Cell Death Effects of the Phthalate 2-Ethyl-1-Hexanol on Human Linfoblast Cells

Abstract

Phthalates have been used in a wide variety of consumer goods. Their versatility as plasticizers has translated into worldwide use in a vast array of consumer products. These compounds can leach into matrices, such as food and liquids that can be routed for human exposure. One of the most used phthalates is Diethylhexyl phthalate (DEHP). Diethylhexyl phthalate and its metabolite 2-ethyl-1-hexanol (2-EH) have demonstrated biological effects which merit further evaluation. In this work, we expand on our previous work with DEHP and screen the 2-EH metabolite for different cell death endpoints such as growth inhibition, apoptosis, autophagy, caspase activation, DNA fragmentation, and cell cycle arrest using fluorophores and the NC3000 instrument. Significant results (p < 0.05) revealed higher toxicity for the 2-EH metabolite when compared to DEHP. Also, 2-EH presented apoptosis induction with characteristic hallmarks, such as loss of mitochondrial membrane potential, caspase activation, DNA fragmentation and cell cycle arrest at the S phase. In addition, the presence of autophagosome was detected through L3CB protein staining. We conclude that 2-EH presents differences in cell death endpoints that interestingly differ from the DEHP parent compound. Further studies are needed to establish the molecular pathways responsible for the observed effects.

Keywords: 2-Ethyl-1-Hexanol; Apoptosis; Autophagy; Caspase; Diethylhexyl Phthalate; Lymphoblast; Phthalates.

Figure 1.

DEHP and its metabolites. MEHP and 2-EH are the principal metabolites from DEHP.

Figure 2.

24 exposure dose response 2-EH. This compound presented average toxicity of 4.4 μM. Cell death effects present dose-dependent toxicity. Figure 2 presents results from 5 doses with average data from 3 experiments; error bars indicate standard deviation.

Figure 3.

Annexin V apoptosis assay. After a 24-hour exposure period our results reveal statistically significant (p < 0.05) apoptosis induction by all the test compounds. DEHP was the lowest inducer of apoptosis but still presented significant induction when compared to the negative vehicle control.

Figure 4.

Mitochondrial membrane potential. Negative samples reflect an average of 3380 Fluorescence Standard Units (FSU) while the camptothecin control presented an average of 2130 FSU. The phthalate samples reflect values of 1797 FSU for DEHP and 556 FSU for 2-EH. The 2-EH sample presents the least number of cells with intact mitochondrial membrane potential.

Figure 5.

Cells with fragmented DNA. the negative control presented an average of 14.2% and 45.6% for the positive camptothecin control. DEHP presented an average of 25.6% and 2-EH (75.3%) presented a majority of cells with fragmented DNA. DEHP value was not statistically significant (p < 0.05) when compared to the negative control.

Figure 6.

Caspase 3 activation. Only camptothecin and the 2EH samples presented caspase 3 activation. DEHP presented 10.6% of cells with active caspase 3 comparable to the negative control (8.6%). Only the positive control camptothecin (74.6%) and 2-EH (82.0%) presented statistically significant (p < 0.05) caspase 3 activation.

Figure 7.

Caspase 8 activation. Negative vehicle sample averaged 9.6% of caspase 8 active cells with the positive camptothecin averaging 37.6% activation. DEHP samples presented 25.0% and 84.0% for 2-EH. Both phthalate compounds activated caspase 8.

Figure 8.

Caspase 9 activation. The negative control presented a low 14.5% of active caspase 9. Camptothecin samples averaged 62% and the phthalate treated samples also presented high activation of caspase 9 with an average 30% for DEHP and 83.25% for 2-EH.

Figure 9.

Cell cycle arrest. All the treated samples show some level of arrest at the S (synthesis) phase of the cell cycle. Camptothecin presents 48.0% of cells arrested at the S phase. DEHP and 2-EH presented 36.0% and 34.0% of cells arrested at the S phase respectively.

Figure 10.

Autophagy activation. The negative control presented the lowest average FSU value of 852. Samples exposed to the test compounds presented statistically significant differences (p < 0.05) when compared to the negative control. Chloroquine averaged 1120 FSU while the DEHP and 2-EH averaged 1287 and 1984 FSU respectively.

Figure 11.

Detection of cathepsin B. Negative sample presented an average of 24,810.3 FSU and the 2-EH sample an average of 24,278.0 FSU. Positive control chloroquine samples presented an average of 36,947.5 FSU and the DEHP samples 46,342.2 FSU. Of the phthalate compounds, only DEHP was positive for cathepsin B staining.