Comment on “Glyphosate impairs male offspring reproductive development by disrupting gonadotropin expression” by Romano et al. 2012

Abstract

Romano et al. (2012) recently published the results of a study in which pregnant rats were treated with a glyphosate-based herbicide formulation from gestational day 18 to postnatal day 5. Various parameters related to sexual behavior and reproductive development were assessed in the resulting male offspring. Based on their results, the study authors claim that glyphosate exposure in the perinatal period causes changes in males that are associated with hypersecretion of androgens. There are a number of issues with this study, however, that preclude such conclusions. First, as a premise for this study, the authors incorrectly assert that glyphosate is a ‘‘potential endocrine chemical disruptor.’’ As evidence, they cite three in vitro studies of altered aromatase activity conducted in a single laboratory (Benachour et al. 2007; Gasnier et al. 2009; Richard et al. 2005), and their own earlier work suggesting reduced serum testosterone concentrations in response to treatment (Romano et al. 2010). These studies, however, were conducted using glyphosate-based herbicide formulations rather than the active ingredient alone. Exposures were at high concentrations; for instance, in the study by Benachour et al. (2007), the concentration of glyphosate in the tissue culture medium was reported to be 1.27 mM (approximately 200,000 lg/L), which is not relevant to concentrations in target tissue concentrations or environmental media. Further, although sporadic positive responses were reported, aromatase activity was generally unaffected in the few experiments that tested glyphosate by itself. It should be additionally noted that the EPA OPPTS guideline for conducting the aromatase assay (U.S. Environmental Protection Agency 2009a) clearly warns that all glassware and apparatus used need to be free of detergent residue; this is because microsomes are extremely sensitive to the surfactants found in detergents. Surfactants are a major component of the various herbicide formulations tested in these studies and are the likely cause for the observed aromatase inhibition. Therefore, the claim that glyphosate is an endocrine disruptor is not substantiated. Second, it is not clear to what agent(s) the animals were exposed. The study authors indicate that Roundup Transorb (480 g/L glyphosate, 648 g/L isopropylamine salt, and 594 g/L inert ingredients) was the product administered. However, they report no additional details regarding the composition of the herbicide formulation used such as the identity and amount of surfactant(s) in the formulation. This is important because in other studies, Levine et al. (2007) have shown that steroidogenesis in Leydig cell cultures is inhibited by the presence of surfactants, which perturb membrane function of mitochondria. An appropriate control group that was exposed to the surfactant(s) only was not included in the study nor was there a group exposed to glyphosate only. As such, the study results are confounded. Third, the authors did not describe any methods to control for potential litter effects, and it is not clear whether this was done or not. Because the dams were the unit of exposure in these experiments, the exposed litter (the dam) is the appropriate unit of analysis. This is particularly important when assessing these experiments because dosing of the dams occurred from gestational day 18 through postnatal day 5; however, glyphosate does not cross well