The In Vitro Proinflammatory Properties of Water Accommodated Sediment Extracts from a Creosote-Contaminated US Environmental Protection Agency Superfund Site.

Abstract

The southern branch of the Elizabeth River near Portsmouth, Virginia, USA, is one of the most creosote-polluted subestuaries in North America and the former location of the Atlantic Wood US Environmental Protection Agency Superfund site. We previously demonstrated that adult Atlantic Wood 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hepatoblastoma and hepatocellular carcinoma, as well as suppressed circulating antibody responses compared to a historical reference site.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 Atlantic Wood fish, including elevated expression of hepatic cyclooxygenase-2 (COX-2), suggesting a proinflammatory environment. To further examine the potential of Atlantic Wood contaminants to modulate innate immune function(s), the present study used RAW264.7 mouse macrophages as an in vitro model to develop new approach methodologies 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 Atlantic Wood sediment extracts (water accommodated fractions) induced nitric oxide and IL-6 secretion as well as inducible nitric oxide synthase and COX-2 proteins at levels comparable to or higher than those induced by LPS treatments alone. Extracts also increased phagocytic activity by macrophages. Using a limulus lysate assay, we show that bacterial endotoxin levels in Atlantic Wood extracts are higher than in reference extracts and that polymyxin-B chelation ameliorates proinflammatory effects. These findings illuminate the reality of sediment constituents other than toxic compounds previously associated with developmental abnormalities and carcinogenesis in killifish from the Atlantic Wood 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. Environ Toxicol Chem 2021;40:1576-1585. © 2021 SETAC.