Atrazine stimulates hemoglobin accumulation in Daphnia magna: is it hormonal or hypoxic?

Hemoglobin accumulation in daphnids is an important adaptive response that is regulated by at least two distinct molecular pathways: an endocrine pathway stimulated by terpenoid hormones and an oxygen-sensing pathway involving the hypoxia-inducible factor. We found that the herbicide atrazine elevated hemoglobin levels in Daphnia magna and hypothesized that atrazine induced hemoglobin in daphnids through the hormonal regulatory pathway. This hypothesis was tested by modeling the combined effects of atrazine and the terpenoid hormone mimic pyriproxyfen on hemoglobin mRNA levels assuming the same mechanism of action (concentration addition model) and alternatively, assuming different mechanisms of action (response addition model). Model predictions were then compared to experimental assessments of the combined action of these two chemicals on hemoglobin mRNA levels. Changes in hemoglobin expression were evaluated using real-time RT PCR with primers specific to each of three D magna hemoglobin genes (dhb1, dhb2, and dhb3). Both atrazine and pyriproxyfen significantly elevated levels of the hb2 gene product, while having little effect on hb1 and hb3 gene products. Induction of dhb2 by combinations of atrazine and pyriproxyfen did not conform to the concentration addition predictions. Rather, dhb2 induction by these binary combinations was highly consistent with response addition model predictions. These results indicate that atrazine does not induce hemoglobin through the terpenoid hormone-signaling pathway. Results from this study demonstrate that mixtures modeling can be used to assess a chemical's mechanism of action and that atrazine likely stimulates hemoglobin accumulation through the oxygen-sensing pathway.