cysts, absent testes) were reported in 13 of the DES-exposed subjects, 4 of the volunteers and 4 of the fertile normal controls (176). Whitehead and Leiter (177) reported genital abnormalities in 29 of 48 men exposed to DES. Hypertrophy and squamous metaplasia of the prostatic utricle was found more frequently in aborted male fetuses that had been exposed to DES than in nonexposed controls (178), suggesting that DES-exposed males may have an increased risk of prostatic hyperplasia and/or cancer when aging. The data connecting DES exposure to several structural abnormalities of the male reproductive tract are convincing and leave little space for speculation on confounding factors. However, no association was found between first-trimester exposure to sex hormones, other than DES, and external genital abnormalities in a recent metaanalysis of 14 studies (179). In a large cryptorchidism study, no association between the disorder and exposure to estrogens during the pregnancy could be found (180).
Semen Quality
Gill et al. (181) studied semen samples from 88 men exposed to DES and 85 men exposed to placebo, who were offspring of the mothers from the 1953 study performed by Dieckmann and co-workers (167). Sperm concentration of men exposed to DES was significantly lower than in the controls (83 million/ml vs 123 million/ml, p<0.02). There was no difference in semen volume, whereas the total sperm count, sperm motility grade, the total number of motile sperm, the percentage of sperm with normal morphology, and the quality score were all statistically lower in men exposed to DES. Azoospermia was found only in men exposed to DES, and 20.5% compared to 3.5% of men who received placebo had a sperm concentration in semen of less than 20 million/ml. The groups did not differ in their testosterone, FSH, or LH levels (173). In a later study on the same men (20 controls declined to participate), sperm concentrations still differed significantly, whereas other semen characteristics were similar between the groups (182). Similar results were obtained in another study (176) in which the mean sperm concentration of men exposed to DES was 66.4 million/ml compared to 101.7 million/ml in normal volunteers (p<0.05). In this study, the zona-free hamster egg penetration assay was also performed: sperm from 14 of 17 men exposed to DES failed to penetrate more than 14% of the eggs (which is the reference value for the normal fertility range), suggesting infertility, whereas only 2 of 12 unexposed volunteers and none of 11 fertile normal controls had an abnormal test result. In the study performed by Whitehead and Leiter (177), only 33% of the men exposed to DES had normal semen quality. However, Andonian and Kessler (183) found no difference in semen quality between 24 men exposed to DES and 24 age-matched control men. Again, the large 1953 study population that has been followed prospectively appears the most valid for evaluation of semen quality. On the basis of that finding, DES exposure resulted in a significant decrease in semen quality.
Semen quality and fertility are not in direct correlation. In the latest follow-up study of the Dieckmann cohort, no difference in the fertility between men exposed to DES and their controls were found (175). This is compatible with the earlier findings (181) that the majority of the men exposed to DES had sperm concentrations well above the limit at which fertility is supposed to be disturbed (20 million/ml), although the mean sperm concentrations of exposed men were lower than those of controls.
Testicular Cancer
There is no conclusive evidence to indicate an increased risk of testicular cancer in men exposed to DES (184), although the incidence of cryptorchidism is a well-known risk factor for testicular cancer and has been observed more frequently in this group (171). Two patient cases with seminoma in men exposed to DES have been reported (185), but epidemiological studies have failed to show a statistically significant relationship between DES exposure and testicular cancer. There have been a few case-control studies that evaluated prenatal hormonal risk factors for testicular cancer (186-191). In the first study (186), 131 testicular cancer patients, under age 40, and their matched controls were analyzed. In 6 cases of cancer the mothers had been treated with hormones during pregnancy, whereas only one mother of the control cases had received any hormones. The difference was not statistically significant, but if another factor, nausea, was combined with hormone treatment, they formed a significant risk factor (relative risk 4.33).
In the case-control study of Depue et al. (187), 108 testicular cancer patients, under age 30, were studied. Mothers of 9 cancer patients had been treated with hormones (2 with DES, 1 with estrogen, 1 with progestin, and 5 had pregnancy tests consisting of a single injection of an estrogen-progestin preparation), whereas 2 controls had either estrogen treatment or a pregnancy test. The relative risk (8.00) was significantly increased in the men exposed to hormones (p=0.02). However, the exposures were very heterogeneous, and single pregnancy tests can hardly be compared to long-term DES treatment.
In a similar study comprising 202 cancer cases and 206 controls, Brown et al. (189) found no excess risk associated with the use of hormones during pregnancy: mothers of 4 cancer patients and 5 control mothers had received hormone treatment. Two mothers in each group had been treated with DES, 1 control with estrogen, 1 case with progesterone, 1 in each group had a hormone pregnancy test, and 1 control had an unidentified hormone treatment. However, it should be noted that 19 mothers in this study were medicated for bleeding problems, but only 2 (both case mothers) mentioned a specific hormone used; 13 of the treated were case mothers and 6 were control mothers.
In a case-control study of 273 testicular cancer patients from northern California (190), no association was found with the mother's hormone exposure or DES exposure. Mothers of 9 cases and 10 controls had been treated with hormones (odds ratio 0.9). Four of the case mothers and 2 control mothers were exposed to DES.
The case-control study of Schottenfeld et al. (188) was based on questionnaires received from 190 testicular cancer patients (The Sloan Kettering Cancer Hospital, New York), 166 hospital controls, and 143 neighborhood controls. There was no statistically significant association between hormone treatment and cancer: 5.8% (n=11) of cases had been exposed to DES or other hormones, whereas 2.1% (n=3) of the neighborhood controls and 2.5% (n=4) of the hospital controls had received exogenous hormones. Similarly, a case-control study of 79 testicular cancer patients from the Connecticut Tumor Registry failed to show any increased cancer risk in men exposed to DES (191).
The studies above have been described in detail because they illustrate two major problems. First, DES treatment may have been initiated at various times during pregnancy; therefore, the presumed critical period during which adverse effects of estrogens might occur may have been missed in some of these studies. Second, the investigated populations of testicular cancer patients have been too small to determine if a significant difference truly exists between DES-exposed and nonexposed men. When we combined the data presented above in a metaanalysis, a marginally significant increase in testicular cancer incidence for the individuals exposed to hormones (including all hormones) was found; Mantel-Haenszel estimates of the common odds ratio was 2.1 with 95% confidence intervals of 1.3 to 3.3. Exposure to DES was a significant risk factor for testicular cancer on the basis of our metaanalysis: odds ratio was 2.6 with 95% confidence limits of 1.1 to 6.1. It would be most important to obtain additional information on the incidence of testicular cancer in men born to mothers who participated in the double-blind, placebo-controlled DES trial in the 1950s (167).
Summary
Exposure to DES during pregnancy results in an increased risk for several male reproductive disorders, such as cryptorchidism, urethral abnormalities, epididymal cysts, and testicular hypoplasia. In addition, the semen quality of DES sons is worse than that of controls. Incidence of testicular cancer is approximately doubled among DES sons compared to the general population, but whether this represents a true increase of the cancer risk is equivocal.
Effects of Synthetic Estrogens on the Testis in Animal Models
Synthetic estrogens, such as DES, ethinyl estradiol, and estradiol benzoate have been thoroughly studied in several animal models because of their pharmaceutical applications. There are comprehensive reviews covering this topic (164,192,193).
Mechanism of Action
The effects of estrogens depend on the dose, time, and probably the duration of exposure. Estrogens also act at several levels in the reproductive system, i.e., they influence specific neuronal areas in the brain, they modulate gonadotropin secretion from the pituitary gland, and they directly affect the reproductive organs. Estrogens probably exert most or all of their effects through a specific receptor; such receptors are present in the brain, pituitary, gonads, and accessory sex organs at one or another time during fetal, prepubertal, or adult life (143). However, the precise localization and temporal expression of estrogen receptors during differentiation and development of the testis and male reproductive tract are poorly described and further, more definitive, studies are needed. The effects of DES are not unique to this compound but are probably shared by all estrogens (164). Many of the synthetic estrogens are more effective in lower doses than endogenous estradiol because they are not bound by sex hormone-binding globulin (SHBG), which normally binds approximately 95% of circulating estradiol, rendering it biologically inactive. Estrogens are metabolized rapidly in the testes, e.g., by specific sulfotransferases, after which they cannot bind to their receptor (194). If the active center of the enzyme is occupied by a xenobiotic, metabolism of endogenous estrogens may be disturbed and high levels of active hormone may be available. This is particularly important during fetal development when the levels of ambient estrogens are high.
Adverse Effects of Neonatal Estrogen Treatment
Long-lasting suppression of spermatogenesis and atrophy of reproductive organs in neonatally estrogen-treated rats and mice have been described by many workers since the 1950s (164). A single injection of estradiol benzoate (250 µg on day 5) resulted in a marked delay of the onset of puberty in rats (195). When mice were treated with repeated doses of estradiol on days 1 to 5 after birth, the testes were irreversibly damaged, and subsequent treatment with testosterone and gonadotropins failed to maintain spermatogenesis in the majority of these estrogenized mice (196). These two studies are cited because they indicate that estrogens given neonatally act directly on both the testes and the pituitary gland. It is noteworthy that the neonatal period in rodents corresponds in many ways to the second and third trimesters of pregnancy in the human.
Adverse Effects of Prenatal Estrogen Treatment
Prenatal (day 11 and 12 postcoitum) exposure of mice from the Sv-Sl CP strain (a strain in which the males are susceptible to testicular teratomas) to ethinyl estradiol resulted in an increased incidence of cryptorchidism (p=0.0001), and 19 of 224 exposed male animals developed testicular teratoma compared to 4 out of 107 controls (the odds ratio of 2.4 was not significantly different) (197).
McLachlan and co-workers have performed a large series of studies on the effects of prenatal exposure of mice to DES (192,193). In most of the studies, pregnant mice were treated with 0.01, 1, 10, or 100 µg/kg/day DES or corn oil on days 9 to 16 of pregnancy (time of sexual differentiation). The high doses are closely equivalent to those used for pregnant women (173). Male offspring from these pregnancies suffered from the same structural and functional anomalies reported in men exposed to DES, i.e., epididymal cysts, cellular atypia in the prostate, cryptorchidism, testicular hypoplasia, poor semen quality, and subfertility (192). In addition, Sertoli-cell hyperplasia, interstitial testicular tumors, squamous metaplasia of the seminal vesicles, and rete testis adenocarcinoma were found frequently in the male offspring of mice exposed to DES during pregnancy (198). The analogy between the findings in the human and the mouse illustrates how informative and relevant the results from animal studies are.
Estrogen Effects on the Müllerian Ducts
Müllerian inhibiting substance secreted by Sertoli cells is responsible for regression of the Müllerian ducts. Analysis of male mouse embryos exposed to DES revealed delayed and incomplete regression of the Müllerian ducts (192). In vitro organ culture experiments verified the inhibitory action of DES on Müllerian duct regression (136). Estrogen receptors were found both in the Müllerian ducts and Sertoli cells at the time of regression (143). Estrogens could either affect the Müllerian ducts directly or influence the expression of the MIS gene in the Sertoli cells. Some of the structural abnormalities observed after birth may arise as a result of incomplete regression of the Müllerian ducts (198).
Estrogen Effects on the Developing Testis
Reports on various experimental animals (e.g., sheep, rats, mice) describe how exposure to exogenous estrogens during the neonatal period causes drastic reductions in the secretion of FSH from the pituitary gland and the presumption is that similar effects would occur before birth (199). As FSH plays a vital role in controlling multiplication of Sertoli cells at this time (138) the prediction would be that estrogen-induced suppression of FSH levels would lead to a slower rate of Sertoli cell multiplication. As the number of Sertoli cells formed in fetal/neonatal life is an important factor influencing the maximum level of sperm production in adult life, the consequences of such a change in terms of sperm counts is obvious; moreover, such an effect is irreversible once Sertoli cell multiplication stops in early postnatal life. There is abundant evidence from man (hypogonadotropic hypogonadism) and from animal species that suppression of FSH levels in early postnatal life results in just such changes [reviewed by Sharpe (138)]. Recent evidence from the fetal sheep (165) also shows that suppression of FSH secretion in the fetal male during the second half of gestation results at birth in testes that contain 40% fewer Sertoli cells than occurs in control animals.
It is therefore hypothesized that prolonged exposure of the developing male, during both fetal and postnatal life, to exogenous estrogens (perhaps even at low levels) could reduce Sertoli cell number and thus reduce sperm output (and sperm counts) in adult life. Experiments involving exposure of rats to various xenoestrogens during the period of Sertoli cell multiplication have shown that in adult life such exposure results in small (8-12%) but highly significant reductions in testis size and a corresponding decrease in daily sperm production (200). These effects have been achieved after exposure to relatively low levels of the chemicals (alkylphenols, phthalates; 1 mg/liter in drinking water of pregnant rats) under test. For example, butylbenzyl phthalate has been found to occur in butter and margarine at concentrations as high as 47.8 mg/kg (201). Such findings suggest that there is the theoretical possibility that human exposure to such chemicals might have contributed to the decline in sperm counts in men described earlier.
Summary
Diethylstilbestrol treatment of experimental animals in utero results in increased incidence of cryptorchidism; urethral abnormalities; testicular hypoplasia; poor semen quality; and infertility, abnormalities in accessory sex organs, rete testis adenocarcinoma, interstitial cell hyperplasia, and tumors. Thus, the outcome of DES exposure of experimental animals is highly analogous to the findings in humans. Recent data in the rat suggest that perinatal exposure to xenoestrogens, such as butylbenzyl phthalate, results in decreased size of the testes and daily sperm production in adult life.
Environmental Chemicals with Known Estrogenic Effects
Estrogenic effects are not restricted to a small group of therapeutic agents but appear in several groups of compounds that are used daily in industry, agriculture, or in the home (79,80,202-204). The major groups of environmental chemicals, such as organochlorine pesticides, PCBs, dioxins, alkylphenol polyethoxylates, phytoestrogens, and other xenoestrogens, currently known to have estrogenic effects in vertebrates or in assays in vitro are discussed here. A major problem is the determination of those chemicals that are estrogenic (or otherwise endocrine-disrupting, i.e., disturbing normal endocrine homeostasis). At present, tens of thousands of man-made chemicals are being used, yet the effects on the endocrine system have been studied for only a few of these. The estrogenic activity of the majority of chemicals (e.g., alkylphenols, phthalate esters, bisphenol-A) has been detected by accident, not by intent, although recently some screening of chemicals used in large volumes has been attempted (204). Hence, it is highly possible that other estrogenic chemicals remain unidentified. However, it should be remembered that many of the chemicals to which man is exposed have been tested (often in two- or three-generation studies) before being approved for use; and hence, if any of these chemicals were a strong estrogen, this would probably have been discovered. This is especially the case for chemicals that are currently approved for use in food production, such as food additives and pesticides, and for new chemicals that have been produced in large amounts from the early 1980s in the European Union (EU). The current legislation demands extensive documentation for safety by regulatory agencies before a chemical can be used in foods or commercial products. However, many chemicals were introduced before these strict regulations were enforced. Thus, the present situation is that man and wildlife are exposed to a very wide range of chemicals. For the majority of these we do not know whether they are estrogenic, whether their effects are additive, or even what the true exposure to these chemicals is.
A xenoestrogen can induce its estrogenic effect in multiple ways: it may act directly through estrogen receptors, or it may disturb estrogen metabolism, thus increasing the levels of the endogenously produced ligand. Different estrogenic and antiestrogenic ligands form functionally different complexes with the estrogen receptor, and their transcriptional effects depend on the cell type and promoter (205). Thus, the same compound may potentially have an estrogenic effect in one system or at one concentration, and an antiestrogenic effect in another system or at another concentration. Furthermore, effects of many compounds influencing other hormone systems (e.g., antiandrogens) may mimic those of estrogens.
A number of chemicals, mainly pesticides and many of which are currently being used, have been implicated as environmental hormones possessing endocrine-disrupting properties (80). In the public debate on male reproductive disorders, this has misled many to suppose that all of these chemicals are estrogenic. In fact, many of these compounds have not been adequately tested for estrogenic activity. However, for many others, a large toxicological database exists, including data on reproductive toxicity, effects on steroid-metabolizing enzymes, and effects on hormone-producing tissues. A short summary of the most relevant toxicologic effects known for a number of xenoestrogens and other environmental chemicals that have been implicated as environmental hormones is given in "Appendix A." It also outlines the safety assessment procedures and principles applied world-wide by regulatory agencies.
Below is a short examination of each of the groups of chemicals that are known to be estrogenic.
Organochlorine Pesticides
Organochlorine pesticides include dichlorodiphenylethanes (DDT, DDD, DDE, dicofol, perthane, methoxychlor), cyclodienes (chlordane, oxychlordane, trans-nonachlor, heptachlor, heptachlorepoxide, aldrin, and dieldrin), hexachlorobenzene, and hexachlorocyclohexanes (206). Many of these, most notably DDT, were used in large quantities until the 1960s when the use of DDT was banned or restricted in Western countries. Hexachlorobenzene, however, was used in the United States until 1985. DDT products are still used widely in many developing countries. Despite restrictions on their use, these compounds are still circulating in the environment because many of them bioaccumulate and become concentrated in body lipids (biomagnify). The breakdown and elimination of these compounds is very slow; therefore, their effects can be persistent, lasting for generations (DDT has a half-life of >60 years in the environment). Long-term exposure to small amounts of organochlorine contaminants leads to the accumulation of considerable burdens in animal and human tissues (207,208). It is therefore not the amount of DDT to which a mother is exposed during pregnancy that is critical but rather her lifetime exposure that will determine the level of exposure of the fetus and the breast-fed infant.
Commercial DDT contains several isomers of which p,p´-DDT is the most prevalent (75-80%), whereas the proportion of the most estrogenic isomer o,p´-DDT is 10 to 25% (89,209). The o,p´-isomers are less stable than the p,p´ configurations and are therefore found only in low concentrations in nature (210). However, p,p´-DDT was also reported to have estrogenic actions both on the rat uterus (211) and in the MCF-7 breast cancer cell line (212). The estrogenic activity of DDT isomers compared to that of estradiol is very weak (103-106 times less potent). However, the long half-life and bioaccumulative properties of DDT indicate that levels of human exposure may be sufficient to induce estrogenic effects in certain circumstances. This is particularly true for the period from the 1940s to 1960s when DDT was used widely including in direct application to humans.
Antiandrogenic (demasculinizing) and estrogenic (feminizing) effects often manifest themselves in the same way, although through distinct receptors (213). Therefore the recent discovery that p,p´-DDE, the main metabolite of DDT in the body, is a potent antiandrogen (214) may explain some of the estrogenic effects observed in the environment; many of these effects may occur due to an antiandrogen activity of a xenobiotic.
Fry and Toone (115) induced feminization in male California gulls (Larus californicus) by injecting eggs with DDT in amounts that were comparable to those found in seabird eggs in southern California in the late 1960s. A skewed sex ratio in favor of females in large gull populations suggested the possibility of a causal relationship with the estrogenic action of DDT (86). The effects of DDT metabolites and dicofol in reptiles have been discussed in the section "Changes in Male Reproduction in Wildlife. Estrogenic Effects on Developing Animals." In mammals, the effects of DDT compounds on male reproductive function are less apparent (215).
Methoxychlor is estrogenic in the E-SCREEN assay (204). It was also found to be estrogenic in vivo in rats (216). Methoxychlor or DDT exposure of neonatal rats did not affect male reproductive organ weights in adulthood (217), and neither induced epididymal cysts (218), which were found frequently in mice exposed to DES (192). However, exposure throughout gestation and lactation in rodents resulted in slightly smaller testes and epididymides and in lower sperm counts in male offspring than in controls (219,220). It was suggested that the inability of the neonatal rodent to metabolize methoxychlor to its active estrogenic form might explain the discrepancy between these studies (220).
Chlorinated cyclodienes induce liver enzymes that hydroxylate testosterone (221). Chlordane disturbed spermatogenesis and caused dose-related damage to the testes of mice fed for 30 days with 0.08 mg or 0.25 mg of the active ingredient (222). Mating studies of dieldrin-exposed rats suggest male-dependent disturbances in fertility (223). In the E-SCREEN assay, chlordane and heptachlor were not estrogenic, but the heptachlor derivative 1-hydroxy chlordane was (212). In addition, dieldrin was shown to be estrogenic (81).
Hexachlorobenzene was also reported to induce liver enzymes hydroxylating androgens (221). Long-term studies have demonstrated liver and kidney anomalies in exposed animals but indicate no effect on fertility (224).
Hexachlorocyclohexanes (HCHs) comprise several isomeric forms; these compounds are also called benzene hexachloride (BHC). 
-HCH has the common name lindane and is the most acutely toxic of the isomers (215). The most persistent and bioaccumulating isomer is ß-HCH, which accounts for 90% of the total HCH found in human milk (225). Lindane was reported to have both estrogenic and antiestrogenic effects in female rats (226). In male weanling rats fed with ß-HCH (0, 2, 10, 50, or 250 mg/kg) for 13 weeks, liver enzyme induction occurred at doses >2 mg/kg; testis weights decreased at doses >50 mg/kg; and testicular atrophy resulted from a dose of 250 mg/kg (227).
In hamsters, a single injection of the weakly estrogenic chlordecone (Kepone) in neonatal males reduced testicular and epididymal weight (228). Estrogenicity of chlordecone was also demonstrated in rats (229) and birds (230).
Polychlorinated Biphenyls
Polychlorinated biphenyls are industrial chemicals used since 1929 as heat transfer and hydraulic fluids, adhesives, flame retardants, dielectric fluids for capacitors and transformers, and waxes (231). PCBs consist of 209 congeners, which are found in different mixtures in commercial products. Before the production of PCBs was banned in the United States in 1977, hundreds of millions of kilograms were produced, and a large proportion of the synthesized product is still in the environment because of bioaccumulation and slow biotransformation.
The biological effects caused by the various congeners differ, not only in potency but also qualitatively. Several non-ortho- and mono-ortho-substituted PCB congeners induce effects similar to those caused by chlorinated dioxins and dibenzofurans; i.e., the toxicity is probably mediated through interaction with the aryl hydrocarbon (Ah) receptor. Other PCB congeners presumably act by different mechanisms. In addition, there are PCB congeners that are intermediate in this respect; i.e., they elicit a mixed spectrum of effects. Our knowledge of possible interactions between the various groups of PCBs is still very limited (232).
Both estrogenic and antiestrogenic effects have been reported for different PCB congeners (233). The estrogenic potency appears to depend on the percentage of chlorine: less-chlorinated PCBs (Aroclors 1221, 1232, 1242, and 1248) have estrogenic activity whereas more chlorinated congeners do not (209). The stability of the compounds increases with higher chlorination. Less-chlorinated compounds were shown to transfer more readily across the placenta than were the highly chlorinated PCBs (234). PCBs are hydroxylated in animals, and these hydroxybiphenyls are quite active as estrogenic compounds [i.e., more than 1/100 of estradiol activity (235)]. Antiestrogenic effects have been found in MCF-7 breast cancer cells with 3,3´,4,4´-tetrachloro-biphenyl (a dioxinlike PCB), a form known to bind to the Ah receptor, mediating the effect (233). Reproductive failure of seals in the Wadden Sea has been attributed to PCBs (236), and has been supported by laboratory studies (121). However, this relationship may not necessarily have been a consequence of the estrogenicity of the PCBs.
Dioxins and Furans. Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) consist of 75 and 135 different congeners, respectively (237). The most toxic congener is 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), commonly referred to as dioxin. These compounds are formed as unwanted by-products in the manufacture of chlorinated hydrocarbons. Other sources include incineration processes, paper and pulp bleaching, and emissions from steel foundries and from motor vehicles (238).
Most of the animal studies on dioxins have been performed with TCDD [reviewed by Peterson et al. (239)]. Dioxins act through an Ah receptor that is also involved in mediating the antiestrogenic effects of TCDD (240,241). However, the role of the Ah receptor has not been established for several of the toxic effects that are found in males exposed to TCDD (242). There is considerable literature documenting the toxic effects of dioxins on the male reproductive system (239,242). Prenatal and lactational exposure of male rats to TCDD profoundly disturbed the developing male reproductive organs: anogenital distance was shortened, testicular descent was delayed, and the weights of all sex organs were reduced (243). Furthermore, spermatogenesis was inhibited, sexual behavior was feminized and demasculinized, and the regulation of LH secretion was feminized (244-246). Perinatal suppression of plasma testosterone concentrations appeared to be involved in the changes described. The effects were elicited by a single maternal oral dose of TCDD on day 15 of pregnancy [ED50 approximately 0.16 µg/kg; at this dose, TCDD had no discernible effect on the mother (242)]. Most of the effects were found at the lowest dose level tested (0.064 µg/kg), whereas in other studies, reproductive toxicity had been observed with doses of <0.001 µg/kg/day (247,248). The mechanism of action of dioxins and furans may not be primarily estrogenic or antiestrogenic (e.g., antiestrogenic effects of TCDD cannot be reversed by high estrogen concentrations), but it is certainly a hormonelike effect. This necessitates the close surveillance of humans exposed to TCDD.
Alkylphenol Polyethoxylates
Alkylphenols and related compounds are present in surface waters and aquatic sediments (249-251). They are products of the microbial breakdown of alkylphenol polyethoxylates (APEs) that are widely used in industrial surfactants (250,251). These effective, nonionic surfactants are used in detergents, paints, herbicides, pesticides, and cosmetics, to name a few major groups of products. Over 300 million kilograms of APEs are produced annually. After sewage treatment, approximately 60% of the APEs are released into the aquatic environment as short-chain APEs (e.g., nonylphenoldiethoxylate [NP2EO]), alkylphenol carboxylic acids (e.g., nonylphenoxycarboxylic acid [NP1EC]), and alkylphenols (e.g., nonylphenol [NP]; and octylphenol [OP]) (252-254). Alkylphenols are relatively persistent and bioaccumulate in the lipids of living organisms (255,256).
NP and OP were shown to be estrogenic both in vivo and in vitro in mammalian systems (202,257). The effects were estrogen receptor mediated. OP was more potent than NP, reaching approximately 1/1000 of the potency of estradiol. In the trout hepatocyte assay, many other APEs were also shown to be weakly estrogenic (111).
Phytoestrogens
Phytoestrogens are natural compounds present in plants and are ingested daily in milligram quantities. The active substances are isoflavones (genistein and daidzen) and coumestans (coumesterol) (258). Fungal metabolites, such as zearalenone, found in foodstuff (e.g., grain) also belong to the phytoestrogens. Reproductive disorders in sheep are well documented after the eating of red clover containing high amounts of genistein (259). Some of the food products rich in phytoestrogens include rye, wheat, cabbage, sprouts, spinach, and soybean. Soybean is by far the richest source of plant estrogens and is used ubiquitously in the food industry as a protein source, including infant milk formula substitutes. Phytoestrogens may not bioaccumulate or biomagnify but they are readily metabolized and excreted (260).
Phytoestrogens have been shown to have estrogenic effects both in vitro and in vivo (212,258,261). Feeding of rams with clover that is rich in isoflavone resulted in decreased sperm counts (262,263). The effects are receptor mediated and depending on the dose are either estrogenic or antiestrogenic in an adult animal (264,265). Because of these potential antiestrogenic effects, high doses of isoflavones in the diet have been proposed as being beneficial for reducing the risk of hormone-dependent cancers (266). However, little consideration has been given to neonatal and childhood life when exposure to phytoestrogens would be presumed to have nonbeneficial effects.
Other Xenoestrogens
Bisphenol-A, a plastic monomer that was released from polycarbonate flasks during autoclaving, was shown to have an estrogenic effect on MCF-7 breast cancer cells (267). Notably, bisphenol-A is used extensively as a plasticizer, e.g., it is used in the lacquer coating of food cans that are then heated for sterilization purposes (268). Other common chemicals used in the plastic industry include the phthalate esters, butylbenzyl phthalate and di-n-butylphthalate, which were shown to act as weak estrogens on breast cancer cells (203). In the E-SCREEN assay, di-n-butylphthalate was not estrogenic (204). Neither of the phthalate esters had antagonist effects, suggesting that their action may be cumulative (203). Phthalates are the most abundant man-made environmental pollutants; and human intake per day by various routes, especially through the diet, is measured in tens of milligrams (201). A food antioxidant, butylated hydroxyanisole (BHA), was also estrogenic in breast cancer cell assays (203). However, the estrogenic potency of BHA and phthalate esters was lower than that of octylphenol or o,p´-DDT.
Summary
Numerous environmental chemicals, such as many organochlorine pesticides, PCBs, alkylphenol polyethoxylates, phthalates, and phytoestrogens are known to have estrogenic effects in vertebrates or in assays in vitro. However, only a few of the tens of thousands of man-made chemicals have been tested for estrogenic or other endocrine activity, and therefore, it is highly possible that other estrogenic chemicals remain unidentified. A major problem at present is how to fill this gap in our knowledge rapidly and cost effectively.
Exposure of Humans to Environmental Chemicals with Estrogenic Activity and Their Effects on Male Reproductive Health
The major routes of exposure to man-made chemicals are thought to be
- Dietary: pesticides, (including chemicals used in the formulation of commercial products), food additives (including synthetic flavoring substances), contaminants (such as PCBs, dioxins, metals, industrial chemicals, especially those that are biomagnified in food chains), packaging and wrapping materials (e.g., plastics, food wraps)
- Environmental: from the pollution of air and water
- Domestic: from contact with household products, cosmetics, clothing, and probably many others
- Occupational: inhalation, dermal contact and ingestion of active compounds, depending on the occupation.
Occupational exposure has not been considered in any detail in this report, which is aimed at describing the situation for the general population. However, valuable information on possible association between exposure to chemicals and effects on humans may originate from studies in occupational settings where high exposures have taken place. Because of better documentation and higher exposure, such studies are more likely to reveal adverse effects of chemicals on humans than are the studies on the general population (269).
The diet is usually regarded as the most important source of foreign chemicals. Very preliminary estimates of some exposures in Denmark are included in "Appendix A." It should not be overlooked that the exposure via routes other than food, such as air, drinking water, and particularly the skin (e.g., detergents) may be highly significant. Current knowledge on actual exposures is rather limited, in particular with regard to the domestic exposure to chemical products. A close examination of all possible exposures to chemicals suspected to be environmental hormones has not been possible within the constraints of this report. Humans may be exposed to xenoestrogens in multiple ways. Direct administration of synthetic hormones, such as DES or ethinyl estradiol is obvious, but the same hormones may be found in meat and dairy products in some countries (270). Cow's milk has a high concentration of endogenous estrogens during late gestation, and this milk is also collected for human consumption. The ratio of estrogens in this milk to that in plasma is generally greater than one (271), and therefore much higher than in human breast milk. Occupational exposure in the pharmaceutical industry is a possibility for a small minority. Estrogens occur in measurable amounts in sewage effluent used for irrigation (272). Phytoestrogens are ingested in large amounts, and weakly estrogenic alkylphenolic compounds are applied to the skin and ingested daily. Important issues to consider are the quantity of estrogenic compounds present, their potency, their capability to bioaccumulate and biomagnify, and their additive, syne
Table 1.
Table 1. Continued
Table 1. Continued
Table 1. Continued
