2001 Progress Report: Endocrine Disruption in Marine Gastropods by Environmental Chemical Mixtures

EPA Grant Number: R827401
Title: Endocrine Disruption in Marine Gastropods by Environmental Chemical Mixtures
Investigators: McClellan-Green, Patricia D.
Institution: Duke University
EPA Project Officer: Carleton, James N
Project Period: September 1, 1999 through August 31, 2002
Project Period Covered by this Report: September 1, 2000 through August 31, 2001
Project Amount: $430,672
RFA: Endocrine Disruptors (1999) RFA Text |  Recipients Lists
Research Category: Economics and Decision Sciences , Endocrine Disruptors , Health , Safer Chemicals


The goal of this study is to determine the biochemical mechanisms responsible for tributyltin (TBT) induced imposex and examine the interrelationship between TBT and other environmental xenobiotics in the development of these sexual abnormalities. The following objectives will be addressed in this study: (1) investigate the biochemical and/or molecular mechanisms by which TBT alters the regulation of steroid hormones and neurohormones to induce imposex in marine mollusks; (2) determine the effects of other environmental contaminants on TBT's ability to induce imposex; and (3) determine whether the mechanisms identified in laboratory studies on imposex plays a significant role in field-induced organisms.

Progress Summary:

The occurrence of sexual abnormalities, pseudo-hermaphroditism or imposex, in marine mollusks has been linked to low levels of TBT in the environment. Rarely, however, is TBT the sole contaminant in harbors. Other contaminants such as polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs), which are known to interact with the steroid-hormone system in vertebrates, also are found in harbors.

During the first 2 years of this project, we have confirmed the results of our preliminary studies of imposex induction by TBT and the influence mediated on this process by other contaminants such as PAHs. We have examined the effects of TBT and other contaminants on steroid metabolism, and we are continuing to work on the identification of the neuropeptide hormones involved in imposex induction in the mud snail. Also, we have initiated studies to determine whether the biochemical processes identified in the laboratory exposures occur in naturally exposed organisms.

(1) Imposex Induction. The results are of our studies over the past 3 years indicate that exposure to TBT significantly induces imposex in females mud snails (~40 percent) over control snails (~6 percent) and that co-exposure to 3-MC reduces this induction (~22 percent). These results confirmed our earlier findings that TBT does induce imposex in female snails, and that this effect is lessened by co-exposure to the PAH, 3-methylcholanthrene. One factor noted by our laboratory is that exposure through diet increases the rate of imposex induction over that of water exposure only. It would be very interesting to look at tissue distribution, uptake rates, metabolism, and elimination of TBT in these animals in the future.

(2) Steroids. Next, we examined the role of TBT on P450 mediated steroid metabolism. This was accomplished by examining testosterone and androstenedione metabolism as well as measuring the levels of testosterone, androstenedione, and progesterone in the tissues following a 45-day exposure. In each incidence, TBT exposure for 45 days decreased the level of free and conjugated steroids produced (see Table 1) in breeding snails. The steroid levels of nonbreeding snails currently are being assayed.

Table 1. Effect of contaminants on steroid levels in Ilyanassa obsoleta following a 45-day exposure

Free Steroid Levels (Breeding)
Progesterone(ng/mL) Androstenedione(nmol/L) Testosterone(pmol/L)
Control 0.059 + 0.21 2.560 + 2.32 0.440 + 0.88
TBT 0.283 + 0.20 0.530 + 1.10 ND
3-MC ND 0.044 + 0.06 3.610 + 7.23
TBT + 3-MC 0.087 + 0.001 ND ND

Table 2. Summary of aromatase activity

Aromatase Activity (pmol/mg/hour)
Laboratory Samples   Control 14.767 + 2.81
    TBT 9.543 + 1.13
    3-MC 6.049 + 0.916
    TBT + 3-MC 9.967 + 6.456
Field Samples Low Imposex Area Normal Female 0.224 + 0.137
    Male 0.454 + 0.263
  High Imposex Area Normal Female 0.0828 + 0.034
    Imposex Female 0.0752 + 0.047

(2a) P450 Aromatase. As shown in our YR01 report, conditions for determination of P450 aromatase activity in Ilyanassa were optimized based on the methods of Lephart and Simpson (1991). This included identifying the optimum temperature, time, substrate concentration, and cellular fractions for use in these studies. A tentative Km of 354 nM androstenedione was calculated for Ilyanassa using a double reciprocal plot of the kinetic data. A summary of Aromatase activity is given in Table 2. As shown, exposure to both TBT and/or 3-MC inhibits the level of aromatase activity. In addition, snails held in the laboratory for 45 days have a higher level of activity than that observed in animals collected from the field.

(2b) Testosterone. Initial studies on P450-mediated testosterone metabolism involved optimizing the conditions for these studies based on the methods of Purdon and Lehman-McKeeman (1997). This process included identifying the optimum temperature, time, substrate concentration, and cellular fraction to use for these studies. Results indicate that there is a change in the type of metabolites produced in in vitro assays. Initially, low levels of 6b and 2a OH-testosterone were the predominant metabolites. Following exposure to TBT, 6b, 15a, 16a, 16b, 2a hydroxy-testosterone and several unknown metabolites are produced. However, it should be noted that testosterone hydroxylation does not appear to be the predominant pathway. Analysis of in vivo metabolism of testosterone indicated that very little hydroxytestosterone is found in the mud snail. However, there is an increase in the presence of apolar compounds that elute from the column. These compounds can be separated into five separate peaks. It is likely that these apolar metabolites are identical to the fatty acid conjugates reported previously by Gooding and LeBlanc (2000).

Figure 1. Testosterone metabolites identified in in vivo extracts of Ilyanassa obsolete

(3) Neuropeptide Hormones. The goal of this portion of the project is aimed at determining the role of neuropeptide hormones in the induction of imposex by TBT in female mud snails.

(3a) PMF. Initial studies focused on identifying a putative PMF and determining the role this factor plays in accessory sex organ development in female snails. Four different molluscan neuropeptides were investigated for their ability to induce penal development during a 2-week exposure period. These neuropeptides included APGWamide, FMRFamide, LSSFVRIamide, and conopressin. In addition, snails also were injected with TBT or with testosterone. Results of these studies have identified APGWamide as the possible PMF in Ilyanassa (Oberdorster and McClellan-Green, 2000).

These studies were expanded to mimic the water-borne studies conducted with TBT and 3-MC. Snails were injected with EtOH, TBT, 3-MC, APGWamide, estradiol, and mixtures of the compounds. The results indicate that 3-MC at both low and high concentrations rescues the imposex rates of exposed snails. They also indicate that high levels of E2 reduce the imposex rates of TBT exposed snails.

A rabbit polyclonal antibody was produced to the APGWamide peptide. This antibody was produced for use in studies designed to determine whether TBT altered its pattern of expression in female mud snails. Initial immunoblot analysis (western blots) of whole tissue extracts proved unsuccessful. It is possible that the signal is too weak to be detected in this manner. In other gastropod species, the putative PMF is thought to be produced by no more than four individual ganglia and accumulate in the heme lacunae near the right tentacle. Currently, we are preparing tissue slices of male, normal female and imposex female mud snails for use in whole tissue immunohistochemical staining.

(3b) Egg Laying Hormone. Dr. Greg Nagle has sent us his antibody to the Egg Laying Hormone (ELH). This antibody has been used in various marine species, e.g.,. Aplysia, and preliminary experiments indicate that it is crossreactive to peptides in Ilyanassa. We will continue this work during the next breeding season (January-April) to determine what role TBT may play in the regulation of this neuropeptide hormone and whether it influences egg-laying.

(4) Metabolism of TBT. Snails have been exposed to TBT and 3-MC singly and in combination. Subsamples of these snails from both spring and fall exposures have been sent for TBT and DBT analysis. Currently, we are awaiting these results. Analyses of these samples are being conducted via the methods of Unger et al., (1998).

Implications: Thus far, our research has confirmed our hypothesis that the induction of imposex by TBT occurs through a multifaceted process. The necessity for initiation by the expression of a PMF appears to be true, and the maintenance of imposex via steroid hormones fits nicely in our model.

Future Activities:

During the coming year, we will complete the analysis of PAH co-exposure on TBT metabolism. We will continue our studies on the role of neuropeptide hormones in imposex induction and determine whether TBT affects the expression of these compounds. Also, we should complete the immunohistochemical studies using mud snail tissue slices. In addition, we will examine the metabolism of androstenedione, progesterone, pregnenolone, and testosterone in field collected samples to determine how their steroid levels compared to laboratory results. We have deployed cages containing approximately 1,500 snails at three separate locations in Carteret County, NC. These three locations normally exhibit low, intermediate, and high levels of imposex in Ilyanassa. These areas have been mapped and identified using GPS coordinates. Sediment contaminants have been analyzed for each of these sites.

Journal Articles on this Report : 8 Displayed | Download in RIS Format

Other project views: All 16 publications 8 publications in selected types All 8 journal articles
Type Citation Project Document Sources
Journal Article Burger J, Fossi C, McClellan-Green P, Orlando EF. Methodologies, bioindicators, and biomarkers for assessing gender-related differences in wildlife exposed to environmental chemicals. Environmental Research 2007;104(1):135-152 R827401 (2001)
not available
Journal Article Halgren RG, Fielden MR, Fong CJ, Zacharewski TR. Assessment of clone identity and sequence fidelity for 1189 IMAGE cDNA clones. Nucleic Acids Research 2001;29(2):582-588 R827401 (2001)
R827402 (2001)
R827402 (Final)
not available
Journal Article McClellan-Green P, Romano J, Oberdorster E. Does gender really matter in contaminant exposure? A case study using invertebrate models. Environmental Research 2007;104(1):183-191 R827401 (2001)
not available
Journal Article Oberdorster E, McClellan-Green P. The neuropeptide APGWamide induces imposer in the mud snail, Ilyanassa obsoleta. Peptides 2000;21(9):1323-1330. R827401 (2001)
not available
Journal Article Oberdorster E. Peptide hormones versus steroid hormones: Case studies from snail and turtle populations. Environmental Hormones: the Scientific Basis of Endocrine Disruption 2001;948:75-79 R827401 (2001)
not available
Journal Article Oberdorster E, McClellan-Green P. Mechanisms of imposex induction in the mud snail, Ilyanassa obsoleta: TBT as a neurotoxin and aromatase inhibitor. Marine Environmental Research 2002;53(3-5):715-718, Special Issue. R827401 (2001)
not available
Journal Article Oberdorster E, Romano J, McClellan-Green P. The neuropeptide APGWamide as a penis morphogenic factor (PMF) in gastropod mollusks. Integrative and Comparative Biology 2005;45(1):28-32 R827401 (2001)
not available
Journal Article Rittschof D, McClellan-Green P. Molluscs as multidisciplinary models in environment toxicology. Marine Pollution Bulletin 2005;50(4):369-373 R827401 (2001)
not available

Supplemental Keywords:

marine, dose-response, sensitive populations, metals, organics., RFA, Health, Scientific Discipline, Toxics, Ecosystem Protection/Environmental Exposure & Risk, Environmental Chemistry, Ecosystem/Assessment/Indicators, Chemical Mixtures - Environmental Exposure & Risk, Health Risk Assessment, Chemistry, pesticides, Endocrine Disruptors - Environmental Exposure & Risk, endocrine disruptors, Ecological Effects - Environmental Exposure & Risk, Ecological Effects - Human Health, Children's Health, Biology, Endocrine Disruptors - Human Health, ecological risk assessment, ecological exposure, xenobiotic, androstenedione, dose response, endocrine disrupting chemicals, steroid, chemical mixtures, marine gastropods, biochemistry, children's environmental health, progesterone

Progress and Final Reports:

Original Abstract
  • 2000
  • Final