Introduction The dichlorofluorescein (DCF) assay is a popular method for measuring cellular reactive oxidant species (ROS). Although caveats have been reported with the DCF assay and other compounds, the potential for artifactual results due to cell-free interactions between the DCF compound and toxicants has hardly been explored. We evaluated the utility of the DCF assay for measuring ROS generation by the toxicants mono-(2-ethylhexyl) phthalate (MEHP), and tetrabromobisphenol A (TBBPA). Methods DCF fluorescence was measured spectrofluorometrically after a 1-h incubation of toxicants with 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA). MEHP was incubated with carboxy-H2DCFDA in cell-free solutions of Hank’s buffered salt solution (HBSS), or in Royal Park Memorial Institute (RPMI) medium with or without fetal bovine serum. TBBPA was incubated with carboxy-H2DCFDA in cell-free HBSS and with human trophoblast cells (HTR8/SVneo cells). Results MEHP did not increase fluorescence in solutions of carboxy-H2DCFDA in HBSS or RPMI medium without serum. However, MEHP (90 and 180 μM) increased DCF fluorescence in cell-free RPMI medium containing serum. Furthermore, serum-free and cell-free HBSS solutions containing 25 μM TBBPA exhibited concentration-dependent increased fluorescence with 5–100 μM carboxy-H2DCFDA (p<0.05), but not 1 μM carboxy-H2DCFDA. In addition, we observed increased fluorescence in HTR8/SVneo cell cultures exposed to TBBPA (0.5–25 μM) (p<0.05), as we had observed in cell-free buffer. Discussion MEHP demonstrated an interaction with serum in cell-free generation of DCF fluorescence, whereas TBBPA facilitated conversion of carboxy-H2DCFDA to the fluorescent DCF moiety in the absence of serum. Because TBBPA increased fluorescence in the absence of cells, the increased DCF fluorescence observed with TBBPA in the presence of cells cannot be attributed to cellular ROS and may, instead, be the result of chemical activation of carboxy-H2DCFDA to the fluorescent DCF moiety. These data illustrate the importance of including cell-free controls when using the DCF assay to study toxicant-stimulated cellular production of ROS.

Polybrominated diphenyl ethers (PBDEs) are widely used flame retardant compounds. Brominated diphenyl ether (BDE)-47 is one of the most prevalent PBDE congeners found in human breast milk, serum and placenta. Despite the presence of PBDEs in human placenta, effects of PBDEs on placental cell function are poorly understood. The present study investigated BDE-47-induced reactive oxygen species (ROS) formation and its role in BDE-47-stimulated proinflammatory cytokine release in a first trimester human extravillous trophoblast cell line, HTR-8/SVneo. Exposure of HTR-8/SVneo cells for 4 h to 20 μM BDE-47 increased ROS generation 1.7 fold as measured by the dichlorofluorescein (DCF) assay. Likewise, superoxide anion production increased approximately 5 fold at 10 and 15 μM and 9 fold at 20 μM BDE-47 with a 1-h exposure, as measured by cytochrome c reduction. BDE-47 (10, 15 and 20 μM) decreased the mitochondrial membrane potential by 47–64.5% at 4, 8 and 24 h as assessed with the fluorescent probe Rh123. Treatment with 15 and 20 μM BDE-47 stimulated cellular release and mRNA expression of IL-6 and IL-8 after 12 and 24 h exposures: the greatest increases were a 35-fold increased mRNA expression at 12 h and a 12-fold increased protein concentration at 24 h for IL-6. Antioxidant treatments (deferoxamine mesylate, (±)α-tocopherol, or tempol) suppressed BDE-47-stimulated IL-6 release by 54.1%, 56.3% and 37.7%, respectively, implicating a role for ROS in regulation of inflammatory pathways in HTR-8/SVneo cells. Solvent (DMSO) controls exhibited statistically significantly decreased responses compared with non-treated controls for IL-6 release and IL-8 mRNA expression, but these responses were not consistent across experiments and times. Nonetheless, it is possible that DMSO (used to dissolve BDE-47) may have attenuated the stimulatory actions of BDE-47 on cytokine responses. Because abnormal activation of proinflammatory responses can disrupt trophoblast functions necessary for placental development and successful pregnancy, further investigation is warranted of the impact of ROS and BDE-47 on trophoblast cytokine responses.

Tetrabromobisphenol A (TBBPA) is a widely used flame retardant. Despite the presence of TBBPA in gestational tissues and the importance of proper regulation of inflammatory networks for successful pregnancy, there is no prior study on the effects of TBBPA on inflammatory responses in gestational tissues. The present study aimed to investigate TBBPA activation of inflammatory pathways, specifically cytokine and prostaglandin production, in the human first trimester placental cell line HTR-8/SVneo. TBBPA enhanced release of interleukin (IL)-6, IL-8, and prostaglandin E2 (PGE2), and suppressed TGF-β release in HTR-8/SVneo cells. The lowest effective concentration was 10 μM TBBPA. A commercial immune response PCR array revealed increased expression of genes involved in inflammatory pathways stimulated by TBBPA in HTR-8/SVneo cells. Because proper regulation of inflammatory mediators in the gestational compartment is necessary for normal placental development and successful pregnancy, further investigation on the impact of TBBPA-stimulated responses on trophoblast function is warranted.

Developing effective, accessible, and affordable training programs for emergency response personnel working with Deaf and hard of hearing persons, some of whom belong to the older adult population, will require a collaborative effort among emergency response agencies, public health organizations, and members of the affected communities. (Disaster Med Public Health Preparedness. 2018;12:606-614).