The In Vitro Pro-Inflammatory Properties of Water Accommodated Sediment Extracts from a Creosote-Contaminated EPA Superfund Site.

The Southern branch of the Elizabeth River near Portsmouth Virginia, USA, is one of the most creosote-polluted sub-estuaries in North America and former location of the Atlantic Wood Industries (AW) EPA superfund site. We previously demonstrated that adult AW killifish collected in situ had severe hepatic lesions, including hepatoblastoma and hepatocellular carcinoma, as well as suppressed circulating antibody responses compared to a historical reference site. Moreover, several innate immune functions were higher in AW fish, including elevated expression of hepatic cyclo-oxygenase-2 (COX-2), suggesting a pro-inflammatory environment. To further examine the potential of AW contaminants to modulate innate immune function(s) this study used RAW264.7 mouse macrophages as an in vitro model to develop new approach methodologies (NAM) for rapid screening. Lipopolysaccharide (LPS)-stimulated nitric oxide secretion by macrophages is a rapid, sensitive, and predictive in vitro system for screening potentially immunotoxic contaminants as single compounds or as complex mixtures. Compared to the reference site, filter-sterilized AW sediment extracts (water accommodated fractions) induced nitric oxide and IL-6 secretion as well as inducible-nitric oxide synthase (iNOS) and COX-2 proteins at levels comparable to, or higher than induced by LPS treatments alone. Extracts also increased phagocytic activity by macrophages. Using a limulus lysate assay, we show that bacterial endotoxin levels in AW extracts are higher than in reference extracts and polymyxin-B chelation ameliorates pro-inflammatory effects. These findings illuminate the reality of sediment constituents other than toxic compounds previously associated with developmental abnormalities and carcinogenesis in killifish from the AW site. Perhaps these data also suggest the presence of contaminant-adapted consortia of sediment microbes at many heavily polluted sites worldwide compared to less contaminated sites. This article is protected by copyright. All rights reserved.