Alterations in Steroidogenesis in Alligators (Alligator mississippiensis) Exposed Naturally and Experimentally to Environmental Contaminants D. Andrew Crain, Louis J. Guillette, Jr., Andrew A. Rooney, and Daniel B. Pickford Department of Zoology, University of Florida, Gainesville, FL 32611 USA Many environmental cont ants aer the reproduction of animals by altering the develop- ment and function of the edocrine system. The ability of environmental contamints to alter the endocrn s of alligators was studied both in a descriptive study in which juvenile alli- gators from; a historicallycontaminated lake were compar to anima fro a control lk d in an esx dy in: wh ing c a w in ovo to severa examining s hormone conc rtions, - me (AM) ar activity, and gonadal histopahol In the d e udy, juvenile alligars from the conta- minated lake had significanty lower plasma testosterone concentrations (29.2 pgtmil compared to 51.3 pg/mi), whereas plasma 17f-esradiol conc ons did not v*y en compard to controls. GAM r activity w t in t:he alig sm the contami- nated lake (7.6 pmollg/hr to t1.4 In e al study, t endocrine-disr n tandd caused sex revenal from male to female, with a correspondig increwe in aoma it Vimoolin had no apparent effect on male or female alligators. Among the herbicides tested, atrazine induced GAM aromatase activity in male haIg ts at was ndether haracteristic of males nor females, although testicular differentiation was not altered. Exposure to 2,4- dichlorophenoxyacetic acid had no effect on the do parameters t were m Together, these studies show that ex e to some e l h (su as atazine) can ater s i t he enon anoted forLa Apopka, Florida, ligators can not be fll explain by tis mechanism. Key words: alligator, aromatase, atrazine, contaminants, 2,4-D, endocrine disruption, hormones. Environ Healdb Perspect 105:528-533 (1997) Environmental contaminants alter the reproduction of a number of wildlife species by changing the normal endocrine environ- ment that mediates sexual differentiation and function (1). Many of these endocrine alterations are thought to occur by direct interactions between the contaminants and hormone receptors (2), but the specific mechanisms by which most environmental contaminants cause endocrine disruption are unknown. Although all vertebrates are potentially susceptible to reproductive dis- ruption by endocrine-disrupting contami- nants (EDCs), many ectothermic verte- brates are particularly sensitive due to the processes mediating the organization of the reproductive system (3). Unlike birds and mammals, many fish, amphibians, and rep- tiles exhibit environmental sex determina- tion, by which the gender of the undifferen- tiated embryo is determined by an environ- mental variable. In many reptiles, the tem- perature of egg incubation determines the sex of the offspring (4). Exposure of devel- oping reptile embryos to exogenous chemi- cals can mimic the effects of temperature on sex determination. For example, when red- eared turtle (Trachemys scripta) embryos are incubated at a male-producing temperature and exposed to 1 7,-estradiol (E2) during the window of developmental sex determi- nation, phenotypically female turtles are produced (5,6). This estrogen-induced sex reversal appears to be dose dependent (7) and suggests that other steroidal agonists, steroidal antagonists, and steroidogenic disruptors could alter normal sexual differ- entiation. Indeed, Wibbels and Crews (8) found that steroid hormones are not exclu- sive in their ability to alter normal sex determination, as many estrogen agonists and steroidogenic modifiers mimic and/or reverse the effects of temperature on the differentiation of primary sex organs in red-eared turtles. The specific mechanisms by which temperature determines gender are unknown, but it is hypothesized that tem- perature stimulates or suppresses pivotal steroidogenic enzymes (9). These enzymes then propagate a cascade of events leading to the organization of a testis or ovary. This hypothesis is supported by work con- ducted on the steroidogenic enzyme aro- matase. Aromatase converts androgens to estrogens by binding the C19 androgen substrate and catalyzing several reactions, thus leading to a phenolic ring characteris- tic of estrogens (10). The pivotal role of aromatase in temperature-dependent sex determination is supported by several lines of evidence. First, several studies indicate that aromatase activity is increased in prospective females during periods coincid- ing with thermosensitivity (11-13). Second, high doses (50-100 pg per egg) of testosterone cause feminization of T scripta at a male-producing temperature (8,14). Because testosterone is the precursor to E2, this phenomenon is thought to be mediated by the enzyme aromatase. Third, adminis- tration of an aromatase inhibitor induces male sex determination in both a female unisexual (parthenogenetic) lizard and a turtle with temperature-dependent sex determination (15). Collectively, these studies suggest that aromatase is an enzyme critical to thermosensitive sex determina- tion and is capable of modification by extrinsic factors. In consideration of these studies, we propose that the endocrine-altering effects of some environmental contaminants may be mediated via changes in the expression or activity of the aromatase enzyme. Two studies, one descriptive and one experimen- tal, were conducted to test this hypothesis. First, juvenile alligators from a control lake and a lake historically contaminated with a number of persistent organochlorines were analyzed for plasma steroid hormones and in vitro gonadal-adrenal aromatase activity. Second, embryos from a control lake were exposed to several known hormonal modi- fiers and two common herbicides, and Address correspondence to D.A. Crain, Department of Zoology, 223 Bartram Hall, University of Florida, Gainesville, FL 32611 USA. D.B. Pickford's current address is Department of Physiology, Pharmacology, and Toxicology, University of Manchester, Manchester, England. We thank A. Woodward, Florida Game and Freshwater Fish Commission, and F. Percival, National Biological Service, for assistance in collect- ing juvenile animals and eggs. J. Joss and M. Smith kindly provided information about aromatase assay techniques, D. Spiteri assisted with the histology, and K. Bjorndal and D. Evans provided helpful edi- torial comments. G. Masson of the U.S. Fish and Wildlife Service provided intellectual and financial assistance. Funding for this project was provided through an EPA Graduate Student Fellowship (U- 914738-01-0), an EPA Grant (CR821437), and an NIEHS Grant (PR471470). All lab work was con- ducted in full compliance with guidelines of the University of Florida Institutional Animal Care and Use Committee, and field work was conducted under permit from the Florida Game and Freshwater Fish Commission.. Received 23 September 1996; accepted 5 February 1997. Volume 105, Number 5, May 1997 * Environmental Health Perspectives 528 Articles * Endocrine alterations in alligators hatchlings were analyzed for egg chorioal- lantoic fluid (CAF) hormones, plasma steroid hormones, and in vitro aromatase activity. Using these studies, we sought to determine whether aromatase function could explain endocrine alterations in alli- gators exposed to EDCs. Materials and Methods Animals and Treatments For the descriptive study, eggs were collect- ed from six nests on Lake Apopka, Florida (contaminated lake), and six nests on Lake Woodruff National Wildlife Refuge, Florida (control lake), during the first week of July 1995. Lake Apopka is designated as one of Florida's most polluted lakes (16) due to extensive agricultural activities around the lake, a sewage treatment facility associated with the city of Winter Garden, Florida, and a major pesticide spill from the Tower Chemical Company (Clermont, FL). The pesticide spill, which occurred in 1980, consisted primarily of dicofol, but had significant amounts of DDT, DDE, and DDD in the mixture (17). Analysis of alligator eggs taken from Lake Apopka in 1984 and 1985 revealed significant residues of toxaphene, dieldrin, p,p'-DDE, p,p'- DDD, trans-nonachlor, and polychlorinat- ed biphenyls (18). A previous study found evidence of estrogenic contamination among the female alligators of Lake Apopka (19) and, as we wanted to mini- mize the number of eggs taken from Lake Apopka, we incubated eggs only at a female-producing temperature (30?C) (20). After hatching, alligators were housed at the Sante Fe Teaching Zoo (Santa Fe Community College, Gainesville, FL) in outdoor semiaquatic enclosures. At 9 months of age, the female alligators were transported to the laboratory for tissue col- lection. For the experimental study, eggs were collected from seven nests on Lake Woodruff, Florida, during the first week of July, 1995. Eggs were transported to our lab and placed in an incubator at 30?C. One egg from each clutch was opened to stage the embryos. Staging was based on criteria defined by Ferguson (21). Five days after collection (and prior to the tempera- ture-sensitive period when sex determina- tion occurs), eggs were separated into two groups such that half of the eggs from one clutch were incubated at 300C (female-pro- ducing temperature) and half at 330C (male-producing temperature). Eggs were maintained at approximately 90% humidi- ty using sphagnum moss as incubation material. Within each incubation group, eggs from each clutch were distributed among six treatment groups of varying dosages (Table 1). One treatment group served as a control and three groups served as endocrine-disrupting standards: E2; tamoxifen, which acts as an estrogen in embryonic alligators but an antiestrogen in hatchlings (22); and vinclozolin, a potent antiandrogen in rodents (23). The two remaining treatment groups were the mod- ern-use herbicides atrazine and 2,4- dichlorophenoxyacetic acid (2,4-D). Treatments were applied topically to the eggshell in 50 pl of 95% ethanol, a tech- nique frequently used to transport com- pounds inside reptilian eggshells (7,8). Using this method, Crews et al. (7) found that greater than 89% of the applied com- pound is incorporated into the embryo. The treatments were applied at stage 21 of embryonic development, the beginning of the critical period of gonadal differentia- tion (20). Upon pipping, CAF was collected and frozen at -72?C until steroid hormone analysis. Total protein content in the CAF samples was determined using a commer- cially available Bradford assay kit (Bio-Rad, Hercules, CA), and CAF steroid hormone concentrations are presented per micro- gram protein. This was necessary due to differential hydration states of the CAF samples. Aromatase assay. Hatchlings were indi- vidually housed for 10 days prior to tissue collection. Following the collection of blood from the dorsal post-cranial sinus, a lethal injection of sodium pentobarbital (0.4 mg/g) was administered in the sinus. Animals are anesthetized within 30 sec using this method. The right gonadal-adrenal mesonephros (GAM) complex was immedi- ately removed for the aromatase bioassay. Aromatase activity was measured indirectly based on the release of tritium from lp-3H- androstenedione during aromatization of the substrate into estrogen (24). Briefly, the tissue was placed in 400 pl culture media (RPMI-1640; Sigma Chemical Co., St. Louis, MO) supplemented with 0.8 mM tri- tiated androstenedione (DuPont NEN Research Products, Boston, MA; # NET- 926). After a 6-hr incubation at 32?C, 300 pl of the media was transferred to a new tube. Chloroform (1.5 ml) was added and the tube was vortexed and then centrifuged for 15 min at 2,000g. A 200-pl aliquot of the aqueous phase was added to a new tube. Five percent charcoal/0.5% dextran (200 pl) was added and the tube was vortexed and then immediately centrifuged for 15 min at 2,000g. Scintillation fluid (5 ml) was added to 300 pl supernatant and the tube was counted on a Beckman scintillation counter (Beckman Instruments, Schaumburg, IL). Aromatase activity is proportional to the amount of tritium in the scintillation vial and is calculated as a percentage of the total substrate added. After subtracting the non- specific tritium release, the disintegrations per minute (dpm) of the sample tubes are converted to a percentage of the total dpm added. This percentage is multiplied by the mass of the substrate added. After adjusting for extraction loss, the value obtained repre- sents the amount of substrate converted to tritiated water, which is proportional to aro- matase activity. Assay sensitivity was defined as twice the mean counts per minute (cpm) of blank tubes, which corresponds to 0.15 pmol/g/hr for the average-weight GAM (0.032 g). GAMs from three additional control female alligators were used to determine the specificity of the aromatase assay. The left GAM was incubated as above, whereas the right GAM was exposed to media sup- plemented with the aromatase inhibitor 4- hydroxy androstenedione (100 pM). Alligators exposed to the aromatase inhibitor had significantly lower GAM aro- matase activity (p = 0.45 pmol/g/hr) com- pared to the individuals incubated normal- ly (p = 3.15 pmol/g/hr). Histology. Histology was conducted to determine histological sex in order to docu- ment which compounds induced sex rever- sal. A complete histopathological examina- tion of the GAMs was beyond the scope of this study. The left GAM was preserved in Bouin's fixative, serial sectioned at 7 pm Table 1. Experimental treatments and dosages that were applied to different groups of eggs Treatment Effect Doses (ppm) Control 8 None None; diluent only 17P-Estradiol Natural estrogen 0.014, 0.14, 1.4, 14 ppm Vinclozolin Androgen antagonist in rodents 0.14, 1.4, 14 ppm Tamoxifen Estrogen agonist/antagonist 0.14,1.4,14 ppm 2,4-D Common herbicide, ? 0.14,1.4,14 ppm Atrazine Common herbicide, ? 0.14, 1.4,14 ppm Abbreviations: 2,4-D, 2-4-dichlorophenoxyacetic acid; ?, effects unknown. A sample size of five eggs was included in each dose-treatment group. aEach chemical was solubilized in 95% ethanol prior to topical application on the egg; thus, two control doses were used-one with 95% ethanol and one without. There was no difference between these controls for any of the variables measured. Environmental Health Perspectives * Volume 105, Number 5, May 1997 529 Articles * Crain et al. following paraffin embedding, and stained with a modification of Harris' trichrome staining procedure (25). Gonads were inspected and scored as testis or ovary by two independent researchers. Histological criteria originally reported by Forbes (26) and recently reestablished by Guillette et al. (19) were used to determine sex. In brief, criteria for testis included reduced cortex and medullary sex cord proliferation, whereas criteria for ovaries included hyper- trophied cortex, medullary reduction, the presence of lacunae in the medulla, and germ cells in the cortex. Radioimmunoassays. E2 and testos- terone (T) concentrations were measured in plasma of the 9-month-old descriptive study animals and in plasma and CAF of all hatchlings that provided ample fluids. Radioimmunoassays for E2 and T were performed as previously described (27) with the following modifications in sample extraction. CAF (750 pl) was mixed overnight (15 hr) with 2 ml of 95% ethanol. The suspension was centrifuged at 1,200g for 20 min. Duplicate aliquots of supernatant (500 pI) for each sample were dried under constant air stream. Extraction efficiency averaged 92% for T and 94% for E2 with this method. For plasma extrac- tion, plasma (125 RI) was mixed with 4 ml ethyl ether for 1 min. The aqueous layer was frozen in a dry ice-methanol bath (-250C), and the ether phase decanted into an assay tube. The aqueous pellet was reex- tracted with ether and the ether added to the assay tube. The ether was dried with constant air stream. Extraction efficiency was consistent and averaged 95% for T and 94% for E2. Cross-reactivities of the T antisera (T3-125, Endocrine Sciences, Calabasas Hills, CA) to other ligands are as follows: dihydrotestosterone, 44%; A-1- testosterone, 41%; A--1-dihydrotestos- terone, 18%; 5 a-androstan-30, 17,-diol, 3%; 4-androsten-3P,17p-diol, 2.5%; A-4- androstenedione, 2%; 51-androstan- 30,17p-diol, 1.5%; estradiol, 0.5%; all other ligands <0.2%. Cross-reactivities of the E2 antisera (E26-47, Endocrine Sciences) to other ligands are as follows: estrone, 1.3%; estriol, 0.6%; 16-keto-estri- ol, 0.2%; all other ligands <0.2%. For the plasma radioimmunoassays (RIAs), interas- say variance was 15.0% for T and 12.6% for E2, and intraassay variance was 3.6% for T and 3.7% for E2. For the CAF RlAs, interassay variance was 11.8% for T and 16.1% for E2, and intraassay variance was 4.68% for T and 3.5% for E2. Statistics. Hormone concentrations were estimated from raw data with the commercially available Beckman EBA/RIA ImmunoFit software program (Fullerton, CA). Statistics were performed with the software packages StatView (Abacus Concepts, Inc., Berkeley, CA) and Superanova (Abacus Concepts, Inc.). For the descriptive study, an unpaired t-test was used for between-lake comparisons. In the experimental study, a two-factor analy- sis of variance (ANOVA) was used to test the effects of treatment and dose on aro- matase activities. Where the interaction of treatment and dose was not significant, the factor of dose was removed from the analy- sis and a one-factor ANOVA was used to test the effects of treatment on hormone concentrations and aromatase activity. Fisher's protected LSD was used as a post- hoc test to discriminate which groups dif- fered significantly. Results Descriptive Study-Female Juvenile Alligators Results of the aromatase enzyme assay are expressed both as fmol/hr and pmol/g/hr. The former is used in other studies of alli- gator gonadal aromatase activity (13,24) and is presented here for comparative pur- poses only. A comparison of the female juvenile alligators found that GAM aro- matase activity was significantly elevated in Lake Woodruff alligators compared to Lake Apopka alligators (Table 2, Fig. 1B). Mean concentrations of E2 were not differ- ent between lakes (p = 0.5178), but con- centrations of T were significantly lower in Apopka animals compared to Woodruff animals (p = 0.05; Fig. IA). Experimental Study-Neonatal Alligators Four response variables were measured for the alligators treated in ovo: sex reversal, CAF hormone concentrations, hatchling plasma hormone concentrations, and GAM aromatase activity. Tables 3 and 4 summa- rize these results. One-hundred percent sex reversal-male to female-was noted for all dosages of E2 and tamoxifen. No other treatments caused sex reversal. Two-way ANOVA revealed that dose had no influence on plasma hormone con- centration or gonadal aromatase activity in the eggs incubated at male and female tem- peratures. Dose did have an influence on CAF E2 concentrations, but this was due only to the E2 treatment group. Therefore, dose was removed from the statistical analysis, and a one-way ANOVA was used to determine if differences existed among treatment groups. The treatment group had no significant influence on CAF hor- mone concentrations, with the exception of tamoxifen treatments on eggs incubated at a male-producing temperature. These sex- reversed females had significantly more CAF T compared to control males (p = 0.024). Neither CAF T concentrations (p = 0.92) nor CAF E2 concentrations (p = 0.77) were different between control males and control females. Plasma E2 concentrations were not sig- nificantly different among treatment groups, but plasma T was different among treatment groups incubated at a male-pro- ducing temperature. Tamoxifen-(p = 0.028) and E2-(p = 0.027) treated animals, which were sex reversed, had elevated plas- ma T concentrations compared to control males. The ratio of E2/T was not signifi- cantly different for eggs incubated at male or female temperatures. Neither plasma E2 (p = 0.72) nor plasma T (p = 0.09) were significantly different between control males and control females. No differences in aromatase activity were detected among treated eggs at the female-producing temperature, but treat- ment groups in the male-temperature regime were significantly different. Tamoxifen-treated eggs had significantly more aromatase activity compared to con- trol males (p = 0.012). Among the other eggs incubated at a male temperature, both E2- and atrazine-treated hatchlings appeared to have elevated aromatase activi- ty. Although not significantly different from control males (E2, p = 0.15; atrazine, p= 0.65), further analysis revealed that these E2- and atrazine-treated animals were also not significantly different from control females (E2, p = 0.65; atrazine, p = 0.13). Discussion There are numerous mechanisms through which environmental contaminants poten- tially can cause endocrine alterations (1), and this study has documented that one such mechanism is the alteration of the steroido- genic enzyme aromatase. Results from the descriptive study indicate that GAM aro- matase activity is significantly different between control and contaminated juvenile alligators, but this difference does not corre- spond with the alterations in circulating hor- mones in these animals. A previous study has shown that juvenile female alligators from Lake Apopka have significantly higher con- centrations of plasma E2 when compared to juvenile females from Lake Woodruff (19). One hypothesis for this increase in plasma E2 is that aromatase activity is increased in the contaminant-exposed Lake Apopka alliga- tors. However, the current study shows that female alligators from Lake Apopka have a significantly lower mean aromatase activity when compared to females from Lake Woodruff. These results are consistent with Volume 105, Number 5, May 1997 * Environmental Health Perspectives 530 Articles - Endocrine alterations in alligators data from in vitro cultures of gonads from Lake Apopka and Lake Woodruff juvenile alligators, which show that ovaries from Lake Apopka animals produce significantly less E2 in vitro than do Lake Woodruff animals (28). Although this study did not detect a dif- ference in plasma E2 between female alliga- tors from Lakes Apopka and Woodruff, sig- nificantly less T was present in the plasma of the female Lake Apopka alligators. Several studies have noted a decreased plas- ma T concentration in male and female juvenile alligators from Lake Apopka (19, 29). The extent to which such decreases in plasma T alter the physiology of alligators is unknown, but a recent study has shown that mean phallic size in alligators from Lake Apopka is significantly smaller than that of males from Lake Woodruff (29). Because alligator phallic development and size are androgen dependent (30), it is probable that the decrease in plasma T con- centrations contributes to an inhibition of penis growth in males. The effects of decreased T on females are unknown. Results from the experimental study indicate that treatment with exogenous chemicals can alter the endocrine system of developing alligator embryos. Among the endocrine-disrupting standards, E2 and tamoxifen treatments caused the develop- ment of ovaries in embryos incubated at a male-producing temperature. However, neither atrazine nor 2,4-D had such obvi- ous endocrine-altering effects. In an attempt to assess the impact of the treat- ments on the developing embryo, we mea- sured hormone concentrations in the CAF. The urinary wastes of developing oviparous embryos are stored in a membrane-bound sac called the allantois. As development proceeds, the allantoic membrane fuses with the chorion; the fluid inside is termed CAF. A previous study measured sex steroids in 15 03 0 10 E -. ._Z 0 @c 5yX E E0 03 0- = 00 0 E2 ....m....a,e. O Aromatase Figure 1. (A) Steroid hormone concentrations and (B) gonadal-adrenal mesonephros aromatase activity for 9-month-old female alligators from Lakes Apopka and Woodruff, Florida. Aromatase activity is significantly higher for animals from Lake Woodruff. this fluid and indicated that the measure- ment of these steroids could provide an assessment of the embryonic hormonal environment (31). Unlike the study by Gross et al. (31), we were unable to detect any difference in steroid hormones between control males and females. The only differ- ence in CAF hormones was for T concen- trations between the tamoxifen treatment and controls at a male-producing tempera- ture. Therefore, measurement of steroid hormones in CAF does not appear to be a usefil technique for assessing the embryon- ic hormonal environment of alligators. Measurement of aromatase activity indicates that the enzymatic activity of GAM tissue from hatchlings incubated at a male-producing temperature can be altered by exposure to exogenous compounds. None of the treatment groups had an effect on aromatase activity of hatchlings incubat- ed at a female-producing temperature, but several differences were noted among alli- gators incubated at a male-producing tem- perature. All dosages of E2 and tamoxifen caused ovarian differentiation at the male- producing temperature, and aromatase activity in these groups was increased accordingly (although only the tamoxifen- treated animals had aromatase activity that was significantly different from the control males). The aromatase activity of the Table 2. t-Test results from gonadal-adrenal mesonephros aromatase activities of alligators from Lakes Apopkaa and Woodruff,b Florida fmol/hr (? 1 SE)C pmol/g/hr (? 1 SE) Lake Apopka (n= 12) 204.5 ? 21.792 7.549 + 1.264 Lake Woodruff (n = 14 364.4 47.82 11.424 1.159 df 24 24 t-value -2.877 -2.262 p-value 0.0083 0.0331 SE, standard error; df, degrees of freedom. aContaminated lake. bControl lake. cAromatase activity is expressed as both fmol/hr and pmol/g/hr; statistical analysis was performed on the latter, and the former is presented only to facilitate comparison with other studies (13,24). Table 3. CAF hormones, plasma hormones, and gonadal-adrenal mesonephros aromatase activity for hatch- ling alligators treated in ovo with various compounds and incubated at a female-producing temperature (30?C). CAF E2 CAF T Plasma E2 Plasma T Plasma Aromatase Treatment (ng/pg protein) (ng/pg protein) (pg/ml) (pg/ml) E/Ta (pmol/g/hr) Control 126 ? 38 235 ? 99 14.1 ? 5.1 33.3 ? 6.1 0.54 ? 0.22 3.3 ? 0.78 E2 443 ? 165 110 ? 25 13.0 ? 2.1 25.9 ? 3.2 0.56 ? 0.08 3.2 ? 0.64 Vinclozolin 104 ? 36 122 ? 29 25.3 ? 5.7 27.9 ? 3.8 0.99 ? 0.22 3.7 ? 1.17 Tamoxifen 196 ? 123 197 ? 88 27.9 ? 6.7 34.3 ? 5.2 0.84 ? 0.19 5.0 ? 0.89 2,4-D 305 ? 137 732 ? 331 10.7 ? 1.7 25.1 ? 3.2 0.48 ? 0.08 4.9 ? 1.19 Atrazine 32 ? 8 65 ? 15 18.7 ? 5.0 28.5 ? 2.6 0.67 ? 0.17 3.8 ? 0.75 Abbreviations: CAF, choriollantoic fluid; E2, 17p3-estradiol; T, testosterone; 2,4-D, 2-4-dichlorophenoxyacetic acid. Variation is represented as ? 1 standard error. Histological examination of the gonads revealed that all animals incubated at a female-producing temperature had ovaries. There were no significant differ- ences between treatment group and control females for any of the response variables. aThe range of E2/T ratios are different from those previously presented for alligators by Guillette et al. (19). This is expected because different antibodies (with different sensitivities and specificities) were used in the two studies. Table 4. CAF hormones, plasma hormones, and gonadal-adrenal mesonephros aromatase activity for hatch- ling alligators treated in ovowith various compounds and incubated at a male-producing temperature (330C). CAF E2 CAF T Plasma E2 Plasma T Plasma Aromatase Treatment (ng/pg protein) (ng/pg protein) (pg/ml) (pg/ml) E2/T (pmol/g/hr) Control 74 ? 15 83 ? 17 11.7 ? 2.5 19.1 ? 3.4 0.57 ? 0.11 0.51 ? 0.17 E a 328 ? 159 145 ? 52 14.0 ? 4.3 37.5 ? 4.2b 0.44 ? 0.09 2.70 ? 0.66 Vinclozolin 77 ? 22 165 ? 46 14.9 ? 3.3 25.0 ? 4.8 0.62 ? 0.07 0.43 ? 0.20 Tamoxifena 282 ? 116 427 ? ll9b 18.3 ? 3.7 37.6 ? 4.5b 0.55 ? 0.11 4.34 ? 1.71b 2,4-D 140?57 285?81 8.6?0.6 28.6?5.6 0.47?0.10 0.44?0.06 Atrazine 69 ? 18 132 ? 28 16.7 ? 4.1 24.3 ? 3.7 0.65 ? 0.13 1.21 ? 0.60 Abbreviations: CAF, chorioallantoic fluid; E2, 17-,B estradiol; T, testosterone; 2,4-D, 2,4-dichlorophenoxy- acetic acid. Variation is represented as ? 1 standard error. aAll embryos treated with E2 and tamoxifen were identified as females after histological examination of the gonads. bSignificant differences between the treatment group and control males. Environmental Health Perspectives * Volume 105, Number 5, May 1997 531 Articles - Crain et al. ovaries from these sex-reversed animals was not significantly different from ovaries of control female alligators. Interestingly, atrazine treatments did not induce sex reversal at a morphological level but did stimulate testicular aromatase activity that was not significantly different from that of ovaries from control animals. Neither vinclozolin nor 2,4-D treatments had any effect on testicular aromatase activ- ity. This suggests that although sex reversal was not induced by atrazine, atrazine altered steroidogenesis in the hatchling alli- gators such that more estrogen was pro- duced. This increase in circulating estrogen was not detected by radioimmunoassay, although this is expected because there is no difference in control male and female hormone concentrations. A more sensitive and precise assay for E2 and T could poten- tially uncover differences in these individ- ual hormones. Using in vivo and in vitro techniques, Connor et al. (32 concluded that chloro-s- triazines do not interact with the estrogen receptor and, thus, any estrogenic or antie- strogenic effects must occur at mechanistic levels other than the estrogen receptor-lig- and interaction. The data presented in this study suggest that atrazine may affect organisms at the level of steroidogenic enzyme activity. The ability of atrazine to alter the activity of steroidogenic enzymes has been noted previously in several studies. For instance, the female offspring of rats treated with atrazine during pregnancy and lactation show increased pituitary 5a- reductase and 3a-hydroxysteroid dehydro- genase activity (33). Conversely, atrazine significantly decreases pituitary 5a-reduc- tase, 3a-hydroxysteroid dehydrogenase, and 3p-hydroxysteroid dehydrogenase activity in adult male rats (34,35). The aromatase enzyme is of the cytochrome P450 enzyme family; therefore, it is likely that many com- pounds (such as atrazine) increase aro- matase activity in a similar manner to that of P450 enzymes involved in detoxification. Atrazine could cause greater endocrine disruption in alligators hatched in the wild than that revealed by the present controlled laboratory experiment. As explained in the introduction, the temperature of egg incu- bation determines the sex of many reptiles, including alligators. Previous studies have indicated that steroid hormones and incu- bation temperature exhibit synergism, such that steroid hormones exert a greater effect at intermediate temperatures that produce both males and females (5,14). For instance, males incubated at a male-pro- ducing temperature close to the tempera- ture threshold for female development are more sensitive to the effects of E2 (5). The present experimental design used two tem- peratures that would produce either 100% females (300C) or 100% males (330C). However, if eggs were incubated at a tem- perature closer to that which produces 50% males and 50% females (pivotal tem- perature), the effect of aromatase and the resultant steroid environment could be magnified. Future studies should explore this hypothesis. Previous studies have shown that E2 and tamoxifen treatments promote ovarian differentiation in reptiles incubated at male-producing temperatures (36,37). Although it is not known if such animals are capable of successful reproduction dur- ing adulthood, the present study finds no significant difference in aromatase activity between the female control animals and the sex-reversed tamoxifen- and E2-treated ani- mals. This would suggest that at least at hatching, exogenously sex-reversed animals are capable of normal steroid production. The present study has indicated that induction and suppression of aromatase enzyme activity are potential modes of con- taminant-induced endocrine disruption in a species with temperature-dependent sex determination. Such disruption may not be limited to species with environmental sex determination, as aromatase function can also be affected in species with genetic sex determination. For example, the newt Pleurodeles walti exhibits a ZZ/ZW system of genetic sex determination in which females are the heterogametic sex and, although sex determination is under gamet- ic control, the sexual differentiation of the gonads can be modified by alterations in temperature (11). When ZW females are incubated at 320C, aromatase activity in the gonadal-mesonephric complex is decreased to male-like levels. This suggests that although sex determination is genetic, the steroidal environment can be easily manipulated by exposure to various extra- neous factors, including compounds that modify steroidogenic enzymes. Indeed, the addition of aromatase inhibitors modifies the sexual differentiation of animals with genetic sex determination. When treated with an aromatase inhibitor, genetically female chickens are masculinized but eggs fertilized by these males are not viable (38). This infertility is species dependent, as genetically female Chinook salmon (Oncorhynchus tshawytscha) develop into functional males if exposed to an aromatase inhibitor for as little as 2 hr (39). This study attempted to determine 1) if the endocrine alterations previously observed for wild alligators could be explained by alterations in aromatase activity and 2) if endocrine disruptions, including alterations in aromatase activity, could be induced by embryonic exposure to herbicides used extensively in the habitat of these wild alliga- tors. Aromatase activity was significantly lower in female Lake Apopka animals com- pared to control animals, a result that sup- ports data from in vitro culture of Lake Apopka alligator gonads (28). This increased aromatase activity does not, however, explain increases in circulating E2 that have been described previously for wild Lake Apopka females (19). Embryonic exposure to 2,4-D had no effect on the endocrine parameters measured, but atrazine exposure caused aro- matase activity that was characteristic of nei- ther males nor females. Thus, atrazine expo- sure may induce endocrine alterations in embryonic alligators. 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