TDCIPP Exposure Research Articles

Whole-Life-Stage Characterization in the Basic Biology of Daphnia magna and Effects of TDCIPP on Growth, Reproduction, Survival, and Transcription of Genes.

Toxicity tests of chemicals have mainly focused on the partial life-cycle evaluation of model animals. Limited information is available for the evaluation of effects of chemicals from a whole-life-stage exposure perspective. The objective of this study was to perform a whole-life-stage characterization in the basic biology of Daphnia magna (D. magna) and evaluate the effects of a known organophosphate ester (OPE) contaminant, tris(1,3-dichloro-2-propyl) phosphate (TDCIPP), on growth, reproduction, survival, and transcription of genes. The whole-life-stage characterization in growth, reproduction, and survival of D. magna was conducted, and representative sampling time points for the three developmental stages were identified (day 6, day 32, and day 62). Transcriptomic profiles for these three stages were compared, and stage-specific PCR arrays of D. magna were developed. The whole-life-stage exposure to environmentally relevant or greater concentrations of TDCIPP significantly inhibited growth and reproduction of D. magna and decreased survival at the later stage of the exposure experiment (≥32 days). Such adverse effects were not observed in the early stage of the exposure (<32 days), suggesting that short-term toxicity tests, such as the standard 21-day test, might underestimate the environmental risk of TDCIPP. Furthermore, expressions of genes selected at day 6, day 32, and day 62 were significantly changed after TDCIPP exposure, and the changes in the expressions of partial genes were correlated to the inhibitory effects on growth, reproduction, and survival.

Tris(1,3-dichloro-2-propyl) phosphate disrupts axonal growth, cholinergic system and motor behavior in early life zebrafish

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) could have neurotoxic effects and alter motor behaviors in zebrafish (Danio rerio) larvae, however, the underlying mechanisms are still unknown. In this study, zebrafish embryos were subjected to waterborne exposure of TDCIPP at 100, 300, 600, 900μg/L from 2 to 120-h post-fertilization (hpf). Behavioral measurements indicate that TDCIPP exposure significantly elevated spontaneous movement, and altered swimming behavior response of larvae to both light and dark stimulation. Interestingly, in accordance with these motor effects, TDCIPP significantly decreased expression of the neuron-specific GFP in transgenic (HuC-GFP) zebrafish larvae as well as decreased expression of the neural marker genes elavl3 and ngn1, inhibited the axonal growth of the secondary motoneurons and altered the expressions of axon-related genes (α1-tubulin, shha and netrin2) in zebrafish larvae. Furthermore, TDCIPP exposure at 900μg/L significantly increased the activity of acetylcholinesterase (AChE) enzyme, and decreased the total acetylcholine (ACh) concentration. Our data indicate that the alteration in motor neuron and inhibition of cholinergic system could together lead to the TDCIPP induced motor behavior alterations in zebrafish larvae.

Estimated tris(1,3-dichloro-2-propyl) phosphate exposure levels for US infants suggest potential health risks.

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) has been widely used as a flame retardant and is commonly detected in environmental samples. Biomonitoring studies relying on urinary metabolite levels (i.e. bis(1,3-dichloro-2-propyl) phosphate (BDCIPP)) demonstrate widespread exposure, but TDCIPP intake is unknown. Intake data area critical component of meaningful risk assessments and are needed to elucidate the potential health impacts of TDCIPP exposure. Using biomonitoring data, we estimated TDCIPP intake for infants. Infants aged 2-18 months were recruited from central, North Carolina (n=43, recruited 2014-2015), and spot urine samples were analyzed for BDCIPP. TDCIPP intake rates were estimated using daily urine excretion and the fraction of TDCIPP excreted as BDCIPP in urine. Daily TDCIPP intake estimates ranged from 0.01-15.03 μg/kg-day for children included in our assessment, with some variation depending on model assumptions. The U.S. Consumer Products Safety Commission (CPSC) previously established an acceptable daily intake of 5μg/kg-day for non-cancer health risks. Depending on modeling assumptions, we found that 2-9% percent of infants had TDCIPP intake estimates above this threshold. Our results indicate that current TDCIPP exposure levels could pose health risks for highly exposed infants.

Assessment of tris (1, 3-dichloro-2-propyl) phosphate toxicology in PC12 cells by using digital gene expression profiling

Tris (1,3-dichloro-2-propyl) phosphate (TDCIPP), one of the most universally used organophosphate flame retardants (OPFRs), is an environmental pollutant. However, limited information is available regarding its toxicity and environmental health risk. In the present study, PC12 cells provided a useful model for the evaluation of the toxic effects of TDCIPP. Exposure to 7.5, 15, 30, or 60 μM TDCIPP for 72 h inhibited cell viability, and enhanced cellular apoptosis and oxidative stress. To further explore the underlying mechanisms, digital gene expression (DGE) technology was used to identify early transcriptional changes following TDCIPP exposure. Expression of the transcripts of 161 genes was significantly altered upon treatment with TDCIPP. Functional and pathway analysis of the transcriptional profile demonstrated that genes showing significant TDCIPP-associated changes in expression were involved in the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, extracellular matrix–receptor interactions, protein digestion and absorption, and microRNAs in cancer. Using quantitative real-time PCR, we validated the differential expression of selected genes. These results showed that the expression profiles of cells exposed to 60 μM TDCIPP were consistent with the DGE data. Furthermore, western blotting showed that treatment with TDCIPP reduced the Bcl-2/Bax ratio and attenuated PI3K/Akt/Myc signaling. Taken together, these data suggest that TDCIPP exposure can reduce cell viability and induce apoptosis in PC12 cells by inhibiting activation of the PI3K/Akt/Myc signaling pathway. These observations provide valuable preliminary information regarding the mechanisms of TDCIPP-induced toxicity in PC12 cells and indicate that further study of the toxicity of other environmental OPFRs is warranted.

The involvement of autophagy and cytoskeletal regulation in TDCIPP-induced SH-SY5Y cell differentiation

Exposure and toxicity to organophosphate-based flame retardants are an increasing health concern. Neurons appear to be particularly vulnerable to the effects of these chemicals. For example, in vitro studies have shown that tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) induces apoptosis and autophagy in neural cells. In the present study, we investigated the cell biological mechanisms of TDCIPP-induced neurotoxicity using undifferentiated human SH-SY5Y neuroblastoma cells as a model. Interestingly, TDCIPP treatment promoted differentiation in SH-SY5Y cells, which displayed various alterations including neurite elongation, an expansion of the numbers of neurite-bearing cells, and an increase in expression of cytoskeletal components normally enriched in neurons. Furthermore, the upregulation of microtubule-associated protein light chain 3, the degradation of p62/sequestosome 1, and the formation of autophagosomes occurred in treated cells, suggesting that TDCIPP exposure induces autophagy. However, pretreatment with the autophagy inhibitor 3-methyladenine suppressed TDCIPP-induced autophagy and reduced expression of the aforementioned cytoskeletal components. This correlated with a reduction in neurite outgrowth and numbers of neurite-bearing cells. Taken together, these results indicate that autophagy might promote TDCIPP-induced SH-SY5Y cell differentiation, which leads to an increase in expression of cytoskeletal components and neurite outgrowth. This study offers key insights into the mechanisms of neurotoxicity associated with this commonly used organophosphate.

Tris(1,3-dichloro-2-propyl)phosphate Induces Genome-Wide Hypomethylation within Early Zebrafish Embryos.

Tris(1,3-dichloro-2-propyl)phosphate (TDCIPP) is a high-production volume organophosphate-based plasticizer and flame retardant widely used within the United States. Using zebrafish as a model, the objectives of this study were to determine whether (1) TDCIPP inhibits DNA methyltransferase (DNMT) within embryonic nuclear extracts; (2) uptake of TDCIPP from 0.75 h postfertilization (hpf, 2-cell) to 2 hpf (64-cell) or 6 hpf (shield stage) leads to impacts on the early embryonic DNA methylome; and (3) TDCIPP-induced impacts on cytosine methylation are localized to CpG islands within intergenic regions. Within this study, 5-azacytidine (5-azaC, a DNMT inhibitor) was used as a positive control. Although 5-azaC significantly inhibited zebrafish DNMT, TDCIPP did not affect DNMT activity in vitro at concentrations as high as 500 μM. However, rapid embryonic uptake of 5-azaC and TDCIPP from 0.75 to 2 hpf resulted in chemical- and chromosome-specific alterations in cytosine methylation at 2 hpf. Moreover, TDCIPP exposure predominantly resulted in hypomethylation of positions outside of CpG islands and within intragenic (exon) regions of the zebrafish genome. Overall, these findings provide the foundation for monitoring DNA methylation dynamics within zebrafish as well as identifying potential associations among TDCIPP exposure, adverse health outcomes, and DNA methylation status within human populations.

Multigenerational effects of tris(1,3-dichloro-2-propyl) phosphate on the free-living ciliate protozoa Tetrahymena thermophila exposed to environmentally relevant concentrations and after subsequent recovery

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is considered a re-emerging environmental pollutant, and exposure to environmentally relevant concentrations has been shown to cause individual developmental toxicity in zebrafish and the water flea (Daphnia magna). However, multigenerational effects during exposure to TDCIPP and after subsequent recovery were unknown. In the present study, individuals of a model aquatic organism, the ciliated protozoan, T. thermophila were exposed to environmentally-relevant concentrations of TDCIPP (0, 300 or 3000 ng/L) for 60 days (e.g., theoretically 372 generations) followed by a 60-day period of recovery, during which T. thermophila were not exposed to TDCIPP. During exposure and after exposure, effects at the molecular, histological, individual and population levels were examined. Multigenerational exposure to 300 or 3000 ng TDCIPP/L for 60 days significantly decreased numbers of individuals, sizes of individuals, expressed as length and width of bodies, number of cilia, and depth and diameter of basal bodies of cilia, and up-regulated expressions of genes related to assembly and maintenance of cilia. Complete or partial recoveries of theoretical sizes of populations as well as sizes of individuals and expressions of genes were observed during the 60-day recovery period. Effects on number of cilia and depth and diameter of basal body of cilia were not reversible and could still be observed long after cease of TDCIPP exposure. Collectedly, and shown for the first time, multigenerational effects to T. thermophila were caused by exposure to environmentally relevant concentrations of TDCIPP. Also, there were multi-generational effects at the population level that were not caused by carry-over exposure to TDCIPP. The “permanent” alterations and their potential significance are discussed.

Acute Exposure to Tris(1,3-dichloro-2-propyl) Phosphate (TDCIPP) Causes Hepatic Inflammation and Leads to Hepatotoxicity in Zebrafish.

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) has been frequently detected in environmental media and has adverse health effect on wildlife and humans. It has been implicated to have hepatotoxicity, but its molecular mechanisms remain unclear. In the present study, adult male zebrafish were exposed to TDCIPP and global hepatic gene expression was examined by RNA-Seq and RT-qPCR in order to understand the molecular mechanisms of TDCIPP-induced hepatotoxicity. Our results indicated that TDCIPP exposure significantly up-regulated the expression of genes involved in endoplasmic reticulum stress and Toll-like receptor (TLR) pathway, implying an inflammatory response, which was supported by up-regulation of inflammation-related biomaker genes. Hepatic inflammation was further confirmed by histological observation of increase of infiltrated neutrophils and direct observation of liver recruitment of neutrophils labeled with Ds-Red fluorescent protein of Tg(lysC:DsRed) zebrafish upon TDCIPP exposure. To further characterize the hepatotoxicity of TDCIPP, the expression of hepatotoxicity biomarker genes, liver histopathology and morphology were examined. The exposure to TDCIPP significantly up-regulated the expression of several biomarker genes for hepatotoxicity (gck, gsr and nqo1) and caused hepatic vacuolization and apoptosis as well as increase of the liver size. Collectively, our results suggest that exposure to TDCIPP induces hepatic inflammation and leads to hepatotoxicity in zebrafish.

The Flame-Retardant Tris(1,3-dichloro-2-propyl) Phosphate Represses Androgen Signaling in Human Prostate Cancer Cell Lines.

The effects of six organophosphate flame retardants (OPFRs) tris(2-butoxyethyl) phosphate, tris(2-chloroethyl) phosphate, tris(1-chloro-2-propyl) phosphate, tris(methylphenyl) phosphate, tris(1,3-dichloro-2-propyl) phosphate (TDCIPP), and triethyl phosphate on the activities of androgen receptor (AR), estrogen receptor (ER), and aryl hydrocarbon receptor (AhR) were assessed in human prostate and endometrial cancer cells. OPFRs had no effect on ER or AhR target gene activation in ECC-1 cells. The effect of TDCIPP on mRNA and protein accumulation of AR target genes was examined further. AR-inducible gene and protein expression were significantly altered by TDCIPP exposure and repressed PSA levels in conditioned media of prostate cancer cells. We demonstrated that TDCIPP has no affinity for the AR ligand binding domain (AR-LBD) and exerts its antiandrogenic effects in a noncompetitive fashion. Thus, the clinical relevance of TDCIPP exposure on prostate cancer detection and progression to a therapeutically refractile state ought to be investigated further.

Defensive and adverse energy-related molecular responses precede tris (1, 3-dichloro-2-propyl) phosphate cytotoxicity.

To understand the potentially adverse effects of human exposure to tris (1, 3-dichloro-2-propyl) phosphate (TDCIPP) and explore the underlying molecular mechanisms, combined transcriptomic and metabolomic approaches were employed to investigate the molecular responses of two human cell lines exposed to different concentrations of TDCIPP. Comparative analyses of transcriptional and metabolic profiles of HepG2/C3A and A549 cells were performed after exposure to 1, 10 and 100 μM TDCIPP for 24 and 72 h. Stress responses (e.g. xenobiotic metabolism and ABC transporter pathways) were observed at the transcriptional level after 24-h exposure to a sub-cytotoxic concentration (10 μM). Transcription of an energy metabolism-related pathway (oxidative phosphorylation) was down-regulated more severely at 100 μM TDCIPP exposure, accompanied by the suppression of pathways relevant to cell proliferation (e.g. cell cycle and DNA replication), while no significant cytotoxic effects were observed. Functional metabolic changes were observed after 72 h in HepG2/C3A cells exposed to 100 μM TDCIPP that corresponded to changes detected at the transcriptional level after 24 h. Taken together, defensive responses to chemical exposure and energy-related changes both precede the cytotoxic effects of TDCIPP in HepG2/C3A cells.