Monitoring estrogenic effects: a genomics approach. (Science Selections).

Genomics, the revolutionary field that promises to one day reveal the genetic code of every living organism, is opening up unforeseen opportunities for advances in many areas of the life sciences. In this issue, a team led by Patrick Larkin of the University of Florida in Gainesville and EcoArray LLC describes a genomics approach to monitoring toxic chemicals in the environment and uncovering their effects on organisms at the molecular level [EHP 111:839-846]. Larkin and colleagues hope to produce an easy-to-use biomarker test capable of detecting metabolic pathways affected by environmental chemicals, and ultimately to formulate specific gene profiles that will permit identification of particular chemical contaminant exposures. In this article, the team describes an expression profiling model system for endocrine-disrupting compounds (EDCs) that mimic estrogens.

Natural and synthetic estrogens are found in pharmaceuticals, industrial by-products, and pesticides, and can cause human health effects including vaginal cancer and reproductive tract abnormalities. Because estrogen is a female reproductive hormone, genes in the estrogen pathway are normally not highly expressed in males. However, when male fish are exposed to natural or synthetic estrogens, the result is an increase in the expression of female-specific genes. The estrogen pathway has been highly conserved during vertebrate evolution--it is shared by many different organisms---so changes due to exposure in fish may presage effects in other animals, including humans.

The team created a gene array by cloning 30 genes--some involved in the estrogen pathway and some controls--from sheepshead minnows. The genes had been previously identified by differential display reverse transcriptase-polymerase chain reaction, a method that screens thousands of RNA messages to identify genes that are turned on or off by specific treatments. The team used microarray analysis to discover which of the 30 preselected genes were significantly changed by exposure of the fish to estrogenic compounds. They also measured changes in levels of gene expression when the fish were exposed to different concentrations of 17[alpha]-ethinyl estradiol, a synthetic estrogen found in birth control pills (which can end up in waterways via sewer systems).

Once they had their array in place, the team exposed male sheepshead minnows to a constant concentration of either strong or weak environmental estrogens. The strong estrogens included 17[beta]-estradiol (the normal estrogen found in vertebrates), 17[alpha]-ethinyl estradiol, and diethylstilbestrol (a synthetic estrogen formerly used to prevent miscarriage that caused cancer, reproductive tract abnormalities, and infertility in the children of women who took it). The weak environmental estrogens included p-nonylphenol (a breakdown product of alkylphenol ethoxylates, which are used in various products such as washing and cleaning agents, emulsifiers, wetting agents, and foaming and foam-reducing agents) and the organochlorine pesticides methoxychlor and endosulfan. Single-stranded DNA for the 30 genes was bound to multiple membranes.

To analyze genes that were differentially expressed in the livers of control and treated fish, the team extracted mRNA and converted it to cDNA, which during this process was labeled by the addition of a tracer amount of radiolabeled nucleotides. The cDNA was then incubated with the membranes and bound proportionately to the 30 genes present thereon. The intensity of the radioactivity in the spots was directly related to the amount of mRNA present in the sample and, when compared to controls, was used to determine whether the expression of a gene was elevated or decreased as a result of exposure to the EDC.

There was an increase in expression of certain genes as a result of exposure. One endocrine receptor (ER[alpha]) was upregulated by every test compound. Four genes involved in the formation of egg cells were upregulated by every compound except endosulfan. A gene that plays an important role in blood clotting also was upregulated by the same five compounds. Interestingly, the gene for ubiquitin-conjugating enzyme 9, whose metabolic role is to tag enzymes that have completed their cellular functions and defective proteins for removal from the cell, was upregulated by p-nonylphenol. The expression of three genes involved in other processes was downregulated by the five compounds. Exposure to different concentrations of 17[alpha]-ethinyl estradiol also revealed that the microarray method is dose-sensitive, and that exposure thresholds vary for different genes. These findings could enable calculation of gene-dependent dose-response curves for evaluating the seriousness of chemical contamination in environmental cleanup efforts.

The scientists plan to expand the expression profiling method to compounds that mimic other reproductive hormones such as androgen and progesterone. They are also going to make microarrays for different game fish species used for food as well as other fish species that are used as standards for monitoring environmental chemicals.

One hurdle for this technology is obtaining reproducible results. Successful replication depends on the accuracy of the DNA amplification of each gene, the correct identification of which genes are bound to the membrane and where, and the RNA extraction efficiency, because RNA degrades rapidly and can become contaminated with DNA. These technical steps also require careful laboratory techniques and multiple replicate experiments to ensure consistent results.

As the methodology expands to include more genes, chemicals, and organisms, the management and analysis of huge volumes of data will become another hurdle. Bioinformatics will become increasingly important as these EDC expression profiling data sets expand. This genetic biomarker assay is an exciting application of genomics tools for toxicology with promise for finding genes that are affected by EDCs, for understanding mechanisms that lead to disease, for applying that knowledge to environmental monitoring and cleanup, and for the rational design of new compounds that will be safer for human health and the environment.