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Johns Hopkins Bloomberg School of Public HealthCAAT

Abstract for TestSmart -- Endocrine Disruptors

Where are We Today? Current Understanding of the Mechanisms of Action of Endocrine Disrupting Chemicals

John A. McLachlan1,2, Jennifer Fox2, Ann Cheek2,3 and Matthew Burow1,2
1Department of Pharmacology, Tulane University Health Sciences Center; 2Environmental Endocrinology Laboratory, Center for Bioenvironmental Research, Tulane and Xavier Universities, New Orleans; 3LA Department of Biology, Southeastern Louisiana University, Hammond, LA

The term "endocrine disruption" has been applied to a variety of adverse effects of chemicals and other factors on the immune, nervous and endocrine system. While consideration of the various systems is important and provides a comprehensive view of potential problems, the breadth of the term does not readily lend itself mechanistic studies.

A useful term, "environmental hormones", defined as "exogenous chemicals that block or mimic vertebrate hormones, or closely related molecules" provides a working definition for which mechanisms can more readily be derived. Another important variation on the definition of environmental hormones is "those chemicals which interact with members of the steroid-thyroid-retinoid receptor gene family". This latter definition would include those members of the family called "orphan" receptors. Recent evidence that one such orphan receptor, SXR, specifically recognizes xenobiotics suggests that nuclear receptor activation remains an important consideration in the determination of hormonally active environmental chemicals. The demonstration that foreign chemicals can alter enzyme activities important in the formation or processing of steroid hormones and resulting in endocrine disruption suggests at least a third mechanistic consideration.

Some time ago, our lab suggested a "mechanism based" assay for endocrine disrupting chemicals called functional toxicology. (1) In it we proposed that chemicals could be screened in vitro by a battery of tests utilizing the nuclear receptor gene family. Thus, chemicals would have a biologic function assigned to them based on the activation of a specific receptor system. One could imagine extending the screen to detect xenobiotics that activated receptors for hormones, neurotransmitters and other important biomolecules. While not definitive, such a short screen coupled with metabolic activation, would be a very useful guide to the potential activity of chemicals. Currently, many receptor-based cell culture assays are under evaluation.

While many mammalian cell culture assays have been shown to reliably reflect biological responses to natural and synthetic estrogens, in vitro assays do not, as yet, determine if the hormonal response will result in long-term phenotypic alteration. Since a special concern in endocrine disruption involves the developing organism, the role that hormones play in phenotypic determination represents a mechanistic "hotspot". An important question remains; can environmental chemicals working through epigenetic processes imprint genes and cell fate? This remains an important challenge for assay development.

We have shown in vivo that mice exposed to estrogenic chemicals early in development exhibit persistent alterations in uterine gene expression(2). One proposed mechanism is the methylation induced imprinting of such genes by chemicals at the appropriate time of development(3). We are currently working to establish methylation-based assays for gene expression as an in vitro system, which may be more sensitive to developmental changes in phenotype. To this end, we have constructed cell culture systems that respond to an estrogen signal by activating cell signaling pathways. While still under construction, these in vitro systems may provide the appropriate indicator of gene imprinting via DNA methylation.

We have also taken advantage of the fact that natural phytoestrogens are actually evolutionarily conserved signaling molecules. A well-studied signaling system is that of symbiosis between legumes and rhizobial nitrogen fixing bacteria. Recent results from our lab demonstrate that some of the same synthetic chemicals reported to be endocrine disrupting in vertebrate animals or cells also disrupt the symbiotic signals between plants and bacteria (Fox et al, submitted). As with endocrine disruption, the disruption of symbiotic signaling has important biological consequences. Whether such bacterial systems represent viable assays systems remains to be determined.

Finally, as assays for detection of hormonally active chemicals become more sensitive, consideration of what this may mean at the population level take on increased importance. We report the results of experiments to evaluate the correlation between an established biomarker for estrogen action (vitellogenesis) and a population-based outcome (reproductive capacity) in fish (Cheek et al, in press). Interestingly, we found that the reproductive capacity of a breeding fish population was adversely affected by endocrine disrupting chemicals at doses lower than that required for measurable stimulation of vitellogenin. These results raise the possibility that some biomarkers of effect may be less sensitive than the effect itself. Such information will be useful in the formulation of assays for endocrine disrupting chemicals.

The wise use of mechanisms discovered for the action of natural and synthetic hormones should continue to improve the environmental assays to detect endocrine disrupting chemicals and anticipate their potential effects.

References

  1. McLachlan, J.A. (1993). Functional toxicology: a new approach to detect biologically active xenobiotics. Environ Health Perspect 101:386-387.
  2. Nelson, K.G., Sakai, Y., Eitzman, B., Steed, T. and McLachlan, J. (1994). Exposure to diethylstilbestrol during a critical developmental period of the mouse reproductive tract leads to persistent induction of two estrogen-regulated genes. Cell Growth Differ 5:595-606
  3. Li, S., Washburn, K.A., Moore, R., Uno, T., Teng, C., Newbold, R.R., McLachlan, J.A., Negishi, M. (1997). Developmental exposure to diethylstilbestrol elicits demethylation of estrogen-responsive lactoferrin gene in mouse uterus. Cancer Res 57:4356-4359