Final Report: An in vivo Model for Detection of Reproductive Effects of Endocrine Disruptors

EPA Grant Number: R825298
Title: An in vivo Model for Detection of Reproductive Effects of Endocrine Disruptors
Investigators: Hinton, David E. , Teh, Swee J. , Washburn, Barbara S.
Institution: University of California - Davis , The University of Texas at El Paso
EPA Project Officer: Hahn, Intaek
Project Period: November 1, 1996 through October 31, 1999
Project Amount: $519,729
RFA: Endocrine Disruptors (1996) RFA Text |  Recipients Lists
Research Category: Economics and Decision Sciences , Endocrine Disruptors , Health , Safer Chemicals


The principal objective of this project was to develop and validate the use of a short term in vivo model, using the small, aquarium fish medaka (Oryzias latipes), to identify adverse effects of exposure to endocrine-disrupting chemicals.

Summary/Accomplishments (Outputs/Outcomes):

There is a need for in vivo models to improve our ability to detect those environmentally persistent compounds that adversely affect reproduction by acting as steroid hormone agonists or antagonists. An in vivo model using the small aquarium fish, medaka (Oryzias latipes), for detection of reproductive effects of endocrine disruptors was developed and refined. A feature common to many laboratory and field studies with various fish species is a higher prevalence of hepatocellular neoplasia in females than in males. During female sexual maturation, endogenous estrogens stimulate substantial increases in liver synthetic activity, including production of vitellogenin and choriogenin, made possible by proliferation of hepatocytes. There is no evidence for enhanced hepatic synthetic ability of males with attainment of sexual maturation.

Quantitative features of this gender specific growth were measured in hatchlings, immature- and sexually-mature control medaka. After exposure to the carcinogen, diethylnitrosamine (DEN), as 21-day-old hatchlings, resultant tumor incidence and time to endpoint (latency period) demonstrated female growth to be a promotional stimulus, positively modulating carcinogenesis. Next, DEN initiation was followed by administration of estradiol in an initiation-promotion assay and this estrogen was shown to promote hepatocarcinogenesis. Control male medaka, exposed to estradiol alone, showed elevated liver weights similar to those of sexually mature females. The xenoestrogen, b-hexachlorocyclohexane, was considered a weakly positive modulator of hepatocarcinogenesis. Xenobiotics with mechanism of action like that of estrogen may promote growth of previously initiated cells and thereby positively modulate the tumor forming process. Given that epizootics of liver neoplasia in wild fishes have been reported within the US, Europe, and Canada, this finding may have ecological significance.

Husbandry conditions including: light duration, temperature and stocking density were all shown to exert effects on growth and sexual maturation; and, effects were particularly obvious in females. A shortened photoperiod profoundly affected medaka reproduction. Whereas, decreased temperature reduced, but did not arrest, fertility; reduced photoperiod was associated with decreased fecundity. These findings have important implications for culture of medaka and indicate the need for standardization and detailed reporting of husbandry conditions. In addition, by strict maintenance of temperature and light duration, breeding (spawning) pairs of adult medaka can be calibrated for embryonated egg production and the effects of exposure on reproduction can be readily quantified using the pairs as their own controls.

We next set about to determine that developmental stage of medaka most susceptible to exogenous hormone (estradiol and testosterone) exposure. Embryo medaka (at stage 10 of development?early organogenesis), and medaka hatchling/larvae at 1-, 7-, or 21-days of age, were exposed to either estradiol or testosterone. Newly hatched medaka proved most sensitive to hormone exposure and effects of brief exposure at that time were seen, 5 months later, as: skewed sex ratios (estradiol exposure?toward female), reduced reproductive success, and reduced viability of offspring.

Because few studies have examined the effects of environmentally persistent compounds that interact with the androgen receptor, we compared the effects of exposure to the mammalian antiandrogen, vinclozolin, to those produced by exposure to a known xenoestrogen, p-tert-octylphenol, either alone or in combination. Embryonic exposure to either compound alone or as a mixture, resulted in ovotestis (intersex) formation in individuals sampled at one month after hatching but prior to sexual maturation. Later samplings, after sexual maturation, revealed intersex only in fish exposed to the mixture. Apparently, intersex?induced by exposure to either compound alone?reverted to the normal condition during puberty. However, exposure to either compound alone was associated with reduced reproductive capacity in actively spawning adults. When adult males were exposed to vinclozolin alone, intersex was observed. This study is the first to report intersex gonadal morphology and adverse effects on reproduction in a teleost fish model caused by a putative antiandrogenic compound.

Our calibrated model with established daily egg production determined that low, environmentally realistic levels of octylphenol (10µg/L), vinclozolin (1µg/L) or a mixture of the two caused changes in reproductive capacity. This was seen as a significant reduction in the number of fertilized eggs (F1). With increased duration of exposure to adults, resultant daily mean embryo/larval viability diminished significantly. Resultant viable fry (F1) were continuously exposed to compound or mixture for an additional 6 days. After rinsing in clean water, groups were cultured until sexually mature (6.5 months later), then evaluated for reproductive capacity (production of F2). While breeding groups comprised of F2 medaka produced viable embryos, each showed reduced reproductive capacity compared to control groups, and, reduction proved statistically significant for the mixture and for the vinclozolin exposure groups. We have no explanation for this multigenerational effect but regard this as fundamentally important. Finally, F1 adults were euthanized, fixed and examined histologically. Histologic evaluation of gonads revealed immature ova (intersex) within testes of 25 percent of fish exposed to either octylphenol or the mixture (octylphenol and vinclozolin) with the latter being more severely affected. Individuals of the vinclozolin alone and control groups were free of intersex. However, all vinclozolin exposed male fish displayed germ cell necrosis. Interstitial cell hypertrophy was observed in testes of fish exposed to the mixture. All ovaries of all groups were histologically normal. We regard these results as highly significant with respect to the potential for adverse ecological effect. Reduced reproductive capacity may be encountered as a result of exposure to adults and these effects are seen in the absence of overt toxicity and at concentrations far below the level needed to result in toxicity within one of the established U.S. EPA three species tests. Furthermore, the reduced reproductive capacity in this and the subsequent generation, may eventually affect overall reproduction and ability to sustain the population. Although the medaka is native to southeastern Asia; and, forms a combined laboratory and field model, this fish is not native to the United States (US). The application of this model in the US is as a defined laboratory in vivo screen that can be used to rapidly detect endocrine disruptors and establish their effect on reproduction. Subsequent studies with species of interest will likely follow this screen.

The mechanism(s) of action of potential endocrine disruptors in the medaka model must be determined. If common mechanism(s) of action are seen in this and other species, the model will have utility as a warning of potential danger of specific compounds for endocrine disruptive effects in resident species of fish, wildlife and possibly in man. For this reason, molecular investigations in medaka and in trout (Oncorhynchus mykiss)?as a necessary prelude to medaka investigations?were carried out.

We examined liver cytochrome P450 enzymes associated with endogenous steroid metabolism in the medaka model and identified, cloned, and characterized CYP3A, an abundant, constitutively-expressed cytochrome P450. Catalytic activities of hepatic CYP3A isoforms include; hydroxylation of steroids hormones including testosterone, progesterone, cortisol, androstenedione, and estradiol. Altered steroid metabolism is one direct mechanism of endocrine disruptive agents. These investigations led to the isolation of two novel CYP3A isoforms with unique expression patterns. Screening of a cDNA library to medaka liver resulted in the identification of two full length cDNA clones containing 1758 and 2306 base pair inserts with open reading frames encoding two peptides of 500 and 501 amino acids respectively. Comparison of the nucleic acid and deduced amino acid sequences using BLAST homology searches indicate that these gene products belong to the cytochrome P450 superfamily and display a high degree of sequence homology to the CYP3A subfamily. Both sequences have been submitted to the cytochrome P450 nomenclature committee and designated CYP3A38 and CYP3A40. Northern blot analysis identified two abundant CYP3A related transcripts in livers of male and female adults and demonstrated significant gender differences in CYP3A expression between the two isoforms. Western blot analysis confirmed the mRNA data and suggests that: (1) CYP3A is constitutively expressed in medaka liver at high levels; (2) liver contains multiple isoforms of CYP3A; and (3) expression of CYP3A appears to be gender specific. CYP3A38 cDNA expressed in HEK 293 cells metabolized testosterone to 6?-OH and 16?-OH forms. Because overall activity of the mammalian expression system was low, both CYP3A genes have been cloned in baculovirus expression system. Studies are currently in progress to ascertain both substrate specificities and kinetics of steroid metabolism.

Both male and female medaka demonstrated a significant reduction in CYP3A protein and RNA with estrogen treatment. No differences were observed with testosterone. Similarly, estrogen treatment significantly reduced CYP3A specific enzyme activities in males and females. A significant decrease in CYP3A expression was additionally observed following exposure to octylphenol, a model xenoestrogen. These results indicate that estrogen and potentially estrogen-like xenobiotics may act as a modulator of cytochrome P450 expression. Whether or not gender differences initially observed in northern and western blots are due to estrogen suppression in females is yet to be determined. In a separate set of experiments, expression studies using RT-PCR and western blotting were conducted to examine the ontogeny of each CYP3A isoform. Protein and RNA were isolated from embryonic, larval and adult stages of medaka and examined for the expression of both CYP3A38 and CYP3A340. Western blots demonstrate the presence of two abundant cytochrome P450 3A like proteins in later stages of development including larva and adults and only a single 3A protein for unfertilized eggs and embryonic stages 10-35. RT-PCR analysis was conducted using gene specific oligonucleotide for CYP3A38 and CYP3A40. Results were consistent with the protein data and suggest that CYP3A40 is expressed early in embryonic development continuing throughout adult stages while CYP3A38 is expressed only in larva and adults.

Androgen biotransformation assays were additionally conducted with microsomes for select medaka embryonic, larval and adult stages. Testosterone metabolite profiles differed considerably between embryonic and adult stages specifically in the formation of both 16?-OH and 11-keto- testosterone. This data is consistent with expression pattern observed with CYP3A38. Preliminary studies additionally indicate that androgen biotransformation is sensitive to disruption by endocrine modulating compounds, nonyl phenol and vinclozolin. For the last part we isolated several genomic clones for both CYP3A genes. These studies have focused on identifying promoter regions associated with gene regulation/differential expression pattern of CYP3A38 and CYP3A40. Currently we are in process of cloning and sequencing these genomic sequences. Work is continuing.

Common biomarkers, estrogen receptor (ER), vitellogenin (Vtg) and eggshell zona radiata protein (Zr-protein) assessed molecular aspects of endocrine disruption in trout. In teleosts, the physiological action of estrogens is predominantly mediated by interaction with high affinity cellular receptors such as the ER. Estrogen mimics (xenoestrogens) may act as ER ligands binding to this receptor, and resulting in modulation of normal endocrine signaling pathways. Differential time-response (2, 6, 12, 24, 48 and 72 hours) of ER, vitellogenin (Vtg) and eggshell zona radiata (Zr) gene and protein expressions were investigated in juvenile rainbow trout treated with nonyphenol (NP; 1, 5 and 25 mg/kg) and estradiol-17? (E2; 5 mg/kg). While Zr gene expression showed an induction only at 24 hours post-exposure, the plasma protein levels showed a time-dependent increase in the 25 mg NP treated group. Vtg transcripts showed an apparent time-dependent increase without a concomitant increase in protein levels in the 25 mg NP/kg treated fish. A significant time-dependent increase in the Vtg and Zr gene expressions without the corresponding increases in ER gene transcription was observed in the E2 ?treated fish at 2, 6 and 12 hours post-exposure. Induction of ER gene transcripts was observed from 24 hours and did not change significantly at 48 and 72 hours. The time-dependent increases in Vtg and Zr gene expressions persisted to 72 hours in the E2 ?treated fish. In this group, the induction of plasma Vtg levels was observed at 48 and 72 hours, while plasma Zr-protein was induced at 24, 48 and 72 hours, after exposure. We conclude that the E2- and NP-induced Vtg and Zr gene expressions at the early time intervals are not attributable to an increase in the transcriptional activity of the ER gene. Hence, Vtg and Zr- gene transcription does not coincide with a concatenate increase in ER suggesting that basal ER transcripts may be sufficient for transcriptional activation or that an alternate ER independent mechanism may be associated with Vtg and Zr transcription. These studies are continuing and we plan to follow these particular trout investigations with studies in medaka.

The in vivo model is ready to apply and as more molecular tools become available, the scope of investigations will broaden enabling mechanistic considerations of whole animal responses.

Journal Articles on this Report : 6 Displayed | Download in RIS Format

Other project views: All 9 publications 6 publications in selected types All 6 journal articles
Type Citation Project Document Sources
Journal Article Cooke JB, Hinton DE. Promotion by 17 beta-estradiol and beta-hexachlorocyclohexane of hepatocellular tumors in medaka, Oryzias latipes. Aquatic Toxicology 1999;45(2-3):127-145. R825298 (Final)
not available
Journal Article Teh SJ, Hinton DE. Gender-specific growth and hepatic neoplasia in medaka (Oryzias latipes). Aquatic Toxicology 1998;41(1-2):141-159. R825298 (Final)
R825433 (Final)
  • Full-text: ScienceDirect-Full Text HTML
  • Abstract: ScienceDirect-Abstract
  • Other: ScienceDirect-Full Text PDF
  • Journal Article Koger CS, Teh SJ, Hinton DE. Variations of light and temperature regimes and resulting effects on reproductive parameters in medaka (Oryzias latipes). Biology of Reproduction 1999;61(5):1287-1293. R825298 (Final)
    R825433 (Final)
  • Abstract from PubMed
  • Full-text: Biology of Reproduction-Full Text HTML
  • Abstract: Biology of Reproduction-Abstract
  • Other: Biology of Reproduction-Full Text PDF
  • Journal Article Koger CS, Teh SJ, Hinton DE. Determining the sensitive developmental stages of intersex induction in medaka (Oryzias latipes) exposed to 17β-estradiol or testosterone. Marine Environmental Research 2000;50(1-5):201-206. R825298 (Final)
    R825433 (Final)
  • Abstract from PubMed
  • Full-text: ScienceDirect-Full Text PDF
  • Abstract: ScienceDirect-Abstract & Full Text HTML
  • Other: ResearchGate-Abstract & Full Text PDF
  • Journal Article Kullman SW, Hamm JT, Hinton DE. Identification and characterization of a cDNA encoding cytochrome P450 3A from the fresh water teleost medaka (Oryzias latipes). Archives of Biochemistry and Biophysics 2000;380(1):29-38. R825298 (Final)
    R834514 (2011)
    R834514 (Final)
  • Abstract from PubMed
  • Full-text: ScienceDirect-Full Text HTML
  • Other: ScienceDirect-Full TextPDF
  • Journal Article Kullman SW, Hinton DE. Identification characterization and ontogeny of a second cytochrome P450 3A gene from the fresh water teleost medaka (Oryzias latipes). Molecular Reproduction and Development 2001;58(2):149-158. R825298 (Final)
    not available

    Supplemental Keywords:

    water, exposure, effects, health effects, pathology, histology, molecular biology, endocrine disruption, fish, animal model, xenoestrogen, anit-androgen, reproduction., RFA, Health, Scientific Discipline, Environmental Chemistry, Health Risk Assessment, Endocrine Disruptors - Environmental Exposure & Risk, endocrine disruptors, Biochemistry, Children's Health, Biology, Endocrine Disruptors - Human Health, adverse outcomes, male, embryo survival, fish, anti-androgen, endocrine disrupting chemicals, in vivo model, animal models, developmental processes, xenoestrogen, reproductive processes, biological effects

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    Progress and Final Reports:

    Original Abstract
  • 1997
  • 1998