Effects of Dibutyl Phthalate as an Environmental Endocrine Disruptor on Gonadal Sex Differentiation of Genetic Males of the Frog Rana rugosa.

To examine the effects of dibutyl phthalate (DBP) on gonadal sex differentiation, genetically male tadpoles of Rana rugosa were exposed to dilute solutions of DBP at concentrations of 0.1, 1, or 10 [micro]M during days 19-23 after fertilization, which is the critical period of gonadal sex differentiation in R. rugosa. Tadpoles were necropsied on day 40. The genetically male tadpoles were produced from crossings between males (ZZ) of one local population, in which females are the heterogametic sex, and females (XX) of another local population, in which males are the heterogametic sex. As positive control groups, tadpoles were exposed to dilute solutions of 17[Beta]-estradiol ([E.sub.2]) at concentrations of 0.01, 0.1, or 1 [micro]M during the same period. The internal structure of the gonads was histologically examined in a total of 30 control tadpoles, 86 [E.sub.2]-treated tadpoles, and 90 DBP-treated tadpoles. The gonads of the control tadpoles all showed the typical structure of testes. In contrast, 0.01, 0.1, and 1 [micro]M [E.sub.2] treatments caused the undifferentiated gonads of 18, 63, and 100% of the tadpoles, respectively, to develop into gonads of complete or partial ovarian structure. After 0.1, 1, and 10 [micro]M DBP treatment, 0, 7, and 17% of tadpoles, respectively, were similarly affected. These findings suggest that DBP was about 1,000-fold less potent than [E.sub.2]. Nevertheless, DBP is an environmentally dangerous hormone that disrupts the pathways of testicular differentiation in ganetically male animals. Key word. environmental endocrine disruptors, estrogens, frogs, gonadal sex differentiation, phthalate ester toxicity, plasticizers, sex chromosomes, sex-determining systems. Environ Health Perspect 108:1189-1193 (2000). [Online 15 November 2000] http://ehpnet1.niehs.nih.gov/docs/2000/108p1189-1193ohtani/abstract.html

In recent years there has been growing concern that the estrogenicity or antiandrogenicity of certain chemical compounds released into the environment may have a harmful influence on the development and function of the male reproductive system in several animal species, including humans. The estrogenic or antiandrogenic effect of the chemical compounds bisphenol A (1,2), nonylphenol (3,4), DDT (5), polychlorinated biphenyls (PCBs) (6), various phthalate esters (7,8), parabens (9), and others, has been ascertained by in vitro and in vivo assays. Fact-finding inquiries have reported that serious effects on feral animals may have been caused by nonylphenol (10), DDT (11), and PCBs (12) in particular.

Dibutyl phthalate (DBP), one of the phthalate esters, was widely used as a plasticizer of polyvinyl chloride resins. The estrogenic potency of DBP is uncertain because different methods of analysis give different results. An in vitro yeast-based estrogen assay indicated that its estrogenic potency is 1,000,000-fold lower than that of 17[Beta]-estradiol ([E.sub.2]), which is one of the most potent endogenous estrogens (13), whereas Milligan et al. (14) detected no estrogenic ability in DBP by an in vivo assay using ovariectomized mice. The risk of DBP to experimental animals may be different from one species to another because of their different levels of resistance to the chemical. Furthermore, the risk may be different according to the method, duration, and magnitude of exposure in the screening trial. In any case, when the estrogenic or antiandrogenic impact of DBP is examined in vertebrates, the genetic sex of the experimental animals used should be standardized to male. From these points of view, the Japanese wrinkled frog, Rana rugosa, is a suitable experimental animal for the following reasons: a) in the gonadal sex differentiation of R. rugosa, exogenous estrogen induces ovarian formation in genetic males, and the estrogen-sensitive period is clearly defined during days 20-22 after fertilization; b) eggs from each spawn are numerous (about 700-2,000 eggs); c) the method of exposure is simple because tadpoles are aquatic; and d) genetically all-male tadpoles are easily produced. The sex of R. rugosa is under the control of sex chromosomes, and the sex-determining systems differ from one local population to another (15). Accordingly, crossings between males (ZZ) of the ZW/ZZ-type of sex-determining system and females (XX) of the XX/XY-type produce only genetically male (XZ) embryos.

The aim of this study was to ascertain whether DBP can alter the intrinsic mode of testicular formation in genetically all-male R. rugosa. We also examined the rate of gonadal sex reversal caused by various concentrations of [E.sub.2] and compared the effect of DBP with that of [E.sub.2].

Materials and Methods

Production of genetically all-male tadpoles. To produce all-male tadpoles, three females and three males were used. The three females had the XX/XY-type of sex-determining system and belonged to the west Japan strain, named in our laboratory, of which the original frogs were collected from Hiroshima city. The three males had the ZW/ZZ-type of sex-determining system and belonged to the north Japan strain, of which the original frogs were collected from Kanazawa city. We induced ovulation of each female by injecting 0.5 mL Ringer's solution containing two pulverized pituitary bodies of Rana catesbiana into her abdominal cavity 10 hr before her eggs were used. The eggs were squeezed out from the anesthetized females onto glass slides and were inseminated by adequate sperm suspension prepared by crushing testes, removed from anesthetized male frogs, in a small amount of dechlorinated tap water with tweezers. Embryos produced from this insemination were subdivided into eight groups: three for dibutyl phthalate treatment, three for [E.sub.2] treatment, one for the vehicle control, and one for the control. Each group comprised 50 embryos.

Reagents for exposure. As stock solutions, 28 mg dibutyl phthalate (DBP; Sigma, St. Louis, MO) and 3.8 mg [E.sub.2] (Sigma) were dissolved separately in 10 mL absolute ethanol. Then, 1, 0.1, and 0.01 mL of these stock solutions were diluted with 1,000 mL of dechlorinated tap water to give final concentrations of 10, 1, and 0.1 [micro]M DBP and 1, 0.1, and 0.01 [micro]M [E.sub.2], respectively. We based the concentration of the DBP on the fact that 100 [micro]M DBP-treated tadpoles always died within 5 min. For the vehicle control, 1,000 mL 0.1% ethanol solution was prepared.

Method of exposure. We exposed 50 male tadpoles in the 1,000 mL respective diluted solutions, which were poured into 2-L enameled containers, from the beginning of day 19 to the end of day 23 after fertilization. The tadpoles were reared at approximately 25 [degrees] C temperature under white fluorescent light and fed on boiled spinach. Rearing water was changed every 2 or 3 days.

Preparation for microscopic examination. On day 40 after fertilization, the earliest time when ovarian structure was distinguished from testicular structure and the undifferentiated state of gonads, a total of 90 tadpoles treated with DBP, 86 treated with [E.sub.2], and 30 vehicle controls were fixed with Nawaschin fixing solution (solution A: 2 g chromic acid and 198 mL distilled water; solution B: 80 mL formalin and 20 mL acetic acid; equal parts of each solution were mixed just before use). The remaining tadpoles had been reared to examine the effects of DBP on the next generation of offspring. Then their gonads were removed with the mesonephros and embedded in paraffin after dehydration through an ethanol series. Samples sectioned successively to a thickness of 10 [micro]m were stained with Mayer's Alum hematoxylin.

Results

Tadpoles produced from the crossings of females with two X chromosomes and males with two Z chromosomes were all genetically male. Of 30 tadpoles in the vehicle control, 28 showed the typical structure of testes in which germ cells intermingled with medullary somatic cells roughly uniformly (Table 1, Figure 1A). The remaining two tadpoles in this group had many meiotic germ cells in the peripheral area of the gonads, although the inside showed normal testicular structure (Figure 2A). In the offspring of R. rugosa collected from Kanazawa, the existence of meiotic germ cells in the gonads of tadpoles is usually a criterion for ovarian differentiation because meiotic germ cells in the testes only begin to appear in young frogs that have completed metamorphosis (26). However, in the offspring of R. rugosa collected from Hiroshima, meiotic germ cells are frequently found as testes-ova in the testes of tadpoles (16). Therefore, we do not regard the presence of meiotic germ cells in the testicular structure of Hiroshima frogs as a sign of feminization.

Table 1. Number of genetically male Rana rugosa tadpoles with gonads
showing various degrees of ovarian and testicular structure induced by
DBP and [E.sub.2] treatment.

                                  Internal appearance of gonads

                                                        Testicular
                                            Ovarian        (many
                               Ovarian        and         meiotic
Concentration                throughout    testicular   germ cells)

0.1 [micro]M DBP(*)               0            0             1
1 [micro]M DBP(*)                 0            2             8
10 [micro]M DBP(*)                1            4            14
0.01 [micro]M [E.sub.2](*)        1            4            10
0.1 [micro]M [E.sub.2]            5            14            3
1 [micro]M [E.sub.2]             28            0             0
Vehicle control(*)                0            0             2

                             Testicular
                             (few if any
                              meiotic
Concentration                germ cells)     Total

0.1 [micro]M DBP(*)              29            30
1 [micro]M DBP(*)                20            30
10 [micro]M DBP(*)               11            30
0.01 [micro]M [E.sub.2](*)       13            28
0.1 [micro]M [E.sub.2]            8            30
1 [micro]M [E.sub.2]              0            28
Vehicle control(*)               28            30

(*) No significant differences ([[Chi]-square]-test, p [is less than]
0.05) between 0.1 [micro]M DBP, 1 [micro]M DBP, and vehicle control
and between 10 [micro]M DBP and 0.01 [micro]M [E.sub.2].

[ILLUSTRATIONS OMITTED]

Exposure to [E.sub.2]

The gonads of 28 tadpoles treated with 1 [micro]M [E.sub.2] all showed the typical structure of the ovary in which the majority of germ cells were in the prophase of meiotic division and an ovarian cavity was formed in the center (Table 1, Figure 1B). Of 30 tadpoles treated with 0.1 [micro]M [E.sub.2], 5 had gonads showing ovarian structure throughout. The gonads of 14 other tadpoles were composed of testicular and ovarian structure in their anterior and posterior parts, respectively (Figure 3), although the proportion of testicular and ovarian structure varied in each gonad. The gonads of 3 other tadpoles had far more meiotic germ cells in their peripheral parts than those of the vehicle control tadpoles, in addition to the testicular structure of the interior (Figure 2B). The remaining 8 tadpoles had typical testes with few if any meiotic germ cells. Of 28 tadpoles treated with 0.01 [micro]M [E.sub.2], 1 had gonads showing ovarian structure throughout, 4 had gonads showing the coexistence of testicular and ovarian structure, 10 had gonads with many meiotic germ cells in the peripheral parts, and the gonads of the remaining 13 showed testicular structure throughout.

[ILLUSTRATIONS OMITTED]

Exposure to DBP

Exposure of tadpoles to 10 [micro]M DBP induced obvious ovarian formation in the gonads of genetic males. Of 30 tadpoles, 1 formed typical ovaries and 4 others had gonads consisting of testicular and ovarian structure (Table 1, Figure 4). In 30 tadpoles treated with 1 [micro]M DBP, the gonads of 2 tadpoles also showed partial ovarian structure. However, no tadpoles treated with 0.1 [micro]M DBP showed any signs of feminization in their gonads like those of the remaining 25 and 28 tadpoles treated with 10 and 1 [micro]M DBP, respectively.

[ILLUSTRATIONS OMITTED]

Discussion

Chemical compounds that mimic estrogenic activity in in vitro assays must be directly examined for their estrogenic or antiandrogenic action on living animal species. Genetic males of oviparous animals should be used for such studies to avoid the effect of maternal estrogen. Willingham and Crews (17) performed an outstanding experiment using all-male embryos of the red-eared slider turtle, Trachemys scripta. In this turtle, gonadal sex is determined by incubation temperature during embryonic development. An incubation temperature of 26 [degrees] C results in all male offspring, whereas an incubation temperature of 31 [degrees] C results in all female offspring (18). Willingham and Crews (17) incubated the turtle eggs at a male-producing temperature and administered the PCB Aroclor 1242 and seven kinds of pesticide compounds at concentrations detected in alligator eggs from Lake Apopka, Florida, to examine their potential estrogenicity. They tested these compounds both singly and in combination. They found that significant sex reversal is induced by trans-nonachlor, cis-nonachlor, Aroclor 1242, p,p'-DDE, and chlordane when used singly.

In R. rugosa, all male offspring or all female offspring can be produced easily. Gonadal sex is determined by the combination of sex chromosomes, unlike sex determination in the red-eared slider turtle (15). The sex chromosomes consist of three variations of subtelocentric, telocentric, and metacentric types caused by two pericentric inversions and conduct the different sex determining systems (19,20). In western and eastern Japan, R. rugosa has homomorphic sex chromosomes of subtelocentric (X and Y) and telocentric (X and Y) types, respectively, in both sexes. R. rugosa in central Japan has heteromorphic sex chromosomes consisting of metacentric (X) and subtelocentric (Y) types in males, whereas in northern Japan it has heteromorphic sex chromosomes consisting of metacentric (W) and subtelocentric (Z) types in females. The complexity of X- and W-metacentric chromosomes is thought to result from the different potential of the female-determining factor, because the combination of Z- and metacentric X-chromosomes produces the same number of females and males, whereas that of Z- and subtelocentric X-chromosomes gives rise to all males (21). Naturally, the combination of Z- and metacentric W-chromosome produces all females. Such complicated sex-determining systems enable R. rugosa to produce all female and all male offspring; that is, female embryos with XX chromosomes and male embryos with ZZ chromosomes can change their gonadal sex permanently under the influence of exogenous testosterone and estradiol, respectively. The crossing of normal XX females with sex-reversed XX neomales produces all female offspring, and that of sex-reversed ZZ neofemales and normal ZZ males produces all male offspring. In addition, the offspring developed gynogenetically from XX females are all female, whereas the gynogenesis of ZW females produces only all male offspring because WW embryos die at an early stage of development. In this study, the sample of all male offspring was produced by crossing females with two subtelocentric X-chromosomes and males with two Z-chromosomes.

DBP, widely used as the plasticizer of polyvinyl chloride resins, was screened for its estrogenic properties for the first time by Jobling et al. (7) using mammalian estrogen screens in vitro, and they proved that DBP is estrogenic. Harris et al. (13) found, using an in vitro yeast-based estrogen assay, that the estrogenic ability of DBP is 1,000,000-fold less potent than [E.sub.2]. However, no estrogenic action by DBP was detected in an in vivo assay using ovariectomized mice (14). In contrast, the developmental toxicity of DBP was evaluated using pregnant rats given DBP at 250-750 mg/kg/day, by Ema et al. (22), Mylchreest et al. (23,24), and Gray et al. (25). These researchers found that the male offspring display an unusually high incidence of reproductive tract malformations--decrease of anogenital distance, testicular and epididymal atrophy, widespread germ cell loss, absence of prostate gland and seminal vesicles, etc. Because these lesions are similar to those elicited by the antiandrogen flutamide, which disrupts androgen signaling necessary for male sexual differentiation, Mylchreest et al. (23,24) concluded that the effect of DBP is not estrogenic but antiandrogenic.

In this study we investigated whether the toxicity of DBP alters the process of gonadal sex differentiation in genetically male R. rugosa tadpoles. The critical period of gonadal differentiation in this frog is days 20-22 after fertilization because exogenous [E.sub.2] was effective only during this period in altering the testicular formation of male tadpoles with two Z-chromosomes (26). The results of 1 [micro]M [E.sub.2] and 0.5 [micro]M aromatase inhibitor treatment were different between the western and the northern Japan frog populations in the gonadal differentiation of R. rugosa (26). The genetically male tadpoles with subtelocentric X- and Y-chromosomes formed impermanent ovaries when treated with [E.sub.2] alone and together with aromatase inhibitor, whereas those with two Z-chromosomes formed permanent ovaries after [E.sub.2] treatment alone, but [E.sub.2] in combination with aromatase inhibitor did not alter the testicular formation of male tadpoles (26). These findings suggest that the gonadal sex reversal of ZZ male tadpoles induced by [E.sub.2] is not necessarily due to the activation of estrogen receptors. Consequently, we cannot identify whether the effect of DBP is estrogenic or antiandrogenic until the target receptors for DBP have been examined. When genetically male tadpoles were exposed to DBP during days 19-23 after fertilization, 10 and 1 [micro]M DBP induced complete or partially developed ovaries in the gonads of 17% and 7% of tadpoles, respectively. The level of gonadal alteration induced by 10 [micro]M DBP is similar to that brought about by 0.01 [micro]M [E.sub.2]. This result indicates that DBP is about 1,000-fold less potent than [E.sub.2]. This result is similar to the results obtained in mammalian-based gene expression assays performed by Zacharewski et al. (27). According to Zacharewski et al., 10 [micro]M DBP exhibited 36% activity, compared with the 100% response using 0.01 [micro]M [E.sub.2]. The current findings highlight the danger of DBP as an environmental endocrine disruptor.

REFERENCES AND NOTES

(1.) Nagel SC, vom Saal FS, Thayer KA, Dhar MG, Boechler M, Welshons WV. Relative binding affinity-serum modified access (RBA-SMA) assay predicts the relative in vivo bioactivity of the xenoestrogens bisphenol A and octylphenol. Environ Health Perspect 105:70-76 (1997).

(2.) Steinmetz R, Brown NG, Allen DL Bigsby RM, Ben Jonathan N. The environmental estrogen bisphenol A stimulates prolactin release in vitro and in vivo. Endocrinology 136:1760-1786 (1997).

(3.) Soto AM, Justicia H, Wray JW, Sonnenschein C. p-Nonylphenol: an estrogenic xenobiotic released from modified polystyrene. Environ Health Perspect 92:167-173 (1991).

(4.) Sumpter JP, Jobling S. Vitellogenesis as a biomarker for estrogenic contamination of the aquatic environment. Environ Health Perspect 103(suppl 7):173-178 (1995).

(5.) Kelce WR, Stone CR, Laws SC, Gray LE, Kemppainen JA, Wilso EM. Persistent DDT metabolite p,p'-DDE is a potent androgen recepter antagonist. Nature 375:581-585 (1995).

(6.) Krishnan AV, Stathis P, Permuth SF, Tokes L, Feldman D. Bisphenol-A: an estrogenic substance is released from polycarbonate flasks during autoclaving. Endocrinology 132:2279-2286 (1993).

(7.) Jobling S, Reynolds T, White R, Parker MG, Sumpter JP. A variety of environmentally persistent chemicals, including some phthalate plasticizers, are weakly estrogenic. Environ Health Perspect 103:562-587 (1995).

(8.) Sharp RM, Fisher JS, Millar MM, Jobling S, Sumpter JP. Gestational and lactational exposure of rats to xenoestrogens results in reduced testicular size and sperm production. Environ Health Perspect 103:1136-1143 (1995).

(9.) Routledge EJ, Parker J, Odum J, Ashby J, Sumpter JP. Some alkyl hydroxy benzoate preservatives (parabens) are estrogenic. Toxicol Appl Pharmacol 153:12-19 (1998).

(10.) Purdom CE, Hardiman PA, Bye V J, Eno NC, Tyler CR, Sumpter JP. Estrogenic effects of effluents from sewage treatment works. Chem Ecol 8:275-285 (1994).

(11.) Guillette LJ Jr, Gross TS, Masson GR, Matter JM, Percival HF, Woodward AR. Developmental abnormalities of the gonad and abnormal sex hormone concentrations in juvenile alligators from contaminated and control lakes in Florida. Environ Health Perspect 102:680-688 (1994).

(12.) DeGuise S, Martineau D, Beland P, Fournier M. Possible mechanisms of action of environmental contaminants on St. Lawrence beluga whales (Delphinapterus leucas). Environ Health Perspect 103:73-78 (1995).

(13.) Harris CA, Henttu P, Parker MG, Sumpter JP. The estrogenic activity of phthalate esters in vitro. Environ Health Perspect 105:802-811 (1997).

(14.) Milligan SR, Balasubramanian AV, Kalita JC. Relative potency of xenobiotic estrogens in an acute in vivo mammalian assay. Environ Health Perspect 106:23-26 (1996).

(15.) Miura I, Ohtani H, Kashiwagi A, Hanada H, Nakamura M. Structural differences between XX and ZW sex lampbrush chromosomes in Rana rugosa females. Chromosoma 105:237-241 (1996).

(16.) Takase M. Differences in genetic backgrounds affecting gonadal differentiation between two local populations of the Japanese wrinkled frog (Rana rugosa). Anat Embryol (Bed) 198:141-148 (1998).

(17.) Willingham E, Crews D. Sex reversal effects of environmentally relevant xenobiotic concentrations on the red-eared slider turtle, a species with temperature-dependent sex determination. Gen Comp Endocrinol 113:429-435 (1999).

(18.) Crews D, Bergeron JM. Role of reductase and aromatase in sex determination in the red-eared slider (Trachemys scripta), a turtle with temperature-dependent sex determination. J Endocrinol 143:279-289 (1994).

(19.) Miura I, Ohtani H, Hanada H, Ichikawa Y, Kashiwagi A, Nakamura M. Evidence for two successive pericentric inversions in sex lampbrush chromosomes of Rana rugosa (Anura: Ranidae). Chromosoma 106:178-182 (1997).

(20.) Miura I, Ohtani H, Nakamura M, Ichikawa Y, Saitoh K. The origin and differentiation of the heteromorphic sex chromosomes Z, W, X and Y in the frog Rana rugosa, inferred from the sequences of a sex-linked gene, ADP/ATP translocase. Mol Biol Evol 15:1612-1619 (1998).

(21.) Ohtani H, Miura I, Hanada H, Ichikawa Y. Alteration of the sex determining system resulting from structural change of the sex chromosomes in the frog Rana rugosa. J Exp Zool 286:313-319 (2000).

(22.) Ema M, Miyawaki E, Kawashima K. Further evaluation of developmental toxicity of di-n-butyl phthalate following administration during late pregnancy in rats. Toxicol Lett 98:87-93 (1998).

(23.) Mylchreest E, Cattley RC, Foster PM. Male reproductive tract malformations in rats following gestational and lactational exposure to di(n-butyl) phthalate: an antiandrogenic mechanism? Toxicol Sci 43:47-60 (1998).

(24.) Mylchreest E, Sar M, Cattley RC, Foster PM. Disruption of androgen-regulated male reproductive development by di(n-butyl) phthalate during late gestation in rats is different from flutamide. Toxicol Appl Pharmacol 156:81-95 (1999).

(25.) Gray LE Jr, Wolf C, Lambright C, Mann P, Price M, Cooper RL, Ostby J. Administration of potentially antiandrogenic pesticides (procymidone, linuron, iprodione, chlozolinate, p,p'-DDE, and ketoconazole) and toxic substances (dibutyl- and diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during sexual differentiation produces diverse profiles of reproductive malformations in the male rat. Toxicol Ind Health 15:94-118 (1999).

(26.) Ohtani H, Miura I, Ichikawa Y. Unpublished data.

(27.) Zacharewski TR, Meek MD, Clemons JH, Wu ZF, Fielden MR, Matthews JB. Examination of the in vitro and in vivo estrogenic activities of eight commercial phthalate esters. Toxicol Sci 46:282-293 (1998).

Address correspondence to Y. Ichikawa, Department of Health Science, Faculty of Human Life and Environmental Science, Hiroshima Women's University, Hiroshima 734-8558, Japan. Telephone: [phone]. Fax: [phone]. E-mail: [e-mail]

We thank J.N. Raybould for his corrections to the manuscript.

We are grateful for the support of the Extensive Research Program of Hiroshima Prefectural Government.

Received 22 March 2000; accepted 15 August 2000.

Hiromi Ohtani,(1) Ikuo Miura,(1) and Youko Ichikawa(2)

(1)Laboratory for Amphibian Biology, Faculty of Science, Hiroshima University, Higashihiroshima, Japan; (2)Department of Health Science, Faculty of Human Life and Environmental Science, Hiroshima Prefectural Women's University, Hiroshima, Japan