Download Endocrine disruption in aquatic and terrestrial invertebrates

ABSTRACT
Endocrine Disruption in Aquatic and Terrestrial Invertebrates – Linking
Gene Expression with Physiological Effects
I Johnson (WRc-NSF) and P Kille (Cardiff University)
In recent years major programmes of environmental research investigating the
effects of endocrine disrupters on vertebrate species in the freshwater and marine
environments have been conducted However, to date less attention has been
focussed on endocrine disruption effects on invertebrates and top predators. This is
clearly an issue, particularly for the invertebrates, since these taxonomic groups
account for approximately 95% of the known animal species, and represent more
than 30 different phyla and are key groups which need to be considered in hazard/
risk assessment strategies.
To address this data gap endocrine disruption in two key invertebrate taxonomic
groups has been addressed. This involved

Identification of sex-specific and/or sex determining genes in test species
Gammarus pulex and Eisenia sp by homology with existing genome data.
Genes associated with the process of moulting in Gammarus were also
considered;

Development of optimised procedures for measurements of biomarker gene
expression in the test species;

Generation of information on ‘normal’ background levels of biomarker gene
expression during the life cycle of the test species. For Gammarus pulex this
involved consideration of both the changes in responses during the development
of the organism and also through the moult cycle (the two being coincident);

Evaluation of the biomarker responses (up/down regulation) following exposure
of test species to a range of known or suspected endocrine disrupters in
screening studies;

Evaluation of the extent to which the biomarker responses are evidently linked to
effects on growth, development and reproduction in the test species.
The studies have expanded the current knowledge of the endocrinology of the
sentinel species through the use of novel genomic techniques. The short-term
screening and longer-term reproduction studies have also provided data on the
effects that a range of known or suspected endocrine disrupters (oestradiol,
ethinyloestradiol, testosterone, bisphenol A, nonylphenol, fenoxycarb, propoxur) have
on the development and reproduction of the species tested. Hypotheses have also
been developed regarding how endocrine disrupters may cause up or down
regulation of gene expression of particular markers and subsequent physiological
effects.
The following conclusions have been drawn from the results of the studies
conducted:
Gammarus pulex studies

The identification of markers of endocrine disruption in Gammarus pulex has
proved to be complicated due to the complex inter-relationships between the
processes of moulting and reproduction. However, the work carried out in this
study has provided the initial insight to the molecular genetic components of the
endocrine system in Gammarus. Over 500 novel sequences have been
generated and key moult and reproduction responsive genes including those
encoding cuticle formation protein and vitellin respectively have been isolated. In
addition genetic assays have been developed that allow the sensitive and
accurate quantification of these molecular indices establishing the moult
regulated cycle of cuticle synthesis and the female specific nature of vitellin
expression. Finally, a start has been made on revealing the integrated network of
nuclear receptors responsible for regulating the endocrine system in Gammarus
through the isolation of a putative moult regulating transcription factor.

Given the complex inter-relationships of the hormone mediated processes of
moulting and reproduction in Gammarus it is probable that effects of endocrine
disrupting chemicals will only be addressed using a array of relevant biomarkers
of gene expression. This array will need to include markers from both male and
female organisms and a moult marker. This area of research is being continued
by the partners through a NERC Case studentship.

Exposure to low levels of the androgen testosterone (10-100 ng l-1) has been
found to markedly inhibit the reproductive capability of Gammarus in both shortterm studies using pre-copula pairs and longer-term studies initiated with
juveniles. The reduction in juvenile production was concentration-dependent and
no juveniles were produced at the highest testosterone concentration. It is
postulated that this may be due to impairment of the reproductive capability of
females, though the exact mechanism of action is unclear.

Exposure to low levels of the moult inhibitor fenoxycarb (1-100 g l-1) has been
found to markedly inhibit the reproductive capability of Gammarus in short-term
studies using pre-copula pairs. However, changes in reproduction observed in
longer-term studies initiated with juveniles were not evident.

The oestrogens oestradiol and ethinyloestradiol and the industrial chemicals
bisphenol A and nonylphenol
were not found to cause effects on the
reproduction of Gammarus pulex following exposure of precopula pairs.

Overall, the amphipod Gammarus pulex has been shown to be a potentially
useful indicator species for assessing the effects of known or suspected
endocrine disrupting chemicals.
Eisenia sp studies

Initially three potential markers for endocrine disruption were identified in the
earthworms but problems were encountered with the development of assays for
two markers (vitellogenin and a ‘sperm’ marker). A robust assay has been
developed for gene expression of annetocin (a protein involved in the release of
cocoons) and oestrogen receptor binding sites have been identified upstream of
the gene start codon. A hypothesis has been advanced for the activation of the
annetocin gene and the role of the protein in cocoon production.

Statistically significant effects on the reproduction of Eisenia sp were only evident
in sexually mature (clittelate) organisms exposed to the insecticide propoxur,
though these effects were probably not endocrine mediated.

No statistically significant effects on reproduction (as the number of hatched
cocoons and live worms) were evident in organisms exposed to the oestrogens
oestradiol and ethinyloestradiol, the androgen testosterone and the industrial
chemicals bisphenol A and nonylphenol.

Earthworms, such as Eisenia sp, are an important test species for assessing the
effects of substances or mixtures on terrestrial ecosystems. Adverse long-term
effects are usually evaluated based on impairment of reproductive output. The
species also potentially represents a key indicator species for assessing the
effects of endocrine disrupting chemicals. However, from this study it has become
evident that the hermaphroditic nature of earthworms means that they may not
represent the most appropriate terrestrial species for assessing the effects of
EDCs. This stems from the fact that the organisms appear to have inherent
homeostatic mechanisms to compensate for internal fluctuations in oestrogen
and androgen concentrations following exposure to endocrine disrupting
chemicals. An alternative species which could be useful in this role is the
collembolan (springtail) Folsomia candida.