Japan and South Florida: Nukes in Common. Geniusofdespair

I like to relate world news to how it impacts us here in Miami when I can since we are called "Eye on Miami." Thankfully Gimleteye is doing that today, so I don't have to.

We started warning about the Housing Crash (437 posts in all) before it was recognized by the mainstream media. We have 86 posts on offshore oil drilling and we were against it long before the massive oil spill (that is the oil spill that just about everyone in State Government has forgotten about).

We have 106 post for Nuclear Power in our archives. This is a subject that concerns us now and always has. Both writers here are environmentalists that focus on trying to keep Florida environmentally safe for people, plants, animals, and sea life. We do not want to foul South Florida and we raise an alarm when we see people that do. Those claiming "Jobs" as an excuse to foul Florida (only because it resonates) like Barney Bishop and Rep. Tom Rooney (see today's Herald story), should be made to spend 24-7 in the St. Lucie Estuary so they can too experience the gene altering, polluted waters that our fish are subjected to. Please read: "Assessment of Reproductive Endocrine Disruption in Fish from the St. Lucie Estuary, Florida" -- I cannot get the link to work so I am printing the results of the study:

Results Vtg purification

Strong induction of vitellogenin by injection of estradiol was evident in all four species as verified by anion exchange purification and SDS-PAGE (Figure 2). This figure shows the elution profile of male and estrogenized male plasma being released by a salt gradient starting at 1.6 min. Estrogen induced the synthesis of a highly expressed protein, not present in the male as displayed in the ion exchange chromatograph (at 2.5 min). Separation of plasma proteins by SDS-PAGE yielded 2 induced proteins (100-120 kDa) in the estrogenized plasma and purified Vtg, not found in the male. Ion exchange purification of 0.5 ml of induced plasma yielded about 2-3 mg of purified Vtg protein to be used as a standard for each species.

The reactivity and specificity of different Mabs to the purified fish Vtg’s by Western Analysis are presented in Figure 3. Although these Mab’s were made against different families of fish Vtg’s there are quite a few that cross-reacted with the fish in this study. This is evident by the appearance of blue bands in each blot strip. This result indicates that there is conservation of epitopes even though these fish are distantly related. Notice that some do not appear to recognize Vtg’s intact high molecular weight proteins (120-100 kDa), but do react with smaller proteins (50 kDa), which are less desirable for ELISA use. There appears to be some degradation of the Vtg protein, evident by the smearing in the Western blot. This may have occurred while the proteins were in the fish itself or during the purification procedure. Aprotinin, a protease inhibitor, was added to the purified Vtg to minimize proteolysis. A summary of the Western Analysis results is presented in Table 1.

ELISA development

Based on the Western results, four Mabs (1C8, 3G2, 5F8, 2D10) were chosen to determine if they would work by direct ELISA (Table 2). This technique is needed to quantify Vtg in each species, whereas Western results are mainly qualitative. Very often Mab’s that work by Western do not recognize Vtg by ELISA, since in the latter procedure the protein is in a native conformation and the epitope may be internal. Proteins were denatured and linearized by SDS- electrophoresis. A Table of the relative reactivity of each Mab with each Vtg is summarized in Table 2. One Mab (2D10) developed against swordfish egg yolk reacted strongly and specifically with all four test species by both Western and direct ELISA. In addition, this Mab did not recognize male plasma. Hence it was used to quantify Vtg in the field samples using species-specific Vtg’s as standards.

Field sampling

A total of 48 Irish pompano, 68 Mangrove snapper were caught after 3 days of sampling at Jupiter and St. Lucie Estuary. Unfortunately, fishing success in each area was not equal. We attempted to capture 20 of each species from each site and anticipated
6 a 1:1 sex ratio. But after histological analysis, this was not the case. Microscopic screening of the histology samples indicated that the Irish pompano were close to spawning with large gonads and high GSI (range: 0.1-9.0%). Over 53% of the mangrove snappers captured had very small gonads consisting of an unweighable ribbon (<0.01g). These samples were considered to be “immature” and appeared to contain undifferentiated gonial cells. All of the mangrove snapper females were non-vitellogenic as shown by histology, showing no evidence of yolk accumulation. The raw data collected from each site is listed in Appendix 1.

Many of the male and immature fish from both sites contained Vtg, which indicated exposure to estrogen or estrogen-like compounds as summarized in Table 3. Male and immature fish do not normally contain vitellogenin. Sixty percent (18/30) of all the Irish Pompano males and 33.3% of the mangrove snapper males (3/9) captured during sampling had appreciable levels of vitellogenin in their blood (range: 0.006-0.290 mg/ml).

A substantial number (42-100%) of the immature mangrove snappers from both St. Lucie and Jupiter areas also contained significant plasma Vtg concentrations (>0.005 mg/ml). It is difficult to ascertain if there is a significant trend in spatial distribution of estrogenic “hot zones”, due to the low numbers of males captured in some areas. Surprisingly, there was evidence of estrogen exposure throughout both sampling sites and different areas of each site. Jupiter inlet was chosen as a relatively clean reference site; however, this site was also impacted by estrogenics.

Discussion

Vitellogenesis is a very important process in egg laying vertebrates. The quantity and quality of this egg yolk precursor can greatly affect larval growth and survival. In addition to providing nutrients (protein, fat, carbohydrate) and building blocks necessary for embryo development, it is also an important carrier protein. Due to the diverse chemical make-up of vitellogenin, a glycolipophosphoprotein, it will bind materials (e.g. minerals, vitamins, hormones) that are critical for development. It will also bind pollutants which can be inadvertently carried into the egg, concentrated, and cause reproductive failure. Previous studies have shown that the phosphate groups on the Vtg protein backbone can carry needed iron, but also harmful toxicants such as copper, cadmium, and mercury (Shackley, 1981). Excess selenium in the diet or water can replace sulfur groups in cysteine and methionine and cause reproductive problems depending on the degree of substitution (Kroll & Doroshov 1990; Lemly, 1985). Organic compounds such as DDT and PCB have been shown to be carried into the egg by the lipid residues on Vtg (Plack et al, 1979; Monteverdi and Di Giulio, 2000).

Estrogen and estrogen mimics can also affect offspring survival by interfering with the maternal reproductive system. Endocrine disrupting chemicals act in the liver to alter gene transcription. In addition to increasing plasma Vtg concentration and zona radiata proteins, estrogen-like compounds cause plasma ferritin, transthyretin, and retinal binding protein levels to decrease (Funkenstein et al., 2000, Larkin et al., 2003). Other 7 genes are also affected. These changes in the balance of genes and proteins may have profound effects on egg quality and offspring success.

In addition to altering the reproductive cycle, estrogens can have a direct effect on the juvenile fish themselves. Fish are gonochoristic, and have the ability to be either male or female dependent on their environmental conditions (e.g. temperature). If fish are exposed to estrogens during a critical developmental window, all-juvenile fish will be turned into females or hermaphroditic gonads will be created. What effect this has on future reproductive performance and population stability is not yet known.

The St. Lucie area has historical documentation of abnormal deformities and evidence of depressed immunity, which may be the result from exposure to chemical compounds (Browder & Kandroshov, 2001). Studies on the Indian river, an major input to the esturary, have documented cancer in mollusks (Van Beneden et al. 1993) and disease in sea turtles (Hirama, 1999). Estuaries are very important nurseries for fish and other wildlife due to rich natural inputs, abundance of foodstuffs, and areas to hide from predators. However, the low flows and slow turnover make it an area for toxicants to accumulate, get magnified in the food chain, and persist. These external signs are a clear indication that some stressors are compromising the populations. One of them appears to be exposure to endocrine disrupting compounds.

There are a number of potential sources of these estrogenic compounds. Treated sewage can significantly induce vitellogenin in fish depending on the proximity to the plants output and the amount of dilution (Folmar et al, Harries et al 1997). There are a variety of pesticides and industrial chemicals currently in use that are known to act as estrogens (Hemmer et al, 2001) and induce Vtg synthesis. The St. Lucie estuary receives input from the Indian River and Lake Okeechobee. Both inputs could contain significant agricultural and municipal run-off. Analysis of fish tissues, sediment, and water could verify the identity and concentrations of potential estrogenic chemicals.
The objectives of this study were to develop tools and determine if there were estrogenic compounds in the St. Lucie and Jupiter water sheds. Indeed there is evidence that these fish are being exposed to estrogenic compounds, among others. Statistical significance of plasma Vtg concentration and location is still under analysis. The source and type of these inducers are yet unknown. Future studies will be needed to determine where the chemicals are coming from and what they are. This may aide in management to reduce or eliminate these sources.

Possible Future studies

This preliminary study was directed towards determining if fish in the St. Lucie estuary and Jupiter inlet were being exposed to estrogenic compounds. The next stage of this study should be directed towards determining where the inputs are coming from and the identity of the contaminants. The sources of estrogen inputs could be studied using caged male Sheepshead minnows. This species is tolerant of variable salinities, and could be placed at various sites to determine input sources and “hot” zones. Our lab has 8 already developed an ELISA for this species and is currently working on “gene chips” which identify genetic markers for hormonally active compounds and endocrine disruptors (Larkin et al., 2003). Soil extracts and water samples concentrated on hydrophobic membranes (e.g. C18) could be collected from suspected areas and brought into the lab and used to expose male fish to ascertain their estrogenicity and the identities of the hormonally active compounds.

Acknowledgements

The National Ocean and Air Administration (NOAA) and the collaboration of Tony Pait and Joan Browder supported this study. The authors would like to thank the following persons for their assistance: Mark Perry of the Florida Oceanographic Society (Stuart, Florida), the fisherman /researchers- Michael & Walter Kandrashov, Stacy Foster & Michael Bresette from the Stuart Florida Power Plant. We would like to dedicate this study to William Kandroshov who saw the deterioration of fish numbers and health in the St. Lucie area and for the last decade worked towards making the public aware of this problem and the estuary’s plight.
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