Sprague Dawley rats to very high The findings of a key paper (Scidoses of the anti-androgenic ence 2005, DOI 10.1126/scifungicide vinclozolin during the ence.1108190) with potentially critical developmental window profound implications for the fuwhen sex determination occurs. ture of environmental health reThe exposure resulted in male search and regulation are being reproductive problems that inchallenged by a small group of cluded fewer sperm with less government and industry scientists who say they cannot replicate its results. This paper and subsequent publications on epigenetics from molecular biologist Michael Skinner’s lab at Washington State University link exposure to an endocrine-disrupting chemical in the womb with health problems that extend for four generations. “If these findings are repro- Rat studies: when it comes to epigenetic response, ducible and robust, then appears to matter. the implications of this motility than normal. Nearly all study are huge,” says epigenetics males in the four subsequent researcher Bernard Robaire at generations studied were afMcGill University (Canada). fected. The cause appeared to be “We would have to redefine the an epigenetic effect related to mission of our regulatory agencies. changes in DNA methylation. That is why it is absolutely essential “Prior to our observations, most for these results to be replicated in transgenerational effects stopped other labs. The fact that these other at the second generation,” says researchers do not appear able to Skinner. replicate the study is very imporRobaire summarized the implitant,” Robaire adds. cations of the findings, should Epigenetics refers to heritable they prove generally applicable to changes in gene function that people, as raising the possibility cannot be explained by changes that your pregnant grandmother’s in DNA sequence. DNA methylaexposure to chemicals may cause tion and histone modification disease in you and your grandare two of the mechanisms inchildren (Nat. Med. 2008, DOI volved in epigenetic regulation. 10.1038/nm1108-1186). Others Numerous studies have shown agree. “The impact on human that nutrition can cause epigehealth, should the Skinner data netic changes in animals and be replicated and expanded in people. Other studies of chemiother labs with other chemicals at cal exposure have reported epilower doses, could be enormous,” genetic effects in the offspring of says Jerry Heindel, a scientific the exposed parents. Thus far, program administrator at the Nathough, Skinner’s lab is almost tional Institute of Environmental alone in linking exposure to enHealth Sciences. “This would be a vironmental chemicals with efnew paradigm that changes everyfects on the epigenetic code that thing and puts more emphasis on persist for many generations. The 2005 paper reported the results of exposure of pregnant 10.1021/es902777p
2009 American Chemical Society
Published on Web 09/23/2009
reducing exposures during pregnancy to lower incidence of disease.” The Skinner group has expanded upon this original report with nine additional peer-reviewed papers. Some of their further findings link fetal exposure to vinclozolin with a higher incidence of prostate and kidney disease, increased immune-system abnormalities, and tumor development. They report that female offspring show uterine hemorrhage, anemia, or both late in pregnancy, as well as an increase in mammary and other tumors. The lab also has described behavioral effects in which female rats three generations after exposure preferred as mates strain those males without a history of exposure. A 2006 paper identifying a series of genes epigenetically altered by vinclozolin exposure (Endocrinology, DOI 10.1210/en.20060987) was retracted in June 2009 by the authors. The published retraction points to “the inability to locate and confirm the data” generated by one author and states, “The coauthors were not aware of this falsification of data.” Other researchers in the field do not see this incident as casting doubt on the remainder of the work from Skinner’s lab. But recent abstracts and presentations by U.S. Environmental Protection Agency (EPA) scientists (Biol. Reprod. 2008, 78: 228. 735) and peer-reviewed papers by industrial labs in Germany (Reprod. Toxicol. 2008, DOI 10.1016/j. reprotox.2008.04.001) and Japan (Toxicol. Appl. Pharmacol. 2009, DOI 10.1016/j.taap.2009.03.004) have not been able to detect any transgenerational effects in similar experiments involving vinclozolin. Earl Gray, a reproductive toxicologist with EPA, has a long history of studying the antiandrogenic properties of JUPITERIMAGES
Key environmental epigenetics paper challenged
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vinclozolin on male rat pups (Toxicol. Appl. Pharmacol. 1994, DOI 10.1006/taap.1994.1117). He has found that high doses of vinclozolin given to pregnant rats demasculinize their male pups when the pregnant mothers are exposed after sexual differentiation has occurred and androgen receptors are activesthat is, at gestational day 13-17. The Skinner lab doses pregnant rats earlier, as the gonads are being formedsgestational day 8-15. During this time, natural epigenetic changes affect the germ cells, and it is possible that an environmental agent could interfere with this process and the germ line, Skinner says. But Gray says that his attempt to replicate the Skinner lab results has been “entirely negative”. His experiment differs from the Skinner lab procedure because Gray gave vinclozolin as an oral dose, rather than as an intraperitoneal (IP) injection, as in the 2005 paper from Skinner’s lab. There is a good reason for the change, argues Gray. “An IP injection with such a high dose is a terrible way to expose a pregnant rat,” he says. “You’re going to drop the chemical right on top of the uterus and fetus. After necropsy you see particles of the chemical, so there’s no way to quantify the exposure to the fetus.” Toxicologist Bennard van Ravenzwaay, who works at BASF, vinclozolin’s manufacturer, also believes that IP injection is not an appropriate way to conduct this
experiment. Using oral exposure, he has not found multigenerational effects. Nevertheless, even studies that have used IP injection to replicate the Skinner lab results have failed. In a peer-reviewed paper published this spring, researchers from Sumitomo Chemical Co., Ltd., used IP injection in their attempt to replicate the Science paper results, but failed. Van Ravenzwaay’s group is also trying to replicate the experiments using IP injection. They presented preliminary results of an IP exposure experiment at the Society of Toxicology meeting in March 2009. “We can certainly state that there is no evidence of a transgenerational effect,” he says. “We did see some minor anti-androgenic effects in the pups exposed as fetuses, but these were not passed on to the next generation.” Van Ravenzwaay adds that he has had no luck in his attempts to correspond with Skinner about the studies. The type of rat used in the experiments is likely to be a factor, Skinner says. In his lab, he notes, inbred rats do not appear to show as big an epigenetic response as outbred rats, which are more genetically diverse. Skinner believes that outbred rats may have a bigger epigenetic response because they are generally more robust and vigorous than inbred strains. However, Robaire says that his demethylation experiments involving pharmaceuticals achieve the best results when he uses inbred rats. “Using inbred rats
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with the same genetics increases the signal-to-noise ratio and gives us better results,” he says. “Our primary focus of the research,” says Skinner, “is not in the toxicology of vinclozolin, but instead its use as a pharmacological agent to induce the transgenerational phenomena to study the mechanisms involved.” His lab has studied another well-known anti-androgenic chemical, flutamide, which does not elicit the same response in rats as vinclozolin. “Right now, we don’t know what it is about vinclozolin that causes this response,” he adds. Heindel says that the fact that others have not reproduced Skinner’s results may not be so surprising. “The published manuscripts from Mike Skinner’s lab have been shown to be reproducible within his lab. If researchers use different doses or routes of exposure or different strains of animals or different diets, then they are not reproducing the Skinner experiments. So, if they don’t get the same results, that does not affect the validity of the Skinner experiments.” But Robaire says the verdict is still out. “The Japanese lab repeated exactly the same dose, exactly the same delivery method, and could not find the results. People are still groping to resolve this information. Michael Skinner is a very solid scientist, but it’s possible that this effect may turn out to be unique to one subspecies of rat,” he says. —REBECCA RENNER
