AT A GLANCE
Research biologist, Gamete and Early Embryo Biology Branch, Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory (NHEERL), US EPA
PhD, biological sciences, University of Warwick, Coventry, W. Midlands, England; 1995
Served as a postdoctoral research fellow in the molecular toxicology laboratory at the school of biological sciences in the University of Surrey, Guildford, Surrey, England, from 1995-1998
Postdoctoral research fellowship in the Reproductive Toxicology Division, NHEERL working with David Dix, from 1998¯2000
Interests: Elucidation of molecular mechanisms of toxicological effects using molecular biology techniques, gene expression during spermatogenesis
At the US Environmental Protection Agency, “we are not just a bunch of politicos,” joked biologist John Rockett during a recent address at the MacroResults Through Microarrays Conference in Boston. There is “increasing use of genomic technology, in particular array technology, when companies submit a chemical for approval” by the EPA, he said. In fact, the agency is considering requiring companies to submit microarray results with their approval applications. But meanwhile, EPA scientists are increasingly using microarrays in their toxicology studies, he said.
In his talk, a summary of which follows,Rockett outlined the principal goals of the EPA’s genomic studies. The first goal, he said, is to identify gene expression patterns associated with exposure to specific classes of chemicals. Second, the group hopes to identify the biomarkers indicating toxicity, including those from “surrogate” tissues. These surrogate tissues would be ones that are accessible to study and have been shown to have similar levels of a certain biomarker as a non-accessible tissue. Once these biomarkers and expression profiles are known, the group would like to delineate toxicants’ mode-of-action in multiple species and in different toxic occurrences. Lastly, the group would like to compare across species in order to determine common hazards and the mode of action for human and ecological exposures.
Minnowing out the Genes
One of the group’s initial array studies involves the Sheepshead Minnow, Cyprinodon variegates, which has been previously established as a test species by the EPA, as it is sexually dimorphic, easily cultured, has a well documented life history, and is ubiquitous in Atlantic and Gulf estuaries.
In the study, Rockett and his colleagues looked at the effect of estrogen-like compounds on the Sheepshead minnow. These estrogen-like compounds — such as estradiol, ethynylestradiol, diethylsilbestrol, methoxychlor, and nonylphenol — come from pesticides and are consequently present in many water systems that include runoff from agriculture, Rockett said. The presence of these chemicals has in some cases been thought to have resulted in endocrine disruption in numerous species, leading to effects such as gonadal atrophy among alligators. This possible association has led to research studying the effects of these chemicals on different populations of animals, such as the Sheepshead minnow.
By treating some of the minnows with estradiol, Rockett and his colleagues sought to find differentially expressed genes that could serve as biomarkers of endocrine disruption. They then extracted total RNA from the livers of control and estradiol-treated fish, and PCR-amplified cDNA clones, which they then spotted onto low-density nylon membranes. Out of 60 genes spotted on an array, they found a handful that appeared reliably to be differentially expressed. In subsequent experiments with arrays of RNA from minnows treated with various doses of ethynylestradiol, they were able to detect dose-dependent changes in gene expression. They found that natural, pharmaceutical, and xenoestrogens (the aforementioned ones in pesticides), all produced identical changes in expression.
In future experiments, Rockett and his group plan to assess the sensitivity and reproducibility of the Sheepshead minnow array, and do time-course experiments to determine gene expression profiles of the minnows over the life cycle of both sexes in response to different environmental conditions. Also, they plan to look at fish samples from contaminated sites to determine expression variability in natural fish populations.
Human Surrogate Tissues
With humans, unlike minnows, it is not possible to just extract RNA from the livers of large numbers of specimens and to see what genes are up- or down-regulated. So, in looking at the effects of environmental toxicants on human health, Rockett and his colleagues are searching for surrogate tissues with gene expression patterns that correlate with that of the target tissue.
For example, “one might examine a patient’s peripheral blood leukocytes in order to determine if there is a problem with their thymus,” he said. Other surrogate tissues that are sources of good quality RNA include blood, hair follicles; milk, cord blood, and placenta from postpartum females; semen from adult males; and urine. The problem is that many toxicants are highly tissue-specific in their action, limiting the usefulness of surrogate tissues. But some surrogate tissues could be reliable markers of what is happening in the adjoining tissue: For example, semen may be a good match for monitoring events occurring in the testis, while peripheral blood leukocytes may be a good biomarker for the thymus, spleen, tonsils, bone marrow, or glandular tissues.
Based on these ideas, the EPA is looking at semen as a biomarker for the potential effect of toxicants on the human male reproductive system, and is also looking at peripheral blood leukocytes as a surrogate tissue for the effect of estradiol on the uterus.
In the human semen experiment, the group hybridized RNA taken from testis samples and semen samples to 27,000-spot microarrays. They identified 7,000 genes that were differentially expressed in testis as compared to other tissues, and 3,000 of these that were also expressed in sperm of normal fertile men. In the future, said Rockett, these mRNA patterns could serve as biomarkers to identify infertile men. “Current semen quality measures are relatively poor indicators of fertility because they are subjective and rely predominantly on physiological and morphological criteria,” he said. “Microarrays could be more informative in this respect.”
With the rat uterine experiment, Rockett and colleagues dosed 12 ovariectomized Long-Evans rats with a form of estradiol or a corn oil control. After the third dose, the animals were harvested, with the uteri being extracted and the leukocytes being collected. Total RNA was prepared from these different samples, and hybridized to Clontech Rat Toxicology 1.2 arrays that had 1,185 genes each. They found 193 genes detectable in both the leukocyte and uterine samples, and 18 that were significantly altered in both. They then verified these results in six genes with qRT-PCR.
In the future, the group plans to look at changes in gene expression over time and with different doses of estradiol, given that these 18 correlated genes might not remain correlated at different dose levesl or during different points in the life cycle. To do this work, the group is using pre-fabricated high density oligo arrays for rodent and human, and is also looking to integrate laser capture microdissection and high throughput proteomics, as well as better bioinformatics. In addition to tools like analysis of variance and regression, Rockett said he would like to see pathway analysis instead of individual gene analysis. “I think it’s pathway analysis that’s important,” he said.