Arsenic, benzene, trichloroethene (TCE), formaldehyde, and polycyclic aromatic hydrocarbons (PAHs) may have endocrine-disrupting effects at lower doses than those required for hematotoxicity or cancer induction. There is some evidence that they can modulate steroid hormone levels in both the corticosteroid and sex steroid pathways, potentially causing multiple adverse health effects. Here we propose to examine the disrupting effects of these common Superfund contaminants on glucocorticoid and sex steroid hormones and their tissue-specific effects in cell lines in vitro, mice in vivo, and perhaps most importantly in humans with well characterized exposures.  Endogenous cortisol is produced in response to stress and acts on the glucocorticoid receptor (GR) triggering metabolic and other disruptions. Environmental chemicals can also impact the GR pathway and are defined as “stressogens.”  An example is arsenic, which alters GR signaling, but the mechanisms by which this impacts different cell types expressing GR are unclear.  Cumulative risk assessment of non-genetic environmental stressors is a priority of EPA and we have developed novel approaches to do so under the exposome paradigm.  Our first central hypothesis is that arsenic is a stressogen that alters GR activity, alone and in conjunction with other chemicals, with tissue-specific biological consequences.  We propose to use an exposomic approach to understand the dose-dependent effects of arsenic, alone and in conjunction with other Superfund contaminants, on the glucocorticoid pathway in liver, adipocyte and blood cells in vitro; in mice in vivo; and in exposed humans (Aims 1-3).  We will use a sensitive GR activity bioassay to screen Superfund contaminants that alter GR activity and will measure total glucocorticogenic (G) activity in media from exposed cells and in small volumes of human plasma from subjects exposed to arsenic.  Use of the GR bioassay directly with plasma is novel. Our preliminary screening data show that arsenic is a GR antagonist and the PAH benzo(a)pyrene, an agonist.  Arsenic, as well as TCE, benzene, formaldehyde and PAHs also cause adverse reproductive effects potentially by modulating natural estrogen and androgen sex hormones.  Thus, our second central hypothesis is that these contaminants modulate estrogen receptor (ER) and androgen receptor (AR) activity by altering natural sex hormone levels. We will screen chemical effects on the production of these hormones (steroidogenesis) in validated mammalian cell models, and total estrogenic (E) and androgenic (A) activity and endogenous hormone levels in plasma from exposed populations (Aims 4-5).  Our preliminary data show that TCE increases E but not A activity in exposed males probably through aromatase induction. Our overall goal is to understand the dose-dependent effects of key Superfund chemicals on steroid hormone pathways, to support comprehensive and cumulative risk assessment.