Tris(1,3-dichloro-2-propyl) phosphate disrupts cellular metabolism within human embryonic kidney (HEK293) cells.

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is a widely used, additive flame retardant that migrates from end-use products, leading to ubiquitous exposure of humans around the world. However, little is known about whether TDCIPP disrupts the physiology of human embryonic cells. Therefore, the objective of this study was to determine whether TDCIPP alters cell viability, cellular metabolism, cytosine methylation, and reactive oxygen species (ROS) levels within human embryonic kidney (HEK293) cells. Relative to vehicle controls, TDCIPP (0.015-0.1225 µM) resulted in a concentration-dependent increase in cell viability, a finding that was driven by an increase in relative ATP abundance. Interestingly, TDCIPP (0.061-0.98 µM) increased the rate of glycolysis - an adaptive mechanism consistent with the Warburg effect exhibited by tumorigenic cells. Moreover, relative to vehicle-treated cells, TDCIPP (0.245-15.63 µM) exposure for 48 h (but not 24 h) resulted in a significant, concentration-dependent decrease in ROS in situ, and TDCIPP (0.245 µM) exposure significantly increased carnosine within the histidine metabolism pathway. However, TDCIPP did not affect global 5-methylcytosine (5-mC) methylation (0.015-15.63 µM), cell membrane integrity (0.061-0.98 µM), nor the abundance of mitochondria (0.061-1.95 µM). Overall, our findings with TDCIPP point to a novel mechanism of action that may be relevant to human embryonic stem cells.

Tris(1,3-dichloro-2-propyl) phosphate disrupts the trajectory of cytosine methylation within developing zebrafish embryos.

Tris (1,3-dichloro-2-propyl) phosphate (TDCIPP) is an organophosphate ester-based flame retardant widely used within the United States. Within zebrafish, initiation of TDCIPP exposure at 0.75 h post-fertilization (hpf) reliably disrupts cytosine methylation from cleavage (2 hpf) through early-gastrulation (6 hpf). Therefore, the objective of this study was to determine whether TDCIPP-induced effects on cytosine methylation persist beyond 6 hpf. First, we exposed embryos to vehicle or TDCIPP from 0.75 hpf to 6, 24, or 48 hpf, and then conducted bisulfite amplicon sequencing of a target locus (lmo7b) using genomic DNA derived from whole embryos. Within both vehicle- and TDCIPP-treated embryos, CpG methylation was similar at 6 hpf and CHG/CHH methylation were similar at 24 and 48 hpf (relative to 6 hpf). However, relative to 6 hpf within the same treatment, CpG methylation was lower within vehicle-treated embryos at 48 hpf and TDCIPP-treated embryos at 24 and 48 hpf - an effect that was driven by acceleration of CpG hypomethylation. Similar to our previous findings with DNA methyltransferase, we found that, even at high µM concentrations, TDCIPP had no effect on zebrafish and human thymine DNA glycosylase activity (a key enzyme that decreases CpG methylation), suggesting that TDCIPP-induced effects on CpG methylation are not driven by direct interaction with thymine DNA glycosylase. Finally, using 5-methylcytosine (5-mC)-specific whole-mount immunochemistry and automated imaging, we found that exposure to TDCIPP increased 5-mC abundance within the yolk of blastula-stage embryos, suggesting that TDCIPP may impact cytosine methylation of maternally loaded mRNAs during the maternal-to-zygotic transition. Overall, our findings suggest that TDCIPP disrupts the trajectory of cytosine methylation during zebrafish embryogenesis, effects which do not appear to be driven by direct interaction of TDCIPP with key enzymes that regulate cytosine methylation.

Using a variant of the optomotor response as a visual defect detection assay in zebrafish.

We describe a visual stimulus that can be used with both larval and adult zebrafish (Danio rerio). This protocol is a modification of a standard visual behavior analysis, the optomotor response (OMR). The OMR is often used to determine the spatial response or to detect directional visuomotor deficiencies. An OMR can be generated using a high contrast grated pattern, typically vertical bars. The spatial sensitivity is measured by detection and response to a change in grating bar width and is reported in cycles per degree (CPD). This test has been used extensively with zebrafish larvae and adults to identify visual- and/or motor-based mutations. Historically, when tested in adults, the grated pattern was presented from a vertical perspective, using a rotating cylinder around a holding tank, allowing the grating to be seen solely from the sides and front of the organism. In contrast, OMRs in zebrafish larvae are elicited using a stimulus projected below the fish. This difference in methodology means that two different experimental set-ups are required: one for adults and one for larvae. Our visual stimulus modifies the stimulation format so that a single OMR stimulus, suitable for use with both adults and larvae, is being presented underneath the fish. Analysis of visuomotor responses using this method does not require costly behavioral tracking software and, using a single behavioral paradigm, allows the observer to rapidly determine visual spatial response in both zebrafish larvae and adults.

Acute developmental exposure to 4-hydroxyandrostenedione has a long-term effect on visually-guided behaviors.

Estrogenic and anti-estrogenic endocrine disrupting compounds (EDCs) are recognized as critical modulators of neural development, including sensory system development. Using the zebrafish model, we tested the effect of transient developmental exposure to a known anti-estrogenic EDC on adult visually-guided behavior. In particular, we exposed zebrafish aged 24-hour post-fertilization (hpf), 72 hpf, or 7-days post-fertilization (dpf) to the aromatase inhibitor 4-hydroxyandrostenedione (4-OH-A) for 24h. After this time, the fish were removed from treatment, placed into control conditions, and reared until adulthood (3-4months) when visually-guided optomotor responses (OMR) were assessed. Our results show significant decreases in positive OMR in adults exposed to 4-OH-A at 72 hpf and 7 dpf. These deficits were not accompanied by changes in overall swimming behaviors and startle responses, suggesting 4-OH-A specifically effected the visual system. Overall, this study identified long-term, quantifiable effects in visually-guided adult behaviors resulting from transient developmental exposure to the anti-estrogenic EDC, 4-OH-A. Further, these effects were noted when 4-OH-A exposure occurred after hatching, suggesting estrogen signaling is important for visual system maturation.