[Pharmwaste] Health impacts of estrogens in the environment, considering complex mixture effects

[Tenace, Laurie]

"An important finding from this study was that some of the responses to the
single compound differed from the responses of fish exposed to the mixture."

http://www.environmentalhealthnews.org/newscience/2007/2007-0905philbyetal.ht
ml

Filby, AL, T Neuparth, KL Thorpe, R Owen, TS Galloway and CR Tyler. Health
impacts of estrogens in the environment, considering complex mixture effects.
Environmental Health Perspectives, in press.
 September 5, 2007

   

-----------------------------------------------------------------------------
---
Synopsis by Dr. Ed Orlando and Wendy Hessler
 
New experiments reveal that the synthetic estrogen used by women for birth
control causes wide ranging health effects in minnows, but that the effects
differed when the drug was tested alone compared with when it was mixed with
wastewater effluent. 
   
 

The estrogen, called 17α-ethinylestradiol, caused feminization of male fish,
and altered DNA integrity, immune cell number and ability to breakdown
pollutants. The study highlights the need for more research on the potential
health effects of exposure to complex mixtures, such as wastewater effluent,
as they may differ from effects of exposure to single compounds known to be
in those mixtures.

Context: Water flowing out of wastewater treatment plants carries estrogenic
chemicals out into the environment. Over the past 15 years scientists have
discovered natural and synthetic estrogenic chemicals in sewage water
effluent, including 17α- ethinylestradiol (EE2) from birth control pills can
affect reproduction and development of fish living in the waters downstream.

They cause male fish to start producing egg yolk (measured by detection of a
yolk protein, vitellogenin), change blood concentrations of hormones, alter
the expression of genes important for synthesizing hormones, decrease the
size of testes and alter secondary sex characters, including coloration and
behavior.

Lab studies have identified EE2 as one of the most powerful components in the
wastewater. In one experimental study in a Canadian lake, EE2 caused the
collapse of the an entire fish population.

This study is the first to examine the impact of EE2 on an array of health
endpoints when it interacts with the complex mixture of chemicals present in
wastewater effluent.
   What did they do? Filby et al. exposed adult fathead minnows for 21 days
to different mixtures of EE2 alone, wastewater effluent and the two as a
mixture and then looked at a wide array of health endpoints to detect
effects. The effluent, called wastewater treatment plant effluent, or WWTPE,
was collected from wastewater treatment plants in the United Kingdom.

They used two different WWTPE exposures, one that was strongly estrogenic and
one that was only weakly estrogenic. Some minnows were exposed to the
strongly estrogenic water. In a second experiment, another set of minnows
were divided into two groups: Both were exposed to a weakly estrogenic WWTPE,
but in addition, one of the groups also was exposed simultaneously to EE2.
The final experimental group was exposed only to EE2. 

The yeast estrogen screen (YES) assay was used to determine relative
estrogenicity of the potent, weak and weak effluent plus EE2 treatments.  The
concentrations were 21.3 ± 9.12, 6.18 ± 0.96 and 11.1 ± 3.39 nanograms
estradiol equivalents per liter (ng E2/L), respectively. 

While the same amount of EE2 was used for the EE2 treatment only and the weak
estrogen plus EE2, direct measurements of the actual concentrations revealed
11.9 ± 0.47 for the EE2 treatment and 4.46 ± 1.00 ng/L in the weak effluent
plus EE2 treatment. The authors suggested that something in the weak effluent
may have bound with the EE2, or that microbes could have digested it. 

This water analysis showed that adding EE2 to the weak effluent increased the
mixture's estrogenicity by 1.8-fold, to 11.1 nanograms estradiol equivalents.


Five health effects were measured in the exposed fish: 1) growth (tissue mass
and insulin-like growth factor-1 expression), 2) genotoxicity (via DNA strand
breakage in blood and/or gonad cells), 3) immunotoxicity (total white blood
cell types (lymphocytes, granulocytes, monocytes and thrombocytes) and
phagocyte activity), 4) metabolic responses (a measure of CYP1A enzyme
activity and the expression of cyp1a, cyp3a and gst genes) and 5) endocrine
effects (vitellogenin and estrogen and androgen receptor gene expression).

The liver enzyme genes, cyp1a, cyp3a, and gst are responsible for making
certain pollutants easier to clear from the body. Gene expression was
measured in the liver and/or gonads.
 

What did they find? In addition to the expected endocrine disruption (i.e.,
altered vitellogenin gene expression and protein induction and altered
hormone receptor expression), effluent exposure was also associated with
increased DNA strand breakage, a decreased number of lymphocytes, an
increased number of granulocytes and thrombocytes, and altered expression of
liver metabolizing enzyme genes. No effects on growth or phagocytosis were
found in fish and so these endpoints were not measured during the second
experiment.

For many of these endpoints, the impacts were greater in the strongly
estrogenic effluent. Some were specific to either female or male fish.

An important finding from this study was that some of the responses to the
single compound differed from the responses of fish exposed to the mixture.

Male fish exposed to all four treatments - the potent effluent, the weak
effluent, the weak effluent plus EE2 and EE2 alone - had increased amounts of
vitellogenin protein and gene expression compared to controls. In females,
potent or weak WWTPE, had no effect on vitellogenin protein or gene
expression, yet EE2 alone slightly increased vitellogenin protein. The
authors suggest males were more sensitive to the effects of WWTPE estrogens
than the females, which normally have higher blood estrogen levels.

The weak WWTPE plus EE2 mix changed the immune response in fish when compared
to the EE2 only exposure. Lymphocytes decreased, and thrombocytes (platelet
precursors) and granulocytes increased in both females and males. For the EE2
alone, there was no effect on any of the white blood cell numbers.

The ability to metabolize natural and foreign chemicals circulating in the
blood differed. Fish exposed to either potent or weak WWTPE had increased
liver CYP1A enzyme (EROD) activity and cyp1a gene expression.

When male fish were exposed to the weak WWTPE and EE2 mixture or EE2 alone,
the effects on both endpoints were reversed; that is, both EROD and cyp1a
were inhibited relative to the weak WWTPE exposed fish. In females, there was
a similar response in EROD activity.  Although there was no significant
difference in cyp1a gene expression from the weak WWTPE exposure, cyp1a
expression from EE2 alone was inhibited. 

The authors suggest that components of the WWTPE are by themselves enough to
activate the metabolizing enzyme (EROD activity) and induce cyp1a gene
expression, but that EE2 is inhibiting this biological effect. This is an
important finding as the metabolizing of many pollutants, including
polyaromatic hydrocarbons, PCBs, and dioxins, would potentially be affected
by the presence of EE2.

What does it mean? Sewage effluent contains a variety of estrogens combined
with other pollutants in a complex chemical mix that can affect normal fish
development, reproduction, metabolism, immune function, DNA integrity and
endocrine function. These effects vary with the potency of, and the
substances found in, the chemical concoctions. Importantly, the common
synthetic estrogen, EE2, elicited different effects on the exposed fish by
itself than it did when added to a weak effluent mix.

This study is important because the researchers thoroughly examined broad
health effects from exposure to WWTPE. The findings are compelling in that
they go beyond endocrine effects to identify changes to the immune, metabolic
and genetic systems brought on by exposure to a complex mixture of chemicals.


More importantly, the mixtures altered how an individual estrogen affected
the fish. Exposing fish to an estrogen alone or the same estrogen in a mix
caused different health effects in the minnows.  The results cast doubt on
the accuracy of chemical safety testing regimes that rely solely on testing
individual chemicals instead of mixtures, which is how exposure occurs in the
environment.

The authors' conclude that "the effects of estrogens when administered alone
cannot always predict their effects in complex environmental mixtures and
these interactive effects have far-reaching implications for the use of some
biomarkers (such as EROD) in environmental monitoring. A greater
understanding of the mechanisms of interactive chemical effects is essential
to fully understand the impacts of environmental mixtures like effluents for
exposed organisms."

In Westernized countries, male fish held in cages or collected from the wild
downstream of WWTPE discharge will have elevated vitellogenin protein,
altered secondary sex characteristics and possibly be intersex from exposure
to ubiquitous estrogens. In fresh and estuarine waterways, researchers also
find diseased fish (e.g., increased parasite load and
microorganism-associated lesions) or the more profound occasional die-offs.
These very same waters receive WWTPE and other industrial and agricultural
effluents.  More than 10 years of research into the effects of WWTPE has
proven a connection between exposure to these effluents and altered
reproduction and development of fishes. Government regulatory agencies are
beginning to notice and take appropriate steps toward protecting wildlife and
human health.

The research by Filby et al. begins to link (now) obvious endocrine system
effects from exposure to broader health consequences. The findings show that
effluent mixtures can have different effects on health and reproduction than
exposure to single pure compounds found in the mix. These results offer
important insights as to how mixtures affect health and could guide
discussions about proper regulation of WWTPE and perhaps other pollutant
mixtures.

 



Laurie J. Tenace
Environmental Specialist
Florida Department of Environmental Protection
2600 Blair Stone Road, MS 4555
Tallahassee, Florida 32399-2400
PH: (850) 245-8759
FAX: (850) 245-8811
Laurie.Tenace at dep.state.fl.us 

Mercury web pages:
http://www.dep.state.fl.us/waste/categories/mercury/default.htm

Unwanted Medications web pages:
http://www.dep.state.fl.us/waste/categories/medications/default.htm




Please Note:  Florida has a very broad public records law.  Most written
communications to or from state officials regarding state business are public
records available to the public and media upon request.  Your e-mail is
communications and may therefore be subject to public disclosure.